ISSN 0254-4725

Manuals of food
quality control

FAO
FOODAND
NUTRITION
PAPER

8. Food analysis: quality, adulteration

and tests of identity

Fflt l i r t H l M M U

n^X

Food
and
Agriculture
Organization
of
the
United
Nations

Manuals of food
quality control
,

8. Food analysis: quality, adulteration

and tests of identity

FAO
FOODAND
NUTRITION
n u ni i un

paper

1 4 / 8

prepared with the support of the

Swedish International Development Authority (SIDA)

Food
and
Agriculture
Organization
of
the
United
Nations

Rome, 1986

Reprinted 1997

The designations employed and the presentation of material in this

publication do not imply the expression of any opinion whatsoever on
the part of the Food and Agriculture Organization of the United
Nations concerning the legal status of any country, territory, city or
area or of its authorities, or concerning the delimitation of its frontiers
or boundaries.

M-82
ISBN 92-5-102412-X

All rights reserved. No part of this publication may be reproduced, stored in a

retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying or otherwise, without the priorpermission of the copyright owner.
Applications for such permission, with a statement of the pu rpose and extent of the
reproduction, should be addressed to the Director, Information Division, Food and
Agriculture Organization of the United Nations, Viale delle Terme di Caracalla,
00100 Rome, Italy.
FAO 1986

FOREWORD

The control of food safety and quality is an integral part of n a t i o n a l

programmes for development.
National food control systems are designed to
protect the health and welfare of the consumer, to promote the development of
trade in food and food products, and to protect the interests of the fair and
honest food p r o d u c e r , processor or marketer against dishonest and unfair
competition.
Emphasis is placed on the prevention of chemical and biological
hazards which result from contamination, adulteration or simple mishandling of
foods.
Also important are the maintenance of general food quality and the
control of the use of food additives and food processing procedures.
In order
must :

to

establish

a workable

food

control

system,

national

government

1.

Enact food control legislation.

2.

Promulgate regulations to enforce that legislation.

3.

Create an agencv to conduct the enforcement.

4.

Establish food inspection

agencies concerned.

5.

Provide physical facilities including a food control laboratory.

and

analysis

staff

within

the

agency

or

To assist the national governments of developing countries in this process, FAO,

with the support of the Swedish International Development Authority (SIDA) has
published the series Manuals of Food Quality Control. These are incorporated as
part of the FAO Food and Nutrition Paper Series No. 14, and include:
No. 14/1

The Food Control Laboratory

No. 14/2

Additives, Contaminants, and Techniques

No. 14/3

Commodities

No. 14/4

Microbiological Analysis

No. 14/5

Food Inspection

No. 14/6

Food for Export

No. 14/7

Food Analysis: General Techniques, Additives,

Contaminants, and Composition

No. 14/8

Food Analysis:

(out of print)

(out of print)

Quality, Adulteration, and Tests of Identity

In addition, FAO, WHO and UNEP jointly have published many guidelines and other
documents designed to further assist developing countries in forming adequate
food control systems. These publications include:
Methods of Sampling and Analysis of Contaminants in Food
A Report of the Second Joint FAO/WHO Expert Consultation,
Rome - 1978
Guidelines for Establishing or Strengthening National Food
C o n t a m i n a t i o n M o n i t o r i n g Programmes - FAO Food Control
Series No. 5 - 1979

iii

Guidelines for the Study of Dietary Intakes of Chemical

Contaminants - WHO Offset Publication No. 87 - 1985
Guide
to
Codex
Recommendations
concerning
Pesticide
Residues, Part 2 - Maximum Limits for Pesticide Residues,
Second Preliminary Issue - Rome - 1985
Recommended Practices for the Prevention of Mycotoxlns in
F o o d , Feed and their Products - FAO Food and Nutrition
Paper No. 10, Rome - 1979
Food Standards, Codes of Practice and Methods of Analysis
Recommended by the Codex Alimentarius Commission - Joint
FAO/WHO Food Standards Programme (several titles)
Food Additive Evaluations and Specifications of Purity and
Identity - Reports and Monographs of the Joint FA0/WH0
Expert Committee on Food Additives (several titles)
The above publications, and others, are available to persons and organizations.
FAO is also interested in receiving comments regarding this volume and
suggestions for future improvement. Please send to:
The Chief
Food Quality and Standards Service
Food Policy and Nutrition Division
Food Agriculture Organization of the
United Nations
Via delle Terme di Caracalla
00100 Rome, Italy
FAO wishes
Development
of Mr. J.
preparation

to acknowledge the generous support of the Swedish International

Authority (SIDA), in the preparation of this volume, and the efforts
Weatherwax and Mr. P.G. Martin who were responsible for the
of the text.

iv

SPECIAL HOTE
The methods and analytical procedures described in this
Manual are designed to be carried ont by properly trained
personnel in a suitably equipped laboratory. In comson vith
any laboratory procedures, the methods quoted frequently
involve hasardous materials.
For the correct and safe execution of these methods it is
essential that laboratory personnel follow standard safety
procedures for the handling of hazardous materials.
While the greatest care has been exercised in the
preparation of this information, FAO expressly disclaims
any l i a b i l i t y to u s e r s of t h e s e p r o c e d u r e s for
consequential damages of any kind arising out of or
connected with their use.
The methods are also not to be regarded as official because
of their inclusion in this Manual. They are simply methods
which have been found by experience to be usable in the
average laboratory.

COMTEITS

1.

SCOPE OF THIS M A H U A L OP POOD A N A L Y S I S

2.

MILK AHD DAIRY PRODUCTS

2.1

Analysis

Dried Milk
Discussion

Analysis

2.3

2.4

Whole Milk
Discussion

2.2

Composition
Routine Analysis
Detection of Adulteration
Off-flavors
Mastitis
Antibiotics
Sugars
Non-bovine Milk
Milk Fat (Gerber Method)
Milk Fat (Rose-Gottlieb Method)
Total Solids (Rapid Method)
Total Solids (Weight Method)
Freezing Point
Milk Acidity
Nitrates in Milk
Phosphatase Test
Turbidity Test

2
3
3
4
5
6
6
7
8
10
12
14
15
20
22
23
26

Composition
Routine Analysis
Equivalence
. . .
Dried Milk Moisture
Dried Milk Solubility
Dried Milk Lactate

27
27
28
30
31
32

. .

Evaporated and Condensed Milk

Discussion
- Composition
- Routine Analysis
Equivalence
Analysis
- Total Solids
Sucrose

Butter and Ghee (Butter Oil)

Discussion
- Composition
Routine Analysis
Analysis
_ - Moisture in Butter . . .
Foreign Fats in Butter (Hydroxamic Acid Index) .
Foreign Fats in Butter (Reichert-PolenskiRirschner Values)
Foreign Fats in Butter as an Ingredient
(R-P-K Semimicro Method)
....
- Vegetable Fat in Butterfat (TLC Method)
- Vegetable Fat in Butterfat (GLC Method)
. . . .

vii

34
34
35
37
38

41
42
43
44
47
54
57
63

2.5

2.6

2.7

3.

3.2

3.3

3.4

Cheese and Other

Discussion
Analysis
-

Composition
Routine Analysis

68
69

frrodncta
Composition
Routine Analysis
Phosphatase Activity (in Dairy Products) . . . .

71
71
73

Text References

SUGARS

3.1

4.

Ice Crea*
Discussion

AID

77

HOMEY

85

Sucrose (White Sugar)

Discussion

Analysis

Sugar Products
Discussion
Analysis

Honey
Discussion

Analysis

Composition
Routine Analysis
Polarimetry
White Sugar (Polarization Method)
Invert Sugar in White Sugar
(Knight and Allen Method)
Invert Sugar in White Sugar
(Berlin Institute Method)
Conductivity Ash
Loss on Drying
Colour Index

94
96
99
101

Composition
Routine Analysis
Sample Preparation - Sugars (Alcohol Extraction)
Sample Preparation - Sugars (Clarification)
. .
Sugars Identification (TLC Method)
Sugars Analysis (GLC Method)
Invert Sugars with Added Sucrose

103
103
105
106
107
108
110

Text References

Adulteration
Discussion
Analysis

92

Composition
114
Routine Analysis
115
Sample Preparation - Honey (Clarification) . . . 118
Moisture in Honey
119
Diastase Activity of Honey
121
Acidity and Lactone in Honey
123
Proline in Honey
124
Dextrose in Honey
125
Apparent Sucrose in Honey
126
Hydroxymethylfurfural in Honey
127
129

FISH AHD SHELLFISH

4.1

85
85
86
90

134

Routine Analysis . . .
Fish Spec es Identification

viii

134
136

4.2

4.3

5.

Text References

Routine Analysis . .
Total Volatile Bases
Trimethy lamine (TMA) in Fish
Indole
.
Histamine
Boric Acid

. . . .

5.2

5.3

6.2

6.3

AND

154
156
. 157
159

162
163
165
169

174

FRUITS

176

Fresh Vegetables and Fruits

Discussion
- Routine Analysis

176

Canned Vegetables and Fruits

Discussion
- Composition
Routine Analysis
Can Examination
Tomato Products
Analysis
- Drained Weight
Fill of Container
Soluble Solids (Tomato Products)

177
178
179
182
183
184
185

Juices
Discussion

Analysis

6.4

Routine Analysis
Meat Content
Hydroxyproline
Nitrate and Nitrite

Text References

VEGETABLES

6.1

154

Freah and Frozen Meat

Discussion
- Routine Analysis
Analysis
- Total Volatile Bases
Thiobarbituric Acid Value
Free Fatty Acids and Peroxide Value

Meat Products
Discussion
Analysis
-

139
140
141
143
146
149

151

MEAT AND MEAT PRODUCTS

5.1

6.

Decomposition
Discussion Analysis
-

Composition
Routine Analysis
Fruit Content (Formol Number Method)
Organic Acids
Vitamin C

Text References

187
188
189
191
194

195

ix

7.

CEREALS, CEREAL PRODUCTS AND PULSES

7.1

7.2

7.3

7.4

8.

197

Whole Grain (Unmilled) Products

Discussion
- Routine Analysis
Analysis
- Glycosidic Cyanide
- Talc on Rice or Barley

197
198
200

Flours and Milled Products

Discussion
- Routine Analysis
- Microscopic Identification
Analysis
- Acidity in Flour (Water Extract)
- Acidity in Flour (Alcohol Extract)
- Ash in Flour
Iron in Flour

Bread
Discussion
Analysis

Routine Analysis
Moisture in Bread

223
224

Text References

225

HERBS AHD SPICES

8.1

8.2

8.3

8.4

8.5

Herbs
Discussion

201
201
. 218
219
220
221

226

Spices - Seeds
Discussion
Analysis
-

Composition

226

Composition
Routine Analysis . . .
Identification
Umbelliferous Seeds (TLC Identification) . . . .
Non-volatile Extract
Volatile Oil

227
227
228
237
238
239

Spicea - Pods and Fruits

Discussion
- Composition
Identification
Analysis
- Capsaicin

241
241
244

Spices - Roots, Barks and Flowers

Discussion
- Composition
Identification
Analysis
- Colouring Power of Saffron

246
246
251

Text References

252

9.

255

OILS AKD FATS

9.1

Vegetable Oils
Discussion
Analysis

9.2

9.3

9.4

10.

Margarine
Discussion

Analysis

Animal Fats
Discussion
Analysis

Text

Composition
Routine Analysis
Saponification Value
Iodine Value (Hanus Method)
Unsaponifiable Matter
Acid Value
Peroxide Value
Titre
Soap Test in Edible Oils
Arachis (Groundnut) Oil Test
Cottonseed Oil Test
Sesame Oil Test
.
Teaseed Oil Test
Identification of Oils and Fats
(GLC of Fatty Acid Methyl Esters)
Alternative Methyl Ester Preparation Method
(Neutral Oils and Fats)
Alternative Methyl Ester Preparation Method
(Acidic Oils and Fats)

255
256
258
259
261
263
264
266
268
269
271
272
273

Composition
Routine Analysis
Monoglycerides in Margarine
Vitamin A in Margarine

284
285
286
288

Composition
Free Fatty Acids
Thiobarbituric Acid Value

291
292
293

fteferences

274
280
282

294

BEVERAGES

296

10.1 Alcoholic - Distilled

Discussion
- Composition
Routine Analysis
Analysis
- Ethanol
- Methanol
- Higher Alcohols
- Acidity

296
296
298
301
302
304

10.2 Alcoholic - Fermented

Discussion
- Composition
Routine Analysis

306
307

xi

10.3 Tea
Discussion
Analysis

10.4 Coffee
Discussion
Analysis

Composition
Routine Analysis
Microscopic Examination
Caffeine in Tea
Extractives from Tea
Stalk in Tea

Composition
Routine Anaysis
Microscopic Examination of Coffee
Caffeine in Coffee

10.4 Text References

of Tea
*

309
309
311
313
315
316

317
317
319
320

324

APPEHDIX - Abbreviations Used in the Manual

x ii

325

1.

SCOPE OF THIS MAIUAL 07 FOOD ANALYSIS

This Manual covers nine food groups which include most of the foods consumed
throughout the world.
Individual foods within a food group (such as 'Dried
M i l k 1 within 'Milk and Dairy Products') are discussed regarding their
composition and/or routine analysis, as well as other appropriate features.
Compositional i n f o r m a t i o n m a y include published analytical data as w e l l as
international standards such as those of the Codex A l i m e n t a r i u s , and some
individual country legal standards. All these are to serve only as a guide in
the event that local standards are not available.
The routine analytical
information is also to serve as a guide and often includes alternative
procedures as well as background literature sources.
The analytical methods given for each food are generally those with specific
application to the food. Some are proximate analyses while others include
tests) for c o n t a m i n a t i o n or adulteration.
Note that all general analytical
methods for contaminants and additives are found in the Manual, "Food Analysis:
Techniques, Additives, C o n t a m i n a n t s , Composition." . Also note that for all
analytical procedures, the following precautions apply:
1.
Use only distilled water or the equivalent.
suitable).

(Deionized water is often

2.
Use the
necessary.

available

3.
Follow
empirical.
4.

best

grade

the method

of

reagent

chemicals

and

purify

instructions exactly as m a n y of the procedures

if

are

Use all laboratory safety procedures and equipment.

The reference section at the end of each food group gives both the references
listed in the text, as well as some general references which may provide
background information.
The first edition of this Manual was written in 1977 by Mr. Peter G. Martin
presently of Lyne, Martin and Radford, Public Analysts, Reading, Berkshire,
England.
The present revised edition has been prepared with Mr. M a r t i n ' s
support and assistance by Mr. John R. Weatherwax, retired Laboratory Director
for the United States Food and Drug A d m i n i s t r a t i o n , Los Angeles, California,
USA.

2.
2.1

MILK AMD DAIRY PRODUCTS

WHOLE MILK

COMPOSITION
The c o m p o s i t i o n of bovine m i l k varies w i d e l y depending on a large number of
factors including breed, season, stage of lactation, milking interval, health
of the cow, and level and type of feed. Quantities of milk large enough (say,
over 10,000 litres) and from sources divergent enough to obliterate these
variations will tend towards a typical composition given by various authorities
as follows:

Richmond(1)

Davis(l)

Pearson(2)

Webb et al(3)

Fat

3.75

3.67

3.61

3.5 - 3.7

Protein

3.20

3.42

3.29

3.5

4.70

4.78

4.65

4.9

0.75

0.73

0.75

0.7

Lactose,
hydrated
Ash

Webb et al (3) have collated the compositional data for milks from 15 countries
and m a n y breeds and the averages of the analytical results fell w i t h i n the
following ranges:

Total

solids:

11.52 - 14. 56%

Fat :

3.75 -

5. 52%

Protein :

2.87 -

3. 76%

Lactose hydrate:

4.34 -

4. 98%

Ash :

0.66 -

0. 72%

The fat content of milk is especially variable. The milk fat of certain breeds
such as Jersey and Guernsey rises to 8% or higher, w h i l e that from Friesians
may be close to 3%. Normal milk from individual cows or small herds may have a
composition outside the range quoted above.
The mineral and citric acid composition of milk is typically as follows:

mg/100 ml whole milk:

About 79-80%
derived from
milk, the ash
above 0.6% of

Ca

Mg

la

123

12

95

58

141

Cl

110

30

Citric
Acid
160

of the n i t r o g e n in m i l k is present as casein, the rest being

albumin, globulin, proteoses and non-protein nitrogen. In normal
is 8Z of the solids-not-fat (SNF) and the albumin is not normally
the whole milk.

The composition of the ash of milk lies within the following ranges:

% of ash:

Fe

K20

CaO

MazO

MgO

23-30

20-27

6-12

2.3-3.1

23

0.05-0.4

25
21-20

C1

13.6-16.4

S0

0-4

ROUTINE AHALYSIS
The chemical tests that must be carried out immediately on receipt of the milk
at the laboratory are the determinations of fat and solids-not-fat.
If these
The
are b e l o w the expected v a l u e s , the freezing point m a y be d e t e r m i n e d .
freezing-point test is invalidated by the presence of formaldehyde, often used
as a preservative for samples, but not by the mercuric chloride/salt mixture
r e c o m m e n d e d by Harding and Royal (4). If this is used, the s a m p l e s m u s t be
clearly labelled "Poison".
The test is affected by the acidity and corrections
have to be m a d e if this is higher than 0.18% expressed as lactic acid. If the
acidity exceeds 0.30% as lactic acid, the f r e e z i n g - p o i n t test is not valid.
Nitrates do not n o r m a l l y occur in m i l k and t h e r e f o r e d e t e c t i o n of these is
confirmation of the addition of water.
Routine tests are designed to check that the composition of milk is normal and
that it has not been subject to adulteration.
The bacteriological quality can
be checked by the methylene blue test or a similar dye reduction test, but more
e x t e n s i v e b a c t e r i o l o g i c a l tests m a y also be required.
E x a m i n a t i o n for
a n t i b i o t i c s , dirt, added alkali and added p r e s e r v a t i v e is i m p o r t a n t .
The
phosphatase test is used to check if the milk has been adequately pasteurised,
and the turbidity test to check if it has been sterilized.
Kempinski (5) gives
a m e t h o d s i m i l a r to the turbidity test for the d e t e c t i o n of UHT (ultra heat
treated) milk.
Analysis of the ash can be useful in detecting abnormality.
Anhydrous lactose,
p r o t e i n s , and ash occur in the p r o p o r t i o n 13:9:2 (Vieth's r a t i o ) in the m i l k
from healthy cows and therefore their determination is useful in checking that
the m i l k is not a b n o r m a l .
The a d d i t i o n of w a t e r to the m i l k does not alter
this ratio.
Instrumental methods of milk testing are reviewed by Harding (6) and
(7). C o r r a d i n i , C. (8) and Haave, I.J.J. (9) d i s c u s s the f o r m a t i o n of
UHT m i l k that has been stored a long time. The gel f o r m s by the slow
c o a g u l a t i o n of the casein. For the a n a l y s i s of sour m i l k suspected
adulterated, see Hanson (10) and Davis and Macdonald (1).

Bergmann
a gel in
enzymic
of being

DETECTION OF ADULTERATION
The natural acidity of m i l k i m m e d i a t e l y it c o m e s from the cow is about 0.130.14% expressed as lactic acid, although mainly derived from phosphate, casein
and to a less extent citrate and CO2.
The lactic level is about 2 mg% (0.002%)
in very fresh milk.
As the m i l k ages, the m i l k b a c t e r i a p r o l i f e r a t e and
produce lactic acid. Once the total acidity reaches about 0.18%, incipient
souring can be detected by smell and taste.
If storage conditions and hygiene
are poor, there may have been an attempt to mask this process by the addition
of alkali, w h i c h w i l l tend to produce low a c i d i t y , high pH, high s o d i u m and
h i g h l a c t a t e , a l t h o u g h not n e c e s s a r i l y outside the natural range.
The
freezing-point depression will also be greater than normal.
The older m e t h o d s to detect n e u t r a l i z a t i o n of m i l k such as those of T i l l m a n s
and L u c k e n b a c k (11 ) (12) (13 ), W o i d r i c h and Schmid (14) and H a n k i n s o n and
Anderson (15) depend on determining the buffering capacity of the milk by one
or m o r e a c i d - a l k a l i t i t r a t i o n s after a d d i t i o n of uranyl n i t r a t e or ferric
hydroxide.
A d e q u a t e m e t h o d s for the determination of lactate are available,
and comparison of total acidity with lactate should usually be sufficient to
detect neutralizers.
Iwaida et al (16) have used Davidson's method for lactic
Davidson's procedure, modified by Lawrence (18),
acid (17) for this purpose.

depends upon oxidation of the lactic acid to acetaldehyde and forming a colour
with p-hydroxydiphenyl, in the presence of concentrated sulphuric acid. It can
be used with milk powders and condensed milks as well as fresh milk. The AOAC
procedure has been developed from Hillig's method (19), involving extraction of
the lactic acid with ether and development of a colour with ferric chloride
after the removal of interferences.
Steffen (20) has described an enzymic
method.
Roy and Basak(21) investigated the subject of neutralisers in milk in detail.
pH of ash of milk, titratable acidity of the soluble ash and direct titrations
of the filtrate (or centrifgate) after coagulation of the proteins can detect
added carbonate or bicarbonate (or hydroxides) of alkali metals. But in all
these parameters those from unadmixed milk have to be subtracted or taken into
consideration. As these vary in pure milk within a range, a generally agreed
mean value has to be determined. Small additions cannot be detected as these
will be concealed within the range of natural variation. TLC and ion-exchange
methods devised in the study can be used for detection. Recovery of added
sodium carbonate in the TLC and ion-exchange is poor but the titrimetric
determination yields a recovery of the added sodium carbonate of 90 + 5Z.
Addition of salts of any sort to milk will lower the freezing-point.
Samples
with a correct freezing-point but low solids-not-fat may be genuine but poor
quality possibly from diseased animals, or watered and neutralized.
Further
analysis will be required. Theoretically, addition of 0.84Z sodium bicarbonate
depresses the freezing-point 0.1850C but in practice this is probably less,
p a r t l y due to i o n i z a t i o n of lactic acid and partly to loss of CO2 on
neutralization.
OFF FLAVOURS
Milk freshly drawn from a healthy udder has a "cowy" flavour distinguishable
from various taints of bacterial origin due either to mastitis or subsequent
bacterial growth or contamination. Although souring is the commonest offflavour derived from bacterial action, this may also cause bitterness, sweet
curdling and odours specific to a particular microorganism.
Feeds and weeds, such as some of the Brassicaceae may cause a taint in the
milk. These tend to pass off on standing or with aeration.
The fishy flavour that sometimes occurs in the milk from cows on wheat pasture
has been shown to be due to trimethylamine (Mehta (22)). Ingested land cress,
Coronopug didvmus. is one of the weeds more recently reported to cause an offflavour in milk (see Walker and Gray (23)).
The most important enzymically-induced taint is that of rancidity.
Milk kept
at low temperatures in the presence of copper and sometimes even in its absence
may develop an oxidized flavour. Irradiation and direct sunlight can induce a
taint variously described as "flat", "burnt" or "emery". Milk that has been
heated over about 80C has a cooked or scorched taste and smell.
The cause of a complaint in relation to a sample of milk may be one of the
above, or may be the accidental contamination of the milk with kerosene, soap,
chlorine disinfectants or other chemicals. Occasionally, microorganisms can
produce off-flavours which might be thought to have chemical origins. For
example, sterilized milk may have a carbolic flavour, and atypical strains of
Streptococcus 1ac t i s can produce caramel or malt flavours. The flavour of
vinegar or some fruits may be due to the action of bacteria or yeasts. Milk
easily picks up flavours from the surrounding atmosphere due to the large
surface area offered by the fat globules.
There is an article on off-flavours in milk by Kratzer (24) showing that in
over 18,000 samples the commonest cause of taint was the feed.

MASTITIS
The term "abnormal milk" in its restricted sense refers to milk from an udder
showing m a s t i t i s or other disease but m a y be used to refer to any m i l k of
unusual composition.
Milk from cows with mastitis has an altered composition.
The solids-not-fat,
fat, lactose, casein, calcium and potassium levels fall and the pH, s o d i u m ,
soluble protein and chloride levels rise. Dzhorov et al(25) have shown that
serum albumin and immunoglobulins increase while beta lactoglobulins and alpha
lactalbumin decrease compared with normal milk.
It is important to r e m e m b e r that the presence of m a s t i t i s does not alter the
freezing-point of the milk, which must show the same osmotic pressure as the
cow's blood.
The main contribution to the freezing-point depression is from
lactose and chlorides.
As the lactose level falls due to m a s t i t i s , the
proportion of chloride, bicarbonate and particularly sodium increases to
compensate.
Zagaevskii (26) describes the use of a reagent claimed capable of
detecting down to 1-3% of mastitis milk in bulk supplies.
Tests designed to detect mastitis in individual cows, or individual quarters of
the udder are beyond the scope of this Manual.
The reader is referred to
Schalm et al (27). The food inspector may have been able to obtain information
about the prevalence of disease in the a n i m a l s from which a milk sample was
derived. Thus the occasions when it is necessary to carry out laboratory tests
for m a s t i t i s in relation to statutory m i l k samples are relatively few.
H o w e v e r , the analyst must be aware of the significance of any information he
receives from the inspector or derives from analysis
The California Mastitis Test and its various modifications (The Milk Quality
Test, the Michigan Mastitis Test, the Brabant Mastitis Test and the Wisconsin
Mastitis Test), the modified Whiteside test and the catalase test are those
most c o m m o n l y used outside the laboratory.
Destruction of excess h y d r o g e n
peroxide with catalase will give a n o m a l o u s results in the catalase test for
mastitis. The total cell count is c o m m o n l y used in the USA; the California
Mastitis test and its modifications are basically measuring the leucocyte count
which is likely to be far in excess of the n u m b e r of bacteria present, at any
rate for all cases except very severe mastitis and is therefore a more readily
measured parameter.
The centrifuged deposit test and the cell count are
alternative ways of measuring essentially the same thing.
The resazurin test
is positive w h e n the n u m b e r of bacteria present is higher than normal.
Schultze et al (28) report a c o m p a r a t i v e study on these tests. A chloride
content above about 0.13 - 0.14% suggests mastitis, but it is not sufficient by
itself as it may indicate the presence of colostrum or m i l k from late in the
lactation period.
Newstead and Ormsby (29) give details of a test for
colostrum that relies on the detection of high levels of immunoglobulin.
Chemical tests to detect mastitis include the measurement of the pH (6.4 - 6.6
for n o r m a l fresh milk), the rennet test, the Koestler ratio and the casein
number (Rowland and Zeid-el-dine (30)). Very bad samples of mastitis milk will
fail to clot with rennet and will reduce resazurin rapidly.
The Koestler
ratio,
100 x chloride
lactose
is about 2.3 in normal milk and above 3 in mastitis milk. The determination of
this ratio is usually adequate for the detection of m a s t i t i s m i l k but the
casein number may also be determined if desired. This is,
casein N% x 100
Total NX
and is 70-80 for n o r m a l m i l k , falling as low as 70-74 in m a s t i t i s milk.

Until c o n f i d e n c e is gained, it is safer to d e t e r m i n e the nitrogen in the

filtrate and the total nitrogen to ensure the three figures agree before
concluding from the results that mastitis is present.
It must be remembered
that these remarks apply to milk that is entirely or substantially derived from
diseased quarters of the udder.
ANTIBIOTICS
V a r i o u s dye reduction tests such as those using m e t h y l e n e blue, resazurin,
triphenyl tetrazolium chloride, and brilliant black have been used to assess
the b a c t e r i o l o g i c a l quality of milk. M i l k of poor quality, containing large
numbers of bacteria, has a relatively low and decreasing reduction potential
and hence reduces these dyes more quickly. In using these dye reduction tests
to detect antibiotics, the milk is pasteurised to destroy most of the organisms
present and then inoculated with an organism which will be able to cause dye
reduction within a certain time under the conditions of test.
The presence of
an inhibitor is shown by failure to do so. The inhibitor may be an antibiotic,
d e t e r g e n t d i s i n f e c t a n t , pesticide residue or preservative. Any antibiotics
other than penicillin are likely to be difficult to identify.
Circumstantial
evidence from the inspector may assist in deciding what further tests should be
carried out.
It is usual to add penicillinase to another portion of the
sample.
A n o r m a l dye reduction in the treated portion indicates that the
inhibitor is penicillin.
If the inhibitor is not penicillin and in the absence of evidence as to the
likely cause, it is best to carry out some of the simpler tests first. For
example, test for peroxide, quaternary ammonium compounds, formaldehyde, free
chlorine and phenolic disinfectants.
Iodophors will increase the iodine
content of the milk. It is useful to check the pH also. If these tests prove
n e g a t i v e , the m i l k m a y be examined for pesticide residues. Wheeler(31) has
reviewed the use of iodophors.
The following may prove useful if the analysis has to be taken further. Hamann
et al (32) g i v e a T L C - G L C m e t h o d for i s o x a z o l y l p e n i c i l l i n s and for
chloramphenicol.
Rybinska (33) used a plate assay method and Micrococcus
f1avus test organism to detect down to 0.05 IU/ml of bacitracin in milk. Kumar
et al (34) found that if the phosphatase test was carried out using FolinC i o c a l t e u reagent to detect the phenol produced, some insecticides, such as
P r o p o x u r , Carbaryl, Te t r am e thy 1 th iur am disulphi-de (Thiram), Phosphamidon and
Dichlorvos interfered in the test.
Thus a discrepancy between the phosphatase
test done in this w a y , and using p-nitrophenyl phosphate or pheno1phtha1ein
m i g h t indicate the presence of these compounds.
Reinbold (35) uses TLC to
detect tylosin in biological m a t e r i a l s including milk. A g g a r w a l (36) found
that Thiram affected the m e t h y l e n e blue test (for milk quality, not for
a n t i b i o t i c s ) even at the 0.05 m i c r o g r a m / m l level, and that the test was much
m o r e sensitive to this residue than the others tested. Standard Methods for
the Examination of Dairy Products(168) contains a chapter on the detection of
inhibitors.
Kramer et al (37) describes methods for specific antibiotics.
For a general review of the determination of antibiotic residues in milk, see
Feagan (38).
Cox and M c N a m a r a (39) and Richard and Kerherve (40) describe
d e t e c t i o n of d i s i n f e c t a n t s on a paper disc and an acid titration method for
antibiotics.
T h e p u b l i c a t i o n of K r a m e r et al (37) g i v e s m e t h o d s for
penicillin, bacitracin, the tetracyclines, streptomycin, neomycin, polymixin,
erythromycin and novobiocin.
SUGARS
The only n a t u r a l l y occurring sugar in m i l k is lactose, although glucose and
galactose may be present due to bacterial or fermentative breakdown of lactose.
L a c t o s e may be d e t e r m i n e d c o 1 o r i m e t r i c a 11 y as in Nickerson et al (41), and
Fernandez and Ramirez (42). Colorimetric and polarimetric methods are compared
by Wilson et al (43). The Bertrand method has been modified to a colorimetric
finish by Rukina and Rastegaeva (44).
GLC (jaynes and Asan (45)), liquid

chromatography
been u s e d .

(Hobbs

and

Lawrence

(46)) and

an e n z y m i c

method

(Bahl

(47)) have

For the qualitative analysis of sugars in milk, the TLC method on the filtrate
o b t a i n e d a f t e r p r o t e i n p r e c i p i t a t i o n and c l a r i f i c a t i o n by
potassium
f e r r y o c y a n i d e / z i n c a c e t a t e , or TLC on c e l l u l o s e m a y be used ( V a h d e h i t a and
L o p e z (48)).
D o u g a l l and M o r g a n (49) u s e T L C to d e t e c t a d u l t e r a t i o n w i t h
s u c r o s e or r e c o n s t i t u t e d s w e e t e n e d d r i e d s k i m m i l k .
M a d r i d V i c e n t e (50)
d e s c r i b e s a r a p i d c o l o u r t e s t for g l u c o s e in m i l k ; t h e t e s t of M i t t a l and Roy
(51) appears to be m o r e sensitive.
Their paper includes a rapid test for added
a m m o n i a or u r e a s u l p h a t e .
A m e t h o d by PC is g i v e n by G u p t a and M a t h e w (52).
D h a r et al (53) p o i n t out that if s u c r o s e h a s b e e n a d d e d to m i l k , it m a y h a v e
b e e n p a r t i a l l y h y d r o l y s e d by the a c i d i t y p r e s e n t n a t u r a l l y .
It is t h e r e f o r e
more reliable to calculate the m i l k solids other than m i l k fat by m u l t i p l y i n g
the protein (N x 6.38) by 24/9 rather than subtracting the fat and sucrose from
the total solids.
The factor 24/9 is derived from Vieth's ratio.
NOH-BOVIHE

MILK

T h e m i l k of g o a t s or o t h e r a n i m a l s m a y be a n a l y z e d in the s a m e w a y as c o w s '
milk.
For details of the c o m p o s i t i o n of m i l k from different a n i m a l s , see Webb
(3). G o a t s ' m i l k is s o m e w h a t m o r e v a r i a b l e in c o m p o s i t i o n than c o w s ' m i l k .
T h e r e a d e r is r e f e r r e d to the p a p e r by J a m e s (54) for d e t a i l s of s o m e r e c e n t
goat m i l k analyses.
Also see Jaouen (55) and Rao and Bector (56).
P r u t h i et al (57) d i s c u s s the R e c k n a g e l p h e n o m e n o n in b u f f a l o m i l k .
In t h i s
p h e n o m e n o n the observed specific gravity of m i l k increases slowly during the
first 12 hours after milking.
This is possibly due to changes in the casein or
in the physical condition of the fat.
The observed gravity may increase by as
m u c h as 0.0013.
D a v i d e and D o m i n g o (58) h a v e p r o d u c e d a s e r i e s of p a p e r s on
Carabao milk.
The po1 y a c r y 1am ide gel e l e c t r o p h o r e s i s of b u f f a l o m i l k is
d e s c r i b e d by M i n c i o n e et al (59).
See a l s o the b o o k by S w a i s g o o d (60) on the
gel electrophoresis of m i l k proteins.
For recent papers on ewes m i l k see AlShabibi et al (61), W i l l i a m s et al (62) and W a g n e r (63).
For camels' milk and
its products see Rao, Gupta and Dastur (64) and Vaghela (65).
Milk from some a n i m a l s m a y cause an allergic reaction in sensitive individuals
and t h e r e f o r e m i s d e s c r i p t i o n can r e p r e s e n t a h e a l t h h a z a r d .
M i l k from a
particular species m a y c o m m a n d a higher price, so that passing off cheaper m i l k
would be e c o n o m i c a l l y attractive.
M o n a c e l l i a n d C a n t a g a l l i ( 6 6 ) a n d C a r i n i a n d B u s c a ( 6 7 ) u s e an a g a r
i m m u n o d i f f u s i o n test to detect c o w s ' m i l k in e w e s ' m i l k and e w e s ' m i l k cheese.
Foissy (68) finds electrophoresis best for this purpose, and also applied it to
the p r e s e n c e of c o w s ' m i l k in g o a t s ' m i l k (see a l s o A s c h a f f e n b u r g and D a n c e
(69), G o m b o c z et al ( 1 6 9 ) and M i t c h e l l (170)).
E g u a r e s (70) d e s c r i b e s the use
of h y p e r i m m u n e antisera, the test giving a precipitate in the presence of c o w s '
milk.
Earlier m e t h o d s are reviewed by Bret (71).
A series of papers describes
the use of po1 y a c r y 1 am id e g e l e l e c t r o p h o r e s i s ( P i e r r e and P o r t m a n n (72 )(7 3 );
P o r t m a n n and Pierre (74)). Adulteration of c o w s ' m i l k with buffaloes' m i l k has
b e e n d e t e c t e d by s t a r c h gel e l e c t r o p h o r e s i s ( M a j u m d e r and G a n g u l i (75) and
M a j u m d e r et al (76)).
Guha et al (77) did not find the gel diffusion technique
using Hansa test serum adequate to do this.

MILK FAT
(Gerber Method)
PRINCIPLE
The m i l k is m i x e d w i t h H j S O ^ and a m y l alcohol in a special G e r b e r tube
permitting solution of the protein present and release of the fat.
The tubes
are c e n t r i f u g e d and the fat rising into the calibrated part of the tube is
measured as a percentage of the sample.
The method is suitable as a routine or
screening test.
APPARATUS
1.

Gerber butyrometer

2.

Gerber centrifuge, 50-cm diameter.

3.

Milk

pipette

tubes, with lock-stoppers and a key.

10.75 ml. (See "interpretation" section

below.)

REAGENTS
1.

Sulphuric

2.

Amyl

acid Sp. gr. 1.815 jf 0.003 (about 90% m/m).

alcohol.

PROCEDURE
Measure 10 ml of sulphuric acid into a Gerber tube, preferably by use
of an a u t o m a t i c d i s p e n s e r , w i t h o u t w e t t i n g the n e c k of the tube.
Mix the milk sample gently but thoroughly and fill the milk pipette
above the graduation line. Wipe the outside of the pipette and allow
the milk level to fall so that the top of the meniscus is level with
the m a r k .
Run the m i l k into the Gerber tube w i t h o u t w e t t i n g the
neck, leave to drain 3 seconds and touch the pipette tip against the
base of the neck of the Gerber tube.
Add 1 ml a m y l a l c o h o l .
Close with a stopper, shake until
homogeneous, inverting to complete admixture of the acid. Centrifuge
for 4 minutes after the centrifuge has reached 1100 rpm.
Allow the
c e n t r i f u g e to c o m e to rest, r e m o v e the Gerber tubes and place in a
w a t e r - b a t h at 65C.
Read off the p e r c e n t a g e of fat after three
minutes, adjusting the height in the tube as necessary by movement of
the lock-stopper with the key.
The Gerber tubes must always be emptied without delay and the highly,
acid waste disposed of appropriately.
The tubes may be cleaned with
chromic acid.
INTERPRETATION
The ISO Standard 2446:1976 suggests that the volume of the pipette is chosen by
carrying out both this and the Rose-Gottlieb determination on a large number of
milk samples and, from a statistical analysis, computing the volume of sample
to be taken a p p r o p r i a t e to the n a t i o n a l average fat content of m i l k in the
country of use.
This is probably not worthwhile in an enforcement laboratory
where the Rose-Gottlieb method must be used on any doubtful samples.
However,
comparison of the results by the two methods on such samples over a period of
t i m e could be used to adjust the v o l u m e taken or c o m p u t e a c o r r e c t i o n factor
for use with the Gerber.
Volumes used in some countries are as
India, 10.75 ml (Ghouse and Jain
The Netherlands, 10.66 ml

follows:

(78))

Hungary, 10.8 ml
U.K., 10.94 ml
U.S.A., 11.0 ml

An 11 m l p i p e t t e ( w h i c h d e l i v e r s a b o u t 10.9 m l m i l k ) w a s used in the o r i g i n a l

Gerber method.
The r e s u l t is e x p r e s s e d m / m and the v o l u m e t a k e n m u s t
c o r r e s p o n d as c l o s e l y as p o s s i b l e to 11.25 g as each unit g r a d u a t i o n on the
tube corresponds to 0.1125 g of fat.
The Gerber method does not work on milks preserved with formaldehyde.
Stoppers
of b u t y l and n i t r i l e r u b b e r are s a t i s f a c t o r y , but n e w s t o p p e r s of n a t u r a l
r u b b e r a b s o r b fat to s o m e e x t e n t . Hornogen i z a t i o n c a u s e s the fat to s e p a r a t e
with more difficulty and centrifuging more than once m a y be required.
On the
other hand, holding the tubes too long at 65C results in ester ification of the
amyl alcohol with the consequent increase in the volume of the fat layer.
To
o v e r c o m e this use a 40C w a t e r - b a t h , c e n t r i f u g e u n t i l a c o n s t a n t r e a d i n g is
obtained and multiply the reading by 1.02.
REFERENCES
G e r b e r , N., 1892.

L ' I n d u s t r i e L a i t i e r e ^ O , 397 ;_52, 413; 1893 , 1 , 1 ; 6., 43.

ISO, 2446-78 and 488-93.

M a r t h , E.H., 1978.
Standard M e t h o d s
American Public Health Association.

for the E x a m i n a t i o n

of D a i r y

Products,

MILK FAT
(Rose-Gottlieb Method)
PRINCIPLE
T h e s a m p l e is t r e a t e d w i t h a m m o n i a a n d e t h a n o l , t h e l a t t e r to p r e c i p i t a t e
p r o t e i n a n d t h e f o r m e r to d i s s o l v e t h e p r e c i p i t a t e , a n d t h e f a t is e x t r a c t e d
w i t h d i e t h y l ether and p e t r o l e u m ether. The mixed ethers are evaporated and
the residue w e i g h e d .
This m e t h o d is c o n s i d e r e d
suitable for reference
purposes.
S t r i c t a d h e r e n c e to d e t a i l is n e c e s s a r y in o r d e r t o o b t a i n r e l i a b l e
results.
APPARATUS
1.

Extraction

2.

100-ml

tube

and siphon

flat-bottomed

(see d i a g r a m ) .

flask with G/G joint,

REAGENTS
1.
Ammonia
SG 0.880.
should be used).
2.

Petroleum

3.

Diethyl

4.

Denatured

5.

Mixed

ether

(six-fifths

of a m m o n i a

SG 0.910

B R 4 0 - 60C.

ether, peroxide
alcohol

ether.

the amount

free.

(95% e t h a n o l ) .

Equal volumes

of p e t r o l e u m

and diethyl

ethers.

PROCEDURE
A c c u r a t e l y w e i g h a b o u t 10 g of h o m o g e n e o u s s a m p l e into an e x t r a c t i o n
tube.
T h i s is c o n v e n i e n t l y d o n e on a t o p - p a n b a l a n c e a c c u r a t e to a
m i l l i g r a m b y s t a n d i n g t h e t u b e in a p l a s t i c b e a k e r o r o t h e r l i g h t
container.
F o r w e i g h i n g o n an a n a l y t i c a l b a l a n c e it m a y b e n e c e s s a r y
to a t t a c h a p i e c e o f w i r e to t h e n e c k o f t h e t u b e a n d h i t c h t h i s to
the p a n - h o o k of the b a l a n c e .
Add 1 m l a m m o n i a and m i x thoroughly.
A d d 10 m l a l c o h o l a n d a g a i n m i x
thoroughly.
A d d 25 m l of diethyl e t h e r , close the tube with a w e t t e d
g r o u n d - g l a s s s t o p p e r or w e l l - f i t t i n g rolled c o r k , shake v e r y g e n t l y
and r e l e a s e the p r e s s u r e w i t h o u t loss of e t h e r , and r e p e a t a c o u p l e
of t i m e s u n t i l the tube c a n b e s h a k e n v i g o r o u s l y w i t h o u t r i s k of
pressure build-up.
Shake v i g o r o u s l y for o n e m i n u t e .
A d d 25 m l of
p e t r o l e u m e t h e r , r i n s i n g the s t o p p e r and n e c k w i t h s o m e of i t , w e t
the s t o p p e r w i t h w a t e r again and shake v i g o r o u s l y for half a m i n u t e .
Weigh a dried 100-ml flat-bottomed ground-glass flask.
Leave the
e x t r a c t i o n t u b e to s t a n d h a l f an h o u r or m o r e u n t i l the l a y e r s a r e
c l e a r l y s e p a r a t e d , i n s e r t t h e s i p h o n t u b e so t h a t t h e o r i f i c e i s 2 - 3
m m a b o v e t h e a q u e o u s l a y e r and b l o w g e n t l y so t h a t the e t h e r e a l

10

e x t r a c t s i p h o n s into the w e i g h e d f l a s k .
R a i s e the s i p h o n a l i t t l e
b u t do n o t r e m o v e it.
R i n s e the tip of it w i t h a b o u t 5 m l of m i x e d
ethers and, without shaking, siphon to the flask.
Use a further 5 ml
of m i x e d e t h e r s to r i n s e the c o r k of the s i p h o n and t h e n e c k of the
t u b e and a g a i n t r a n s f e r w i t h o u t s h a k i n g the t u b e , t h e n r e m o v e the
s i p h o n f r o m the t u b e .
R i n s e the tip of the s i p h o n and the n e c k of
the flat-bottomed flask.
The evaporation of the solvent in the flask
can be started while the second extraction is in progress.
Add 15 m l of d i e t h y l e t h e r to the tube and s h a k e v i g o r o u s l y for one
minute,
t a k i n g the s a m e p r e c a u t i o n s as b e f o r e .
Add 15 m l of
petroleum ether and shake vigorously for half a m i n u t e , transfer the
ethers and rinse as before.
Extract a third time with 15 ml of each
solvent and rinse as before.
Evaporate all the solvent in the flask,
c o m p l e t i n g the p r o c e s s on the w a t e r - b a t h and f i n a l l y in the o v e n ,
drying to constant weight.
Leave in the desiccator to cool at least
an hour and do not wipe the flask just before weighing.
(it may have
been necessary to wipe it on removing from the water-bath).
W e i g h , t h e n add p e t r o l e u m e t h e r to d i s s o l v e the
d e c a n t t a k i n g c a r e to l e a v e any s e d i m e n t in the
f l a s k w i t h p e t r o l e u m e t h e r u n t i l all the fat is
sediment remains.
D r y in the o v e n and w e i g h
difference in w e i g h t s represents the weight of fat
milk.

fat and c a r e f u l l y
flask.
R i n s e the
r e m o v e d , b u t any
as b e f o r e .
The
extracted from the

For the most accurate work it m a y be checked that the residue from a
f o u r t h e x t r a c t i o n is n e g l i g i b l e and a b l a n k e x t r a c t i o n m a y be d o n e
using 10 ml of water in place of the sample.
CALCULATION

fat

m / m

weight of fat
weight of milk

100

INTERPRETATION
The a v e r a g e fat c o n t e n t of m i l k f r o m a d e q u a t e l y fed c o w s is a b o u t 3.5%, b u t
varies widely depending on a n u m b e r of factors including breed, feed, season,
m i l k i n g i n t e r v a l , etc. " E u r o p e a n s t a n d a r d m i l k " c o n t a i n s 3.5% of f a t , and in
the U.K. b e l o w three percent is assumed to indicate adulteration or abstraction
of fat u n t i l p r o v e d to the c o n t r a r y .
T h e r e is a f e d e r a l m i n i m u m of 3.25% in
the U.S.A., but some individual States set higher standards.
IDF, ISO and AOAC have jointly agreed upon a reference m e t h o d , published also
in C A C / M 1 - 1 9 7 3 of the C o d e x A l i m e n t a r i u s C o m m i s s i o n .
The w o r d i n g h e r e is
slightly different.
The I D F / I S O / A O A C m e t h o d u s e s a d i f f e r e n t type of fatextraction flask (ISO 1 2 1 1 : 1 9 7 0 ) .
The N M K L v e r s i o n of the m e t h o d (no. 10, 1 9 7 7 ) r e c o m m e n d s that c o a g u l a t e d
samples be re-dispersed by adding a k n o w n amount of concentrated a m m o n i a .
If
the fat h a s a c t u a l l y s e p a r a t e d , r e - e m u 1 s i f i c a t i o n is a c h i e v e d by c a r e f u l l y
shaking at 40C with 0.5 ml of chloroform per 100 m l of m i l k or cream.
REFERENCES
ISO/R,

1211-70.

Official Methods of Analysis of the AOAC, 1984.

16.064.

Code of Principles Concerning Milk and Milk Products, International Standards

Methods of Sampling and Analysis for Milk Products, 7th Ed., CAC/Ml-1973.

11

TOTAL SOLIDS
(Rapid Method)
PRINCIPLE
T h e d e n s i t y of the m i l k is m e a s u r e d u s i n g a m i l k h y d r o m e t e r ( l a c t o m e t e r ) and
the total solids are calculated.
APPARATUS
1.

Milk

hydrometer.

2.
Cylinder,
hydrometer.

at

least 4 m m

greater in diameter

than the bulb of the

PROCEDURE
The m i l k s h o u l d be w a r m e d to and k e p t at 40-45C for five m i n u t e s ,
c a r e f u l l y b u t t h o r o u g h l y m i x e d , a v o i d i n g i n c l u s i o n of b u b b l e s , and
t h e n c o o l e d to 20 +_ 2C and the d e n s i t y d e t e r m i n e d as soon as
possible.
F i l l the c y l i n d e r to o v e r f l o w i n g by p o u r i n g the s a m p l e over the
lactometer bulb, avoiding bubble formation.
Allow the lactometer to
float in the m i l k without permitting more than a few m m of the stem
a b o v e the m i l k s u r f a c e to b e c o m e w e t t e d . Read the scale at the top
of the meniscus, the eye being horizontal to it, move the hydrometer
a f e w m m v e r t i c a l l y and c h e c k the r e a d i n g .
R e m o v e the l a c t o m e t e r ,
r e c o r d the r e a d i n g and d e t e r m i n e and r e c o r d the t e m p e r a t u r e of the
milk.
W a r m i n g the s a m p l e to 40-45C is to e n s u r e the fat is in a liquid
condition.
This can therefore be omitted if the sample has been warm
for some time previously.
Lactometer slide rules allow for density
determinations between about 15 and 25C.
CALCULATION
The f o r m u l a SNF = 0.250 + 0.22F + 0.72 a p p l i e s if the t e m p e r a t u r e of
the sample is 20C and if the fat of the milk is in the liquid state,
w h e r e F = % m / m fat, D = (1000 x d e n s i t y - 1 0 0 0 ) and SNF = s o l i d s not-fat.
If the m i l k w a s n o t at 20C the d e n s i t y m u s t be c o r r e c t e d
before application of the formula, adding to "D" 0.24 for each degree
above 20 and subtracting for each degree below.
For e x a m p l e , if the
( G e r b e r ) is 3.65%

density

reading

Density at 18C
D, (1000 x density - 1000)
D at 20 C
Therefore, solids-not-fat

is

1.032

at

18C

and

the

fat

1.032
32 at 18C
32 - (2 x 0.24) = 31.52
(0.25 x 31.5) + (0.22
x 3.65) + 0.72 = 9.39

INTERPRETATION
T h i s m e t h o d is r a p i d and is o n l y a p p r o x i m a t e .
It is c o n v e n i e n t to use it in
c o n j u n c t i o n w i t h the G e r b e r or B a b c o c k m e t h o d s for fat.
C h e c k any s u s p e c t
samples by determination of the total solids gravimetrically and the fat by the
method of Rose-Gottlieb.

12

T h e r e are t w o w a y s of i m p r o v i n g the a c c u r a c y of the d e t e r m i n a t i o n of t o t a l

solids using this m e t h o d .
O n e w a y is to d e t e r m i n e the g r a v i t y and a l s o the
weighed total solids on ten or twelve samples and to calculate the correction
to be added to, or subtracted from, the gravity reading before the correction
for temperature is made. This corrects for any error in the calibration of the
lactometer.
A n o t h e r w a y is to c a r e f u l l y check, the c a l i b r a t i o n of
the
lactometer against a solution of k n o w n gravity.
B.S. 734, Part 2, 1959 uses a
reference lactometer and solutions of sodium carbonate.
O ' K e e f e (79) h a s s u g g e s t e d that the a b o v e f o r m u l a (SNF = 0.25D + 0.22F + 0 . 7 2 )
o v e r e s t i m a t e s the s o 1 id s - n o t - f a t and t h a t the o l d e r f o r m u l a of SNF = 0.25D +
0.21F + 0.66 is m o r e c o r r e c t .
T h e f o r m u l a h a s a l s o b e e n d i s c u s s e d by L u n d e r
(80) and Aparicio Gallego (81).
See also A r t e m ' e v and Panasenkov (82), Uzonyi
and V a r g a (83), D o z e t et al (84) and S l a n o v e c et al (85).

REFERENCES
Official Methods
British

Standard,

of Analysis

of the AOAC,

734:1959.

13

1984.

16.033.

TOTAL SOLIDS
(Veight Method)
PRINCIPLE
M i l k is dried

in the oven under

standard

conditions and

the residue

weighed.

APPARATUS
1.
Dish and lid,
2.5 cm d e e p .
2.

Ventilated

metal,

oven at

flat-bottomed,

about

7-8 cm

diameter by 1-

100C.

PROCEDURE
H e a t the c l e a n dry e m p t y dish and lid in the o v e n , cool in a
Add 3 - 4 g m i l k , r e p l a c e the lid and w e i g h
d e s i c c a t o r and w e i g h .
again.
Place the dish without the lid on a boiling water-bath until
the water is evaporated from the sample, wipe the undersurface of the
dish, and place in the oven at 102C for 2-1/2 hours.
Put the lid on
the dish, cool in the desiccator and weigh.
Continue heating and reweighing at hourly intervals until successive weighings do not vary
by m o r e t h a n 0.5 m g .
CALCULATION
A total solids m/m =

weight of residue
of milk
weight

100

REFERENCES
Official Methods of Analysis of the AOAC, 1984.
British

Standard,

16.032.

1741:1963.

Society of Public Analysts, 1954.

Analyst JO,

14

105-6; 1885, 1 0 ,

216.

FR E E Z I M G P O I H T
PRINCIPLE
T h e m i l k is s u p e r c o o l e d ,
seeded w i t h ice c r y s t a l s
freezing-point determined
in t h e s t a n d a r d
Hortvet
f o l l o w i n g the e x a c t i n s t r u c t i o n s for u s e .

if n e c e s s a r y a n d
the
apparatus,
carefully

APPARATUS
H o r t v e t c r y o s c o p e (see d i a g r a m ) .
The original e q u i p m e n t was ethercooled,
a procedure
now
considered
unduly
hazardous.
Any
r e f r i g e r a t e d b a t h t h a t c a n b e k e p t a t -3C i s s u i t a b l e ( e . g . t h a t o f
T e m p l e (86)); 20% e t h y l e n e g l y c o l
in w a t e r
is a
convenient
refrigerant.
Simple
manual
cryostats
are
still
available
commerc ially.
STANDARDIZATIOH

OF

THERMOMETER

T h e b o r e o f the t h e r m o m e t e r m a y n o t b e u n i f o r m a l o n g i t s l e n g t h a n d
therefore requires calibration.
Calibrated t h e r m o m e t e r s can be
obtained from certain standards organizations, but uncalibrated ones
m u s t be c h e c k e d a g a i n s t s t a n d a r d s u c r o s e or salt s o l u t i o n s .
T h e r e is
a t e n d e n c y n o w to u s e t h e s a l t a l t h o u g h t h e H o r t v e t f r e e z i n g - p o i n t
t e n d s to b e h e l d f o r a s h o r t e r t i m e t h a n w i t h s u c r o s e s o l u t i o n s .
C a r e f u l l y p r e p a r e p u r e s u c r o s e s o l u t i o n s , 7 . 0 , 7 . 5 , 8 . 0 , 8.5 a n d
8.75% m / v a t 20C. D e t e r m i n e t h e f r e e z i n g - p o i n t o f e a c h s o l u t i o n t w o
or t h r e e t i m e s .
T h e s t a n d a r d v a l u e s a r e g i v e n in t h e
following
table :
Z m/v sucrose
a t 20C

Freezing-point
d e p r e s s i o n , C

7.0
7.5

-0.422
-0.454
-0.487
-0.520
-0.537

8.0
8.5
8.75

If t h e r e s u l t s d i f f e r f r o m t h e s e v a l u e s , t h e f r e e z i n g - p o i n t s
of
s a m p l e s m u s t b e c o r r e c t e d b y a d d i n g or s u b t r a c t i n g t h i s d i f f e r e n c e ,
i n t e r p o l a t i n g if n e e d b e .
For e x a m p l e , s u p p o s e the e x p e r i m e n t a l l y
d e t e r m i n e d v a l u e f o r 8.0% s u c r o s e is -0.489C a n d f o r 8.5% s u c r o s e is
-0.524C, and the f r e e z i n g - p o i n t ( u n c o r r e c t e d ) of the s a m p l e s is 0.504C, 0 . 0 0 3 m u s t b e a d d e d to t h i s g i v i n g a f r e e z i n g - p o i n t
( c o r r e c t e d ) o f 0.507C.
If

salt

solutions

are

to b e u s e d , t h e y m a y

HaCl g/kg water

be prepared

Freezing-point

6.8920
8.5978
8.8772
10.2060

as

follows:

*C

-0.422
-0.525
-0.540
-0.621

T h e s a l t s h o u l d b e d r i e d b y h e a t i n g a t 500C b e f o r e
in a n e f f i c i e n t d e s i c c a t o r .
Note that a l t h o u g h the
are m / v the salt s o l u t i o n s are m / m .
The w e i g h t s of
h a v e n o t b e e n c o r r e c t e d for b u o y a n c y b u t t h e e r r o r is
in p r a c t i c e .

15

u s e , and c o o l e d
sugar
solutions
s a l t to be u s e d
not significant

260
250tJ

Spacer H

240:2

S c c t ! o n

l h r o u e h

"

Section through Y - Y
N O T E . Control t h e r m o m e t e r r.ot s h o w n .
All dimensions are in millimetres.

lY-x t d i a
60int
75int dia
nominal
3 3 t

Diagram of Sample Tube and Sample Tube Holder

of HortveC Apparatus

PROCEDURE
C o o l the a p p a r a t u s by a s u i t a b l e m e a n s in o r d e r to b r i n g the
t e m p e r a t u r e of the o u t e r j a c k e t to -2.5C.
P o u r a l c o h o l into the
s a m p l e tube c a v i t y so that the level is w e l l a b o v e that of the m i l k
in the s a m p l e t u b e s .
M a i n t a i n the a l c o h o l l e v e l d u r i n g use as
required.
Distil some de-ionized water from a little permanganate, or otherwise
prepare high quality distilled water, rejecting the first part of the
distillate, which m a y contain dissolved gases. The water may be kept
in a s t o p p e r e d f l a s k in a d e e p f r e e z e w i t h o u t its f r e e z i n g - p o i n t
altering.

16

F i l l the s a m p l e t u b e w i t h the h i g h p u r i t y w a t e r to 12 m m a b o v e the

top of the t h e r m o m e t e r bulb, place the tube in the sample tube cavity
and s t a r t the s t i r r e r m o t o r so that the s t i r r i n g r a t e is a b o u t 30
s t r o k e s p e r m i n u t e (or s t i r b y h a n d ) .
The bung carrying
the
t h e r m o m e t e r and the tube must be scrupulously clean as particles such
as d u s t c a n act as n u c l e i for i c e - c r y s t a l f o r m a t i o n and p r e v e n t
supercooling.
For e x a m p l e , poor hygiene may result in mould growth
in the stirrer channel and mould pieces falling into the sample tube.
K e e p i n g the o u t e r j a c k e t at -2.5C, s u p e r c o o l the w a t e r b l a n k to
-1.0C to -1.2C, s t o p s t i r r i n g and seed w i t h i c e - c r y s t a l s to i n d u c e
the water to freeze.
M o m e n t a r y erratic stirring may be sufficient to
start crystallization.
In any c a s e s t o p s t i r r i n g as s o o n as the
temperature begins to rise.
It is convenient to keep a jagged-ended
c o p p e r t u b e , or a w i r e w i t h a c o r k h a n d l e , in the d e e p f r e e z e to
facilitate addition of ice to the tube.
As the temperature nears the expected freezing-point, stir (complete
up and d o w n m o t i o n ) t h r e e t i m e s and s h a r p l y tap the t h e r m o m e t e r
alternative sides near the top of the m e r c u r y thread, seven times in
all w i t h a c o r k or r u b b e r g a v e l .
Take a reading.
About thirty
seconds after stirring, repeat the stirring, tapping and reading, and
t h e n a third t i m e a b o u t h a l f a m i n u t e l a t e r .
T h e s e c o n d and t h i r d
readings should not differ by m o r e than 0.002 - 0.003C.
If they do,
or if the t e m p e r a t u r e b e g i n s to fall a g a i n , the r e s u l t m u s t be
discarded.
T h e m e t h o d r e l i e s on the h e a t p r o d u c e d by c r y s t a l l i z a t i o n b e i n g
c o m p e n s a t e d by the h e a t l o s s e s to the s u r r o u n d i n g c o o l i n g j a c k e t s ,
hence the importance of adhering to the exact experimental procedure.
T h e t a p p i n g of the t h e r m o m e t e r is d e s i g n e d to p r e v e n t the m e r c u r y
t h r e a d f r o m g e t t i n g s t u c k if t h e r e is any r o u g h n e s s on the i n n e r
glass surface at that point.
S o m e f o r m of m a g n i f i e r m a y be u s e d to read the t h e r m o m e t e r to the
nearest 0.001C by interpolation, but care m u s t be taken with some of
the h e a v i e r o n e s that can be a t t a c h e d to the t h e r m o m e t e r that the
l a t t e r is n o t m o v e d f r o m the v e r t i c a l , ( u n l e s s a s p a c e r is u s e d )
bringing the bulb close enough to the side wall to alter the rate of
heat loss mentioned above.
The b l a n k and s a m p l e s s h o u l d be d o n e at least in d u p l i c a t e and
preferably in triplicate.
The blank w i l l vary slightly, tending to
r i s e w i t h a t m o s p h e r i c p r e s s u r e , c h a n g e s in w h i c h are s u f f i c i e n t to
slightly affect the v o l u m e of the bulb.
The v o l u m e of the bulb also
tends to change with age, thus affecting the blank.
R e p l a c e the w a t e r b y a s a m p l e , a d j u s t the c r y o s t a t so t h a t as the
m i l k s u p e r c o o l s , the o u t e r j a c k e t is at -3C. The w o r k is h a s t e n e d
by u s i n g p r e c o o l e d m i l k ( w h i c h m u s t n o t h a v e ice in it) a n d / o r b y
a l l o w i n g the j a c k e t to be b e l o w -3C d u r i n g the i n i t i a l s t a g e of
cooling the milk.
A l l o w the m i l k to s u p e r c o o l 1.2 b e l o w
the
e x p e c t e d f r e e z i n g - p o i n t (i.e., to a b o u t -1.65C) and d e t e r m i n e the
freezing-point as for the w a t e r blank.

CALCULATION
F

% m/m added water

17

x (100 - T )

Where :
Fg

freezing-point of genuine milk

(see "interpretation" below).

Fg

freezing-point of sample.

total solids of sample (% m/m).

INTKRPRETATIOH
The freezing-point of milk is usually at least 0.530C below the
of water.
This is spoken of as a freezing-point depression.

freezing-point

The F Q to be used in the f o r m u l a above should be that of m i l k taken from the

cow or herd by a supervised milking not more than 72 hours after the taking of
the questioned sample.
The milk from such a supervised milking is known as an
appeal-to-cow or appeal-to-herd sample.
The usual procedure is to notify the
inspector as soon as possible and he will probably decide to take a sample from
a s u p e r v i s e d m i l k i n g . He m a y be required by law to do it w i t h i n a specified
t i m e (usually b e t w e e n 24 and 72 h o u r s ) and in any case it should be taken as
soon as possible to avoid the possibility that other factors have affected the
milk.
If the freezing-point of the "appeal-to-cow" sample (which may in fact
be an "appeal-to-herd" sample) is substantially the same as the original one,
the m i l k is regarded as genuine even though the f r e e z i n g - p o i n t d e p r e s s i o n is
less than 0.530C.
W h a t is m e a n t by "is s u b s t a n t i a l l y the s a m e " has to be
defined.
It is p r o b a b l y r e a s o n a b l e to regard a d i f f e r e n c e of 0.010C or m o r e
as i n d i c a t i n g the p r e s e n c e of added w a t e r in the s a m p l e s h o w i n g the lower
freezing-point depression.
The p r e c i s i o n and accuracy of the analyst's o w n
r e s u l t s over a period of t i m e m a y enable h i m to c o m e to his o w n c o n c l u s i o n s
about what represents a signficant difference between two samples.
The absence of an "appeal-to-cow" sample should not mean that an adverse report
cannot be made.
It means that a minimum value for genuine milk must be chosen.
The analyst should determine the average freezing-point depression for milk in
his area.
H e n n i n g s o n (87) and his c o l l a b o r a t o r s in the U.S.A. found that genuine m i l k
from herds may have a freezing-point depression as low as 0.513C and that 5.3%
of the herd samples examined had freezing-point depressions less than 0.530C.
The spread of these r e s u l t s is s i m i l a r to that from i n d i v i d u a l c o w s and it
would appear that the conclusions of Wood (88) that the average freezing-point
for bulked-milk is -0.544C and the minimum -0.540C, are valid as long as the
bulked milk (perhaps better called pooled milk) is derived from enough herds to
remove the effect of inter-herd variations.
As the yield per cow and the size
of herd have i n c r e a s e d , the 200 gallons (originally regarded as the m i n i m u m
v o l u m e r e p r e s e n t i n g bulked m i l k ) s e e m s to now be too s m a l l to be regarded as
representative of pooled milk. Thus, the analyst must take these factors into
account when choosing the appropriate value.
When no "appeal-to-cow" sample
is a v a i l a b l e , .the r e g u l a t o r y analyst m u s t choose an a c c e p t a b l e m i n i m u m
freezing-point depression preferably based on at least a few hundred analyses
from his area.
When the freezing-point depression is less than about 0.510C
there is no doubt that water has been added, but the analyst still has to use a
m i n i m u m value for genuine milk in order to calculate the approximate percentage
of added w a t e r . For i n d i v i d u a l c o w s 0.530C has frequently b e e n taken w h i l e
the AOAC 12th Edition used 0.525C as a presumptive limit irrespective of size
of herd.
It is generally considered most appropriate to report the result as
"not less than x % added water".
An a u t o m a t i c t h e r m i s t o r cryoscope is quicker than the Hortvet for
purposes.
Results for regulatory purposes may be checked against the
or it may be preferred to accept thermistor results as official.
For
of its use see A O A C (13th Ed.) and M o o r e (89), Anon (90), and H o f f e l n e r

18

routine
latter,
details
(91).

The effects of pasteurization and uperisation, which raise the freezing-point

a b o u t 0.005C are d i s c u s s e d by D i 1 1 i e r - Z u 1 auf (92), and Di 1 1 i e r - Z u l a u f and
D o y o t t e (93).
Milk samples for freezing-point tests may be preserved by the addition of 1.5
ml per 100 ml of a solution containing 1% m/v H g C ^ and 0.85% m/v NaCl (Harding
and Royal (4)) rather than formaldehyde, the presence of which invalidates the
freezing-point test.
An i n c r e a s e in a c i d i t y l o w e r s the f r e e z i n g - p o i n t and h e n c e t e n d s to m a k e
s a m p l e s a p p e a r to c o n t a i n less added w a t e r .
0.0034C is s u b t r a c t e d from the
f r e e z i n g - p o i n t d e p r e s s i o n for e v e r y 0.01% by w h i c h the a c i d i t y e x p r e s s e d as
l a c t i c acid e x c e e d s 0.18%.
If the a c i d i t y e x c e e d s 0.30% the f r e e z i n g - p o i n t
test is invalid.
Some care should be taken in interpreting the results if the
acidity exceeds about 0.23 - 0.24%.
Binder (94) suggests subtracting from the
f r e e z i n g - p o i n t d e p r e s s i o n 0.0002C for e a c h 0.1 S o x h l e t - H e n k e l (0.00225 %
l a c t i c a c i d ) a b o v e 7 S o x h l e t - H e n k e l and a d d i n g that a m o u n t for each 0.1 S - H
below.
P a n e t s o s and G e o r g a k i s (95) s u g g e s t 0.0008C per 0.1 S - H and state
that o v e r 12 S - H (0.25% lactic a c i d ) the c o r r e c t i o n s b e c o m e u n r e l i a b l e .
B i n d e r ' s f i g u r e c o r r e s p o n d s to 0.000888C per 0.01% lactic acid and h e n c e is
a b o u t 4 t i m e s l o w e r than that of P a n e t s o s and G e o r g a k i s .
The e x p r e s s i o n of
m i l k acidity other than as % lactic acid is dealt with under "acidity".
W e l b o r e n and v a n der V e l d e n (96) h a v e s u g g e s t e d a s c r e e n i n g m e t h o d for added
water.
P r e n t i c e (97) r e p o r t e d an e x t e n s i v e s t u d y c o m p a r i n g the H o r t v e t
apparatus with other cryoscopes.
For further data and discussion on the freezing-point determination, see:
Luck
and D r e s n e r (98), B i n d e r (99), A s p e r g e r (100), B o u c h e z and W a e s
(101),
H e n n i n g s o n ( 1 02 ) (103)( 1 0 4 ) , J a m o t t e and D u c h a t e a u (105), F r e e m a n et al (106),
and Dermott (107).
REFERENCES
Official Methods of Analysis of the AOAC, 1984.
Hortvet, J., 1922.
British

16.104.

Journal of the AOAC 5, 470-97.

Standard Method, BS 3095, Parts

19

1-3, 1980 and

1981.

MILK ACIDITY
PRINCIPLE
The sample is titrated with sodium hydroxide to a pheno1phtha1ein
using milk containing rosaniline as a comparison standard.

endpoint

APPARATUS
1.

Two 100 ml poreclain evaporating

2.

Burette, 5 ml.

dishes.

REAGENTS
1.

0.1N sodium hydroxide

solution.

2. Rosaniline acetate solution.

Dissolve 0.12 g of rosaniline
acetate in 95% ethanol, containing 0.5 ml of glacial acetic acid, and
dilute to 100 ml. Store in the dark. Dilute 1 ml to 500 ml with 95%
ethanol-water, 1+1.
3. P h e n o 1 p h t h a 1 e i n .
Dissolve 1 g phenolphthalein in 110 ml 95%
ethanol and add 0.1 N sodium h y d r o x i d e dropwise until a faint pink
colour is obtained. Dilute to 200 ml with distilled water.
PROCEDURE
Pipette 10 ml of milk into each evaporating dish. To one add 1 ml of
To the
the dilute rosaniline solution and stir with a glass rod.
other add 1 ml of phenolphthalein solution and titrate with 0.1N
sodium hydroxide, stirring the sample, until the colour is the same
as that of the rosaniline comparison standard.
CALCULATION

ml NaOH
% m/v lactic acid =

1000

100
x 0.1 x 90 x i 0 (ml of milk taken)

= ml NaOH x 0.09
where % m/v

= percent mass in volume.

INTERPRETATION
The various standard methods for determination of acidity in milk give slightly
d i f f e r e n t a n s w e r s even if used by the same operator.
These differences
(discussed by O'Connor (108)) tend to be greater if no colour standard is used
(e.g. AOAC method).
The acidity of freshly drawn milk is mainly due to citric
acid, phosphates, carbon dioxide and casein, and is about 0.13-0.14% expressed
as lactic acid. The increase in acidity on ageing is due to the production of
lactic acid as a result of bacterial action.
Sourness begins to b e c o m e
noticeable when the acidity exceeds about 0.18% as lactic acid.
The acidity of milk is also often expressed

in degrees, defined as follows:

Degrees Soxhlet-Henke1 (S-H) = ml of N/4 alkali required

of m i l k (hence S-H/44.4 = % of lactic acid).

to neutralize

Degrees Dornic (D) = ml of 0.046N calcium hydroxide solution

water at 15C) required to neutralize 100 ml of milk.

20

100 ml

(saturated

lime

Degrees English = ml of N/9 alkali required to neutralize

100 ml of milk.

Degrees R i c h m o n d (R) = No. of m l of 0.1N alkali required to n e u t r a l i z e 100 m l

of milk.
Hence 0.14% lactic acid = 30D = 7S-H = 17R - 14 English.
Luck et al (109) have investigated the relation between acidity and pH.
Stiles
et al (110) compared a number of methods and found the one given above the most
accurate.
Citric acid m a y be d e t e r m i n e d by the IDF m e t h o d for cheese (see
N o w a k and L a s k o w s k i (111)).
The acidity of m a s t i t i s m i l k m a y be as low as
0.10% as lactic acid while very rich milks may contain as much as 0.16% before
lactic fermentation starts.
The acidity of pasteurized milk does not normally
exceed 0.15% as lactic acid nor that of sterilized milk 0.16%.
REFERENCE
British Standard

1741:1963.

21

NITRATES IN MILK
PRINCIPLE
U n d e r the c o n d i t i o n s of t e s t , d i p h e n y 1 a m i n e is o x i d i z e d by n i t r a t e to the
i n t e n s e l y b l u e q u i n o n e - i m m o n i u m salt via d i p h e n y l b e n z i d i n e .
This is a
qualitative test only.
APPARATUS
Note:
C a r e m u s t be t a k e n to rinse all g l a s s w a r e to e n s u r e the
a b s e n c e of e v e n t r a c e s of n i t r a t e .
The test m u s t not be c o n d u c t e d
near
sources
of n i t r o u s
fumes
s u c h as r e a g e n t b o t t l e s
of
c o n c e n t r a t e d n i t r i c acid.
The filter p a p e r s used m u s t also be
checked for nitrates and washed prior to use if necessary.
REAGENTS
1.
Diphenylamine solution - weigh 0.085 g diphenylamine and dissolve
in 50 m l w a t e r .
S l o w l y add 4 5 0 m l of c o n c e n t r a t e d s u l p h u r i c acid
with shaking, keeping the solution cool.
2.
P r e c i p i t a t i n g r e a g e n t - d i s s o l v e 20 g of m e r c u r i c c h l o r i d e and
5 g of a m m o n i u m c h l o r i d e in w a t e r , add 20 m l of c o n c e n t r a t e d
hydrochloric acid and dilute to 100 ml with water.
PROCEDURE
To 5 m l of m i l k in a t e s t - t u b e add 6 or 7 d r o p s of the p r e c i p i t a t i n g
reagent and shake occasionally for about 2 minutes.
Pipette 2 ml of
the d i p h e n y 1 a m i n e s o l u t i o n into the b o t t o m of a n o t h e r t e s t - t u b e
without allowing any of the solution to touch the walls of the tube.
Place a filter paper in this tube and incline it so that the filtrate
from the precipitated milk runs gently down the side of the tube and
f o r m s a layer on top.
W h e n about 1 m l of f i l t r a t e has c o l l e c t e d ,
remove the filter and examine the fi 1 1 r a t e / d i p h e n y 1 a m i n e i n t e r f a c e
over a w h i t e surface.
In the a b s e n c e of n i t r a t e s , t h e r e is no
colour, and some yellow or b r o w n colour may appear when the tube is
rotated.
In the p r e s e n c e of n i t r a t e s a b l u e c o l o u r d e v e l o p s e i t h e r
i m m e d i a t e l y or on r o t a t i o n of the tube.
C a r r y out a b l a n k w i t h
genuine milk.
The test d e t e c t s d o w n to about 0.1 m i c r o g r a m s/m 1 in
the filtrate.
INTERPRETATION
It is g e n e r a l l y a c c e p t e d that n i t r a t e does not occur in n o r m a l m i l k , but is
o f t e n p r e s e n t in d r i n k i n g w a t e r , so the test, w h e n p o s i t i v e , s e r v e s as
confirmation of the addition of water to milk.
There is no reason to apply the
test if n i t r a t e is k n o w n to be a b s e n t from the local w a t e r s u p p l y .
Reraond
(112) discusses
t h e p r e s e n c e of n i t r a t e
in m i l k f r o m o t h e r
causes.
Q u a n t i t a t i v e e s t i m a t i o n of n i t r a t e and n i t r i t e in m i l k is not u s u a l l y
j u s t i f i e d , but' if n e e d e d , the m e t h o d of M a n n i n g , et al (113) or R e s m i n i and
Volonterio (114) may be used.
See also Ling (115) and Davis and MacDonald (1).
REFERENCE
This

is a method

of great antiquity, apparently due to Fuch.

Richmond, H.D., 1894.

Lorrigo, 1930.

Analyst JJ?, 73-87.

Analyst _55, 433.

22

See for

example:

PHOSPHATASE

TEST

PRINCIPLE
Any p h o s p h a t a s e p r e s e n t in the m i l k s p l i t s the s u b s t r a t e , p - n i t r o p h e n y l
p h o s p h a t e , to g i v e p - n i t r o p h e n o l , w h i c h is h i g h l y c o l o u r e d in a l k a l i n e
solution.
APPARATUS
1.

A Lovibond

comparator

with

stand

for w o r k

in r e f l e c t e d

2.

A Lovibond

3.

Two fused glass cells, 25 mm

4.

A water bath or incubator capable of being maintained

light.

comparator disc APTW or APTW7.

depth.
at 37.5C +

0.5.C.
5.

A pipette to deliver 5 ml.

6.

A supply of 1.0 ml straight-sided

7.

A 1000 ml graduated

8.

A 100 ml measuring

cylinder.

9.

Test tubes nominal

size 150/16 mm with rubber

pipettes.

flask.

stoppers to

fit.

CARE OF APPARATUS
1. After use, each test tube must be emptied, rinsed in water, well
w a s h e d in hot w a t e r c o n t a i n i n g soda, r i n s e d in d i s t i l l e d w a t e r and
f i n a l l y dried.
2.
If after this treatment a test tube does not appear to be clean,
the treatment must be repeated and in addition after being rinsed in
warm water it must be soaked in 50 percent hydrochloric acid and then
rinsed again in warm water before being rinsed in distilled water and
f i n a l l y dried.
3.
New glassware must be cleaned by soaking in chromic acid solution
p r e p a r e d by s l o w l y and c a r e f u l l y a d d i n g 4 v o l u m e s of c o n c e n t r a t e d
sulphuric acid to 5 volumes of 8 percent
potassium dichromate.
The
solution must be kept covered and must be discarded when it becomes
green.
After cleaning in chromic acid solution, new glassware must
be rinsed in warm water, rinsed in distilled water and finally dried.
4.
P i p e t t e s m u s t be w e l l rinsed in cold w a t e r and then c l e a n e d by
s o a k i n g for 2 4 h o u r s in c h r o m i c acid s o l u t i o n in a 250 m l g l a s s
cylinder or other suitable container. The pipettes must then be well
rinsed in warm water, rinsed in distilled water and finally dried.
5.
G l a s s w a r e used for the test m u s t not be used for any o t h e r
p u r p o s e a n d m u s t be k e p t a p a r t f r o m o t h e r a p p a r a t u s in t h e
laboratory.
REAGENTS
1. B u f f e r s o l u t i o n :
D i s s o l v e 3.5 g of a n h y d r o u s s o d i u m c a r b o n a t e
and 1.5 g of s o d i u m b i c a r b o n a t e in d i s t i l l e d w a t e r , and m a k e up to
1 L with water. Store in a refrigerator and discard after one month.

23

2. Substrate: D i s o d i u m p-nitropoheny1 phosphate.

strate must be kept in the refrigerator.

The solid sub-

3. Buffer-sub8trate solution:
Weigh 0.15 g of the substrate into a
100 ml m e a s u r i n g cylinder, and m a k e up to 100 ml with the buffer
solution.
The solution must be stored in a refrigerator and
protected from light.
It m u s t give a reading of less than the
standard marked 10 on the comparator disc APTW or APTW7 when viewed
in t r a n s m i t t e d light through a 25 m m cell in the c o m p a r a t o r
(distilled water being used for comparison). The solution must be
discarded after one week.
PRECAUTIONS
The following precautions must be taken:
1. Milk
tested .
2.

which

shows

evidence of taint or souring

should

not

be

All glassware must be clean immediately before use.

3. A fresh pipette m u s t be used for each sample of milk.

must not be contaminated with saliva.
4.

The test must not be carried out in direct sunlight.

5.

Distilled water must be used

Pipettes

throughout.

6. The sample of milk should be examined as soon as possible after

arrival at the laboratory.
If not examined immediately, it must be
kept at a t e m p e r a t u r e of b e t w e e n 3C and 5C until examined.
The
sample must be brought to room temperature immediately before being
tested.
PROCEDURE
Pipette 5 ml of the b u f f er-sub s t r a te solut.ion into a test tube,
stopper the test tube and bring to a temperature of 37C. Add 1 ml
of the milk to be tested, replace the stopper in the tube and mix the
contents w e l l by shaking.
Incubate for exactly 2 hours at 37C.
Incubate one blank prepared from boiled milk of the same type as that
undergoing the test with each series of samples.
(Where the samples
consist of highly coloured milk, such as homogenized milk, prepare a
separate blank of such milk).
After incubation remove the test tube
from the water bath and m i x the contents of the tube.
Place the
blank on the left hand side of the comparator stand and the test
s a m p l e on the right. Take readings in reflected light by looking
down onto the two apertures with the comparator facing a good source
of daylight (preferably north light).
If artificial light is needed
for m a t c h i n g , use a "daylight" type of illumination.
Revolve the
disc until the test sample is matched.
Record readings falling
between two standards by affixing a plus or minus sign to the figure
for the nearest standard.
INTERPRETATION
The test is considered to be satisfied by m i l k which gives a reading of 10 p g
or less of p-nitrophenol/ml of milk.
Properly pastuerized milk will give no
discernible colour.
REFERENCES
A s c h a f f e n b u r g , R. and M u l l e n , J.E.C., 1949.
67.

24

Journal of Dairy Research J_6, 58-

Official Methods of the AOAC,

1984.

16.111-.129.

The Milk (Special Designation) Regulations,

25

1963,

S.I. 1571

of the U.K.

T U M I D I T Y TEST
PRINCIPLE
The heating required by sterilization together with a m m o n i u m
sulphate
e f f e c t i v e l y d e n a t u r e s and precipitates albumin as well as casein and the
filtrate of a sample treated in this way is therefore clear.
APPARATUS
1.

Conical flasks of 50 ml capacity.

2.

Graduated cylinders of 25 ml capacity.

3.

Test tubes, 150/16 mm.

4.

Filter funnels, 6 cm diameter.

5.

Beakers, 400 ml capacity.

6.

12.5 cm folded filter papers, Whatman No. 12 or equivalent.

REAGENTS
1.

Ammonium sulphate, AR or equivalent purity.

PROCEDURE
W e i g h 4 _+ 0.1 g of a m m o n i u m sulphate into a 50 ml conical flask.
Pipette 20 +_ 0.5 m l of the m i l k sample into the conical flask and
shake the flask for 1 minute to ensure that the a m m o n i u m sulphate
dissolves.
Leave the m i x t u r e for not less than 5 m i n u t e s and then
filter through a folded filter paper into a test tube. When not less
than 5 ml of a clear filtrate have collected, place the tube in a
beaker of w a t e r which has been kept boiling and keep there for 5
m i n u t e s . Transfer the tube to a beaker of cold w a t e r , and when the
tube is cool, examine the contents for turbidity by moving the tube
in front of an electric light shaded from the eyes of the observer.
INTERPRETATION
The m i l k is considered sterilized when
filtrate showing no sign of turbidity.

a sample

treated

as above gives a

Unlike p h o s p h a t a s e , peroxidase once destroyed by heating (sterilization,

boiling or heating more intensely than pasteurization) cannot be regenerated as
far as is k n o w n and can thus be used as another test for sterilized milk or
m i l k products. The method of detection employed by Guha and Roy (116) is as
follows:
Shake 5 ml milk, curd or milk products (dispersed in water) with 5-10
mg p - p h e n y 1 e n e d i a m i n e and 2 drops of a 10 v o l u m e solution of H22- A violet
colour indicates the presence of peroxidase.
REFERENCE
Ascha ffenburg , R., 1950.

Journal of the Society of Dairy Techno1og y _3, 236.

26

2.2

DRIED MILK

COMPOSITION
Codex
1.

compositional
Milkfat

and

standards

for dried milk products are as

follows

water:

Whole milk powder

Minimum milkfat content:
Maximum milkfat content:
Maximum water content:

26% m/m
less than 49% m/m
5% m/m

Partly
Minimum
Maximum
Maximum

more than 1.5% m/m

less than 26% m/m
5% m/m

skimmed milk powder

milkfat content:
milkfat content:
water content:

Skimmed milk powder

Maximum milkfat content:
Maximum water content:

1.5% m/m
5% m/m

Additives :
Stabilizers
Sodium, potassium and
salts of :
hydrochloric acid
citric acid
carbonic acid
orthophosphoric acid
polyphosphoric acid

Maximum

level

calcium
5000 mg/kg singly
or in combination
expressed as
anhydrous substances

Emulsifiers in instant milk

powders only
Mono- and di-glycerides
Lecithin

2500 mg/kg
5000 mg/kg

Anticaking agents in milk

powders intended to be dispensed
in vending machines
Tricalcium phosphate
Silicates of aluminium,
calcium, magnesium and
sodium-aluminium
Silicon dioxide (amorphous)
Calcium carbonate
Magnesium oxide
Magnesium carbonate
Magnesium phosphate,
tribasic

10 g/kg singly or in
combination

ROUTINE ANALYSIS
The most important determinations to assess the quality of dried m i l k are the
bacteriological examination, m o i s t u r e , solubility index, lactate, and acidity.
R a n c i d i t y v a l u e s s h o u l d be d e t e r m i n e d on s a m p l e s o t h e r than s k i m m e d .
The
constituents other than m o i s t u r e m u s t be in the same proportions as in liquid
m i l k and Vieth's ratio still applies.
Roller-dried m i l k should be examined for
b u r n t p a r t i c l e s w h i c h m a y be i n a d v e r t e n t l y i n c l u d e d in the p r o d u c t d u r i n g
manufacture.
The b a c t e r i o l o g i c a l e x a m i n a t i o n is m o r e i m p o r t a n t t h a n the
chemical and should at least include tests for faecal Streptococci, total plate
c o u n t and p r e s u m p t i v e c o l i f o r m s .
If the l a t t e r is p o s i t i v e , t e s t s s h o u l d be

27

m a d e for S t a p h y l o c o c c i , S a l m o n e l l a and Clostridia. Identity and composition

m a y be checked from the fat, p r o t e i n , lactose and ash and HAI or RPK on the
fat.
The fat content may be determined by the method of Rose-Gottlieb, taking up to
1.5 g of s a m p l e d e p e n d i n g on the expected fat content and d i l u t i n g to 10 m l
w i t h w a t e r , then p r o c e e d i n g as for liquid milk. P a t r a t i i and S o c h n e v a (117)
discuss the various methods available.
P r o t e i n is c a l c u l a t e d from the K j e l d a h l n i t r o g e n , using a factor of 6.38 for
all m i l k p r o d u c t s .
L a c t o s e can be d e t e r m i n e d on a cleared solution by a
polarimetric, gravimetric or volumetric method.
A b o u t 2 g is taken for the ash d e t e r m i n a t i o n .
It is n e c e s s a r y to r e m o v e the
dish from the m u f f l e about an hour after smoke is no longer given off. Break
up the ash w i t h a g l a s s rod, add w a t e r , e v a p o r a t e , dry and c o n t i n u e ashing.
The r e s i d u e m a y be used to d e t e r m i n e the a l k a l i n i t y of the ash.
10 ml 0.2 N
HC1 is added and the mixture warmed to complete solution.
After cooling 2 ml
of 40% n e u t r a l c a l c i u m chloride solution and 0.5 ml of p h e n o 1 p h t h a 1 e i n are
added and the m i x t u r e is titrated w i t h 0.2 N NaOH to a faint pink e n d - p o i n t
l a s t i n g for 30 seconds. The result is expressed as ml of 0.2 N HC1 per 100 g
of original powder.
Cans of dried milk are often packed under inert gas (which should not contain
m o r e than 5% o x y g e n ) and this m a y result in a s w e l l i n g of the a l u m i n i u m foil
seal if stored at a h i g h t e m p e r a t u r e .
This is not to be confused with gas
production due to bacterial spoilage.
EQUIVALENCE
A claim may be made as to the number of pints or litres of liquid cows' milk to
which the tin or container of dried milk is supposed to be equivalent. This is
calculated from the analytical data assuming values for liquid milk.
Suggested assumed values for Standard Liquid Milk are:

Type of Dried Milk

Full Cream
Three-quarters
Half cream
Quarter cream
Skimmed

cream

Z Fat

Z Total Milk
Solids

3.6
2.7
1.8
0.9

12.4
11.6
10.8
9.9

Z SolidsHot-Fat

9.0

Density
at 20C
1.032
1.033
1.034
1.034
1.0355

The equivalent pints may be calculated from the fat, total solids, solids-notfat, or even from the p r o t e i n or lactose provided the a p p r o p r i a t e factor is
calculated.
For e x a m p l e , the label c l a i m s the c o n t e n t s of the can are
equivalent to Z litres of half-cream milk.
1 litre of standard half-cream milk weighs 1,034 g.

This contains

1.8
100

x 1,034 = 19.4 g of fat.

Fat in sample (by experiment) = 14.3%.

Contents of sample can = 507 g.
14.3
Then the can contains

x 507 = 72.5 g

28

14.3 x 507
Equivalent
to
4

Or in general:

where

P
W

19.4 x 100

14.3 x 507
=

1940

, .
.J
,.
litres of liquid milk

P x W
400 x P

= weight of the parameter in the whole contents of

conta iner.
= weight of whole contents of container.

P^ = weight of the parameter in the standard volume

(litre or pint) of liquid milk.

29

DRIED MILK MOISTURE

PRINCIPLE
The sample is dried
as moisture.

to constant

weight at 102C and the loss in weight

reported

APPARATUS
1.

Metal dishes with close-fitting

lids.

PROCEDORE
D r y a d i s h and lid in the o v e n and cool in the d e s i c c a t o r .
Exactly
weigh approximately 1 g of sample into the dish and dry in the oven 2
hours with the lid alongside.
Place the lid on the dish, transfer to
the desiccator and quickly weigh when the dish has completely cooled.
Heat in the oven half-an-hour and re-weigh.
Repeat until succeeding
weights do not differ by more than 0.5 mg.
CALCULATION

% moisture m/m

weight lost x 100

;
weight of sample

INTERPRETATION
S p r a y d r i e d p o w d e r s u s u a l l y h a v e a m o i s t u r e c o n t e n t of about 3% and r o l l e r
dried powders about 5%. The latter are less commonly found than formerly as an
article of commerce.
Results over 5% would justify further examination, as the
moisture may be sufficient to sustain mould or bacterial growth.
Difficulties in obtaining consistent results may be due to small differences in
oven temperature, oven design etc.
These are discussed by E m m e n s et al (118),
M o r i s s e y ( 1 1 9 ) and A n d e r s o n and B e r l i n (120) h a v e s h o w n that the total
m o i s t u r e , including the water of crystallization of alpha-lactose monohydrate
m a y be d e t e r m i n e d by K a r l F i s c h e r or a z e o t r o p i c d i s t i l l a t i o n w i t h t o l u e n e
( m e t h o d of D e a n and Stark).
D r y i n g at 65C for six h o u r s u n d e r a v a c u u m of
a b o u t 100 torr p a r t i a l p r e s s u r e g i v e s a v a l u e c o r r e s p o n d i n g to the "free"
moisture.
The AOAC method involves drying under vacuum at 100C.
REFERENCE
International

Dairy Federation, FIL-IDF

26:1964.

DRIED MILK

SOLUBILITY

PRINCIPLE
The p o w d e r is s h a k e n w i t h w a t e r and the total s o l i d s of the s u s p e n s i o n
determined before and after centrifuging.
The amount of powder remaining in
suspension after centrifuging expressed as a percentage of the total amount in
suspension is taken as a measure of the solubility.
APPARATUS
1.

Centrifuge with 50 ml

tubes.

PROCEDURE
Shake 4 g of p o w d e r w i t h 32 m l w a t e r at 50C for 10 s e c o n d s in a 50
ml centrifuge tube and keep the tube in water at 50C for 5 minutes.
Centrifuge the suspensions from half-cream and full-cream samples for
10 m i n u t e s at 2 0 0 0 r p m .
Cool in a r e f r i g e r a t o r and r e m o v e the fat
layer after p r i s i n g it from the w a l l s of the tube w i t h a n e e d l e .
Warm to room temperature, break up the deposit with a glass rod and
shake vigorously until the suspension appears homogeneous.
For all t y p e s of s a m p l e , p i p e t t e 2 m l from the t u b e , w e i g h into a
tared metal dish with lid and determine the total solids by drying on
a w a t e r b a t h and t h e n in the o v e n 1 - 1 / 2 h o u r s .
C e n t r i f u g e for 10
m i n u t e s at 2000 r p m and d e t e r m i n e the total s o l i d s of 2 m l of
supernate.
CALCULATION
% solubility
where

Tj

T2

S^
S2

=
=

100 T, S 2
ijr^gy^ *

weight of suspension taken for total

solids determination before centrifuging
weight of suspension taken for total
solids determination after centrifuging
weight of dried solids remaining after
evaporation of Tj^
weight of dried solids remaining after
evaporation of T2.

INTERPRETATION
Spray dried powders are almost completely soluble, roller dried to the extent
of 80% or m o r e .
The r e s u l t s d e p e n d on the e x a c t c o n d i t i o n s of test and the
a c i d i t y of the p o w d e r so it is a d v i s a b l e to c o m p a r e d o u b t f u l s a m p l e s w i t h a
spray dried p o w d e r k n o w n to be r e a s o n a b l y fresh.
The p o w d e r s b e c o m e less
soluble with age, thereby affecting the quality.

REFERENCES
D a v i s , J.G. and M a c D o n a l d , F.J., 1953.
London.
British Standard
Bonduelle,

1743:Part

C. and Luquet,

Richmond's Dairy Chemistry,

2:1980.
F.M.,

1971.

Technique

31

Laitiere

718.

18-19.

Griffin,

DRIED MILK LACTATE

PRINCIPLE
The sample is reconstituted and clarified and lactate in the filtrate converted
to acetaldehyde.
The colour developed b e t w e e n the acetaldehyde and ph y d r o x y d i p h e n y l is determined spec t r ophotometr ical ly and the lactate content
calculated from a standard graph.
APPARATUS
1.

Spectrophotometer reading at 570 nm.

2.

Graduated pipette or burette accurate to _+ 0.05 ml.

REAGEHTS
1.
Copper sulphate solution, 25% - dissolve 250
sulphate pentahydrate in water and dilute to 1000 ml.

of

cupr ic

2.
Acid copper sulphate solution - add 0.5 ml of 25%
sulphate solution to 300 ml of concentrated sulphuric acid.

copper

3.
C a l c i u m h y d r o x i d e suspension - grind in a mortar 300
c a l c i u m h y d r o x i d e with water to a total of 900 ml.
Store
tightly stoppered bottle.
4.
p-H y d r o x y d i p h e n y 1
h y d r o x y d i p h e n y l in 5 ml of
necessary.
Dilute to 50 ml
in a cool place and discard

g of
in a

s o l u t i o n - d i s s o l v e 0.75 g of p 5% aqueous NaOH, shaking and w a r m i n g as

in a graduated flask.
Store in the dark
if discoloured or turbid.

5.
Lithium lactate standard - dissolve 0.1067 g in water and dilute
to 100 ml.
1 ml contains 0.1 mg equivalent lactate.
Prepare fresh.
6.
Fresh dried milk containing
g of solids-not-fat.

less than 30 mg lactic acid per 100

PROCEDURE
For the test use a weight of sample equal to 1000/(SNF-10) where SNF
(solid8-not-fat) is the percentage calculated by subtracting fat and
moisture from 100. Thus a little over 11 g of skimmed milk powder is
usually used, weighed accurately to the nearest 0.1 g.
Add this
weight to 100 ml water and mix thoroughly. Pipette 5 ml into a 50 ml
graduated flask and dilute to about 35 ml. Prepare a blank at the
same time by adding about 35 ml of water to another flask.
Add
slowly, with swirling, 5 ml of 25% copper sulphate solution and leave
to stand 20 m i n u t e s . Dilute to 50 ml, shake vigorously, filter and
Pipette 1 ml of filtrate
discard the first part of the filtrate.
into a test tube (25 x 150 mm).
Add exactly 6 ml of acid copper
sulphate solution, heat in a boiling waterbath 5 minutes then cool in
running water.
Add 2 drops of p-hydroxydiphenyl solution and shake
thoroughly.
Keep in water at 30 +_ 2C for 15 m i n u t e s , in boiling
water for 90 seconds and then cool to ambient temperature in running
water. M e a s u r e the absorbance at 570 nm against the blank. If the
reading is higher than the most concentrated standard, dilute an
aliquot of the filtrate and repeat the test with 1 ml of this diluted
solution.

32

PREPARATION OP STANDARDS
M i x a weight of fresh l o w - a c i d s k i m m e d m i l k p o w d e r with 100 ml
of w a t e r , the w e i g h t calculated from 1 0 0 0 / ( S N F - 1 0 ) as for samples.
Pipette 5 ml of the reconstituted m i l k into each of 5 v o l u m e t r i c
flasks and add 0, 1, 2, 3 and 4 m l r e s p e c t i v e l y of standard lactate
solution.
The standards correspond to 0, 20, 40, 60 and 80 mg of
added lactic acid per 100 g of s o 1 id s-no t-f a t. Dilute the contents
of each flask to about 35 ml with water and proceed as for a sample.
Plot m e a s u r e d a b s o r b a n c e against m g of lactic acid. The standards
must be m e a s u r e d against the same blank as the samples. The blank
w h e n m e a s u r e d against w a t e r should not give an a b s o r b a n c e reading
corresponding to more than 20 mg of lactic acid per 100 g of solidsnot-fat. Agreement of duplicate determinations should be better than
10%.
CALCULATION
The c a l i b r a t i o n curve gives the a b s o r b a n c e s for
lactic acid. 1 m l of filtrate is derived from 1/50
weight of powder taken.

0 - 0.4 mg of
x 5/100 of the

Hence mg lactic acid per 100 g of so1 ids-not-fat = mg

(read from graph) x 50/1 x
100/5
x
100/weight taken.

in

aliquot

INTERPRETATION
The lactate content tends to decrease slightly on storage, but may generally be
taken as an i n d i c a t i o n of the acidity of the m i l k b e f o r e processing. A high
lactate value would indicate the possibility that the dried milk was made using
soured whole milk.
REFERENCE
ISO 3495:1975.

33

2.3

EVAPORATED AND CONDENSED MILK

COMPOSITION
Traditionally, evaporated milk refers to the unsweetened product and condensed
to the s w e e t e n e d , a l t h o u g h there have b e e n r e c o m m e n d a t i o n s to discard this
terminology.
Some typical analyses

(taken from Pearson

(2))

Sweetened Condensed Milk

Full Cream
Skimmed
1
2
3
4

Water
Milk Solids
Fat
Lactose monohydrate
Protein
Sucrose
Ash
Acidity
(as lactic acid)
Specific gravity
The Codex Alimentarius
are as follows:

25 .0
33 .3
10 .6
12 .2
8 .5
41 .5
1 .93

25.1
32.9
9.6
13.0
8.4
41.9
1.86

26.6
27.2
0.2
14.9
9.6
46.1
2.45

27.0
26.3
0.2
14.2
9.5
46.5
2.31

0 .32
1 .30

0.28

0.35

0.30

Commission

standards

Evaporated
Milk
Minimum milkfat
content % m/m
Minimum total milk
solids content % m/m

Evaporated Milk
Full Cream
Skimmed
6
5
7
%
%
%
66.1
33.8
9.2
13.3
9.1
-

76.6
23.3
0.7
12.5
8.3

2.05

1.94

1. 7<

0.35
1.08

0.40
1.08

0.41

for evaporated

Evaporated
Skimmed
Milk

67.4
32.5
9.1
12.7
8.7

and condensed

Sweetened
Condensed
Milk

milk

Skimmed
Sweetened
Condensed
Milk

8.0

7.5

25.0

20.0

28.0

24.0

Sodium, potassium and

calcium salts of hydrochloric, citric, carbonic, orthophosphoric
and polyphosphoric acid,
expressed as anhydrous
substances

2000 mg/kg
singly,
3000 mg/kg
in combination

2000 mg/kg
singly,
3000 mg/kg
in combination

2000 mg/kg
singly,
3000 mg/kg
in combination

2000 mg/kg
singly,
3000 mg/kg
in combination

Carrageenan

150 mg/kg

150 mg/kg

It is not suggested that these standards are necessarily the only compositional
limits that need to be defined for the purposes of national legislation.
Some
national standards have somewhat higher values for milkfat and milk solids.
ROUTINE ANALYSIS
A proximate analysis - total solids, fat, lactose, protein, ash and sucrose in
sweetened milks should be done on routine samples.
The acidity should account
for the r e m a i n d e r .
If there appears to be a d i s c r e p a n c y in the analysis, it
m a y be w o r t h w h i l e to look for starch, or other fillers or t h i c k e n e r s , and if
the p r o t e i n is high in r e l a t i o n to the lactose and ash (Vieth's ratio), for
gelatin.

34

T h e f a t m a y b e d e t e r m i n e d b y t h e R o s e - G o 1 1 1 i e b m e t h o d , d i l u t i n g 2 - 2.5 g ,
a c c u r a t e l y w e i g h e d , w i t h 8 m l of w a t e r and p r o c e e d i n g as for m i l k s a m p l e s .
A
r a p i d r e s u l t m a y b e o b t a i n e d b y t h e G e r b e r m e t h o d f o r l i q u i d m i l k u s i n g 10.75
B u t g r o m e t e r r e a d i n g x 5 = % fat m / m .
m l of a 20% m / v s o l u t i o n of the s a m p l e .
It is c o n v e n i e n t to u s e t h e 2 0 % m / v s o l u t i o n so p r e p a r e d f o r a l l o f t h e
proximate analysis.
L i v i o (121) d i s c u s s e s u s e of the M o j o n n i e r m e t h o d .
P r o t e i n and ash m a y be d e t e r m i n e d by r o u t i n e m e t h o d s .
Ashing
c o n d e n s e d m i l k m a y be f a c i l i t a t e d b y a d d i t i o n of a d r o p of o i l .

of

sweetened

L a c t o s e m a y b e d e t e r m i n e d b y t h e L a n e and E y n o n t i t r a t i o n , u s i n g 25 m l o f m i x e d
F e h l i n g ' s s o l u t i o n and c a l c u l a t i n g the r e s u l t as l a c t o s e m o n o h y d r a t e .
10-12 g
i s d i l u t e d w i t h 2 0 0 m l o f h o t w a t e r in a 2 5 0 m l g r a d u a t e d f l a s k a n d l e f t t o
s t a n d at l e a s t 30 m i n u t e s .
A f t e r c o o l i n g , 4 m l of z i n c a c e t a t e s o l u t i o n is
a d d e d , m i x e d and t h i s is f o l l o w e d b y 4 m l o f p o t a s s i u m f e r r o c y a n i d e s o l u t i o n .
T h e m i x t u r e is d i l u t e d to 2 5 0 m l a n d f i l t e r e d a n d t h e t i t r a t i o n c a r r i e d o u t
using
the f i l t r a t e .
T h e s t r e n g t h of the p r o t e i n
prcipitants
and
the
c o r r e c t i o n s f o r t h e v o l u m e o f p r e c i p i t a t e a r e t h e s a m e a s t h o s e u s e d in t h e
d e t e r m i n a t i o n of s u c r o s e in c o n d e n s e d m i l k .
N o w a k a n d L a s k o w s k i ( 1 1 1 ) f o u n d t h a t t h e I S O / I D F m e t h o d for c i t r i c a c i d in
c h e e s e and p r o c e s s e d c h e e s e w o r k s e q u a l l y w e l l w i t h o t h e r m i l k p r o d u c t s .
P h o s p h o r u s c a n b e d e t e r m i n e d b y t h e m e t h o d g i v e n in I D F 4 2 : 1 9 6 7 .
EQUIVALENCE
The e q u i v a l e n t v o l u m e of a n o r m a l m i l k m a y be c a l c u l a t e d and c o m p a r e d w i t h any
c l a i m on the l a b e l .
For the p u r p o s e of c a l c u l a t i n g the e q u i v a l e n t p i n t s the
c a n s h o u l d b e w e i g h e d u n o p e n e d , t h e c o n t e n t s t r a n s f e r r e d to a s a m p l e j a r w i t h
an a i r t i g h t s e a l , t h e c a n w a s h e d a n d d r i e d a n d r e w e i g h e d , in o r d e r to o b t a i n
the exact w e i g h t of the c o n t e n t s .
A n y s m a l l s l i v e r s of m e t a l p r o d u c e d d u r i n g
o p e n i n g m u s t b e i n c l u d e d in t h e w e i g h t o f t h e e m p t y c a n .
Old tins m a y r e q u i r e
w a r m i n g to a b o u t 4 0 b e f o r e o p e n i n g t o f a c i l i t a t e m i x i n g t h e c o n t e n t s .
The
s a m p l e s h o u l d a l s o b e e x a m i n e d f o r m e t a l l i c c o n t a m i n a t i o n ( t i n a n d l e a d ) if t h e
can appears etched.
N e x t , d i l u t e the c o n t e n t s o f the c a n w i t h t h e r e q u i s i t e a m o u n t of w a t e r to m a k e
m i l k of n o r m a l c o m p o s i t i o n .
T h e v o l u m e so o b t a i n e d m a y b e c a l l e d b y a p h r a s e
A c l a i m m a y b e m a d e in t h e l a b e l r e l a t i n g to t h i s
s u c h as " e q u i v a l e n t p i n t s " .
v o l u m e and the a c c u r a c y of a n y such c l a i m m u s t be c h e c k e d .
The c o m p o s i t i o n of
" n o r m a l m i l k " is a s s u m e d , e i t h e r b y b e i n g s t i p u l a t e d in r e g u l a t i o n s , o r b y
taking known average values.
For the p u r p o s e of the w o r k e d e x a m p l e " n o r m a l
m i l k " w i l l b e t a k e n to c o n t a i n 3.6% f a t a n d 12.4% t o t a l m i l k s o l i d s .
These
f i g u r e s h a v e b e e n c a l c u l a t e d u s i n g T M S = 0.25D + 1.22F + 0.72 and d e n s i t i e s of
1 . 0 2 9 , 1.031 and 1.0325 ( w h e r e T M S = t o t a l m i l k s o l i d s , D = 1,000 x d e n s i t y 1,000 and F = % fat).
One pint ("imperial"
ounces weight.
Total milk solids
= 72.56 g.
Total milk
=

T M S x WfoQ

Equivalent

solids

p i n t ) of

present

in a c a n

this

MS

= 20

fluid

( 1 2 . 4 / 1 0 0 ) x 20.64

of c o n d e n s e d

.
( w h e r e W is t h e w e i g h t

pints =

milk

of

T M S in c a n
in o n e s t a n d a r d

milk

the

= 2.559

x 1.032

ounces

20.64

weight

(TMS)

contents

pint

ounces

in

grams)

% TMS x W=
100/72.56

% TMS x W
725Z

By a n a l o g y , f a c t o r s c a n b e c a l c u l a t e d f o r l i t r e s a n d A m e r i c a n p i n t s , if t h e
c l a i m is m a d e in e i t h e r o f t h e s e u n i t s .
A l s o , factors can be c a l c u l a t e d using
a s s u m e d v a l u e s f o r f a t , m i l k - p r o t e i n , l a c t o s e or s o l i d s - n o t - f a t .
A m e r i c a n and

35

Imperial fluid ounces are slightly different, but ounces weight are the
One U.S. pint 0.8327 " i m p e r i a l " pints.
Assumed

Composition of Standard Milk

Fat
Ful1-Cream
Half-Cream
Skimmed

(semi-skimmed)

Solidsnot-fat

Total
milk solids

SG

8.8
9.0
9.0

12.4
10.8
9.0

1 .032
1 .034
1 .0355

3.6
1.8
-

36

same;

TOTAL SOLIDS
(Evaporated and Condensed

Milk)

PRINCIPLE
The s a m p l e is dried to c o n s t a n t w e i g h t at 100C, u n d e r s t a n d a r d c o n d i t i o n s ,
which must be followed closely in order to obtain reproducible results.
APPARATUS
1.
F l a n g e d m e t a l dish about
close fitting lid.

7.5 cm d i a m e t e r

and 2.5 cm d e e p

with

2.
G l a s s s t i r r i n g rod w i t h a f l a t t e n e d end, the o t h e r end bent to
rest over the dish edge.
3.

Steam bath.

REAGENTS
1.
Acid-washed
mesh one.

sand,

retained

by an 85 mesh sieve but passing

a 30

PROCEDURE
P l a c e a b o u t 25 g of sand into a d i s h and dry to c o n s t a n t w e i g h t at
98-100C.
Dry a lid and s t i r r i n g rod at the s a m e t i m e .
P l a c e the
lid with the stirring rod on it, on the dish before removing it from
the oven, cool in a desiccator and weigh after 45 minutes. Allow the
sand to slide to one side of the dish and add about 3 g of well-mixed
u n s w e e t e n e d or 1.5 g of s w e e t e n e d m i l k and w e i g h r a p i d l y . Add 3 m l
of w a t e r for u n s w e e t e n e d , 5 m l for s w e e t e n e d m i l k , m i x w i t h the
stirring rod, and then mix the diluted milk with the sand. Leave the
s t i r r i n g rod in the d i s h , h o o k i n g the end over the e d g e of the d i s h .
P l a c e the d i s h on a s t e a m b a t h such that the f l a n g e is at the l e v e l
of the s t e a m - b a t h cover but the t w o m e t a l s u r f a c e s should be
s e p a r a t e d by s u p p o r t i n g the f l a n g e of the d i s h on a p o r c e l a i n or
rubber ring. Stir the sand-milk mixture at first to avoid caking and
to keep the mass aerated.
Once the aqueous phase has evaporated, lay
the s t i r r i n g - r o d on the sand.
L e a v e the d i s h on the s t e a m - b a t h
twenty minutes in all, then transfer to an oven at 98-100C, with the
lid alongside.
Leave in the oven 1-1/2 hours, replace the lid, cool
in a desiccator 45 minutes and weigh.
If possible cool each dish in
a separate desiccator.
(Ensure that the d e s i c c a n t is e f f e c t i v e . )
Remove the lid and place both dish and lid in the oven for a further
hour, cool and weigh as before. Repeat until successive weighings do
not differ by more than 0.5 milligram.
CALCULATION
Total Solids =

Weight of residue
;
Weight of sample

x 100

INTERPRETATION
T h i s m e t h o d is s u b j e c t to e r r o r s s i m i l a r to those m e n t i o n e d under m o i s t u r e
d e t e r m i n a t i o n in s k i m m e d m i l k p o w d e r , due to the p r e s e n c e of l a c t o s e .
It is
for this r e a s o n that the m e t h o d m u s t be f o l l o w e d e x a c t l y in o r d e r to o b t a i n
reproducible results.
F r a n z e n (122) r e c o m m e n d s 4 - 6 a n a l y s e s per s a m p l e for
r e l i a b l e results.
REFERENCE
International Dairy Federation

15:1961.

37

SUCROSE
(Condensed M i l k )
PRINCIPLE
The optical rotation of a cleared solution of the sample is measured before and
a f t e r m i l d h y d r o l y s i s w h i c h i n v e r t s the s u c r o s e b u t has a l m o s t no e f f e c t on
l a c t o s e or o t h e r s u g a r s .
The p e r c e n t a g e of s u c r o s e p r e s e n t is c a l c u l a t e d by
use of a formula.
APPARATUS
1.
Polarimeter with sodium or mercury green light (Mercury vapour
lamp with prism or special Wratten Screen 66A) reading 0.05 of angle
or better or saccharimeter with International sugar scale with white
l i g h t p a s s e d t h r o u g h a f i l t e r of 1.5 c m of 6% p o t a s s i u m d i c h r o m a t e ,
or s o d i u m l i g h t , and r e a d i n g 0.1 cm or b e t t e r on the I n t e r n a t i o n a l
sugar scale.
Both should be fitted with 2 dm tubes.
2.

Waterbath

at 60C + 1.

REAGENTS
1.
Zinc a c e t a t e s o l u t i o n .
D i s s o l v e 21.9 g of the d i h y d r a t e in
w a t e r , add 3 m l of g l a c i a l a c e t i c acid and d i l u t e to 100 m l w i t h
water.
2.
P o t a s s i u m f e r r o c y a n i d e (II) s o l u t i o n .
trihydrate in water and dilute to 100 ml.
3.

Dilute

ammonia

solution, 2N

4.

Dilute acetic acid solution, 2N

5.

Hydrochloric

Dissolve

10.6 g of the

(3.5%).
(12%).

acid, 6.34N.

PROCEDURE
Accurately weigh approximately 40 g of well-mixed sample, add 50 ml
of water at 80-90C and mix.
Transfer to a 200 ml volumetric flask,
rinsing with quantities of water at about 60C until the total volume
is 1 2 0 - 1 5 0 m l .
M i x , cool and add 5 m l of 2N a m m o n i a s o l u t i o n .
Mix
and leave to stand 15 minutes.
This completes lactose mutarotation.
Add exactly the v o l u m e of 2N acetic acid required to neutralize the
ammonia.
Add 12.5 ml of zinc acetate solution, swirling at the same
time and then 12.5 m l of potassium ferrocyanide solution, continuing
swirling.
Dilute to volume at 20C. Avoid inclusion of air bubbles.
If this inadvertently occurs, apply gentle suction.
Shake, leave to
stand a few minutes and filter through a dry 15 cm filter paper (e.g.
W h a t m a n No. 4), rejecting the first 25-30 ml of filtrate.
Determine the optical rotation of the filtrate at 20C.
There is no
need to correct the reading for temperature as long as the solution
is b e t w e e n 18 and 22C.
I n v e r t ( h y d r o l y s e ) an a l i q u o t by p i p e t t i n g 40 m l into a 50 ml
graduated flask, adding 6.0 m l of 6.34 N HC1 and immersing the flask
up to its n e c k in a w a t e r b a t h at 60C for 15 m i n u t e s .
M i x by a
r o t a r y m o v e m e n t d u r i n g the first five m i n u t e s .
Cool to 20C and
d i l u t e to 50 ml.
M i x and leave to stand at 20C for one hour.
Determine the optical rotation.
The temperature must be between 18
and 22C.
If it is not exactly 20C use the correction factor 0.0037
x ( T - 2 0 ) w h e r e T is the e x p e r i m e n t a l t e m p e r a t u r e , a d d i n g if the
temperature is above 20C and subtracting if it is below.

38

METHOD

VALIDATIOH

Add 18.00 g of p u r e s u c r o s e to 100 g of l i q u i d m i l k (or 110 g of

liquid s k i m m e d milk).
T h i s c o r r e s p o n d s to 4 0 g of c o n d e n s e d m i l k
containing 45% sucrose.
Calculate the sugar content using the weight
of liquid m i l k and its protein and fat values to calculate the v o l u m e
of precipitate, but using 40.00 g as the weight taken in the formula
to c a l c u l a t e s u c r o s e c o n t e n t .
T h e r e s u l t s h o u l d be w i t h i n 0.1% of
45%.

CALCULATION
C o r r e c t i o n in m l
ciari ficat ion,

for

the v o l u m e

of the p r e c i p i t a t e

formed

during

v = (W/100) (1.08F + 1.55P)

where W = weight of sample in g
F = % fat in sample
P = % protein (N x 6.38) in sample

D - (5/4 x I)
% sucrose =

(V - v )
x

(L x W )

where D = polarimeter reading before inversion

I = polarimeter reading after inversion
V = volume in ml to which sample was diluted before
filtration (200ml)
v = volume of precipitate as calculated above
L = length in dm of polarimeter tube (2 dm)
Q = the inversion divisor factor, which varies with
wavelength of incident light, concentration and
temperature
For sodium light and angular
Q = 0.8825 + 0.0006
For mercury

(C - 9) - 0.0033

light and angular

Q = 1.0392 + 0.0007

degrees

degrees

(C - 9) - 0.0039

For white light and International

Q = 2.549 + 0.0017

(T - 2 0 )

sugar

degrees

(C - 9) - 0.0095

(T = temperature of inverted

(T - 2 0 )

(T - 20)

solution when

read)

C is the p e r c e n t a g e of t o t a l s u g a r s in the i n v e r t e d s o l u t i o n as
polarised, equal to 9.00 if exactly 40 g of condensed m i l k of n o r m a l
composition is used.
With this weight and the temperature at exactly
20C, s o d i u m l i g h t , a n g u l a r d e g r e e s and a 2 dm t u b e , the f o r m u l a
simplifies to:

(D -

- ) (2.833 - 0.00612F - 0 . 0 0 8 7 8 8 )

The c o r r e c t i o n 0.0006 ( C - 9 ) is o n l y a c c u r a t e if C is c l o s e to 9, but

the c o r r e c t i o n is s m a l l e n o u g h to be i g n o r e d w i t h n o r m a l s a m p l e s .
Duplicates should agree within about 0.3%.

39

2.4

BUTTER A M D GHEE

(Butter Oil)

COMPOSITIOM
The fat c o n t e n t of b u t t e r is u s u a l l y over 8 0 % but m a y be a l i t t l e under in
salted b u t t e r s . It is m o s t c o n v e n i e n t l y d e t e r m i n e d by d i f f e r e n c e (100% less
water, curds and salt). It can also be determined by direct ether or petroleum
ether extraction of the residue from the moisture determination, filtration and
evaporation.
This requires more careful manipulation to ensure that no fat is
left in the f i l t e r nor d r a w n to the o u t s i d e of the d i s h by c a p i l l a r i t y ,
especially if diethyl ether is used.
The moisture is usually about 12-15% and
some countries have an upper limit of 16%. The CAC recommends a m i n i m u m of 80%
m / m fat, m a x i m a of 2% milk solids other than milkfat and 16% water.
The CAC standard at present recommends that the colours annatto, beta-carotene
and curcumin be permitted if added according to good manufacturing practice,
and
the n e u t r a l i z i n g
salts calcium
hydroxide,
and
the
carbonate,
orthophosphate, bicarbonate and hydroxide of sodium may be added only for the
p u r p o s e of pH a d j u s t m e n t up to a m a x i m u m , s i n g l y or in c o m b i n a t i o n , of 2 0 0 0
mg/kg expressed as anhydrous substances.
The ash is about 0.1% in u n s a l t e d b u t t e r and the salt is less than h a l f of
t h i s , b u t m a y be 0.5 - 5% in s a l t e d b u t t e r .
The a m o u n t of salt that m a y be
added is limited by the solubility in the aqueous phase and cannot exceed 2-3%
in countries that impose an 80% m i n i m u m fat content.
If salt has been added,
mention of this addition should be included in the name.
The value for free fatty acids as oleic acid is normally 0.2-0.3% in the fresh
product, and the peroxide value less than 10 meq of oxygen per kilogram of fat.
0.5% and 20 meq/kg respectively may reasonably be taken as upper limits.
The CAC s t a n d a r d r e q u i r e s g h e e (butter o i l ) to c o n t a i n at least 99.3 m / m of
b u t t e r f a t and a m a x i m u m of 0.5% m / m of w a t e r .
For the a n h y d r o u s p r o d u c t the
corresponding figures are 99.8% and 0.1%.
Butter oil for certain manufacturing
purposes only (e.g. biscuit making), excluding use to prepare recombined milk
or milk products, may contain propyl, octyl or dodecyl gallates in combination
with BHA or BHT up to a m a x i m u m of 200 mg/kg in combination, but the gallates
must not exceed 100 mg/kg.
The r e f r a c t i v e i n d e x , s p e c i f i c g r a v i t y , t i t r e , s a p o n i f i c a t i o n v a l u e , i o d i n e
v a l u e , V a l e n t a , and the C r i s m e r and C T D t e s t s all i n d i c a t e a d u l t e r a t i o n if
found to be o u t s i d e the n o r m a l r a n g e for b u t t e r oil but the v a l u e s for m a n y
severely
o t h e r fats and fat m i x t u r e s also lie w i t h i n this r a n g e , thus
restricting the usefulness of these tests.
The CTD test, favoured by Hart and
Fisher, developed out of the Valenta and Crismer tests and may be found in the
AOAC (13th edition, 1980).
The methodology for the other tests is given in the
section on oils and fats.
Typical values for butter are as follows:

Specific gravity at
Refractive

3 7.8 C
^ gc

index at 40C

Max

M in

Mean

9.913

9.910

9.912

1.4580

1.4522

1.4588

= Zeiss butryo

scale

45.5

40.0

43.5

Saponification

value

243.0

209.0

228 .5

Iodine value

50.0

26.0

36.0

Mean molecular weight of

fatty acids

267.0

258.0

260.0

Titre

variable, usually

41

32-38C

T h e r e f r a c t i v e i n d e x used to be of v a l u e , but this is no l o n g e r true as m a n y

m a r g a r i n e s h a v e v a l u e s in the s a m e range.
The v a r i o u s c o l o u r tests for
p a r t i c u l a r o i l s g i v e n in the s e c t i o n on o i l s and fats m a y also be a p p l i e d to
butter and of course, if positive, indicate adulteration.
The only proviso is
that Halphen's test for cottonseed oil, and also the Villavecchia-Fabris test
for sesame seed oil m a y be slightly positive for genuine butter samples if the
animals have been feeding on these products.
The feeding of unsaturated fats
in m i c r o - e n c a p s u l a t e d f o r m to c o w s f a c i l i t a t e s the m a n u f a c t u r e of a m o r e
spreadable butter with lower RPK, HAI and butryic acid values.
ROUTINE

AHALYSIS

Butter should first be examined for moisture, salt, curds and fat. Butter oil
( g h e e ) and the fat f r o m b u t t e r s h o u l d be c h e c k e d for i d e n t i t y , r a n c i d i t y and
added antioxidants.
The residual moisture in butter oil may be determined by
d r y i n g in the o v e n or by K a r l F i s c h e r t i t r a t i o n .
It m a y also be n e c e s s a r y to
look for trace metals (especially copper, but also Fe, Mn, Cr, Ni, etc. promote
r a n c i d i t y ) , a d d e d c o l o u r and u n d e s i r a b l e p r e s e r v a t i v e s such as b o r i c acid.
Ghee m a d e from buffalo milk normally has a higher Reichert value.
For the means of checking the identity, and a more detailed examination if the
identity is suspect, see the analysis sections on the detection of foreign fats
in b u t t e r f a t .
T h e r e is v e r y l i t t l e i n f o r m a t i o n on the p r e v a l e n c e of the
adulteration of butter, but a report from one country for the year 1970 showed
that of 50 samples, 20 contained over 16% moisture, 10 contained less than 80%
fat, 13 samples contained non-milk fat and two contained starch.
T e s t s for f i l t h , s e d i m e n t and p e s t i c i d e r e s i d u e s are g i v e n in the A O A C .
Details for the routine analysis of butter, including pH of serum, titratable
a c i d i t y , e x t r a n e o u s m a t t e r , c o p p e r and iron are g i v e n in the A u s t r a l i a n
S t a n d a r d AS 1 9 3 9 - 1 9 7 5 and in BS 769:1961. BS 5 0 8 6 : 1 9 7 4 d e s c r i b e s the rapid
methods.
T i m m e n and B l u t h g e n (123) d e s c r i b e a p h o t o m e t r i c m e t h o d for the
d e t e r m i n a t i o n of copper and iron in butterfat and Roschnik (124) describes an
atomic absorption method for copper.
Parodi (125) found that examination of the ratios of fatty acids as determined
by G L C w a s o n l y p a r t i a l l y s u c c e s s f u l in i d e n t i f y i n g a d u l t e r a t e d b u t t e r f a t
samples.
H o w e v e r , P a r o d i (126) found that a c o m b i n a t i o n of fatty acid
analysis, sterol analysis, triglyceride distribution patterns, softening point
v a l u e s and IR m e a s u r e m e n t of trans u n s a t u r a t i o n w a s a d e q u a t e to d i s t i n g u i s h
samples.
a b n o r m a l and t h o s e m o d i f i e d by f r a c t i o n a t i o n , from a d u l t e r a t e d
Hendrickx and Huyghebaert (127) discuss the examination of the sterols by GLC,
TLC and IR, the fatty acids by GLC and the determination of the monglyceride
c o n t e n t for the c h a r a c t e r i s a t i o n of m i x t u r e s p r e p a r e d from a n i m a l fats,
vegetable oils and synthetic triglycerides including tributyrin such that the
RPK and s a p o n i f i c a t i o n v a l u e s w e r e s i m i l a r to g e n u i n e b u t t e r .
See also
Hendrick and Huyghebaert (128), Kuzdal-Savoie (129) and Parodi (130) and (131).
To detect foreign fats in butterfat, two of the following three tests should be
carried out: the hydroxamic acid index, GLC of the volatile fatty acids and the
R e i c h e r t - P o 1 enske-Kir schner procedure.
If the b u t t e r is g e n u i n e by these
tests,
that m a y be c o n s i d e r e d
adequate
for r o u t i n e p u r p o s e s , but the
possibility of adulteration is not rigorously excluded and it. is preferable to
also carry out at least a test for phytosterols.
A c o n s i d e r a b l e a m o u n t of a t t e n t i o n has b e e n d i r e c t e d in r e c e n t y e a r s to
triglyceride analysis.
T h i s is c a r r i e d out on p a c k e d or c a p i l l i l a r y GLC
columns and is described by T i m m s (171), Buro'n Arias et al (172), Traitler and
Prevot (173) and Wathelet et al (174).

42

MOISTURE

IH BUTTER

to constant

weight

PRINCIPLE
The weighed sample is dried
calculated as moisture.

at 100C, and the weight loss is

APPARATUS
1.
M e t a l d i s h , f l a t - b o t t o m e d , a b o u t 7.5 cm d i a m e t e r , 2.5 cm
preferably w i t h a lip.
2.

Oven at

3.

Analytical balance reading

deep,

100C.
to 0.1 mg.

PROCEDURE
K e e p the s a m p l e at 32-35C in an a i r t i g h t c o n t a i n e r and
vigorously until a h o m o g e n e o u s lump-free e m u l s i o n is obtained.

shake

It is convenient to put in the dish a glass rod with a flattened end

and l o n g e n o u g h so t h a t the o t h e r end c a n r e s t on the r i m .
Dry in
the o v e n , c o o l at l e a s t h a l f an h o u r in the d e s i c c a t o r and w e i g h .
Add 3 - 4 g of b u t t e r to the d i s h and r a p i d l y and a c c u r a t e l y w e i g h .
Stir in a little alcohol to facilitate evaporation, leave the dish on
a boiling w a t e r b a t h , stirring occasionally, until no water is visible
on the bottom of the dish. Wipe the outside of the dish and transfer
to the o v e n . D r y to c o n s t a n t w e i g h t (less t h a n 2 m g d i f f e r e n c e in
successive weighings.
CALCULATION

% moisture

Weight loss in oven

:
Weight of sample

x 100

REFERENCES
F A O / W H O C o d e of P r i n c i p l e s C o n c e r n i n g M i l k and M i l k P r o d u c t s , I n t e r n a t i o n a l
Standards and Standard Methods of Sampling and Analysis for Milk Products.
H u n t e r , M., K i r c h , G. and H a m m o n d ,
Science and Technology _3, 123.

H.

1973.

43

New

Zealand

Journal

of

Dairy

FOREIGN FATS IB BUTTER

(Hydroxamic Acid Index)
PRINCIPLE
F a t t y acid e s t e r s form h y d r o x a m a t e s and these give a red colour with ferric
chloride. The hydroxamates of the lower fatty acid esters such as those of the
b u t y r a t e s found in b u t t e r f a t are m u c h m o r e soluble in w a t e r than the
h y d r o x a m a t e s of the h i g h e r fatty acids found in other oils and fats.
The
c o n d i t i o n s of the test are arranged so that for pure butter the solution
c o n t a i n i n g the w a t e r - s o l u b l e h y d r o x a m a t e s should have about the same color
intensity as that containing an aliquot from all the hydroxamates.
APPARATUS
1.
G r a d u a t e d c y l i n d e r s , 25 ml and 50 ml capacity, or glass tubes
h a v i n g g r a d u a t i o n s at 25 ml and 50 ml. G l a s s w a r e c o m m o n l y used in
blood and urine analysis is suitable.
2.
P h o t o e l e c t r i c c o l o r i m e t e r , w i t h 525 nm filter and
cells, or spectrophotometer.
3.

Volumetric

pipettes 20 m l , 10 m l , 1 m l , and 0.5 ml

absorption

capacity.

REAGENTS
1.
H y d r o x y l a m i n e h y d r o c h l o r i d e solution.
Mix equal w e i g h t s of
h y d r o x y l a m i n e h y d r o c h l o r i d e and water.
W a r m with stirring or
s w i r l i n g on a steam bath until salt is dissolved.
Cool to room
temperature.
This reagent must be prepared with care. Use only the
m i n i m u m amount of heat necessary to dissolve the salt. Prepare fresh
daily.
2.
Alcoholic potassium hydroxide solution.
Add 6.25 g KOH pellets
to 100 m l i s o p r o p a n o l .
S w i r l the m i x t u r e over a steam bath to
d i s s o l v e the KOH. Cool to room t e m p e r a t u r e .
This solution can be
stored in a ground glass stoppered b o t t l e in a r e f r i g e r a t o r for
several days, but should be discarded after it has become yellow. If
a stored solution is used, warm it to room temperature before using
in test.
3.
I s o p r o p a n o 1 - w a t e r - a c e t o n e solution.
M i x equal v o l u m e s of
isopropanol and water. Add 1% by volume of acetone to the mixture.
4.
A c e t i c a c i d - c h l o r o f o r m solution.
acid to one litre with chloroform.

Dilute 6 ml glacial

acetic

5.
Iron solutions.
(1) Dissolve 0.90 g anhydrous ferric chloride
in 5 ml concentrated HC1 by warming over a steam bath. Dilute to 100
m l w i t h w a t e r and f i l t e r t h r o u g h a p a p e r (No. 1 W h a t m a n or
equivalent).
Store in a r e f r i g e r a t o r . (This reagent can be stored
successfully for two months under refrigeration).
Alternatively, dilute 1.6 ml of 60% solution with 5 ml HC1 and dilute
to 100 m l w i t h w a t e r . (2) Dilute a portion of iron solution No. 1
w i t h e q u a l v o l u m e of 2.25% HC1, m a d e by d i l u t i n g 5 ml c o n c e n t r a t e d
HC1 to 100 ml with water. Dilute as needed.
PROCEDURE
Fat may be isolated from butter by churning and filtering
or by ether extraction.
Use ghee (butter oil) directly.

44

procedures,

Place a p p r o x i m a t e l y 0.1 g melted fat in a 50 m l tube or cylinder, and

add 0.50 m l h y d r o x y l a m i n e h y d r o c h l o r i d e s o l u t i o n . W h i l e the fat is
still m e l t e d ,
add 5 m l a l c o h o l i c K O H s o l u t i o n , m i x i n g
during
addition.
Place the tube in a 30C water bath for 30 minutes.
At the end of the 30 minute reaction period remove the tube from the
b a t h and d i l u t e the c o n t e n t s to 50 m l w i t h a c e t i c a c i d - c h l o r o f o r m
solution.
Shake this mixture vigorously by placing the palm of the
hand over the end of the tube and m a k i n g ten up and down strokes in 5
seconds.
After shaking, the contents of the tube will appear turbid,
because of finely dispersed inorganic salt.
Clarify the solution by
i n s e r t i n g a b a l l of g l a s s w o o l the d i a m e t e r of the tube into the
latter at the level of liquid and slowly pushing it to the bottom of
the tube with a 1 ml pipette, while holding the index finger over the
upper end of the pipette.
Using this same 1 m l pipette transfer two
1 m l a l i q u o t s of the c l a r i f i e d s o l u t i o n to t w o 50 m l t u b e s (1 m l to
each tube). Dilute each 1 m l aliquot to w i t h i n a few m l of the 50 m l
graduation with isopropanol-water-acetone solution.
Put these tubes
a s i d e t e m p o r a r i l y u n t i l the e x t r a c t of t h e w a t e r - a c e t o n e s o l u b l e
hydroxamic acids is prepared.
T r a n s f e r a 10 m l a l i q u o t of the a c i d i f i e d r e a c t i o n s o l u t i o n , from
w h i c h the t w o 1 m l a l i q u o t s h a v e a l r e a d y b e e n r e m o v e d , to a 50 m l
tube.
To this 10 ml add 20 ml acetic acid-chloroform solution and 20
ml distilled water, each from v o l u m e t r i c pipettes or burettes.
Shake
the tube vigorously 30 times in up and d o w n strokes, in ten seconds,
u s i n g the p a l m of the h a n d to s t o p p e r t h e tube.
Set the tube a s i d e
to a l l o w the aqueous layer to separate from the heavy organic layer.
Tap the tube gently to shake d o w n the droplets of chloroform clinging
to the air meniscus of the aqueous layer.
W i t h a volumetric pipette
t r a n s f e r 10 m l of the c l e a r a q u e o u s u p p e r l a y e r to a 25 m l t u b e and
dilute to within a few m l of the 25 ml graduation with isopropanol.
With a volumetric pipette, add 0.50 m l iron solution No. 1 to each of
the two 50 ml tubes containing the 1 ml aliquots previously diluted
to n e a r l y 50 m l .
D i l u t e to 50 m l w i t h i s o p r o p a n o l - w a t e r - a c e t o n e
solution.
Add 0.50 m l iron s o l u t i o n No. 2 to the 25 m l tube
containing the previously diluted 10 ml aqueous extract and dilute to
25 ml with isopropanol.
Mix the contents of each of the three tubes
by slowly inverting each tube three times, using the palm of the hand
to s t o p p e r the t u b e s .
E a c h of the t h r e e t u b e s s h o u l d n o w e x h i b i t
about the same intensity of red colour if the sample being analyzed
is pure m i l k fat.
Adulterated m i l k fat or n o n - m i l k fats will yield
an aqueous extract tube which is m u c h lighter in colour than the two
50 ml tubes representing the total hydroxamic acids from the fat.
If
no milk fat is present in the sample the aqueous tube will exhibit no
Place the coloured solutions in appropriate absorption
red colour.
cells and measure their absorbances in a c o l o r i m e t e r , using a 525 nm
filter with distilled water as blank.
(The iron solution should have
b e e n a d d e d to e a c h of the t u b e s at a b o u t the s a m e t i m e , and the
colorimeter readings should be made not m o r e than 15 minutes apart.)
Using the colorimeter readings, taken from the absorbance scale and
not from the percent transmittance scale of the instrument, calculate
the H y d r o x a m i c Acid Index.

CALCULATION

Hydroxamic

Acid

100 x colorimeter reading of

water extratable acids
Index = i 0 x average of two colorimeter
readings of total

45

acids

100 X 0.296
Example:

HAI
10

0.275 + 0.277

The HAI is r o u g h l y equivalent to

soluble fatty acids in the fat.

the

molecular

percentage

of

water-

INTERPRETATION
T h e h y d r o x a m i c acid i n d e x is n o r m a l l y in the r a n g e 9 - 1 2 .
If the b u t t e r is
d i l u t e d w i t h o t h e r f a t s the i n d e x is l o w e r and in the a b s e n c e of b u t t e r the
value is b e l o w 2 or 3.
According to one source the test is not reliable if the
proportion of b u t t e r present in the fat is b e l o w about 20%.

REFERENCE
B a s s e t t e , R. and K e e n e y , M., 1956.

Journal of the AOAC 39 (2), 469-74.

46

FOREIGN FATS IN BUTTER

(Reictaert-Polenske-Kirschner V a l u e s )
PRINCIPLE
Butter is distinguished from other fats by the presence of the glyceryl esters
of r e l a t i v e l y l o w m o l e c u l a r w e i g h t fatty a c i d s , e s p e c i a l l y b u t y r i c but also
c a p r o i c , c a p r i c , c a p r y l i c , lauric and m y r i s t i c .
T h e s e acids are w h o l l y or
partially steam-volatile and water-soluble.
The fat is saponified with sodium hydroxide, the melt acidified and distilled
under standard conditions.
The d i s t i l l a t e is f i l t e r e d and the s o l u b l e a c i d s
t i t r a t e d ( R e i c h e r t value).
The i n s o l u b l e a c i d s are d i s s o l v e d in a l c o h o l and
titrated (Polenske value).
The titrated soluble acids are treated with silver
s u l p h a t e and the f i l t r a t e is a c i d i f i e d and r e - d i s t i l l e d and the d i s t i l l a t e
titrated (Kirschner value).
The Reichert value reflects the amount of butryic
and c a p r o i c acid p r e s e n t , the K i r s c h n e r , b u t r y i c alone and the P o l e n s k e ,
chiefly caprylic, capric and lauric with some contribution from myristic and
even palmitic acids.
The procedure should be carried out without
value, which is usually about 0.5 ml.

a sample

in order to obtain a blank

APPARATUS
1.

Distillation

2.

Burettes.

apparatus.

See diagram.

REAGENTS
1.

Sodium hydroxide

solution, 50%, m/m + glycerol

(1+9)

2.
Dilute sulphuric acid, 25 ml per litre, and adjust so that 40 m l
exactly neutralizes 2 ml of the sodium hydroxide solution.
3.

Pumice powder or anti-bumping

4.

Phenolphthalein

granules.

solution, 0.5% in denatured

ethanol.

5.
Barium hydroxide approximately 0.1 N, a c c u r a t e l y s t a n d a r d i z e d .
Shake 20 g of the o c t a h y d r a t e w i t h a litre of w a t e r u n t i l the
crystals dissolve and leave a couple of days for the barium carbonate
to s e t t l e out.
S t o r e in a b o t t l e w i t h a g u a r d - t u b e c o n t a i n i n g
powdered soda-lime to prevent ingress of carbon dioxide.
Standardize
the s o l u t i o n a g a i n s t 0.1 N h y d r o c h l o r i c acid or p o t a s s i u m h y d r o g e n
phthalate.
(NaOH m a y be used i n s t e a d of b a r i u m h y d r o x i d e if the
Kirschner value is not going to be determined.)
6.

Silver sulphate, finely

powdered.

PROCEDURE
W e i g h 5 g (j+O.Ol) of the oil (obtained by m e l t i n g the b u t t e r and
f i l t e r i n g ) into the d i s t i l l i n g f l a s k , and add 20 m l of the g l y c e r o l
caustic mixture.
The weighing may be done by attaching the flask to
the pan h o o k of an a n a l y t i c a l b a l a n c e by a p i e c e of w i r e and
c a r e f u l l y a d d i n g the s a m p l e u n t i l the tare + 5 g is a t t a i n e d .
Sapobify by gently heating over a small flame with constant swirling,
until the liquid no longer foams and becomes clear.
Allow the flask
to cool to a b o u t 90C, add 90 m l of r e c e n t l y b o i l e d d i s t i l l e d w a t e r
of about the s a m e t e m p e r a t u r e and m i x .
The liquid should r e m a i n
clear. Add 0.6 to 0.7 g of the p u m i c e and t h e n 50 m l s u l p h u r i c acid
s o l u t i o n (1 N).

47

DETERMINATION OF REICHERT AND POLENSKE VALUES

dimensions in mm
DISTILLATION APPARATUS

hole (tl

trou

C o n n e c t the f l a s k i m m e d i a t e l y to the d i s t i l l a t i o n a p p a r a t u s and w a r m

it g e n t l y u n t i l t h e free f a t t y a c i d s f o r m a c l e a r s u r f a c e l a y e r .
S t a r t h e a t i n g a n d r e g u l a t e t h e f l a m e so as to c o l l e c t in t h e
m e a s u r i n g f l a s k 1 1 0 m l o f d i s t i l l a t e in 1 9 - 2 1 m i n u t e s , t a k i n g t h e
m o m e n t w h e n the f i r s t d r o p f o r m s in the c o n d e n s e r as the b e g i n n i n g of
t h e d i s t i l l a t i o n p e r i o d . R e g u l a t e the w a t e r f l o w i n g in t h e c o n d e n s e r
so as to m a i n t a i n the t e m p e r a t u r e of the w a t e r l e a v i n g the c o n d e n s e r
a t 2 0 +_ 1C.
If t h e t e m p e r a t u r e o f t h e c o o l i n g w a t e r e x c e e d s 20C as in t r o p i c a l
a n d s u b t r o p i c a l a r e a s , a n d if n o s p e c i a l a r r a n g e m e n t s c a n b e m a d e ,
t h e m e a s u r i n g f l a s k s h o u l d s t a y in t h e w a t e r - b a t h a t 20 +_ 1C f o r
a b o u t 1 h o u r . W h e n e x a c t l y 110 m l of d i s t i l l a t e h a v e b e e n c o l l e c t e d ,
r e m o v e the b u r n e r i m m e d i a t e l y and s u b s t i t u t e a s m a l l b e a k e r for the
measuring flask.
M i x the c o n t e n t s of t h e m e a s u r i n g f l a s k b y g e n t l y
s h a k i n g a n d i m m e r s e t h e f l a s k in a w a t e r b a t h at 20 _+ 1C f o r 10 to
15 m i n u t e s , t h e 1 1 0 m l m a r k o n t h e f l a s k b e i n g 1 c m b e l o w t h e l e v e l
of the w a t e r in the w a t e r b a t h and the flask b e i n g t u r n e d from t i m e to
time .
S t o p p e r the
shaking.

flask

and

mix

by

inverting

it

4 or

5 times

without

F i l t e r t h e 1 1 0 m l o f d i s t i l l a t e throug;h a d r y m e d i u m s p e e d f i l t e r
p a p e r ( d i a m e t e r 80-90 m m ) w h i c h fits snugly into the funnel.
The
f i l t r a t e s h o u l d be c l e a r . The filter should be of such a size that
15 m l p o u r e d i n t o it w i l l f i l l it c o m p l e t e l y .

48

Determination

of T o t a l

Soluble

Fatty Acida

(Reichert

Value)

P i p e t t e 1 0 0 ml o f t h e f i l t r a t e i n t o a c o n i c a l f l a s k o f 3 0 0 m l , a d d
0 . 5 ml o f p h e n o l p h t h a l e i n i n d i c a t o r s o l u t i o n and t i t r a t e w i t h t h e
s t a n d a r d i z e d aqueous a l k a l i s o l u t i o n to a p i n k c o l o u r p e r s i s t e n t for
1 / 2 to 1 m i n u t e .
C a l c u l a t e t h e R e i c h e r t v a l u e a c c o r d i n g to t h e
formula b e l o w .
R e t a i n the n e u t r a l i z e d f i l t r a t e for the d e t e r m i n a t i o n
of the K i r s c h n e r v a l u e .
C o n d u c t a b l a n k t e s t w i t h o u t f a t and i n s t e a d o f s a p o n i f y i n g o v e r a
n a k e d f l a m e , h e a t on a b o i l i n g w a t e r b a t h f o r 15 m i n u t e s .
Not m o r e
t h a n 0 . 5 ml of the s t a n d a r d i z e d a l k a l i s o l u t i o n s h o u l d be r e q u i r e d
for the t i t r a t i o n of the b l a n k .
I f the volume e x c e e d s t h i s ,
prepare
fresh reagent solutions.
Determination

of

Insoluble

Volatile

Fatty Acids

(Polenske

Value)

R i n s e t h e f i l t e r w i t h t h r e e s u c c e s s i v e 15 ml p o r t i o n s o f d i s t i l l e d
w a t e r at a t e m p e r a t u r e o f 20 +_ 1 C , e a c h h a v i n g p r e v i o u s l y p a s s e d
through the c o n d e n s e r , the s m a l l b e a k e r and the m e a s u r i n g f l a s k .
P l a c e the
of 2 0 0 ml

f u n n e l and
capacity.

filter

in

the

neck

of

a dry

clean

conical

flask

D i s s o l v e the i n s o l u b l e f a t t y a c i d s by r e p e a t i n g the w a s h i n g p r o c e d u r e
but u s i n g 15 ml p o r t i o n s of e t h a n o l ( 9 5 - 9 6 % , p r e v i o u s l y n e u t r a l i z e d ) .
T i t r a t e the combined e t h a n o l i c w a s h i n g s w i t h the s t a n d a r d i z e d aqueous
a l k a l i s o l u t i o n u s i n g 0.5 ml of p h e n o l p h t h a l e i n i n d i c a t o r
solution,
to a p i n k c o l o u r p e r s i s t e n t f o r 1 / 2 to 1 m i n u t e .
Calculate
the
P o l e n s k e v a l u e as o u t l i n e d b e l o w .
D e t e r m i n a t i o n of
(Kirschner V a l u e )

Volatile

Fatty

Acida

with

Soluble

Silver

Salts

Add 0 . 5 g of f i n e l y p o w d e r e d s i l v e r s u l p h a t e to the n e u t r a l i z e d
s o l u t i o n from the R e i c h e r t d e t e r m i n a t i o n .
L e a v e the f l a s k in the
dark one hour w i t h o c c a s i o n a l shaking and f i l t e r the c o n t e n t s through
a dry f i l t e r , in the d a r k .
T r a n s f e r 1 0 0 ml o f t h e f i l t r a t e to a d r y
P o l e n s k e f l a s k , add 35 ml of cold r e c e n t l y - b o i l e d d i s t i l l e d w a t e r , 10
ml of the d i l u t e s u l p h u r i c a c i d s o l u t i o n and a l i t t l e pumice powder
or about 30 cm of a l u m i n i u m w i r e about 1 mm t h i c k wound into a c o i l
a b o u t 5 mm a c r o s s .
C o n n e c t t h e f l a s k to t h e s t a n d a r d d i s t i l l a t i o n
a p p a r a t u s and d i s t i l
as f o r a R e i c h e r t d e t e r m i n a t i o n .
M i x and
f i l t e r , o m i t t i n g i m m e r s i o n in a w a t e r b a t h at 20<>C.
T i t r a t e 100 ml of
the f i l t r a t e w i t h 0.1 N b a r i u m h y d r o x i d e s o l u t i o n .
C a l c u l a t e the
K i r s c h n e r v a l u e as b e l o w .
CALCULATION
R e i c h e r t v a l u e (RV) = 1.1 x ml of 0.1 N b a r i u m h y d r o x i d e r e q u i r e d for
neutralization.
T h e a l k a l i w i l l n o t n o r m a l l y b e e x a c t l y 0 . 1 N , so
the t i t r e must be m u l t i p l i e d by a s u i t a b l e f a c t o r a f t e r d e d u c t i o n of
the b l a n k t i t r e .
Report the r e s u l t rounded to the f i r s t d e c i m a l .
P o l e n s k e v a l u e ( P V ) = ml o f
n e u t r a l i z e the a l c o h o l - s o l u b l e
titre
Kirschner

value

0.1 N b a r i u m
acids.

x 1.21 x
T -

w h e r e C i s n u m b e r o f ml
the R e i c h e r t
titration.

of

0.1

(100

49

hydroxide

required

to

hydroxide

required

in

+ C)

barium

REPEATABILITY OF RESULTS
The difference between results of duplicate determinations (results
obtained simultaneously or in rapid succession by the same analyst)
should not exceed 0.5 for the Reichert and K i r s c h n e r values and 0.3
for the Polenske value.
The results vary slightly with atmospheric pressure.
The following
formula may be applied to results obtained at an elevated altitude:
(p = atmospheric pressure in mm of Hg)
Corrected RV

10

Corrected PV

PV

(RV - 10) log 760

log p

INTERPRETATION
The R e i c h e r t v a l u e of butter is g e n e r a l l y over 24, p r o b a b l y a l w a y s so for
butter produced in bulk from properly husbanded animals.
Samples for which the
value exceeds 28, with the Polenske in proportion, may be accepted as genuine.
A v a l u e b e l o w 28 j u s t i f i e s further tests, and b e l o w 24 r a i s e s the suspicion
that the sample is adulterated.
A very small proportion of samples, less than
1%, m a y s h o w v a l u e s d o w n to about 20 or even l o w e r , but the P o l e n s k e and
Kirschner values will be in proportion for such samples. Bulking of production
of course tends to mask some of the natural variation. Conversely, butter from
a s m a l l n u m b e r of a n i m a l s , or ones fed a b n o r m a l l y m a y have a c o m p o s i t i o n
outside the usual range (one of the problems of interpretation of the freezingpoint of milk).
For example, the Reichert value may be reduced by exposure of
the a n i m a l s to c o l d , and is l o w e r in the m i l k f a t t o w a r d s the end of the
l a c t a t i o n period.
C o w s fed on b e e t r o o t leaves or turnips appear to give
milkfat with an abnormally high Polenske value.
B u t t e r c l a r i f i e s q u i c k l y during the s a p o n i f i c a t i o n stage of the Reichert
process, margarine usually more slowly.
The acids from coconut oil distil as
oily drops, those from palm oil as white flakes.
The relation between the three values, Reichert, Polenske and Kirschner
fairly closely with the following table:
(see Williams (128))
Reichert Value
23.7
24.1
24.5
25.0
25.4
25.9
26.3

26.8

Polenske Value

1.6

1.8

20.0
20.3
20.7

1.9

21.1

2.0
2.1
2.2

21.5
21.9
22.3
22.7
23.0
23.4
23.8
24.2
24.6
25.0
25.4
25.8

1.7

27.2
27.7

2.3
2.4
2.5

28.1

2.6

28.5
28.9
29.4
29.8
30.3
30.7
31.1
31.6
32.0

Kirschner Value

2.7

2.8
2.9
3.0
3.1
3.2
3.3
3.4
3.5

26.1
26.5
26.9
27.3

50

accord

Read f r o m the a b o v e t a b l e the P o l e n s k e v a l u e c o r r e s p o n d i n g to the R e i c h e r t

value obtained experimentally.
If the Polenske value obtained e x p e r i m e n t a l l y
is m o r e than 0.5 a b o v e the v a l u e read from the t a b l e , the p r e s e n c e of c o c o n u t
or palm kernal oil or their products m a y be assumed.
N e x t , read f r o m the t a b l e the P o l e n s k e v a l u e c o r r e s p o n d i n g to the K i r s c h n e r
value obtained experimentally.
The P o l e n s k e v a l u e o b t a i n e d e x p e r i m e n t a l l y
s h o u l d not v a r y by m o r e than 1.0 ( e i t h e r w a y ) f r o m that read f r o m the t a b l e .
The a d d i t i o n of l e s s t h a n 5% of c o c o n u t o i l c a u s e s the P o l e n s k e v a l u e to fall
outside this limit.
The f o l l o w i n g t w o t a b l e s t a k e n f r o m
values m a y vary in genuine butter.

Davis

Reichert-Polenske
of

Samples

and

Macdonald

Values

Reichert
Average
22
23
24
25
26
27
28
29
30
31

15
22
43
56
35
26
22
15
26
30

1.50
1.60
1.65
1.70
1.90
1.95
2.05
2.20
2.10
2.25

Correlation of Reichert and Polenske

.chert :
1.3
1.4
1.5
1.6
1.7
1.8
1.9
2.0
2.1
2.2
2.3
2.4
2.5
2.6
2.7
2.8
2.9
3.0
3.1
3.2
3.3
3.4
3.5
3.6
3.7
3.8
3.9

36

35

34

33

32

31

30

29

28

27

1
3
1
1

1
3
3
4
2
2

1
1
4
1
3
3
1

29

2
3
3
1

1
3

8
5
7
7
9
8
3
2
3
2

67

3
4
6
8
8
7
4
8
7
1
1

1
3
1
5
7
12
6
6
4
5
10
5
6
11
8
6
2
2
2
1
1

4
8
6
17
15
15
11
4
7
2
6
5
6
2
5
4
5
3
2
2

62 104 166 130

51

Polenske
Maximum

Minimum

1.7
1.8
2.0
2.3
2.4
2.9
3.1
2.9
2.9
3.2

1.2
1.4
1.4
1.4
1.5
1.6
1.6
1.8
1.7
1.6

Values
26

25

24

23

1
1

3
2
5
9
10
10
16
9
8
7
6
5
4
17
13
13
11
8
9
1

(1) s h o w h o w

1
2
4
12
9
6
1
2
1
1

1
2
5
2

2
1

1
1

1
1

1
1

43

10

1
2
1
7
10
20
28
44
32
35
46
35
31
22
32
30
37
42
39
48
37
21
17
8
2
1
1
629

52
C

0n>
1-t

Cfl

T3
I
n>
CO

these

It is i m p o r t a n t to carry out the d e t e r m i n a t i o n a few t i m e s to ensure that

c o n s i s t e n t r e s u l t s are obtained b e f o r e any d e d u c t i o n is m a d e as to the
likelihood of adulteration, particularly in the case of borderline values.
The
result must be confirmed by tests for phytosterols, iso-oleic acid, etc.
Results on difficult samples must be interpreted with care. Addition of animal
fats up to 10 or 20% m a y not l o w e r the RPK v a l u e s b e l o w the accepted m i n i m a .
There is the possibility of addition of triacetin or tributyrin to increase the
R e i c h e r t v a l u e and only e x t e n s i v e analysis would reveal that there was a
d i s c r e p a n c y in the p a t t e r n of results.
GLC of the intact t r i g l y c e r i d e s has
been used (Breckenridge and Kuksis (133)) (Parodi (126)) and appears promising
as a means of assessing the proportion of butterfat in fat mixtures.
Reichert
and K i r s c h n e r v a l u e s of cultured b u t t e r s w i t h an a r t i f i c i a l l y induced high
acidity are lower than normal.
RPK v a l u e s for the fat from the m i l k of n o n - b o v i n e a n i m a l s are not as well
e s t a b l i s h e d as those for c o w ' s m i l k . The values reported by v a r i o u s w o r k e r s
fall w i t h i n the ranges given in the following tables:
(See W i l l i a m s (132))
(Note:
'a' indicates a single value and 'b' a typical value.)
Reichert

Polenske

Kirschner

Goat

17 - 29

1.9 - 9.8

15. 6 b

Ass

about 14

Buffalo

24 - 37

1.5 - 1.8

Ewe

22.8 - 23.4

1.5 - 2.1

17.6 a

5.9 a

2.6

6.2a

Mare

Typical analyses for some other oils are as follows:

Reichert

Polenske

Kirschner

Palm Kernel

5.2 - 6.5

9.7 - 10.7

0.8

Coconut

6.5 - 8

15 - 17

1.6 - 1.9

Babassu

6.lb

11.4b

8.4

15.9'

1.2

1.6'

Margosa or Neem oil

(Azadriachta

indica)

Shea nut oil, shea butter

Macassar
oil, Schleicher
trijiga, "kusum"
or "paka"

8.3 b

0.25 b

2.6 b

0.7 b

17.0

3.0

5.0

15.5

Other oils and fats may generally be assumed to give negligible values.
To distinguish cow butteroil from buffalo butteroil, Latif and Mazloumu (134))
used fractional crystallisation from acetone and determination of the Reichert,
Polenske and Kirschner values on the fractions.
REFERENCES
IDPAC II.D.9.
(IUPAC Method II.D.10. d e s c r i b e s the d e t e r m i n a t i o n of fatty
acids with soluble magnesium and insoluble silver salts in order to calculate
the properties of butter, coconut and palm kernel oils in mixtures).

52

International
British

Dairy Federation

Standard

37:1966.

769:1961.

S o c i e t y of P u b l i c A n a l y s t s , 1 9 3 6 .
AOCS, Volume

1 , Cd

Garcia-Olmedo,

Analyst

6J., 4 0 4 - 8 .

5-40.

R. and H e l l i n , B.C., 1 9 7 1 .

53

Anales

de

B r o m a t o l o g i a _1, 4 1 - 5 9 .

FOREIGN FATS IN BOTTER AS AN INGREDIENT

(Reichert-Polenske-Kirschner Values, Semi-Micro M e t h o d )
PRINCIPLE
See

'Foreign Fats

in

Butter'.

APPARATUS
1.

Distillation

apparatus.

See

diagram.

REAGENTS
As for m a c r o - m e t h o d ,
except
for i t e m
a p p r o x i m a t e l y 0.02N accurately s t a n d a r d i s e d .
as for 0.1 N.

6, b a r i u m
hydroxide,
S t a n d a r d i s e and s t o r e

PROCEDURE
Separate or extract the butterfat from sample and w e i g h exactly 1 g
of the fat i n t o t h e d i s t i l l i n g f l a s k , add 0.5 m l s o d i u m h y d r o x i d e
s o l u t i o n and 3.5 m l g l y c e r o l .
S a p o n i f y by g e n t l e h e a t i n g o v e r a
m i c r o - b u r n e r or s m a l l f l a m e w i t h c o n s t a n t s w i r l i n g .
Do n o t h e a t
a f t e r s a p o n i f i c a t i o n is c o m p l e t e , this b e i n g s h o w n b y the s u d d e n
change of the m i x t u r e to a clear single-phase liquid.
It m a y require
a little practice before this point is easily detected.
If there is
a n y s i g n of c h a r r i n g , r e j e c t and r e p e a t w i t h a f r e s h s a m p l e .
Cover
with a small watchglass.
As s o o n as the m e l t is cool e n o u g h for
w a t e r to b e a d d e d w i t h o u t l o s s , add 19 m l of r e c e n t l y b o i l e d h o t
w a t e r , initially drop by drop. Once the soap is dissolved, add 10 ml
of dilute sulphuric acid and about 0.05 g powdered pumice.
C o n n e c t the f l a s k to the d i s t i l l a t i o n a p p a r a t u s and h e a t g e n t l y to
m e l t the fatty acids.
Then heat strongly, adjusting the distillation
r a t e to c o l l e c t 21 m l of d i s t i l l a t e in 5 to 7 m i n u t e s .
R e m o v e the
burner and i m m e d i a t e l y substitute a small beaker for the 21 m l flask.
Reichert

Value:

S t o p p e r the f l a s k , m i x g e n t l y and stand in w a t e r at 15C for 10

minutes.
F i l t e r t h r o u g h a s m a l l f i l t e r p a p e r (e.g. 4.25 cm W h a t m a n
No. 4 or e q u i v a l e n t ) and t i t r a t e 20 m l of the f i l t r a t e w i t h 0.02 N
b a r i u m h y d r o x i d e u s i n g p h e n o 1 p h t h a 1 e i n as i n d i c a t o r .
R e t a i n the
n e u t r a l i s e d filtrate.
Prepare
and
procedure.
Reichert
Polenske

conduct

value

blank

1.1 x

determination

through

the

entire

(titre-blank)

Value:

R e m o v e the splash-head of the distillation apparatus,' and rinse the

condenser w i t h 3 m l of cold w a t e r , using it to also rinse the beaker,
the 21 m l f l a s k and the f i l t e r p a p e r in turn and f i n a l l y r e j e c t i n g
the filtrate.
Repeat with a further 3 ml of water.
P a s s 3 m l of n e u t r a l e t h a n o l d o w n the c o n d e n s e r , u s i n g it to r i n s e
t h e b e a k e r , t h e 21 m l f l a s k and the f i l t e r - p a p e r , c o l l e c t i n g the
filtrate in a small conical flask.
Repeat with two further portions
o f 3 m l of n e u t r a l e t h a n o l so as to c o l l e c t all of the w a t e r i n s o l u b l e but a 1 c o h o 1 - s o 1 u b 1 e a c i d s in the c o n i c a l flask.
Titrate

54

the alcohol solution with 0.02 N barium hydroxide

phenolphthalein as indicator.

using

Polenske value = titre - blank

Kirschner Value:
To the neutral filtrate from the Reichert value, add 0.1 g of finelypowdered silver sulphate, and leave for one hour in the dark, shaking
occasionally.
Filter into a dry flask. Pipette 20 ml into a Reichert
d i s t i l l a t i o n f l a s k , add 7 m l d i s t i l l e d w a t e r , 2 m l of d i l u t e
sulphuric acid and a little powdered pumice or short lengths of fine
a l u m i n i u m w i r e to r e d u c e b u m p i n g .
D i s t i l 21 m l in 5 to 7 m i n u t e s ,
c o o l , m i x and filter (4.25 cm W h a t m a n No. 4 or e q u i v a l e n t ) and
t i t r a t e 20 m l of the f i l t r a t e w i t h 0.02 N b a r i u m h y d r o x i d e u s i n g
phenolphthalein as indicator.

Kirschner value =

titre x 1.21 x (20 + C)

20

w h e r e C is the n u m b e r of m l of 0.02 N b a r i u m h y d r o x i d e r e q u i r e d
the Reichert value.

for

CALCULATION
See the individual value calculations.
The barium hydroxide solution
will not usually be exactly 0.02 N so that the titre obtained must be
multiplied by the appropriate factor before being used to calculate
the above values.
INTERPRETATION
This test is intended for the examination of samples such as bread and butter,
cream buns and items of flour, chocolate and sugar confectionery claiming to
contain butter or milk derivatives, from which it may be difficult to obtain as
much as 5 g of fat.
The results, together with the HAI, are normally adequate
to c h a r a c t e r i z e the fat as b u t t e r or not.
The i n t e r p r e t a t i o n g i v e n in the
section on the detection of foreign fats in butterfat should be applied with a
certain amount of caution as both methods are empirical.
The original authors
(Dyer et al (135) claim that the Reichert values agree with those by the macrom e t h o d , P o l e n s k e v a l u e s are s l i g h t l y l o w e r by the s e m i - m i c r o m e t h o d .
It is
thus a l w a y s p r e f e r a b l e to use the m a c r o - m e t h o d if e n o u g h fat is a v a i l a b l e .
There is no difficulty in interpretation if the only question is whether or not
the sample is butter.
REFERENCE
Dyer, B., Taylor, G. and Hamence , J.H., 1941.

55

Analyst _66 , 355.

Distillation Apparatus for Semi-Micro Determination

of eictaert-Polenske-Kirsctaner Values

56

VEGETABLE FAT IR BTTERFAT

(Thin-Layer Chromatographic M e t h o d )
PRINCIPLE
S t e r o l s are p r e c i p i t a t e d as their d i g i t o n i d e s from the s a p o n i f i e d fat.
The
s t e r y l a c e t a t e s are p r e p a r e d , s e p a r a t e d by r e v e r s e - p h a s e T L C and v i s u a l i z e d
u s i n g p h o s p h o m o 1ybdic acid. The p r e s e n c e of p h y t o s t e r y l a c e t a t e s i n d i c a t e s
that the sample contains vegetable fat.
APPARATUS
1.

Micro

filter such as:

Glass m i c r o filter for s t e r o l a c e t a t e

precipitates
A: T o p p o r t i o n of fitter, capacity 1 ml. B: Lower p o r t i o n
of filter. G r o u n d surfaces b e t w e e n A and B h o l d filter pad
A and B are held together by springs. D. C: filter flask E:
wire t w i s t e d a r o u n d s t o p p e r to h o l d lower end of springs.

2.

TLC equipment

(plates, tank, etc.)

3.

Micropipettes or microsyringes,

4.

Chromatography

10

yl.

spray.

REAGENTS
1.
Potassium hydroxide solution.
distilled water.
2.
Digitonin solution.
ethanol 95% v/v.
3.

Ethanol, 95% v/v.

4.

Ethanol, 80% v/v.

5.

Diethyl

6.

Acetic

7.

Penta"

Dissolve

D i s s o l v e 400 g of KOH in 600 ml

digitonin

in

100

ether.
anhydride.
or light petroleum ether

57

(boiling

range

40-60C).

ml

of

8.
Copper
sulphate
solution.
Dissolve
70 g of
c o p p e r s u l p h a t e p e n t a h y d r a t e in w a t e r and d i l u t e to
water.
9.

Anhydrous

sodium

crystallized
1 litre with

sulphate.

10.
P h o s p h o m o l y b d i c acid s o l u t i o n .
(This r e a g e n t m u s t be f r e s h l y
prepared.
If f a i n t b l u e c o l o u r e d b a n d s are o b t a i n e d w h e n s p r a y i n g
the c h r o m a t o p l a t e , the reagent m u s t be discarded and a fresh solution
p r e p a r e d , p r e f e r a b l y f r o m pho s p h o m o 1 y b d i c a c i d , r e c r y sta 11 i zed in
n i t r i c a c i d (d=1.2) if n o t s u f f i c i e n t l y p u r e . )
D i s s o l v e 10 g of
p h o s p h o m o l y b d i c a c i d P 2 0 5 ' 2 4 M o O j ' n l ^ O in 50 m l e t h a n o l (95%, v/v).
C o n t a c t of t h i s s o l u t i o n w i t h m e t a l l i c o b j e c t s , s u c h as s p a t u l a s ,
etc., m u s t be avoided.
11.
Reference standards solution.
T h i s m u s t be f r e s h l y p r e p a r e d .
D i s s o l v e 98 m g cholosteryl acetate and 2 mg soybean oil phytosteryl
acetates in 10 m l diethyl ether.
PROCEDURE
P r e p a r e the a c e t i c a c i d - a c e t o n i t r i 1e m o b i l e p h a s e for T L C ,
as
follows:
M i x 100 m l glacial acetic acid and 300 m l acetonitrile and
s h a k e w i t h 18 m l u n d e c a n e ( b o i l i n g r a n g e 1 9 0 - 2 2 0 C ) in a s e p a r a t o r y
funnel.
(Conduct the operation in a fume-cupboard as acetonitrile is
toxic.)
L e a v e the 2 l a y e r s to s e p a r a t e 16 h o u r s at 22-23C.
The
acetic a c i d - a c e t o n i t r i l e layer, saturated with undecane, is used as
the m o b i l e phase.
(Note:
m i x t u r e s already used for chromatographic
separation m u s t be discarded.)
P r e p a r e the TLC t a n k by i n t r o d u c i n g e n o u g h of the a c e t i c a c i d a c e t o n i t r i l e ( m o b i l e p h a s e ) to o b t a i n a l a y e r of a b o u t 1 cm d e p t h .
L i n e the w a l l s of the t a n k w i t h f i l t e r p a p e r and c l o s e the t a n k
t i g h t l y w i t h t h e lid.
L e t the t a n k e q u i l i b r a t e at 2 2 - 2 3 C for 24
hours.
Prepare

the test

samples as

follows:

a.
Butter:
M e l t a b o u t 50 g of the b u t t e r s a m p l e in a d r y i n g o v e n
at a t e m p e r a t u r e b e l o w 50C until the fat and water layers separate.
Separate the fat layer by dcantation and clarify the fat in the oven
at a t e m p e r a t u r e of a b o u t 40C b y f i l t e r i n g it t h r o u g h a d r y p a p e r
f i l t e r , t a k i n g c a r e that t h e f i l t e r is n o t w e t t e d b y the a q u e o u s
phase .
b.
M i l k and C r e a m :
Centrifuge the sample to obtain a cream of 40%
fat.
C h u r n t h e c r e a m in a l a b o r a t o r y c h u r n .
C o l l e c t the b u t t e r
lumps and proceed as described under butter.
c.
Cheese:
R u b the s a m p l e in a m o r t a r w i t h a n h y d r o u s s o d i u m
s u l p h a t e u n t i l a g r a n u l a r m a s s is p r o d u c e d .
E x t r a c t the m a s s w i t h
pentane or light petroleum ether (a continuous extraction apparatus
m a y be used) and evaporate the solvent in a boiling water-bath.
d.
C o n d e n s e d M i l k , E v a p o r a t e d M i l k , Ice C r e a m : Add to the sample
t w i c e its v o l u m e of boiling water and heat the mixture on a boiling
w a t e r - b a t h u n t i l t h e t e m p e r a t u r e is 75C. Add an a m o u n t of c o p p e r
sulphate solution equal to one-tenth of the v o l u m e of the m i x t u r e and
c o n t i n u e h e a t i n g until the p r e c i p i t a t e c o a g u l a t e s .
F i l t e r the
precipitate through a paper filter and wash it with w a r m water until
the filtrate is colourless.
Carefully drain the precipitate, rub it
in a m o r t a r w i t h anhydrous sodium sulphate and proceed as described
under c h e e s e .

58

e.
Dried Milk:
Rub the sample in a mortar with some water so as to
obtain a clotted mass.
Allow it to stand for about 15 minutes.
Then
add anhydrous sodium sulphate and rub again until a granular m a s s is
produced.
Extract the m a s s with pentane or light petroleum ether (a
c o n t i n u o u s e x t r a c t i o n a p p a r a t u s m a y be u s e d ) and e v a p o r a t e the
solvent on a boiling water-bath.
P r e p a r e s t e r y l a c e t a t e s as f o l l o w s :
W e i g h to the n e a r e s t 0.1 gm
a b o u t 15 g of the fat in a c o n i c a l f l a s k of 5 0 0 m l c a p a c i t y .
Add 10
m l of p o t a s s i u m h y d r o x i d e s o l u t i o n and 20 m l of e t h a n o l (95% v/v).
A t t a c h an a i r c o n d e n s e r to the f l a s k , h e a t it on a b o i l i n g w a t e r b a t h , w i t h s w i r l i n g , u n t i l the s o l u t i o n h a s b e c o m e c l e a r , and
c o n t i n u e b o i l i n g for h a l f an h o u r .
Add 60 m l of w a t e r and t h e n 180
m l of e t h a n o l (95% v / v ) , and r a i s e the t e m p e r a t u r e to a b o u t 40C.
A d d 30 m l of the a l c o h o l i c d i g i t o n i n s o l u t i o n (1%), s w i r l and a l l o w
to cool.
P l a c e the f l a s k in a r e f r i g e r a t o r at a b o u t 5C for a b o u t
twelve hours or overnight.
Collect the precipitate of sterol digitonide by filtration through a
Wash
m e d i u m speed filter paper in a Buchner funnel (diameter 8 cm).
the p r e c i p i t a t e w i t h w a t e r at a b o u t 5C u n t i l the f i l t r a t e s t o p s
f o a m i n g , t h e n o n c e w i t h 2 5 - 5 0 m l of e t h a n o l (95% v / v ) and o n c e w i t h
25-50 ml of diethyl ether.
D r y the f i l t e r p a p e r w i t h the p r e c i p i t a t e on a w a t c h - g l a s s in a
drying oven at 102 +_ 2C for 10-15 minutes.
Fold the filter paper in
two, allowing the precipitate to come off as a pellicle and transfer
the precipitate into a weighing bottle or other convenient container.
Transfer 100 _+ 5 mg of the sterol digitonide to a test tube, add 1 m l
of a c e t i c a n h y d r i d e and h e a t the t u b e in a g l y c e r o l b a t h at 130 145C u n t i l the p r e c i p i t a t e h a s d i s s o l v e d .
Do n o t use d i r e c t h e a t ,
since spattering m a y occur.
C o n t i n u e h e a t i n g for t w o m i n u t e s and
a l l o w to c o o l to a b o u t 80C.
Add 4 m l of e t h a n o l (95% v / v ) , m i x ,
h e a t s l i g h t l y to d i s s o l v e a n y s t e r y l a c e t a t e w h i c h m a y tend to
c r y s t a l l i z e out.
F i l t e r the s t i l l w a r m s o l u t i o n t h r o u g h a s m a l l
m e d i u m s p e e d f i l t e r p a p e r p r e v i o u s l y m o i s t e n e d w i t h e t h a n o l and
c o l l e c t the f i l t r a t e in a n o t h e r test tube.
C a r e f u l l y h e a t the
filtrate in the test tube until it boils gently.
Keep the solution boiling and add drop by drop, from a pipette while
shaking vigorously, 1 to 1.5 ml of water until the steryl acetate is
just about to precipitate but still remains in
solution.
Avoid
superheating.
Add a f e w d r o p s o f 95% e t h a n o l to r e d i s s o l v e any
precipitated steryl acetate.
A l l o w to cool in air for two hours and
finally in ice-water for half-an-hour.
Filter the crystallized steryl acetates on a small disc of hardened
fast f i l t e r p a p e r by s u c t i o n in a g l a s s m i c r o f i 1 1 e r i n g d e v i c e and
r i n s e the c r y s t a l s w i t h 1 m l of e t h a n o l (80% v/v). D r y the c r y s t a l
c a k e on the p a p e r in a d r y i n g o v e n f i r s t at a b o u t 30C and then at
102 _+ 2C for 1 0 - 1 5 m i n u t e s . The c a k e m a y be s t o r e d in a d e s i c c a t o r
ready for c o m p l e t i o n of the test the following day.
D i s s o l v e the c a k e in a b o u t 2 m l of d i e t h y l e t h e r so as to g i v e a
c o n c e n t r a t i o n of a b o u t 10 y g / y l .
( D i g i t o n i n , M W 1229 f o r m s an
e q u i m o l a r a d d u c t w i t h s t e r o l s and as the M W of c h o l e s t e r o l , for
e x a m p l e , is 3 8 6 , s t a r t i n g w i t h 100 m g of a d d u c t and a s s u m i n g 8 0 % of
t h e a c e t a t e is r e c o v e r e d , t h e y i e l d w o u l d b e a b o u t 20 m g of
cholesteryl acetate).
T h i s is the s t e r y l a c e t a t e s o l u t i o n
for
spotting on the TLC plate.
Prepare just before use.

59

N e x t , p r e p a r e the T L C p l a t e as f o l l o w s :
Coat a glass plate w i t h a
s l u r r y of d i a t o m a c e o u s e a r t h in d i s t i l l e d w a t e r u s i n g a s u i t a b l e
s p r e a d e r so as to o b t a i n a l a y e r of 0.18 m m u n i f o r m
thickness.
Prepare the slurry from d i a t a m a c e o u s earth containing 13% gypsum or
use a proprietary p o w d e r (e.g. Kieselguhr G., Merck), using 1 part to
2 parts of w a t e r by weight.
After the plates have air-dried, activate by heating in a drying oven
at 1 0 0 C f o r 2 5 m i n u t e s .
Allow
t h e p l a t e to c o o l to
room
temperature.
M a r k o n e of the s i d e s of the p l a t e s , p e r p e n d i c u l a r to the d i r e c t i o n
of c o a t i n g , as t h e b o t t o m s i d e b y s c r a t c h i n g a s m a l l s i g n in the
layer.
T a k e the p l a t e b e t w e e n t h u m b and f o r e f i n g e r of b o t h h a n d s , w e a r i n g
rubber gloves, and dip it horizontally and carefully for some seconds
in a shallow tray, containing a solution of 10% v/v undecane (BR 1902 2 0 C ) in p e t r o l e u m e t h e r (BR 4 0 - 6 0 C ) or the u n d e c a n e l a y e r left
f r o m p r e p a r i n g the m o b i l e p h a s e .
A l t e r n a t i v e l y , the p l a t e m a y be
sprayed directly w i t h the undecane solution.
R e m o v e the e x c e s s u n d e c a n e s o l u t i o n by h o l d i n g the p l a t e in a
v e r t i c a l p o s i t i o n , w i t h t h e b o t t o m s i d e on t o p , o v e r the t r a y for
about ten seconds.
The degree of impregnation should be 0.08 - 0.09
gram of u n d e c a n e per gram of diatomaceous earth.
Store the plate at
a constant temperature of 20-24C in a draught-free place for 50-60
m i n u t e s to evaporate the light petroleum.
During this evaporation period remove with a brush, pentagonal pieces
f r o m t h e t h i n l a y e r and a l s o s t r i p s of a b o u t 1.5 cm w i d t h a l o n g the
e d g e s of the p l a t e in the s a m e d i r e c t i o n as u s e d for a p p l y i n g the
thin layer, using an appropriate template as indicated in the figure
b e l o w , the p e n t a g o n a l f i g u r e s b e i n g s i t u a t e d on the b o t t o m of the
plate.
Use the plate i m m e d i a t e l y .
(Note:
T h e c h r o m a t o p l a t e s m u s t be h a n d l e d
especially during heating procedures.)

in a c l e a n

atmosphere,

N o w , spot by m e a n s of a m i c r o - p i p e t t e , 10 p i of the freshly prepared

solution of the steryl acetates in diethyl ether containing 10 p g per
pi, at the centre of the bridges of the chromatoplate as indicated in
the figure.
If m o r e than 5% phytosteryl acetates are expected to be
present in the steryl acetates sample, a smaller amount, e.g. 2.5 - 5
pi of t h e s t e r y l a c e t a t e s s o l u t i o n in d i e t h y l e t h e r , s h o u l d be
spotted on the starting point, otherwise the bands will not separate
clearly.
Open
the TLC tank and,
in o r d e r n o t to d i s t u r b t h e
vapour
e q u i l i b r i u m , place the c h r o m a t o p l a t e quickly in a vertical position
in the vessel and close it i m m e d i a t e l y with the lid.
Develop by ascending c h r o m a t o g r a p h y at a temperature of' 22-23C for
about 1.5 hours.
Discontinue development when the solvent front has
travelled to a height of about 16 c m , as measured from the baseline.
D r y the d e v e l o p e d c h r o m a t o p l a t e in air for 1-3 h o u r s and then in a
drying oven at 100C for 45 minutes.
Allow the chromatoplate to cool
to room t e m p e r a t u r e and spray u n i f o r m l y with the phosphmolybdic acid
solution.

60

THE DETECTION OF V E G E T A B L E FAT IN MILK FAT by thin-layer chromatography of steryl acetates

CHROMATOPLATE T E M P L E T

dimensions In m m

solvent front

200

KO

rA , vis i i Ay

/
s t a r t points

IS

11
-200-

T h e b a c k g r o u n d of the chroraatoplate s h o u l d be y e l l o w .
A greenish
c o l o u r m a y i n d i c a t e i n t e r f e r e n c e of e x t r a n e o u s v a p o u r .
The
cholesteryl acetate band will appear at a distance of 5-6.5 cm from
the s t a r t i n g p o i n t .
H e a t t h e c h r o m a t o p 1 ate
in a d r y i n g o v e n at
100C for 5 - 1 0 m i n u t e s u n t i l the b a n d s are c o l o u r e d to m a x i m u m
intensity.
A reference test must also be conducted simultaneously, on the same
c hr om a t op 1 a t e as u s e d for the s a m p l e t e s t , u s i n g a spot of 10 y 1 of
the r e f e r e n c e s o l u t i o n . A m a j o r b a n d of c h o l e s t e r y l a c e t a t e and a
m u c h smaller one of beta-sitosteryl acetate, with a lower m i g r a t i o n
r a t e , s h o u l d be c l e a r l y d i s c e r n i b l e .
If the a c e t a t e s w e r e not p u r e
e n o u g h for an a d e q u a t e
separation,
the d i g i t o n i d e s
should
be
recrystal1 i sed and the TLC repeated.
R e c r y s t a 11 i s a t i o n m a y be c a r r i e d o u t as f o l l o w s :
R e d i s s o l v e the
crystal cake by heating it over a m i c r o - b u r n e r in a short Pyrex glass
tube w i t h 1 m l of e t h a n o 1 (95% v/v).
A l l o w to c o o l f i r s t in air for
15 m i n u t e s and then in i c e - w a t e r for five m i n u t e s .
R e f i l t e r the
crystallized
steryl
acetates
and
dry
as
described
above.
R e c r y s t a 11 i s a t i o n m u s t be r e p e a t e d up to four or five t i m e s as
necessary to obtain a pure product.
IHTERPRETTIOH
If a s m a l l band w i t h the s a m e m i g r a t i o n rate as b e t a - s i t o s t e r y l a c e t a t e is
observed on the sprayed chromatoplate, the presence of phytosteryl acetates is
i n d i c a t e d and the fat s a m p l e u n d e r i n v e s t i g a t i o n , from w h i c h the s t e r y l
acetates have been obtained, is considered to contain vegetable fat.

61

The presence of at least 1% beta-sitosteryl

can be d e m o n s t r a t e d by this method.

acetate

The sensitivity of the detection of vegetable

as t h i s d e p e n d s u p o n t h e n a t u r e of the fat
phytosterol content of such fat.

Dairy Federation 32:1965

and

62

mixtures

fat in m i l k fat cannot be given

a d d e d and e s p e c i a l l y u p o n the

REFERENCE
International

in steryl acetate

38:1966.

VEGETABLE FAT IH BUTTERFAT

(Gas Chromatographic Method)
PRINCIPLE
S t e r o l d i g i t o n i d e s p r e p a r e d as g i v e n in the TLC m e t h o d are d i s s o l v e d in a
m i x t u r e of f o r m a m i d e and d i m e t h y 1 f o r m am ide.
The l i b e r a t e d s t e r o l s are
extracted with pentane and separated by gas-liquid chromatography.
If, on the
chromatogram, a peak with the retention time of beta-sitosterol is obtained,
the p r e s e n c e of v e g e t a b l e fat in the fat s a m p l e under i n v e s t i g a t i o n is
demonstrated.
Peaks of other phytosterols may support this conclusion.
(Note:
Formamide is a suspected teratogen and must be handled carefully).
APPARATUS
1.
Gas c h r o m a t o g r a p h ,
fitted w i t h h y d r o g e n f l a m e
ionization
d e t e c t o r , s i l v e r or g l a s s i n j e c t i o n s y s t e m , or d i r e c t - o n - c o l u m n
injection device, and recorder.
2.
Gas c h r o m a t o g r a p h i c c o l u m n , g l a s s , U - s h a p e d or c o i l e d , l e n g t h
100-200 cm, inside d i a m e t e r 3 - 4 m m .
(Note:
since s o m e t y p e s of
s t a i n l e s s s t e e l s c a u s e false r e s u l t s by d e t e r i o r a t i o n of s t e r o l s ,
only glass is recommended).
3.
Ml.

M i c r o - s y r i n g e , c a p a b l e of d e l i v e r i n g a v o l u m e of up to 5 or 10

REAGENTS
1.
Mixture of equal volumes
(See note under "PRINCIPLE").
2.

of

formamide

and

dimethyl

formamide.

n-Pentane.

3.
Column packing:
2-4% loading of a methyl silicone gum rubber,
s t a b l e up to at least 300C, on a f l u x - c a l c i n e d d i a t o m a c e o u s e a r t h
acid washed and silanized, mesh size 80/100 or 100/120.
4.
Sensitivity test
f r e s h l y prepared.

solution:

1 mg

cholesterol

5.
Peak resolution
test
solution:
p h y t o s t e r o l s and 0.1 m g c h o l e s t e r o l in
prepared.
6.
Reference test solution:
n-pentane, freshly prepared.

1 mg

0.9
1 ml

in 1 ml

n-pentane,

mg
rape seed
oil
n-pentane,
freshly

soyabean oil phytosterols in 1 ml

PROCEDURE
Dissolve about 10 mg sterol digitonide, prepared as described in the
T L C m e t h o d for d e t e c t i o n of p h y t o s t e r o l s , in 0.5 m l of a m i x t u r e of
e q u a l v o l u m e s of f o r m a m i d e and d i m e t h y l f o r m a m i d e in a s m a l l test
Shake the s o l u t i o n , w h e n
t u b e , if n e c e s s a r y w i t h g e n t l e h e a t i n g .
c o o l , w i t h 2.5 m l n - p e n t a n e .
Let the l a y e r s s e p a r a t e and use the
clear upper pentane layer (containing the liberated sterols) for gas
chromatographic analysis.
Establish the gas chromatograph operating conditions as follows:
set
the c o l u m n t e m p e r a t u r e at 220-250C.
T e m p e r a t u r e of i n j e c t i o n
system, if it can be separately heated should be 20-40C above column
temperature.
N i t r o g e n f l o w rate:
30-60 ml/min.
Disconnect
detector and equilibrate new columns under these conditions for 16-24
hours.
C o n n e c t d e t e c t o r , i g n i t e f l a m e and r e g u l a t e h y d r o g e n and

63

oxygen or air flow rates so as to obtain appropriate flame height and

detector sensitivity.
Start the recorder at a suitable chart speed,
a d j u s t z e r o s e t t i n g and a t t e n u a t o r .
If the b a s e line is s t e a d y the
a p p a r a t u s is ready for use.
S e n s i t i v i t y test:
I n j e c t 3 - 5 yl of the s e n s i t i v i t y test s o l u t i o n .
O n l y o n e p e a k o f c h o l e s t e r o l w i l l a p p e a r on t h e g a s c h r o m a t o g r a m
( F i g u r e 2.1). A d j u s t a t t e n u a t o r so as to o b t a i n a p p r o x i m a t e l y full
scale d e f l e c t i o n on the recorder.

GLC of MILKFAT STEROLS

Figure

2.1

Peak r e s o l u t i o n test:
I n j e c t 3 - 5 p i of the p e a k r e s o l u t i o n t e s t
solution.
P e a k s of c h o l e s t e r o l , b r a s s ica s t e r o 1, c a m p e s t e r o l and
b e t a - s i t o s t e r o 1 w i l l a p p e a r on t h e c h r o m a t o g r a m
(Figure
2.2).
M e a s u r e the r e t e n t i o n d i s t a n c e s (distance from sample injection to
m a x i m u m p e a k h e i g h t ) of t h e p e a k s , d q H for c h o l e s t e r o l , dg for
bras sicasterol, d for campesterol and dg for beta-sitosterol and the
peak base w i d t h s (retention d i m e n s i o n b e t w e e n intersections of base
line with tangents to the points of inflection on the front and rear
sides of the p e a k ) w q j j for cholesterol and w
for brassicaste rol.
g

2
Peak R e s o l u t i o n

(PR)

(d B -

dCR)

.
W

PR shall be at least

1.

CH

C a l c u l a t e the r e l a t i v e r e t e n t i o n t i m e s ( c h o l e s t e r o l
b r a s s i c a s t e r o l , c a m p e s t e r o l and beta-sitosterol.

64

1.00)

for

CLC of RAPE SEEDOIL STEROLS .r.d CHOLESTEROL

Figure

2.2

Reference test:
Inject 3-5
p 1 of the r e f e r e n c e test s o l u t i o n .
Peaks of campesterol, stigmasterol and beta-sitosterol will appear on
the c h r o m a t o g r a m ( F i g u r e 2.3). M e a s u r e t h e r e t e n t i o n d i s t a n c e s of
the peaks, d for campesterol, d g f o r stigmasterol, and dg for betasitosterol.
Cholesterol

1,.00 (about

Brassicasterol

1,.13 - 1 .15

Campesterol

1..32 - 1 .34

Stigmasterol

1,.44 - 1 .46

Beta-sitosterol

1 .66
,
- 1 .68

Next, inject 3-5 pi of the sample solution and switch the attenuator
to a four times (usually two steps) lower attenuation factor.
Record
the c h r o m a t o g r a m .
If on t h e c h r o m a t o g r a m a p e a k w i t h the r e l a t i v e
retention time of beta-sitosterol and a height of at least 2% of full
scale is observed, the presence of beta-sitosterol is indicated and
the fat sample under investigation from which the sterols have been
i s o l a t e d , is c o n s i d e r e d to c o n t a i n v e g e t a b l e fat. The p r e s e n c e , on
the gas c h r o m a t o g r a m ,
of p e a k s of o t h e r p h y t o s t e r o l s such as
campesterol or stigmasterol w i l l support the conclusion.

65

CLC of SOYABEAN OIL STEROLS

Figure 2.3
INTERPRETATION
The p r e s e n c e of at least 0.5% beta-s itostero 1 in sterol m i x t u r e s can be
demonstrated by this method.
The limit of detection of vegetable fat in milk
fat cannot be given since this d e p e n d s on the beta-s ito stero 1 content of the
fat used for a d m i x t u r e , i.e. upon the n a t u r e of the fat or m i x t u r e of fats
added to the milk fats. Note that very small amounts of beta-sitosterol may be
found in g e n u i n e b u t t e r , due to v e g e t a b l e oil used as a diluent for added
color.
Some papers relate GLC to RPK values.
For example, Huyghebaert and Hendrickx
(127)(128)(136).
For interpretation of GLC data, the reader should consult the
excellent review by Dickes and Nicholas (137) from which much of the following
i n f o r m a t i o n is taken.
The r e v i e w by Kuzda 1-Savoie (129) and the paper by
Parodi (130) are also important.
A n u m b e r of r a t i o s of one group of fatty acids to another is given
following table, compiled from Dickes and Nicholas's (137) data:

66

in the

Ratio of Fatty
c

Butterfat
Margar ine
Coconut

oil

Palm oil

4:C6+8
1.8

12:C10

Acids

14:C12

1.0-1.6

1 8 unsat.

3.2-6.0

: C

15

sat

2.4-3
2.4-3
3

8-8.5
7.9-8.2
13.5-17.1

examined by a n u m b e r of workers, including Shehata et

Stampbach (139), Tandan and Ganguli (140) and S c h w a r t z

This has been

al (138), H u l s t k a m p and
and Bright (141).

T h e p r e s e n c e o f a d d e d t r i b u t r y i n w o u l d h a v e to b e a s s e s s e d f r o m
the
c h r o m a t o g r a p h y of the g l y c e r i d e s t h e m s e l v e s , w i t h o u t p r i o r s a p o n i f i c a t i o n .
K u k s i s and M c C a r t h y (142) p o i n t o u t that a d d i t i o n of a j u d i c i o u s m i x t u r e of
lard and p a l m or c o c o n u t oil c o u l d r e s u l t in g a s c h r o m a t o g r a p h i c r e s u l t s
s i m i l a r to t h o s e f r o m b u t t e r , b u t of c o u r s e s u c h a s a m p l e s h o u l d g i v e a
positive phytosterol test.
S e p a r a t i o n of t r i g l y c e r i d e s by a r g e n t a t i o n - T L C p r i o r to GLC h a s b e e n used b y
Shehata et al (143).
They later used a preliminary separation on silicic acid
columns.
Sebastian and Rao (144) have investigated TLC methods for detecting
adulteration in butterfat.
I n d i c a t o r s are s o m e t i m e s r e q u i r e d to be put in b u t t e r not i n t e n d e d for h u m a n
c o n s u m p t i o n and t h e s e h a v e b e e n d i s c u s s e d by G u y o t in a s e r i e s of p a p e r s
(145)(146 ).
REFERENCES
International
IUPAC

Dairy Federation 32:1965

and

II.D.6.

67

54:1970.

2.5

ICE CREAM

COMPOSITION
It is important to m a i n t a i n adequate standards of composition for ice-cream as
it is o f t e n c o n s u m e d b y c h i l d r e n .
There are c o n s i d e r a b l e d i f f e r e n c e s among
n a t i o n a l standards.
The traditional product is m a d e from milk, whether fresh or processed, sugar,
and b u t t e r f a t , w i t h various e m u l s i f i e r s and e m u l s i o n stabilizers added.
The
International Dairy Federation produced its own International Standard in 1969.
T h i s l a y s d o w n c o m p o s i t i o n a l s t a n d a r d s for i c e - c r e a m and m i l k i c e s ( e d i b l e
i c e s ) p r o d u c e d f r o m m i l k and m i l k p r o d u c t s .
T h i s s t a t e s that " e d i b l e ices
produced from m i l k and m i l k produces are preparations, the solid or pasty state
of w h i c h has been obtained by freezing and which are intended to be consumed in
that s t a t e . "
The IDF

standards

are as

follows;
Fruit icecream (fruit
or pulp 15%)
(10% for lemon)

Edible
Ices
Butter fat
minimum
Total solids
minimum

Milk
Ices

Milk
with

Ices
eggs

Ice-cream
with eggs

8% m/m

6% m/m

3% m/m

3% m/m

8% m/m

32% m/m

30% m/m

28% m/m

28% m/m

32% m/m

7% m/m

7% m/m

Liquid egg yolk

or equivalent as
dehydrated yolk

W a t e r should only be used to reconstitute m i l k included as an ingredient.

Milk
of a n i m a l s o t h e r t h a n c o w s s h o u l d be d e s i g n a t e d .
The only other permitted
i n g r e d i e n t s are e g g s , n u t r i t i v e s w e e t e n e r s ,
f l a v o u r s and the
following
additives :
A l g i n i c a c i d and a l g i n a t e s .
A l g i n i c acid and its s o d i u m and
c a l c i u m c o m p o u n d s w i t h a t o t a l m a x i m u m c o n t e n t of 3 3 % of
m i n e r a l substances (sodium, potassium and calcium compounds of
o r t h o - and p y r o - p h o s p h o r i c acid, tartaric acid and citric a c i d )
Carrageenan
Gelatin
Pectin
Agar
T r a g a c a n t h gum
K a r a y a gum
A r a b i c gum
Guar gum
Carob seed gum
Carboxymethylcellulose
P r o p y l e n e glycol alginate

1%

(for products

containing

1%

Glycerine
G l y c e r y l mono- and
Lecithin
Sucrose esters

fruit)

di-stearate
0.6%

C i t r i c , tartaric, a s c o r b i c , lactic and malic

Starch and hydrolyzed starch derivates

68

acids

3%

(Note:
The percentages given are m a x i m u m
with other items in a bracket.
The over-run,
not e x c e e d 2
Gayakwad and
samples from

for the item alone or in combination

defined as the ratio, volume in litres/ mass in kilograms, should

(2.25 for p r o d u c t s c o n t a i n i n g over 35% t o t a l solids).
Thatti,
Laxminarayana (147) have published the results of analysis of 270
the Bombay market.

ROUTINE ANALYSIS
The bacteriological examination of ice-cream is very important as the product
is i m p l i c a t e d in o u t b r e a k s of food p o i s o n i n g from t i m e to t i m e .
Chemical
analysis should be carried out on a separate sample, as the bacteriologist has
to k e e p to a t i m e t a b l e for the m e t h y l e n e b l u e test and the s a m p l e is m e l t e d
Samples for chemical analysis should be tested immediately on
before testing.
arrival or stored in the deep-freeze as low sucrose values may be obtained due
to its conversion to dextran by Leuconostoe mesenteroides.
I c e - c r e a m should be a n a l y z e d for c o m p o s i t i o n , t o t a l s o l i d s , fat, s u c r o s e ,
lactose, protein (N X 6.38), ash and for metallic contamination, particularly
lead and zinc.
It m a y a l s o be n e c e s s a r y to t e s t f o r t h e p r e s e n c e o f
preservatives, artificial sweeteners, antioxidants in the fat, starch, flavours
and colours.
The type of fat present may be ascertained in the first instance
by d e t e r m i n a t i o n of the h y d r o x a m i c acid index (HAl) or Re i c h e r t - P o 1 e n s k e K i r s c h n e r (RPK) v a l u e .
The i o d i n e v a l u e of the fat is u s e f u l for i n d i c a t i n g
the presence of hydrogenated fat.
The level of milk solids other than milkfat can be calculated from the lactose,
p r o t e i n or c a l c i u m (1.88% as CaO in dry m i l k s o 1 id s - n o t - f a t ) but the r e s u l t s
must be interpreted with care as gelatin, lactose, calcium alginate thickener,
etc., may have been added. This can be seen from the ratio between them if one
of the f i g u r e s is not in line.
TLC is a c o n v e n i e n t w a y of c h e c k i n g that
sucrose and lactose are the only sugars present.
When ice-cream is manufactured, air is beaten into it to improve the texture.
The volume is thus increased and the increase is expressed by the "over-run",
defined as the percentage increase in volume, and calculable from the formula
1/d - 1/c
Over-run

(%) =

x 100 =

c - d

x 100

where c = SG of ice-cream before melting

d = SG of ice-cream, melted and deaerated.
For ice-cream sold by volume the overrun thus determines the weight sold per
unit price and is therefore of importance to both manufacturer and consumer.
M o s t of the a n a l y s e s can be c a r r i e d out by s l i g h t m o d i f i c a t i o n s of s t a n d a r d
m e t h o d s and w i l l not be d e s c r i b e d h e r e in full.
If the s a m p l e is taken from
the d e e p - f r e e z e , m e l t at b e l o w 45C, s h a k e and cool to r o o m t e m p e r a t u r e .
In
certain mixed products the ice-cream portion may have to be separated before
analysis.
Fat can be determined by Rose-Gottlieb, or Gerber methods. The various methods
are discussed by Park and McKeon (148).
For the Rose-Gottlieb method, use 5 g
of s a m p l e , 2 m l a m m o n i a and 8 ml of w a t e r .
M a i n t a i n at 65C for f i f t e e n
minutes, add 15 ml of 95% ethanol, mix and cool. The mixture should be free of
lumps, but any remaining can usually be broken up with a glass rod. If this is
impossible discard the determination and recommence with a fresh portion.
25
m l of d i e t h y l ether is added to the h o m o g e n e o u s m i x t u r e and the a n a l y s i s
completed as for milk.

69

The Gerber m e t h o d
is m o s t c o n v e n i e n t l y c a r r i e d out u s i n g an
ice-cream
b u t y r o m e t e r , but those intended for m i l k , cream or cheese are suitable provided
the correct a m o u n t s of sample and water are chosen.
For the routine screening of ice-cream for fat content, the Gerber or Babcock
t u b e u s i n g S a l w i n r e a g e n t m a y be used.
T h i s r e a g e n t is p r e p a r e d by m i x i n g
equal v o l u m e s of glacial acetic acid and 60% perchloric acid.
The procedure for the d e t e r m i n a t i o n of total solids
that given in this M a n u a l for evaporated milk.

is essentially

the same as

S u g a r s c a n be d e t e r m i n e d b e f o r e and a f t e r i n v e r s i o n by L a n e and E y n o n ' s

v o l u m e t r i c m e t h o d a f t e r c l e a r i n g w i t h z i n c f e r r o c y a n i d e or lead a c e t a t e and
sodium oxalate.
M i x 10 g with 150 ml water, clear and filter, wash the filter
a n d d i l u t e to 2 5 0 m l .
U s e an a l i q u o t for i n v e r s i o n .
If T L C h a s s h o w n o n l y
sucrose and lactose to be present, calculate sucrose as follows:
% sucrose = (T - I) 0.95, where:
I = reducing sugars as invert sugar
T = total sugars as invert sugar
U s e the l a c t o s e t a b l e to c a l c u l a t e the r e d u c i n g s u g a r s as X l a c t o s e .
l i q u i d c h r o m a t o g r a p h y of s u g a r s in ice c r e a m is d e s c r i b e d by W a r t h e s e n
K r a m e r (149).

The
and

P r o t e i n m a y be d e t e r m i n e d by the standard Kjeldahl procedure, using a factor of

6.38.
The m e t h o d for total solids is described in detail.
To d e t e r m i n e a s h , d r y a b o u t 10 g a c c u r a t e l y w e i g h e d
incinerate
at 5 5 0 C .
Determine
the c a l c i u m
in
p r e c i p i t a t i o n , flame p h o t o m e t r y or AAS.

on the w a t e r b a t h and
the ash by
oxalate

For acidity, titrate 10 g of ice-cream in a porcelain dish with 0.1N NaOH using
1 m l p h e n o 1 p h t h a l e in s o l u t i o n as i n d i c a t o r .
E x p r e s s the r e s u l t s as l a c t i c
acid.
T h e m i x t u r e m a y t h e n be u s e d for the d e t e r m i n a t i o n of p r o t e i n by the
f o r m o l titration (Crowhurst (150)).

70

2.6

CHEESE A N D OTHER

PRODUCTS

COMPOSITION
T h e C o d e x Al intentar ius C o m m i s s i o n h a s
Standards for the following cheeses:
Amsterdam
Brie
But terka se
Camembert
Cheddar
Cheshire
Cottage Cheese (including
creamed Cottage C h e e s e )
Coulommiers
Cream Cheese
Danablu
Danbo
Edam
Emmentaler
Es rom
Friese (Frisian)
Fynbo

developed

Recommended

Gouda
Gruyere
Gudbrandsdalsost
Harzer Kase
Havarti
Herrgordso8t
Hushallsost
Leidse (Leyden)
Limburger
Maribo
Norveg ia
Provolone
Romadur
Saint Paulin
Samsoe
Svec ia
Tilsiter

(Whey

International

Cheese)

Cheese standards typically prescribe m i n i m a for dry m a t t e r and for fat in the
dry m a t t e r .
The use of a d d i t i v e s m a y be r e s t r i c t e d to b a c t e r i a l and m o u l d
cultures, rennet, salt, emulsifying salts such as phosphates or citrates of the
a l k a l i and a l k a l i n e - e a r t h m e t a l s , n a t u r a l c o l o u r s and s o m e t i m e s s r b a t e ,
nitrate or nitrite as preservatives.
T h e I n t e r n a t i o n a l D a i r y F o u n d a t i o n h a s s t a n d a r d s for c u l t u r e d b u t t e r m i l k ,
y o g h u r t , a c i d o p h i l u s m i l k , k e f i r and k o u m i s s .
W h e t h e r or n o t y o g h u r t as
presented for sale should contain viable bacteria is discussed by Kroger (151)
and D a v i s (152).
D i f f e r e n t t y p e s of c r e a m such as c l o t t e d , d o u b l e , w h i p p e d , or s t e r i l i s e d are
c a t e g o r i s e d by t h e i r fat c o n t e n t .
C r e a m s h o u l d c o n s i s t o n l y of t h a t p a r t of
m i l k r i c h in fat and the a q u e o u s p h a s e s h o u l d h a v e the c o m p o s i t i o n n o r m a l to
s k i m m e d m i l k , a l t h o u g h in s o m e c o u n t r i e s s o m e g r a d e s of c r e a m m a y c o n t a i n
c e r t a i n additives.
ROUTINE
a.

ANALYSIS

Cheese

The major constituents of cheese are m o i s t u r e , butterfat, m i l k and protein

there are internationally agreed procedures to d e t e r m i n e these.

and

Protein may be determined by the Kjeldahl process.

Moisture can be determined
b y d r y i n g in the o v e n .
U s e of the K a r l F i s c h e r and D e a n and S t a r k m e t h o d s is
discussed
by S t r a n g e (153).
Arentzen (154)(155) discusses
sampling
difficulties.
For r o u t i n e p u r p o s e s the fat m a y be d e t e r m i n e d by the G e r b e r or V a n G u l i k
methods.
Reference m e t h o d s include the Rose-Gottlieb for whey cheeses and the
S c h m i d - B o n d z y n s k i - R a z 1 aff for o t h e r c h e e s e s . The l a t t e r m e t h o d is c o m p a r e d
with the Weibull-Stoldt and Van Gulik m e t h o d s by W i n k l e r (156).
The IDF h a s d e v e l o p e d a m e t h o d for p h o s p h a t a s e a c t i v i t y in p a s t e u r i z e d
stabilized cheese as a m e a n s of checking that pasteurization during processing
was adequate.

71

The e n z y m i c m e t h o d of Bahl (157)(158) can be used for lactose.

Novak and
L a s k o w s k i (159) d i s c u s s the c o l o r i m e t r i c m e t h o d s of M a r i e r and Boulet and
R i c h a r d s for s m a l l a m o u n t s (less than 100 m i c r o g r a m s ) of lactose in cheese.
Taylor (160) gives an enzymic method.
b.

Fermented Milk

M e t h o d s of a n a l y s i s g e n e r a l l y a p p l i c a b l e
fermented milks.

to m i l k products are suitable

for

Ney and Wirotama (161) describe the electrophoretic detection of gelatin added
to yoghurt.
Quarg (known as tvorog in eastern Europe) is a fermented cheeselike product of r e l a t i v e l y low total solids content.
Papers on quality and
composition include those by Solms (162), and Czulak and Hammond (163).
Khoa is another
me thods .
c.

fermented

cheese-like product, and may be analyzed by the same

Milk Formulae for Infants

These also may be analyzed by methods usually applicable to milk products. The
determination of lactose, sucrose and starch in infant milk formulae is dealt
with the Laskowski and Jamiolkowska (164).
d.

Crea

The analysis of cream does not present unusual difficulty.

Butyrometer tubes
for the determination of fat in cream by the Gerber method are available.
The
Rose-Gottlieb method can be used as a reference method for fat in cream. Davis
(165) d i s c u s s e s the l a b o r a t o r y control of cream.
A n e w m e t h o d for the
determination of added water has been reported (166).
e.

General Analysis of Dairy Products

The level of lactose of another major milk consistuent may sometimes be taken
as an adequate indication of the level of a milk product in a compound food.
The p r e s e n c e of w h e y p o w d e r in s k i m m e d
estimating the sialic acid content (167).

milk

powder

can be d e t e r m i n e d

by

O r o t i c acid (uraci 1 - 4 - c a r b o x y 1 ic acid) has been used as a m e a s u r e of the

proportion of non-fat milk solids present, occurring to the extent of 48 - 74.5
mg/100 g in non-fat milk solids (168).

72

PHOSPHATASE ACTIVITY
(In Dairy Products)
PRINCIPLE
Dilution of the liquid dairy product or the reconstituted liquid dairy product
with a buffer at pH 10.6 containing disodium phenylphosphate and incubation at
37C for one h o u r , l i b e r a t e s p h e n o l if a l k a l i n e p h o s p h a t a s e is p r e s e n t in the
product.
The phenol is reacted with dibromoquinonechloroimide and the colour
formed is measured photometrically.
APPARATUS
All glassware, stoppers and sampling tools must be carefully cleaned.
It is desirable to rinse them with freshly boiled distilled water or
to steam them.
Certain types of plastic stoppers may cause phenolic
contamination and their use must therefore be avoided.
Use ware with
glass stoppers when possible.
1.

Water bath capable of being maintained

2.
nm.

Spectrophotometer,

3.
Test
10 ml.

tubes,

suitable

Pipettes, graduated

5.

Glass funnels of convenient

6.

Filter

7.

Volumetric

8.

Litmus

9.

Analytical

1C.

for readings at a wavelength

16 or 18 m m x 150 m m ,

4.

at 37

preferably graduated

of 610

at 5 and

1 and 10 ml.
size, for example 5 cm diameter.

paper.
flasks for the preparation of standard

solutions.

paper.
balance.

REAGENTS
All reagents should be of analytical reagent quality and water should
be freshly boiled distilled water, or water of at least equal purity,
free from CO2.
1.
Barium borate-hydroxide buffer:
D i s s o l v e 50.0 g of b a r i u m
hydToxide (Ba(0H) 2 .
8H2O), free from carbonate, in water to a volume
of 1,000 ml.
D i s s o l v e 22.0 g of b o r i c acid ( H 3 B O 3 ) in w a t e r to a
v o l u m e of 1,000 ml.
W a r m 500 m l of e a c h s o l u t i o n to 50C, m i x the
s o l u t i o n s , s t i r , cool r a p i d l y to about 20C and adjust the pH if
necessary to 10.6 +_ 0.1 by addition of barium hydroxide or boric acid
solution.
Store the solution in a tightly stoppered bottle.
Dilute
the solution before use with an equal volume of water.
2.
Colour development buffer:
Dissolve 6.0 g of sodium metaborate
( N a B 0 2 ) or 12.6 g of N a B 0 2 ' 4 H 2 0 , and 20.0 g of s o d i u m c h l o r i d e (NaCl)
in water and dilute with water to a volume of 1,000 ml.
3.
Colour dilution buffer:
buffer to 100 ml with water.

Dilute

10 ml of the colour

development

4.
Buffer substrate:
D i s s o l v e 0.5 g of disodium phenyl-phosphate
( N a 2 C 5 H 5 P O 4 ' 2 H 2 0 ) in 4.5 m l of the c o l o u r d e v e l o p m e n t b u f f e r , add 2
d r o p s of the s o l u t i o n of 2, 6 - d ib r o m o q u inone - ch 1 o r o im id e and let

73

stand at r o o m t e m p e r a t u r e for 30 m i n u t e s .
E x t r a c t the colour so
formed with 2.5 ml of butan-l-ol and let stand until the butan-l-ol
separates.
R e m o v e the b u t a n - l - o l and d i s c a r d .
R e p e a t this
e x t r a c t i o n if n e c e s s a r y .
T h e s o l u t i o n m a y be s t o r e d in a
r e f r i g e r a t o r for a few days.
D e v e l o p the colour and r e - e x t r a c t
before use.
Prepare the buffer substrate immediately before use by
d i l u t i n g 1 m l of this s o l u t i o n to 100 ml w i t h the b a r i u m b o r a t e hydroxide b u f f e r .
5.
Zinc-copper precipitant:
D i s s o l v e 3.0 g of zinc s u l p h a t e
( Z n S 0 4 * 7 H 2 0 ) and 0.6 g of c o p p e r s u l p h a t e (CuSO^'Sl^O) in w a t e r to a
volume of 100 ml.
6.
2, 6 - d i b r o m o qu i n o n e c h 1 o r o i m i d e s o l u t i o n (Gibbs reagent):
Dissolve 4 0 + 1 mg of 2, 6-dibromoquinonechloroimide (BQC) in 10 ml
of 9 5 % (v/v~) e t h a n o l .
S t o r e in a d a r k - c o 1 o u r e d b o t t l e in a
refrigerator.
Reject if discoloured or more than 1 month old.
7.
Copper sulphate solution:
D i s s o l v e 0.05 g of copper
(CUSO^'5H20) in water to a volume of 100 ml.
8.

sulphate

Sodium hydroxide, 0.5 N solution.

9.
Phenol standard solutions:
Weigh 200 +_ 2 mg of pure anhydrous
phenol, transfer to a 100 ml volumetric flask, fill to the mark with
w a t e r and m i x .
(This s t o c k s o l u t i o n r e m a i n s s t a b l e for s e v e r a l
months in a refrigerator. Dilute 10 ml of this stock solution to 100
ml with water and mix.
1 ml contains 200 p g of phenol.
PROCEDURE
Important
1.

notes:

Avoid direct

sunlight during

the

determination.

2.
C o n t a m i n a t i o n w i t h t r a c e s of s a l i v a or p e r s p i r a t i o n can give
false positive results and must be avoided.
Pipetting in particular
must be done with special care and not by mouth.
Preparation of Sample
M i l k , b u t t e r m i l k and w h e y :
c a r r y out the a n a l y s i s p r e f e r a b l y
directly after sampling.
O t h e r w i s e , k e e p t h e s a m p l e in a
r e f r i g e r a t o r , b u t n o t for m o r e t h a n 2 d a y s .
M i x the s a m p l e
c a r e f u l l y , if n e c e s s a r y w i t h m o d e r a t e h e a t i n g . The t e m p e r a t u r e of
mixing must under no circumstances exceed 30C.
Dried milk, buttermilk powder and whey powder:
Dissolve 10 g of the
p r o d u c t in 90 m l of w a t e r .
The t e m p e r a t u r e applied in d i s s o l v i n g
must under no circumstances exceed 35C.
Neutralization of sour products: Add to a sour product dilute
hydroxide until the test solution is neutral to litmus paper.

sodium

Test Portion
P i p e t t e into each of two test tubes 1 ml of the test s a m p l e ,
one tube as a control or blank.

using

Determination
Heat the blank for 2 minutes in boiling water; cover the tube and the
beaker of boiling water with aluminium foil to ensure that the entire
tube w i l l be h e a t e d .
Cool to room t e m p e r a t u r e .
F r o m this point

74

treat the b l a n k and the test s a m p l e alike. Add 10 ml of the b u t t e r

s u b s t r a t e and m i x .
I m m e d i a t e l y i n c u b a t e in the w a t e r b a t h at 37C
for 60 m i n u t e s , m i x i n g the c o n t e n t s o c c a s i o n a l l y .
Heat in b o i l i n g
w a t e r for 2 m i n u t e s in the s a m e m a n n e r as for the blank.
Cool to
room temperature.
Add 1 m l of the z i n c - c o p p e r p r e c i p i t a n t to each
tube and mix thoroughly.
Filter through a dry filter paper, discard
the first r u n n i n g s , r e f i l t e r if n e c e s s a r y u n t i l the f i l t r a t e runs
c l e a r , and c o l l e c t 5 m l in a test tube.
Add 5 m l of the c o l o u r
development buffer.
Add 0.1 m l of the BQC s o l u t i o n , m i x and a l l o w
the c o l o u r to d e v e l o p for 30 m i n u t e s at r o o m t e m p e r a t u r e .
Measure
the a b s o r b a n c.e a g a i n s t the b l a n k in a s p e c t r o p h o t o m e t e r
at a
wavelength of 610 nm.
Repeat the determination with an approximate dilution of the sample
or the reconstituted sample if the absorbance exceeds the absorbance
of the s t a n d a r d c o n t a i n i n g 20 p g of p h e n o l per tube. P r e p a r e this
d i l u t i o n by m i x i n g 1 v o l u m e of the test s a m p l e w i t h an a p p r o p r i a t e
volume of a part of the same test sample thoroughly boiled in order
to inactivate the phosphatase.
Preparation of Standard

Curve

P r e p a r e a s u i t a b l e r a n g e of d i l u t e d s t a n d a r d s , s t a r t i n g from the
standard phenol, containing 0 (control or blank), 2, 5, 10 and 20 y g
of phenol per millilitre and pipette respectively 1 ml of water and 1
m l of the four p h e n o l s t a n d a r d s o l u t i o n s into each of five test
tubes.
Add to each tube 1 m l of the c o p p e r s u l p h a t e s o l u t i o n , 5 m l of the
colour dilution buffer, 3 m l of water and 0.1 ml of the BQC solution,
and m i x .
A l l o w the c o l o u r to d e v e l o p for 30 m i n u t e s at room
temperature.
Measure the absorbance against the control or blank in
the spectrophotometer at a wavelength of 610 nm.
Prepare the calibration curve by plotting the absorbances against the
quantities of phenol in micrograms.
The standard curve should be a
straight line.
CALCULATION
Convert the absorbance to micrograms of phenol per millilitre of the
liquid p r o d u c t or in the case of dried p r o d u c t s , per m i l l i l i t r e of
reconstituted liquid product, by means of the following formula:
Phosphatase activity = 2.4 x A x D
where:

A is the quantity of phenol in micrograms obtained

D is the dilution factor of any dilution,
(in the case of no dilution, D = 1).

REPEATABILITY
The difference between the results of two determinations carried out
simultaneously or in rapid succession by the same analyst should not
exceed 2 p g of phenol.
If a d i l u t i o n is a p p l i e d this l i m i t r e f e r s
to the results obtained on the diluted sample.
INTERPRETATION
A value of more than 1 is indicative
tion with unpasteurized substances.

of improper

75

pasteurization

or

contamina-

2.7

TEXT

REFERENCES

1.

DAVIS,
J.G. & M a c D O N A L D ,
Churchill & Co.

2.

P E A R S O N , D.
Churchill .

3.

W E B B , B.H., J O H N S O N ,
Chemistry, AVI.

A.H. & A L F O R D ,

4.

HARDING, F. & ROYAL,

IE, 4 5 3 .

J.H.L.

5.

KEMPINSKI,

Deutsche-Milchwirtschaft,

6.

HARDING, F.

7.

BERGMANN, J.
8, 1 6 0 - 3 .

8.

CORRADINI, C.

Milchwissenschaft, 30 (7) 413-416.

9.

HAAVE, I.J.J.

1973.

10.

HANSON, N.W. (Ed.)

Analysis, 2nd Ed.

11.

T I L L M A N S , J. & L U C K E N B A C H , W.
Lebensmitteln, 50 103.

1925.

Zeitschrift

12.

T I L L M A N S , J. & L U C K E N B A C H , W.
267 .

1926.

M i 1 ch w ir t s ch a f t F o r s c h u n g , 3 2 2 5 -

13.

T I L L M A N S , J. & L U C K E N B A C H , W.
1930.
untersuchung und Forschung, 60 342.

14.

WOIDRICH, K. & SCHMID, L.

und Forschung, 102 16 7.

15.

HANKINSON, D.J. & ANDERSON,

Station Bulletin, 234.

16.

IWAIDA, M., EBINE, R. & TANIMURA,

of J a p a n , 14 (3) 2 5 8 - 2 6 3 .

17.

DAVIDSON, J.J.

1949.

Dairy Research,

18.

LAWRENCE,

1970.

Australian

19.

H I L L I G , F.
1942.
Chemists, 25 253.

Journal

20.

STEFFEN,

Schweizerische

21.

B A S A K , A. & R O Y , B.R.

22.

M E H T A , R.S., B A S S E T T E , R. & W A R D , G.
(3) 2 8 5 - 2 8 9 .

1974.

23.

W A L K E R , N.J. & G R A Y , I.K.

Chemistry, 18* (3) 346-352.

Journal

24.

KRATZER, D.D.

1981.

C.

C.

1953.

The C h e m i c a l

1971.
1973.

Richmond's

Analysis

1974.

J.A.

XIX

of Food

1974.

Dairy

8th Ed.

Chemistry.

Livingstone-

Fundamentals

International

Dairy

of

Dairy

Congress,

Vol.

22^ (51/52) III-V.

Dairy Industries, 38 (7) 311-315.

1976.

A.J.

F.J.

Instrumental

Methods Review,

Heieriposten,

Tierzuchter,

28 (4) 156-

62 (49) 1078-1080.

1973.
Standardised, R e c o m m e n d e d & Official
The Society for Analytical Chemistry.

1971.

1967.

1955.

1 975.

E.O.

1939.

A.

fur U n t e r s u c h u n g s

Zeitschrift

Zeitschrift

fur

Storrs

Journal

fur

der

Lebens m i11e1-

Lebensmitteluntersuchung

Agricultural

Experimental

of the Food Hygienic

Food

of the A s s o c i a t i o n

Technology,

of O f f i c i a l

Milchzeitung,

67 1073,

I n d i a n J o u r n a l of D a i r y

Society

Journal

of

198.
Agricultural

1078.

S c i e n c e , 28 1 2 7.
of D a i r y S c i e n c e , 57

Agricultural

Journal of Dairy Science, 50 1384 p 73.

77

of

209-216.

Journal of

1970.

Methods

and

Food

25.

D Z H O R O V , T., R O S T O V ,
N a u k i , _6_ ( 6 ) 1 1 3 - 1 1 9 .

26.

ZAGAEVSKII,

27.

S C H A L M , O.W., C A R R O L L , E.J. & J A I N ,

Febiger.

28.

S C H U L T Z E , W.D., S M I T H , J.W., J A S P E R , D.E., K L A S T R U P , 0., N E W B O U L D , F.H.S.,

P O S T L E , D.S. & U L L M A N , W.W.
1972.
J o u r n a l of M i l k and Food T e c h n o l o g y ,
35_ (1 1 ) 6 3 9 - 6 4 5 .

29.

N E W S T E A D , D . F . & O R M S B Y , J.E.
and T e c h n o l o g y , 5 (2) 5 2 - 5 6 .

30.

R O W L A N D , S.J. &
108, 267.

31.

WHEELER,
175.

32.

HAMANN,
J.,
TOLLE,
A.,
BLUTHGEN,
M i l c h w i s s e n s c h a f t , 3 0 (1) 1 - 7 .

33.

RYBINSKA,
215.

34.

K U M A R , P., S U D ,
( 5 ) 5 5 3 - 5 5 7. .

35.

RE I N B O L D , G.W.
619.

36.

A G G A R W A L , P.K., S U D , R.K. & G U P T A , K.G.

56_ ( 1 2 ) 1 5 6 2 - 1 5 6 4 .

37.

KRAMER,
J., C A R T E R ,
G.G., A R R E T , B., W I L N E R , J., W R I G H T , W . W . &
K I R S C H B A U M , A.
1974.
A n t i b i o t i c R e s i d u e s in M i l k , D a i r y P r o d u c t s &
Animal Tissues:
M e t h o d s , R e p o r t s and P r o t o c o l s , N a t i o n a l C e n t e r for
A n t i b i o t i c & I n s u l i n A n a l y s i s , F o o d & D r u g A d m i n i s t r a t i o n , D e p t . of
H e a l t h , E d u c a t i o n & W e l f a r e , W a s h i n g t o n DC 2 0 2 0 4 .

38.

FEAGAN, J.T.

31.

COX, D. & McNAMARA,

40.

R I C H A R D , J. &
127, 129, 131.

41.

NICKERSON,
S c i e n c e , 59

42.

F E R N A N D E Z , F.J. & R A M I R E Z , E.E.

1974.
R e v i s t a de M e d i c i n a V e t e r i n a r i a
A r g e n t i n a , 55 (3) 1 9 9 - 2 0 0 , 2 0 3 - 2 0 4 , 2 0 7 - 2 0 8 , 2 1 1 - 2 1 2 , 2 1 5 - 2 1 7 .

43.

W I L S O N , A.G., S W E E T S U R , A . W . M . & W H I T E , J.C.D.

D a i r y C o n g r e s s , IE 4 5 5 - 4 5 6 .

44.

R U K I N A , N.I., R A S T E G A E V A , N.A. & N A U C H N Y E T R U D Y .

1974.
Omskii Ordena
L e n i n a Se 1 ' s k o k h o z y a i s t v e n n y i I n s t i t u t i m e n i S.M. K i r o v a , 121 1 1 6 - 1 1 9 ,
136.

45.

J A Y N E S, H.O. & A S A N , T.
(6) 3 3 3 - 3 3 6 .

I.S.

S.M.

1974.

&

VITKOV,

Molochnaya

ZEID-EL-DINE,

1979.

K.

L.

Roczniki

R.K. & G U P T A ,

1971.

1966.

Journal

Dairy
C.

KERHERVE,

T.A., V U J I C I C ,
(3) 3 8 6 - 3 9 0 .

M.C.

M.

1939.

K.G.

of

Dairy

A.,

I.F.

1973.

Revue

LIN,

of

Dairy

Dairy

Lea

Higiney,

of D a i r y

34

(4)

W.

25

28

(2)
6

19 75 .

(2)

(2)

1976.

1974.

and

213,

56

613-

Science,

53-60.
38-45.

Laitiere

Milk

10

169-

Science,

of D a i r y

&

Science

Research,

HEESCHEN,

Journal

A. Y.

of

Mastitis,

T e c h n o l o g y , 34_(12)

Technology,

Journal

78

&

Food

42-43.

Technology,

Zakladu

Abstracts,

&

of

Journal

and

1,

Journal

Journal

1973.

of M i l k

No.

Bovine

Zealand

Panstwowego

1973.

1973.

1971.

Journal

Dairy

L.

Veterinarno-medicinski

Promyshlennost'

New

Science

1975.

1969.

1970.

Australian

1974.

M.

Franaise,

Journal

XIX

Food

of

306,

Dairy

International

Technology,

36

46.

H O B B S , J.S.
Agriculture,

& LAWRENCE,
23.

47.

BAHL, R.K.

48.

V A L D E H I T A , M.T. S. L O P E Z , M.L.
40.

49.

D O U G A L L , B.M. & M O R G A N , D.T.

1973.
Analytical Chemistry, 10 (7) 181.

50.

MADRID

51.

M I T T A L , S.B. & R O Y , M.K.

52.

GUPTA, P.K. & M A T H E W ,

I n d i a , 41 (2) 5 7 - 9 .

53.

D H A R , A.K., ROY, B.R., G U H A , K.C. S. M I T R A ,

Dairy Science, 23 (1) 12-15.

54.

JAMES,

55.

JAOUEN,

56.

R A O , S.R. & B E C T O R ,
1-6.

57.

P R U T H I , T.D., R A V A L , N.P. S. B H A V S A R , N.K.

(1) 2 1 - 2 3 .

58.

D A V I D E , C.L. & D O M I N G O , L.E.

432-438.

59.

M I N C I 0 N E , B., S C U D I E R O , A., A L B 0 N I C 0 , F. & F R A N C I S C I S , G.

e Tecnologa degli Alimenti, 3 (6) 342-345.

60.

S W A I S G O O D , H. ( E d i t o r ) 3 3 p p . ( u n d a t e d ) U r b a n a ,
Dairy Science Association.

61.

A L - S H A B IB I, M., K A R A M , H.A., E L I YA, J., J U M A , H., A B U - A L - M A ' A L I ,

1971.
Alexandria Journal of Agricultural Research, 19 (1) 77-80.

62.

W I L L I A M S , A.P., B I S H O P ,
R e s e a r c h , 43 (2) 3 2 5 - 9 .

D.R. & C O C K B U R N ,

63.

W A G N E R , A.
1973.
(1/2) 1 5 3 - 1 5 8 .

Agronmica

64.

R A O , M.B., G U P T A , R.C. & D A S T U R ,

S c i e n c e , 23 (2) 7 1 - 7 8 .

65.

VAGHELA, L.H.
University.

66.

M O N A C E L L I , R. & C A N T A G A L L I , P.
Provineiali, 26 (10) 360-364.

67.

CARINI, S. & BUSCA, M.

68.

FOISSY, H.

69.

ASCHAFFENBURG,
383-84.

1972 .

1972.

1976.

J.C.

1971.

Revista
1974.

T.V.

of

the

Science

of

Food

and

Acta

1976.

Proceedings

Espaola

Journal

1980.

of

du Lait,

the

the

Society

for

19-22.

S.N.

Institution

1970.

July-Aug

1971.

Analysts,

7-13;

of D a i r y

Sept

J.E.

N.N.

1970.

of Camel's

7-25.

S c i e n c e , 33

79

29

(9/10)

Scienza

Illinois, USA;

American

Journal

Scientiarum

Indian

of

H.N.

Dairy

Hungaricae, 22

Journal

of

Milk, M.Se Thesis,

dei L a b o r a t o r i

Dairy

Bombay

Chimici

610-612.

Milchwirtschaft,
1968.

(1)

1973.

1976.

Bolletino

of

14.

1974. M i l c h w e i s s e n - s c h a f t ,

Academiae

Latte,

R. 5. D A N C E , J.E.

Chemists,

P h i l i p p i n e A g r i c u l t u r i s t , 54

1975.

Osterreichische

of

Indian Journal

of Public

Indian Journal

Some Properties

1975.

of

de Lecheria, 83

of the Association

Le Technicien
B.S.

A n a l e s de B r o m a t o l o g i a , 23 (1 ) 3 5 -

X I X 1st D a i r y C o n g r e s s , IE 4 7 2 - 3 .

1969.

Journal

1973.

1976.

Journal

Analyst, JJ7 (1 156) 559-561 .

VICENTE, A.

G.V.

J.G.

31

(3, Beilage

2) 5-8.

J o u r n a l of D a i r y R e s e a r c h , 35 (3)

70.

EGUARAS, J.L.

1975.

71.

BRET, G.
39 pp.

72.

P I E R R E , A. & P O R T M A N N , A.
107-130.

1970.

Annales de T e c h n o l o g i e A g r i c o l e , 19 (2)

73.

P I R R R E , A. & P O R T M A N N , A.
177-79.

1970.

Annales de T e c h n o l o g i e A g r i c o l e , 19 (2)

74.

P O R T M A N N , A. & P I E R R E , A.
1971.
Annales
l'Expertise Chimique, 64 (696) 159-177.

75.

MAJUMDER, G.C. & GANGULI, N.C.

(3) 189-190.

76.

M A J U M D E R , G.C.,
Animal Sciences,

77.

GUHA, K., DHAR, A.K. & ROY, B.R.

Technology (Mysore), _9_ (2) 90-91.

78.

G H O U S E , M. & JAIN, M.K.

43-47.

79.

O'KEEFE, M.G.

80.

LUNDER,

81.

APARICIO

82.

ARTE M ' E V, A.A. & P A N A S E N K O V , N.S.

1969.
Izvestiya Vysshikh
Zavadenii, Pischewaya Tekhnologiya, (1) 161-164.

83.

UZONYI, G. & VARGA, G.

84.

DOZET, N., STANISIC,

85.

S L A N O V E C , T., A R S O V , A., P L A N K A R , S. & GALVAN, S.

(7) 156-160.

1968.

T.L.

Alimentaria, 11 (65Z) 31-33.

International

Dairy

1972.

Federation

1967.
1970.

GALLEGO,

des

Falsifications

N.C.

1972.

1972.

Journal

Indian

of

Food

Science

1970.

and

Revista Espaola de Lecheria, (78) 193-200.

1973.

Uchebnykh

Tejipar 22 (4) 89-91.

M. & SUMENIC,

S.

1973.

Mljekarstvo, 23 55-62.
1974.

Mljekarstvo

24

87.

HENNINGSON, R.W. 1969. Journal of the Association of Official

Chemists, 52 (1) 142-151.

88.

W O O D , E.C.
(3) 68-70.

Analyst, 62_ 709.

89.

MOORE, S.

90.

ANON.
1971.
(1) 31.

New
~~

91.

HOFFELNER,

1968.

92.

DILLIER-ZULAUF,
1608 & 1710.

93.

D I L L I E R - Z U L A U F , A. & D O Y O T T E , J.P. 1971. M i t t e i l u n g e n aus dem

der Lebensmittelunter8uchung und Hygiene, 62 (1 ) 32-41.

94.

BINDER,

1972.

1970.

of

21 (1) 1-18.

TEMPLE, P.L.

W..

de

Journal

86.

K.

et

Journal of Dairy Research, 34 211.

M.

1974.

(7)

Indian Journal of Dairy Science, 26 (1)

Scienza e Tcnica Lattiero-Casearia,

1937.

Bulletin,

Indian Journal of Dairy Science, 25

JOSHI, V.K. & G A N G U L I ,

42 (10) 779-784.

1973.

Annual

Journal

of

the A s s o c i a t i o n

of Public

Analytical

A n a l y s t s , 10

Milk Industry, _75 (3) 20-22 .

A.

Zealand

Journal

of Dairy

Science

and

Technology,

Osterreichische
1971.

Deutsche

Milchwirtschaft, 23_(15) 261-4.

Mo1kerei-Zeitung,

92

International Dairy Congress (18th Sydney).

80

(40)

1704-

Gebiete

IE:87.

95.

PANETSOS, A. & G E O R G A K I S , S.
870, 872-874.

96.

W E L B O R E N , J.T. S. V E L D E N , H. van der. 1974. M i t t e i l u n g e n aus dem Gebiete

der Leben8mittelunter8uchung und Hygiene, 65 (1) 151-156.

97.

PRENTICE, J.H.

98.

LUCK, H. & D R E S N E R , S.
]_ (4) 221-227.

99.

BINDER, W.
465.

100. ASPERGER,
'
465-466.

1978.

1974.

H.

1970.

Deutsche M i l k e r e i - Z e i t u n g , 91 (21)

Analyst 103 1269-1273.

1975.

South A f r i c a n Journal of Dairy

Technology,

Proceedings of the XIX International Dairy Congress, IE

1974.

Proceedings

101. BOUCHES, D. & WAES, G.

1973.

of the XIX International Dairy

Congress,

Revue de l'Agriculture, 26 (4) 799-812.

102. HENNINGSON y R. W 1970. Journal of the Association of Official Analytical

Chem ists, _53_ (3) 539-542
103. HENNINGSON y R. W 1972. Journal of the As soc iat ion of Official Analyt ical
Chemists, 53 (3) 504-506
104. HENNINGSON y R W 1968. Journal of the Association of Official Analyt ical
Chemists, 51 (4) 816-821.
105. J A M O T T E , P. & D U C H A T E A U , J.
1973.
T e c h n i c i e n du Lait,
21 in January issue and pp 25-27 and 29-34 in February issue.
106. F R E E M A N , T.R., BUCY, J.L. & K R A T Z E R , D.D.
Technology, 34 (4) 212-214.
107. D E R M O T T ,
140-141.

B.J.

1971.

108. O'CONNOR, C.B.

(5) 23-25.

1970.

Journal

Irish

of

1971.

Milk

and

Agricultural

109. LUCK, H., KRIEL, J.B., M O S T E R T ,

Dairy Technology, _5_ (3) 129-133.

J.F.

1973.

110. STILES, M.E., B R E D E N K A M P , B.L.F. & A B B O T T ,

Journal of Dairy Technology, 6 (2) 123-130.
111. N O W A K , E. & L A S K O W S K I , K.
Mleczarskiego, 14 (3) 33-42.
112. REMOND, B.

1975.

1972.

114. RESMINI, P. & VOLONTERIO, G.

E.R.

1963.

116. GUHA, K.C. & R O Y ,

Agriculture, 24 1.

17,

19,

Journal of M i l k and Food

Food

and

Technology,

Creamery

South

C.W.

Roczinki

34

(3)

Review,

23

Journal

of

African

1974.

South

Instytutu

African

Przemyshu

Lait, 55 (547) 390-395.

113. M A N N I N G , P.B., C O U L T E R , S.T.

Science, 51 (11) 1725-30.

115. LING,
Hall.

pp

& JENNESS,

1973.

A Textbook

B.R.

117. PATRAT11, A. P. & SOCHNEVA, L.G.

Issaldevatl'skei, 30 108-110.

1 986.

Journal

of

Dairy

Latte No. 9, 167-169.

of Dairy

1 9 7 3.

R.

Journal

1973.

81

Chemistry

of

the

Trudy,

Vol.

II,

Science

of

Usesoyuznyl

Chapman

&

Food

and

Nauchino-

118. EMMENS, D.B.

119. MORRISEY,

1970.

P.A.

XIX International Dairy Conference, IE 523.

1974.

Irish Agricultural & Creamery Review, 27 (7) 5-7.

120. A N D E R S O N , B.A. & BERLIN, E.

29.

1974.

Journal of Dairy Science, 58_(1) 25-

121. LIVIO, L.T.

1969.

Latte, 43 (8) 561-64.

122. F R A N Z E N , K.
139-144.

1947.

Lebensmitte1chemie

und G e r i c h t l i c h e C h e m i e , 28 (4)

123. T I M M E N , H. & B L U T H G E N , J.
1973.
Zeitschrift
suchung und Forschung, 153 (5) 283-288.

fur

124. ROSCHNIK, M.R.

Mitteilungen
und H y g i e n e , 63 (2) 206-21.

Lebensmitteluntersuchung

aus dem Gebiete der

125. P A R O D I , P.W. & D U N S T A N , R.J. 1971.

26. 60.
126. PARODI,

P.W.

1972.

Lebensmittelunter-

A m e r i c a n Journal of Food

Australian Journal of Food Technology,

Technology,

27 (3) 90-94.

127. HUYGHEBAERT, A. & HENDRICKX, H. 1971.

Milchwissenschaft,

128. HUYGHEBAERT,

Milk Industry, 67 (3) 18-23.

A. & HENDRICKX, H. 1970.

129. K U Z D A L - S A V O I E , S., L A N G L O I S & T R E H I N , J. 1975.

International Dairy Federation, 86 180-184.

26 (10) 613-617.

Annual B u l l e t i n of the

130. PARODI,

P.W.

1973.

Australian Journal of Dairy Technology,

28 (4).

131. PARODI,

P.W.

1980.

Australian Journal of Dairy Technology,

35 (1) 17-22.

132. WILLIAMS, K.A.

Oils, Fats and Fatty Foods, p. 217.

133. B R E C K E N R I D G E , W.C. & K U K S I S , A. 1967.

Metabolism AVI Westport, USA 233 pp.
134. L A F T I , A. & M A Z L O U M U , K.
162-172.

1969.

Indian Journal of Dairy Science, 22 (3)

135. DYER, B., TAYLOR, G. & HAMENCE, J.H.

136. HUYGHEBAERT, A. & HENDRICKX, H.
137. DICKES, G.J. & NICHOLAS, P.V.
A n a l y s t s , J_0^(4) 87-98.

S y m p o s i u m : Dairy Lipids & Lipid

1941.

1970.

1972.

Analyst, 66 355.

Milchwissenschaft,

25^ (99) 506-10.

Journal of the Association of Public

138. SHEHATA, A.Y., de M A N , J.M. & A L E X A N D E R , J.C.

of Food Technology Journal, 3 (3) 85-89.

1970.

Canadian

Institute

139. H U L S T K A M P , J. & S T A M P B A C H , H. 1970. M i t t e i l u n g e n aus dem Gebiete

Lebensmitteluntersuchung und Hygiene, 61 (5-6) 388-394.
140. T A N D A N , K.C. & 'GANGULI, N.C.
(3) 179-183.
141. SCHWARTZ,
274.

1972.

D.P. & BRIGHT, R.S.

142. K U K S I S , A. & M C C A R T H Y
Society, 41 12-21.

1964.

1974.

Indian Journal of Dairy Science. 25

Analytical

Journal

82

der

of

the

Biochemistry,

American

61 (1) 271-

Oil

Chemists

143. K H A L A F A L L A , S.M., E L S A D E K , G.M., S H E H A T A , A.E. & E L - M A G D O U B , M.N.I.

E g y p t i a n J o u r n a l of D a i r y S c i e n c e , 1 (1) 1 3 - 2 0 .
144. SEBASTIAN,

J. & R A O , M.B.

1974.

29

Mi1chwi ssenschaft,

1973.

(9) 5 3 3 - 3 4 .

145. G U Y O T , A.L. 1 9 7 1 .
(3 / 4 ) 3 2 5 - 3 7 8 .

B u l l e t i n d e s R e c h e r c h e s A g r o n o m i q u e s d e G e m b l o u x , 6_

146. G U Y O T , A.L. 1 9 7 2 .
(1/4) 9 1 - 1 1 3 .

Bulletin

des R e c h e r c h e s A g r o n o m i q u e s

1 4 7 . T H A T T I , B.L., G A Y A K W A D , K.S. & L A X M I N A R A Y A N A ,

D a i r y S c i e n c e , 25 (1) 9 - 1 3 .
1 4 8 . P A R K , H . S . & M c K E O N , J.R.
149. W A R T H E S E N ,
626-627.

1978.

J.J. & K R A M E R ,

150. CROWHURST, B.
151. KROGER, M .

1956.

1975.

P.L.

1979.

A n a l y s t , jU

Cheese

Journal

A.G.J.
64 (42)

1972.
Officieel
1003-6.

155. A R E N T Z E N ,
Zuiveboud,

A.G.J.
65 (35)

1972.
825-28.

Officieel

157. BAHL, R.K.

1971.

A n a l y s t , 96_ 8 8 - 9 2 .

158. BAHL, R.K.

1972.

A n a l y s t , 97

1972.

Australian

1 6 1 . NE Y , K.H. & W I R O T A M A , I.P.G.

s u c h u n g und F o r s c h u n g .
1 6 2 . S O L M S , J. & G U I G O Z , Y.
(6) 3 6 5 - 5 7 .

Journal
1 970.

1974.

163. C Z U L A K , J. & H A M M O N D , L.A.

73-74.

Methods

Orgaan.

of

424-426.

Science,

44

(2)

54-55.

Vol

II J.

Churchill,

of O f f i c i a l

Analytical

Koninklijke

Nederlandse

Koninklijke

Nederlandse

Berichte

Roczinki

aus

den

Bundesanstalten

Zeitschrift

fur

25

Przemyshu

(1) 7 - 9 .

L e b e n sm i 1 1 e 1 u n t e r -

Lebensmittel-Wissenschaft

1973.

& T e c h n o l o g i e , _7_

C S I R O F o o d R e s e a r c h Q u a r t e r l y , 33

J.

165. DAVIS, J.G.

Industries,

(May) Dairy

Instytutu

of D a i r y T e c h n o l o g y ,

164. L A S K O W S K Y , K. & J A M I 0 L K 0 W S K A ,
M l e c z a r s k i e g , 12 3 5 - 4 5 .
1971.

Journal

559-561.

L A S K O W S K I , K.
1 4 , (3) 3 3 - 4 2 .
1970.

Food

(5) 5 2 ,

Orgaan.

1 5 6 . W I N K L E R , S.
1974.
Milchwirtschaftliche
W o l f p a s s i n g und R o t h o l z , 4 0 1 6 1 - 2 0 0 .

G.G.

of

of t h e A s s o c i a t i o n

154. A R E N T Z E N ,
Zuiveboud,

160. TAYLOR,

Indian

123-4.

Manufacturing

1 5 3 . S T R A N G E , T.E.
1970. Journal
C h e m i s t s 53 (4) 8 6 5 - 6 8 .

1972.

J o u r n a l of F o o d P r o t e i n , 4 1

D a i r y & Ice C r e a m F i e l d , 158

152. D A V I S , J.G.
1976.
Edinburgh 965-1132.

159. N O V A K , E. &
Mleczarskiego

H.

d e G e m b l o u x , 1_

1970.

166. L E C H N E R , E.
1 97 5.
Zeitschrift
F o r s c h u n g , 159 (1) 3 9 - 4 2 .

fur

83

Roczniki

Instytutu

(4)

Przemyslu

267-275.

Lebens m itte1untersuch ung

und

167. SERRINI, G., LANZA-SERRINI, G. & MONFRINI, C. 1981. Determination of Free

Sialic Acid in Skimmed M i l k , Rennet Whey P o w d e r s and their Relative
M i x t u r e s , EUR 7598 EN Directorate-General, Information Market and
Innovation, Btiment Jean Monnet, Luxembourg.
168. ARCHER, A.W.

1973.

Analyst, 98 755.

169. G O M B O C Z , E., H E L L W I G , E. & PETUELY, F. 1981.

mittaluntersuchung und Forschung, 172 178-181.
170. M I T C H E L L , G.E. & MIDDLETON. G.
Technology, 35 (1) 15-16.
171. TIMMS, R.E.

1980.

1980.

Zeitschrift

fur Lebens-

The Australian Journal of Dairy

Journal of Dairy Research, 47 295-303.

172. BURON A R I A S , I., GARCIA TERESA, R. & VALENCIA DIAZ, F.

Aceites, 31 335-340.

1980. Grasas y

173. TRAITLER, H. & PREVOT, A. 1981. Journal of High Resolution

graphy and Chromatography Communications, 4 109-114.
174. W A T H E L E T , J-P., DEROANNE, C. & SEVERIN, M.
Corps Gras, 26^(4) 175-179.

1979.

Chromato-

Revue Franaise

des

Further Reading
A M E R I C A N DRIED M I L K INSTITUTE, INC. 1971.
including Methods of Analysis, Chicago, USA.

Standards for Grades of Dry Milk

ARORA, K.L. & RAJORBIA, G.S.

1978.
Review on the D e t e r m i n a t i o n
Milk, Indian Journal of Animal Research 12 (2) 59-70.
Challenges to C o n t e m p o r a r y Dairy Analytical Techniques,
Chemistry Special Publication No. 49. 1984. London, UK.
DAVIS, J.G.

1950.

Society

of

A Dictionary of Dairying and Supplement Leonard Hill.

DAVIS, J.G. 1965-1975.

FOX. P.F.
Science.

Royal

of Fat in

Cheese Vols I-IV, Churchill, London.

1982 & 1983. D e v e l o p m e n t s

in Dairy C h e m i s t r y Vols 1 & 2 Applied

MARTH, E.H.
1978. Standard Methods for the Examination of Dairy Products 14th
Ed. American Public Health Association, Washington.
M c G A N N , T.C.A. 1979. Review of Analysis of Dairy Products in Carroll, P.M.,
B u r n s , D.T., B r o w n , D.A. & M a c D a e i d , D.A. E u r o a n a l y s i s III R e v i e w s on
Analytical Chemistry, Applied Science Publishers.
SCOTT, R. 1981.

Cheesemaking Practice Applied

84

Science.

3.
3.1

SUGARS AND HOMEY

SUCROSE

COMPOSITION
CAC Standards for white sugar and white

Polarization
(min. 155)

White
Sugar
Spec.A

White
Sugar
Spec.B

99. 7

99.5'

sugar

products:

Powdered
(Icing)
Sugar(a)

Soft
Sugar
Spec.A

Soft
Sugar
Spec.B

99.7

Sucrose + Invert Sugar Content (min.)

88.0%

0.3-12.0%

97.0%

Invert Sugar
Content

0.04%

0.1%

0.04%

Conduct. Ash

0.04%

0.1%

0.04%

Loss on Drying
(3 hr at 105C)(b)

0.1%

0.1%

0.1%

4.5%

3.0%

0.3-12.0%

0.2%

Color
(ICUMSA Units)

60

150

60

60

Sulphur
Dioxide

20

70

20

40

40

(ppm)

Arsenic

(ppm)

Copper
Lead

(ppm)

(ppm)

Sulphated Ash
(Note:

10

10

3.5%

All single values are maxima unless otherwise

noted.)

(a) Icing sugar m a y c o n t a i n up to 5 p e r c e n t s t a r c h as long as no o t h e r a n t i caking agent is used.

If s t a r c h is not p r e s e n t , it m a y c o n t a i n any of the
following, singly or in combination, up to a m a x i m u m of 1.5% - tribasic calcium
phosphate, magnesium carbonate, magnesium stearate, silicon dioxide, amorphous
(dehydrated) silica gel, calcium silicate, m a g n e s i u m trisilicate, sodium
calcium alumino-silicate.
(b) The limit for loss on drying does not apply ,to white sugar in lump or cube
form, to crystal candy sugar (crystal korizato), or to rock sugar (korizato).

ROUTIHE ANALYSIS
Methods of analysis
1.

suitable

for powdered

Loss on drying at 105C for 3 hours

and crystalline

sucrose

are:

(1).

2.
I n v e r t s u g a r s by the K n i g h t and A l l e n m e t h o d , as r e c o m m e n d e d by I C U M S A .
This depends upon titration of excess unreduced copper with EDTA using murexide
as an i n d i c a t o r .
If the invert sugar c o n t e n t e x c e e d s 0.04%, the B e r l i n
Institute method (1) should be used.

85

3.
P o l a r i z a t i o n , c o n d u c t i v i t y ash and c o l o u r can be d e t e r m i n e d by I C U M S A
m e t h o d s r e p r o d u c e d in t h i s c h a p t e r .
ICUMSA have also r e c o m m e n d e d , among
others,
methods
for S O 2 , p H , a r s e n i c , c o p p e r and l e a d .
Except
for
d e t e r m i n a t i o n of invert sugar, m o i s t u r e and polarization, the above m e t h o d s are
s u i t a b l e for o t h e r p o w d e r e d and c r y s t a l l i n e s u g a r s .
M o i s t u r e in a n h y d r o u s
d e x t r o s e and dextrose m o n o h y d r a t e is determined by drying for 4 hours at 100C
and a p r e s s u r e of 100 m m Hg.
The I C U M S A m e t h o d
for s u l p h a t e d ash is
s u b s t a n t i a l l y t h e s a m e as t h e g e n e r a l m e t h o d .
T h e c o p p e r r e d u c t i o n and
p o l a r i m e t r i c m e t h o d s in general used for the d e t e r m i n a t i o n of sugars in foods,
c o r n s y r u p and o t h e r s u g a r s y r u p s are a l s o a p p r o p r i a t e for a s s a y i n g d e x t r o s e
and fructose powders.
The b r o w n sugar with a characteristic taste known as D e m e r a r a m a y have had the
colour stabilized w i t h stannous chloride, titanous chloride or artifial colour.
The extraneous w a t e r - i n s o l u b l e m a t e r i a l in good quality white sugar is usually
l e s s t h a n 25 m g / k g .
It m a y b e d e t e r m i n e d b y m a k i n g a s o l u t i o n of a l a r g e
quantity of sample (e.g. 1 kg) up to 1,800 ml with boiling w a t e r , filtering and
t h e n w a s h i n g w i t h a l i t r e of h o t w a t e r and s o a k i n g in w a t e r on a s i e v e for an
h o u r in o r d e r to r e m o v e t r a c e s of s u g a r f r o m the p e r i p h e r y of the p a p e r .
The
filter is then dried, weighed and examined under the microscope.
T h e lead c o n t e n t of s u g a r s w i l l u s u a l l y be v e r y c o n s i d e r a b l y l o w e r than the
l i m i t of 2 m g / k g .
T r a c e m e t a l s c a n be d e t e r m i n e d d i r e c t l y on s o l u t i o n s of
w h i t e sugars but b r o w n sugars must be ashed in the presence of ash-aid for lead
and digested or ashed according to standard m e t h o d s for arsenic.
Copper can be
d e t e r m i n e d by extracting the diethyl carbodithioate into carbon tetrachloride.
Degrees Brix are defined as the percentage of sucrose by weight.
For example,
a sucrose solution of 50 Brix consists of equal weights of water and sucrose.
O r i g i n a l l y tables w e r e constructed relating degrees Brix to specific gravity, a
the l a t t e r and the B r i x w a s read from the
h y d r o m e t e r w a s u s e d to m e a s u r e
table.
Later the r e f r a c t o m e t e r was used and the Brix read from a similar table
r e l a t i n g it to the r e f r a c t i v e i n d e x .
S t r i c t l y s p e a k i n g , d e g r e e s Brix only
apply to pure sucrose solutions, but in industry the refractive index of juices
and f r u i t p r o d u c t s g e n e r a l l y is d e t e r m i n e d as a q u a l i t y c o n t r o l m e a s u r e and
r e p o r t e d as " a p p a r e n t B r i x " or j u s t "Brix".
The analyst m u s t treat these
f i g u r e s w i t h s o m e r e s e r v e , as t h e r e f r a c t o m e t e r r e a d i n g a l t e r s w i t h
t e m p e r a t u r e , a c i d i t y and the p r e s e n c e of o t h e r s u b s t a n c e s i n c l u d i n g o t h e r
s u g a r s and the " a p p a r e n t B r i x " m a y b e a r a m o d i f i e d r e l a t i o n to the a c t u a l
sucrose c o n t e n t .

POLARIKETRY
S o l u t i o n s of sugars alter the plane of polarised light, to an extent depending
o n c o n c e n t r a t i o n and on the s u g a r .
T h u s the c o n c e n t r a t i o n of a s o l u t i o n of a
p u r e k n o w n s u g a r m a y be d e t e r m i n e d by c o m p a r i s o n of the n u m b e r of d e g r e e s of
angle by w h i c h it turns the light (angular rotation) with the rotation caused
by a standard s o l u t i o n .
N o t e that any o t h e r s u b s t a n c e c a p a b l e of r o t a t i n g
p o l a r i s e d l i g h t , i n c l u d i n g any other sugars, w i l l interfere.
This does not
prevent the use of the technique for the d e t e r m i n a t i o n of sugars in mixtures.
F o r e x a m p l e , s u c r o s e m a y be d e t e r m i n e d in the p r e s e n c e of i n v e r t s u g a r b y
d e t e r m i n a t i o n of the rotation before and after hydrolysis of the sucrose.
The
h y d r o l y s i s c a u s e s a c h a n g e in r o t a t i o n p r o p o r t i o n a l to the p e r c e n t a g e of
sucrose present.
If the reducing power of the mixture is also determined, the
p r o p o r t i o n s of t h r e e s u g a r s in a d m i x t u r e m a y be d e t e r m i n e d .
T h e p r e s e n c e of
salts of alkaline reaction decreases the reading by polarimetry and allowance
for t h i s is m a d e in s o m e of the f o r m u l a s u s e d to c a l c u l a t e r e s u l t s .
For
example,
correction
is m a d e
f o r t h e a m o u n t of s a l t p r o d u c e d b y t h e
n e u t r a l i z a t i o n of h y d r o c h l o r i c acid used for hydrolysis.
If a s u g a r e x i s t s in s o l u t i o n as t w o s t e r e o i s o m e r s in e q u i l i b r i u m ,
but
c r y s t a l l i s e s in o n l y o n e f o r m , t h e n on r e - s o l u t i o n it w i l l r e q u i r e s o m e t i m e
for the e q u i l i b r i u m to be r e - e s t a b l i s h e d .
If the t w o i s o m e r s h a v e d i f f e r e n t

86

rotatory p o w e r , as they usually do, the optical rotation of the solution will
be c h a n g i n g u n t i l e q u i l i b r i u m
is a t t a i n e d .
T h i s p h e n o m e n o n is
called
mutarotation.
In p r a c t i c e , the s o l u t i o n is e i t h e r left o v e r n i g h t b e f o r e
r e a d i n g , or a m m o n i a , w h i c h l i k e any a l k a l i a c c e l e r a t e s the a t t a i n m e n t of
equilibrium, is added, or the neutral solution m a y be boiled.
In 1 8 4 2 , V e n t z k e d e v i s e d a s c a l e s u c h t h a t an a p p r o x i m a t e l y 26 p e r c e n t m / v
s u c r o s e s o l u t i o n read in a 20 cm tube g a v e a r e a d i n g of 100. A f t e r a n u m b e r
of changes over the years, it is now agreed that the polarization of the n o r m a l
solution (26.000 g of pure sucrose dissolved in 100 m l and polarized at 20C in
line in v a c u o ( X =
a 200 m m t u b e , u s i n g p o l a r i z e d l i g h t of the g r e e n
546.2271 n m ) as defined by the International C o m m i s s i o n for Uniform Methods of
S u g a r A n a l y s i s ( I C U M S A ) ) , be a c c e p t e d as the b a s i s of c a l i b r a t i o n of the 100
p o i n t on the I n t e r n a t i o n a l S u g a r S c a l e . The d e t a i l s h a v e c h a n g e d o v e r the
years.
T h e p r e s e n t d e f i n i t i o n s are g i v e n in the I C U M S A m e t h o d s (1).
The
rotation for 100S (international Sugar Degrees) is equivalent to:

01

20.00 C
546. 2271 nm

= 40. 765

For practical polarimetry, wavelengths in the region of 540 to 590 nm are also
permissible for fixing the 100 point.
100S at the wavelength of yellow sodium
light will be equal to a rotation of:
20.00 C
589.4400 nm

34.616

A s a c c h a r i m e t e r is of s o m e w h a t d i f f e r e n t d e s i g n to a p o l a r i m e t e r .
Quartz
c r y s t a l s h a v e a v e r y s i m i l a r r o t a t o r y p o w e r to s u g a r s but in the o p p o s i t e
d i r e c t i o n , h e n c e b y i n s e r t i o n of a q u a r t z w e d g e into the l i g h t p a t h , the
r o t a t i o n due to the sugar m a y be fixed and the p e r c e n t a g e of s u g a r in
s a c c h a r i m e t e r d e g r e e s is read f r o m the s c a l e c a l i b r a t e d a c c o r d i n g to the
d i s t a n c e the w e d g e h a s to be i n s e r t e d in o r d e r to c a n c e l the r o t a t i o n of the
sample.
The instrument is scarcely sensitive to changes in the wavelength of
the l i g h t u s e d , u n l i k e an o r d i n a r y p o l a r i m e t e r , b e c a u s e it w o r k s by t h i s
compensatory m e c h a n i s m .
Therefore, the use of w h i t e light is specified in the
standard method and a dichromate filter is used to remove light from the violet
end of the spectrum, giving better delineation.
The m a i n f a c t o r s a f f e c t i n g the r o t a t i o n of s u g a r s are the w a v e l e n g t h of the
l i g h t u s e d , the c o n c e n t r a t i o n of s u g a r in the s o l u t i o n , the c o n c e n t r a t i o n of
acid and salt p r e s e n t , t e m p e r a t u r e , s o l v e n t , c l a r i f y i n g a g e n t used and if
hydrolysis (inversion) of a higher saccharide to a monosaccharide is involved,
the exact conditions of temperature, time and hydrolysing agent used.
Thus the
m a n y formulae found in m e t h o d s of analysis relate to particular conditions and
the method must be followed in every detail and the correct formula relating to
t h a t m e t h o d u s e d if a c c u r a t e r e s u l t s are to be o b t a i n e d .
It s h o u l d be n o t e d
that c h a n g e of o p t i c a l r o t a t i o n w i t h c h a n g e of w a v e l e n g t h of the l i g h t u s e d ,
like the other factors, is different for different sugars, being greatest for
galactose and least for rhamnose.
The following table is taken from Pearson (2).
The formulae
calculation of the specific rotation of the indicated sugar:

Sugar

Formula

Sucrose

, N20
( o t ) D = 66.462

Dextrose

, x20
(a) D = 52.50 + 0.0188p +

Dextrose

given are for

, 2 0
5461A

(a)

62

87

+ 0.0087c -

-032

9
0.000235c /
?
0.000517p

0.04257c

the

Sagar

Formula

Fructose

(et)

Invert

(a)

sugar

Invert sugar
temperature correction

20

20

138.475 -

0.01837p

-(19.415 + 0.07065c 20

(a)D

0.00054c )

+ ( 0 . 2 8 3 + 0 . 0 0 1 4 c ) ( t - 20C)

where
c = c o n c e n t r a t i o n in g r a m s per 100 m l
p = percentage by weight
t = t e m p e r a t u r e (C) d u r i n g a n a l y s i s
T h e C l e r g e t - H e r z f e l d F o r m u l a is u s e d to c a l c u l a t e t h e p e r c e n t a g e of s u c r o s e in
a s a m p l e b y m e a s u r i n g the r o t a t i o n b e f o r e and a f t e r i n v e r s i o n ( h y d r o l y s i s ) of a
s o l u t i o n to g l u c o s e and f r u c t o s e ( i n v e r t s u g a r , an e q u i m o l e c u l a r p r o p o r t i o n of
the t w o ) .
T h e d e t e r m i n a t i o n w a s u s u a l l y c a r r i e d out u s i n g a s a c c h a r i m e t e r and
t h e r e s u l t s t h e r e f o r e h a d to b e r e f e r r e d to the s a c c h a r i m e t e r s c a l e , b a s e d on a
26 p e r c e n t m / v s o l u t i o n of the s a m p l e and the s p e c i f i c r o t a t i o n of s u c r o s e of
a b o u t 66.5. H o w e v e r , o n i n v e r s i o n the s p e c i f i c r o t a t i o n d o e s n o t f a l l to z e r o
b u t a c t u a l l y b e c o m e s n e g a t i v e , the s p e c i f i c r o t a t i o n of i n v e r t s u g a r b e i n g
a b o u t - 2 0 . 4 a n d t h i s m u s t b e m u l t i p l i e d b y 1 . 0 5 3 a s 1 0 0 g of s u c r o s e g i v e
1 0 5 . 3 g of f r u c t o s e a n d d e x t r o s e o n h y d r o l y s i s .
H e n c e t h e t o t a l c h a n g e is
a b o u t 8 7 . 5 a n d t h e r e a d i n g m u s t b e m u l t i p l i e d b y 6 6 . 5 / 8 7 . 5 so t h a t it c a n b e
r e a d o f f as p e r c e n t a g e s u c r o s e o n t h e s a c c h a r i m e t e r s c a l e .
In t h e o r i g i n a l
C l e r g e t f o r m u l a the r e a d i n g w a s d i v i d e d by 87.5526/66.5
or 1.3166.
As
e x p l a i n e d a b o v e , t h e a n a l y t i c a l c o n d i t i o n s a f f e c t t h e v a l u e s a n d H e r z f e l d in
1 8 8 8 c l o s e l y s t a n d a r d i z e d t h e p r o c e d u r e and d e r i v e d a f o r m u l a of the t y p e :

100 ( P - I )
1 3 3 . 2 + 0 . 0 6 7 6 ( 1 3 - m ) - 0.5

(t-20)

w h e r e 1 0 0 / 1 3 3 . 2 r e p r e s e n t s t h e f a c t o r c o n v e r t i n g to the s a c c h a r i m e t e r s c a l e ,
0 . 0 6 7 6 ( 1 3 - m ) c o r r e c t s f o r t h e t o t a l s o l i d s ( m ) f r o m t h e o r i g i n a l s o l u t i o n in
1 0 0 m l of i n v e r t s o l u t i o n , P is t h e r o t a t i o n of t h e n o r m a l w e i g h t b e f o r e
i n v e r s i o n and I the r o t a t i o n after.
( T h u s if 26 g o f a n h y d r o u s s a m p l e a r e
t a k e n the c o r r e c t i o n is z e r o , as the n o r m a l w e i g h t is d i s s o l v e d in 100 m l and a
50 m l a l i q u o t is i n v e r t e d and d i l u t e d to 100 m l . ) t r e p r e s e n t s t e m p e r a t u r e in
degrees Centigrade.
T h e f a c t o r s h a v e b e e n r e - e x a m i n e d by J a c k s o n and G i l l i s (3) and J a c k s o n and
M c D o n a l d (4) a n d f r o m t i m e to t i m e b y the s u c c e e d i n g C o m m i s s i o n s for U n i f o r m
M e t h o d s of S u g a r A n a l y s i s ( 1 9 6 4 ) ( 1 9 7 8 ) . S e e a l s o N o r r i s h ( 5 ) , I n t e r n a t i o n a l
C r i t i c a l T a b l e s ( 6 ) a n d B r o w n a n d Z e r b a n (7).
T h e s e f o r m u l a s a p p l y if t h e
e x a c t m e t h o d s g i v e n in o f f i c i a l c o m p e n d i a such as t h o s e of the A O A C and I C U M S A
are f o l l o w e d .
In a c i d h y d r o l y s i s m e t h o d s a n a m o u n t o f s a l t , e q u a l to t h a t
as a r e s u l t o f t h e a d d i t i o n a n d s u b s e q u e n t n e u t r a l i z a t i o n
of
produced
h y d r o c h l o r i c a c i d to the h y d r o l y s e d a l i q u o t , is a d d e d to t h a t p o r t i o n w h i c h is
n o t h y d r o l y s e d in o r d e r to c a n c e l out the salt e f f e c t on the r o t a t i o n .
T h e v e r y a c c u r a t e w o r k of J a c k s o n and M c D o n a l d
(4) w a s d o n e
using
s a c c h a r i m e t e r s a n d it is n e c e s s a r y to c a l c u l a t e t h e f a c t o r s f o r u s e w i t h a n
i n s t r u m e n t r e a d i n g in a n g u l a r d e g r e e s .
S i n c e Io on the I n t e r n a t i o n a l S u g a r
S c a l e = 0.34616 a n g u l a r , the r e a d i n g is s i m p l y d i v i d e d b y t h i s f i g u r e and the
r e s u l t s u b s t i t u t e d in t h e f o r m u l a a b o v e .
T h e I n v e r s i o n D i v i s i o n F a c t o r ( Q ) is a n a l t e r n a t i v e m e t h o d of c a l c u l a t i o n
p r o v i d e d the a n g u l a r d e g r e e s are read from a p o l a r i m e t e r i l l u m i n a t e d by a
s o d i u m l a m p . "Q" is d e f i n e d as "the c h a n g e in the s p e c i f i c r o t a t i o n of s u c r o s e

88

on inversion.
It can be calculated from
inversion at 60C and readings at 20C.

saccharimeter

formulas, e.g. for acid

The value of Q is not only affected by the factors that influence the ClergetHerzfeld formulas, but also the wavelength of light used (which is always white
light w i t h the sac char i m e t e r ) and the presence of other substances including
the precipitant used.
Values adopted by the SPA in 1930 were (8):

(Zinc ferrocyanide
precipitant)
Sodium light
Mercury green light
White light (International
Sugar Scale Light)

0.8825
1.0392
2.549

(Phos photungs t ic
acid precipitant)
0.8865
1.0439
2.561

For impure products containing other polarizing substances such as raffinose,

glucose, fructose, amino-acids etc. the determination of sucrose, by use only
of a p o l a r i m e t e r , r e q u i r e s m e t h o d s of m u l t i p l e p o l a r i z a t i o n of w h i c h the
Clerget method is a particular example.
If the p r o d u c t d o e s not c o n t a i n r a f f i n o s e , t w o p o l a r i z a t i o n s are m a d e :
one
d i r e c t and one a f t e r h y d r o l y s i s .
The s u c r o s e c o n t e n t is d e d u c t e d from the
c h a n g e in p o l a r i z a t i o n on h y d r o l y s i s , it b e i n g a s s u m e d that the p o l a r i z i n g
impurities are not affected by the hydrolytic reagents and behave identically
in both measurements.
If the product contains raffinose, this trisaccharide is partially hydrolysed
to fructose and melibiose under the conditions required to hydrolyse sucrose,
and a third polarization is required, either after complete hydrolysis of the
r a f f i n o s e (and s u c r o s e ) , or u n d e r s o m e o t h e r pH c o n d i t i o n s so as to p r o d u c e a
predictable change in, the optical activity of the sugars.
The sucrose can be
d e d u c t e d from t w o s i m u l t a n e o u s e q u a t i o n s i n v o l v i n g the three p o l a r i z a t i o n s .
A g a i n it is a s s u m e d that the r e m a i n i n g p o l a r i z i n g i m p u r i t i e s are o t h e r w i s e
unchanged by the hydrolytic reagents.
The fate of these i m p u r i t i e s , in e i t h e r c a s e , m a y not c o r r e s p o n d to the
h y p o t h e s i s for v a r i o u s r e a s o n s .
O t h e r o p t i c a 1 1 y - a c t i v e c o m p o u n d s m a y be
h y d r o l y s e d or their r o t a t i o n s m a y be c h a n g e d by the h y d r o l y t i c r e a g e n t s ; the
results obtained thus necessarily have an uncertain character.
It is essential
to take into a c c o u n t such f a c t o r s as the n a t u r e of c l a r i f y i n g a g e n t , the
content of dry solids of the solution to be polarized and the temperature and
conditions of hydrolysis.
Since many optically-active compounds contribute to
p o l a r i z a t i o n , t e m p e r a t u r e c o r r e c t i o n s are n e c e s s a r i l y u n c e r t a i n and it is
recommended that, if possible, all polarizations should be carried out at 20C
to avoid any temperature correction.
The s i m u l t a n e o u s e q u a t i o n s for three p o l a r i z a t i o n s can be solved to d e d u c e
r a f f i n o s e c o n c e n t r a t i o n , and f o r m u l a s h a v e a l s o b e e n p r o p o s e d for d e d u c i n g
raffinose from two polarizations if the material is free from reducing sugars,
or if the r o t a t i o n a l c o n t r i b u t i o n of the r e d u c i n g s u g a r s can be e s t i m a t e d
independently.
Since the raffinose content is very much less than the sucrose
c o n t e n t , the u n c e r t a i n t i e s h a v e a r e l a t i v e l y g r e a t e r e f f e c t on the r a f f i n o s e
value and none of these procedures is recommended for estimation of raffinose,
which is best done by the method of Schiweck and Busching (9,10).

89

WHITE SUGAR
(Polarization Method)
PRINCIPLE
An a q u e o u s s o l u t i o n of the sugar s a m p l e (26 g, i.e. the n o r m a l w e i g h t of
s u c r o s e in 100 m l w a t e r ) is polarized by m e a n s of a s a c c h a r i m e t e r w h i c h is
c a l i b r a t e d to read 100S on the " i n t e r n a t i o n a l Sugar Scale" under specified
conditions.
APPARATUS
1.

S a c c h a r i m e t e r , calibrated with quartz plates.

2.
F l a s k s (100 m l ) c o n f o r m i n g to I C M S A class A, w h i c h are
individually calibrated by weighing with water at 20 _+ 0.1C.
Flasks
w h o s e c o n t e n t s fall w i t h i n the range 100.00 _+ 0.01 m l m a y be used
w i t h o u t c o r r e c t i o n . Flasks w h o s e contents fall outside this range
must be used with the appropriate correction to 100.00 ml.
3.
Polarimeter tube,
with cover glasses.
4.

length 200 m m ,

conforming

Water-bath, thermostatically controlled

to ICUMSA Class A,

to 20.0 _+ 0.1C.

PROCEDURE
The n o r m a l w e i g h t (26 _+ 0.002 g) of the sugar s a m p l e is weighed out
and transferred to a flask (100 ml) by washing with distilled or deionized w a t e r (about 80 ml).
The sugar is dissolved by a g i t a t i o n
without heating and water added to just below the calibration mark.
The t e m p e r a t u r e of the sugar solution is adjusted to 20 _+ 0.1C by
m e a n s of a w a t e r bath. The inside w a l l of the neck of the flask is
dried with filter paper and the solution volume adjusted exactly to
100 ml with water (20 +_ 0.1C) using either a hypodermic syringe or a
p i p e t t e w i t h a d r a w n - o u t point.
The flask is then sealed w i t h a
clean, dry stopper and its contents mixed thoroughly by hand-shaking.
The polarimeter tube is thoroughly rinsed twice with two-thirds its
v o l u m e of sugar s o l u t i o n and filled w i t h the sugar solution at 20 _+
0.1C in such a w a y that no air b u b b l e s are entrapped.
The tube is
placed in the s ac char im e t er and polarized at 20
0.1C.
Five
m e a s u r e m e n t s are taken to 0.05S or better.
The average value is
e x p r e s s e d to 0.01S.
In the s a m e w a y the quartz control plate
reading is determined to 0.01S.
CALCULATION
N o r m a l l y it is not convenient to adjust the t e m p e r a t u r e of the
quartz-wedge compensator to 20 _+ 0.1C and consequently the following
equation can be used to apply the necessary correction:
p20

= p

0.00014 (t - 20)]

Where :
P20

is the polarization at 20 _+ 0.1C,

P is the observed polarization at 20 + 0.1C and

t is the temperature in C of the quartz-wedge
compensator.

90

If a f l a s k c o r r e c t i o n is r e q u i r e d t h e n the p o l a r i z a t i o n is c o r r e c t e d
b y a d d i n g t h e f l a s k c o r r e c t i o n to t h e o b s e r v e d p o l a r i z a t i o n .
F l a s k c o r r e c t i o n = A c t u a l v o l u m e of the
H i g h l y c o l o u r e d or t u r b i d s u g a r s o l u t i o n s
b a s i c lead a c e t a t e b e f o r e p o l a r i z a t i o n .
The result
0.01S.

is to b e e x p r e s s e d

as p o l a r i z a t i o n

flask -

100.00

must

clarified

be

with

in S to an a c c u r a c y

of

REFERENCES
T h i s is a n I C U M S A t e n t a t i v e m e t h o d ( 1 9 7 9 ) .
ICUMSA, Report
P r o c e e d i n g s of the 1 6 t h S e s s i o n , 1 9 7 4 , S u b j e c t 1 9 , R e c o r d 6 , 2 6 8 .

of

the

I C U M S A , R e p o r t of the P r o c e e d i n g s of the 1 6 t h S e s s i o n , 1 9 7 4 , S u b j e c t
265 .

19,

I C U M S A , R e p o r t of the P r o c e e d i n g s of the 15th S e s s i o n , 1 9 7 0 , S u b j e c t

95.

11,

91

ISVKRT SUGAR II WHITE SDGAR

(Knight and Allen Method)
PRINCIPLE
The method is suitable for the determination of low invert sugar contents (e.g.
in w h i t e sugar up to 0.02Z).
A c o p p e r - c o m p l e x s o l u t i o n containing sodium
carbonate as the main alkaline agent is used.
When heated in a boiling waterb a t h , the invert sugar r e d u c e s the cupric ions to cuprous oxide.
After
c o o l i n g , the r e s i d u a l cupric ions are titrated w i t h EDTA, using m u r e x i d e as
indicator.
The experimental conditions, including weight of sugar, volume of
w a t e r , v o l u m e of a l k a l i n e copper solution and time of h e a t i n g , are strictly
standardised and the result is obtained from a calibration curve.
APPARATUS
1.

Test tubes (150 x 20 mm).

2.
W h i t e p o r c e l a i n e v a p o r a t i n g dishes,
ensure better detection of the end-point).
3.

Water-bath, maintained

at boiling

used

for

titration

(to

point.

REAGENTS
1.
A l k a l i n e copper solution:
dissolve 25g anhydrous
sodium
carbonate and 25g sodium potassium tartrate tetrahydrate in 600 ml of
distilled water containing 40 ml of N sodium hydroxide solution in a
1L volumetric flask.
Dissolve 6g of cupric sulphate pentahydrate in
about 100 ml of distilled w a t e r and q u a n t i t a t i v e l y transfer to the
alkaline tartrate solution and dilute to the mark and mix.
2.
E D T A s o l u t i o n , 0.005N:
d i s s o l v e 0.9306g EDTA d i h y d r a t e
distilled water and dilute to 1L in a volumetric flask.

in

3.
Murexide indicator:
since murexide solution is not very stable,
the i n d i c a t o r is best prepared in a solid state.
Grind 0.5g
murexide, 0.15g methylene blue and 40g sodium chloride together using
a p e s t l e and m o r t a r .
To avoid c a k i n g , store in a d e s i c c a t o r over
s i l i c a gel.
U s e a p p r o x i m a t e l y O.lg solid indicator for each
titration.
PROCEDURE
D i s s o l v e 5 g of the sugar in 5 ml of cold distilled water in a test
tube.
Add e x a c t l y 2 m l of the alkaline copper solution.
After
thorough mixing place the tube in a boiling water-bath for exactly 5
m i n and then cool i m m e d i a t e l y in a cold w a t e r - b a t h .
Transfer the
s o l u t i o n and w a s h i n g s to a w h i t e e v a p o r a t i n g d i s h and add
approximately O.lg of the indicator.
Titrate the mixture with 0.005N
EDTA solution.
The colour c h a n g e s from green, through grey, to purple which
end-point.
The grey stage appears just before the end-point.
the purple colour is r e a c h e d , it disappears s l o w l y , owing
oxidation of the cuprous oxide at a rate which depends to some
The end-point
on the a m o u n t of cuprous oxide present.
therefore be approached as rapidly as possible.

is the
When
to the
extent
should

CALCULATION
Prepare a calibration curve giving ml 0.005N EDTA solution (abscissa)
against % invert sugar (ordinate).
The curve should be linear up to

0.02g invert sugar/lOOg sucrose. Read the amount of invert sugar

directly from the calibration curve.
REFERENCES
Knight, J. and Allen, C.H. 1960. International Sugar Journal, 62., 344.
ICUMSA, Report of the Proceedings 15th Session, 1970, Subj. 14, Rec. 1
(c), 147.
Emmerich, A., 1967.
Methods, 1979, (1).

Zucker,

603.

93

This

is reproduced

in

ICUMSA

INVERT SUGAR IK WHITE SUGAR

(Berlin Institute Method)
PRINCIPLE
This method
is used for the d e t e r m i n a t i o n of i n v e r t sugar in p r o d u c t s
c o n t a i n i n g not m o r e t h a n 10% i n v e r t s u g a r in the p r e s e n c e of s u c r o s e .
No
d e f e c a t i o n is r e q u i r e d even in the case of d a r k - c o l o u r e d s o l u t i o n s .
To the
s o l u t i o n c o n t a i n i n g i n v e r t s u g a r , Miiller's s o l u t i o n ia added (this c o n t a i n s
sodium carbonate instead of the sodium hydroxide used in Fehling's solution).
T h e m i x t u r e h a s a pH of a b o u t 10.4 ( r e l a t i v e l y l o w in c o m p a r i s o n w i t h o t h e r
m e t h o d s ) and is heated in a boiling water-bath.
Foaming caused by impurities
during direct heating and boiling of the reaction mixture is thus avoided.
The
c u p r i c ions are r e d u c e d by the i n v e r t sugar to c u p r o u s oxide a n d , after
cooling, the liquid is acidified and an excess of standardised iodine solution
added.
W h e n all the c u p r o u s o x i d e has reacted w i t h the i o d i n e , the e x c e s s
iodine is back-titrated with a solution of sodium thiosulphate, using starch as
indicator.
The e x p e r i m e n t a l c o n d i t i o n s , i n c l u d i n g v o l u m e of test s o l u t i o n ,
v o l u m e of Miiller's solution, time of heating and titre of iodine solution, are
strictly standardised.
B e c a u s e the t e m p e r a t u r e is l o w e r than in the other
m e t h o d s and b e c a u s e the pH of the r e a c t i o n m i x t u r e is l o w , the s u c r o s e
correction is small and more closely controlled.
APPARATUS
1.

Erlenmeyer

flasks, 300 ml.

2.
Water-bath,
w i t h v i g o r o u s l y b o i l i n g w a t e r (to e n s u r e that
i m m e r s i o n of f l a s k s d o e s n o t i n t e r r u p t b o i l i n g ) .
The f l a s k s are
p l a c e d in the w a t e r - b a t h so that the w a t e r level is 2 cm above the
surface of the liquid in the flasks.
3.
Burette
graduations.

stand

equipped

with

t w o 50 m l b u r e t t e s ,

with

0.1

ml

REAGENTS
1.
Miiller's solution:
Dissolve 35g cupric sulphate pentahydrate in
about 400 ml of boiling distilled water.
In another beaker, dissolve
173g potassium sodium tartrate tetrahydrate and 68g anhydrous sodium
Cool the two
carbonate in about 500 m l of boiling distilled water.
s o l u t i o n s and pour the s o d i u m c a r b o n a t e s o l u t i o n into the cupric
sulphate solution with stirring.
Dilute the combined solution to 1L.
A f t e r v i g o r o u s s h a k i n g w i t h 2g a c t i v e c a r b o n , filter the s o l u t i o n
through hardened filter paper under vacuum.
If c u p r o u s o x i d e is
precipitated on storage, refilter the solution.
2.

Acetic acid, 5N.

3.
Iodine solution, 0.0333N
than 6g potassium iodide/L).

(this

solution

should

not contain

more

4.
S o d i u m t h i o s u l p h a t e s o l u t i o n , 0 . 0 3 3 3 N ( t h i s s o l u t i o n is
stabilised by the addition of N sodium hydroxide solution, 3 ml/L).
5.
Starch indicator:
D i s s o l v e lg
saturated sodium chloride solution.

soluble

starch

in

100 m l

of

PROCEDURE
Pipette or
of i n v e r t
v o l u m e up
the f l a s k

weigh an amount of sample (cntaining not more than 30 mg

s u g a r ) into an E r l e n m e y e r flask and d i s s o l v e .
M a k e the
to 100 ml.
Add 10 ml of Miiller's solution and mix.
Place
in a b o i l i n g w a t e r - b a t h for 10 m i n (+ 5 sec.).
After

94

h e a t i n g , cool the f l a s k r a p i d l y , w i t h o u t a g i t a t i o n , u n d e r r u n n i n g
w a t e r (a s m a l l b e a k e r b e i n g p l a c e d o v e r t h e m o u t h of the flask).
Acidify the cold solution with 5 m l 5N acetic acid and add an excess
of 0.0333 N i o d i n e s o l u t i o n (20 to 40 m l ) f r o m the b u r e t t e .
Make
both additions without agitation to avoid oxidation of cuprous oxide
M i x the s o l u t i o n .
W h e n the p r e c i p i t a t e h a s c o m p l e t e l y
by air.
d i s s o l v e d , b a c k - t i t r a t e the e x c e s s of i o d i n e w i t h 0.0333N s o d i u m
t h i o s u l p h a t e s o l u t i o n in the p r e s e n c e of a few d r o p s of s t a r c h
indicator which are added as the end-point is approached.
CALCULATION
The d i f f e r e n c e b e t w e e n the v o l u m e of the i o d i n e and t h i o s u l p h a t e
solutions used is the v o l u m e of 0.0333N iodine used in the reaction.
T h i s v a l u e is c o r r e c t e d by s u b t r a c t i o n of the f o l l o w i n g
three
correc t ions :
1.
Blank correction:
the v o l u m e ( m l )
r e q u i r e d in a b l a n k t e s t w i t h d i s t i l l e d
This correction should not exceed
solution.
and should be determined for each n e w batch

of the i o d i n e s o l u t i o n
w a t e r i n s t e a d of s u g a r
0.1 m l for pure reagents
of Miiller's solution.

2.
Cold correction:
the v o l u m e .(ml) of iodine solution required by
the sugar solution after standing 10 m i n at room temperature before
a c i d i f i c a t i o n (this c o r r e c t s for the e f f e c t of r e d u c i n g s u b s t a n c e s
present).
3.
Sucrose correction:
proportionate correction to allow for the
reducing action of the sucrose (0.2 ml iodine solution/g sucrose).
A f t e r t h e s e c o r r e c t i o n s h a v e b e e n m a d e , 1 m l of i o d i n e s o l u t i o n is
equivalent to 1 mg invert sugar.
REFERENCES
S o e n g l e r , 0., T o d t , F. and S c h e u e r , M. 1936. Z. W i r t s c h a f t s gr.
130 and 322.
ICUMSA,

Report

of

the

Proceedings

15th

Session,

95

Z u c k e r i n d . , 86 .

1970, Subj. 14, Rec.

1(b), 147.

CONDUCTIVITY ASH
PRIHCIPLE
T h e s p e c i f i c c o n d u c t i v i t y o f a s u g a r s o l u t i o n o f k n o w n c o n c e n t r a t i o n is
determined.
It is a s s u m e d t h a t t h e c o n d u c t i v i t y h a s i t s o w n s i g n i f i c a n c e and
t h e e q u i v a l e n t a s h is c a l c u l a t e d b y t h e a p p l i c a t i o n o f a c o n s t a n t f a c t o r . T w o
c o n c e n t r a t i o n s m a y b e u s e d , i.e. 2 8 g / 1 0 0 g f o r w h i t e s u g a r a n d o t h e r p r o d u c t s o f
v e r y l o w c o n d u c t i v i t y a n d 5 g / 1 0 0 m l for a l l o t h e r p r o d u c t s .
T h e f a c t o r s used
for t r a n s f o r m i n g m e a s u r e d c o n d u c t i v i t y into ash are p u r e l y c o n v e n t i o n a l and
a p p l i c a b l e o n l y to s u g a r s o l u t i o n s .
APPARATUS
1.

Volumetric

f l a s k s , 1 0 0 m l , 5 0 0 m l and 1 L .

2.

Pipettes, 10ml.

3.
Sugar ash bridge
specifications :
Sugar a s h b r i d g e
indicator):
a.

or null

balance

(a b a a n c e d

bridge

bridge

circuit

the

with

Accuracy of built-in

null

point

e.

Indication:

f.

S c a l e u n i t s : o h m s or s i e m e n s

a n d 0.01 to

_+ 3% o r b e t t e r b u t , f o r l o w
not less
than
0 . 0 0 1 % (0.5

visual.

g.
T e m p e r a t u r e of solution:
m e a s u r e m e n t s h a l l b e 20C.

( = S ) or a s h u n i t s .
the standard

h.
Temperature compensation:
compensating mechanism.
Null balance

sugars

s t a n d a r d s : _+ 1% o r b e t t e r .

d.
Accuracy of measurement:
ash
(conductivity)
values,
PS/cm) .

shall

have

temperature
a

of

temperature

bridge:

a.

Frequency:

b.

R a n g e : 0 to 500 p S / c m .

c.

E l e c t r o d e v o l t a g e : 0 . 2 to 1 0 V .

d.

A c c u r a c y o f b u i l t - i n s t a n d a r d s : _+ 1% o r b e t t e r . .

e.
ash

following

F r e q u e n c y : 50 to 2 0 0 0 H z .

b.
R a n g e : 0.001 to 0.1% ash for w h i t e
0.90% a s h f o r r a w s u g a r s .
c.

with

50 to 2 0 0 0 H z .

A c c u r a c y of m e a s u r e m e n t : + 3% or b e t t e r b u t , f o r l o w
( c o n d u c t i v i t y ) v a l u e s , n o t l e s s t h a n 0.5 p S / c m .

f.

Indication:

visual.

g.

S c a l e u n i t s : o h m s or s i e m e n s

h.

Electrodes:

with

fixed

('"S).

distance.

96

i.

Cell

construction:

of glass

or s y n t h e t i c

j.

T e m p e r a t u r e m e a s u r e m e n t : m e a n s for m e a s u r i n g
ture of the s o l u t i o n to be provided.

k.

Cell c o n s t a n t : w i t h i n

the range

material.

0.2 to 3

the

tempera-

cm-^.

REAGENTS
1.
Purified
water:
twice
distilled
c o n d u c t i v i t y of less than 2 P S / c m .

or

2.
P o t a s s i u m c h l o r i d e , 0.01N: w e i g h 745.5 m g
c h l o r i d e and d i s s o l v e in w a t e r .
M a k e to 1L.
3.
Potassium
s o l u t i o n to 1L.

chloride,
0.0025N:
It has a c o n d u c t i v i t y

de-ionized

with

of d r i e d

potassium

Dilute
2 5 0 m l o f the
of 328 P S / c m at 20C. .

0.01N

4.
P o t a s s i u m c h l o r i d e , 0.0002N:
D i l u t e 10 m l of the 0.01N s o l u t i o n
to 5 0 0 m l .
It h a s a c o n d u c t i v i t y o f 26.6 +_ 0.3 p S / c m at 20C a f t e r
d e d u c t i o n of the specific c o n d u c t i v i t y of the w a t e r used.
PROCEDURE
M e t h o d for 5 g / 1 0 0 m l s o l u t i o n s :
D i s s o l v e 5g of the s a m p l e in w a t e r in
a 1 0 0 m l f l a s k a n d m a k e to v o l u m e at 20C.
In t h e e v e n t o f t h e
or the s o l i d s c o n t e n t o f the
c o n d u c t i v i t y exceeding 500 p S / c m
s o l u t i o n b e i n g less than 5g, w h i t e s u g a r of l o w ash c o n t e n t m u s t be
added in such a w a y as to m a i n t a i n the total solids c o n c e n t r a t i o n at
5g/100ml.
A f t e r t h o r o u g h m i x i n g , t r a n s f e r t h e s o l u t i o n i n t o the
m e a s u r i n g cell and m e a s u r e the c o n d u c t i v i t y at 20 _+ 0.2C.
C h e c k the
m e a s u r e m e n t u s i n g an a p p r o p r i a t e p o t a s s i u m
chloride
reference
solution.
M e t h o d for 2 8 g / 1 0 0 g s o l u t i o n s :
D i s s o l v e 31.3 +_ O.lg of s u g a r in
w a t e r in a 1 0 0 m l v o l u m e t r i c f l a s k and m a k e to v o l u m e at 20C o r
d i s s o l v e 28.0 _+ O.lg of s u g a r in w a t e r to g i v e a s o l u t i o n o f 1 0 0 . O g
weight.
In t h e c a s e o f l i q u i d s ( s y r u p s ) , t h e a m o u n t t a k e n m u s t b e
s u c h t h a t it c o n t a i n s 31.3 or 28.0 g of s o l i d s .
After
thorough
m i x i n g , t r a n s f e r the s o l u t i o n into the m e a s u r i n g cell and m e a s u r e the
c o n d u c t i v i t y at 20 _+ 0.2C.
C h e c k the m e a s u r e m e n t using the 0.0002N
p o t a s s i u m c h l o r i d e solution.
If the d e t e r m i n a t i o n c a n n o t b e m a d e at the s t a n d a r d t e m p e r a t u r e
20C a t e m p e r a t u r e c o r r e c t i o n m a y b e m a d e to t h e f i n a l r e s u l t
f o l l o w s , provided that the range of +5C is not e x c e e d e d :

of
as

F o r t h e m e t h o d at 5 g / 1 0 0 m l , t h e c o r r e c t i o n is 2.3% p e r C (to b e
a d d e d for t e m p e r a t u r e s b e l o w 20C and s u b t r a c t e d for t e m p e r a t u r e s
a b o v e 20C.
F o r the m e t h o d at 2 8 g / 1 0 0 g , the c o r r e c t i o n is 2.6% p e r C (to b e
a d d e d f o r t e m p e r a t u r e s b e l o w 20C and s u b t r a c t e d f o r t e m p e r a t u r e s
a b o v e 20C).

CALCULATIONS
For

the m e t h o d
C = M -

at

5g/100ml:

0.9W

97

where:

C = corrected conductivity (in yS/cm)

M = measured conductivity (in yS/cm) at 20C
W specific conductivity (in yS/cm) of water at
20 C.

% conductivity ash = 0.0018C

(Note:
Any w h i t e sugar added in the s a m p l e
preparation must be taken into account.)

solution

For the method at 28g/100g:

C = M - 0.35W
where:
X

C, M and W are as above.

conductivity ash = 0.0006C

REFERENCES
I C U M S A , Report of the P r o c e e d i n g s 15th Session, 1970, Subj. 16, Rec. 1 and 5,
171.
ICUMSA,

Report of the Proceedings 16th Session, 1974, Subj. 16, Rec. 2,3 and 4,

222.
(Note:

This method

is only tentative for molasses.)

98

L O S S OM

DRYIMG

PRINCIPLE
There is a considerable difference of opinion on t e r m i n o l o g y in sugar analysis.
Loss of w e i g h t m e a s u r e m e n t s have been r e c o m m e n d e d for the d e t e r m i n a t i o n of w h a t
has variously been referred to as water content, m o i s t u r e content, total solids
content, dry substance content, dry m a t t e r content, loss of weight on drying,
loss on drying, etc.
In the light of current technological k n o w l e d g e , the use
of s u c h t e r m s as ' m o i s t u r e c o n t e n t ' and ' w a t e r c o n t e n t ' in t h i s c o n t e x t is
erroneous.
The C o m m i t t e e on Sugars of the Codex A l i m e n t a r i u s C o m m i s s i o n has
agreed upon the suitability of the term 'loss on drying' and this term has also
b e e n a d o p t e d by the E E C a u t h o r i t i e s .
This method measures m a i n l y 'surface
m o i s t u r e ' by air oven drying w i t h uniform conditions for cooling.
APPARATUS
1.
Forced draught oven maintained at a t e m p e r a t u r e of 105
1C as
m e a s u r e d 2.5
0.5cm above the dishes in the test.
The oven is to be
v e n t i l a t e d and the c i r c u l a t i o n fan f i t t e d w i t h an i n t e r l o c k s w i t c h
w h i c h opens w h e n the oven door is opened.
2.

Desiccator

containing

self-indicating

silica

gel.

3.
Dishes w i t h tight-fitting lids.
These should have a d i a m e t e r of
6 to 10 cm and a d e p t h of 2 to 3 c m .
A l t h o u g h they m a y b e m a d e of
glass, platinum or nickel, a l u m i n i u m is r e c o m m e n d e d .
The thickness
of the d i s h e s is o p t i o n a l , e x c e p t that due r e g a r d s h o u l d be paid to
the w e i g h t of the dish in relation to the weight of the sample and to
the loss to be determined.
4.

Surface pattern dial

thermometer.

PROCEDURE
P r e h e a t the o v e n to 105C.
P l a c e the e m p t y d i s h e s , w i t h l i d s o p e n ,
in t h e o v e n for n o t l e s s t h a n 30 m i n .
R e m o v e the d i s h e s f r o m the
o v e n , r e p l a c e the l i d s and p l a c e in the d e s i c c a t o r .
The c o n t a c t
t h e r m o m e t e r is p l a c e d on top of o n e of the d i s h e s .
When
the
t e m p e r a t u r e of the d i s h e s h a s f a l l e n to a m b i e n t + 5C, w e i g h as
r a p i d l y as p o s s i b l e to an a c c u r a c y of j^O . 1 m g.
As rapidly
as
p o s s i b l e , p l a c e 20 to 30g of the s a m p l e in e a c h d i s h , r e p l a c e the
l i d , and w e i g h to an a c c u r a c y of j^O.lmg.
(The d e p t h of s u g a r in the
dish m u s t not exceed 1 cm).
R e t u r n the d i s h e s , w i t h the l i d s o p e n , to the o v e n . D r y for 3 h o u r s
exactly.
T h e r e m u s t be no o t h e r m a t e r i a l s in the o v e n d u r i n g the
drying period.
Replace the lids and r e m o v e the dishes from the oven
and replace in the desiccator w i t h the contact t h e r m o m e t e r on one of
them.
Cool the dishes until the t h e r m o m e t e r indicates a t e m p e r a t u r e
of a m b i e n t + 5C.
W e i g h to an a c c u r a c y of
O.lmg.
(No a t t e m p t
s h o u l d be m a d e to d r y to c o n s t a n t w e i g h t and c a r e m u s t be t a k e n to
ensure that there is no physical loss of sugar at any stage.)
CALCULATIOH
Loss in weight
the sample:

is expressed

as a percentage

IOO(W2-W3)
Loss on drying =
w

99

W l

of the original w e i g h t

of

where:

W^ weight of dish.
W 2 " weight of dish + sugar before drying and
W j = weight of dish + sugar after drying.

Duplicate results are acceptable if neither is outside the limits of

_+ 10% of the m e a n v a l u e for the test.
Tests in w h i c h either
duplicate exceeds this limit should be repeated.

REFERENCES
I C U M S A , Report

of the P r o c e e d i n g s 16th Session, 1974, Subj. 19, Rec. 2.

268.
ICUMSA, Report of the Proceedings 16th Session, 1974, Subj. 19, 263.
ICUMSA, Report of the Proceedings 16th Session, 1974, Subj. 19, 252.

100

COLOUK

INDEX

PRINCIPLE
The a b s o r b a n c e of a filtered s o l u t i o n of the s a m p l e is d e t e r m i n e d at 420 nm
(560 n m for d a r k e r c o l o u r e d p r o d u c t s ) u s i n g a s p e c t r o p h o t o m e t e r and the
absorbance index is calculated and converted to ICUSMA colour units.
APPARATUS
1.

Spectrophotometer with 10 cm absorption

2.

Membrane

cells.

filters, 50 mm with poresize of 0.45 y .

PROCEDURE
P r e p a r e a s o l u t i o n of the sugar to be tested using d i s t i l l e d
Use the following concentrations:
White

sugar

Darker

water.

50g/100g

sugar

A s high
as
practicable,
c o n s i s t e n t w i t h reasonable
f i l t r a t i o n r a t e s and
cell
depths
Original density,
unless
dilution is required to obtain
reasonable filtration rates or
cell depths

Liquors, syrups
and juices

F i l t e r the s o l u t i o n u n d e r v a c u u m .
W h i t e sugar s o l u t i o n s and light
coloured liquors should be filtered through a membrane filter of pore
Darker
size 0.45 p a c c o r d i n g to the m e r c u r y e x t r u s i o n m e t h o d .
s o l u t i o n s should be f i l t e r e d w i t h a n a l y t i c a l g r a d e K i e s e l g u h r (1
p e r c e n t on s o l i d s ) o v e r f i l t e r p a p e r .
The first p o r t i o n of the
f i l t r a t e should be d i s c a r d e d if c l o u d y .
A d j u s t the pH of d a r k e r
solutions to 7.0
0.2 with dilute HC1 or NaOH.
Do not adjust the pH
of white sugar solutions.
Remove entrained air under vacuum, or in
an ultrasonic bath (take care to m i n i m i s e evaporation).
P l a c e the s o l u t i o n in a 10-cm a b s o r p t i o n cell (the cell length is
c h o s e n so that the i n s t r u m e n t r e a d i n g w i l l be b e t w e e n 20 and 80
percent transmittancy).
Determine the absorbance of the solution at
420 n m (or 560 n m ) in a s p e c t r o p h o t o m e t e r or e q u i v a l e n t , u s i n g
distilled water as a reference.
CALCULATION

A
Absorbance

Where:

Absorbance

index

-log T
=

A
T
b

=
=
=

absorbance
transmit tance
cell length in cm

concentration of total solids in g/ml

index x 1000 = 'colour' in ICUSMA units.

101

REFERENCES
ICUMSA, Report of the Proceedings 16th Session, 1974, Subj. 22, Rec. 1 and
2, 303.
I C U M S A , Report of the P r o c e e d i n g s 15th Session, 1970, Subj. 22, Appendix
5, 255.
ICUMSA,

Report of the Proceedings 14th Session, 1966, Subj. 22, 129.

102

3.2

SUGAR

PRODUCTS

COMPOSITION

CAC Standards

for reducing

sugar products

are:

Powdered
Dextrose
Dextrose
Dextrose
(Anhydrous) (Monohyd.) (icing)(a)
d-glucose content
(or equivalent)(min)

99. 5%

99. 5%

Total solids

98. 0%

90. 0%

Sulphated

(min)

ash

0. 25%

Sulphur dioxide
Arsenic
Copper
Lead

(ppm)

0. 25%

20%

20%

70%

93.0%

1.0%

1.0%

40(d)

40(e)

20

20

20

(ppm)
(ppm)

(ppm)

(Note:

0. 25%

99. 5%

Dried
Glucose
Glucose
Syrup(b) Syrup(c)

All values are maxima unless otherwise

noted.)

(a)

Icing dextrose may contain any of the following, singly or in c o m b i n a t i o n

up to a m a x i m u m of 1.5%: Tribasic calcium phosphate, m a g n e s i u m carbonate,
m a g n e s i u m s t e a r a t e , a m o r p h o u s silicon d i o x i d e (dehydrated silica gel),
calcium silicate, m a g n e s i u m trisilicate, sodium calcium aluminosilicate.

(b)

D e f i n e d as a p u r i f i e d
saccharides obtained from

(c)

Defined

(d)

In g l u c o s e s y r u p u s e d o n l y for m a n u f a c t u r e of s u g a r c o n f e c t i o n e r y ,
limit is 400 ppm.

(e)

When used
ppm.

ROUTINE

as glucose

concentrated
starch.

syrup from

in the m a n u f a c t u r e

which

aqueous

solution

of

the water has been partially

nutritive

removed.
the

of sugar confectionery only, the limit is 150

ANALYSIS

Glucose syrup or corn syrup is also called liquid glucose, which is a term that
s h o u l d be d i s c o u r a g e d .
It is m a d e by the h y d r o l y s i s of s t a r c h , w h i c h is
frequently obtained from maize (corn), hence the term corn syrup.
It consists
of dextrose, m a l t o s e and higher saccharides.
It is usually sold on the basis
of its d e x t r o s e e q u i v a l e n t and d e n s i t y in d e g r e e s B a u m e .
The
dextrose
e q u i v a l e n t is d e f i n e d as the p e r c e n t a g e of r e d u c i n g s u g a r s in the s y r u p , as
determined by Lane and Eynon titration or another standard method, calculated
as dextrose and expressed as a percentage of the total solids.
Degrees B a u m e
is d e f i n e d as the r a t i o of the t o t a l v o l u m e d i s p l a c e d
in w a t e r by the
h y d r o m e t e r and the v o l u m e displaced by the unit scale length of the h y d r o m e t e r
stem.
It is related to true specific gravity by
0

Be

, _
-

145

T R U E

S G

145
15.5C/15.5C

For a p r o d u c t of a n y p a r t i c u l a r d e x t r o s e e q u i v a l e n t , the t o t a l s o l i d s in the

sample m a y be deduced from degrees B a u m e ' b y use of a table.

103

T r e a c l e , m o l a s s e s and g o l d e n s y r u p are d e r i v e d from the u n c r y s t a l 1 i s a b 1 e

r e s i d u e l e f t o v e r a f t e r r e m o v a l of s u c r o s e d u r i n g the p r o c e s s i n g of c a n e and
b e e t sugar.
I n v e r t s u g a r is o b t a i n e d b y the h y d r o l y s i s ( i n v e r s i o n ) of s u c r o s e and t h e r e f o r e
contains equimolecular proportions of g l u c o s e ( d e x t r o s e ) and
fructose
(laevulo8e).
T h e r e a r e n o w e n z y m e s a v a i l a b l e c o m m e r c i a l l y w h i c h i s o m e r i s e the
g l u c o s e to t h e s w e e t e r f r u c t o s e .
T h e t o t a l s o l i d s o f s y r u p s a r e u s u a l l y d e t e r m i n e d b y d r y i n g a t 70C a n d a
v a c u u m o f a b o u t 2 5 m m H g . T h e t o t a l s o l i d s (X m / m ) m a y a l s o b e o b t a i n e d f r o m
t h e r e f r a c t i v e i n d e x a t 20C a n d u s e o f a t a b l e . C o r r e c t f o r t h e p r e s e n c e o f
i n v e r t s u g a r ( i f t h e s y r u p i s a o t a s u c r o s e s y r u p ) b y a d d i n g 0 . 0 2 2 to t h e R . I .
for e v e r y 1 p e r c e n t invert sugar p r e s e n t .
If c r y s t a l s a r e p r e s e n t , d i l u t e w i t h
an e q u a l w e i g h t of w a t e r .
R e d u c i n g sugars are u s u a l l y d e t e r m i n e d by a copper
r e d u c t i o n m e t h o d s u c h as the L a n e and E y n o n p r o c e d u r e and c a l c u l a t e d as i n v e r t
s u g a r or as the d e x t r o s e e q u i v a l e n t .
T h e r e a r e s e v e r a l q u a l i t a t i v e c h e m i c a l t e s t s for s u g a r s , s u c h as F e h l i n g s ,
B a r f o e d ' s or t h e r e a c t i o n w i t h a n t h r o n e .
TLC does not take m u c h m o r e operator
t i m e and i d e n t i f i e s t h e s u g a r s p r e s e n t .
T h i s s t e p is u s e f u l as it e n a b l e s the
a n a l y s t to c h o o s e t h e a p p r o p r i a t e m e t h o d for t h e q u a n t i t a t i v e a n a l y s e s of
m i x t u r e s of s u g a r s s u c h as p o l a r i m e t r y , g r a v i m e t r y or t i t r i m e t r y or a
c o m b i n a t i o n o f t h e m . T h e s e r e s u l t s m a y b e u s e d to c a l c u l a t e t h e p r o p o r t i o n s o f
k n o w n m i x t u r e s of s u g a r s b y u s e of s i m u l t a n e o u s e q u a t i o n s .
T h e a n a l y s i s o f s u g a r s in food i n v o l v e s t h r e e s t e p s :
r e m o v a l of i n t e r f e r e n c e s ,
h y d r o l y s i s ( f o r a l l b u t r e d u c i n g s u g a r s ) and f i n a l e s t i m a t i o n .
Interferences
a r e u s u a l l y r e m o v e d e i t h e r b y e x t r a c t i o n o f food w i t h 80 p e r c e n t a l c o h o l or b y
p r e c i p i t a t i o n of p r o t e i n and o t h e r m a t e r i a l w i t h c l e a r i n g ( d e f e c a t i n g ) a g e n t s
or b o t h . E x t r a c t i o n w i t h 80 p e r c e n t e t h a n o l or 1 - p r o p a n o l d i s s o l v e s s u g a r s ,
amino
acids,
s a l t s , o r g a n i c a c i d s and other s m a l l m o l e c u l e s ,
leaving
p o l y s a c c h a r i d e s and proteins u n d i s s o l v e d .
T h i s is t h e m e t h o d o f C h o i c e f o r
p r o d u c t s c o n t a i n i n g s t a r c h a n d is n e c e s s a r y for t h e d e t e r m i n a t i o n of s u g a r s in
fruit and v e g e t a b l e products that c o n t a i n p o l y s a c c h a r i d e s p r e c i p i t a b l e b y or
i n s o l u b l e in 8 0 p e r c e n t e t h a n o l .
O r g a n i c a c i d s and a m i n o a c i d s , c o l o u r and
o t h e r s m a l l m o l e c u l e s are then r e m o v e d by c l a r i f i c a t i o n w i t h n e u t r a l lead
a c e t a t e or b y i o n - e x c h a n g e .
O t h e r c l a r i f y i n g a g e n t s s u c h as a l u m i n a c r e a m ,
b a s i c lead a c e t a t e , activated c h a r c o a l , s o d i u m t u n g s t a t e and p h o s p h o t u n g s t i c
a c i d a r e s o m e t i m e s u s e d ; b a s i c lead a c e t a t e and c h a r c o a l t e n d to a b s o r b s u g a r s .
Z i n c f e r r o c y a n i d e is a v e r y g o o d c l e a r i n g a g e n t , p r e c i p i t a t i n g p r o t e i n ( w h i c h
t a k e s d o w n m o d e r a t e a m o u n t s o f f a t w i t h i t ) a n d is t h e a g e n t o f c h o i c e f o r
dairy products.
F a t c a n a l s o b e r e m o v e d , if n e c e s s a r y , b y p r i o r e x t r a c t i o n
with petroleum ether.
T h e c l a r i f i e d s o l u t i o n is r e a d y f o r d e t e r m i n a t i o n o f r e d u c i n g s u g a r s if
p r e s e n t . N o n - r e d u c i n g s u g a r s m u s t f i r s t be h y d r o l y s e d w i t h a c i d or e n z y m e s .
T h e c o n d i t i o n s o f h y d r o l y s i s m u s t b e a d h e r e d to s t r i c t l y o t h e r w i s e i n c o m p l e t e
h y d r o l y s i s or t h e f u r t h e r b r e a k d o w n of the s u g a r s m a y r e s u l t .
If t h e f i n a l
d e t e r m i n a t i o n is b y p o l a r i m e t e r , t h e c o n d i t i o n s o f h y d r o l y s i s a f f e c t t h e
e q u a t i o n to b e u s e d to c a l c u l a t e t h e r e s u l t .
The e s t i m a t i o n on the p r e p a r e d s o l u t i o n m a y be c a r r i e d out by e n z y m i c
p r o c e d u r e s , w h i c h a r e s p e c i f i c , as w e l l as b y p o l a r i m e t r y or r e d u c t i o n of
F e h l i n g s s o l u t i o n , w i t h d e t e r m i n a t i o n of the e n d - p o i n t b y s u b s e q u e n t w e i g h i n g
of the p r e c i p i t a t e d c u p r o u s o x i d e or b y t i t r a t i o n .
M a n y other m e t h o d s are
a v a i l a b l e , b u t the a b o v e a r e p r o b a b l y t h e o n e s m o s t c o m m o n l y u s e d in the food
laboratory.
Sugars m a y also be separated by c o l u m n c h r o m a t o g r a p h y
and
d e t e r m i n e d b y o n e of t h e a b o v e t e c h n i q u e s , or d e t e r m i n e d b y GLC ( u s u a l l y as the
t r i m e t h y s i l y l d e r i v a t i v e s ) , or b y liquid c h r o m a t o g r a p h y .

104

SAMPLE PREPARATION - SUGARS

(Alcohol Extraction)
PRINCIPLE
The sample or filtered sample is mixed with ethanol so that the final
concentration of the latter (taking into account the moisture in the sample) is
80 % v/v. Chalk is added to neutralize acidity. The mixture is boiled on the
water bath, diluted to volume, filtered and the filtrate evaporated to remove
alcohol.
Any enzymes present are inactivated by the alcohol.
REAGENTS
1.

Alcohol, 95 %.

2.

Calcium carbonate, precipitated.

PROCEDURE
Add the ground or finely chopped and weighed sample to hot alcohol to
which enough precipitated chalk has been added to neutralize acidity,
using enough alcohol so that the final concentration, allowing for
water in the sample, is 80 % v/v.
Heat nearly to boiling on a
waterbath for 1 hour, stirring frequently. The solution may be kept
at this stage if it is not convenient to proceed immediately with the
analysis. Decant the solution into a volumetric flask and mix the
solids in a high-speed blender with 80 percent alcohol.
Boil the
blended material on a waterbath for 30 minutes, cool, transfer to the
volumetric flask and dilute to volume with 80 Z v/v ethanol at 20C.
Filter and evaporate an aliquot of the filtrate.
Add water as
necessary to prevent the solution evaporating to dryness.
When the
odour of alcohol disappears, add about 100 ml of water and heat to
80C to soften g u m m y precipitates and break up insoluble masses.
Filter through a thin mat of Celite on a filter paper in a Buchner
funnel previously washed with water until the washings are clear.
After passing the sample solution through the Celite, wash the latter
with water and dilute the combined filtrate and washings to a
suitable volume or use the entire filtrate, which should not be much
in excess of 50 ml.
The solution is n o w ready for clarification by lead acetate or for
hydrolysis and final determination if clarification is not necessary.
If it is necessary to determine starch in the product, boil the
sample with 80 % alcohol initially only for 30 m i n u t e s , filter
through a paper or extraction thimble, collecting the filtrate in a
volumetric flask. Return any insoluble material to the beaker, boil
an hour with 80 percent alcohol and filter through the same paper or
thimble.
If the extract is highly coloured, repeat a third time.
Finally transfer the residue to the paper or thimble, leave to drain
and then dry. Grind the residue to pass a 1 mm sieve, place thimble
(or paper inside a thimble) in a Soxhlet extractor and extract 12
h o u r s w i t h 80 % a l c o h o l .
The c o m b i n e d a l c o h o l e x t r a c t s are
evaporated as above and the residue may be used for the determination
of starch.

105

SAMPLE PREPARATION - SUGARS

(Clarification)
PRINCIPLE
A solution or extract of the sample, free of polysaccharides, is treated with
n e u t r a l lead a c e t a t e , r e m o v i n g c o l o u r , organic acids, a m i n o acids and other
small ionic molecules. Excess lead acetate is removed with oxalate.
REAGENTS
1.

Neutral

lead acetate, saturated

solution in water.

2.

Sodium or potassium oxalate crystals.

PROCEDURE
Weigh a quantity of sample appropriate for the final determination
into a 250 m l v o l u m e t r i c flask.
For e x a m p l e , the Lane and Eynon
titration using 10 ml of mixed Fehling solution requires a quantity
of s a m p l e c o n t a i n i n g 0.15 - 0.75 g of reducing sugars, the B e r l i n
m e t h o d r e q u i r e s 0.01 - 0.08 g of reducing sugars in 250 ml and the
method of Somogyi requires 0.005 - 0.003 g in the same volume.
If it
was necessary to extract the sample with 80 percent alcohol, transfer
to the 250 m l v o l u m e t r i c flask the aqueous extract of the s a m p l e
after all alcohol has been removed by evaporation.
Dilute to 100 - 120 ml with distilled water and add by pipette enough
saturated n e u t r a l lead acetate solution to produce a flocculent
p r e c i p i t a t e , shake t h o r o u g h l y and leave to stand 15 m i n u t e s .
Test
tne s u p e r n a t e w i t h a few drops of lead acetate solution and if a
p r e c i p i t a t e f o r m s shake and leave to stand again.
If no further
precipitate forms, dilute to the mark with water, mix thoroughly and
filter t h r o u g h a dry paper. Add enough solid sodium oxalate to the
filtrate to p r e c i p i t a t e all the lead and re-filter through a dry
paper.
Check for absence of lead in the filtrate by adding a little
sodium oxalate.
Alternatively, add just enough lead acetate to the solution to cause
complete precipitation.
This point is reached when a drop of dilute
sodium oxalate added to the supernate gives a precipitate.
Then add
the same volume of lead acetate solution again. Thoroughly shake and
let the m i x t u r e stand a few m i n u t e s , then filter into a beaker
containing an estimated excess of sodium oxalate crystals.
Wash the
filter until the filtrate no longer gives a p r e c i p i t a t e w i t h the
oxalate.
Check that oxalate is present in excess by adding one drop
of lead acetate solution.
Filter off and w a s h the lead oxalate
p r e c i p i t a t e , c o l l e c t i n g the filtrate and w a s h i n g s in a v o l u m e t r i c
flask. D i l u t e to the m a r k w i t h water and mix. The solution is n o w
ready for hydrolysis or final estimation of reducing sugars.
REFERENCE
Official Methods of Analysis of the AOAC, 1984, 31.021(d).

106

SUGARS IDENTIFICATION
(Thin-Layer Chromatography)
PRINCIPLE
The sugars are extracted into water
thin-layer chromatography.

solution and

separated

and identified

using

APPARATUS

1.

Large shallow Pyrex

tray.

2.

TLC

3.

TLC plates - silica gel 60.

tank.

REAGENTS
1.

0.3N N a H 2 P 0 4 ,

(12 g/L)

2.
Sugar standard solutions:
1% a q u e o u s
glucose, lactose, m a l t o s e and fructose.
3.
Developing
0.3N N a H 2 P 0 4 .

solvent:

40 m l

Butan-l-ol

solutions

+ 50 m l

4.
S p r a y r e a g e n t A: d i s s o l v e 2g d i p h e n y 1 a m i n e
acetone and m a k e up to 100 ml.
5.

Spray reagent

6.

Zinc acetate

7.

Potassium

B:

orthophosphoric

of

acetone

sucrose,

+ 10

ml

and 2g a n i l i n e

in

acid, 85%.

solution, 21.6% w/v.

ferrocyanide

solution, 10.6% w/v.

PROCEDURE
L i n e the tank w i t h c h r o m a t o g r a p h y p a p e r and pour in the d e v e l o p i n g
solvent.
R e p l a c e the lid and a l l o w a t m o s p h e r e in tank to s a t u r a t e
for a b o u t 30 m i n u t e s .
Soak the TLC p l a t e in 0.3 N N a H 2 P 0 4 s o l u t i o n
Remove and dry in an oven at 100C
in the Pyrex tray for one minute.
for 30 m i n u t e s .
M e a n w h i l e take a q u a n t i t y of s a m p l e in a 50 m l
beaker and add 8 m l water and 1 m l each of zinc acetate solution and
potassium ferrocyanide solution.
M i x w e l l and f i l t e r .
Spot the
filtrate onto the prepared plate along with the standards (this can
be done quantitatively with a micro-pipette or qualitatively with an
extruded Pasteur pipette).
Place the plate in the tank and allow to
develop for 10 cm.
Remove and dry in the oven for one minute.
Take
20 m l s p r a y r e a g e n t A in a b e a k e r and add r e a g e n t B d r o p w i s e .
The
m i x t u r e g o e s c l o u d y and then s l o w l y c l e a r s .
S p r a y the d r i e d p l a t e
w i t h the c l e a r s p r a y r e a g e n t in a f u m e c u p b o a r d and d r y in the o v e n
for 1 - 2 m i n u t e s .
T h e s u g a r s s h o w up as b l u e or b r o w n s p o t s .
Each
s u g a r is a s l i g h t l y d i f f e r e n t c o l o u r and a s l i g h t l y d i f f e r e n t R j
value.
If the colours are faint spray again and dry.
B e s t s e p a r a t i o n is o b t a i n e d b y u s i n g the s m a l l e s t p o s s i b l e
(less than 1 m m ) of a fairly concentrated solution (2-10%).

spots

INTERPRETATION
T h i s m e t h o d w i l l d i s t i n g u i s h d e x t r o s e from g l u c o s e s y r u p (corn s y r u p ) , the
latter showing the presence of various higher saccharides and a streak near the
baseline.
G l u c o s e s y r u p g i v e s an e x c e l l e n t s e r i e s of s p o t s on u n b u f f e r e d
silica gel using propanol:ethyl ace ta te : water as solvent.

107

SUGARS ANALYSIS
(Gas Chromatography)
PRINCIPLE
The sugars are extracted and silyl derivatives formed.
are separated and quantitated using gas chromatography.

The derivatized

sugars

APPARATUS
1.

Reactivial 3 ml with magnetic

2.

100 ml volumetric flasks, and beakers.

3.

Thermostatically controlled water bath.

4.

Drying oven at 80C.

5.

Vacuum oven at 70C.

6.

Ultrasonic bath.

7.

Magnetic

stirrer/hot

stirring

elements.

plate.

8.
Gas c h r o m a t o g r a p h w i t h 3% O V - 1 7 c o l u m n and flame
detector.
Operating conditions are typically:
Nitrogen carrier gas
Hydrogen
Air
Column temperature

ionization

30 ml/min
30 ml/min
300 ml/min
180C

REAGEHTS
1.

Standard

sugar samples:

2.

Internal standard:

D(-) Fructose
D(+) Glucose anhydrous
Sucrose
D(+) Lactose monohydrate
Maltose monohydrate

meso-inositol.

3.
Oximation reagent:
dissolve
in pyridine and make up to 50 ml.
4.

Clearing

agents:

5.

Silylating

6.

Propan-2-ol

1.25g of Hydroxyammonium

Carrez I (potassium fer rocyanide

Carrez II (zinc acetate 21.6%)

chloride

10.6%)

agent: bis(trimethylsilyl)-tri fluoroacetamide

(BSTFA)

PROCEDURE
A c c u r a t e l y w e i g h about 1 g of s a m p l e and 0.3 g of internal standard
into a 100 ml beaker. Add 30 ml of hot distilled water and leave for
15 m i n u t e s in a hot w a t e r bath at 80C to dissolve.
Add 0.5 ml
C a r r e z I and 0.5 ml Carrez II.
Filter into a 100 m l v o l u m e t r i c
flask, rinse and make up to volume with distilled water.
Prepare a
s o l u t i o n c o n t a i n i n g each of the r e f e r e n c e sugars and internal
standard (0.5 g of each sugar is a convenient amount), following the
same procedure.

108

P i p e t t e 0.5 m l of the s o l u t i o n into a 3 m l R e a c t i v i a l c o n t a i n i n g a

magnet.
E v a p o r a t e on a m a g n e t i c s t i r r e r / h o t p l a t e set on v e r y l o w
heat under a gentle stream of nitrogen.
W h e n nearly dry, add 0.5 m l
p r o p a n - 2 - o l and c o n t i n u e e v a p o r a t i o n .
P l a c e the v i a l s in a v a c u u m
o v e n at 70C for 1.5 h o u r s .
C o o l and add 0.5 m l of o x i m a t i o n
reagent.
S t o p p e r t i g h t l y and p l a c e in an u l t r a s o n i c b a t h for t w o
m i n u t e s to l o o s e n m a g n e t from the s i d e s of the v i a l .
H e a t for 30
m i n u t e s at 80C in an o v e n ( t i m e and t e m p e r a t u r e m u s t be p r e c i s e l y
controlled).
C o o l at r o o m t e m p e r a t u r e .
Add 1 m l B S T F A , s t o p p e r
t i g h t l y and r e p l a c e in the o v e n for a f u r t h e r 30 m i n u t e s .
Cool at
room temperature.
I n j e c t 1 p 1 of the s o l u t i o n into a gas c h r o m a t o g r a p h , u s i n g the
above operating conditions.
C a l c u l a t e the s u g a r s b y c o m p a r i s o n of
sample and standard c h r o m a t o g r a m s .
CALCULATION
If time and temperature of derivatisation are carefully controlled,
the f a c t o r s c a n be c a l c u l a t e d o n c e f r o m a s e r i e s of
standard
solutions.
Example:

Ksu

Calculation of the Sucrose

weight of Sucrose
weight of m-ino8itol

Weight of Sucrose

(Ksu)

Peak area
Peak area

m-inositol
Sucrose

(mg)

Ksu x mg of m-inositol

X Sucrose

factor

added

Peak area Sucrose

Peak area m-inositol

wt of Sucrose (mg) x 100

wt of sample (mg)

109

INVERT SUGARS WITH ADDED SUCROSE

(Lane and Eynon Method)
PRINCIPLE
T h e s a m p l e s o l u t i o n is m i x e d w i t h a k n o w n v o l u m e of F e h l i n g ' s s o l u t i o n and
water.
A f t e r b r i n g i n g to a b o i l , m e t h y l e n e b l u e i n d i c a t o r is a d d e d a n d t h e
s o l u t i o n is t i t r a t e d w i t h a s t a n d a r d i n v e r t s u g a r s o l u t i o n .
T h e final v o l u m e
is k e p t c o n s t a n t .
T h e s a m p l e i n v e r t s u g a r is c a l c u l a t e d b y d i f f e r e n c e . T h e
t i t r a t i o n is c o n d u c t e d in a b o i l i n g s o l u t i o n in o r d e r to e l i m i n a t e a i r w h i c h
would change the end-point.
The experimental conditions must be strictly
f o l l o w e d to g a i n r e p r o d u c i b l e r e s u l t s . A s t h e f i n a l v o l u m e is k e p t c o n s t a n t b y
the a d d i t i o n o f a p r e d e t e r m i n e d a m o u n t of w a t e r , the t i t r a t i o n
always
c o r r e s p o n d s to t h e s a m e a m o u n t o f i n v e r t s u g a r a n d a l l o w s t h e u s e of a s i n g l e
f o r m u l a instead of tables.
If s u c r o s e is p r e s e n t , t h e r e s u l t m u s t b e
multiplied by a sucrose correction factor.
APPARATUS
1.
Heat-resistant
capacity.

glass

flat-bottomed

flasks

of 3 0 0 to 4 0 0 m l

2.
B u r e t t e , 5 0 m l , g r a d u a t e d in 0.1 m l f o r t h e s u g a r s o l u t i o n . T h e
burette should have a pinch-cock instead of a glass tap and a bent
o u t l e t t u b e in o r d e r to k e e p t h e g r a d u a t e d s e c t i o n o f t h e b u r e t t e o u t
o f t h e s t e a m w h i l e a d d i t i o n s a r e m a d e to t h e b o i l i n g m i x t u r e .
3.

P i p e t t e s , 1 0 , 1 5 , 2 0 , 25 a n d 5 0 m l , c l a s s A .

REAGENTS
1.
Fehling's solution (Soxhlet's modification): This solution does
n o t k e e p i n d e f i n i t e l y a n d t h e r e a g e n t s a r e t h e r e f o r e d i s s o l v e d in t w o
separate solutions, A and B, which are mixed together i m m e d i a t e l y
b e f o r e u s e . T h i s m i x i n g is d o n e b y a d d i n g a v o l u m e o f s o l u t i o n A to
a n e x a c t l y e q u a l v o l u m e o f s o l u t i o n B . It is e s s e n t i a l t h a t t h e
m i x i n g b e c a r r i e d o u t in this o r d e r , o t h e r w i s e the p r e c i p i t a t e of
cupric hydroxide initially formed m a y not redissolve completely.
Solution A: cupric sulphate pentahydrate
a n d d i l u t e d to 1 0 0 0 m l in d i s t i l l e d w a t e r .

(69.28

g ) is

dissolved

S o l u t i o n B : s o d i u m p o t a s s i u m t a r t r a t e t e t r a h y d r a t e (346 g ) a n d s o d i u m
h y d r o x i d e ( 1 0 0 g ) a r e d i s s o l v e d in d i s t i l l e d w a t e r a n d d i l u t e d to
1000 m l .
2.

Invert
sugar
stock
prepared as f o l l o w s :

solution

- 1

invert

sugar

100 ml

D i s s o l v e 2 3 . 7 5 0 g p u r e s u c r o s e in a b o u t 1 2 0 m l d i s t i l l e d w a t e r , a d d 9
ml concentrated hydrochloric
acid a n d a l l o w to s t a n d at r o o m
t e m p e r a t u r e f o r 8 d a y s . M a k e u p t h e s o l u t i o n to 2 5 0 m l a n d c h e c k f o r
c o m p l e t i o n o f h y d r o l y s i s b y a s a c c h a r i m e t e r r e a d i n g ( - 1 1.80 + 0 . 0 5 5
a t 20C). D i l u t e 2 0 0 m l o f t h e 10 p e r c e n t s o l u t i o n o f i n v e r t s u g a r
thus obtained and a d d , with swirling, sufficient N sodium hydroxide
( a b o u t 7 1 . 5 m l ) so t h a t t h e s o l u t i o n , if d i l u t e d to 2 0 0 0 m l , w o u l d
h a v e a n a c i d i t y o f a b o u t 0 . 0 0 1 N w i t h r e s p e c t to h y d r o c h l o r i c a c i d .
A f t e r t h i s a d d i t i o n , a d d a s o l u t i o n of 4 g b e n z o i c a c i d in w a r m
w a t e r , c o o l t h e w h o l e a n d m a k e u p to 2000 m l to g i v e a 1 % s o l u t i o n
o f i n v e r t s u g a r i n a 0.2 % s o l u t i o n o f b e n z o i c a c i d .
This stable
stock solution should be diluted i m m e d i a t e l y before use.

110

3.
Standard invert sugar s o l u t i o n ,
0.25 g / 1 0 0 ml:
P i p e t t e 25 ml
of the stock s o l u t i o n to a 100 ml v o l u m e t r i c f l a s k and d i l u t e to mark
with water.
4.
Methylene
blue
indicator,
1 g/100
ml:
dissolve
1 g
m e t h y l e n e b l u e and d i l u t e to 100 ml u s i n g d i s t i l l e d w a t e r , and
filter.

pure
then

PROCEDURE
T h e F e h l i n g s s o l u t i o n m u s t be s t a n d a r d i z e d as f o l l o w s :
Add 15 ml
w a t e r a n d 39 ml o f t h e s t a n d a r d i n v e r t s u g a r s o l u t i o n to 20 ml o f
Fehlings.
T i t r a t e as b e l o w f o r s a m p l e , u s i n g the s t a n d a r d i n v e r t
sugar s o l u t i o n .
The t o t a l v o l u m e of the s t a n d a r d i n v e r t
sugar
s o l u t i o n r e q u i r e d s h o u l d be 4 0 ml ( 3 9 ml + t i t r a t i o n v o l u m e ) .
If
n e c e s s a r y , a d j u s t F e h l i n g s and r e t i t r a t e .
T h e c o n c e n t r a t i o n of
invert sugar/100 ml.

the

sample

solution

should

be

250

- 400

mg

A p r e l i m i n a r y test should be c a r r i e d out to a s c e r t a i n the volume of

w a t e r to be a d d e d to t h e 20 ml of F e h l i n g ' s s o l u t i o n i n o r d e r to
o b t a i n a f i n a l t o t a l v o l u m e o f 75 ml w h e n the e n d - p o i n t of the
t i t r a t i o n is reached.
U s i n g the sample t i t r a t i o n p r o c e d u r e
the
f o l l o w i n g m i x t u r e is t i t r a t e d :
20 ml of F e h l i n g ' s s o l u t i o n , 25 ml of
the t e s t s o l u t i o n ,
and 15 ml o f d i s t i l l e d w a t e r .
The l a s t
two
a d d i t i o n s correspond to the 4 0 ml of d i l u t e i n v e r t sugar s o l u t i o n .
I f t h e r e d d i s h c o l o u r o f t h e b o i l i n g s o l u t i o n p e r s i s t s a f t e r the
a d d i t i o n o f the m e t h y l e n e b l u e i n d i c a t o r , t h i s i n d i c a t e s t h a t the
t e s t s o l u t i o n is too c o n c e n t r a t e d ; the t e s t s o l u t i o n must t h e n be
d i s c a r d e d and a l e s s c o n c e n t r a t e d s o l u t i o n used.
I f more than 50 ml of test s o l u t i o n , added to 20 ml of the
solution,
are r e q u i r e d to a t t a i n the r e d d i s h c o l o u r ,
this
that a more c o n c e n t r a t e d s o l u t i o n should be u s e d .
The v o l u m e of w a t e r to be a d d e d
ml ( F e h l i n g ' s s o l u t i o n ) - volume
of w a t e r to be added.
The

sample

titration

is

conducted

Fehling's
indicates

i s c a l c u l a t e d as 75 ml ( t o t a l ) - 2 0
( m l ) of test s o l u t i o n = volume ( m l )

as

follows:

F e h l i n g ' s s o l u t i o n (20 m l ) i s p i p e t t e d into a g l a s s f l a s k ; the volume

of d i s t i l l e d w a t e r i n d i c a t e d by the p r e l i m i n a r y test is then a d d e d .
T h e b u r e t t e i s r i n s e d and f i l l e d w i t h t h e t e s t s o l u t i o n .
The w h o l e
volume of the sample s o l u t i o n r e q u i r e d in the p r e l i m i n a r y t e s t ( l e s s
1 ml) i s run i n t o the f l a s k .
A few f r a g m e n t s of pumice are added and
the c o n t e n t s of the f l a s k a r e w e l l m i x e d by g e n t l e s w i r l i n g .
The
f l a s k i s p l a c e d on a w i r e g a u z e o v e r a b u n s e n f l a m e and h e a t e d to
boiling.
The l i q u i d i s k e p t b o i l i n g m o d e r a t e l y f a s t f o r p r e c i s e l y 2 m i n u t e s
and then 3 or 4 drops of m e t h y l e n e b l u e i n d i c a t o r are added d i r e c t l y
i n t o the b o i l i n g m i x t u r e .
The m i x t u r e s h o u l d a s s u m e a d i s t i n c t l y
blue colour.
T h e t i t r a t i o n i s to b e c o m p l e t e d i n 1 m i n u t e b y t h e
f u r t h e r a d d i t i o n of s m a l l i n c r e m e n t s , i n i t i a l l y of 0 . 2 m l , t h e n of
0 . 1 ml and f i n a l l y o f s i n g l e d r o p s u n t i l t h e e n d - p o i n t i s r e a c h e d .
I t i s i n d i c a t e d by the d i s a p p e a r a n c e o f the b l u e c o l o u r of the
i n d i c a t o r and t h e a p p e a r a n c e
of t h e r e d d i s h
c o l o u r due to
the
p r e c i p i t a t e d cuprous o x i d e .

Ill

Note that the t i t r a t i o n should be c o m p l e t e d in 3 m i n u t e s from the

c o m m e n c e m e n t of b o i l i n g .
The h e a t i n g device used for boiling the
r e a c t i o n m i x t u r e during the t i t r a t i o n is of p r i m e i m p o r t a n c e w h e n
a c c u r a t e r e s u l t s are to be g u a r a n t e e d . During the w h o l e time, the
flask should r e m a i n on the w i r e gauze and boil at a m o d e r a t e rate.
The continuous emission of steam from the neck prevents atmospheric
o x i d a t i o n of the F e h l i n g ' s s o l u t i o n or of the indicator.
During
additions of sugar solution to the boiling liquid, the main burette
tube must be kept out of the steam while the jet is brought over the
mouth of the flask.
CALCULATION
In the absence of sucrose:
If C is the c o n c e n t r a t i o n (g/100 m l ) of the product (for e x a m p l e ,
m o l a s s e s ) in the test s o l u t i o n and V is the v o l u m e (ml) of test
solution used in the titration, then:
invert sugar, g/100 g product =

^^^

In the presence of sucrose:

If f is the correction factor deduced from the following table based
on the a m o u n t of sucrose p r e s e n t , the invert sugar content is given
by the formula:
invert sugar, g/100 g product = f x

1000
CV

For amounts of sucrose intermediate between two consecutive figures

in this table, the correction factor is obtained by interpolation.
Sucrose Correction Factor Table
Sucrose in
boiling mixture
(g)

Correction
factor
(f)

0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
10.0

0.985
0.972
0.964
0.956
0.949
0.942
0.936
0.930
0.924
0.918
0.913
0.908
0.903
0.898
0.895
0.891
0.887
0.883
0.880
0.876

112

INTERPRETATION
T h i s m e t h o d c a n be u s e d f o r the d e t e r m i n a t i o n o f i n v e r t s u g a r ,
glucose,
f r u c t o s e , m a l t o s e and l a c t o s e and of i n v e r t sugar in the p r e s e n c e of s u c r o s e .
The range of sugar c o n c e n t r a t i o n s to w h i c h t h i s method can be d i r e c t l y a p p l i e d
v a r i e s f r o m 0 . 1 to 0 . 8 g / 1 0 0 ml f o r g l u c o s e , f r u c t o s e and i n v e r t s u g a r .
For
the d e t e r m i n a t i o n of i n v e r t s u g a r i n the p r e s e n c e o f s u c r o s e , t h i s m e t h o d i s
e s p e c i a l l y s u i t a b l e for those p r o d u c t s w h i c h h a v e a r e l a t i v e l y h i g h r e d u c i n g
sugars c o n t e n t .
T h i s is due to the h i g h v a l u e of the sucrose c o r r e c t i o n .
I n a n y e v e n t , t h i s m e t h o d s h o u l d n e v e r b e u s e d f o r t h e d e t e r m i n a t i o n of t h e
r e d u c i n g s u g a r s c o n t e n t of b e e t m o l a s s e s b e c a u s e o f the h i g h c o l o u r of t h i s
product in r e l a t i o n to i t s low r e d u c i n g s u g a r s c o n t e n t .
Some n o n - s u g a r s p r e s e n t i n m o l a s s e s i n f l u e n c e the r e s u l t s .
However,
the
r e m o v a l o f n o n - s u g a r s b y c l a r i f i c a t i o n a n d s u b s e q u e n t d e - l e a d i n g l e a d s to
i r r e g u l a r r e s u l t s and t h i s procedure is not recommended.
A l s o , c a l c i u m p r e s e n t in m o l a s s e s forms a complex w i t h g l u c o s e and f r u c t o s e ,
r e s u l t i n g i n s l o w e r r e a c t i o n r a t e s and a p p a r e n t l y l o w i n v e r t s u g a r r e s u l t s ,
t h e r e m o v a l o f c a l c i u m i s t h e r e f o r e a c h i e v e d b y a d d i t i o n to t h e u n t r e a t e d
m o l a s s e s of a p o t a s s i u m o x a l a t e s o l u t i o n f o l l o w e d by f i l t r a t i o n .
1 ml or 2 to
4 ml o f a p o t a s s i u m o x a l a t e s o l u t i o n (5 g / 1 0 0 m l ) i s a d d e d to 1 g o f c a n e
molasses.
T h i s procedure may be r e p l a c e d by a d d i n g a c o m p l e x i n g agent such as
E D T A , w h i c h f o r m s a s t r o n g e r c o m p l e x w i t h c a l c i u m t h a n do t h e h e x o s e s , t h u s
a v o i d i n g the f i l t r a t i o n .
An e f f e c t i v e d e c r e a s e in c o l o u r and an improved end4 ml o f an E D T A s o l u t i o n (4 g / 1 0 0 m l ) p e r g o f c a n e
point are o b t a i n e d .
m o l a s s e s i s recommended.
REFERENCES
ICUMSA,

Report

ICUMSA,

Report

of

the

of

the

Proceedings

16th

Proceedings

Session,

16th

1974,

Session,

Subj.

1974,

14,

Subj.

Rec.
14,

2,

189.

Appendix

4,

180.
Emmerich, A., ICUMSA,
Table 21, 182.
ICUMSA,

Report

of

Report

of t h e P r o c e e d i n g s

the P r o c e e d i n g s

E y n o n , L . and L a n e ,

J.H.,

1949.

10th

Session,

International

113

16th S e s s i o n ,

1949,

Subj.

4,

Sugar J o u r n a l ,

1974,

9.
5_1,

169.

Subj.

14,

3.3

HOMEY

COMPOSITION
Honey is the sweet substance produced by honey bees from the nectar of blossoms
or from s e c r e t i o n s of or on living parts of plants, w h i c h they t r a n s f o r m and
combine with specific substances, and store in honey combs.
H o n e y c o n s i s t s e s s e n t i a l l y of d i f f e r e n t sugars, p r e d o m i n a n t l y glucose and
fructose.
Besides glucose and fructose, honey contains protein, amino acids,
enzymes, organic acids, mineral substances, pollen and other substances, and
may include sucrose, maltose, melezitose and other oligo-saccharides (including
dextrins) as well as traces of fungi, algae, yeasts and other solid particles
resulting from the process of obtaining honey. The colour of honey varies from
n e a r l y c o l o u r l e s s to dark b r o w n .
The c o n s i s t e n c y can be fluid, v i s c o u s or
p a r t l y to e n t i r e l y c r y s t a l l i z e d .
The flavour and a r o m a vary, but usually
derive from the plant origin.
There are various subsidiary definitions of honey as follows:
B l o s s o m or n e c t a r h o n e y is the honey w h i c h c o m e s m a i n l y from n e c t a r i e s of
flowers. H o n e y d e w h o n e y is the honey which comes mainly from secretions of or
on living parts of plants. Its colour varies from very light brown or greenish
to a l m o s t black. C o a b h o n e y is honey stored by bees in the cells of freshly
built broodless combs and sold in sealed whole combs or sections of such combs.
E x t r a c t e d h o n e y is h o n e y obtained by centrifuging decapped b r o o d l e s s combs.
P r e s s e d h o n e y is honey obtained by pressing broodless combs with or without the
application of moderate heat.
The average composition of 490 samples of honey, and range values were (11):

Average
Moisture - - - - - - - - percent
Fructose - - - - do
do
Glucose
- - - - do
Sucrose
- - - - Maltose
- - - - do
Higher Sugars
- do
Undetermined - - do
pH
Free Acid
- - - - meq/kg
do
Lactone
- - - - do
Total Acid - - - Lactone/Free Acid
Ash
- - - - - - - - - - percent
Nitrogen - - - - do
Diastase Value - -

17.2
38.19
31.28
1.31
7.31
1.50
3.1
3.91
22.03
7.11
29.12
0.335
0.169
0.041
20.8

Standard
Deviation
1.46
2.07
3.03
0. 95
2.09
1.03
1.97
-

8.22
3.52
10.33
0.135
0.15
0.026
9. 76

Range
13.4-22.9
27.25-44.26
22.03-40.75
.25-7.57
2.74-15.98
.13-8.49
0-13.2
3.42-6.10
6.75-47.19
0-18.76
8.68-59.49
0-.950
.020-1.028
0-.138
2.1-61.2

Honey must have no objectionable taint absorbed during processing or storage,

it must not be fermenting or effervescing and must not have been heated enough
to have seriously affected the enzymes present. The acidity must not have been
changed artificially.
No additives or additions should be permitted.
H o n e y should be heated as little as possible during processing.
If the
diastase activity is reduced and the content of 5 - h y d r o x m e t h y 1 f u r f u r a 1 (HMF)
increased beyond the given limits it is considered that over-heating has taken
place or the sample is adulterated.
The use of the diastase activity to assess
the d e g r e e of h e a t i n g is discussed by Shirotori et al (12). Fini and S a b a t i n i
(13) found that Italian unprocessed honeys contained an average of 13 mg/kg of
HMF (400 samples), while processed commercial samples averaged 59 mg/kg and 27%
w e r e a b o v e the o d e x r e g i o n a l standard limit.
S i m o n y a n (14) e x a m i n e d 20

114

R u s s i a n h o n e y s and f o u n d the H M F l e s s t h a n 10 m g / k g .
H i g h l e v e l s of H M F w e r e
o r i g i n a l l y t a k e n to i n d i c a t e a d u l t e r a t i o n w i t h i n v e r t s u g a r ( F i e h e ' s t e s t ) b u t
h o n e y from t r o p i c a l areas m a y n a t u r a l l y c o n t a i n over 40 m g / k g .
D a l z e l l and
S i n g e r s (15) r e p o r t t h a t s o m e g e n u i n e N e w Z e a l a n d h o n e y d e w h o n e y s did n o t
c o m p l y w i t h the C o d e x E u r o p e a n r e g i o n a l s t a n d a r d .
If it is d e s i r e d to c o n f i r m
t h a t a h o n e y is a d u l t e r a t e d r a t h e r t h a n t h a t it m e r e l y h a s a h i g h H M F l e v e l , a
m o r e e x t e n s i v e a n a l y s i s is n e c e s s a r y .
T h e e f f e c t on h o n e y of h e a t i n g and
s t o r a g e is d i s c u s s e d b y B e r g e l a n d S t u w e ( 1 6 ) , H a s e e t a l ( 1 7 ) a n d B o r u k h a n d
P a n c h e n k o (18).
D e t a i l s of the c o m p o s i t i o n of h o n e y s f r o m A u s t r a l i a a r e g i v e n b y C h a n d l e r et al
(19) and f r o m S i c i l y by F i n i and S a b a t i n i (20). M i n i e r i and C h i a r a m e l l o (21)
h a v e p u b l i s h e d a r e v i e w o n h o n e y w h i c h i n c l u d e s d e t a i l s of c o m p o s i t i o n .
Some
The
e x a m p l e s of a d u l t e r a t i o n are r e p o r t e d by K a t s u t a and N i s h i k a w a (22).
c a r b o h y d r a t e c o m p o s i t i o n o f h o n e y is r e v i e w e d b y S i d d i q u i ( 2 3 ) a n d t h a t o f
R u s s i a n h o n e y is d e s c r i b e d b y G e n s i t s k i i (24).
ROOTIRE

ANALYSIS

H o n e y can be a d u l t e r a t e d w i t h o t h e r n u t r i t i v e s w e e t e n e r s .
W h i t e (25) has
suggested that adulterants are:
c o n v e n t i o n a l ( a c i d or e n z y m e c o n v e r t e d ) c o r n
s y r u p ( C C S ) , h i g h f r u c t o s e c o r n s y r u p ( H F C S ) and i n v e r t s y r u p s f r o m c a n e ( C I S )
or b e e t (BIS). He r e c o m m e n d s t h a t t e s t s s h o u l d be c a r r i e d o u t in the o r d e r
l i s t e d b e l o w , u s i n g o n l y a s m a n y as r e q u i r e d to a t t a i n t h e o b j e c t i v e .
The
l i m i t e d a v a i l a b i l i t y of e q u i p m e n t for t h e c a r b o n - 1 3 i s o t o p e t e s t r e d u c e s t h e
p r a c t i c a l v a l u e of t h i s v e r y u s e f u l i n d i c a t o r of a d u l t e r a t i o n .
Carbon-13

isotope

test

(26)

V a l u e s of less n e g a t i v e t h a n -21.5/oo (4s) are c o n c l u s i v e for the p r e s e n c e of

c o r n o r c a n e s y r u p s , u n l e s s t h e s a m p l e is o f c i t r u s o r i g i n w h i c h c a n b e
a s c e r t a i n e d by f l a v o r and a r o m a or m e t h y l a n t h r a n i l a t e c o n t e n t (27).
For
c i t r u s , t h e c o r r e s p o n d i n g 4 s v a l u e i s - 2 0 / o o . If v a l u e s a r e b e t w e e n t h e s e
l i m i t s and -23.4/ o o , the T L C m e t h o d is r e q u i r e d to c o n f i r m c o r n s y r u p s .
Thin-layer chromatography

(TLC) test

(28)

A p o s i t i v e r e s u l t s h o w s a b o u t 5 - 7 % H F C S or a b o u t 1 - 2 % C C S .
l e s s s e n s i t i v e to s e c o n d or t h i r d g e n e r a t i o n H F C S .

This

test m a y

be

Hydroxyaethy1furfural

(HMF) teat

U s i n g the b i s u l f i t e
adulteration with BIS
m g / 1 0 0 g then product
adulterated.
(Confirm

m e t h o d , t h e n 20 m g / 1 0 0 g or m o r e
indicates prob ab1e
o r C I S . ( C o n f i r m b y c a r b o h y d r a t e a n a l y s i s ) . If 1 0 - 2 0
m a y b e h e a t o r s t o r a g e a b u s e d h o n e y , o r it c o u l d b e
with carbohydrate analysis).

Carbohydrate distribution

(29)

analysis

T h e p r o p o r t i o n s of t o t a l m o n o s a c c h a r i d e s , d i s a c c h a r i d e s and h i g h e r s u g a r s a r e
c h a r a c t e r i s t i c of h o n e y and p r o v i d e c o n f i r m a t i o n of the p r e s e n c e of m o s t
adulterants.
A t l e a s t o n e v a l u e o u t s i d e the l i m i t s of 6 0 - 7 9 % m o n o s a c c h a r i d e s ,
4 - 1 2 % d i s a c c h a r i d e s a n d a b o v e 3.8% f o r h i g h e r s u g a r s , is i n d i c a t i v e
of
adulteration.
T w o or m o r e v a l u e s o u t s i d e a r e c o n c l u s i v e .
Test

for g l u c o s e , fructose and

sucrose

U s e H P L C or a n a l y z e the f r a c t i o n s f r o m the d i s t r i b u t i o n a n a l y s i s , u s i n g g l u c o s e
o x i d a s e for g l u c o s e ,
fructose by d i f f e r e n c e ,
and s u c r o s e by
invertase
hydrolysis.
If g l u c o s e e x c e e d s 4 0 % , s a m p l e is n o t g e n u i n e h o n e y ; if 3 8 - 4 0 % ,
p o l l e n of c o t t o n , b l u e c u r l s , or m a n z a n i t a m u s t b e p r e s e n t if a g e n u i n e h o n e y ;
if a b s e n t , it is p r o b a b l y a d u l t e r a t e d .
Pure h o n e y contains over 31% f r u c t o s e ,
l e s s t h a n 3 8 % g l u c o s e a n d l e s s t h a n 8% s u c r o s e ( u n l e s s f r e s h l y e x t r a c t e d c i t r u s
honey).
R a t i o of f r u c t o s e to g l u c o s e is 1.00 or m o r e .

115

nitrogenous constituents analysis

All pure h o n e y c o n t a i n s at least 15 mg proline per 100 g and at least 65 mg
true protein per 100 g (determined after dialysis).
Because average values are
much higher, this is useful only to confirm gross adulteration.
Presence of honeydew
Honeydew may occur naturally in honey and is not an adulterant.
It originates
from extra-floral secretions on plants, gathered and stored by honeybees.
It
may contribute a molasses-like flavour and appear to respond positively to the
TLC test.
It may be differentiated as follows:
Determine constant direct polarization value.
Values more positive than -2S
(not s p e c i f i c r o t a t i o n ) m a y indicate e i t h e r h o n e y d e w or the presence of corn
syrup or sucrose.
H o n e y d e w w i l l contain over 5% m e l e z i t o s e , less than 8%
sucrose, and may not conform to the normal distribution of sugars.
The methods given for the analysis of sugars in honey are adequate for routine
purposes.
Takahashi and Tokumura (30) describe the use of the Fehling-LehmannSchoorl and Furukolmen methods in conjunction with simultaneous equations to
c a l c u l a t e the p r o p o r t i o n s of g l u c o s e , fructose, sucrose and m a l t o s e .
Bose,
Singh and Mukherjee (31) use an alkaline hypoiodite titration for estimation of
glucose and total reducing sugars.
The use of GLC for the a n a l y s i s of sugars in honey is described by B a t t a g l i n i
and Bosi (32) and H a d o r n , Z u r c h e r and Stracte (33). The sugars found in honey
are discussed in a review by Doner (34).
The chemical analysis of honey has been reviewed by Iwaida (35) in relation to
Japanese standards for the product and by Hase et al (17).
T h e r e are very o c c a s i o n a l l y reports of toxic effects due to the ingestion of
honey and the cause is usually traced to toxic plants from which the bees have
fed.
Clinch and Turner (36) report the presence of a toxin, tutin, in some New
Zealand honeys.
The p r e s e n c e of g r a y a n o t o x i n s in some C a n a d i a n honey is
r e p o r t e d by Scott, C o l d w e l l and W i l b e r g (37) and an outbreak in Russia is
described by Hedveditskova (38).
The geographical origin of honey can be difficult to determine. Gilbert et al
(39) has carried out s t a t i s t i c a l a n a l y s i s on a m i n o - a c i d v a l u e s w i t h some
success.
If bees have collected pollen from species of plants characteristic
of a r e g i o n , m i c r o s c o p i c a l e x a m i n a t i o n of the c e n t r i f u g e d s e d i m e n t from the
h o n e y and i d e n t i f i c a t i o n of the c h a r a c t e r i s t i c pollen present p e r m i t s the
g e o g r a p h i c a l o r i g i n of the h o n e y to be d e t e r m i n e d .
It also enables the
m i c r o 8 c o p i s t to s u b s t a n t i a t e c l a i m s about the b o t a n i c a l origin - that it is
heather honey, orange blossom honey and so on. In practice results have to be
interpreted with extreme care.
The proportion of pollen grains in the sample
from different species of plant is in no way related to the extent to which the
b e e s have fed from those plants.
For e x a m p l e , citrus b l o s s o m sheds little
pollen and although the bees may have derived the major part of the honey from
citrus blossom the proportion of citrus pollen present may be extremely low.
The m e t h o d is still u s e f u l as there should be s o m e citrus pollen present in
h o n e y from that source.
The pollen of E u c a l y p t u s spp has a c h a r a c t e r i s t i c
morphology, but the trees are grown in many parts of the world and the presence
of p o l l e n is no g u a r a n t e e that the honey originated from A u s t r a l i a , the only
area in which Eucalyptus spp are indigenous.
The vegetation from a particular
area or region being characteristic, the pollen analysis in the honey from that
region is also characteristic, even though the proportion of pollen grains of
d i f f e r e n t types p r e s e n t is no r e f l e c t i o n of the ecological pattern of the
vegetation.

116

A g r e a t d e a l of u s e f u l i n f o r m a t i o n a b o u t the f l o r a l , g e o g r a p h i c a l
and
t o p o l o g i c a l s o u r c e s of h o n e y c a n be o b t a i n e d f r o m e x a m i n a t i o n of the p o l l e n .
T h i s r e q u i r e s c o n s i d e r a b l e e x p e r i e n c e g a i n e d f r o m e x a m i n i n g m a n y s a m p l e s of
known source.
T h e o r i g i n o f s o m e h o n e y s c a n b e d e t e r m i n e d v e r y q u i c k l y b y an
experienced m i c r o s c o p i s t , while other samples require very detailed work.
A
f o r m a l e x a m i n a t i o n or an e x a m i n a t i o n of a s a m p l e of d i s p u t e d o r i g i n , s h o u l d
a l w a y s b e d o n e a c c o r d i n g to the I n t e r n a t i o n a l M e t h o d (40).
T h e l i t e r a t u r e on
p o l l e n a n a l y s i s is v e r y e x t e n s i v e .
S o m e r e f e r e n c e s a r e i n c l u d e d at t h e e n d of
this chapter.

117

SAMPLE PREPARATION - HONEY

(Clarification)
PRINCIPLE
A diluted sample of honey is clarified by treatment with alumina cream and
filtering.
The clarified sample can then be used for sugars analysis.
REAGENTS
A l u m i n a c r e a m - p r e p a r e a cold s a t u r a t e d s o l u t i o n of a l u m
(K2SO4AI2 (80^)3.24H2O) in water.
Add a m m o n i u m hydroxide with
constant stirring until the solution is alkaline to litmus, let
p r e c i p i t a t e settle and wash by dcantation with water until w a s h water gives only slight test for sulphate with barium chloride
solution.
Pour off excess water and store residual cream in
stoppered bottle.
SAMPLING
If the s a m p l e is l i q u i d or s t r a i n e d h o n e y and is free from
If granulated,
granulation, mix thoroughly by stirring or shaking.
place the closed container in water-bath without submerging and heat
30 m i n u t e s at 60C; then if necessary heat at 65C until liquefied.
Occasional shaking is essential.
Mix thoroughly and cool rapidly as
s o o n as s a m p l e l i q u e f i e s .
Do not h e a t h o n e y i n t e n d e d
for
hydroxymethylfurfural or diastase determination.
If foreign matter,
such as w a x , sticks, bees, particles of c o m b , etc. is present, heat
sample to 40C in water-bath and strain through cheese-cloth in hotwater funnel before sampling.
If it is comb honey, cut across top of comb (if sealed) and separate
the honey completely from the comb by straining through a sieve with
square openings of 0.5 m m by 0.5 m m .
When portions of comb or wax
pass through the sieve, heat sample as above and strain through
cheesecloth.
If honey is granulated in c o m b , heat until wax is
liquefied; stir, cool and remove wax.
PROCEDORE
Transfer an accurately weighed sample of approximately 25 g from the
well mixed honey to 100 ml volumetric flask, add 5 ml alumina cream,
dilute to volume with water at 20C and filter.
REFERENCE
Codex Alimentarius Commission Regional Standard for Honey, CAC/RS 12-1969.

118

MOISTURE IH HONEY
PRINCIPLE
The method based oil refractometric

examination of honey.

APPARATUS
1.

Refractometer

PROCEDURE
Determine the refractive index of the sample using a refractometer at
a c o n s t a n t t e m p e r a t u r e n e a r 20C.
C o n v e r t the r e a d i n g to m o i s t u r e
c o n t e n t ( p e r c e n t m / m ) u s i n g the t a b l e g i v e n b e l o w .
If the
determination is made at a temperature other than 20C, convert the
reading to standard temperature of 20C, according to the temperature
corrections quoted.
The m e t h o d used should be noted in the test
report.
Table for the Estimation of Moisture
Refractive
Index
(20 C )
1.5044
1.5038
1.5033
1.5028
1.5023
1.5018
1.5012
1.5007
1.5002
1.4997
1.4992
1.4987
1.4982
1.4976
1.4971
1.4966
1.4961
1.4956
1.4951
1.4946
1.4940

Moisture
Refractive
Content
Index
(percent)
(20 C )
13.0
13.2
13.4
13.6
13.8
14.0
14.2
14.4
14.6
14.8
15.0
15.2
15.4
15.6
15.8
16.0
16.2
16.4
16.6
16.8
17.0

1.4935
1.4930
1.4925
1.4920
1.4915
1.4910
1.4905
1.4900
1.4895
1.4890
1.4885
1.4880
1.4875
1.4870
1.4865
1.4860
1.4855
1.4850
1.4845
1.4840
1.4835

Content

Moisture
Refractive
Content
Index
(percent)
(20 C)
17.2
17.4
17.6
17.8
18.0
18.2
18.4
18.6
18.8
19.0
19.2
19.4
19.6
19.8
20.0
20.2
20.4
20.6
20.8
21.0
21. 2

erature corrections to change Refractive

Moisture
Content
(percent)

1.4830
1.4825
1.4820
1.4815
1.4810
1.4805
1.4800
1.4795
1.4790
1.4785
1.4780
1.4775
1.4770
1.4765
1.4760
1.4755
1.4750
1.4745
1.4740

Index:

Temperatures above 20C:

add 0.00023 per C

Temperatures below 20C:

subtract o.00023 per C.

REFERENCES
Chataway, H.D., 1932. Canadian Journal of Research _6, 540 et seq.
Chataway, H.D., 1933. Canadian Journal of Research _9, 435 et seq.
Chataway, H,D., 1935. Canadian Bee Journal 43

119

(8), 215.

21.4
21.6
21.8
22.0
22. 2
22.4
22.6
22.8
23.0
23.2
23.4
23.6
23.8
24.0
24.2
24.4
24.6
24.8
25.0

Wedmore, E.B., 1955. Bee World 2

197 et seq.

(Note: The above m e t h o d is the m o d i f i c a t i o n of

Wedmore.
Also see W h i t e , J.W., 1969, Journal
Official Analytical Chemists 52 (4), 729-37 for a
chemical methods to determine moisture in honey.
drying honey on sand under vacuum ( < 5 0 mm Hg) at

120

C h a t a w a y ' s m e t h o d by
of the A s s o c i a t i o n of
review of physical and
Other methods include
60C.

DIASTASE ACTIVITY OF HONEY

PRINCIPLE
The rate of starch destruction is monitored
color.
Note - do not heat honey to be used

by the intensity of the iodine blue

for diastase activity.

APPARATUS
1.

Water-bath

at 40 +_ 0.2C.

2.

Spectrophotometer

to read at 660 nm.

REAGENTS
1.
Iodine stock solution:
d i s s o l v e 8.88 g of i o d i n e a n a l y t i c a l
grade, in 30-40 ml water containing 22 g potassium iodide, analytical
grade, and dilute to 1 litre with water.
2.
Iodine solution about 0.0007 N:
dissolve 20 g potassium iodine,
a n a l y t i c a l g r a d e , in 3 0 - 4 0 m l w a t e r in a 5 0 0 m l v o l u m e t r i c f l a s k .
Add 5.0 m l i o d i n e s t o c k s o l u t i o n and m a k e up to v o l u m e .
M a k e up a
fresh solution every second day.
3.
A c e t a t e b u f f e r - pH 5.3 (1.59 N):
d i s s o l v e 87 g s o d i u m a c e t a t e
trihydrate in 400 m l w a t e r , add about 10.5 ml glacial acetic acid in
A d j u s t the pH to 5.3 w i t h
a l i t t l e w a t e r and m a k e up to 5 0 0 m l .
sodium acetate or acetic acid as necessary, using a pH meter.
4.
S o d i u m c h l o r i d e s o l u t i o n 0.5 N:
d i s s o l v e 14.6 g s o d i u m
chloride, analytical grade, in boiled-out distilled water and m a k e to
500 ml.
(The keeping time is limited by mould growth.)
5.
Starch solution:
w e i g h o u t t h a t a m o u n t of s t a r c h w h i c h is
e q u i v a l e n t to 2.0 g a n h y d r o u s s t a r c h .
M i x w i t h 90 m l of w a t e r in a
250 ml conical flask.
B r i n g r a p i d l y to the b o i l , s w i r l i n g the
s o l u t i o n as m u c h as p o s s i b l e , h e a t i n g over a t h i c k w i r e g a u z e
p r e f e r a b l y w i t h an i n s u l a t e d c e n t r e .
B o i l g e n t l y for 3 m i n u t e s ,
cover and allow to cool spontaneously to room temperature.
Trasfer
to a 100 m l v o l u m e t r i c flask, place in a water bath at 40C to attain
this t e m p e r a t u r e and m a k e up to v o l u m e at 40C. T h i s is the s t a r c h
s o l u t i o n to be used in the a n a l y s i s .
The starch used should have a
"blue v a l u e " of b e t w e e n 0.5 - 0.55 u s i n g a 1 cm cell.
If o u t s i d e
this range, adjust the weight of the starch taken.
The method of determining this is as follows:
the amount of starch
equivalent to 1 g anhydrous starch as prepared by the sample method
is cooled and 2.5 m l acetate buffer added before making up to 100 ml
in a v o l u m e t r i c flask.
To a 100 m l v o l u m e t r i c flask add 75 ml water,
1 m l N h y d r o c h l o r i c acid and 1.5 m l of 0.02 N i o d i n e s o l u t i o n .
Then
add 0.5 m l of the s t a r c h s o l u t i o n and m a k e up to v o l u m e w i t h w a t e r .
A l l o w to stand for one hour in the dark and read in 1 cm cell using a
s p e c t r o p h o t o m e t e r at 660 nm a g a i n s t a b l a n k c o n t a i n i n g all the
i n g r e d i e n t s e x c e p t the s t a r c h s o l u t i o n .
T h e r e a d i n g on t h e
absorbance scale = "blue value".

PROCEDURE
W e i g h 10.0 g h o n e y into a 50 m l b e a k e r and add 5.0 m l a c e t a t e b u f f e r
s o l u t i o n t o g e t h e r w i t h 20 m l w a t e r to d i s s o l v e the s a m p l e .
Stir
until dissolved.
Do n o t w a r m . Add 3.0 m l s o d i u m c h l o r i d e s o l u t i o n
to a 50 m l volumetric flask and transfer the dissolved honey sample

121

to this and adjust the volume to 50 ml. (Note:

it is essential that
the honey should be buffered before coming into contact with sodium
chloride. )
Warm the starch solution to 40C and pipette 5 ml into 10 ml of water
at 40C and m i x well.
Pipette 1 ml of this solution into 10 m l
0.0007 N iodine solution, diluted w i t h 35 ml of water and mix well.
Read the c o l o u r at 660 nm against a w a t e r blank using a 1 cm cell.
The a b s o r b a n c e should be 0.760 _+ 0.020. If n e c e s s a r y the v o l u m e of
added water is adjusted to obtain the correct absorbance.
Pipette 10 ml honey solution into 50 ml graduated cylinder and place
in 40C +_ 0.2C w a t e r bath w i t h flask c o n t a i n i n g starch solution.
After 15 m i n u t e s , pipette 5 ml starch solution into the honey
solution, mix, and start stop-watch.
At 5 minute intervals remove 1
m l a l i q u o t s and add to 10.00 m l 0.0007 N iodine solution.
Mix and
dilute to standard volume (50 ml). Determine absorbance at 660 nm in
spectrophotometer immediately using 1-cm cell.
Continue taking 1 ml
aliquots at intervals until absorbance of less than 0.235 is reached.
CALCULATIOH
The a b s o r b a n c e is plotted against time ( m i n u t e s ) on r e c t i l i n e a r
paper. A straight line is drawn through at least three points on the
graph to d e t e r m i n e the time w h e n the reaction m i x t u r e reaches an
absorbance of 0.235. Divide 300 by the time in minutes to obtain the
diastase number (DN). This number expresses the diastase activity as
ml of 1% starch solution hydrolysed by the enzyme in 1 g of honey in
1 h at 40C. This diastase n u m b e r c o r r e s p o n d s with the Gothe scale
number.
Diastase
40 C.

activity

= DN = ml starch solution (1 percent)/g honey/h at

REFERENCES
S c h a d e , J.E., M a r s h , G.L. and E c k e r t , J.E., 1 958. Food R e s e a r c h 23, 446.
W h i t e , J.W. and P a i r e n t , F.W., 1959. Journal of the A s s o c i a t i o n
Agricultural Chemists 42, 344.

of

Official

Hadorn, H., 1961. Milleilungen aus dem Gebeite der Lebensmitteluntersuchung

Hyg iene 52^, 6 7.

122

und

ACIDITY AND LACTONE IH HONEY

PRINCIPLE
The s a m p l e is t i t r a t e d w i t h s o d i u m h y d r o x i d e to o b t a i n the free a c i d i t y .
E x c e s s s o d i u m h y d r o x i d e is added to h y d r o l y s e any l a c t o s e p r e s e n t and
i m m e d i a t e l y b a c k - t i t r a t e d w i t h h y d r o c h l o r i c acid.
The total a c i d i t y is
calculated as free acidity plus lactone in m il1iequivalents per kilogram.
APPARATUS
1.

pH meter.

REAGENTS
1.

0.05 N NaOH

2.

0.05 N HC1

PROCEDURE
D i s s o l v e 10 g of s a m p l e in 75 m l of c a r b o n d i o x i d e - f r e e w a t e r in a
250 m l beaker.
Stir with a magnetic stirrer, immerse the electrodes
of the pH meter in the solution and record the pH.
Titrate with 0.05
N N a O H at a r a t e of 5 m l / m i n u t e , u n t i l the pH r e a c h e s 8.5.
Record
the burette reading.
Immediately add by pipette 10 ml of 0.05 N NaOH
and i m m e d i a t e l y b a c k - t i t r a t e w i t h 0.05 N HCl from a 10 m l b u r e t t e
until the pH reaches 8.3.
Also do a reagent blank.
CALCULATION
Free acidity = (ml of 0.05 N NaOH to bring
pH 8.5-blank) x 0.05 x 1000/10

solution to

Lactone = (10.00 - titre of 0.05 N HCl in ml) x 0.05 x

1000/10
Total acidity
=
free acidity + lactone (all results
being expressed as milli-equivalent of acid per
kilogram of honey).
REFERENCES
Codex Alimentarius

Commission Regional

Standard

Official Methods of Analysis of the AOAC, 1984,

123

for Honey, CAC/RS12-1969.

31.168.

PROLINE

IB HONEY

PRINCIPLE
P r o l i n e is d e t e r m i n e d s p e c t r o p h o t o m e t r i c a l l y
form a coloured compound.

after

reaction

with

ninhydrin

APPARATUS
1.

Spectrophotometer.

REAGENTS
1.

Formic

acid

2.

Ninhydrin

- 3% s o i n

( w / v ) in m e t h y l

cellosolve

P r o l i n e S t d . - in w a t e r ( d e i o n i z e d ) 4 0 m g t o 25.0 m l ; t h e n 1 m l
3.
to 2 5 . 0 m l . ( u s e 0.5 m l in p r o c e d u r e )
PROCEDURE
D i s s o l v e 5 . 0 g h o n e y in 5 0 m l w a t e r , q u a n t i t a t i v e l y t r a n s f e r t o 1 0 0
m l v o l u m e t r i c f l a s k , m a k e t o m a r k , s t o p p e r , a n d m i x w e l l . U s e 0.5 m l
per d e t e r m i n a t i o n .
D i l u t e s a m p l e (0.5 m l p r e p a r e d a b o v e ) a n d 0.5 m l
o f s t a n d a r d a r e p l a c e d i n s e p a r a t e 2 0 m l s c r e w - c a p p e d v i a l s a n d 0.25
m l o f f o r m i c a c i d a n d 1.0 m l o f 3 % n i n h y d r i n s o l u t i o n a r e t h e n a d d e d
to e a c h v i a l .
T h e v i a l s a r e t i g h t l y c a p p e d a n d p l a c e d in a b o i l i n g
w a t e r b a t h f o r 15 m i n u t e s .
T h e v i a l s a r e t h e n c o o l e d in a w a t e r b a t h
W h i l e c o o l i n g , a d d 5.0 m l o f 1 : 1
a t 7 0 f o r 5 t o 10 m i n u t e s .
i s o p r o p a n o l - w a t e r solution; c o n t i n u e cooling and develop colour for
at l e a s t 1 / 2 h o u r .
Scan spectra from 600 to 450 n m and take
a b s o r b a n c e r e a d i n g s at m a x i m u m ca 5 1 2 n m . ( R e a d i n g s s h o u l d b e taken
before an hour h a s elapsed).
A b l a n k u s i n g 0.5 m l w a t e r i s a l s o
carried through the procedure.
CALCULATION
mg Proline/100

g h o n e y = AiL x S x
As

Where:

Au = absorbance

of sample

As = absorbance

of standard

= m g std weighed

16 = p r o d u c t
W

aliquot

= ca 40 m g (stock

of dilution

= g honey weighed

aliquot

soin)

factors

= ca 5.0 g

REFERENCE
Official

Methods

of Analysis

of the AOAC,

124

1984, 31.124-.126.

to

DEXTROSE IN HOMEY
PRINCIPLE
D e x t r o s e is e s t i m a t e d i o d o m e t r i c a l l y , total r e d u c i n g
reduction method and the fructose obtained by difference.

sugars

by

copper

APPARATUS
1.

Burette.

REAGENTS
1.

0.1 N iodine

2.
0.2 N s o d i u m b i c a r b o n a t e / c a r b o n a t e s o l u t i o n .
D i s s o l v e 16.8g
sodium bicarbonate and 21.2 g sodium carbonate in water and dilute to
1 litre.
3.

Sulphuric

acid 25%, v/v.

4.

Sodium thiosulphate 0.1 N.

PROCEDURE
D i l u t e 2 g of h o n e y to 250 m l and e s t i m a t e total r e d u c i n g s u g a r s by
one of the standard procedures.
To determine dextrose, to 25 ml of the honey solution add by pipette
a v o l u m e of 0.1 N i o d i n e at least t w i c e that r e q u i r e d for the
reaction followed by 100 ml of sodium bicarbonate/carbonate solution.
L e a v e in the d a r k for 2 h o u r s , a c i d i f y w i t h 12 m l of 25% s u l p h u r i c
acid and titrate with 0.1 N thiosulphate solution.
Carry out a blank
at the s a m e t i m e .
The d i f f e r e n c e b e t w e e n the t w o
titrations
represent the dextrose.
CALCULATION
1 ml 0.1 N iodine = 0.009005 g dextrose
Fructose = total reducing sugars - dextrose.
REFERENCE
Pearson's Chemical Analysis of Foods, 1976, 7th Edition,

125

141.

APPARENT SUCROSE IN HONEY

PRINCIPLE
Sugars are determined by Fehling's titration before
sucrose is calculated by difference.

and

after

inversion,

REAGENTS
1.

Soxhlet modification of Fehling's

solution.

2.

Standard invert sugar

3.

Hydrochloric

4.

Sodium hydroxide solution (5 N aqueous).

5.

Methylene blue solution 2 g/L.

solution.

See method for

Invert Sugars
with Added
Sucrose

acid, 6.34 N.

PROCEDURE
Prepare the honey sample as in "Sample Preparation-Honey".
Dilute 10
m l of this s o l u t i o n to 250 ml w i t h distilled water.
Pipette 50 m l
into a 100 ml graduated flask, together with 25 ml distilled water
and heat to 65C over a boiling w a t e r - b a t h .
Cool the solution
n a t u r a l l y for 15 m i n u t e s and then cool to 20C, n e u t r a l i z e with 5 N
s o d i u m h y d r o x i d e , using l i t m u s paper as indicator, cool again and
adjust the volume to 100 ml (diluted honey solution).
Continue as in
the method for "Invert Sugars with Added Sucrose".
CALCULATION
C a l c u l a t e percent invert sugar (g invert sugar per 100 g h o n e y )
b e f o r e and after inversion using the sam f o r m u l a as for "Invert
Sugars with Added Sucrose." Apparent sucrose content = (Invert sugar
content after inversion minus invert sugar content before inversion)
x 0.95. The result is expressed as g apparent sucrose/100 g honey.
REFERENCE
Walker, H.S., 1917. Journal of Industrial

126

and Engineering

Chemistry _2, 490.

The

HYDBOXYMETHYLFURFURAL

IN HONEY

PRINCIPLE
HMF
A colour is developed reacting HMF with p-toluidine and barbituric acid.
can be formed if honey is heated, so the sample must not be subjected to heat.
APPARATUS
1.

Spectrophotometer

2.

Water bath.

to read at 550 nm.

REAGENTS
1.
Barbituric acid solution:
weigh out 500 mg barbituric acid and
transfer to a 100 ml graduated flask using 70 ml water.
Place in hot
water bath until dissolved, cool and make up to volume.
2.
p-Toluidine solution:
weigh out 10.0 g p-toluidine, analytical
grade, and dissolve in about 50 ml isopropanol by gentle warming on a
w a t e r bath.
T r a n s f e r to a 100 m l g r a d u a t e d flask w i t h i s o p r o p a n o l
and add to 10 m l g l a c i a l acetic acid.
Cool and m a k e up to v o l u m e
Keep the solution in the dark.
Do not use for at
with isopropanol.
least 24 hours.
3.
Distilled water (oxygen free):
Nitrogen gas is
boiling distilled water.
The water is then cooled.

passed

through

4.
Hydroxymethylfurfural (HMF) standard solutions:
dissolve 10 mg
H M F in 1 litre w a t e r (stock s o l u t i o n ) .
P i p e t t e 10 m l , 20 m l , 30 m l
and 40 m l into four 50 m l v o l u m e t r i c flasks.
M a k e to v o l u m e w i t h
w a t e r (dilute s o l u t i o n s ) .
H M F is v e r y h y g r o s c o p i c , so c h e c k the
s t a n d a r d c o n c e n t r a t i o n by m e a s u r i n g the a b s o r b a n c e at 282.5 n m ,
assuming E = 21,215 (Turner, 1954).
PROCEDURE
Weigh 10 g of the honey sample and dissolve without heating in 20 ml
oxygen-free distilled water. Transfer to a 50 ml graduated flask and
m a k e up to v o l u m e (honey s o l u t i o n ) . The s o l u t i o n s a m p l e should be
tested after preparation without delay.
Pipette 2.0 ml of honey solution into each of two 25 m m diameter test
tubes and add 5.0 ml p-toluidine solution to each.
Pipette into one
test tube 1 ml water and into the other 1 m l barbituric acid solution
and shake b o t h tubes. The one w i t h added w a t e r s e r v e s as the w a t e r
bank.
The addition of the reagents should be done without delay and
should be finished in about 1-2 minutes.
Read the absorbance of the sample against the blank at 550 nm using a
1 cm cell immediately the m a x i m u m value is reached.
P r e p a r e a s t a n d a r d curve by p i p e t t i n g 2 m l from each of the four
d i l u t e s t a n d a r d s o l u t i o n s into four test tubes.
Add 5.0 m l ptoluidine to each and continue as with the sample.
CALCULATION
Find the amount of HMF (in micrograms) in the sample by reading from
the standard curve.
This figure divided by 10 equals ppm H M F in the
sample.

127

IHTKRPRETTIOH
For cleanup, pass the dilute sample through a activated charcoal column.
HHF should not be present in genuine honey in large amount unless it has been
abased by heating or has deteriorated due to improper or prolonged storage.
The presence of H M F in significant amounts tends to indicate the presence of
added invert sugars as an adulterant.
40 ppm is the legal limit in some
countr ies .
REFERENCES
Winkler, 0., 1955.
(3), 166-7.

Zeitschrift

T u r n e r , J.H., Rebers,
Chemistry 26, 898-901.

P.A.,

fur Lebensmitteluntersuchung

Bamik,

P.L. & Cotton,

128

R.H.

und Forschung 102

1954.

Analytical

3.4

TEXT

1.

SCHNEIDER,

2.

PEARSON,

3.

JACKSON, R.F. & G I L L I S , C.L.

1920.
The D o u b l e P o l a r i z a t i o n Method f o r
E s t i m a t i o n of S u c r o s e a n d t h e E v a l u a t i o n o f the C l e r g e t D i v i s o r 3 7 5 US
D e p t Comm. B u r . S t d . S c i e . 1 2 5 - 1 9 4 .

4.

JACKSON,
R . F . & Mc D O N A L D , E . J .
O f f i c i a l A g r i c u l t u r a l C h e m i s t s 22

5.

NORRISH, R.S.
1967.
S e l e c t e d T a b l e s of P h y s i c a l P r o p e r t i e s o f S u g a r
S o l u t i o n s , S c i e n t i f i c and T e c h n i c a l Surveys 51 B r i t i s h Food M a n u f a c t u r i n g
I n d u s t r i e s Research A s s o c i a t i o n , L e a t h e r h e a d , E n g l a n d .

6.

BATES,
Tables

7.

BROWN, C.A.
An a1 y s i s .

8.

SOCIETY OF PUBLIC ANALYSTS.

1930.

Analyst

9.

SCHIWECK,

H.

& BSCHING,

L.

1969.

Zucker

22

377.22.

10.

SCHIWECK,

H.

& BSCHING,

L.

1975.

Zucker

28

242.

11.

Composition

12.

S H I R O T O R I , T . , I W A I D A , M. & K A W A S H I R O , I .
S o c i e t y of Food N u t r i t i o n 21 ( 4 ) 2 6 1 - 2 6 4 .

13.

F I N I , M . A . & SABAT IN I , A . G .
(6) 375-379.

14.

SIMONYAN, T.A.
(3) 17-18.

15.

DALZELL, K.W.
(3) 329-332.

16.

BERGEL,

17.

H A S E , S . , O D A T E , M. & K U R A Y A B A S H I ,
T e c h n o l o g y ( J a p a n ) 20 ( 6 ) 2 5 7 - 2 6 4 .

18.

BORUKH,

19.

CHANDLER, B.V.,
Paper D i v . Food

20.

F I N I , M . A . & SAB I T I N I ,
(6) 349-355.

21.

MINIERI,
359-396.

22.

KATSUTA, T. S. N I S H I K A W A .
Laboratory Public Health

23.

S I D D I Q U I , I.R.
1970.
Advances in C a r b o h y d r a t e C h e m i s t r y
25 2 8 5 - 3 0 9 .
Food Research I n s t i t u t e Dept of A g r i c u l t u r e ,

REFERENCES
F.

D.

(Ed).
1981.

F., PHELPS,
7_ 3 3 4 - 3 5 5 .

C.

&

F.P.

ZERBAN,

1972.

&

ICUMSA Methods

The Chemical

&

1962.

G.N.

of

Foods,

Analysis.

1975.

1968.

1973.

CHIARAMELLO,

1974.

S.

1975.

Acta

1971.
Annual
23 81-86.

129

Sugar

(3)

of

Japanese

of

Science

16

18

77-78.

Journal

Medica

126.

Promysh-lennost

of

Food

T.

1974.

&

Technical

d e g l i A l i m e n t i 4_

Veterinaria

Tokyo

Science

30-33.

e Tecnologia

Report

of

degli Alimenti

Journal

Tovarovedenir

Scienza

Methods

Journal

F E N W I C K , D . , O R L O V A , T. & R E Y N O L D S ,
Research 38 CSIRO 3 9 .
A.G.

Critical

Bulletin

e Tecnologia

16

of

115.55.

Zealand

Susswaren

pl45.

Association

Chemical

Kondi terskaya

New

A.

the

USDA T e c h n i c a l

Ed.

International

and

55

8th

of

1 92 7.

Scienza

1975.

1972.

& PANCHENKO,

&

C.F.

Khlebopetarnaya

G.G.

Sugar

Physical

1972.

W.A.

of

1939.
Journal
580-588.

1941.

Honeys.

SINGERS,

& STUWE,

Analysis

SNYDER,

F.W.

of American

I.F.

L.

1979.

20

Metropolitan

(5/6)

Research

and B i o c h e m i s t r y
Ottowa,
Canada.

24.

GENSITSKII, I.P.

25.

W H I T E , J.W.
1985.
H o n e y t e c h Inc., P.O. Box
77868 (Private communication).

26.

ASSOCIATION OF OFFICIAL ANALYTICAL

Analysis 31.158 - 31.161.

27.

W H I T E , J.W. & R O B I N S O N , F. A.
Analytical Chemists 66 (1) 1-3.

28.

ASSOCIATION OF OFFICIAL ANALYTICAL

Analysis 31.156 - 31.15 7.

CHEMISTS.

1984.

Official

Methods

of

29.

ASSOCIATION OF OFFICIAL ANALYTICAL

Analysis 31.153 - 31.155.

CHEMISTS.

1984.

Official

Methods

of

30.

T A K A H A S H I , T. & T O K U M U R A , H.
1968.
Annual
Research Institute Aichi. Prefecture 9_ 125-128.

31.

B O S E , S., S I N G H , H. & M U K H E R J E E , S.

32.

B A T T A G L I N I , M. & B O S I , C.
(4) 2 1 7 - 2 2 1 .

33.

HADORN, H., ZURCHER, K. & STRACTE, C.

1974.
Mitteilungen
der Lebensmitteluntersuchung und Hygiene 65 (2) 198-208.

34.

D O N E R , L.W.
456.28.

35.

I W A I D A , M.
489.

36.

C L I N C H , P.G. & T U R N E R , J.C.

325-326.

37.

S C O T T , P.M., C O L D W E L L , B.B.
Toxicology 9_ (2) 179-184.

38.

MEDVEDITSKOVA, N.D.

39.

G I L B E R T , J., S H E P H E R D , M.J, W A L L W O R K , M.A. & H A R R I S , R.G.

of Agricultural Research 20 (2), 125-135.

40.

LOUVEAUX,
J., M A U R I Z I O ,
A. & V O R W O H L ,
G.
1978.
Methods
of
Melissopalynology published in Bee World 59 (4) 139-157 and available as a
reprint from the International Bee Research Association, Gerrards Cross,
SL 9 0 N R , U.K.

Further

1974.

197 7.

1970.

Voprosy Pitaniya 5 57-60

CHEMISTS.

1983.

1059, Navasota, Texas,

1984.

Journal

1975.

(in Russian).

of

Official

the

Reports

USA

Methods

of

Association

of

of

the

Federal

I n d i a n Sugar 24 (10) 861.

S c i e n z a e t e c n o l o g a d e g l i A l i m e n t i _3.

1 973.

aus dem

Gebiete

J o u r n a l of the S c i e n c e of Food & A g r i c u l t u r e 28 4 4 3 -

J o u r n a l of Food H y g i e n i c S o c i e t y of J a p a n 11 (6) 4 8 6 -

1969.

1975.

&

N e w Zealand J o u r n a l of S c i e n c e 18 (3)

WIBERG,

G.S.

1971.

Food

and

Cosmetic

Voprosy Pitaniya 28 (6) 76-78.

1981.

Journal

Reading

Sugar
"An a 1 y s e n - M e t h o d e n
Fabrikanten".

der

I n t e r n a t i o n a 1en

Association Internationale
Verlag D - 2 0 0 0 Hamburg 22.

des F a b r i c a n t s

Vereinigung

de C o n f i s e r i e

der

Zuckerwaren-

edited

by

Benecke

" I n t e r n a t i o n a l L i s t of S e r i a l s d e a l i n g w i t h Sugar and r e l a t e d S u b j e c t s " .

Institut fur Zuckerindustrie edited by Buchdruckerei Gruss D-1000 Berlin.

130

I G U M S A , 15th Session P r o c e e d i n g s

1970.

I C U M S A , 16th

1974.

Session Proceedings

JUNK, W.R. & PANCOAST, H.M.

P L E W S , R.W.
SOUTHGATE,

1970.

1973.

Analytical

D.A.T.

1976.

H a n d b o o k of S u g a r s ,

Methods

Used

Determination

in S u g a r

of F o o d

AVI.

Refining,

Carbohydrates,

Elsevier.
Applied

Science.

S t a n d a r d A n a l y t i c a l M e t h o d s of the M e m b e r C o m p a n i e s of C o r n I n d u s t r i e s R e s e a r c h
Foundation, Corn Refiners Association Inc.
1001 C o n n e c t i c u t A v e n u e , N.W.,
W a s h i n g t o n DC 2 0 0 3 6 .
C r i t i c a l D a t a T a b l e s of
A v e n u e , N.W., W a s h i n g t o n
L E E , C.K. & K I N G , R.D.
1, A p p l i e d S c i e n c e .
B I R C H , G.G.
Science.

S. P A R K E R ,

the C o r n R e f i n e r s
DC 2 0 0 3 6 .
1978.

K.J.

Association,

Developments

(Eds)

1982.

in F o o d

Inc.,

Analysis

Nutritive

1001

Connecticut

Techniques

Sweeteners,

Volume

Applied

Honey
C R A N E , E.
Pollen

1979.

Honey:

5.
6.

S u r v e y , Bee

Research

Association

UK.

Analysis

B A R T H , O.M.
1970.
Honey Samples:
1.
2.
3.
4.

A Comprehensive

Anales

Academias

Brasil

Ciencia,

Microscopical

Analysis

of

P r e d o m i n a n t P o l l e n 42. (2 ) 3 5 1 - 3 6 6 .
S e c o n d a r y P o l l e n 42_ ( 3 ) 5 7 1 - 5 9 0 .
M i n o r P o l l e n 42 ( 4 ) 7 4 7 - 7 7 2 .
P o l l e n S p e c t r u m of S o m e H o n e y S a m p l e s of the S t a t e Rio' d e J a n e i r o
30. ( 4 ) 5 7 5 - 5 8 2 ( 1 9 7 0 ) .
H o n e y d e w in H o n e y of B e e s 30 ( 4 ) 6 0 1 - 6 0 8 ( 1 9 7 0 ) .
P o l l e n S p e c t r u m of S o m e H o n e y S a m p l e s of the S t a t e s B a h i a and C e a r a
_3i ( 4 ) 4 3 1 - 4 3 4 ( 1 9 7 1 ) .

B A R T H , O.M.
1971.
A p i d o l o g ie
Brasilianischer Honigtauhonige.
B A T T A G L I N I, M . & RIC C I A R D E LLI
Agaraia U n i v e r s i t a di Perugia
Honeys.

2_ (2 ) 1 5 7 - 1 6 7

M i k r o skopi sche

Bestandteile

D ' A L B O R E , G.
1971.
A n n a l i della Facolta di
2 6 2 7 7 - 3 0 3 P o l l e n S p e c t r u m of S o m e S i l i c i a n

B A T T A G L I N I , M . & R I C C I A R D E L L I D ' A L B O R E , G. 1 9 7 2 . A p i c u l t u r a M o d e r n a
6 4 - 8 2 1. B o t a n i c a l O r i g i n o f the H o n e y s of the P r o v i n c i a di P e r u g i a .

62

(4)

B A T T A G L I N I , M . & R I C C I A R D E L L I D ' A L B O R E , G.
1972.
Annali della Facolta di
A r g a r i a U n i v e r s i t a d i P e r u g i a 2 7 21 9 - 2 2 4 P r e l i m i n a r y S t u d i e s o n I t a l i a n
Unifloral Honeys.
B E U G , J.J.
Stuttgart.

1961.

Leitfaden

der

Po11enbestimmung

Gustav

C O W A N , A. C. & M I T C H E L L , T.J. 1 9 6 4 .
The Analyst
E x a m i n a t i o n of the O c c u r r e n c e of H o n e y d e w in H o n e y .
E R D T M A N , G.
1960.
Svensk Botanisk
Method: A Revised Description.

Tidskrift

131

54

89

Fischer

Verlag,

(1 0 5 6 ) 2 2 2 - 2 2 6

561-564

The

An

Acetolysis

FERRAZZI, P.
1974.
Apicultura Moderna 65 (1/2) 21-26
Dandelion Honeys
Piedmont and Lombardy, 1. Melissopalynological Analysis.

from

F O S S E L , A.
1968.
Z e i t s c h r i f t fur B i e n e n f o r s c h u n g _9_ (6) 252-258
Der
Wasserknoterich (Polygonum Amphibium L.) Eine Wenig Beachtete Tracht-pflanze.
FOSSEL, A.
1973.
Apidologie _4_ (1) 81-85
Verbreitung
Bedeutung der S i l b e r w u r z ( D r y a s Octopetala).

und

Pollen-analytische

FOSSEL, A.
1974.
Mitt. Naturwiss. Ver. Steiermark 104 87-118 Die
der Ostalpen, Dargestellt am Beispiel des Steirischen.

Bienenweide

FOSSEL, A. & RUTTNER, F.

1966.
Zeitschrift fur Bienenforschung 8 168-177 Die
K l e i n e W a c h s b l u m e , C e r i n t h e M i n o r L., Eine C h a r a k t e r i sti sche T r a c h t p f l a n z e
Pannonischer und Alpiner Trockengebiete.
F O S S E L , A. & V O R W O H L , G. 1974. Phyton (Austria) 16 (1/4) 49-55
und Fieberklee (Menyanthes Trifoliata).

Honigbienen

G E N I E R , G. 1966. A n n a l e s Abeille 9 (4) 271-321 Le Pollen des E r i c a c e a e dans

les Miels Franais.
GRIEBEL, C.
1930/1931.
Zeitschrift fur Untersuchung der Lebensmittel 59 (1)
63-79; (2/3) 197-211; (5) 441-471.
Zur Pollenanalyse des Honigs 61 256-305.
HODGES, D.
London.

1974.

The Pollen Loads of the Honey Bee

Bee Research

Association,

SAWYER, R.W.
1975.
Journal of the Association of Public Analysts 13 (2) 64-72
Melissopalynology in the Determination of the Geographical and Floral Origin of
Honey.
SAWYER, R.W.
1981.
Pollen
Cardiff Press, Wales.
HYDE, H.A. & ADAMS,
DEANS, A.S.C.
UK.

K.F.

1957.

Identification

1958.

for Beekeepers

University

An Atlas of Airborne Pollen Grains

Survey of British Honey Sources

Bee Research

College

Macmillan.
Association

L O U V E A U X , J.
1970. A n n e x e s M i c r o p h o t o g r a p h i q u e s aux M e t h o d e s O f f i c i e l l e s
D ' A n a l y s e , T o m e III, Atlas P h o t o g r a p h i q u e D ' A n a l y s e P o l l i n i q u e Des M i e l s ,
Service de la R e p r e s s i o n des Fraudes et Du C o n t r o l e de la Q u a l i t , Paris,
France.
H A Y E S , G.
Journal.

1925.

KAPP, R.O.
ERDTMAN, G.

Nectar Producing P l a n t s and their Pollen

1969.

Bee

Pollen and Spores Wm. C. Brown Publishers.

1969.

Handbook of Palynology

M O O R E , P.D. & W E B B ,
Hodder & Stoughton.

J.A.

WODEHOUSE, R.P.

1965.

PRYCE-JONES,

1942-3.

J.

The British

1978.

An

Pollen Grains

Hafner.

Illustrated

Guide

to Pollen

Hafner.

Proceedings of the Linnaen Society

MORRIS, N. & ROGERS, P.E.W.

1976.

Analysis

155 Sess. 129-174.

British Bee Journal Oct 152-153.

132

ALLEN, Rev M. Yate.

1937-38.
The M i c r o s c o p e
Dec 1 2 7 - 1 2 8 , Feb Vol II No. 1 15, M a r V o l II No. 2 2 9 - 3 2 , A p r i l Part III 8 2 - 8 4 , M a y Part IV 9 2 - 9 5 , J u n e P a r t V
134-137, July Part VI 161-164, Aug Part VII 183-186, Nov-Dec Part VIII 2 6 0 - 2 6 3 ,
Part IX 4 8 - 5 0 , P a r t X 9 9 - 1 0 2 , P a r t XI 2 2 8 - 2 3 1 .
D E O D I K A R , G.B., S H E N D E ,
B e e J o u r n a l 2 7 1.

S.G., T H A K A R ,

LOUVEAUX,

J. & V E R G E R O N ,

Ph.

1964.

Annales

KUBISOVA,

S. & NEDBALOVA, V.

1976.

Polnohospodarstvo

KERKVLIET,

J.D. & PUTTEN

KROPACOVA,
703-710.
KROPACOVA,

1969.

Acta

Z A N D E R , E.
1935.
vols
Berlin.
W.W.

KROPACOVA,
Agronmica
PEREZ,

B.S.

JAMES,

G.V.

S. &

1969.

RODRIGUEZ,
1969.

Acta

1972.

l'abeille

und

A.T.

Journal

of

1974.

1970.
the

22^ ( 1 0 )

Agriculturae

V.

17

(4)

Anales

Acta

943-959.

(3)

195-206.

de

of

Public

(4)

20

(3)

Agriculturae

bie

Blutenhonig

of P u b l i c A n a l y s t s

22^ (4)

Analysts

22

7 (3) 8 8 -

Agriculturae

Bromatologia

19

793-797.

Universitatis

Universitatis

Association

133

Indian

Agriculturae

Herkunft-sbestimmung

Acta

1965.

Univers itati s Agriculturae

Acta Universatatis

NEDBALOVA,

K.R.

329-347.

Apidologie

Journal, of the A s s o c i a t i o n

S. & N E D B A L O V A , V .
B r u o 22 (1) 8 9 - 9 9 .
&

1971.

Pollengestaltung

de

1975.

Universatatis

S. & N E D B A L O V A , V.

K U B I S O V A , S., K R O P A C O V A ,
(4) 7 1 9 - 7 2 9 .

HOWELLS,
93.

DER A.P.J.

S. & N E D B A L O V A , V.

S.

KROPACOVA,
455-461.

VAN

C.V. & V I N C H U R K A R ,

Facultas

377-406.
(4)

128-32.

4.
4.1

FISH ADD

SHELLFISH

ADLTERATIOH

R O O T I (TE

ANALYSIS

F i s h a n d s h e l l f i s h t e n d to a c c u m u l a t e h e a v y m e t a l s , o t h e r t o x i c e l e m e n t s a n d
stable fat-soluble organic compounds such as o r g a n o c h l o r i n e p e s t i c i d e s .
The
e x t e n t to w h i c h a n e l e m e n t is a c c u m u l a t e d v a r i e s w i t h t h e s p e c i e s .
Total
m e r c u r y and organochlorine pesticides are contaminants which should be looked
for r o u t i n e l y in f i s h .
GLC p r o c e d u r e s (Pearce et al (1), T e e n y (2), Schafer et
al (3), a n d K a c p u r z a k a n d C h v o j k a (4) c a n be u s e d for t h e d e t e r m i n a t i o n of
m e t h y l m e r c u r y i n f i s h , b u t 8 0 - 9 0 p e r c e n t is p r e s e n t i n t h i s m o r e t o x i c f o r m so
that total mercury determinations
by cold-vapour
atomic
absorption
s p e c t r o p h o t o m e t r y o r t h e d i t h i z o n e c o l o r i m e t r i c p r o c e d u r e a r e a d e q u a t e to
assess this e l e m e n t for routine purposes.
E l e m e n t s that can be accumulated b y
s o m e species of fish and shellfish include m e r c u r y , c a d m i u m , z i n c , copper and
arsenic.
T h r o w e r a n d E u s t a c e (5) d e s c r i b e t h e u p t a k e of h e a v y m e t a l s b y
o y s t e r s a n d s u b s e q u e n t d e t e r m i n a t i o n in the f l e s h .
In c a n n e d fish p r o d u c t s , as o t h e r c a n n e d g o o d s , t h e c o n d i t i o n of the c a n should
be checked.
C o n t a m i n a t i o n by toxic elements m a y be acquired during shelf life,
or from d e t e r i o r a t i o n of the c a n . Canned products should be e x a m i n e d for tin
and lead.
U t h e et al (6) h a v e c o m p a r e d w e t and d r y ashing m e t h o d s for the
d e t e r m i n a t i o n o f a r s e n i c . G a j e w s k a e t a l (7) d e s c r i b e t h e d e t e r m i n a t i o n o f lead
in f i s h b y m e t h o d s w h i c h i n c l u d e t h e u s e o f d i t h i z o n e . N a b r z y s k i ( 8 ) d e s c r i b e s
the d e t e r m i n a t i o n of m e r c u r y , copper and zinc b y dithizone.
M i r e x r e s i d u e s in f i s h m a y b e d e t e r m i n e d b y t h e m e t h o d s o f B o r t h w i c k e t a l ( 9 )
and H a w t h o r n e et al (10). Neff and Anderson (11) describe a UV method for
d e t e r m i n i n g n a p h t h a l e n e s in o i l - c o n t a m i n a t e d f i s h .
A n t i b i o t i c s h a v e o c c a s i o n a l l y b e e n u s e d to d e l a y b a c t e r i a l s p o i l a g e of fish
b e t w e e n catching and landing, a practice not generally recommended.
Bethea and
H i l l i g ( 1 2 ) d e s c r i b e a m i c r o b i o l o g i c a l m e t h o d r e p r o d u c e d in H a r t a n d F i s h e r
( 1 3 ) , f o r t h e d e t e r m i n a t i o n o f c h l o r t e t r a c y c l i n e in s u c h f i s h .
Perna (14) and
McCracken
et al (15) describe
methods
for some
other
antibiotics.
C h l o r a m p h e n i c o l is a n a n t i b i o t i c t h a t h a s c a u s e d c o n c e r n m o r e r e c e n t l y .
The i d e n t i t y under w h i c h fish or shellfish h a s been sold m a y c o m e
into
question.
If t h e f i s h c a n n o t b e i d e n t i f i e d u n e q u i v o c a l l y f r o m t h e m o r p h o l o g y ,
m i c r o s c o p i c a l e x a m i n a t i o n of the scales and disc e l e c t r o p h o r e s i s of the
proteins are used.
T h e AOAC g i v e s a k e y , w i t h p h o t o g r a p h s of s c a l e s , for the
identification of canned salmon.
For shrimps and prawns see the monograph by
F i n c h a r a a n d W i c k i n s ( 1 6 ) a n d t h e R e p o r t o f t h e G o v e r n m e n t C h e m i s t (U.K.) f o r
1975.
The CAC r e c o m m e n d e d standard for quick frozen gutted Pacific s a l m o n gives the
d i f f e r e n t s p e c i e s to w h i c h this t r i v i a l n a m e m a y b e a p p l i e d .
The standards for
c o d , h a d d o c k and ocean perch do l i k e w i s e and specify that the additives
phosphate and ascorbate should
n o t b e p r e s e n t i n e x c e s s o f 0.5 p e r c e n t as P 2 O 5
and 0.1 p e r c e n t as a s c o r b i c acid and t h e i r p r e s e n c e m u s t b e d e c l a r e d .
The drained weight for canned products should be determined
if this is
feasible.
F o r e x a m p l e , the CAC standard for canned s h r i m p s and p r a w n s sets a
m i n i m u m o f 6 0 p e r c e n t m / m d r a i n e d w e i g h t (2 m i n u t e s o n a s i e v e o f 2.8 x 2.8 m m
square holes).
A l t e r n a t i v e l y , f o r p r o d u c t s c a n n e d in o i l , t h e a m o u n t of o i l
m a y be decanted and measured.
The identity of the oil should be checked
a g a i n s t a n y c l a i m m a d e on the label b y c a r r y i n g out the i d e n t i t y tests d e t a i l e d
u n d e r "oils a n d fats", as w e l l as c h e c k i n g for r a n c i d i t y .
S e m i - p r e s e r v e d fish
m a y be e x a m i n e d for salt, total acidity and volatile acidity.

134

Aflatoxins
sauces.

have

been

reported

in

fermented

fish

products,

dried

fish

and

fish

The fish content of products is determined by the Stubbs and More method as for
m e a t p r o d u c t s , b u t the n i t r o g e n c o n t e n t of m a n y fish s p e c i e s
varies
considerably and the d e t e r m i n a t i o n is correspondingly less accurate.
Pearson
g i v e s the l a r g e n u m b e r of n i t r o g e n f a c t o r s p u b l i s h e d by the U.K. A n a l y t i c a l
Methods C o m m i t t e e of the Society for Analytical Chemistry (17).
The p h o s p h o l i p i d c o n t e n t of the fat in f a t t y f i s h is f a i r l y h i g h and the
c h l o r o f o r m - m e t h a n o l method of Bligh and Dyer will therefore give higher results
than an ether extraction.
The c h l o r o f o r m - m e t h a n o l extract is suitable for the
d e t e r m i n a t i o n of r a n c i d i t y v a l u e s if t h e s e are r e q u i r e d .
K i n g and R y a n (18)
have described a method for d e t e r m i n i n g shrimp in shrimp cocktail.
C a n n e d fish m a y c o n t a i n c r y s t a l s of s t r u v i t e (magnesium a m m o n i u m phosphate)
w h i c h is h a r m l e s s , b u t l o o k s l i k e g l a s s and c a n b e the s u b j e c t of c o m p l a i n t .
The c r y s t a l s w i l l d i s s o l v e in d i l u t e acid and g i v e the u s u a l t e s t s of
qualitative inorganic chemistry.
M i c r o s c o p i c e x a m i n a t i o n will d e m o n s t r a t e the
p r e s e n c e of g l a s s w h i c h d o e s n o t s h o w up b e t w e e n c r o s s e d p o l a r o i d s .
It m a y
a l s o be p o s s i b l e to h e a t a g l a s s f r a g m e n t s u f f i c i e n t l y to m e l t an e d g e and
confirm that this has taken place by r e e x a m i n a t i o n under the microscope.
Fish m a y be examined for preservatives by the usual methods.
Sulphur dioxide
is o f t e n u s e d to c o n t r o l o x i d a t i v e b l a c k e n i n g in the p r o c e s s i n g of l o b s t e r
t a i l s and s h r i m p s ( B a r n e t t (19)).
A l i m i t of 30 m g / k g is b e i n g d i s c u s s e d b y
the relevant CAC c o m m i t t e e .
Canned shrimps and prawns may contain formaldehyde
d e r i v e d f r o m t h e d e g r a d a t i o n of d i - and t r i m e t h y 1 a m i n e and t r i m e t h y 1 am ine
oxide.
F i s h oil c o n c e n t r a t e s s u c h as s h a r k l i v e r oil are sold as r i c h s o u r c e s of
v i t a m i n s A and D. D e t e r m i n a t i o n of v i t a m i n A and the usual rancidity tests are
normally, adequate to assess quality and genuineness.
It is w o r t h w h i l e to e x a m i n e s h r i m p s , p r a w n s and s m o k e d - c u r e d fish for a d d e d
colouring matters.
D i s c o l o u r a t i o n of s k i p j a c k tuna has been investigated by
Y a m a n a k a (20) and c o - w o r k e r s , and of fish jelly products by Fujita and M i y a m o t o
(21).
Caviar substitutes with added Black 7984 contain
unidentified
decomposition
products
of
the
colour
(Dragoni
and
Cantoni
(22)).
Discolouration of crabmeat is reviewed by Boon (23).
The CAC standard for canned shrimps and prawns p e r m i t s the following colours to
be added up to a m a x i m u m of 30 m g / k g ( s i n g l y or in c o m b i n a t i o n ) in the f i n a l
product - a m a r a n t h , beta-carotene, erythrosine, Ponceau 4R, Sunset Y e l l o w FCF,
tartrazine.
Calcium sodium EDTA is allowed up to 250 mg/kg and the addition of
citric acid is not specifically limited.
T h e p r o b l e m of "red tide", a t t r i b u t e d to the g r o w t h of the d i n o f 1 a g e 1 1 a t a
Gonyaulax tamarensis, is described by Ahles (24).
The dinoflagellate produces
saxitoxin, and shellfish which have fed on the organism in sufficient quantity
are toxic to h u m a n s , causing "paralytic shellfish poisoning".
The AOAC details
a biological method for its d e t e r m i n a t i o n using mice.
TLC-fluorescence m e t h o d s
are described by Bates and Rapoport (25) and Buckley, et al (26).
T e t r o d o t o x i n from p u f f e r f i s h ( F u g u s p p ) and c i g u a t e r a p o i s o n are the o t h e r
l i k e l i e s t c a u s e s of c h e m i c a l p o i s o n i n g f r o m the i n g e s t i o n of s e a f o o d s (NAS
(27)).
It w a s r e p o r t e d by H a s h i m o t o et al (28) that the C h i n a m a n
fish,
Glabrilutj anus n e m a t o p h o r u s , is one of a fairly large n u m b e r of species w i t h
w h i c h c i g u a t e r a p o i s o n i n g is a s s o c i a t e d .
A l l of t h e s e c a u s e s of c h e m i c a l
poisoning taken together account for no m o r e than a few outbreaks per year of
sufficient seriousness to be reported in the tchnical literature.

135

FISH SPECIES

IDENTIFICATION

PRINCIPLE
An aqueous (for uncooked fish) 6 M or 10 M urea extract (for cooked fish) of
fish m u s c l e is p r e p a r e d and an aliquot of the extract placed at one end of a
tube of acrylamide gel which is polymerized in situ.
The gel is subjected to
an electric potential and then stained, excess stain removed and the pattern of
stained p r o t e i n layers c o m p a r e d w i t h extracts from authenticated samples.
P a t t e r n s of cooked and raw fish of the same species are not identical, as
cooking denatures the protein.
APPARATUS
1.
Analytical
capacity)
2.

disc

Agla semi-micro

electrophoresis

apparatus

(6,

8 or

12

tube

syringe.

REAGENTS
1.
T R I S - g l y c ine Buffer (Stock Solution):
D i s s o l v e 28.8 g of
g l y c i n e and 6.0 g of T R I S - ( 2 - a m i n o - 2 - ( h y d r o x y m e t h y 1 ) - p r o p a n e - 1 , 3 diol) in 1 litre of distilled water and adjust the pH to 8.6 with 1 N
hydrochloric acid.
2.
TRIS-glyc ine Buffer Solution:
Dilute 1 part of Stock
(1) w i t h 13 p a r t s of w a t e r for use as the solvent for
reagents and as the electrolyte for electrophoresis.

Solution
the gel

3.
Gel reagents:
e.g. (a) "Cyanogum 41":
(A mixture of 95 percent
of a c r y l a m i d e m o n o m e r and 5 percent of b i s a c r y 1 a m i d e ; supplied by
B.D.H. Chemicals, Ltd. Poole, Dorset, U.K.); (b) Ammonium Persulphate
Solution:
D i s s o l v e 0.2 g of a m m o n i u m persulphate in 100 ml of th
TRIS-glycine buffer solution; (c) Be t a-d ime thy 1 am ino-pr o p ion i t r i 1 e
S o l u t i o n (1.60 percent):
D i s s o l v e 1.83 ml of b e t a - d i m e t h y 1 a m i n o propionitrile in 100 ml of TRIS-glycine buffer solution.
4.
P r o t e i n - s t a ining Solution:
percent acetic acid solution.

0.1

5.

Wash Solvent:

acid/water

6.

Sucrose Solution:

Methanol/acetic

percent.

Amido

Black

in 7

(21:3:96).

40 percent sucrose in distilled

water.

7.
Urea Solution:
A freshly prepared 10 M solution.
Dissolve 480
g of u r e a in w a t e r and d i l u t e to 1 litre. For 6 M d i s s o l v e 288 g of
urea in water, dilute to 1 litre.
PROCEDURE
Preparation of Protein Extracts:
(a)
R a w Fish:
Extract the m y o g e n proteins by h o m o g e n i s i n g the
muscle (10-25 g) with an equal weight of distilled water. Centrifuge
the h o m o g e n a t e at 3000 rpm for 20 m i n u t e s and decant off the clear
supernatant solution.
Dilute this solution with an equal volume of
40 percent sucrose solution and store at 0C until required.
(Note:
A l l o w frozen m u s c l e to thaw out b e f o r e e x t r a c t i n g the proteins as
above.
W h e r e the fish is breaded as in fish portions and fish
f i n g e r s , r e m o v e the outer layer of bread and cooked flesh. Extract
only the inner raw portion).

136

(b)
Cooked Fish:
B r e a k up 1 0 - 2 5 g of the c o o k e d f i s h m u s c l e and
s u s p e n d in t w i c e t h e v o l u m e ( 2 0 - 5 0 m l ) of 10 M u r e a s o l u t i o n .
Allow
t h e m i x t u r e to s t a n d o v e r n i g h t a t r o o m t e m p e r a t u r e a n d
remove
i n s o l u b l e r e s i d u e b y c e n t r i f u g i n g for 20 m i n u t e s at 3 0 0 0 r p m .
Use
the s u p e r n a t a n t s o l u t i o n d i r e c t l y for e l e c t r o p h o r e s i s .
(Note:
When
c o o k e d f i s h are r e q u i r e d as c o n t r o l s , p r e p a r e t h e m b y h e a t i n g on a
s t e a m b a t h for 30 m i n u t e s in c o v e r e d c a s s e r o l e s .
Extract with urea
s o l u t i o n as in (b) a b o v e ) .
Prepare

the a c r y l a m i d e

gel rods

for e l e c t r o p h o r e s i s

as

follows:

(a)
6.0 p e r c e n t a c r y l a m i d e g e l f o r m y o g e n p r o t e i n
separation:
D i s s o l v e 2.40 g of " C y a n o g u m 4 1 " in 20 m l of T R I S - g l y c i n e b u f f e r
solution.
A d d 10 m l o f t h e 1.60 p e r c e n t w / v
-dimethy1 aminop r o p i o n i t r i 1 e s o l u t i o n a n d 10 m l of t h e 0 . 2 0 p e r c e n t
ammonium
persulphate solution.
M i x , a n d t r a n s f e r t h e s o l u t i o n q u i c k l y to t h e
p r e v i o u s l y s t o p p e r e d g e l l i n g t u b e s (7.5 x 0.5 c m i.d.) w i t h a w i d e
n e e d l e (19 G x 2 in.) s y r i n g e (10 m l ) a n d a d j u s t t h e l e v e l s t o 6.5
cm.
T o o b t a i n a f l a t s u r f a c e at t h e t o p o f t h e g e l s , a p p l y a l a y e r
of w a t e r (2-3 m m ) c a r e f u l l y on t o p of t h e a c r y l a m i d e s o l u t i o n .
Use
an A g l a s y r i n g e w i t h the tip of its n e e d l e p l a c e d j u s t a b o v e the
solution.
S e t a s i d e to p o l y m e r i z e a t r o o m t e m p e r a t u r e .
It is
i m p o r t a n t that p o l y m e r i s a t i o n should take place w i t h i n 20-30 m i n u t e s
as d i f f u s i o n of t h e w a t e r i n t o t h e a c r y l a m i d e s o l u t i o n w i l l r e s u l t in
i n c o m p l e t e p o l y m e r i s a t i o n and an u n e v e n g e l s u r f a c e .
( b ) 7.5 p e r c e n t a c r y l a m i d e g e l s f o r s e p a r a t i o n o f u r e a e x t r a c t s o f
c o o k e d f i s h a r e p r e p a r e d as in ( a ) a b o v e u s i n g 3.0 g of " C y a n o g u m
41".
A f t e r p o l y m e r i s a t i o n is c o m p l e t e , d e c a n t o f f a n y u n p o 1 y m e r i s e d
m o n o m e r s o l u t i o n , r e m o v e the p o l y t h e n e s t o p p e r s and t r a n s f e r the
p r e p a r e d a c r y l a m i d e g e l r o d s to the e l e c t r o p h o r e s i s a p p a r a t u s .
Fill
w i t h the T R I S - g l y c i n e b u f f e r e l e c t r o l y t e .
C a r r y o u t an e l e c t r o p h o r e s i s p r e - r u n for 20 m i n u t e s at 2 0 0 v o l t s to
remove persulphate ions.
T h e n , b y m e a n s of an A g l a
semi-micro
s y r i n g e d e l i v e r 1 0 - 2 0 m i c r o l i t r e s of the p r o t e i n e x t r a c t s to the t o p s
of the g e l s .
In t h i s o p e r a t i o n , d i p t h e s y r i n g e t h r o u g h the u p p e r
e l e c t r o l y t e s o l u t i o n w i t h the tip of the n e e d l e j u s t a b o v e the
s u r f a c e of the a c r y l a m i d e g e l .
( N o r m a l l y , d u p l i c a t e analyses are
p e r f o r m e d o n e a c h e x t r a c t , a l l o w i n g f o r 4 e x t r a c t s to b e e x a m i n e d
s i m u l t a n e o u s l y in o n e a p p a r a t u s .
If d e s i r e d , a s e c o n d
disc
electrophoresis
apparatus
can
easily
be
connected
to
the
transformer).
C a r r y o u t t h e e l e c t r o p h o r e s i s f o r 30 m i n u t e s a t
c o n s t a n t v o l t a g e (280 v o l t s ) at r o o m t e m p e r a t u r e .
(in s o m e i n s t a n c e s
s l i g h t l y i m p r o v e d s e p a r a t i o n , p a r t i c u l a r l y for t h e u r e a e x t r a c t s , c a n
b e o b t a i n e d b y r u n n i n g for 50 m i n u t e s at the s a m e v o l t a g e in a c h i l l r o o m n e a r 0C).
W h e n the run has b e e n c o m p l e t e d , r e m o v e the u p p e r r e s e r v o i r , e m p t y
the b u f f e r s o l u t i o n and p u l l out the t u b e s . R e m o v e the gel c o l u m n s
f r o m the t u b e s by an i r r i g a t i o n m e t h o d u s i n g a s y r i n g e c o n t a i n i n g
d i s t i l l e d w a t e r and fitted w i t h a No. 1 size n e e d l e .
Insert the
n e e d l e b e t w e e n t h e g e l a n d t h e w a l l of t h e t u b e , r o t a t e t h e t u b e
s l o w l y and at t h e s a m e t i m e d i s c h a r g e w a t e r f r o m t h e s y r i n g e .
Repeat
t h i s o p e r a t i o n at t h e o t h e r e n d o f t h e t u b e .
The gel should then
s l i p out of the t u b e .
S t a i n t h e g e l f o r 2 0 m i n u t e s in t h e A m i d o B l a c k s t a i n i n g s o l u t i o n .
R e m o v e e x c e s s of d y e by w a s h i n g s e v e r a l t i m e s w i t h the a q u e o u s
methanol solvent.
A f t e r s t o r i n g the g e l s o v e r n i g h t in the s o l v e n t ,
the p r o t e i n z o n e s s h o u l d a p p e a r as d a r k b l u e d i s c s a g a i n s t a w a t e r clear background.
T r a n s f e r t h e g e l s to g l a s s t e s t - t u b e s a n d s t o r e

137

them in the s a m e solvent. E x a m i n e the gels against an i l l u m i n a t e d

w h i t e screen. This a r r a n g e m e n t is also suitable for p h o t o g r a p h i n g
the gels.
INTERPRETATIOH
This method only works on raw and partially cooked fish from which sufficiently
undenatured proteins are still extractable.
It does not work with fish cooked
under pressure, such as that found in fish paste.
The p r o c e d u r e for cooked fish m a y be s o m e w h a t i m p r o v e d by preparing the 7.5
p e r c e n t a c r y l a m i d e gel in stronger buffer in 6 M urea (1 v o l u m e stock TRISg l y c i n e b u f f e r + 7 v o l u m e s 6 M urea), and sufficient p e r s u l p h a t e to produce
p o l y m e r i s a t i o n in 2 0 - 2 5 m i n u t e s . The tubes m u s t be p e r f e c t l y clean, e.g. by
use of c h r o m i c acid, rinsing t h o r o u g h l y and drying. The run m a y be extended
from 30 to 45 m i n u t e s .
After staining, the excess dye may be r e m o v e d by
repeated washing with 25-30 ml of the aqueous methanol solvent in 50 ml conical
flasks. A l t e r n a t i v e l y , if s a m p l e s are being e x a m i n e d daily, it m a y be m o r e
convenient to use the continuous destaining system shown below:

Elmt'<?n

From pump

-Wing nut

Charcoal (animal)
column

Lid
-Gel
rack

Gel holder

-Rack

-Stainless
steel
-Stand

Stand

Staining & destaining. gel holder

made from a disposable
Petri dish

Solvent

^tank

A recycling filtration system for destaining polyacrylamide disc electrophoresis gels.

In this s y s t e m a p u m p c o n t i n u o u s l y r e c i r c u l a t e s the solvent and on passage

through the charcoal column the Amido Black is absorbed.
Destaining by either
m e t h o d is n o r m a l l y achieved overnight.
If rapid destaining (less than half
h o u r ) is r e q u i r e d an e l e c t r o l y t i c m e t h o d can be used.
In this latter system
the unbound dye is removed by electrophoresis in dilute acetic acid.
REFERENCE
Mackay,

I.M.

1972.

Journal

of the Association of Public Analysts

138

1J), 18.

4.2

DECOMPOSITION

ROTIHE A N A L Y S I S
D u r i n g s t o r a g e of fresh fish a n u m b e r of c h a n g e s take p l a c e due to the a c t i o n
of b a c t e r i a and e n z y m e s as w e l l as p u r e l y c h e m i c a l r e a c t i o n s .
A m o n g the
c h e m i c a l s p r o d u c e d are a m m o n i a ,
trimethylamine,
trimethy1 amine
oxide,
hypoxanthine and histamine.
These, as well as those chemicals responsible for
r a n c i d i t y in fish o i l , can be used to a s s e s s s p o i l a g e .
The a m o u n t s of t h e s e
s u b s t a n c e s f o r m e d d e p e n d s u p o n s p e c i e s of f i s h , b a c t e r i a p r e s e n t ,
time,
t e m p e r a t u r e , pH and p r o b a b l y o t h e r f a c t o r s , so it is n o t p o s s i b l e to fix
general limits for any of these quality parameters.
Also, the presence of one
or m o r e of t h e s e s u b s t a n c e s are o f t e n a n o r m a l q u a l i t y c h a r a c t e r i s t i c in
certain products prepared by fermentation, semi-drying, etc.
H o w e v e r , in fish
sold as f r e s h , the c o n c e n t r a t i o n of t h e s e c h e m i c a l s i n c r e a s e s w i t h t i m e and
results of their d e t e r m i n a t i o n are u s e d to s u p p o r t o r g a n o l e p t i c a n a l y s i s and
physical appearance as to whether a sample is unfit for food.
A l t h o u g h the r a t e at w h i c h the s p o i l a g e c h e m i c a l s are p r o d u c e d w i l l v a r y
according to the factors mentioned above, the general trend is as illustrated
by the following graphs.

The c h a n g e s in c o n c e n t r a t i o n s of
total volatile b a s e s , h y p o x a n t h i n e
and t r i m e t h y l a m i n e with the degree
of s p o i l a g e of cod. The e x a c t f o r m
of
the
changes
differs
for
different species.
From
Connel
i
t
"Control
of Fish Quality". (29)

Cteys in ice

The r e s u l t s a l s o v a r y w i t h the e x a c t m e t h o d of a n a l y s i s , and it is t h e r e f o r e

advisable to d e t e r m i n e each parameter on acceptable samples of the same species
when fresh and again when the samples seem to be starting to decompose.
Histamine levels are s o m e t i m e s high in scombroid fish such as mackerel and tuna
after these have been kept some time.
S h i m i z u et al (30) h a s s u g g e s t e d 100
m g / 1 0 0 gm as a tentative limit.
H i s t a m i n e seems to be largely derived from the
bacterial b r e a k d o w n of histidine present in the fish muscle.
Other indices that have been used include volatile acid n u m b e r , formic acid and
a c e t i c acid ( H i l l i g et al (31)).
I n d o l e is s u g g e s t e d to a s s e s s s p o i l a g e of
shrimps and a m m o n i a to assess spoilage of crabmeat.

139

TOTAL VOLATILE BASES

PRINCIPLE
The spoilage of fish stored in ice is due to bacterial and enzyme action which
results in the production of various volatile compounds such as trimethylamine
(TMA), d i m e t h y l a m i n e , a m m o n i a and volatile acids.
TMA is a reduction product
of T M A oxide during spoilage and the a m m o n i a is formed mainly as a product of
protein breakdown.
T o t a l v o l a t i l e b a s e s (TVB) i n c l u d e s all v o l a t i l e a m i n e s
plus ammonia.
T V B c a n be used as an i n d e x of s p o i l a g e of fish. T h i s m e t h o d
steam distils all volatile nitrogenous compounds from an alkaline solution of
the s a m p l e .
T h e s e are c o l l e c t e d in s t a n d a r d acid.
The u n r e a c t e d acid is
titrated and the TVB calculated.
APPARATUS
1.

Blender.

2.

Centrifuge

3.

Markham distillation

4.

Burette

(optional).

and

apparatus.

pipettes.

REAGENTS
1.

Trichloroacetic

acid - 5%

solution.

2.

NaOH - 2N.

3.

Hydrochloric

acid - 0.01 N.

4.

Rosolic acid

indicator - 1% solution in 10% ethanol.

5.

NaOH - 0.01 N.

PROCEDURE
W e i g h 100 g s a m p l e and blend w i t h 300 m l t r i c h l o r o a c e t i c acid.
F i l t e r or c e n t r i f u g e to o b t a i n a c l e a r e x t r a c t .
P i p e t 5 m l of the
extract into the Markham apparatus.
Add 5 ml 2N NaOH.
Steam distil into 15 ml standard 0.01N HCl containing 0.1 ml rosolic
indicator.
After distillation, titrate excess acid in the receiving
flask using standard 0.01 N NaOH, to a pale pink end point.
Do a procedural blank using 5 ml trichloroacetic
Titrate as before.

acid

with no sample.

CALCULATION

TVB

(mg/100 g sample) =

Where:

Vg
W
N
Vg

(n)

(V

~ V s>

(14)

(30 +

(5)
ml NaOH used for blank titration.
Water content of sample in g/100 g
Normality of NaOH standard solution
ml NaOH used for sample titration

REFERENCE
Pearson's Chemical Analysis of Foods, 8th Ed, 1981, 414-5.

140

>

TRIMETHYLAMINE (TMA) Il FISH

(Colorimetric Method)
PRINCIPLE
Fish stored in ice s p o i l s as a r e s u l t of b a c t e r i a l and e n z y m e a c t i o n w h i c h
r e s u l t s in the f o r m a t i o n of v o l a t i l e b a s e s , i n c l u d i n g t r i m e t h y 1 am ine (TMA).
TMA is the reduction product of trimethylamine oxide.
The amounts of TMA and
t o t a l v o l a t i l e b a s e s (TVB) p r e s e n t in the fish can be used as i n d i c e s of fish
spoilage.
T h i s m e t h o d i n v o l v e s the e x t r a c t i o n of v o l a t i l e b a s e s w i t h t r i c h l o r o a c e t i c
acid.
Bases other than T M A are complexed with formaldehyde.
Toluene is used
to e x t r a c t the T M A from a b a s i c m e d i u m and then r e a c t e d w i t h picric acid to
yield a coloured picrate salt.
This is analyzed spectrophotometrically.
APPARATUS
1.

Blender

2.

Centrifuge

3.

Pyrex glass-stoppered

4.

Spectrophotometer

test-tubes

in the visible

range.

REAGENTS
1.
Trichloroacetic
distilled water.

acid

(TCA),

7.5%

7.5

to

100

ml

with

2.
Toluene, anhydrous:
To r e m o v e i n t e r f e r e n c e s , shake 500 m l
toluene with 100 m l IN H2SO4, distil, and dry with anhydrous Na2S043.

Picric acid

solutions:

a)
S t o c k s o l u t i o n , 2% : D i s s o l v e 2 g in 100 ml of a n h y d r o u s
toluene.
(Caution:
Picric acid is highly sensitive to shock when in
a dry state.
In c o n t a c t w i t h m e t a l s and N H 3 , it p r o d u c e s p i c r a t e s
w h i c h are m o r e s e n s i t i v e to s h o c k than p i c r i c acid*
It is r e a d i l y
a b s o r b e d t h r o u g h the skin and is i r r i t a t i n g to eyes.
Wear heavy
rubber gloves and eye protection).
b) Working solution, 0.02% :
ml with anhydrous toluene.

Dilute

1 ml stock solution to 100

4.
Potassium Carbonate Solution, 50% w / w :
carbonate (K2CO3) in 100 ml water.

Dissolve 100 g potassium

5.
F o r m a l d e h y d e , 20% : Shake 1 litre 4 0 % f o r m a l d e h y d e s o l u t i o n
(HCHO) w i t h 100 g m a g n e s i u m c a r b o n a t e ( M g C O j ) until
nearly
colourless, and filter. Dilute 100 ml to 200 ml with water.
6.
H y d r o c h l o r i c acid (1+3) :
1 v o l u m e of c o n c e n t r a t e d
1.18) plus 3 volumes of distilled water.
7.

Anhydrous sodium sulphate

8.

Trimethylamine

HC1

(S.G.

(Na2S0^).

(TMA) standard

solutions:

a)
Stock solution 1 mg T M A / m l :
Add 0.682 g t r i m e t h y l
ammonium hydrochloride, to 1 ml HC1 (1+3) and dilute to 100 ml with
d i s t i l l e d w a t e r . C h e c k b a s i c N - c o n t e n t of 5 m l a l i q u o t by a d d i n g 6
m l 10% N a O H s o l u t i o n , s t e a m d i s t i l l i n g into 10 m l 4% boric acid and

141

titrate w i t h 0.1N H 2 S O 4 using m e t h y l

as in a protein determination.

red

- methylene

blue

indicator

b)
W o r k i n g s o l u t i o n , 0.01 m g T M A - N / m l :
Add 1 m l
solution to 1 m l HC1 (1+3) and dilute to 100 m l w i t h distilled

stock
water.

PROCEDURE
W e i g h 100 g minced or chopped, well-mixed sample into a blender.
Add
2 0 0 m l of 7.5% T C A s o l u t i o n and b l e n d .
Centrifuge blended solution
at 2000-3000 rpm until supernatant is practically clear.
Pipette aliquot (preferably containing 0.01-0.03 mg T M A - N ) into a 20
x 1 5 0 m m P y r e x g l a s s - s t o p p e r e d test tube and d i l u t e to 4.0 m l w i t h
distilled water.
(Note:
For f r e s h f i s h , u s e 4.0 m l a l i q u o t of
supernate, stale fish, use 1.0-3.0 m l aliquot of supernate.)
At t h e s a m e t i m e , p r e p a r e a b l a n k and s t a n d a r d s . For the b l a n k , u s e
4.0 m l d i s t i l l e d w a t e r .
For s t a n d a r d s , use 1.0, 2,0 and 3.0 m l of
w o r k i n g standard solution (0.1 m g T M A - N / m l ) and dilute to 4.0 ml w i t h
distilled water.
To e a c h t u b e ( b l a n k , s t a n d a r d s , s a m p l e s ) , add 1 m l H C H O (20%), 10 m l
a n h y d r o u s t o l u e n e and 3 m l K 2 C O 3 s o l u t i o n (100 g%).
Stopper testt u b e c o n t a i n i n g a b o u t 0.1 g a n h y d r o u s N a 2 S 0 ^ .
S h a k e w e l l to dry
toluene.
Pipette 5 m l toluene layer and add 5 m l picric acid working
solution (0.02%).
Mix and transfer to a spectrophotometric cell and
m e a s u r e absorbance at 410 nm against the blank.
(Note:
If original
a l i q u o t c o n t a i n s m o r e t h a n 0.03 T M A - N , d i l u t e e x t r a c t w i t h T C A
solution and repeat determination.)

CALCULATION
mg T M A - N / 1 0 0 g sample

A
A^

Where:

mg TMA-N/ml

300

std

solution x ml std solution used

ml of aliquot of sample used

absorbance

of

absorbance
of s am p 1 e

of standard

x 300

sample

approximation total
(100 g + 200 m l ) .

nearest

supernatant

to

absorbance

(ml)

INTERPRETATION
F r e s h f i s h g i v e s T M A v a l u e s of a b o u t 1 m g / 1 0 0 g and s p o i l e d s a m p l e s o v e r 8
m g / 1 0 0 g.
Values around 5 m g / 1 0 0 g could be taken as indications of doubtful
quality.
In fro z e n - s t o r e d g a d o i d f i l l e t s , the T M A i n d i c a t e s the e x t e n t of
m i c r o b i a l spoilage before the m u s c l e w a s frozen.

REFERENCE
Official Methods

of Analysis

of the AOAC, 1984,

142

18.031-.033.

INDOLE
PRINCIPLE
T h e I n d o l e t e s t is c o n s i d e r e d a r e l i a b l e t e s t f o r s h e l l f i s h f r e s h n e s s .
It is
a s s u m e d t h a t t h e o d o u r o f s p o i l e d s h e l l f i s h is c a u s e d in p a r t b y a c c u m u l a t i o n
of i n d o l e , a b r e a k d o w n p r o d u c t of t r y p t o p h a n .
The method involves
steam
d i s t i l l a t i o n , f o l l o w e d b y e x t r a c t i o n of the i n d o l e into c h l o r o f o r m , c o l o u r
d e v e l o p m e n t w i t h p - d i m e t h y l - a m i n o b e n z a l d e l y d e in a c i d m e d i u m and r e - e x t r a c t i o n
of the c o l o u r e d p r o d u c t into the acidic p h a s e .
It is t h e n
quantitated
spectrophotometrically.
APPARATUS
1.

Blender.

2.

Steam

3.

Distillation

4.

Condenser.

5.

Flask.

6.

Separatory

7.

Spectrophotometer

generator.
flask.

funnels:

125 m l ,

500 m l .

in t h e v i s i b l e

range .

REAGENTS
1.
Colour reagent:
D i s s o l v e 0.4 g p - d i m e t h y 1 - a m i n o - b e n z a 1 d h y d e
(use A R g r a d e w h i c h s h o u l d be p a l e y e l l o w in c o l o u r ) in 5 m l a c e t i c
a c i d a n d m i x w i t h 92 m l H 3 P O 4 a n d 3 m l a c e t i c a c i d .
2.
Glacial Acetic acid, purified:
If i t t u r n s p i n k w i t h t h e c o l o u r
r e a g e n t , p u r i f y a s f o l l o w s - a d d i n o r d e r s p e c i f i e d t o 1. L r o u n d b o t t o m f l a s k : 5 0 0 m l g l a c i a l a c e t i c a c i d , 25 g K M n O ^ a n d 20 m l H 2 S O 4 .
D i s t i l in a l l g l a s s s t i l l n o t m o r e t h a n 4 0 0 m l .
3.

Dilute

HCl, 5%:

D i l u t e 5 m l riCl to 1 0 0 m l w i t h

water.

4.
I n d o l e s t a n d a r d s t o c k s o l u t i o n 10 m g %:
A c c u r a t e l y w e i g h 20 m g
i n d o l e into a 200 m l v o l u m e t r i c f l a s k and d i l u t e to v o l u m e
with
alcohol.
K e e p r e f r i g e r a t e d and d i s c a r d a f t e r 2 w e e k s .
5.
I n d o l e s t a n d a r d w o r k i n g s o l u t i o n , 0.1 m g % :
P i p e t t e 1 m l of
i n d o l e s t a n d a r d s t o c k s o l u t i o n into a 100 m l v o l u m e t r i c flask and
d i l u t e v o l u m e to w i t h a l c o h o l .
6.

Chloroform,

7.

Saturated

8.

Alcohol,

AR

Na2S0^

Grade.
solution.

95%.

PROCEDURE
F o r o y s t e r m e a t s w e i g h 50 g .
For d r a i n e d crab m e a t or peeled
w e i g h 2 5 o r 50 g ( d e p e n d i n g u p o n a m o u n t o f i n d o l e e x p e c t e d ) .

prawns

T r a n s f e r w e i g h e d p o r t i o n to a h i g h - s p e e d b l e n d e r .
Add 80 m l w a t e r
( f o r o y s t e r o r c r a b m e a t s a m p l e ) o r 80 m l a l c o h o l (if s a m p l e is
shrimp)
and
blend
for
several
minutes,
until
homogeneous.
Q u a n t i t a t i v e l y t r a n s f e r m i x t u r e to d i s t i l l a t i o n f l a s k a n d r i n s e w i t h

143

m i n i m u m a m o u n t of s a m e s o l v e n t used for p r e p a r i n g m i x t u r e .
Add
g l a s s - b e a d s to d i s t i l l a t i o n f l a s k .
C o n n e c t f l a s k for
steam
distillation.
Gently apply steam until distillation is well started,
taking care not to pass in steam so vigorously as to cause excessive
foaming.
Collect 350 ml distillate in about 45 minutes (if alcohol
w a s used in preparation of sample, collect 450 ml).
W a s h condenser
w i t h a s m a l l a m o u n t of a l c o h o l and d r a i n into r e c e i v i n g
flask
containing distillate.
T r a n s f e r d i s t i l l a t e to a 5 0 0 m l s e p a r a t o r and add 5 m l d i l u t e HCl
(5%) a n d 5 m l s a t u r a t e d N a 2 S 0 ^ .
E x t r a c t w i t h 25 m l C H C l o .
Shake
v i g o r o u s l y for at l e a s t 1 m i n u t e e a c h t i m e .
A l l o w the 2 l a y e r s to
separate.
R e m o v e and save CHCI3 layer.
Re-extract the aqueous layer
In the final
2 m o r e t i m e s w i t h 20 m l and 15 m l p o r t i o n s of C H C I 3 .
e x t r a c t i o n , discard aqueous layer.
C o m b i n e the 25 m l and 20 ml CHCI3 extracts in a 500 ml separator and
w a s h w i t h 4 0 0 m l w a t e r c o n t a i n i n g 5 m l s a t u r a t e d N a 2 S 0 ^ and 5 m l
d i l u t e H C l (5%).
S h a k e v i g o r o u s l y and a l l o w the 2 l a y e r s to
separate.
Filter CHCI3 layer through a cotton plug into a dry 125 m l
separator.
W a s h the 15 ml CHCI3 extract, using the same wash water
as a b o v e .
F i l t e r t h r o u g h a c o t t o n p l u g i n t o the s a m e 125 m l
separator.
A d d 10 m l c o l o u r r e a g e n t to the c o m b i n e d C H C I 3 e x t r a c t s .
Shake
v i g o r o u s l y for exactly 2 m i n u t e s and let the acid layer separate as
c o m p l e t e l y as p o s s i b l e .
T r a n s f e r 9.0 m l acid l a y e r to a 50 m l
v o l u m e t r i c flask and dilute to v o l u m e with glacial acetic acid.
Mix
wel 1.
T r a n s f e r a p o r t i o n of the c o l o u r s o l u t i o n to a p h o t o m e t e r cell and
m e a s u r e t h e a b s o r b a n c e at 5 6 0 n m a g a i n s t a r e a g e n t b l a n k ( b l a n k
consists of 9 m l colour reagent, diluted with glacial acetic acid to
50 m l ) .
(Note:
C o l o u r s o l u t i o n m a y be diluted w i t h g l a c i a l acetic
acid containing 9 m l colour reagent/50 ml solution, provided blanks
are d e t e r m i n e d at the same dilutions).
Include a distillation blank
(i.e., o m i t t i n g a d d i t i o n of i n d o l e ) .
S u b t r a c t a b s o r b a n c e of the
d i s t i l l a t i o n blank from the sample.

STANDARD CURVE
P i p e t t e 0 m l , 1 m l , 3 m l , 5 m l , 7 m l of i n d o l e s t a n d a r d w o r k i n g
s o l u t i o n (1 p g / m l ) i n t o s e p a r a t e d i s t i l l a t i o n f l a s k s .
Add 80 m l
alcohol
and a f e w g l a s s b e a d s .
P r o c e e d as for s a m p l e .
Plot
a b s o r b a n c e at 560 nm against p g / m l indole.

CALCULATION

Indole,

p g / 1 0 0 g sample =

, V ^
(C) (V)

100
-5j-

Where :
Ax

absorbance of

sample

Ag

absorbance of

standard

concentration

of standard

volume of standard

weight

solution

solution used

(g) of sample

144

taken.

( pg/ml)

(ml)

INTERPRETATION
If the indole value exceeds 25
t o b e fresh.

y g / 1 0 0 gm sample, the product is considered

REFERENCE
Official Methods of Analysis of the AOAC, 1984,

145

18.072-.074.

not

HISTAMINE
PRINCIPLE
H i s t a m i n e is formed as a d e c o m p o s i t i o n product in fish. Highest levels are
often found in s c o m b r o i d - t y p e fish.
H i s t a m i n e can cause violent allergic
r e a c t i o n s in sensitive persons.
This method extracts the h i s t a m i n e into
m e t h a n o l , p a r t i t i o n s it into b e n z e n e - b u t a n o l and isolates it using a cotton
acid succinate column. The isolated
histamine is then coupled to a diazotized
aromatic
amine
to f o r m
a coloured
complex.
This
is
determined
spectrophotometric ally.
APPARATUS
1.

Blender.

2.

Volumetric

3.

Fine tip dropper with bulb.

4.

Ice bath and water bath.

5.

pH meter.

6.

Spectrophotometer.

flasks and pipettes.

REAGEHTS
1.

Methanol.

2.

Benzaldehyde

3.

Sodium hydroxide solution, 20%.

4.

Benzene - butanol mixture, (3+2) v/v.

(chloride-free).

5.
Cotton acid succinate (CAS):
dissolve 5 g anhydrous sodium
acetate (fused just before use) and 40 g succinic anhydride in 300 ml
glacial acetic acid in a 500 ml erlenmayer flask.
Cut 10 g asorbent
cotton into strips and i m m e r s e in solution. Attach a drying tube ..
containing a desiccant and heat at 100C for 48 hours.
Filter
reacted cotton (CAS) from solution and wash with water, then (1+9)
HC1, then w a t e r again and finally w i t h alcohol.
Dry the CAS in a
vacuum oven at 100C for 1 hour.
6.

Ethanol, 95%.

7.

Sulphuric acid, 0.40 N (j+ 0.02N - standardized).

8.
D i a z o n i u m reagent:
dissolve 0.1 g p-nitroani 1ine in 0.1N HCl
and dilute to 100 ml with 0.1 N HCl.
Store in refrigerator.
Dissolv e 4 g sodium nitrite in water and dilute to 100 ml.
Also
store in a refrigerator.
Prepare the diazonium reagent just before
use by placing 10 ml of p-nitroaniline in an ice bath for 5 minutes,
then add 1 ml of the N a N 0 2 and mix.
Let stand in the bath an
additional 5 minutes.
It is then ready to use.
9.
Coupling buffer:
dissolve 7.15 g sodium metaborate (NaB0 2 ) and
5.7 g sodium carbonate in water and dilute to 100 ml.
10.

Borax

(Na 2 B 4 0 7 .10H 2 0).

11.

Methyl isobutyl ketone.

146

12.
Barbital buffer:
dissolve 10 g sodium barbital in 1 litre water
and a d j u s t to pH 7.7 u s i n g a c e t i c acid and a pH m e t e r .
Store in a
refrigerator to prevent mould growth.
13. Histamine standard solution - dry histamine 2HC1 2 hours over
H2SO4.
D i s s o l v e 0.1656 g in w a t e r and d i l u t e to 100 ml (1 m g / m l ) .
T h i s is the s t o c k s o l u t i o n .
P r e p a r e the w o r k i n g s t a n d a r d fresh
w e e k l y by d i l u t i n g 10 ml of the s t o c k to 100 m l w i t h w a t e r , then
dilute 5 m l of this plus 5 m l of methanol to 100 m l with water.
This
final working standard is 5 pg/ml.
Store in a refrigerator.
(Note :
a l l w a t e r m u s t be d i s t i l l e d f r o m g l a s s .
Do
d e t e r g e n t s to c l e a n g l a s s w a r e .
Use fresh c h r o m i c acid
solution and rinse with distilled water).

not use
cleaning

PROCEDURE
Place 10 g minced sample in a small blender cup.
Add 50 ml methanol
and blend 2 minutes.
Transfer to a 100 ml volumetric flask, rinsing
with methanol.
Heat in a w a t e r b a t h at 60C for 15 m i n u t e s .
Cool
and dilute to volume.
Mix and filter.
Keep filtrate in a stoppered
flask.
This may be stored in a refrigerator several weeks.
Pipette
5 ml of the filtrate into a 150 m m glass stoppered test tube and add
S t o p p e r and shake
1 drop b e n z a l d e h y d e and 0.1 m l of 20% NaOH.
vigorously at least 25 times.
Let stand 5 minutes.
(if an emulsion
has formed, centrifuge to break it).
Prepare a CAS column by firmly placing a small plug of CAS (about 50
m g ) in a micro column or a 15 ml centrifuge tube with the bottom cut
off.
W a s h the plug w i t h three 15 m l p o r t i o n s w a t e r and t w o 3 m l
portions ethanol.
B l o w out last p o r t i o n of each w a s h w i t h a s m a l l
amount of air pressure.
T r a n s f e r the u p p e r o r g a n i c layer in the tube, u s i n g a f i n e - t i p
dropper with bulb, to the CAS column.
(Note: do not transfer any of
the lower aqueous layer).
Add 15 ml benzene-butanol to the centrifuge tube and again shake 25
times. Let stand 5 m i n and transfer upper layer to the CAS column as
before.
Rinse inside of CAS column with small amount of ethanol and
drain.
Blow out last bit with mild air pressure.
Add 3 ml ethanol,
drain and blow out as before.
Repeat with
washings.

two 3 ml portions

of w a t e r .

Discard

all s o l v e n t s

and

E l u t e h i s t a m i n e from the CAS by p i p e t t i n g 2.0 m l of 0.4N H 2 S 0 ^ into

column.
Drain and collect eluate.
Add 3 ml water.
Drain into same
eluate and blow out last drop.
Cool the eluate in an ice bath for 510 m i n u t e s .
Add 0.5 m l cold d i a z o n i u m r e a g e n t and let stand 5
m i n u t e s (still in ice bath).
P i p e t t e 0.5 m l c o u p l i n g b u f f e r w h i l e
swirling.
Let stand 5 minutes in the ice bath.
Add 0.25 g powdered
borax.
Shake for 30 s e c o n d s and let stand 15 m i n u t e s in the ice
bath.
Pipette 5.0 ml methyl isobutyl ketone and shake vigorously 25
times.
Pour b o t h l a y e r s (do not r i n s e ) into 150 m m test tube. Let stand 10
minutes at room temperature.
Transfer the upper organic layer using
a f i n e - t i p d r o p p e r , into a second 150 m m g l a s s s t o p p e r e d
tube
containing 5 ml of the barbital buffer.
(Note:
do not transfer any
of the lower aqueous layer - the transfer need not be quantitative).
Stopper and shake 25 times.
Let stand 10 minutes.

147

Transfer
cuvette.
ketone.

upper organic layer with a fine-tip dropper, to a 1 cm

Determine absorbance at 475 nm against pure methyl isobutyl

Conduct a procedural blank using 5 ml methanol.

Conduct a standard determination using 5 ml of the 5
standard solution.

pg/ml

working

CALCULATION
Ax - Ag
Histamine ppm

Where:

(50)

As - A b

Ax = absorbance of sample
As = absorbance of standard
absorbance of blank
factor:

25
"JQ"

100
X "y

= 50

INTERPRETATION
There is no established maximum histamine level. However, some agencies have
used a figure of 100 ppm as the level at which histamine would be of concern.
REFERENCE
Official Methods of Analysis of the AOAC, 1984, 18.064-.066.

148

BORIC ACID
PRINCIPLE
B o r i c acid and b o r a x w e r e c o m m o n l y used as p r e s e r v a t i v e s p r i o r to their
p r o h i b i t i o n by m a n y c o u n t r i e s .
B o r o n p r e s e r v a t i v e s are c o n s i d e r e d as less
desirable substances in view of their cumulative nature and their possible use
to m a s k i n c i p i e n t p u t r e f a c t i o n .
B o r i c acid and t u r m e r i c r e a c t to y i e l d a
c h a r a c t e r i s t i c red c o l o u r .
The p r o d u c t i o n of this c o l o u r f o r m s the b a s i s of
the following test.
APPARATUS
1.

Beakers, 200 ml.

2.

Glass wool.

3.

Pipettes.

4.

Test tubes.

REAGEHTS
1.

Concentrated

HCl.

2.
Turmeric paper:
Add 100 m l 8 0 % a l c o h o l to 1.5-2.0 g t u r m e r i c
p o w d e r in 250 m l c o n i c a l flask.
Shake 5 m i n u t e s and filter.
Dip
Hang paper to
sheets of Whatman No. 2 paper into the clear filtrate.
dry.
A f t e r 1 h o u r cut into 6x1 cm s t r i p s and store in t i g h t l y
stoppered container protected from light.
3.
Boric acid standard solution, (10 mg
H3BO3 in water and dilute to 100 ml.

H3BO3/111I):

Dissolve

1.0 g

PROCEDURE
Heat 25 g m i n c e d s a m p l e
beaker (do not overheat).
wool.
P i p e t t e 10 m l of
0.7 m l of c o n c e n t r a t e d
turmeric paper into the
in the air.
If a red colour

is formed

w i t h 50 m l of d i s t i l l e d w a t e r in a 200 m l
Chill then filter through a plug of glass
the cooled f i l t r a t e into a test tube.
Add
HCl s t o p p e r and m i x .
I m m e r s e s t r i p of
acidified filtrate.
Let turmeric paper dry

on the turmeric paper, proceed as follows:

T r a n s f e r 0.0, 0. 1, 0.2, 0.5, 0.75, 1.0, 2.50 and 5.0 m l of s t a n d a r d

H3BO3 solution (20 m g / m l ) to eight 15 ml test tubes.
Dilute each to
10 ml with water and add 0.7 ml HCl.
(These standards represent 0.0,
'0.02, 0.04, 0.10, 0.15, 0.20, 0.50 and 1.0% H 3 B O 3 in m e a t (based on
25 g s a m p l e e x t r a c t e d w i t h 50 m l w a t e r and 10 m l a l i q u o t used for
test. Keep tubes tightly stoppered to prevent evaporation.
Make identification on end of eight pieces of turmeric paper and dip
the unmarked ends into the series of standard solutions.
Dry strips
at r o o m t e m p e r a t u r e (about 1 hour).
Place dried standard strips
about 1 cm apart on a white filter paper background and bring sample
s t r i p b e t w e e n a d j a c e n t s t a n d a r d s for c l o s e c o l o u r m a t c h i n g .
If
c o l o u r falls b e t w e e n 2 s t a n d a r d s , e s t i m a t e the v a l u e .
(Disregard
streaks of colour that m a y develop at edge of test strip).
If colour
intensity of sample strip is beyond range of standards, repeat test
with dilution of meat filtrate.

149

4.3

TEXT

REFERENCES

1.

PEARCE,
I.D.,
BROOKS,
R.R. & REEVES,
R.D.
A s s o c i a t i o n of O f f i c i a l A n a l y t i c a l C h e m i s t s
59

2.

TEENY, F.M.
668-671.

3.

SCHAFER, M . L . , RHEA, U., PEELER,

1975.
J o u r n a l of A g r i c u l t u r a l and

4.

KACPURZAK,
J.L. & CHVOJKA,
O f f i c i a l A n a l y t i c a l Chemists

5.

THROWER,
546-553.

6.

U T H E , J . F . , F R E E M A N , H . C . , J O H N S T O N , J . R . & M I C H A L I K , P.
of the A s s o c i a t i o n of O f f i c i a l A n a l y t i c a l C h e m i s t s 57 ( 6 )

7.

GAJEWSKA, R., NABRZYSKI, M., L I P K A , E. & G A J E W S K I ,

i Chemia T o k s y k o l o g i c z n a _9_ ( 1 ) 1-5, 7-12.
F S TA 8

8.

NABRZYSKI,
M.
FSTA 8 4 R 1 8 2 .

9.

BORTHWICK,
P.W.,
COOK,
G.H.
& PATRICK,
Monitoring Journal,
7 ( 3 / 4 ) 144-145.7.

10.

HAWTHORNE,
J.C.,
FORD,
J.H.
E n v i r o n m e n t a l C o n t a m i n a t i o n and

11.

NEFF,
J.M.
& ANDERSON,
J.W.
1975.
Bulletin
C o n t a m i n a t i o n and T o x i c o l o g y 14 ( 1 ) 1 2 2 - 1 2 8 .

12.

B E T H E A , S. & H I L L I G ,
Agricultural Chemists

13.

HART,

14.

P E R N A , A . & C I E N , B.
1973.
V e t e r i n a r i e 27 5 8 9 - 5 9 0 .

15.

McCRACKEN, A., FIDGEON, S., O ' B R I E N ,

of A p p l i e d B a c t e r i o l o g y 40 ( 1 ) 61-66.

16.

FINCHAM,

1975.

S.J.

F.L.

&

A.S.

Journal

& EUSTACE,

1975.

R.
59

I.J.

Agricultural

1965.
1971.

H.J.

1971.

WICKINS.

Food

of

Technology

Chemistry

Chemia

J.M.

of

Modern

Food

Jr.

Australia

23

the

Identification

8_ ( 2 )

1974.

of

of

25

of

(11)

165-170

Pesticides

Bulletin

of

Environmental

of

Official

Springer-Verlag.

Italiana

& ANDERSON,

J.E.

Bromatologi_a

Association

Analysis

(4)

1974.
Journal
1363-1365.

1974.
258-264.

Societa

J.J.

in

the

Association

Toksykologiczna

Journal

della

the

A.
1976.
11R682.

& MARKIN.
G.P.
T o x i c o l o g y 11 ( 3 )

Atti

1976.

Food

1976.
Journal
(1) 153-157.

1973.

F.
48

and

of

J . T . , H A M I L T O N , C.H. & C A M P B E L L ,
Food C h e m i s t r y 23 ( 6 ) 1 0 7 9 - 1 0 8 3 .

Bromatologia

FISHER,

&

of

1 9 7 6.
Journal
(3) 655-657.

D.

delle

1976.

Commercial

Scienze

Journal

Shrimps

and

London.
17.

21

ANALYTICAL METHODS

COMMITTEE.

1966.

ANALYTICAL METHODS

COMMITTEE.

1971.

The An a1 y s t 96

ANALYTICAL METHODS

COMMITTEE.

1973 .

The A n a l y s t

18.

KING, F.J.
Analytical

19.

BARNETT,

20.

YAMANAKA,
H.
1975.
B u l l e t i n of
F i s h e r i e s (Nihon S u i s a n G a k k a i - s h i ) ,

D.

The A n a l y s t

& RYAN, J . J .
1976.
Journal
C h e m i s t s 59 ( 3 ) 6 4 4 - 6 4 9 .
1976.

Australian

Fisheries

of

35

the

(2)

98_
Association

of

Official

17-18.

the J a p a n e s e S o c i e t y of
Scientific
41 ( 3 ) 3 5 7 - 3 6 3 , 5 7 3 - 5 7 8 FSTA 8 2 R 1 0 0 .

151

21.

FUJITA, Y. & M I Y A M O T O , M.
1975.
Bulletin of the Japanese Society of
Scientific F i s h e r i e s (Nihon Suisan G a k k a i - s h i ) 41 (12) 1263-1269 FSTA 8
9R566.

22.

D R A G O N I , I. & CANTONI,
128. F S TA 8 1R42.

23.

BOON, D.D.

24.

A H L E S , M.D.
4R185.

25.

BATES, H.A. & RAP0P0RT, H.

Chemistry 2_3 (2) 237-239.

26.

BUCKLEY, L.J., IKAWA, M. & SASNER, J.J.

and Food Chemistry 24 (1) 107-111.

27.

T o x i c a n t s occurring naturally
Sciences, Washington, D.C.

28.

H A S H I M O T O , Y., K A M I Y A , H., KINJO, K. & YOSHIDA, C. 1975. Bulletin of the

Japanese Society of Scientific Fisheries (Nihon Suisan Gakkai-shi) 41 (8)
903-905.
FSTA 8 6R325.

29.

CONNELL, J.J. 1976. Control of Fish Quality

Long Garden Walk, Farnham, Surrey, U.K.

30.

S H I M I Z U , W. & H I B I K I , S.
1955.
Bulletin of the Japanese Society
Scientific Fisheries
(Nihon Suisan Gakkai-shi) 21 (5) 357-360.

31.

H I L L I G , F., S H E L T O N , L.R. & L O N G H R E Y , J.H.

1962.
Journal
Association of Official Agricultural Chemists 45 (30 724-731.

1975.
1974.

C.

1975.

Industrie

Alimentari

Journal of Food Science 4<D (4) 756-761.

14

(9)

126-

FSTA 8 1R28.

A m e r i c a n Journal of Public Health 64 (8) 807. FSTA 7

1975.

Journal

in

of Agricultural

1976.

foods.

and

Food

Journal of Agricultural

1973.

National

Academy

of

Fishing News Books Ltd., 1

of

of

the

Farther Reading
REPORT OF THE GOVERNMENT CHEMIST.
HATTULA, M.L.
(3) 331-337.

19 74.

1975.

H.M.S.O., London.

Bulletin of Environmental Contamination and Toxicology

EGAN, H., KIRK, R.S. & SAWYER, R.

8th Ed. Churchill Livingstone.
D R E W , R.E.
813-816.

1975.

Journal

1981.

12

Pearson's Chemical Analysis of Foods

of the Fisheries Research Board of Canada 32 (6)

WADA, T., SAWADA, T., HASEGAWA, K. & YAMANAKA, H. 1974. Bulletin of the Tokai
Regional Fisheries Research Laboratory (Tokai-ku Suisan Kenkyusho Kenkyu
Hokoku), 78 59-661
FSTA 8 11R636.
ZITKO, V., HULZINGER, 0. & CHOI, P.M.K.
1974.
Bulletin of
Contamination and Toxicology 12 (6) 649-653. FSTA 7 12R689.
W O L F R A M , J.H.
1975.
Order No. 75-19879.

Dissertation

Abstracts

International

SCHUTZ, D.E., CHANG, G.W. & BJELDANES, L.F. 1976.

of Official Analytical Chemists 59 (6) 1224-1225.

Environmental

B 36 (3) 1205,

Journal of the Association

R e c o m m e n d e d I n t e r n a t i o n a l Standard for Quick Frozen Gutted Pacific

1970. Codex Alimentarius Commission, Recommended Standard 36.

152

Salmon.

R e c o m m e n d e d I n t e r n a t i o n a l S t a n d a r d for Q u i c k F r o z e n f i l l e t s of C o d a n d
1971. Codex A l i m e n t a r i u s C o m m i s s i o n , R e c o m m e n d e d Standard 50.

Haddock.

R e c o m m e n d e d I n t e r n a t i o n a l S t a n d a r d for Q u i c k F r o z e n f i l l e t s of O c e a n
1971. Codex A l i m e n t a r i u s C o m m i s s i o n , R e c o m m e n d e d Standard 51.
R e c o m m e n d e d I n t e r n a t i o n a l S t a n d a r d for C a n n e d S h r i m p s
Alimentarius C o m m i s s i o n , Recommended Standard 37.

153

and P r a w n s .

1970.

Perch.

Codex

5.
5.1

MEAT AMD MEAT PRODUCTS

FRESH AMD FROZEN MEAT

ROUTINE ANALYSIS
The quality of fresh meat is normally verified by veterinary inspection carried
out at the a b a t t o i r s .
L a b o r a t o r y w o r k includes e x a m i n a t i o n for p e s t i c i d e
residues, drugs and anabolic agents. The identity and freshness of samples may
be questioned.
Chemicals such as ascorbic, erythorbic and nicotinic acids are sometimes used
to maintain a fresh appearance of the meat.
Ascorbic and nicotinic acids are
usually diluted with sucrose or glucose for sprinkling on meat, so examination
of an extract for these sugars by TLC is a useful preliminary test. The method
of S c h m a l l et al (l)(2) may be used for the q u a n t i t a t i v e d e t e r m i n a t i o n of
ascorbic and erythorbic acids. M e a n s of 111 end e r i s ing" m e a t w i t h p r o t e i n a s e s
such as papain (from papaya fruit), bromelain (from pineapples) and ficin (from
figs), i n c l u d e i n j e c t i o n into the c i r c u l a t i o n just b e f o r e slaughter.
Such
tenderising increases the level of free tyrosine.
Diethylstilboestrol is a growth promoter in meat-producing animals and in food.
R e s i d u e s are not a l l o w e d in foods in countries such as the USA because of the
c a r c i n o g e n i c p o t e n t i a l of the drug.
Residues in a n i m a l tissues m a y be
d e t e r m i n e d by the GLC m e t h o d s of D o n o h o et al (3) or K o h r m a n and M a c G e e (4).
S i m p l e r p r o c e d u r e s include the q u a l i t a t i v e paper c h r o m a t o g r a p h i c m e t h o d of
S m i t h and M c N e i l (5) and the T L C - f 1 u o r i m e t r y p r o c e d u r e of Ponder (6).
The
paper c h r o m a t o g r a p h i c t e c h n i q u e has a l i m i t of d e t e c t i o n of 10 ppb, w h i c h is
above the carcinogenic level in mice.
Mouse uterine assay was originally used
(Umberger et al (7)(8)). Kling and Lazar (9) have developed a radioimmunoassay
technique.
The technique of Heffter et al (10) may also be used.
In problems of identity the veterinarian or meat inspector and the analytical
The veterinarian will often be able to identify
chemist should work together.
b o n e s and cuts of r a w m e a t , o b v i a t i n g the n e c e s s i t y for laboratory work.
If
this is required for confirmation, precipitin tests and gel electrophoresis can
be used on raw and frozen meat and the latter technique may give useful results
as long as cooking has not been so severe as to d e n a t u r e c o m p l e t e l y all the
proteins present.
Identification of cooked meats can be difficult and the best
a p p r o a c h is via GLC and U.V. of the fat. Pig fat contains less stearate than
beef or sheep, but a little more linoleate. Horsefat contains more linolenate
and linoleate than the other three fats mentioned.
Q u a l i t a t i v e tests for c o l o u r , starch, sulphur dioxide and n i t r a t e should be
carried out on minced meat.
It may be worthwhile to examine other fresh meat
for p r e s e r v a t i v e s .
M i n c e d m e a t m a y c o n t a i n an e x c e s s i v e a m o u n t of fat, or
gristle or offals such as brains, feet, gut, chitterlings (smaller intestines),
manifolds, udders, sweetbreads (pancreas, sometimes thymus) tripe (rumen and
r e t i c u l u m ) m e l t s , lites (lungs), spinal cord, uteri, pigs' m a w s (stomach),
calves veils (inner lining of fore stomach) and skirt (diaphragm).
P r o x i m a t e a n a l y s i s is of seme a s s i s t a n c e in assessing general quality, for
example, minced meat should not contain more than a reasonable amount of fat.
The a n a l y s i s of the a m i n o - a c i d s m a k i n g up the p r o t e i n s m a y assist in
d e t e r m i n i n g the p r o p o r t i o n of p r o t e i n from a n i m a l or v e g e t a b l e sources. 3methyl histidine appears to occur only in protein from animal sources (Rangeley
and L a w r i e (11)). A d d i t i o n of v e g e t a b l e oils or fats to m e a t can be detected
both in cooked and uncooked products by examination of the sterols, by GLC of
the fats and by examination of the U.V. absorption.
L i v e r is h i g h in iron so the iron m a y be used s an a p p r o x i m a t e indication of
the a m o u n t of liver in a m i x t u r e , but the iron content has also been used to
a s s e s s the a m o u n t of blood added to h a m b u r g e r s (Hankin (12)).
Liver and
k i d n e y , as part of their p h y s i o l o g i c a l functions, process and in some cases
154

accumulate exogenous substances and their metabolites.

For e x a m p l e , analysis
of a n i m a l k i d n e y s and l i v e r s for h e a v y m e t a l s , e s p e c i a l l y c a d m i u m , lead and
arsenic, m a y be a useful guide to environmental contamination.
These organs
are appropriate ones to use w h e n screening for residues of veterinary drugs and
their metabolites.
Frozen chicken carcases m a y have been treated with salt and/or polyphosphates
as during spin-chilling.
This is said to improve the texture and also results
in the retention of more water than in untreated carcases.
The polyphosphate
is quickly hydrolysed to orthophosphate, but the total level of phosphate (P)
compared with nitrogen (N) appears a fairly reliable indication that the former
has been added.
P / N v a l u e s do n o t e x c e e d 0.1 in u n t r e a t e d b i r d s ( H a m e n c e and
K u n w a r d i a (13)).
The EEC m e t h o d for w a t e r in f r o z e n c h i c k e n is a l s o of
a s s i s t a n c e in a s s e s s i n g q u a l i t y .
E x t r a c t i o n of p h o s p h a t e s f r o m food is
d e s c r i b e d by D o r o and R e m o l i (14)).
TLC can be used i n s t e a d of PC (lida and
Y a m a b e (15)).
See also Togonai and Tanaka (16), Pesino et al (17) and Van Hoof
et al (18).
Freshness is measured by extract release volume and total volatile bases.
The
usual rancidity values on the fat m a y also be informative.
The total volatile
b a s e s i n c r e a s e as the lean p o r t i o n of the m e a t d e t e r i o r a t e s . T h e TVB test is
not reliable on smoked or cured products.
H a n k i n (12) d e s c r i b e s the d e t e r m i n a t i o n of i r o n to a s s e s s b l o o d added to
hamburger.
A l e v e l of 2 - 3 . 5 p e r c e n t m a y be a d d e d to m a s k the p r e s e n c e of
excess fat, iron levels being significantly higher than in normal meat if added
blood exceeds 1 percent.
In the p a p e r c i t e d , it is s u g g e s t e d t h a t i r o n is
d e t e r m i n e d v i a the c o l o u r w i t h t h i o g 1 y c o 1 1 i c acid but AAS w o u l d a l s o be
suitable.
T h e d e t e r m i n a t i o n of a d d e d b l o o d in g r o u n d b e e f is d e s c r i b e d by
K a r a s z et al (19).

155

TOTAL VOLATILE BASES

PRINCIPLE
The sample is distilled from m a g n e s i u m oxide under standard conditions and
volatile bases are titrated with boric acid.
APPARATUS
1.

Kjeldahl macro-distillation unit.

REAGENTS
1.

Magnesium oxide, solid.

2.

Antifoam.

3.

Boric acid, 2% aqueous

silicone preparation or octyl alcohol.

solution.

4.
M e t h y l red indicator. Dissolve 0.016 g m e t h y l red and 0.083 g
bromocresol green in 100 ml neutral denatured ethanol.
5.

Sulphuric acid, 0.1 N.

PROCEDURE
Add 100 ml of water to 10 g of the minced sample in a food blender
and homogenise for one minute. Wash into the distillation flask with
a further 200 ml of water. Add 2g magnesium oxide and a drop or two
of a n t i f o a m solution. Bring to the boil in exactly 10 m i n u t e s and
distil for exactly 25 m i n u t e s , using the same rate of heating, into
25 ml of 2 percent boric acid solution with added indicator in a 500
ml conical flask.
The tube on the end of the condenser should dip
b e l o w the boric acid solution.
Disconnect the splash-head, stop
heating and wash the condenser down with distilled water and titrate
the contents of the conical flask with 0.1 N H2SO4.
Carry out a
blank determination.
CALCULATION
Total volatile bases (as mg N per 100g flesh) = 14(titre-blank)
INTERPRETATION
The method is valid only for fresh or frozen meats and not for bacon and other
cured meats. Meat should give a value of less than 20 mg percent calculated on
a fat-free basis, values over 30 mg percent are considered to correspond to
staleness and a p p r e c i a b l e production of t r i m e t h y l a m i n e .
H o w e v e r , it is
important to compare with values obtained from satisfactory samples of the same
meat.
REFERENCE
P e a r s o n ' s , The C h e m i c a l Analysis of Foods, 7th Ed., 1976, 376-386, Churchill
Livingstone.

156

THI0BARBITURIC ACID (TBA) VALUE

PRIHCIPLE
Oxidized lipids are formed as fats b e c o m e rancid. Thiobarbituric acid will
react with these lipids to form a red-colored complex which can be determined
spectrophotometrically.
Malonaldehyde is one of the end products of oxidative
rancidity, and is believed to be involved in the reaction with TBA.
Therefore,
the TBA value is expressed as mg malonaldehyde per kg sample. The TBA test is
applicable to fatty foods (e.g. meat) as well as fats and oils.
APPARATUS
1.

Distillation apparatus (flask, condenser, receiver).

2.

Glas sbeads.

3.

Electric mantle.

4.

Pipette.

5.

Glass stoppered test tube.

6.

Sptrophotometer.

REAGENTS
1.

Hydrochloric acid - 4N.

2.

Antifoam liquid.

3.
Thiobarbituric acid reagent - dissolve 0.2883 g in 100 ml of 90%
glacial acetic acid.
PROCEDURE
Macerate 10 g of minced sample with 50 ml water for 2
then transfer to distillation flask, using 47.5 ml water
Add 2.5 ml 4N HC1. (pH should be 1.5). Add antifoam and
beads. Distil at a rate so that 50 ml of distillate is
10 minutes from the time boiling commences.

minutes and
for washing.
a few glass
collected in

Pipette 5 ml of the distillate into a glass-stoppered tube. Add 5 ml

TBA reagent. Shake and heat in boiling water for 35 minutes.
Prepare a blank similarly, using 5 ml water, for 35 minutes.
Cool the sample and blank tubes and measure the absorbance of the
sample against the blank at 538 nm using 1 cm cells.
CALC ULATIOH
TBA value (as mg malonaldehyde per kg sample) *= 7.8 x A
where A = absorbance of sample vs blank
(Caut ion: the method
factor to be valid).

must

be

followed

exactly

for

the

7.8

IHTERPRETATIOH
The TBA value increases as the fat in meats b e c o m e s rancid.
The value for
fresh meats, h o w e v e r , will vary. It is therefore necessary to establish TBA

157

v a l u e s for f r e s h m e a t s and for t h o s e m e a t s found to be g o i n g

o r g a n o l e p t i c tests.
These base values can then be used to evaluate
samples.

rancid
results

by
on

Journal

of

REFERENCE
T a r l a d g i s , B.G., W a t t s , B.M., Y o u n a t h a n , N.T. and D u g a n , L.
the A s s o c i a t i o n of the A m e r i c a n Oil C h e m i s t s Society 3_7, 44.

158

1960.

FREE FATTY ACIDS AND PEROXIDE VALUE

PRINCIPLE
Fat s p o i l a g e can be a s s e s s e d by e s t i m a t i n g the
peroxide value on a c o m m o n chloroform extract.

free

fatty

The FFA in the s a m p l e e x t r a c t is d i l u t e d w i t h a l c o h o l

titration with sodium hydroxide.
The FFA are expressed as
extracted fat. This method may be used for determining FFA
fat) and d r i p p i n g (edible b e e f and m u t t o n fat), as w e l l
extracted from fresh meats.

acids

(FFA) and

and n e u t r a l i z e d by
% oleic acid on the
in lard (edible pig
as t a l l o w and fats

The analysis for peroxide value depends on the reaction of potassium iodide in
acid solution with the peroxide oxygen followed by titration of the liberated
iodine with sodium thiosulphate.
The amount of iodine liberated is expressed
as m i l 1 i - e q u i v a l e n t s of p e r o x i d e o x y g e n per kg e x t r a c t e d fat.
This method
determines all substances which oxidize potassium iodide under the conditions
of the test.
These substances are generally assumed to be peroxides or other
s i m i l a r p r o d u c t s of fat o x i d a t i o n .
The test m a y be used to d e t e r m i n e the
peroxide value of fats extracted from fresh meat.
APPARATOS
1.

Erlenmeyer

flasks, 250 ml.

2.

Filter paper, Whatman No. 1 or equivalent.

3.

Water-bath with temperature

4.

Drying oven maintained

5.

Glass desiccator, charged with any efficient

regulator.

at 100 _+ 2C.
desiccant.

REAGENTS
1.

Chloroform.

2.

Anhydrous sodium

sulphate.

3.

Sodium hydroxide

standard

4.

Glacial acetic

5.

Potassium

6.

Sodium t h i o s u l p h a t e

7.

Phenolphthalein

8.

Starch solution as indicator, 1% (m/v) freshly

solution

(0.002N)

acid.

iodide, saturated

solution.

standard

indicator,

Freshly

prepared.

s o 1 u t i o n ( 0.0 IN)

1.0% solution

in 95%

ethanol.
prepared.

9.
E t h a n o l , 95% n e u t r a l i z e d w i t h 0.1 N s o d i u m h y d r o x i d e u s i n g
phenolphthalein solution as indicator.

1%

PROCEDURE
T r i m as m u c h fat as p o s s i b l e from the s a m p l e . M a c e r a t e 50 g of the
fat mechanically with 200 ml chloroform, filter.
Re-filter through a
paper containing anhydrous sodium sulphate and keep the filtrate in a
stoppered flask.

159

Pipette 20 ml of the filtrate into a tared evaporating dish.

E v a p o r a t e off the chloroform on a w a t e r - b a t h and then dry in an air
oven at 100'C for 3 hours.
Cool the dish in desiccator before
weighing.
(This is to determine the fat content).
D e t e r m i n e the FFA as follows: Pipette 20 ml of filtrate into a 250
m l conical flask. Add 20 ml of neutralized ethanol. Titrate with
0.02N sodium hydroxide solution using phenolphthalein as indicator.
Shake vigorously during the titration.
N e x t , d e t e r m i n e the peroxide value as follows:
Pipette 20 ml of
filtrate into a 250 ml glass-stoppered Erlenmeyer flask.
Add 15 ml
glacial acetic acid and 0.5 ml of saturated potassium iodide.
Allow
the solution to stand with occasional shaking for exactly 1 minute
and then add 30 ml of distilled water. Titrate with 0.01 N sodium
t h i o s u l p h a t e adding it gradually and with constant and vigorous
shaking.
Continue the titration until the yellow colour has almost
disappeared.
Add a b o u t 0.5 m l of s t a r c h i n d i c a t o r s o l u t i o n .
Continue the titration, shaking the flask vigorously near the end
point to liberate all the iodine from the chloroform layer. Add the
thiosulphate dropwise until the blue colour has just disappeared.
Conduct a blank d e t e r m i n a t i o n of the reagents daily.
The blank
titration m u s t
not exceed 0.5 ml of the 0.01N sodium thiosulphate
solution.
CALCULATION
Fat content in 20 ml of chloroform extract:
M = Ml - M2
Where:

Ml 3 mass in grams of dish and contents

M2 = mass in grams of empty dish

FFA (as oleic acid on extracted

% FFA

Where:

fat), % (m/m):

VI x N x 28.2
g

VI = volume in millilitres of sodium hydroxide

N normality of sodium hydroxide
28.2 = mil1iequivalent weight of oleic acid (include
factor of 100 for %)

(Note:
FFA are frequently expressed in terms of acid value instead
of X oleic acid. The acid value is defined as the n u m b e r of mg of
K0H n e c e s s a r y to neutralize 1 gram of extracted fat. To convert %
oleic acid to acid value, multiply the former by 1.99).
Peroxide v a l u e ,
extracted fat:

Where:

m il1iequivalents

of

peroxide

oxygen

per

V2 x N2 x 1000
M

PV

V2

volume in millilitres of sodium thiosulphate

solution used

N2

"

normality of sodium
used.

160

thiosulphate

solution

kg

of

ISTERPRETATIOM
A n i m a l fats largely consist of g l y c e r i d e s w h i c h are esters of the trihydric
alcohol glycerol with fatty acids of v a r i o u s types.
These acids are of the
long-chain v a r i e t y , having 14 to 18 carbon a t o m s in each chain. They m a y be
fully s a t u r a t e d , such as stearic and p a l m i t i c acid, or u n s a t u r a t e d , such as
oleic acid and linoleic acid where one or more reactive double bonds occur in
the side chain.
Unsaturated fatty acids are much more unstable chemically than are saturated
acids as o x i d a t i o n can readily occur at the site of the double b o n d s causing
the fatty acid chain to break down into fragments. This causes the development
of rancid off-odours and off-flavours.
Such oxidation is caused by atmospheric
oxygen and increases with increasing temperature.
It is catalysed by metals
such as copper and iron.
The extent to which rancidity has developed in a fat
is m e a s u r e d by its peroxide value.
Most fresh beef samples give a peroxide
value of 0-1. A value of 5. could be considered a maximum acceptable level.
A n i m a l tissues contain e n z y m e s called lipases w h i c h have the ability to
hydrolyse fats, splitting fatty acid molecules from the glycerol molecule. The
extent to which this occurs can be determined by measuring the free fatty acid
content.
In a good quality p r o d u c t , the free fatty acid content should not
exceed 1.2%, expressed as oleic acid in the extracted fat.
REFERENCES
Sully, B.D., 1954, Analyst 79

IUPAC Standard Methods for the Analysis of Oils, Fats and Derivatives, 6th Ed.,
Pergamon Press, 1979, Method ll.D.l.

161

5.2

MEAT PKODUCTS

ROUTIKE ANALYSIS
The d e t e r m i n a t i o n of the m e a t content of a meat product is made on the
assumption that a specific lean defatted meat has an average nitrogen content,
referred to as the nitrogen factor. For example, lean defatted raw beef is
taken to have an average nitrogen content of 3.55 percent.
If a sample is
found to contain 1.775 percent of nitrogen, the lean defatted beef content is
taken to be 50 percent.
Addition of the amount of fat in the sample gives its
total meat content.
Other a t t e m p t s to assess m e a t content include d e t e r m i n a t i o n of particular
amino-acids derived from protein such as 3-methylhistidine (Rangeley and Lawrie
(11)) and methionine.
The reliability of these values as assessments of meat
content is not yet well-established.
M e a t products m a y have to be examined for preservatives, especially SO2,
n i t r i t e and nitrate.
Other constituents that may have to be determined or
looked for are salt, added colour, starch, cereal and other fillers, textured
v e g e t a b l e protein, dried m i l k , soya bean m e a l , added phosphates and ascorbic
acid.
Sometimes, products contain excessive amounts of gristle or connective
tissue.
The level of h y d r o x y p r o l i n e present can be used to estimate the
p r o p o r t i o n of rind and gristle in a m e a t product. Protein from collagen and
skin contains 11-14 percent.
The EEC Intervention Board uses the formula
hydroxyproline x 8 = gristle/collagen.
Canned products should be examined for
lead, tin and arsenic. The lead content of meat products such as corned beef
in tins sealed by spot soldering may be high near the seal only. Tests that
are routine for canned foods such as v a c u u m , water capacity of the can,
h e a d s p a c e and drained weight (where appropriate) may also be made on canned
meat products.
Bacterial spoilage due to Clostridium species, Streptococcus faecalis, etc. may
be due to a m i x t u r e of poor hygiene and inadequate pickling.
Analysis for
salt, pH, nitrate and nitrite may be helpful.
The A M C (20) considered that the proportion of creatine and creatinine was a
suitable parameter for assessment of the quality of a meat extract and that in
addition the moisture, ash, chloride and total nitrogen should be determined in
routine analysis. The report gives a method for the estimation of creatine and
creatinine, republished in Official, Standardized and R e c o m m e n d e d Methods of
Analys i s .
The presence of offal and textured vegetable proteins in comminuted meat is
investigated mainly by histological techniques at the present time (Hole et al
(21)).
Most textured vegetable proteins contain more magnesium (around 6000
m g / k g ) than m e a t (300-3000 mg/kg).
Soya bean protein isolate from North
A m e r i c a is required to contain titanium diobxide as a marker but soya bean
products of lower protein content are not.
The immunochemical techniques of
double
diffusion,
single radial diffusion,
h a e m a g g 1utin ation
and
immunoelectrophoresis suffer from the disadvantage of requiring soya specific
agglutinin and tend to show cross-reactions with some other legumes. They are
s c r e e n i n g , q u a l i t a t i v e methods.
Po1 yacry1 am ide gel electrophoresis and
isoelectric focusing of extracts prepared with urea, 2 - m e r c a p t o e t h a n o l or
sodium dodecyl sulphonate work well on uncooked products but less well if the
meat is cooked.
Soya products used in meat include defatted flour (about 50 percent protein),
protein concentrate (70-73 percent protein) and soya protein isolate (over 90
percent protein). Soya flour may be detected m i c r o s c o p i c a l l y by looking for
"beaker cells", but these will be absent from protein isolate.
The proportion
of soya flour present may be assessed from the hemi-cellulose level (Bennett
(22)), or pentoses and pentosans (Fredholm (23)), or by the levels of fibre,
m a n g a n e s e and m a g n e s i u m ( F o r m o , H o n o l d and M a c L e a n (24)) or by gel

162

e l e c t r o p h o r e s i s ( P a r s o n s and L a u r i e (25), Guy et al (26)). The v a l i d i t y of a

method of analysis m a y be affected by the type of soya product present and by
the other constituents of a meat product.
F o r m o , et al (24) r e p o r t the f o l l o w i n g
percent protein) and ground beef:

Average
Concentration
Textured
soyflour

Ground
beef

figures

for

textured

Standard
Deviation
Textured
soyflour

soy

flour

Coefficient
of Variation

Ground
beef

Textured
soyflour

Ground
bee f

Magne s ium

2049
mg/kg

151
mg/kg

186
mg/kg

7.4
mg/kg

6.3%

4.9%

Mangane se

33.4
mg/kg

0. 125
mg/kg

3.4
mg/kg

0.025
mg/kg

10.1%

20%

Fibre

2.03%

0.01%

0.17%

13.3%

MEAT

(50

CONTENT

T h e m o i s t u r e , f a t , p r o t e i n and ash are d e t e r m i n e d and the c a r b o h y d r a t e or

cereal filler obtained by difference.
The amount of nitrogen derived from the
f i l l e r ( a s s u m e d to be p r e s e n t h e r e i n to the e x t e n t of 2 p e r c e n t , or 12.5
percent as protein) is subtracted from the total nitrogen and the rest assumed
to be d e r i v e d from the m e a t .
T h e t o t a l m e a t c o n t e n t is t h e n c a l c u l a t e d f r o m
the meat-derived nitrogen and fat contents, as follows:
nitrogen
Nitrogen factor

Lean defatted meat =

100

T h e n i t r o g e n f a c t o r is the n i t r o g e n c o n t e n t of lean d e f a t t e d m e a t of the

species present in the sample under analysis.
C o m m o n l y accepted figures are as
follows (Meat Products S u b - C o m m i t t e e of the Society for Analytical Chemistry,
U.K. (2 7) (28) (29) (,3 0 ) (3 1) (32) (33)):
Beef
Veal
Pork
Tongue
(Ox and Pig)
Ox Liver
Pig Liver
Kidney

3.55
3.35
3.45
3.0
3.45
3.65
2.7

Total meat = lean

Chicken, breast
dark meat
whole
Turkey,
breast
dark meat
whole
Blood

defatted meat plus

3.9
3.6
3.7
3.9
3.5
3.65
3.2

fat

Alternatively, it m a y be considered that lean m e a t contains 10 percent of fat

(interstitial).
T h e r e f o r e , lean m e a t = 1.1 x lean d e f a t t e d m e a t and the
remaining fat is regarded as free fat.
The amount of interstitial fat in lean
meat varies considerably but 10 percent is taken as an average for the purposes
of the calculation.
T h e r e are a n u m b e r of c o n s i d e r a t i o n s to be b o r n e in m i n d w h e n u s i n g this
calculation.
The topic has been reviewed by Pearson (34).
Incorporation of a
c e r t a i n a m o u n t of m e a t e x t r a c t or o t h e r n i t r o g e n s o u r c e s m a y lead to an

163

e r r o n e o u s l y h i g h a s s e s s m e n t of t h e m e a t c o n t e n t f r o m t h e n i t r o g e n f i g u r e .
M o n o s o d i u m g l u t a m a t e , s o d i u m g u a n y l a t e and s i m i l a r flavour e n h a n c e r s i n c r e a s e
the n i t r o g e n c o n t e n t in this w a y .
T h e n i t r o g e n f a c t o r s a p p l y to the f r e s h m e a t w i t h t h e n o r m a l a m o u n t of
accompanying water.
P r e s s e d and cured m e a t s such as c a n n e d b e e f , cured pates
and s a l a m i i n g r e d i e n t s h a v e had w a t e r r e m o v e d d u r i n g p r o c e s s i n g and t h e r e f o r e
c o n t a i n a h i g h e r p r o p o r t i o n of p r o t e i n .
For e x a m p l e , a factor of 4.7 has b e e n
s u g g e s t e d for t r a d i t i o n a l corned b e e f .
It is a d v i s a b l e to c a r r y out q u a l i t a t i v e tests for c a r b o h y d r a t e such as adding
i o d i n e to d e t e c t t h e p r e s e n c e o f s t a r c h .
It m a y b e p o s s i b l e to m a k e
a s s u m p t i o n s about the n i t r o g e n c o n t e n t of c a r b o h y d r a t e f i l l e r . The c a l c u l a t i o n
i s modified accordingly.
For e x a m p l e , if r u s k of an a s s u m e d or k n o w n n i t r o g e n
c o n t e n t of 2 p e r c e n t ( e q u i v a l e n t to 12.5 p e r c e n t p r o t e i n ) is p r e s e n t :
Carbohydrate

(C) = 100 - ( m o i s t u r e + fat + p r o t e i n + a s h )

N i t r o g e n a t t r i b u t a b l e to filler 0 . 0 2 C
Lean defatted meat =

(N - 0 . 0 2 C )
i t r o g e n factor

1 0 0

M e a t a n a l y s i s for the p r e s e n c e of a d u l t e r a t i o n b y other p r o t e i n s o u r c e s (e.g.

s o y a f l o u r , t e x t u r e d v e g e t a b l e p r o t e i n (TVP)) w h i c h g i v e an e l e v a t e d a p p a r e n t
m e a t c o n t e n t has b e e n an area of great a n a l y t i c a l i n t e r e s t in r e c e n t y e a r s . A
r e v i e w of soya p r o t e i n d e t e r m i n a t i o n s w a s carried o u t by O l s m a n and H i t c h c o c k
(35).
Soya can be d e t e c t e d and q u a n t i f i e d m i c r o s c o p i c a l l y , u s i n g h i s t o l o g i c a l
staining techniques.
(Flint and M e e c h (36)). A m o r e r e c e n t m e t h o d , the E L I S A
t e c h n i q u e ( E n z y m e - 1 i n k e d I m m u n o a s s a y ) is b e c o m i n g m o r e w i d e l y a c c e p t e d as a
r o u t i n e q u a n t i t a t i v e t e s t for Soya P r o t e i n ( H i t c h c o c k et al (37), C r i m e s et al
( 3 8 ) , G r i f f i t h s et a l ( 3 9 ) ) .
T h e a d d i t i o n of o f f a l s to m e a t p r o d u c t s can be d e t e c t e d h i s t o l o g i c a l l y (Hole et
al (21), F l i n t and M e e c h (36)) and this m e t h o d can p r o v i d e a s e m i - q u a n t i t a t i v e
a s s e s s m e n t . G e l e l e c t r o p h o r e s i s m e t h o d s c a n b e u s e d to i d e n t i f y o f f a l s b u t
m o r e r e s e a r c h is r e q u i r e d for q u a n t i t a t i v e a n a l y s i s .
F o r e i g n m e a t s (i.e. o t h e r m e a t t y p e s p r e s e n t in m e a t p r o d u c t s ) h a s for m a n y
y e a r s r e l i e d u p o n U n l e n l u t h s t e s t ( C a s t l e d i n e a n d D a v i s (40)) w h i c h is a
q u a l i t a t i v e m e t h o d f o r u n c o o k e d p r o d u c t s and is n o n - s p e c i f i c in m a n y c a s e s .
E l e c t r o p h o r e s i s m e t h o d s h a v e b e c o m e m o r e w i d e l y used for i d e n t i f y i n g m e a t types
( H o y e m and T h o r s o n ( 4 1 ) , C o d u r i a n d R a n d , (42)). Q u a n t i f i c a t i o n of f o r e i g n
m e a t in m e a t p r o d u c t s b y t h i s t y p e o f m e t h o d is as y e t in i t s e a r l y s t a g e s o f
development.

164

HYDROXTPROLINE
PRINCIPLE
T h e s a m p l e is h y d r o l y s e d in a h y d r o c h l o r i c acid s o l u t i o n c o n t a i n i n g stannous
c h l o r i d e . A f t e r n e u t r a l i z a t i o n , f i l t r a t i o n and d i l u t i o n , the h y d r o x y p r o l i n e is
o x i d i z e d by c h l o r a m i n e - T , f o l l o w e d by the f o r m a t i o n of a red c o m p o u n d w i t h 4d i m e t h y l a m i n o b e n z a l d e h y d e . T h i s is m e a s u r e d s p e c t r o p h o t o m e t r i c a l l y .
APPARATUS

1.

M e c h a n i c a l m e a t m i n c e r , laboratory size, fitted with a plate

w i t h h o l e s n o t e x c e e d i n g 4 m m in d i a m e t e r .

2.
Round or f l a t - b o t t o m e d h y d r o l y s i s f l a s k , c a p a c i t y about 200 m l ,
w i d e - n e c k e d , e q u i p p e d w i t h an a i r - c o o l e d or w a t e r - c o o l e d c o n d e n s e r .
3.
E l e c t r i c h e a t i n g d e v i c e (for e x a m p l e h e a t i n g m a n t l e , h o t p l a t e
e l e c t r i c a l l y h e a t e d sand bath).

or

4.
Filter
suitable).

is

paper discs, diameter

12.5 c m

(e.g. S a n d S N o . 187

5.

pH m e t e r .

6.

A l u m i n i u m or p l a s t i c s

7.

W a t e r b a t h , t h e r m o s t a t i c a l l y c o n t r o l l e d at 60 _+ 0.5C.

foil.

8.
S p e c t r o p h o t o m e t e r , c a p a b l e of b e i n g used at a w a v e l e n g t h of 558
+_ 2 n m .
REAGENTS

1.
Stannous chloride solution:
D i s s o l v e 7.5 g of stannous c h l o r i d e
d i h y d r a t e ( S n C l 2 . 2 H 2 0 ) in w a t e r , d i l u t e to 5 0 0 m l a n d a d d 5 0 0 m l o f
h y d r o c h l o r i c acid.
2.
H y d r o c h l o r i c a c i d , 6N s o l u t i o n :
c h l o r i c acid and w a t e r .

M i x e q u a l v o l u m e s of

3.
S o d i u m h y d r o x i d e , 10N s o l u t i o n :
Dissolve
h y d r o x i d e in w a t e r . Cool and d i l u t e to 100 m l .

40

4.
S o d i u m h y d r o x i d e , IN s o l u t i o n : D i s s o l v e 4 g of s o d i u m
in w a t e r . Cool and d i l u t e to 100 m l .

of

hydrosodium

hydroxide

5.
B u f f e r s o l u t i o n , p H 6.0:
D i s s o l v e in w a t e r :
50 g of c i t r i c
a c i d m o n o h y d r a t e , 12 m l o f a c e t i c a c i d , 1 2 0 g o f s o d i u m a c e t a t e
t r i h y d r a t e , 34 g of s o d i u m h y d r o x i d e , a n d d i l u t e to 1 0 0 0 m l w i t h
w a t e r . M i x this s o l u t i o n w i t h 200 m l of w a t e r and 300 m l of p r o p a n l - o l . This s o l u t i o n is stable for several w e e k s at 4C.
6.
Chloramine-T
toluenesulphonamide,
s u c c e s s i v e add 10 m l
pH 6.0. P r e p a r e this

reagent:
Dissolve
1.41 g o f
N-chloro-ps o d i u m salt ( c h l o r a m i n e - T ) in 10 m l of w a t e r and
of p r o p a n - l - o l and 80 m l of the b u f f e r s o l u t i o n
solution immediately before use.

7.
Colour reagent:
D i s s o l v e 10.0 g of 4 - d i m e t h y l a m i n o b e n z a l d e h y d e
in 35 m l of 60% p e r c h l o r i c acid s o l u t i o n and then s l o w l y add 65 m l of
propan-2-ol.
P r e p a r e this s o l u t i o n on the d a y of u s e .
(Note:
P u r i f i c a t i o n o f t h e 4 - d i m e t h y l a m i n o b e n z a l d e h y d e is n e c e s s a r y .
P r o c e e d as f o l l o w s :
P r e p a r e a s a t u r a t e d s o l u t i o n of the 4d i m e t h y l a m i n o b e n z a l d e h y d e in h o t 7 0 Z e t h a n o l . C o o l , f i r s t a t r o o m
165

t e m p e r a t u r e , and finally in a refrigerator.

After about 12 hours
filter on a Buchner funnel. Wash w i t h a little 70% ethanol.
Again
dissolve in hot 70% ethanol.
Add cold water and agitate thoroughly.
Repeat this procedure until a sufficient quantity of m i l k - w h i t e
crystals has been formed.
Place in the refrigerator overnight.
Filter on the Buchner funnel, wash with 50% ethanol and vacuum dry
over phosphorus pentoxide.
8.
Hydroxyproline standard solution:
Prepare a stock solution by
d i s s o l v i n g 100 m g of h y d r o x y p y r r o 1 i d i n e - a 1 p h a - c a r b o n ic acid
(hydroxyproline) in water.
Add 1 drop of hydrochloric acid solution
and dilute to 100 ml with water. On the day of use, dilute 1 ml of
the stock solution to 100 ml with water in a volumetric flask. Then
prepare four standard solutions by diluting 10, 20 and 40 ml of this
solution to 100 ml with water to obtain hydroxyproline concentrations
of 1, 2, 3 and 4 y g / m l respectively.
PROCEDURE
Prepare the sample as follows:
Raw meat and raw meat products:
Reduce intact meat to small cubes (approximately 0.5 cm"') by cutting
Either
it while it is cold (just b e l o w 0C) using a sharp knife.
place the sample in a container and seal the latter hermetically, or
vacuum pack the sample in a heat-resistant plastic film; then heat so
as to maintain a temperature of at least 70C for at least 30 minutes
in the geometrical centre of the sample; cool and proceed as follows:
(Note: in this w a y , the raw connective tissue is softened and less
resistant to homogenization by mincing). Cooked meat and cooked meat
products:
Make the sample homogeneous by passing it at least twice
through the meat mincer and mixing. Keep the homogenized sample in a
completely filled, airtight, closed container and store it in such a
way that d e t e r i o r a t i o n and change in c o m p o s i t i o n are prevented.
Analyse the sample as soon as possible, but always within 24 hours.
Weigh, to the nearest 1 mg about 4 g of the prepared sample into the
h y d r o l y s i s flask.
Ensure that none of the sample adheres to the
s i d e - w a l l of the flask.
Add some boiling chips and 100 +_ 1 ml of
stannous chloride solution.
Heat to gentle boiling and maintain for
16 hours under reflux (conveniently overnight). (Note: if desired
the hydrolysis may alternatively be accomplished in two periods, each
of 7 to 8 hours on consecutive days.
This alternative procedure has
been proved experimentally
to y i e l d r e s u l t s that are n o t
significantly different from those obtained with a single hydrolysis
period of 16 hours).
Filter the hot hydrolysate through filter paper into a 200 ml
volumetric flask. Wash the filter three times with 10 ml portions of
h o t h y d r o c h l o r i c acid s o l u t i o n and add the w a s h i n g s to the
hydrolysate.
M a k e up to the m a r k w i t h w a t e r .
(Note:
the
h y d r o l y s a t e can be kept at this state for at least one week under
refrigeration).
Using a pipette, transfer into a beaker a v o l u m e V ml of the
hydrolysate so as to obtain a hydroxyproline concentration.within the
range 1 to 4 p g / m l after dilution to 250 ml. (Note:
In most cases,
V w i l l be of the order of 5 to 25, depending on the amount of
connective tissue present in the sample). With the aid of the pH
m e t e r , adjust the pH to 8 +_ 0.2 by dropwise addition first of 10 N
sodium hydroxide solution then, when approaching the required pH, of
1 N sodium hydroxide solution.
Remove the tin hydroxide precipitate
by filtering the solution through a filter paper washing the
precipitate on the filter at least twice with 50 ml portions of water
and collecting the filtrate and washings in a 250 ml volumetric
flask. Make.up to the mark with water and mix.

166

Transfer 4.00 ml of this solution into a test tube and add 2.00 ml of
the chloramine-T reagent. Mix and leave at room temperature for 20 +_
1 minute.
Add 2.00 ml of the colour r e a g e n t , mix t h o r o u g h l y and cap the tube
with aluminium or plastics foil.
Transfer the tube quickly into the
w a t e r bath, controlled at 60^+0.5C and heat for exactly 20 m i n u t e s .
Cool under running tap w a t e r for at least 3 m i n u t e s .
M e a s u r e the
absorbance at 558 +_ 2 nm in a glass cell using the spectrophotometer.
Carry out in duplicate the same procedure, substituting water for the
diluted hydrolysate.
(Note:
if the absorbance of the blank exceeds
0.040, a fresh colour reagent should be prepared and, if n e c e s s a r y
the 4-dimethylaminobenzaldehyde should be repurified).
Carry out the same procedure again but s u b s t i t u t i n g 4.00 m l of each
of the four diluted standard hydroxyproline solutions for the diluted
hydrolysate.
Plot the measured absorbance values, corrected for the
blank value,
against
the c o n c e n t r a t i o n s
of
the
standard
hydroxyproline solutions and construct the best fitting straight line
through the plotted points and the origin.
CALCULATION
C a l c u l a t e the h y d r o x y p r o l i n e c o n t e n t ,
percentage by weight, from the formula:

H,

of

the

sample,

as

5h
H

w x V

Where :
h = hydroxyproline concentration, in micrograms per millilitre,
of the diluted hydrolysate
w = weight, in grams, of the test portion
V = volume, in millilitres, of solution taken for
to 250 ml.

dilution

INTERPRETATION
Duplicate determinations should be run to determine the repeatability of the
analysis. The v a l u e s on d u p l i c a t e d e t e r m i n a t i o n s should not differ by m o r e
than 5% of the arithmetic mean of the two values.
The presence of hydroxyproline is an indication of excess collagen-containing
skin and connective tissue in the meat product. Pearson's Chemical Analysis of
Foods, 8th Ed., gives the following table of hydroxyproline content of tissue:
Hydroxyproline
Content (Z)

Tissue (fat free)

Col1 agen

13.4 - 14.5

Tendon

11.2 - 13.2

Tendon with muscle

12.3 - 13.3

Cooked

11.0 - 12.0

skin

0.002- 0.07

Skeletal muscle

none

Plant materials

167

HITRATE AMD NITRITE

PRINCIPLE
Sodium nitrite in a product produces the characteristic red colour c o m m o n l y
associated with cured meats. The nitrite itself combines with the red m e a t
p i g m e n t , m y o g l o b i n , to form the relatively stable pink compound which is
carried through the smoking and cooking operation.
Nitrate is added to provide a reservoir of nitrite. The nitrate is active only
after conversion to nitrite.
This conversion is brought about by certain
bacteria normally present in meat.
An aromatic primary amine will react with an acidified solution of nitrite to
produce a diazonium salt.
If this salt is then condensed or coupled with
another primary aromatic amine (N-l-naphthylethyenediamine"2 HC1) an aminoazo
compound (a red dye) is formed which is measured in a spectrophotometer at 538
nm.
This method determines nitrite directly and the nitrate
reduction of the nitrate to nitrite using a cadmium column.

indirectly,

after

APPARATUS
1.

Beakers.

2.

Glass column and glass wool.

3.

Volumetric flask.

4.

Pipettes.

5.

Spectrophotometer.

REAGENTS
1.
Zinc rods,
cadmium column.

ca 15 cm x ca 6 mm

- 3 to 5 rods are needed

2.
C a d m i u m sulfate solution, 30 g/L
SO^)3.8H20 in water and dilute 'to 1 litre.
3.

- dissolve

37

of

per

(Cd

Hydrochloric acid, 0.1 N.

4.
A m m o n i u m b u f f e r s o l u t i o n , pH 9.6 - 9.7.
D i l u t e 20 ml
concentrated HC1 with 500 ml water. Add 10 g Na EDTA dihydrate and
55 ml concentrated NH^OH. Dilute to 1 litre. Check pH.
5.
Sodium nitrate standard solution - dissolve 1.2329 g NaN3 in
water and dilute to 100 ml in a volumetric flask. This is the stock
Pipette 5 m l of this solution into a litre v o l u m e t r i c
solution.
flask. Dilute to v o l u m e with water. This is the working solution
(61.6 y g / m l ) and must be prepared on the day of use.
6.
Su 1 ph an i 1 am id e solution, 0.2%. Dissolve 2 g sul phani 1 am ide in
800 ml water, w a r m i n g if necessary.
Cool and add 100 ml of
concentrated HC1. Dilute to 1 litre.
7.
C o u p l i n g r e a g e n t , 0.01X. D i s s o l v e 0.25 g N - l - n a p t h y l ethylenediamine dihydrochloride in water and dilute to 250 ml. Store
in tightly closed b r o w n - g l a s s bottle.
Solution may be kept in a
refrigerator for up to one week.
8.

Hydrochloric acid, (1+1).

169

9.
Borax solution.
Dissolve
water.
Cool before using.

50

g NajB^O^lOl^O

10.

Activated

11.
3H2O

P o t a s s i u m f e r r o c y a n i d e s o l u t i o n , 1%.
in w a t e r a n d d i l u t e to 1 l i t r e .

12.
Zinc
dihydrate
litre.

in

1 litre

warm

charcoal.
D i s s o l v e 10 g K ^ F e ( C N ) ^ .

a c e t a t e s o l u t i o n , 21.6%.
D i s s o l v e 216 g zinc acetate
a n d 30 m l g l a c i a l a c e t i c a c i d in w a t e r a n d d i l u t e to 1

13.
S o d i u m n i t r i t e s t a n d a r d s o l u t i o n - d i s s o l v e 1.0 g o f N a N 2 i n
w a t e r a n d d i l u t e to 100 m l (1% s o l u t i o n ) in a v o l u m e t r i c f l a s k .
This
is the s t o c k s o l u t i o n .
P i p e t t e 5 m l of t h i s i n t o a l i t r e v o l u m e t r i c
f l a s k a n d d i l u t e t o v o l u m e w i t h w a t e r (50 p g / m l ) .
T h i s is t h e
w o r k i n g s o l u t i o n and m u s t b e p r e p a r e d f r e s h e a c h d a y .
PROCEDURE
Preparation

and p r e - t r e a t m e n t of cadmium

column:

a.
P l a c e 3 t o 5 z i n c r o d s in t h e c a d m i u m
sulphate
solution
c o n t a i n e d i n a b e a k e r (1 l i t r e o f c a d m i u m s u l p h a t e s o l u t i o n is
s u f f i c i e n t for p r e p a r i n g o n e c a d m i u m c o l u m n ) .
b.
R e m o v e the s p o n g y m e t a l l i c c a d m i u m d e p o s i t f r o m the z i n c
e v e r y 1 o r 2 h o u r s b y s w i r l i n g t h e m in t h e s o l u t i o n o r r u b b i n g
against each other.
c.
A f t e r 6 to 8 h o u r s , d e c a n t t h e s o l u t i o n a n d w a s h
t w i c e w i t h 1 l i t r e of w a t e r , t a k i n g c a r e t h a t the
c o n t i n u o u s l y c o v e r e d w i t h a l a y e r of l i q u i d .

rods
them

the d e p o s i t
cadmium
is

d.
T r a n s f e r the c a d m i u m d e p o s i t w i t h 400 m l of h y d r o c h l o r i c acid
s o l u t i o n to a l a b o r a t o r y m i x e r , b l e n d f o r 10 s e c o n d s a n d r e t u r n t h e
c a d m i u m s l u r r y to t h e b e a k e r .
e.
O c c a s i o n a l l y stir up the c a d m i u m d e p o s i t
L e a v i n g it o v e r n i g h t in h y d r o c h l o r i c a c i d .

with

a glass

f.
S t i r to r e m o v e b u b b l e s a n d d e c a n t t h e s o l u t i o n .
c a d m i u m s l u r r y t w i c e , e a c h t i m e w i t h 1 l i t r e of w a t e r .
cadmium under liquid).
g.
Fit a glass w o o l plug
to c o n t a i n t h e c a d m i u m .

to the b o t t o m

Wash
(Keep

of the g l a s s c o l u m n

h.
W a s h the c a d m i u m into the g l a s s c o l u m n
h e i g h t o f t h e c a d m i u m b e d is a b o u t 17 c m .

with

water

rod.

the
the

intended

until

the

i.
D r a i n t h e c o l u m n o c c a s i o n a l l y d u r i n g f i l l i n g , t a k i n g c a r e n o t to
a l l o w t h e l e v e l of t h e l i q u i d to f a l l b e l o w t h e t o p o f t h e c a d m i u m
b e d . E l i m i n a t e i n c l u s i o n s of g a s (by s t i r r i n g w i t h a w i r e or t h i n
glass rod).
j.
F i l l t h e r e s e r v o i r c o m p l e t e l y w i t h w a t e r and c h e c k the f l o w of
the liquid.
It s h o u l d f l o w o u t at a r a t e n o t e x c e e d i n g 3 m l p e r
m i n u t e to a v o i d the r i s k o f i n c o m p l e t e r e d u c t i o n .
k.
W a s h t h e c a d m i u m c o l u m n s u c c e s s i v e l y w i t h (1) 25 m l of 0.1N H C 1 ;
(2) 50 m l of w a t e r ; a n d (3) 25 m l of the a m m o n i a b u f f e r s o l u t i o n .
T h e c o l u m n is n o w r e a d y for u s e .

170

Check of cadmium column reducing

efficiency:

a.
Pipette 20 m l of sodium nitrate working standard solution (61.6
y g / m l ) and s i m u l t a n e o u s l y add 5 m l of a m m o n i a b u f f e r s o l u t i o n into
the r e s e r v o i r on top of the c a d m i u m c o l u m n .
(Note:
61.6
yg/ml
nitrate is equivalent to 50.0 y g/ml nitrite).
Collect the effluent
in a 100 ml v o l u m e t r i c flask.
b.
W h e n the r e s e r v o i r is n e a r l y e m p t y ( a b o u t 20 m i n ) , w a s h the
w a l l s w i t h a b o u t 15 m l of w a t e r ; r e p e a t the s a m e t r e a t m e n t w i t h
another 15 ml portion of water (takes about 5 m i n , each time).
After
t h i s p o r t i o n h a s r u n i n t o the c o l u m n as w e l l , c o m p l e t e l y fill the
reservoir with water.
c.
After nearly 100 ml of effluent has been collected, remove the
f l a s k f r o m u n d e r the c o l u m n and d i l u t e to the m a r k w i t h w a t e r .
Pipette 10 ml of the eluate into a 100 ml volumetric flask.
d.
Add 10 m l sulphanilamide solution followed by 6 m l of (1+1) HC1,
mix and leave the solution for 5 minutes at room temperature in the
dark.
Add 2 m l coupling reagent, m i x and leave the solution for 3 to
10 minutes at room temperature in the dark.
Dilute to the m a r k with
water.
e.
M e a s u r e the a b s o r b a n c e of the s o l u t i o n in a 1 cm cell u s i n g a
s p e c t r o p h o t o m e t e r at a w a v e l e n g t h of 538 m m .
O b t a i n the n i t r i t e
concentration of the eluate from the calibration curve.
f.
If the n i t r i t e c o n c e n t r a t i o n of the e l u a t e as d e t e r m i n e d f r o m
t h e c a l i b r a t i o n c u r v e is b e l o w 0.9 y g of s o d i u m n i t r i t e p e r
m i l l i m e t e r (i.e. 90% of the c r i t i c a l v a l u e ) , the c a d m i u m
column
should be rejected.
Preparation of calibration

curve:

a.
Pipette the following amounts of the NaN2 working standard (50
y g / m l ) i n t o s i x 100 m l v o l u m e t r i c f l a s k s : 1 m l , 2 m l , 4 m l , 5 m l , 10
m l , 20 ml. Make each flask to v o l u m e using water.
b.
P i p e t t e 10 m l of e a c h of the a b o v e i n t o six 100 m l v o l u m e t r i c
flasks.
In a s e v e n t h f l a s k , p i p e t t e 10 m l w a t e r .
(These r e p r e s e n t
yg/ml,
N a N 0 2 c o n c e n t r a t i o n s of 0, 0.5, 1.0, 2.0, 2.5, 5.0 and 10.0
respectively).
c.
Add w a t e r to all s e v e n f l a s k s to a v o l u m e of a b o u t 60 m l .
Add
10 m l of s u l p h a n i l a m i d e s o l u t i o n , f o l l o w e d by 6 m l of (1+1) H C 1 to
e a c h f l a s k , m i x and l e a v e the s o l u t i o n for 5 m i n u t e s at r o o m
temperature in the dark. Add 2 m l of coupling reagent, m i x and leave
the s o l u t i o n for 3 to 10 m i n u t e s at r o o m t e m p e r a t u r e in the d a r k .
Dilute each flask to the mark w i t h water.
d.
Prepare a calibration
concentration ( y g / m l ) .

curve

by

plotting

absorbance

against

Prepare the sample by finely mincing and mixing.

(Start analysis as
soon as possible after preparation - the minced sample may be stored
only overnight in a closed, well-filled container in a refrigerator).
W e i g h , to the n e a r e s t 1 m g , 10 g of the s a m p l e i n t o a 2 0 0 m l
v o l u m e t r i c f l a s k w i t h the aid of a f u n n e l and 100 m l of hot w a t e r .
Add 5 m l of borax-solution and 0.5 g activated charcoal.
Heat, w i t h
r e p e a t e d a g i t a t i o n , for 15 m i n u t e s on a b o i l i n g w a t e r b a t h .
Allow
the flask and its contents to cool to room temperature.

171

After not less than an hour, add successively 2 ml each of potassium

ferrocyanide and zinc acetate solutions.
Mix thoroughly after each
a d d i t i o n . D i l u t e to 200 ml w i t h w a t e r , m i x and a l l o w the flask to
stand for 30 m i n u t e s at room t e m p e r a t u r e .
C a r e f u l l y decant the
s u p e r n a t a n t liquid and filter it through a No. 44 filter paper to
obtain a clear filtrate.
Pipette 20 ml of the filtrate into the reservoir on top of the column
and simultaneously add 5 ml of ammonia buffer solution.
Collect the
effluent from the column in a 100 ml volumetric flask.
When the reservoir is nearly empty, wash the walls with about 15 ml
of w a t e r ; r e p e a t the s a m e t r e a t m e n t with another 15 m l portion of
water.
A f t e r t h i s p o r t i o n h a s r u n i n t o t h e c o l u m n as w e l l ,
c o m p l e t e l y fill the reservoir with water. After n e a r l y 100 m l of
effluent has been collected, remove the flask from under the column
and dilute to the mark with water.
Pipette an aliquot of the eluate (but not more than 25 ml) into a 100
m l v o l u m e t r i c flask.
At the s a m e time, pipette 25 m l of s a m p l e
f i l t r a t e (containing less than 100 pg N 0 ) into a 50 ml v o l u m e t r i c
flask.
For b o t h , p r o c e e d
calibration curve".

as

specified

above

under,

"preparation

CALCULATION
Reducing efficiency of Cd column:
Z efficiency =
Where:

C =
S =
1.23 =

C x 50 x 1.23 x 100
S

yg/ml nitrite from calibration curve

yg/ml nitrate working standard (61.5 yg/ml)
factor to convert nitrite to nitrate

Ha nitrite (HaN0 2 ) without reduced nitrate

Where:

C x 100 x 200
g x y

ppm NaN0 2

(HaHC^)

C m yg/ml nitrite from calibration curve

S = sample weight in grams
V = ml aliquot of filtrate taken

Ha nitrite (HaN0) including reduced nitrate (HaN(>3)

ppm

NaN02

Where:

Ha nitrate

C =
S =
V =

reduced

C x 100 x 200 x 5
g~y

pg/ml total nitrite from calibration curve

sample weight in grams
ml aliquot of eluate taken

(HaHC^)

ppm nitrate = (NaN0 2 /NaN0 3

Where:

NaNOj

calculation - NaN0 2 calculation) x 1.23

1.23 = factor to convert nitrite to nitrate.

172

of

REFERENCES
ISO 2918:1975, Meat and Meat Products - Determination of Nitrite

Content.

ISO 3091:1975, Meat and Meat Products - Determination of Nitrate

Content.

173

5.3

TEXT REFERENCES

1.

SCHMALL,
1486.

2.

S C H M A L L , M., P I F E R , C.W. &

1954.
A n a l y t i c a l C h e m i s t 26

3.

D O N O H O , A.L., J O H N S O N , W.S., S I E C K , R.F. & S U L L I V A N , W.L.

J o u r n a l of the A s s o c i a t i o n of O f f i c i a l A n a l y t i c a l C h e m i s t s
785-792.

4.

K O H R M A N , K.A. & M A C G E E , J .
1977.
A n a l y t i c a l C h e m i s t s 60 5 - 8 .

5.

SMITH,

6.

P O N D E R , C.
C h e m i s t s _57

7.

UMBERGER,
806-15.

E.J.,

8.

UMBERGER,
S c i e n c e 18

E.J., C U R T I S ,
221-6.

9.

K L I N G , R.E. & L A Z A R , A.
Sept.

10.

HEFFTER,
Rundschau

A., V O C K S , R.
68 (11) 3 6 7 .

11.

RANGELEY,
59.

W.R.D. & L A W R I E ,

12.

H A N K I N , L.
C h e m i s t s 48

13.

H A M E N C E , J.H. & K U N W A R D I A , G.H.

1974.
P o l y p h o s p h a t e s and M o i s t u r e
Meat Products
J o u r n a l o f t h e A s s o c i a t i o n o f P u b l i c A n a l y s t s 12 8 5 - 9 2 .

in

14.

D O R O , B. & R E M O L I ,
(1) 2 3 .

S.

1963.

Bolletino

14

15.

IIDA,

T.

1971.

Journal

16.

T O G O N A I , Y. & T A N A K A , K.
(Japan) .

17.

P E S I N O DE B A T T I S T I S , P. & M A R Z A D O R I , F.
20 (1-2) 4 1 - 7 .

18.

VAN
HOOF,
J.,
Diergeneeskundig

19.

K A R A S Z , A.B., A N D E R S E N , R. & P O L L M A N ,
A s s o c i a t i o n of O f f i c i a l A n a l y t i c a l C h e m i s t s

20.

AMC

21.

HOLE,
337.

M., P I F E R ,

W.G. & M c N E I L ,

N.H.,

WOLLISH,

E.E.

GASS,

1972.
of

G.H.

&

of

the

J.M.

G.H.

Chemist

25

R. & G A I N E R ,

H.

Association

Chemist

Association

GASS,

Analytical

DUSCHINSKY,

Analytical

R.A.

of

G.

of

Official

1958.

1976.

the

1974.

Journal

Association

of

Food

Shokukin

Eiseigaku

1974.

54

143-

Provinciali

413.

Zasshi

G.

1_1

Agricultural

Acta. Medica

P. & V A N D E N B R A N D E ,
4 3 (5) 2 6 0 - 7 3 .

Animal

Lebensmittel-

Technology

Chimico

Chromatography

of

63

By-Lines No. 2

of O f f i c i a l

Laboratorio

Analytical

Journal

Deutsche

of

Official

Endocrinology

1959.

1972.

of

1973.
56
(4)

44_ 1 0 8 4 - 1 0 8 7 .

Food & Drug A d m i n i s t r a t i o n

WALTHER,

MOLLAERT,
Tijdschrift

E.G.,

CURTIS,

1976.

1965.
Journal
1122-26.

1951

&

1953.

Journal

the

J.M.

&

E.G.

WOLLISH,
1521.

1974.
Journal
919-923.

T. & Y A M A B E ,

Analyst

C.W. &

L5

(5)

331-6

Veterinaria

1974.

R.
19 76 .
Journal
59 (6) 1 2 4 0 - 3 .

Vlaam s

of

the

76. 3 2 9 - 3 3 3 .

ROBERTS,

M.P.

&

BRYCE

174

JONES,

K.

1964.

Analyst

89

332-

22.

BENNETT, O.L.
1948.
Chemists 31 513-9.

23.

FREDHOLM, H.

24.

F O R M O , M.W., H O N O L D , G.R. & M A C L E A N , D.B.

1 974.
Journal
Association of Official Analytical Chemists 57 (4) 841-6.

25.

P A R S O N S , A.L. 5. L A W R I E , R.A.
62.

26.

GUY, R.C.E., J A Y A R A M , R. & W I L L C O X ,

Food Agriculture 24 (12) 1551-63.

27.

Analyst 1961 86 557-60.

28.

Analyst 1963 88 422-3, 583-4.

29.

Analyst 1964 89 630 1.

30.

Analyst 1965 90 256-7, 579-80, 581.

31.

Analyst

32.

Analyst 1967 92 326-7.

33.

Analyst 1968 93 476-7, 478-9.

34.

P E A R S O N , D.
1975.
The E x a m i n a t i o n of M e a t
Reference to the Assessment of the Meat Content.

35.

OLSMAN, W.J. & HITCHCOCK, C. Development in Food Analysts Techniques, Vol

2 225-60 (Ed. R.D. King) Applied Science Publishers, 1980.

36.

FLINT, F.0. & MEECH, M.V.

37.

H I T C H C O C K , C.H.S., BAILEY, F.J., C R I M E S , A.A., DEAN, D.A.G. & DAVIS,

1981 Journal of Scientific Food Agriculture 32 157.

38.

C R I M E S , A.A., B A I L E Y ,
Proc edures 164.

39.

G R I F F I T H S , N.M., B I L L I N G T O N , M.J. & G R I F F I T H S , W.

Association of Public Analysts 19 113.

40.

C A S T L E D I N E , S.A. & DAVIES, D.R.A.

Public Analysts _6_ 39.

41.

HOYEM, T. & THORSON, B.

18. 737.

42.

CODURI, R.J. & RAND, A.G. 1972.

Analytical Chemists 55 461.

1967.

Journal

of the Association

of Official

Agricultural

Food Technology 7A 93-4.

1 972.

Journal of Food Technology

C.J.

1973.

Journal

of

of

the

(4) 455-

Scientific

1966 91 538-9.

1978.

Analyst 103 252.

F.J. & H I T C H C O C K ,

1970.

Products w i t h Special
Analyst 100 73-81.

1968.

C.H.S.

1981.

1981.

P.J.

Analytical

Journal of the

Journal of the A s s o c i a t i o n of

Journal of Agricultural and Food

Chemistry

Journal of the A s s o c i a t i o n of Official

Further Reading
LAWRIE, R.A.

1979.

Meat Science Pergamon.

W I G G I N S , G.S. & W I L S O N , A. 1976.

Wolfe Medical Publications Ltd.
W I L S O N , N.R.P. (Ed) 1981.
Control
Elsevier.

A Colour A t l a s of M e a t & Poultry

Meat & M e a t P r o d u c t s

175

Inspection

: Factors Affecting

Quality

6.
6.1

VEGETABLES AMD FRUITS

FRESH VEGETABLES AND FRUITS

ROUTIHE AHALYSIS
Although fresh produce is among the commonest of foods, regulatory control does
not usually involve compositional analysis.
Visual inspection is adequate to
detect gross adulteration, so routine laboratory analysis of the fresh article
is restricted to testing for contaminants in most cases. Vegetables and fruits
may be examined for potentially toxic elements such as copper, lead, cadmium,
arsenic and pesticides.
M y c o t o x i n s can also occur in these products (e.g.
aflatoxin in figs, patulin in apples).
Analysis for such contaminants should
p r e f e r a b l y b e d o n e as p a r t of s y s t e m a t i c c o n t a m i n a n t s s u r v e y .
The
determination of starch and sugars is often of relevance to dietary surveys.
Potatoes at certain stages of their growth occasionally contain an undesirably
h i g h level of solanine, which is not entirely destroyed by cooking.
If the
tuber has been exposed to light w h i l e g r o w i n g it w i l l be green due to the
presence of c h l o r o p h y l l , but this is not necessarily accompanied by a high
solanine content. Baker, L a m p i t t and M e r e d i t h (1) describe a method for the
determination of solanine.
The c o m p o s i t i o n and analysis of onions are described by Sherratt (2). The
yellow patches occasionally seen on onions show under the microscope as needle
crystals.
They are composed of quercetin and are generally considered to
denote prolonged storage.
The p r o x i m a t e c o m p o s i t i o n of vegetables change greatly with ripening.
The
p r o x i m a t e c o m p o s i t i o n also varies due to variety, climate, soil, stage at
harvest and m a n y other factors so it is often very difficult to relate
composition to quality parameters of interest to the regulatory chemist.
The
quality of peas and canned corn is judged by the percentage of alcoholinsoluble solids.
Fresh fruit should be examined for pesticides, especially fungicides, including
d i t h i o c a r b a m a t e s , b i p h e n y l , 2 - h y d r o x y b i p h e n y l , thiabendazole and benomyl.
Compounds of copper, arsenic and lead may also have been used as fungicides on
s o m e crops. Citrus fruits and dried fruits m a y be treated with m i n e r a l oil,
w h i c h can be r e m o v e d w i t h s o l v e n t , w h i c h is then e v a p o r a t e d and the
unsaponifiable matter weighed.
Fruits are s o m e t i m e s treated with colouring
matter in an attempt to improve the appearance.
Proximate analysis of fruit and vegetable products is generally carried out by
standard methods. Homogeneity of the sample can be difficult to achieve and it
may be necessary to blend or otherwise homogenise quite large amounts.
Reports
should always state how the sample was prepared, whether it was peeled, readyto-eat, cooked or uncooked, etc. For the determination of total solids, ISO/R
1026-1969 recommends drying at 70C and 20-25 mm mercury to 'constant weight 1
(change of less than 0.001 g/hr).
For the v a c u u m drying m e t h o d , a slow current of air (about 40 L/hr at NTP),
dried by sulphuric acid, is recommended.
Determinations on 10 percent sucrose
solution and 1 percent lactic acid are suggested as checks on the method.

176

6.2

CAMMED VEGETABLES AID FRUITS

COMPOSITIOH
The various Codex recommended standards for canned fruits generally set limits
for drained w e i g h t , m i n i m u m fill, cut-out strength of the syrup and quality
criteria such as m a x i m a for b l e m i s h e d , b r o k e n or o t h e r w i s e d e f e c t i v e fruit.
The standards also indicate acceptable labeling and in some cases limits for
additives such as firming agents, flavours, acidifying agents, a n t i - f o a m i n g
agents, colouring m a t t e r s , a n t i - c l o u d i n g agents and antioxidants.
Some
standards recommend a maximum for tin of 250 mg/kg. Codex standards for canned
fruit packing syrups are:
Minimiim Cnt-ont Strength of Syrup in Degrees Brix

Slightly
sweetened
syrup
Peache s
Grapefruit
Pineapple
Plums
Raspberries
Pears
Strawberries
Mandarin oranges
Fruit Cocktail

Extra
light
syrup

Light
syrup

Heavy
syrup

14
16
14
15
15
14
14
14
14

18
18
18
19
20
18
18
18
18

10

12

10
11
11
10
10
10
10

The f o l l o w i n g drained w e i g h t s tandard s have b e e n r e c o m m e n d e d

Alimentarius Commission:

22
-

22
25
26
22
22
22
22

by

the

Minimum Z drained weight

Product
Canned mushrooms
Canned mature processed

Extra
heavy
syrup

53 (27.5 if packed in sauce)

peas

60

Canned green peas

60

Canned sweet corn (whole kernel)

61

Canned green beans and canned

wax beans

50 for "whole" and "sliced lengthwise"

style; 55 for other styles

Canned asparagus, peeled

60 for long shoots; 58 for all other

styles

Canned asparagus, unpeeled

57 for long shoots and shoots; 55 for

all other styles

Canned

tomatoes

50

Canned

peaches

In extra
heavy and
heavy syrup

In light and
extra light
syrup

Solid
pack

Clingstone type

57

59

84

Freestone type

54

56

82

177

Miniatm Z drained weight

Product
Canned

grapefruit

Canned

pineapple

50
58 for all styles other than crushed or
chips; 63 for crushed or chips style
r e g u l a r pack; 73 for crushed or chips
style heavy pack; 78 for crushed or
chips style solid pack.

Canned plums, whole style

50 for whole; 55 for halves

Canned

raspberries

37

Canned

pears

50 for w h o l e ; 60 for diced; 53 for all

other styles

Canned

strawberries

35

Canned mandarin oranges

55 for w h o l e s e g m e n t s ;
segments and pieces

58

for

broken

RODTIHE ANALYSIS
S a l t , sugars and " c o n d i t i o n e r s " such as the p h o s p h a t e s , s u l p h a t e , citrate or
chloride of calcium or double sulphates of aluminium with an alkali metal may
be added to vegetables during canning and mention of this addition on the label
may be required.
Since calcium and, at a lower level alumimium, mostly occur
n a t u r a l l y in food p r o d u c t s , careful c o m p a r i s o n of the n o r m a l range for the
untreated article with the results of analysis would be necessary before any
statement could be made that addition without declaration had taken place. The
p r o p o r t i o n of total solids and a 1 c o h o 1 - i n solub le solids in canned processed
peas are higher than in canned garden peas and therefore these parameters are
used to check label claims.
The syrup s t r e n g t h of the fill liquor is of i m p o r t a n c e .
The final syrup
strength, known as the "cut-out" strength, may be determined by refractometer
G e n e r a l l y , the syrup used in
or by d e n s i t y , e.g. w i t h a W e s t p h a l balance.
c a n n i n g w i l l have a h i g h e r o s m o t i c p o t e n t i a l than the cellular liquid in the
fruit and water will therefore be drawn out of the fruit, diluting the syrup.
The drained w e i g h t of the fruit w i l l thus tend to be l o w e r than the "filled
weight", that is, the weight of fruit put into the can at the time of filling.
The d e g r e e to w h i c h these c h a n g e s take place d e p e n d s on the strength of the
syrup used, the type of fruit, its r i p e n e s s and succulence and the ratio of
fruit to syrup.
The c u t - o u t strength and drained w e i g h t from fixed initial
syrup strength and filled weight will therefore vary over a considerable range.
C o n v e r s e l y , the c a n n e r has to put in s u b s t a n t i a l l y m o r e fruit in order to be
sure of conforming to a minimum drained weight.
It is therefore important to
take the a v e r a g e d r a i n e d w e i g h t of at least ten cans.
Codex r e c o m m e n d e d
standards include definitions of different syrups in terms of degrees Brix.
If erythrosine has been used as a colour, the can contents should be examined
for the presence of fluorescein, formed by interaction between erythrosine and
the tin and iron present.
Canned g r a p e s m a y c o n t a i n argol crystals (Kagan et al (3)), crude p o t a s s i u m
bitartrate, which can be mistaken for glass by the layman.
Similarly, calcium
t a r t r a t e has b e e n reported in cherries (Dickinson and F o w l e r (4)).
Orange
spots on mandarins may be narirgin. The mould Byssochlamys fulva may withstand
p r o c e s s i n g and w i l l then break d o w n the pectin in fruits, e.g. s t r a w b e r r i e s ,
causing their disintegration.

178

CAH

EXAMIHATION

Samples may be submitted because the condition of

In such a case, it
that the contents are spoiled.
c a n s , as b a c t e r i o l o g i c a l e x a m i n a t i o n m a y r e q u i r e
easier to carry out chemical tests if two or three

the can leads to a suspicion

is important to have several
h a l f a d o z e n and it is m u c h
extra cans are available.

T h e can is f i r s t t e s t e d for v a c u u m , w h i c h s h o u l d be a b o u t 2 0 0 - 3 0 0 m m b e l o w
atmospheric pressure and never less than 75 m m b e l o w atmospheric pressure.
If
it is zero, the contents should be tested for tin.
A positive pressure as in a
swollen can is likely to be due to hydrogen derived from chemical action on the
can itself or to carbon dioxide due to product decomposition.
Hydrogen lights
w i t h a popping noise.
Carbon dioxide m a y be absorbed into lime w a t e r or into
sodium hydroxide solution.
I d e n t i f i c a t i o n of the gas m a y be used to o b t a i n
c o n f i r m a t i o n of bacteriological results.
Once the can is open, the can coating
is i n s p e c t e d to see if it is i n t a c t .
T h e can m u s t be c h e c k e d for p i n - h o l e s ,
e s p e c i a l l y if t h e r e is r u s t on the i n t e r n a l s u r f a c e .
Cans with p i n - h o l e s
should always be removed from sale, as they could b e c o m e contaminated at any
time, if not already so.
Outside, rust tends to be around the seams and where
the l a b e l is g l u e d . C o m p l e t e d e t i n n i n g is i n d i c a t e d if the i n s i d e of the can
rusts quickly after emptying and being left to dry.
The bacteriological examination is the most important in the case of swollen
( b l o w n ) c a n s b u t the c o n t e n t s s h o u l d a l s o b e e x a m i n e d for t i n , lead and pH.
I n t e r p r e t a t i o n of b a c t e r i o l o g i c a l r e s u l t s is b e y o n d the scope of t h i s b o o k .
S u f f i c e it to say t h a t t h e s w e l l m a y b e " h a r d " or " s o f t " , and h a r d s w e l l s a r e
a l m o s t i n v a r i a b l y due to b a c t e r i o l o g i c a l c o n t a m i n a t i o n , w h i c h m a y b e due to
C l o s t r i d i u m spp and t h e r e f o r e s u c h c a n s s h o u l d o n l y b e o p e n e d u n d e r the
supervision of a competent bacteriologist.
Slight swelling may also be due to
o v e r f i l l i n g of the c a n ,
so s w o l l e n c a n s s h o u l d n o t b e r e p o r t e d
as
unsatisfactory without proper examination.
Overfilling is a quality control
p r o b l e m of m o r e i n t e r e s t to the c a n n e r t h a n i n s p e c t i o n a u t h o r i t i e s .
On the
other hand, the v o l u m e of gas in the can, called the "headspace", should not be
excessive.
At l e a s t 90 p e r c e n t fill ( h e a d s p a c e m a x i m u m 10 p e r c e n t ) is a
reasonable general standard but there are exceptions.
Sound cans will usually be examined for headspace and vacuum and the contents
for drained w e i g h t , pH, acidity and specific gravity of the liquor and metallic
c o n t a m i n a n t s , m o s t importantly lead, arsenic and tin but also c a d m i u m , zinc,
m e r c u r y and c o p p e r .
In the a b s e n c e of n a t i o n a l l e g i s l a t i o n , m o r e than 2 5 0
m g / k g of t i n or 20 m g / k g of c o p p e r m i g h t b e c o n s i d e r e d e x c e s s i v e
and
occasionally products containing less than these amounts m a y be of bitter or
metallic taste.
L i m i t s for tin in certain canned foods have been r e c o m m e n d e d
by the Codex A l i m e n t a r i u s Commission.
Most metal food cans (other than a l u m i n i u m ) are m a d e
alloy and a tin coating on the inside surface.

of mild

steel

w i t h a tin

Schematic Representation of the Structure of Tinplate

0.19-0.5 mm mild

steel

^ S n coating 0.15 - 2.1 micrometres (1.1 g/m^ - 15.1 g/m^)

thinnest acceptable for food is 0.38 m i c r o m e t e r s (2.8 g/m^)
Oxide film about
(
Oil layer

.002 micrometers

i
[
)

removed during
processing or
storage

Tinning is never perfect and some areas of mild

steel base will always be exposed to the product.
Alloy

(FeSn2)

179

For beer and b e v e r a g e s , acidified beetroot, berry fruits, etc. the tin is
l a c q u e r e d , the underlying mild steel having a low corrosion resistance. Tin
may be lacquered for many other products or just for effect. Detinning by acid
foods takes place more slowly on tin with a more complete alloy layer.
L a c q u e r s are usually sprayed on at the rate of 6-9 g/m^ (which needs two
s p r a y i n g s ) w h e n the tin is flat and, w h e r e it is important that the product
does not interact with the can (beer and beverages), the seam is given a "wipe"
after can fabrication.
There are often lines on tinplate which can be used to
identify its type.
The tinned mild steel sheet may be used as it is or coated
w i t h l a c q u e u r , e n a m e l or plastic.
Cans coated with these last three but
without tinning are also used for certain products.
The three main types of reaction of food with tinplate are:
1.

Corrosion of the tin coating.

2.

Pitting of the mild steel base.

3.

Staining.

The various problems encountered with tinned food cans are discussed below:
Corrosion
C o r r o s i o n of the tin is essential because it provides e l e c t r o c h e m i c a l
p r o t e c t i o n to the m i n u t e areas of base steel that are exposed to the product
through pores and unavoidable scratches in the tin coating. Normally, etching
should occur evenly over the wetted internal surface of the can; in the first
month or so the mirror surface of the tin coating should change to one in which
the shape of the individual tin crystals may be seen with the eye.
Definition
of the crystal boundaries in the tin should become clearer over the first year
at t e m p e r a t u r e s of about 25C, but no detinned areas should be visible.
Detinned areas are identified by their grey surface, by resistance to
scratching w i t h a needle and by a tendency to rust rapidly when exposed to
water and air.
Detinned areas should not be seen in cans stored for less than
1-1/2 to 2 years.
E x a m i n a t i o n , w i t h a lens or m i c r o s c o p e , of cans undergoing uniform etching
should shov no evidence of corrosion of the base steel until detinning is
extensive. However, localized areas of detinning may occur around the spots of
solder that are s o m e t i m e s found near the side seams of cans. These detinned
areas should not be confused with pitting corrosion and they do not cause early
failure of the can.
Rapid Detinning
U n u s u a l l y rapid detinning in plain cans will result in the a c c u m u l a t i o n of
unacceptable levels of tin and iron in the product and sometimes in the early
development of hydrogen swells.
Rapid detinning is caused by the use of plate
w i t h a t i n c o a t i n g m a s s t h a t is too l i g h t , or by a p r o d u c t t h a t is
i n t r i n s i c a l l y too corrosive or contains corrosion accelerators. Nitrate in
products with pH > 6 has been implicated in incidents of rapid detinning, but
dissolved oxygen, some dyes, anthocyanins and amine oxides also accelerate
corrosion.
During the detinning process these compounds (termed depolarizers)
are c h e m i c a l l y reduced, so loss of colour is evidence of the depolarizing
action of some substances. Sulphur dioxide m a y affect the can and therefore
should be absent from fruit used for canning.
If it is desired to try to
establish the cause of unexpectedly corroded cans, it may be worthwhile to look
for sulphur dioxide.
Plain cans are unsuitable for foods containing active depolarizers.
If
possible, such foods should be packed in lacquered cans or the product should
be modified to remove or inactivate the depolarizer.

180

Waterline

Detinning

U n e v e n d e t i n n i n g is a n o t h e r r e s u l t of p r o d u c t - c o n t a i n e r i n c o m p a t i b i l i t y or of
unsatisfactory canning practice.
If t h e c a n is d e t i n n e d at t h e i n t e r f a c e
b e t w e e n p r o d u c t and h e a d s p a c e w i t h i n a w e e k or so of p r o c e s s i n g , it is l i k e l y
t h a t t h e c a n c o n t a i n e d e x c e s s i v e a m o u n t s o f o x y g e n w h e n it w a s c l o s e d .
The
h e a d s p a c e v o l u m e m a y be l a r g e , the v a c u u m m a y be poor or the p r o d u c t m a y h a v e
been inadequately deoxygenated.
If t h e c a n s a r e l e a k y , a i r w i l l e n t e r t h e
h e a d s p a c e . A l t e r n a t i v e l y , the tin c o a t i n g m a y be too l i g h t .
W a t e r l i n e d e t i n n i n g (as t h i s c o n d i t i o n is o f t e n c a l l e d ) w i l l u s u a l l y b e
f o l l o w e d b y e v e n e t c h i n g of t h e r e s t of t h e c a n o n c e the o x y g e n h a s b e e n
c o n s u m e d . H o w e v e r , d e t i n n i n g w i l l be s o m e w h a t faster b e c a u s e of the i n c r e a s e d
a r e a of e x p o s e d s t e e l and the s h e l f l i f e of t h e c a n s w i l l b e m a r g i n a l l y
s h o r t e n e d . R e d r u s t is s o m e t i m e s f o u n d at t h e h e a d s p a c e e n d o f c a n s c l o s e d
w i t h e x c e s s i v e l e v e l s of r e s i d u a l o x y g e n , b u t , should the can be shaken b e f o r e
e x a m i n a t i o n , the r u s t m a y be d i s l o d g e d or d i s s o l v e d b y the p r o d u c t .
The o n l y
e v i d e n c e of the p r e v i o u s e x i s t e n c e of rust w i l l be isolated areas of d e t i n n i n g
w i t h s h a l l o w pits in the b a s e s t e e l .
Side-sea* Detinning
P r e f e r e n t i a l d e t i n n i n g of the side s e a m u s u a l l y o c c u r s in cans m a d e from plate
g i v e n c a t h o d i c d i c h r o m a t e p a s s i v a t i o n t r e a t m e n t (the m o s t c o m m o n ) . The p a s s i v e
s u r f a c e of the p l a t e r e s i s t s t h e a t t a c k of the p r o d u c t e x c e p t w h e r e the tin
c o a t i n g w a s r e f l o w e r e d and the p a s s i v e f i l m d i s r u p t e d b y the heat of s o l d e r i n g ;
this area is then p r e f e r e n t i a l l y d e t i n n e d . This c o n d i t i o n is u s u a l l y seen w i t h
m i l d l y c o r r o s i v e p r o d u c t s , s u c h as t o m a t o s o u p .
It h a s c a u s e d v e r y l a r g e
commercial losses.
The p r o b l e m m a y be o v e r c o m e b y u s i n g plate p a s s i v a t e d by
t h e s o d i u m d i c h r o m a t e d i p t r e a t m e n t w h i c h g i v e s e v e n e t c h i n g of t h e w e t t e d
s u r f a c e s of the c a n , or by using lacquered c a n s .
Fitting

Corrosion

If the tin d o e s n o t c o r r o d e for o n e r e a s o n or a n o t h e r , t h e n the s p o t s o f t h e

m i l d steel b a s e that are e x p o s e d to the p r o d u c t w i l l p r o b a b l y o r r o d e i n s t e a d .
This m a y occur due to p r o t e c t i v e s u b s t a n c e s in the p r o d u c t being p r e f e r e n t i a l l y
a b s o r b e d o n to t h e t i n . T h i s c a n h a p p e n w i t h s a u e r k r a u t . T h e r e s u l t i n g p i t s
in the s t e e l c a n u s u a l l y b e s e e n b y t h e aid of a h a n d - l e n s or l o w - p o w e r
microscope.
T h e y m a y a p p e a r to b e c o v e r e d b y an i n t a c t l a y e r of t i n , b u t
s c r a t c h i n g w i t h a sharp n e e d l e w i l l u s u a l l y s h o w that this is no longer plated
to the layer b e n e a t h . This type of c o r r o s i o n p r o d u c e s h y d r o g e n w h i c h m a y h a v e
r e s u l t e d in s w e l l i n g of the c a n . T h e m a n u f a c t u r e r m a y h a v e to u s e l a c q u e r e d
cans in order to o v e r c o m e this p r o b l e m .
Sulphide

Staining

B l a c k s u l p h i d e staining due to FeS u s u a l l y o c c u r s at isolated points on the can

m a i n l y in the h e a d s p a c e r e g i o n . It can be a v o i d e d b y using l a c q u e r e d c a n s , or
cans w i t h l a c q u e r e d e n d s .
It looks o b j e c t i o n a b l e and t h e r e f o r e m a y g i v e rise
to c o n s u m e r c o m p l a i n t s b u t is in fact h a r m l e s s .
Iron s u l p h i d e s t a i n s rub off
v e r y easily.
T i n s u l p h i d e s t a i n is a l s o h a r m l e s s a n d is b l u e - b j . a c k ( s o m e t i m e s b r o w n ) and
a d h e r e s f i r m l y to the tinned s u r f a c e .
It can be r e m o v e d b y r u b b i n g w i t h a w e t
e r a s e r or b y c a t h o d i c t r e a t m e n t in 5% N a 2 C 3 at 6 v o l t s .
T h i s s e r v e s to
d i s t i n g u i s h this t y p e of staining from d e t i n n i n g . L o c a l i z e d s u l p h i d e s t a i n i n g
m a y be seen in m e a t p r o d u c t s .
R e a c t i o n B e t w e e n L a c q u e r e d C a n s and

Products

If the l a c q u e r l a y e r is n o t c o n t i n u o u s , the a c i d i c f o o d u s u a l l y p r o c e s s e d in
this type of can w i l l soon a t t a c k the u n d e r l y i n g m e t a l .
This w i l l be a p p a r e n t
f r o m the a p p e a r a n c e of t h e l a c q u e r f i l m .
O b j e c t i o n n e e d o n l y b e t a k e n if
181

swelling has occurred, the product has acquired a metallic taste, the tin level
i s excessive, the can has rusted inside and contaminated the contents, or pinholes have formed.
TOMATO PRODUCTS
Tomato concentrates are usually marketed canned or in tubes. The Recommended
Codex International Standard for Processed Tomato Concentrates designates those
of 8-14% natural tomato soluble solids as puree and those more concentrated as
pastes.
Under this standard, a lot will be considered as m e e t i n g the
a p p l i c a b l e m i n i m u m natural tomato soluble solids r e q u i r e m e n t when: (a) the
average of the values from all containers or sub-samples tested meets at least
the m i n i m u m percentage r e q u i r e m e n t for the concentration as declared or as
required for the product n a m e or description; and (b) individual test values
are at least 92.5% of such m i n i m u m declared or required percentage of
concentration.
The recommended standard permits addition of seasoning such as
salt, spices, onion and lemon juice as an acidulent but not sugars or other
sweeteners.
Sodium b i c a r b o n a t e m a y be added to reduce acidity or citric,
malic, L-tartaric and lactic acids added to increase it, provided the final pH
is not m o r e alkaline than 4.3. Tin m u s t not exceed 250 mg/kg. The product
must be of proper colour, texture and flavour and not otherwise defective. The
p e r c e n t a g e of natural tomato soluble solids m u s t be declared and the term
'concentrated tomato puree 1 may only be applied to products containing at least
18 percent of such solids.
The determination of soluble solids is by refractive index. Determination of
p o t a s s i u m and lycopene can be of assistance in verifying that the soluble
solids are derived from tomato.
Pearson's Chemical Analysis of Foods states
that the dry solids of tomato purees and pastes contain about 9.5 percent ash,
13.8 percent protein, 50-65 percent total sugar (as invert sugar), 5.8-13.4
percent total acidity (as citric acid) and 4.8 percent potassium (as K0).
D a r b i s h i r e (28) reported a m e a n value of 1420 mg/kg of lycopene in the dry
solids of various tomato purees but the natural variation is quite wide. There
should be no dark specks or scale-like particles sufficient to noticeably
affect the appearance. The total solids m a y be determined by drying at 70C
and less than 50 m m mercury.
The metal contaminant of most importance is
copper although it m a y also be useful to d e t e r m i n e lead, tin and arsenic.
Sugars are d e t e r m i n e d by boiling the sample gently with w a t e r , diluting to
v o l u m e and filtering.
Hydrolysis and titration of the filtrate follows
standard procedures.
The sucrose content is usually very low.
The papers of
B i g e l o w et al (5), W i l l i a m s (6) and Goose and Binsted (7) give useful
additional information.
Jarvis (8) has suggested a chemical method for the
estimation of mould in tomato products.
The method is based on the estimation
of chitin, w h i c h is hydrolysed to chitosan, the latter deaminated to 2, 5anhydromannose which is estimated colorimetrica11y.
To d e t e r m i n e lycopene,
v i g o r o u s l y shake 50 ml of an 0.2% aqueous suspension of puree with 25 ml of
p e t r o l e u m ether (BR 80-100C) and then shake m e c h a n i c a l l y for 15 minutes.
Measure the extinction of the organic phase at 505 nm.
lcm
lycopene = 2820.

DRAISED HEIGHT
PRINCIPLE
The sample is drained on a standard mesh sieve.
The weight of m a t e r i a l
remaining in the sieve is expressed as a percentage of the can contents.
APPARATUS
Sieve with square openings 2.8mm x 2.8mm (no. 6 B.S.). Use a sieve
with square openings 11.2 x 11.2mm for canned tomatoes. Use a 20cm
sieve if the total weight of the produce is under 1.5 kilos and a
30cm sieve if it is over.
PROCEDURE
Weight the full can, open and pour the entire contents on a circular
sieve for which a tare has been established.
Without shifting the
product, incline the sieve so as to facilitate drainage.
In the case
of products with a cavity such as peach halves, invert if necessary
so that liquid can drain from the cavity but o t h e r w i s e the product
should not be touched. Drain 2 minutes, weigh either drained solids
or free liquid direct, and weigh the dried empty can.
For products in canned sauce, it is usual to d e t e r m i n e the "washed
drained weight".
Use a sieve with square openings 0.3 x 0.3mm.
Wash
the contents of the can on to the sieve, and rinse with running cold
water and then running hot water until free of adhering substances.
Spread the product out on the sieve, leave it to drain 5 minutes, dry
the underside of the sieve and weigh.
REFEREHCES
CAC/RM 36/37 1970.
CAC/RM 44 1972.

183

FILL OF COSTAIHER
PRINCIPLE
This method determines the percent total volume of a container occupied by the
contained food.
It is designed p r i m a r i l y for cans but can be used for wide
mouth glass containers also.
APPARATUS
Head space gauge.
One can be conveniently made from a straightedge
and a s m a l l ruler.
Place the straightedge across the top of the
opened c o n t a i n e r , resting on the container edge. Use the ruler to
measure the distance from the bottom of the straightedge to the top
of the food in the container.
PROCEDURE
Open the container (use a can opener for cans and remove lid for
jars) and m e a s u r e the distance from the container top to the food
using the h e a d s p a c e gauge. This is usually done at the center, but
if the food surface is uneven, then make several m e a s u r e m e n t s at
different points and average them.
Pour out and discard the food.

Hash, dry and weigh the container.

Fill the container with water to within 5 m m of the top (using the
headspace gauge).
Weigh the container and water.
N e x t , d r a w off w a t e r from the container until the water is at the
same level as m e a s u r e d for the food.
Again weigh container and
water. (Note that the water temperature should be the same during
both weighings).
CALCULATIOH
W2-T
The % fill of container

Where:

T
W1
W2

=
=
=

W1_T

x 100

tare weight of the container

container plus water, first weight
container plus water, second weight

REFERENCE
USA Code of Federal Regulations, revised April 1, 1984. Title 21, Part 130.12,

184

SOLUBLE SOLIDS
(Tonato Products)
PRINCIPLE
The sample is treated with pectic enzyme, filtered and the refractive index
the filtrate determined.
The result is expressed as percentage of sucrose
20 C.
APPARATUS
1.

Refractometer, sensitive to 0.0001.

2.
Filters.
Cut stems off 75mm ID glass or plastic funnels about
lcm from the apex at 90 angle and firepolish the ends. Place the
funnels in 150ml jars, about 5 5 m m ID. If 150ml beakers are used,
close the pouring spout with tape to prevent evaporation.
REAGENTS
1.
Pectic e n z y m e , in a d i a t o m a c e o u s earth base e.g. Pectinol R-10
(Rohm & Hass Co.) Klerzyme* analytical (Wallerstein Co.) or Spark-L*
(Miles Laboratories Inc.). Prepare a 0.4-1% aqueous solution, mix
thoroughly and allow to settle. Use the clear supernate.
PROCEDURE
Weigh 100g sample at room temperature and add a weighed amount (0.21.0g) of The dry enzyme preparation. Mix with a spatula immediately
to avoid evaporation and transfer to a filter containing a 12.5cm
paper ( W h a t m a n 2V or equivalent).
T a m p so that the sample is in
close contact with the paper and cover with the top or bottom half of
a petri dish to form a loose seal w i t h the top of the funnel.
Discard samples that take more than an hour to filter. For those,
m i x 0.2-lg dry enzyme with 100g fresh sample and incubate 30-60
minutes at about 40C in a closed container.
Cool nearly to room
temperature before opening, r e - m i x and transfer to the filter.
Samples that still filter too slowly should be filtered after
dilution.
For samples that contain 35% solids or more and those slow to filter,
add 100g enzyme solution to 100g sample and mix with a spatula
i m m e d i a t e l y to avoid e v a p o r a t i o n , or in a sealed
blender.
Alternately blend and shake to dislodge and break up lumps sticking
to container.
Examine the mixture carefully for lumps and continue
mixing until homogeneous. Transfer to the filter and cover with a
petri dish.
Check that the refractometer reads 1.3330 with water at 20C.
T r a n s f e r a d r o p of the s u b s t a n t i a l l y c l e a r f i l t r a t e to the
refractometer prism.
If it is not possible to read at 20C or if
condensation occurs at this temperature, read at room temperature and
correct according to the table. Read as percentage sucrose.
Repeat
the d e t e r m i n a t i o n with another drop of filtrate.
Readings should
agree within 0.1% sucrose.
If not, repeat readings on successive
portions of filtrate until agreement is obtained.
Erratic readings
indicate evaporation of sample or faulty mixing and/or filtration.
Determine the refractive index of a 1% solution of dry enzyme on the
refractometer as % sucrose.

185

CALCULATION
Soluble

solids of sample Z sucrose (test reading) - 1.15xBxC

Where: 1.15 = correction for insoluble solids in the sample

assuming 12% of the total solids to be insoluble
B Z enzyme preparation added to sample
C = reading as sucrose obtained on 1Z enzyme

solution

If a dilute sample is used:

Soluble solids of sample = 2 [Z sucrose (test reading) - 0.55 x
D x C] + E
Where: 0.55 = correction for insoluble solids, as above
D = % enzyme preparation to sample and E is a
correction factor according to the following
table
Natural tomato soluble solids
as Z sucrose, corrected for
enzyme x 2 (that is, 2 (test
reading - 0.55 x D x C))
25.0
30.0
35.0
40.0
45.0
50.0

Correction, E
0.3
0.4
0.5
0.7
0.8
0.9

Note that c o r r e c t i o n for salt m a y be m a d e if the s a m p l e contains

added salt and use of the following formula leads to a lower figure:
Soluble solids as Z increase at 20C (corrected

for salt)

= (R-N) 1.016 where R is the uncorrected value and N is Z total

chloride expressed as NaCl.
REFERENCES
Official Methods of Analysis of the AOAC, 1984, 32.023-.025.
Lamb, F.C., 1969.
52, 1050-1054.

Journal of the Association of Official Analytical

Chemists,

Lamb, F.C., 1972.

55, 809-810.

Journal

Chemists,

of the Association of Official Analytical

186

6.3

JUICES

COMPOSITIOH
Orange juice is defined by the Codex as the unfermented but fermentable juice,
intended for direct c o n s u m p t i o n , obtained by a mechanical process from the
endocarp of sound, ripe oranges (Citrus sinensis Osbeck).
The juice m a y
contain up to 10% m/m of mandarin juice (Citrus reticulata Blanco). The juice
may have been concentrated and later reconstituted with water, provided the
quality of the reconstituted product is equivalent to that of pure juice.
The soluble solids exclusive of added sugar shall not be less than 10% m/m as
determined by refractometer at 10C, uncorrected for acidity and read as Brix
on the International Sucrose Scale.
Sucrose, dextrose or dried glucose syrup
may be added as long as the total added sugars do not exceed 5%. The product
shall have the characteristic colour, aroma and flavour of orange juice.
Natural volatile orange juice components may be restored to any orange juice
from which natural orange juice components have been removed. The addition of
concentrate to juice is permitted.
Only concentrates from orange and mandarin
may be used. There shall be only traces of volatile acids present, the ethanol
content shall not exceed 3 g/kg nor the essential oils content 0.4 ml/kg.
Limits for toxic elements are as follows:
Metal

M a x i m level

Arsenic (As)

0.2 mg/kg

Lead

0.3 mg/kg

(Pb)

Copper (Cu)

5 mg/kg

Zinc (Zn)

5 mg/kg

Iron (Fe)

15 mg/kg

Tin (Sn)

250 mg/kg

Total metal content

precipitable by potassium
hexacyanoferrate (II)

20 mg/kg,
expressed as Fe

The product must occupy not less than 90% v/v of the water capacity of the
container.
The complete list of ingredients must be declared on the label in
descending order of proportion, and mention must be made of reconstitu-tion if
preparation involved that p r o c e s s .
For j u i c e r e c o n s t i t u t e d from the
concentrate, soluble solids must be at least 11% m/m.
The contents of sugars as a percentage of total sugars expressed
usually in the following ranges in genuine orange juice:
Sucrose:
Glucose:
Fructose:

as invert are

30.6 - 56.0%
22.6 - 33.0%
18.1 - 37.2%

The sucrose/invert sugar ratio does not exceed 1.3/1.

The amino-acid pattern
and carboxylic acid patten are useful indications of adulteration.
Synthetic
carotenoids and glycine are possible additives used to hide adulteration with
added water and sugar. Addition of peel and pulp is indicated by an abnormally
high chloramine value and pentose equivalent.
Beet-sugar is a possible source
of betaine. Serine averages about 38 mg/100g in juice and levels in pulp and
peel are similar.

187

ROUT1MB ANALYSIS
The sugars present in fruit juices m a y be detected by TLC and a suitable
combination of the routine methods devised for the quantitative estimation of
those present.
A c i d i t y is d e t e r m i n e d by t i t r a t i o n to pH 8.2, u s i n g
p h e n o l p h t h a l e i n or a pH meter. The result is expressed as percentage m / m of
citric acid.
T h e o r g a n i c a c i d s p r e s e n t m a y be d e t e c t e d by p a p e r
chromatography. Ethanol may be determined as in beer, but the sample should be
neutralized first.
Fruit juices should be examined for preservatives, sulphur dioxide being the
p r e s e r v a t i v e m o s t usually present, and for v i t a m i n C and trace metals.
As
extensive a compositional analysis as possible should be carried out. Acidity,
determined by titration to phenolphthalein or a potentiometric end-point, is
expressed as the acid characteristic of the fruit - citric in citrus, malic in
apples and pears, tartaric in grape products, etc.
Adulteration can be
e x t r e m e l y sophisticated and the analyst is strongly advised not to report
a d v e r s e l y on s a m p l e s without careful m e t h o d s validation and a thorough
u n d e r s t a n d i n g of the interpretation of the results of fruit juice analysis.
A n a l y s i s r e v i e w s i n c l u d e t h o s e of H e r r m a n n (9), W a l l r a u c h (10) and
Wucherpfennig (11).
The r e v i e w of M e a r s and Shenton (12) and the paper by Sawyer (13) are very
useful as an introduction to the problems of the detection of adulterations of
orange and grapefruit juices. There is evidence of the manufacture of products
specifically intended for the adulteration of orange juice (Kefford (14), Anon
(15), Koch and Hess (16)). More recent papers on the adulteration of this
juice include those of Benk (17)(18), Benk and B e r g m a n n (19), Rother (20),
K a t s o u r a s (21), W e i t z et al (22), van Gils and van den Bergh (23) and the
review of Koch (24).
G e n e r a l l y speaking, a single analytical parameter is not reliable as an
indication of adulteration and it is for this reason that as many constituents
as possible should be determined. The difficulty is accentuated by the wide
Variation is less in fruits of specified variety, locality
natural variation.
and soil c h a r a c t e r i s t i c s , but these details are rarely available to the
e n f o r c e m e n t analyst.
For e x a m p l e , in genuine orange juice the analytical
p a r a m e t e r s show relationships within certain limits. The data is therefore
susceptible to statistical examination, either by use of the X ^ distribution
(Lifschitz, Stepak and Brown (25)) or regression analysis (Coffin (26)).
Brown
(27) d i s c u s s e s the use of combined non-independent, one sided tests of
significance.

188

FRUIT CONTEST
(Formol Hoaber Method)
PRIHCIPLE
By the addition of formaldehyde, one H + is liberated per molecule of a m i n o acid. It is titrated with alkali. The secondary amino-group of histidine does
not react; those of proline and hydroxy-proline react to about 75%.
Tertiary
nitrogen and guanidine-groups undergo no reaction.
APPARATUS
1.

pH meter.

REAGENTS
1.

Sodium hydroxide, 0.25N.

2.
Formaldehyde solution: pure formalin of at least 35% is brought
exactly to pH 8.1 with dilute sodium hydroxide as determined by means
of the pH meter.
3.

Hydrogen peroxide, pure, 30%.

PROCEDURE
25 ml fruit juice (for lemon juice 10 ml + 10 distilled water) or the
corresponding amount of concentrate diluted to this v o l u m e are
neutralized in a beaker with 0.25 N sodium hydroxide to pH 8.1 on the
pH meter. 10 ml of the formaldehyde solution is then added. After
ca. 1 minute the solution is titrated p o t e n t i o m e t r i c a 1 1 y to pH 8.1
with 0.25N sodium hydroxide.
If more than 20 ml 0.25N sodium hydroxide are required, the titration
is to be repeated using 15 ml formaldehyde solution instead of 10 ml.
When sulphur dioxide is present the sample is treated with a few
drops of 30% hydrogen peroxide before neutralization.
CALCULATIOH
The amount of alkali used in the titration, expressed as ml 0.1 N
alkali and referred to 100 ml fruit juice or 100 g concentrate is
equal to the formol number of the sample under test. Calculate to
whole numbers (without decimals).
INTERPRETATION
As fruits ripen the formol number of the juice tends to decrease, as a rule.
Conversely, on storage of the juice a slight increase may be noted.
Various
factors can lead to a lowering of the formol number of a fruit juice, e.g.
treatment with ion-exchangers or addition of ascorbic acid.
In the literature the formol number may also be found defined as ml N alkali
for each 100 ml sample, which corresponds to values 10 times smaller than those
given by the preceding method of calculaion.
For orange juice, the % fruit is * ,
1.4

where F is the formol number.

189

REFEREMCES
A y r e s , A.D.,
Packaging

Charley,
413.

S c h r o d e r , H., 1 9 5 6 .
104. 386.

V.L.S.

&

Zeitschrift

Swindells,

R.,

1961.

Food

Processing

fur L e b e n s m i 1 1 e 1 u n t e r s u c h u n g u n d

190

and

Forschung

ORGAHIC ACIDS
PRINCIPLE
The organic acids present in fruit juices can be separated by descending
paper c h r o m a t o g r a p h y .
After drying the paper the acid spots obtained are
sprayed with an acridine solution.
Under the UV-lamp they can be detected by
their y e l l o w fluorescence.
By a d d i t i o n a l spraying of the paper w i t h an
alcoholic solution of b r o m o p h e n o l blue they can also be detected as y e l l o w
spots on a blue b a c k g r o u n d .
For the i d e n t i f i c a t i o n of u n k n o w n acids it is
recommended to use developing solvents of varying composition.
APPARATUS
1.
Glass chromatography tank with two solvent troughs, 40 cm
30 cm wide and 55 cm high, sealable.
2.

Micro-pipette, 20 microlitre

3.

Spray assembly.

4.
Chromatography
Whatman equivalent.

paper,

long,

capacity.

Schleicher

and

Schuell,

5.

Warm air drier.

6.

Ultraviolet lamp (spectral range: 280-360 nm) .

No.

2043b

or

REAGENTS
1.
Solvent mixture I:
mix together 7.5 parts n-butanol, 2.5 parts
tert. amyl alcohol, 3 parts formic acid (98%, anhydrous), and 3 parts
water.
This is to be m a d e at least 24 hours before use in a
separating funnel. The l o w e r aqueous layer is separated and kept.
It will be used later for equilibrating the chromatography tank.
2.
Solvent mixture II:
mix together 95 parts n-butanol
with water) and 5 parts formic acid (98%, anhydrous).

(saturated

3.
Solvent m i x t u r e III: m i x t o g e t h e r 500 g phenol, 6.7 ml
acid (98%, anhydrous) and 167 ml water.
Note:
4.

formic

all three solvent mixtures are stable for one week.

Spray reagents:
a.
Alcoholic bromophenol blue solution, 0.4 g/L (this solution
is to be neutralized with 0.1 N NaOH until the appearance of the
first blue-green colouration).
b.
Acridine
ethanol.

solution:

250 mg acridine dissolved

in 200 ml

5.
Cation exchanger Dowex 50, 12% cross-linked, 50-100 mesh BSS, H +
form.
6.
Standard acids (tartaric acid, malic
acid, succinic acid, etc., each 5 g/L).

acid,

citric

acid,

lactic

PROCEDURE
10 ml of the fruit juice is vigorously shaken three times, each for 1
minute and each time with lg cation exchanger Dowex 50 (H+), without
i n t e r m e d i a t e filtration.
Filter and use the filtrate for spotting.

191

Sugar in fruit j u i c e s can interfere in the separation if present in

too high a concentration.
It is therefore recommended that, in such
cases,
10-20 M l , at the m o s t 40 Ml of the filtrate be spotted on
the paper.
(Note that chromatography papers are very sensitive to contact with
acids and alkalis.
Stringent cleanliness is therefore to be observed
when cutting and marking papers, e.g. wash hands, use clean supports,
etc. ) .
The w i d t h of paper chosen can be varied according to the n u m b e r of
samples or standard acids to be examined.
For example, with a width
of 20 c m , 8 starting points can be applied.
A distance from the
m a r g i n of 3 cm on each side should i n v a r i a b l y be observed w h a t e v e r
the width of the paper.
Spot the individual sample and standard solution using a blood-sugar
pipette.
10-20 M 1 of each solution is applied to the starting point
in 4 p o r t i o n s of about equal size (ca 7 cm from the upper edge).
After each application dry the moist spot on the paper using a warmair drier.
L i n e the c h r o m a t o g r a p h y tank w i t h filter paper on the inner
Add 10-20 ml of the aqueous phase to the tank bottom.

side.

If e s p e c i a l l y r e p e a t a b l e c h r o m a t o g r a m s are r e q u i r e d , and if time

p e r m i t s , it id r e c o m m e n d e d to hang the spotted c h r o m a t o g r a m in the
e m p t y c h r o m a t o g r a p h trough o v e r n i g h t .
Next day the solvent is
introduced.
D e v e l o p by the d e s c e n d i n g technique.
The time of
running is at least 10-15 hours according to the choice of solvent.
Remove the developed chromatogram from the tank and dry for 3-4 hours
in a stream of cold air (chamber with ventilator).
When the solvent
contains phenol, the drying is to be carried out for one hour with a
hot-air drier at 70C.
Spray the dried c h r o m a t o g r a m on each side w i t h acridine solution.
A f t e r a short d r y i n g - o f f p e r i o d , v i e w under the U V - l a m p . The acid
spots can be d i s t i n g u i s h e d as sharply d e f i n e d , b r i g h t l y g l o w i n g
y e l l o w spots. It is r e c o m m e n d e d that the o u t l i n e s of the spots be
lightly marked with a hard pencil.
In order to make the acid spots also visible by ordinary light, it is
r e c o m m e n d e d that the c h r o m a t o g r a m be a d d i t i o n a l l y sprayed on both
sides w i t h b r o m o p h e n o l blue solution. The acid spots can then be
distinguished as yellow zones on a blue background.
IHTERPSETATIOR
The RF-values serve as an aid in the characterization of the individual acids.
These values are, however, influenced by many factors, so that they are not to
be taken as absolute values, but as reference points in the characterization.
U n e q u i v o c a l r e s u l t s w i l l be o b t a i n e d if the acids to be identified are run
together with the standard acids presumed present.
The approximate RF-values
for the different acids run in solvent mixtures I, II and III can be taken from
the following tables.

192

Acids
Galacturonic acid
Gluconic acid
Quinic acid
Tartaric acid
Ascorbic acid
Citric acid
Malic acid
Chlorogenic acid*
-Ketoglutaric acid
Lactic acid
Succinic acid
Caffeic acid*
Glutaric acid
Fumaric acid

Solvent I
(n-butanol:
tert-amylalcohol
: formic acid:
water)
RP-Values

Solvent II
(n-butanol, H2O
saturated :
formic acid)
RF-Values

Solvent III
(phenol:
formic acid:
water)
RF-Values

0.08

0.04
0.05
0.14
0.18
0.28
0.32
0.39
0.41
0.56
0.67
0.67
0.69
0.75
0.83

0.23
-

0.22
0.32
0.32**
0.48
0.55
0.58
0.69
0. 78
0.80
0.82
0.85
0.89**

* These acids can be distinguished by their blue fluorescence

chromatogram is viewed under the UV-lamp
** Visible in UV-light as dark violet spots before

0.52
0.20
0.41
0.24 and 0.28
0.42
0.71
0.60
0.72
0.66
0.62
0.79
0.63
if the unsprayed

spraying.

Note that if acids present only in traces are to be detected (provided that the
sugar content is b e l o w 20 g/L) juice v o l u m e s up to 50 p i m a y be applied
directly to the paper.
The ion exchange treatment described also sets free inorganic acids that may be
present; after d e v e l o p m e n t they are usually found near the starting point.
This fact must be taken into account in the identification of unknown acids.
Volatile acids such as formic acid, acetic acid, etc. cannot be separated by
acid solvents, they volatilise during the sample application.
For semi-quantitative determinations suitable standard acids are applied at
different c o n c e n t r a t i o n s .
C o n c l u s i o n s as to the a m o u n t present can then be
drawn by a comparison of the spot size of the test substance with those of the
c o n c e n t r a t i o n series. More accurate values can be obtained if the spots are
marked out w i t h pencil under the U V - l a m p , and then cut out and w e i g h e d .
Recording spot w e i g h t s on the one hand and c o n c e n t r a t i o n s on the other in a
system of c o - o r d i n a t e s , gives a curve from w h i c h the c o n c e n t r a t i o n of the
required acid can be derived with good accuracy.
As an alternative to paper chromatography, thin-layer chromatography can also
be applied.
In the latter case plates may be prepared from cellulose powder.
The various solvent mixtures can be employed in the separation.
REFERENCES
Tanner, H. 1959. Scheweizerische Zeitschrift

fur Obst und Weinbau 68,

270-273.

Tanner, H. 1964. Scheweizerische Zeitschrift fur Obst und Weinbau 73,

150-154.

Tanner, H. & Rentschler, H. 1958. Mitteilungen Klosterneuberg VIII A, 113-120.

L e h o n g r e , G., Tanner, H. & R e n t s c h l e r , H. 1957. M i t t e i l u n g e n aus der
der Lebensmitteluntersuchung und Hygiene ^8, 40-46.

Gebiete

R e n t s c h l e r , H. & T a n n e r , H. 1954. M i t t e i l u n g e n
mitteluntersuchuag und Hygiene
142-158.

Lebens-

193

aus der Gebiete

der

VITAMIN C
PRINCIPLE
This m e t h o d was designed for fruit juices but can be applied s u c c e s s f u l l y to
most liquid samples, or samples that can be easily dissolved. The exception is
blackcurrant juice which is too densely coloured.
REAGENTS
1.
Standard indophenol solution - dissolve 0.05g 2,6-dichlorop h e n o 1 - i n d o p h e n o l in w a t e r , dilute to 100 m l and filter.
Prepare
freshly.
2.
Standard ascorbic acid solution - dissolve 0.0500g of pure
ascorbic acid in 60 m l 20% metaphosphoric acid and dilute with water
to exactly 250 ml.
3.

20% metaphosphoric

4.

Acetone.

acid.

PROCEDURE
S t a n d a r d i z e the indophenol solution as f o l l o w s :
Pipette 10 ml of
standard ascorbic acid solution into a small flask and titrate with
i n d o p h e n o l s o l u t i o n until a faint pink colour persists for 15
seconds.
Express the concentration as mg ascorbic acid equivalent to
1 ml of the dye solution (i.e. 10 ml ascorbic acid solution = 0.002g
ascorbic acid).
If 0.002g a s c o r b i c acid requires V ml dye solution to n e u t r a l i z e it
then 1 ml dye solution = 0-002

g ascorbic acid.

P i p e t t e 50 m l of u n e o n c e n t r a t e d j u i c e (or the e q u i v a l e n t of
concentrated juice) into a 100 ml volumetric flask, add 25 ml of 20%
m e t a p h o s p h o r i c acid as stabilizing agent and dilute to v o l u m e .
P i p e t t e 10 m l into a s m a l l flask and add 2.5 ml acetone.
Titrate
with the indophenol solution until a faint pink colour persists for
15 seconds.
(The acetone may be omitted if sulphur dioxide is known
to be absent.)
CALCULATION

c
Where :

(mg/

V = ml
= mg

REFERENCES
Official Methods of Analysis of the AOAC, 1984. 43.064-.068.
Deutsch, M.J. 1967. Journal of the Association of Official Analytical
798-806.

194

Chemists,

6.4

TEXT

REFERENCES

1.

BAKER,
Science

2.

SHERRATT,

3.

KAGAN,
I.S.,
GRISHINA,
Uchebnykh Z a v e d e n i i ( 5 )

I.P.
&
172-4.

4.

DICKINSON,

H.D.

5.

BIGELOW,
W.D.,
SMITH,
H.R. &
Research Laboratory B u l l e t i n
Washington.

6.

W I L L I A M S , H.A. 1 9 6 8 .
and 1 9 7 2 10 1 0 3 .

7.

GOOSE, P.G. & BINSTED,

Food Trade P r e s s .

8.

JARVIS,

9.

HERRMANN,

10.

WALLRAUCH,

11.

WUCHERPFENNIG,

12.

MEARS,

13.

SAWYER,

14.

KEFFORD,

15.

ANON.

1972.

16.

KOCH,

J.

& HESS,

17.

BENK,

E.

1971.

Industrielle

18.

BENK,

E.

1971.

Mineralbrunnen

19.

BENK, E.
282-284.

20.

ROTHER,

21.

KATSORAS,

22.

W E I T Z , J . , B R O E K E , R. t e n , G O D D I J N , J . P . , G O R I N , N. & S C H U T Z , G . P . 1 9 7 1 .
Z e i t s c h r i f t fur L e b e n s m i t t e l u n t e r s u c h u n g und Forschung 145 ( 6 ) 3 3 5 - 3 8 .

23.

GILS,
W.F. Van &
mitteluntersuchung

24.

KOCK,

25.

L I F S C H I T Z , A . , STEPAK, Y. & BROWN,

of O f f i c i a l A n a l y t i c a l C h e m i s t s 54

26.

COFFIN, D.E. 1968.

C h e m i s t s 51 1 1 9 9 .

L.C.,
LAMPITT,
L.H. & MEREDITH,
of Food & A g r i c u l t u r e _6_ 197-202.
J.G.

D.

B.

1943.

&

FOWLER,

1977.
K.

R.

J.

1963.

of

the A s s o c i a t i o n

Tomato

Obst

Flussiges

J.F.

&

H.

Journal

1969.

Paste

18

42
Obst

72

D.

K.G.

1975.

R.

Deutsche
Obst

1971.

21

Izvestiya

Vysshikh

1950.
Tomato
Products:
Canners
Association,

of P u b l i c

Other

Analysts

Tomato

6_ 69

Products,

581-91.
410-415.

(6)

214-6,

218,

220-6.

o f Food T e c h n o l o g y 8

Food

Agriculture

Quarterly

29

14

357-89.

302.

65.

Hemika

Lebensmittel-Rundschau

und

Gemusevrerwertung

(9)

278

&

280.

Industrielle

Obst

Mineralbrunnen
1971.

the

15.

1971.

BERGMANN,

of

225-9.

43

Scientific

and

12

(10)

(6)

Food P r e s e r v a t i o n

Gordian

1971.

of

Journal

706.

GREENLEAF,
C.A.
27-L,
National

R.G. & SHENTON, A.G. 1 9 5 9 . J o u r n a l

R.

1964.

80

Food T e c h n o l o g y

Flussiges

1976.

L.P.

Analyst

Ernahrungs-Umschau

1975.
K.

ZYABKO,

1955.

1973.

of

1955.

200-202.

Journal

Journal

1971.

S.

Analyst

O.B.

21_ 3 9 0 ,

Hronika

36

392

(8/9)

BERGH,
J . A . Van d e n .
und Forschung 152 ( 5 )

Flussiges

Obst

Journal

4j2_ ( 6 )

of

&

185.

(23)

692-694.

Gemuseverwetung

56

10

394.
185-90.

1974. Z e i t s c h r i f t
288-91.

fur

Lebens-

217-24.
M.B. 1 9 7 1 .
1266.

the

und

56

67

Journal

Association

195

of

of t h e

Official

Association

Analytical

27.

BROWN, M.B. 1975. Biometrics n

(4) 987-92.

28.

DARBISHIRE, O.B. 1965. Analyst 90^ 439-40.

Farther Reading
KEFFORD,

J.F. & CHANDLER, B.V. The Chemical Constituents of Citrus Fruits.

Methods of Analysis of the International Federation of Fruit Juice

10 Rue de Lige, 5-75009, Paris 9e, France.

Producers,

NAGY, S. & A T T A W A Y , J.A. 1980. Citrus Nutrition & Quality ACS S y m p o s i u m

American Chemical Society.

196

143

7.

7.1

CEREALS, CEREAL PRODUCTS AND PULSES

WHOLE 6RAID (UHMILLED) PRODUCTS

ROUTIIE ANALYSIS
As cereals, pulses and their products form the larger part of the diet of most
people, it i s most important that the quality of these foods is m a i n t a i n e d .
For unmilled products this is more a matter of inspection than analysis, but
samples received at the laboratory should be examined for filth (including uric
acid), rodent hairs and excreta, infestation, d a m a g e (breakage, shrivelling,
fungal attack, insect damage), m o i s t u r e , pesticide residues, m y c o t o x i n s ,
fumigant residues and heavy metals.
Rodent urine on grain shows fluorescence
under ultraviolet light.
Mercury compounds are still used as seed dressings
and dressed seed occasionally become available for human consumption.
Cadmium
and zinc are absorbed from the soil by rice and other cereal and root crops and
may reach undesirable levels if the plants are grown in soils containing highlevels of cadmium or zinu.
Selenium levels can be high in the soil in certain
areas and in such areas crops may need to be analysed for selenium.
Some pulses contain toxicants naturally, such as cynogenetic glycosides (lima
bean), trypsin inhibitors (e.g. soybean, lima bean, navy bean, black-eyed pea),
goitrogens (soybean) and h e a m a g g l u t i n i n s (Phaseolus vulgaris, soybean).
The
latter three toxicants are proteinaceous and are therefore denatured and
rendered h a r m l e s s by heat. Cynogenetic glycosides occur in a wide range of
plants, including almonds and other fruits of the Rosaceae, sorghums and Kaffir
corns, cassava and elderberry (Sambucus nigra).
Added organic colouring
material can be extracted from pulses or pulse flour with 80 percent ethanol.
After evaporation of the ethanol, the identification may be completed in the
usual way.
The general methods of ISO for oleaginous seeds may be used for soya. ISO 664
describes preparation of the sample for analysis.
ISO 659-1968 describes
d e t e r m i n a t i o n of the oil by hexane extraction using a soxhlet or similar
extractor, grinding with sand after 4 hours and six hours of a total eight hour
extraction time.
ISO 658 describes the determination of impurities by sieving
and hand-picking. Moisture and volatile matter is determined as loss in weight
at 103C (665-1968) after reduction of the seeds to below 2 mm.
The ISO basic reference method for the determination of moisture in cereals and
cereal products requires that the sample be first ground so that particles are
1.7 m m or less, less than 10 percent over 1 m m and over 50 percent less than
0.5 m m . Drying is at 50C and 10-20 m m m e r c u r y in the presence of phosphorus
pentoxide until constant weight is achieved (over 100 hours).
The routine
method requires drying at 130C for 2 hours (1-1/2 hours for flours). It is
important to allow the dish to cool completely before weighing (30-45 minutes).
When examining samples for contaminants it can be preferable to wash the whole
grain (with organic solvents for pesticide residues or with dilute nitric acid
for mercury) rather than use a milled sample. A few samples of washed grains
should be ground and analyzed to check the effectiveness of the washing.
Methods for the determination of residues of fumigants are given by Malone (1)
and a panel of the U.K. (2). Aluminium phosphide is finding increasing use as
a source of phosphine for fumigation. Muthu, Kashi and Majumder (3) describe a
method of analysis.

197

GLYCOSIDIC CYANIDE
PRINCIPLE
The glycosides are hydrolysed and the cyanide steam distilled.
determined by argentimetrie titration.

The cyanide is

APPARATUS
1.
Mechanical grinding mill, easy to clean, enabling samples to be
ground w i t h o u t b e c o m i n g heated and w i t h o u t a p p r e c i a b l e change in
moisture content.
2.

Sieve with 1 mm apertures.

3.

Analytical

4.

Incubator, adjusted

balance.
to operate at 38 +_ 2C.

5.
Steam distillation apparatus, provided with a 1 litre flask with
G/G neck.
This r e m o v a b l e flask should be able to be stoppered
hermetically with a G/G stopper.
The end of the condenser should be
provided with an extension drawn out to a point.
REAGENTS
1.

O r t h o p h o s p h o r ic

acid,concentrated.

2.

Sodium hydroxide

3.

Silver nitrate, 0.01 N standard volumetric

4.

Potassium iodide (Kl) solution, 50 g/L.

pellets.
solutions.

5.
A m m o n i a s o l u t i o n , a p p r o x i m a t e l y 6 N obtained by diluting
concentrated ammonia solution with an equal volume of water.
PROCEDURE
Grind about one twentieth of the laboratory sample in the previously
w e l l cleaned m e c h a n i c a l grinding m i l l (in order to c o m p l e t e the
c l e a n i n g of the m i l l ) , and reject these grindings.
Then grind the
rest to p a r t i c l e s w h i c h w i l l pass through the sieve c o m p l e t e l y .
Collect the grindings, mix thoroughly and carry out the determination
without delay.
W e i g h , to the nearest 0.1 g, a p p r o x i m a t e l y 20 g of the prepared
sample. Transfer to the 1 litre distillation flask and add 200 ml of
d i s t i l l e d w a t e r and 10 m l of o r t h o p h o s p h o r i c a c i d .
Stopper
hermetically, mix well and leave the flask for 12 hours (overnight)
in a i n c u b a t o r at 38C.
Fit the flask to the d i s t i l l a t i o n apparatus and distil- into 20 ml
water containing 0.5 g NaOH.
Collect 100-120 ml of distillate.
T r a n s f e r the d i s t i l l a t e to a 250 ml v o l u m e t r i c flask and dilute to
the m a r k w i t h d i s t i l l e d water. Pipette 100 ml into a beaker. Add 2
ml of potassium iodide solution and 1 ml of ammonia solution.
T i t r a t e w i t h silver n i t r a t e solution until p e r m a n e n t turbidity
appears.
For the easy recognition of the end point of the titration,
it is r e c o m m e n d e d that a black background should be used. M a k e a

198

second titration with another 100 ml portion of distillate and take

the m e a n of the t w o titra'tions.
C a r r y out t w o
complete
determinations on the same prepared sample.
C a r r y out a b l a n k test u n d e r the s a m e c o n d i t i o n s as in
determination but replacing the distillate by distilled water.

the

CALCULTIOH
Under the conditions of the reaction:
1 ml of 0.01 N silver nitrate
solution corresponds to 0.54 mg of hydrocyanic acid (HCN).
The content of glycosidic hydrocyanic acid,
of HCN per 100 g of sample, is equal to

0
54 (V
U.54

- WV,
1 )) xx

ioo

xx

100
=
i
^

135

expressed

in milligrams

l)

Where :
m

is the mass, in g, of the test portion

V Q is the v o l u m e , in m l , of 0.1 N silver nitrate solution used

for the determination.
V^ is the v o l u m e , in ml, of 0.1 N silver nitrate solution used
for the blank test.
INTERPRETATIOH
If the amount of HCN determined is less than 1 ml of titrant volume, consider
the sample practically free from glycosidic cyanide.
REFERENCE
ISO 2164:1975.

199

TALC 0> KICK OK BAKLEY

PRIRCIPLE
The talc is floated off, filtered, digested, ignited and weighed.
KEAGEKTS
1.

10% ammonia

solution.

2.

Hydrogen peroxide, 3%.

3.
Hydrochloric:chromic acid mixture.
Carefully dissolve 10 g of
chromic trioxide in 100 ml of water and add to 900 ml of concentrated
hydrochloric acid.
PROCEDURE
Shake 20 g of sample with the dilute a m m o n i a and dilute hydrogen
peroxide solutions.
Heat to about 60C so that the gas formed causes
the particles of talc to come away from the surface. Decant off the
liquid containing the talc, wash the grains several times with water
and add these washings to the decanted liquid. Heat the liquor with
the h y d r o c h l o r i c / c h r o m i c acid m i x t u r e to oxidize suspended m e a l ,
filter off the talc, wash, ignite and weigh.
INTERPRETATIOR
In unpolished rice, the talc residue does not normally exceed 0.025%.
REFEREHCE
Kozizan, R. 1906. Zeitschrift Untersuchung Nahrung Genussmittel xi, 641-650.

200

7.2

FLOURS AND MILLED PRODUCTS

ROUTINE ANALYSIS
The identity of any flour or other milled cereal product should be checked by
microscopical examination, comparing the sample with authentic specimens.
The quality of flour can usually be adequately assessed by determination of
moisture, ash, acid-insoluble ash, acidity, nitrogen, gluten (for wheat flour)
and filth. The proportion of fibre is an indication of the extraction rate,
the higher the rate of extraction (80 percent, 85 percent, etc.) the closer the
flour i s to w h o l e m e a l (100 percent).
In the case of wheat flour the lowest
extraction (i.e., whitest flour) is n o r m a l l y 72 percent.
Flour m a y be
fortified with iron, chalk and vitamins such as thiamine and nicotinic acid.
Self-raising flour is m a d e by addition of an acid phosphate and sodium
bicarbonate. The baking process may be assisted by the addition to flour of
bleaching agents, improvers and other additives including ammonium persulphate,
ammonium
chloride,
cysteine,
acetone
peroxide,
sulphur
dioxide,
a z o d i c a r b o n a m i d e , potassium b r m a t e , nitrosyl chloride, chlorine, chlorine
dioxide, benzoyl peroxide, potash alum, magnesium carbonate, sodium aluminium
The use of some
sulphate, calcium sulphate and di- and tri-calcium phosphate.
of these additives is under review and subject to legislation in many
countries.
The main sources of m e t h o d s of analysis for flours are the International
Association for Cereal C h e m i s t r y (ICC), the A m e r i c a n Association of Cereal
C h e m i s t s , Kent-Jones and Amos (1967) and Pearson (4). Among the m a n y ICC
methods may be mentioned n u m b e r s 110 and 111 for nicotinic acid, 117 and 119
for thiamine, and 122 and 123 for starch.
Flours commanding a higher price may be admixed with cheaper ones. The methods
of Griffiths (5), Tillmans, Holl and Janivala (6) and Tillmans (7) and the AACC
method 06-10 may be used for the detection of rye flour in wheat flour. The
presence of soy flour is usually detected by the urease test, this enzyme being
peculiar to soya beans among the flours of c o m m e r c e .
While a positive test
certainly indicates the presence of soya, the enzyme may have been inactivated
during processing and hence it is preferable also to carry out a microscopical
e x a m i n a t i o n , the flat "hour-glass" cells of the epidermis of soya being very
characteristic.
Sometimes rice and barley are "faced" with materials such as talc and glycerol
or oil containing a trace of blue pigment to improve the appearance.
The
effect is solely cosmetic and the practice should be discouraged. The total
ash of rice is usually between 0.2 and 0.4 percent.
MICROSCOPIC IDENTIFICATION
This is a useful physical analytical procedure to identify the source of flours
or starches. The starch grains may be examined in mounts in water but some
grains may distort in time.
This can be avoided by mounting in alcohol.
Addition of a drop of 2 percent aqueous solution of chromium trioxide makes the
striations of starch grains more easily seen. The presence of starch in a
powder such as flour m a y be confirmed by adding a drop of very dilute (say
0.001 N) iodine to the slide, the grains staining an intense blue. Different
characteristic patterns of starch grains can be seen between crossed polaroids.
The m i c r o s c o p i s t should be aware of the form of the various layers in the
entire grain so that he can recognize the various elements in the flour and
pick out any that are foreign.
A grain may be softened by soaking in water or
glycero1methanol (1:1) overnight and then sections made as thin as possible by
embedding the grain in pith and cutting with a razor. The diagram of the wheat
caryopsis typifies the m o r p h o l o g i c a l elements of cereal grains. On another

201

p o r t i o n the s t a r c h is s o l u b i l i z e d w i t h h y d r o c h l o r i c acid or d i a s t a s e or by
boiling with chloral hydrate or glacial acetic acid and the residue centrifuged
and examined, mounted in dilute glycerol or chloral hydrate.
T h e f o l l o w i n g d e s c r i p t i o n s and i l l u s t r a t i o n s w i l l a s s i s t the m i c r o s c o p i c
identification of cereal and pulse starches, flours and some grains:
1.

Wheat

Crains

A t r a n s v e r s e s e c t i o n of a g r a i n of w h e a t ,
exhibits the following layers (Figure 7.1):

groove

examined

under

the

microscope,

lOO^m

starchy
endosperm

100 jim
aleurone
layer

embryo

scutellum

epidermis
cuticular layer

tube cell
pericarp

cross cells

100 im

F i g u r e 7.1. C a r y o p s i s (A) of W h e a t (Triticuia) and P a r t s of its

Pericarp in Longitudinal Section (B) and Surface V i e w s (C,D)

(a)
O u t e r e p i d e r m i s of the p e r i c a r p , c o m p o s e d of t a b u l a r c e l l s w h i c h , in
s u r f a c e v i e w , are p o l y g o n a l , e l o n g a t e d and h a v e t h i c k e n e d , p i t t e d w a l l s .
In
the u p p e r part of the g r a i n it b e a r s s i m p l e , u n i c e l l u l a r , c o n i c a l h a i r s , the
lumen of which is s o m e w h a t abruptly enlarged at the base.

202

(b)
H y p o d e r m a , c o n s i s t i n g of c e l l s w h i c h , t o w a r d s the e x t e r i o r , c l o s e l y
resemble those of the outer epidermis, but in the inner part vary in form and
o f t e n lignify.
(c) A l a y e r of t r a n s v e r s e
thickened, pitted walls.

cells;

these

are t a n g e n t i a l l y e l o n g a t e d

and

have

(d) I n n e r e p i d e r m i s of the p e r i c a r p , c o n s i s t i n g of s m a l l c e l l s w i t h r o u n d e d
s e c t i o n , b u t e l o n g a t e d in s u r f a c e v i e w ; t h e i r t u b e l i k e a p p e a r a n c e h a s g a i n e d
for them the n a m e of tubular cells.
These four layers constitute the pericarp
of the g r a i n .
(e)
Seed-coat or b r o w n layer composed of two layers of cells closely
to one another, and of a y e l l o w or y e l l o w i s h - b r o w n colour.
(f)

Hyaline

layer:

a layer of rectangular

cells with

small, n a r r o w

applied

lumen.

(g) Proteid or aleurone layer; a single layer of cubical cells w i t h very

walls, and filled with a granular substance.
(h)
Endosperm,
c o n s i s t i n g of p o l y g o n a l c e l l s
characters of w h i c h have already been described.
2.

Wheat

filled

with

thick

starch,

the

Flour

To w h a t e v e r d e g r e e of f i n e n e s s the f l o u r m a y h a v e b e e n r e d u c e d it a l w a y s
c o n t a i n s p o r t i o n s of the p e r i c a r p and s e e d - c o a t s in a d d i t i o n to the s t a r c h ,
although the latter, of course, constitutes by far the greater portion.
The diagnostic

characters

(a)

and

The

shape

of wheat

size of the

flours

large

are

starch

(Figure

7.2):

grains.

(b) The h a i r s , w i t h l u m e n e n l a r g e d at the b a s e , b u t in the u p p e r p a r t

narrower than the wall.
(c) The thick-walled,
of the hypoderma.
(d)
The
transverse

pitted

t h i c k - w a 1 1 ed ,
cells.

cells

pitted

Figure 7.2.

203

Wheat

Flour

rather

3.

Wheat

Starch

W h e a t s t a r c h ( F i g u r e 7.3) is o b t a i n e d from the f r u i t s of s e v e r a l s p e c i e s of

T r i t i c u m , as, for i n s t a n c e , T_. s a t i v u m , Lam., etc.
Like some other starches
d e r i v e d f r o m c e r e a l g r a s s e s it c o n s i s t s p r i n c i p a l l y of a m i x t u r e of a large
n u m b e r of v e r y s m a l l g r a i n s w i t h o t h e r s of m u c h l a r g e r s i z e ; i n t e r m e d i a t e
grains are comparatively rare.
The small grains very from 2 p to 8 U in diameter, averaging about 6 y or 7 M ;
they are rounded or oval in outline, seldom polygonal or pointed.
The large g r a i n s in s u r f a c e v i e w
appear sometimes rounded, sometimes
s l i g h t l y i r r e g u l a r or o v a l , b u t
when, by touching the coverslip with
the needle they are made to present
their edges to an observer, they are
seen to be f l a t t e n e d or l e n t i c u l a r
in shape.
T h e y s e l d o m e x h i b i t any
c o n c e n t r i c s t r i a e or evident hilum.
They m a y a t t a i n as m u c h as 45 M in
d i a m e t e r , but they a v e r a g e o n l y 25
to 35 p .
In side v i e w they are
e l l i p t i c a l or s o m e t i m e s
spindles h a p e d , and e x h i b i t a l o n g i t u d i n a l
l i n e t h a t is a l w a y s s i m p l e and
usually straight or slightly wavy.

O
vSo?
FIGURE 7.3.

4.

Rye

&

O
Wheat

Starch

Flour

The anatomical structure of the rye grain closely resembles that of wheat. The
principal diagnostic features of the flour are to be found in the appearance of
the h a i r s and in the s i z e and c h a r a c t e r s of the s t a r c h g r a i n s .
The h a i r s of
rye h a v e a b o u t the s a m e s h a p e as t h o s e of w h e a t ; they d i f f e r , h o w e v e r , in the
l u m e n , for in the rye this gradually enlarges from apex to base, whereas in the
w h e a t it is n e a r l y l i n e a r in the u p p e r p a r t , and then s u d d e n l y e n l a r g e s and
b e c o m e s b u l b - s h a p e d at the base.
T h e r e is also a d i f f e r e n c e in the shape of
the
lignified
cells
of
the
h y p o d e r m a ; these are usually longer
than the transverse cells, whereas
in w h e a t they are s h o r t e r .
The
transverse cells are also m o r e
frequently rounded at the ends, and
h a v e t h i n n e r w a l l s t h a n they h a v e
in wheat.
The diagnostic characters
flour are (Figure 7.4):

of

rye

(a)
T h e h a i r s w i t h less a b r u p t l y
e n l a r g e d l u m e n and t h i n n e r w a l l s
than in wheat.
(b)
The transverse cells which
have thinner walls and fewer pits,
t h e y are m o s t l y s h o r t e r than the
h y p o d e r m a cells, and often rounded
at the e n d s , w h e r e the w a l l s are
also rather thicker.

204

(c) T h e s t a r c h g r a i n s w h i c h are r a t h e r l a r g e r t h a n t h o s e of w h e a t and

show a stellate h i l u m and concentric striae.
5.

Rye

often

Starch

Rye starch (Figure 7.5) is contained in the fruits of the rye, Scala cereale,
Linn.
L i k e w h e a t s t a r c h it c o n s i s t s of a m i x t u r e of v e r y s m a l l g r a i n s and
l a r g e o n e s , t o g e t h e r w i t h a c e r t a i n n u m b e r of i n t e r m e d i a t e size.
The large
g r a i n s a r e d i s c o i d in s h a p e , and o f t e n e x h i b i t i r r e g u l a r p r o t u b e r a n c e s , in
c o n s e q u e n c e of w h i c h t h e i r side v i e w is o f t e n less r e g u l a r l y f u s i f o r m or
elliptical than it is in wheat starch.
They average 40 y in diameter, but m a y
a t t a i n 50 and are t h e r e f o r e l a r g e r t h a n the g r a i n s of w h e a t
starch.
S o m e t i m e s the c o n c e n t r i c s t r i a e are i n d i s t i n c t , s o m e t i m e s t h e y a r e e a s i l y
visible.
In the centre there is often a c a v i t y w i t h f r o m t h r e e to five r a y s
and in s u c h a c a s e the h e l u m is said to
be s t e l l a t e . A m o n g s t the g r a i n s of
m e d i u m and s m a l l s i z e , h a t - s h a p e d and
bell-shaped ones are to be found; these
a r e v e r y s e l d o m s e e n in w h e a t s t a r c h .
The small grains of rye starch are also
r a t h e r l a r g e r t h a n the c o r r e s p o n d i n g
grains of wheat starch; they vary from 3
to 10 y in
diameter.

Figure 7.5.

6.

Barley

Rye

Starch

Flour

In m a n y respects barley resembles wheat and rye in its anatomical structure.

One of the p r i n c i p a l d i f f e r e n c e s is to be f o u n d in the h a i r s w h i c h h a v e a
l a r g e r l u m e n t h a n t h o s e of e i t h e r w h e a t or rye.
T h e w a l l s of the e p i d e r m a l
cells of the pericarp are not pitted, nor are they so thick as those of the two
latter grains.
The aleurone layer invariably consists of two or three rows of
cells, whereas in the wheat and rye there is only one row.
T h e g r a i n s of b a r l e y s t a r c h are s m a l l e r than t h o s e of w h e a t and m u c h s m a l l e r
t h a n t h o s e of rye. T h e y are l e s s r e g u l a r in s h a p e , and f r e q u e n t l y r e n i f o r m ,
b u t t h e s e c h a r a c t e r s are d i f f i c u l t e v e n for an e x p e r t to d e t e r m i n e , and the
detection of barley flour w h e n mixed with wheat or rye flour is very difficult.
There is, h o w e v e r , one peculiarity that m a y facilitate the identification of
b a r l e y flour.
The b a r l e y g r a i n is e n c l o s e d b e t w e e n t w o p a l e a e , and t h e s e
adhere so firmly to the pericarp of the fruit that it becomes very difficult to
e f f e c t t h e i r c o m p l e t e r e m o v a l , e s p e c i a l l y f r o m the g r o o v e on the v e n t r a l
surface of the grain.
The result is that barley flour often contains traces of
the d e b r i s of the p a l e a e , and t h e s e a r e e a s i l y i d e n t i f i e d by the v e r y
remarkable sinuous w a l l s of the outer epidermal cells, and by the little hairs
on the i n n e r e p i d e r m i s .
The d o u b l e or t r i p l e l a y e r of a l e u r o n e c e l l s s h o u l d
also be searched for.
The diagnostic

characters

of barley

flour are (Figure

7.6):

(a)

The epidermal cells of the paleae with thickened, sinuous

(b)

The hairs on the inner epidermis of

(c)

The thin-walled

(d)

The aleurone

walls.

paleae.

epidermal cells of the pericarp,

layer of two or three rows of

205

cells.

which are not

pitted.

(e) The starch grains, which are rather

m o r e irregular in shape.

smaller

Figure 7.6.

7.

Barley

Barley

than those of wheat, and

often

Flour

Starch

Barley
starch
(Figure
7.7) m a y
be
o b t a i n e d f r o m the f r u i t s
of H o r d e u m
d i s t i c h o n . L i n n , and o t h e r s p e c i e s of
Hordeum.

' ' MqQ

B a r l e y s t a r c h c o n s i s t s of a m i x t u r e of
l a r g e and s m a l l g r a i n s , w i t h a f e w of
intermediate
size.
T h e y are
rather
s m a l l e r than the grains of wheat starch,
and
are a l s o d i s t i n g u i s h e d
by t h e i r
o u t l i n e , which is less regular and often
bears protuberances.
In s u r f a c e v i e w
the large grains are seldom round; they
a r e m o r e o f t e n s l i g h t l y e l o n g a t e d or
e l l i p t i c a l , s o m e t i m e s r e n i f o r m , bulb- or
pear-shaped.
In d i a m e t e r they vary from
2 0 y to 35 y , m a n y
b e i n g b e t w e e n 20 y
F i g u r e 7.7. B a r l e y S t a r c h
and 2 5 y . T h e y h a v e no a p p a r e n t h i l u m ,
b u t s o m e of t h e m e x h i b i t
concentric
striations.
T h e y are s e l d o m f i s s u r e d at the h i l u m and w h e n that is the c a s e
the f i s s u r e is m u c h l e s s c o n s p i c u o u s t h a n it is in r y e s t a r c h , and n e v e r
stellate.
T h e g r a i n s of m e d i u m s i z e v a r y from 10 y to 15
in d i a m e t e r , the
s m a l l ones are about the same as those of wheat or rye-starch.

206

8.

Maize

Floor

The a n a t o m y of the g r a i n of m a i z e is a n a l o g o u s to that of the fruit of o t h e r

cereal grasses.
It is characterized by the following particulars:
Below the
e p i d e r m i s of the p e r i c a r p are t w o l a y e r s of c e l l s that e x h i b i t m a r k e d
differences, one having pitted and
relatively
slightly
thickened
w a l l s , those of the o t h e r b e i n g
s m o o t h and r e l a t i v e l y
strongly
thickened.
Within these layers is
one of irregular cells with lacunae
( t r a n s v e r s e c e l l s ) , and n e x t to
this the t u b u l a r c e l l s that form
the
inner
epidermis
of
the
pericarp.
The t u b u l a r c e l l s are
smaller, more numerous, and closer
t o g e t h e r than they are in w h e a t .
D u r i n g the r i p e n i n g of the g r a i n
the s e e d - c o a t s d i s a p p e a r
almost
entirely.
The diagnostic characters
flour are ( F i g u r e 7.8):

mm?

hypoderm a

(c)
The n u m e r o u s
cell s .
Figure 7.8.

9.

Maize

of maize

small

tubular

Maize Flour

Starch

M a i z e s t a r c h (Figure 7.9) is o b t a i n e d from the fruits of Zea m a y s , Linn.

The
grains of maize starch exhibit a certain difference in shape depending whether
they are derived from the mealy centre of the endosperm or from the translucent
horny periphery.
<33. <-9
Those from the centre of the grain, having
b e e n s u b j e c t e d in a less d e g r e e to m u t u a l
pressure, are irregularly rounded in shape,
or at least not markedly angular; some are
nearly round, others are elongated, oval or
pear-shaped.
The h i l u m is a l w a y s r a t h e r
large and c o n s p i c u o u s ; the g r a i n s m e a s u r e
from 10 y to 25 y in diameter, the average
being about 13 y to 15 y .
Figure 7.9.

Maize

Starch

The grains from the horny part of the endosperm exhibit an angular contour due
to the m u t u a l p r e s s u r e to w h i c h they h a v e b e e n s u b j e c t e d .
Their a p p e a r a n c e
u n d e r the m i c r o s c o p e v a r i e s c o n s i d e r a b l y a c c o r d i n g to the p o s i t i o n in w h i c h
they lie. T h e y are a l w a y s e a s i l y r e c o g n i z e d by their r e g u l a r s h a p e , a n g u l a r
o u t l i n e , m o r e or less u n i f o r m size, and by the p r e s e n c e of a d i s t i n c t h i l u m ,
which is sometimes rounded, but more often fissured or stellate.
The diameter
of these grains averages from 14 y to 15 y but may sometimes reach 25 y or 26y,

207

10.

Rice Flour

I n t h e r i c e f l o u r of c o m m e r c e t h e r e i s o n l y a v e r y s m a l l p r o p o r t i o n o f the
s e e d - c o a t s of the g r a i n , and the c h i e f c h a r a c t e r s are t h e r e f o r e to be found in
t h e s i z e and s h a p e o f the g r a i n s o f s t a r c h o f w h i c h i t a l m o s t
entirely
consists.
T h e s e a r e as f o l l o w s :
(1)
Small
simple
polyhedral
grains.
(2) Large
or
small
compound g r a i n s , oval or rounded
^ / T v , - ^
, aj P<> o
in s h a p e , and v a r y i n g in s i z e
y ^ J f ^ J JQ
'S&Vaj e_aS
^SO^O^o0
according
to
the
number
of
constituent
grains.
(3)
Fragm e n t s of the a b o v e of v a r y i n g
shape.
(4)
Masses
of
starch
from the c e l l s of the e n d o s p e r m ,
or
masses
of
several
cells
together (Figure 7.10):
The v e g e t a b l e d e b r i s to be found
in r i c e f l o u r c o n s i s t s of only a
few very narrow tubular
cells
c l o s e l y a t t a c h e d to a l a y e r of
very small elongated cells.
A
k n o w l e d g e of t h e s e c h a r a c t e r s is
v e r y d e s i r a b l e as the a d u l t e r a t i o n of w h e a t f l o u r w i t h r i c e
flour
is
frequent
at
times.

Figure 7.10.

11.

Rice

Flour

Kice Starch

R i c e s t a r c h ( F i g u r e 7 . 1 1 ) is
obtained
from the f r u i t s of 0 r y z a s a t i v a , L i n n .
L i k e oat s t a r c h ,
it c o n s i s t s of both
s i m p l e and compound g r a i n s .
The s i m p l e
g r a i n s are t o l e r a b l y u n i f o r m in s i z e and
shape;
they range
from 4 p
t o 6 |i ,
sometimes
reaching
8 VJ
,
and
are
generally angular.
The compound g r a i n s
a r e o v o i d or r o u n d e d i n s h a p e , b u t v a r y
v e r y much i n s i z e ,
according
to
the
number of c o n s t i t u e n t g r a i n s that they
contain.
Rice
starch
closely
resembles
oat
starch.
The
grains
are,
however,
uniformly rather
s m a l l e r and
never
s p i n d l e - or l e m o n - s h a p e d .
When t r e a t e d
with
water
the compound grains
are
readily
dissociated
into
their
constituent grains,
and so i t h a p p e n s
t h a t the f o r m e r are s e l d o m found in the
r i c e s t a r c h of c o m m e r c e .

< 3

o Y

O'O

o o w ^ o o t Q o
o

O >

Figure 7.11.

208

Rice

UoO

Starch

12.

Oat Floor

The oat grain is also enclosed between two paleae which may furnish valuable
means of identifying the flour.
The grain may also be distinguished from the
three foregoing grains by the following characters:

Figure 7.12.

13.

Oat Flour

a) By the elongated shape of the hairs

which are also often g e m i n a t e .
(b) By
the cells of the outer e p i d e r m i s of the
pericarp, which have very thin walls and
fairly n u m e r o u s pits.
(c)
By the
irregular polygonal shape of the cells of
the h y p o d e r m a , of w h i c h , h o w e v e r , there
is but little to be found.
(d) By the
cells of the seed coat, of which there is
only a single r o w ; they are p o l y g o n a l or
fusiform in shape, pale yellowish-brown
in colour:
their w a l l s are s m o o t h and
s e l d o m p i t t e d , and the c e l l s o f t e n
exhibit an irregular arrangement.
(e) By
the starch which consists of small simple
rounded grains associated w i t h a n u m b e r
of large oval c o m p o u n d grains and the
isolated angular c o n s t i t u e n t grains of
the latter (Figure 7.12).

Oat Starch

Oat starch (Figure 7.13) is contained in the fruits of Avena sativa, Linn. It
consists of two kinds of g r a i n s , s i m p l e and c o m p o u n d .
The s i m p l e grains
They are mostly rounded in outline, very few
average about 10 y in diameter.
are angular, but some are spindle-shaped or lemon-shaped. The latter should be
specially noted as they form a distinctive feature of oat starch.
The compound grains are oval or
rounded and more or less regular
in shape, ranging usually from
35 M to 45 y in l e n g t h , but
a t t a i n i n g as m u c h as 50 y .
They consist of a varying number
(5 to 200) of grains c o m p a c t e d
together.
The
constituent
grains vary in shape according
to t h e p o s i t i o n
they
have
occupied in the c o m p o u n d grain.
Those
from
the
centre
are
angular, w h i l s t those from the
periphery are curved on one side
and angular on the other; they
are g e n e r a l l y rather
smaller
than the simple grains.

* 0 6k

floV
9 V 7T<?
Figure 7.13.

14.

Oat Starch

Pea Flour

The pea
differs
instead
sections
60 y or

(Pisum sat ivum. Linn.) m u c h r e s e m b l e s the lentil in s t r u c t u r e , but

in the shape of the palisade cells, w h i c h are square at the apex
of conical.
These are thickened by s i m i l a r bars visible in surface
viewed from above, but not when viewed from below (size of the cells,
m o r e in length, 12 y to 15 y in width). The p a r e n c h y m a of the seed-

209

coat is c o m p o s e d of cells s i m i l a r in shape to those of the lentil, but

exhibiting conspicuous intercellular spaces. The cells of the epidermis of the
cotyledons are elongated, but in varying directions instead of parallel to one
a n o t h e r as in the lentil. The starch grains are rather larger than those of
the lentil (30 y to 47 y ) and m a n y of them bear rounded p r o t u b e r a n c e s .
The
h i l u m is c o m p a r a t i v e l y seldom f i s s u r e d , and even then the fissure is not
b r a n c h e d as it is in bean starch; the concentric striae are less regular, and
often indistinguishable.
The d i a g n o s t i c c h a r a c t e r s of
pea flour are (Figure 7.14):
(a) The p a l i s a d e
square ends.

cells

(b) The characteristic

mal cells.

with

hypoder-

(c) The e p i d e r m a l cells of the

cotyledons not parallel.
(d) T h e s t a r c h g r a i n s w i t h
r o u n d e d s w e l l i n g s , less distinct unbranched hilum and less
evident striae.

Figure 7.14.
15.

Pea Flour

Lentil Flour

The lentil (Lens e s c u l e n t s . M o e n c h ) , r e s e m b l e s m o s t l e g u m i n o u s

structure.
The seed-coat is composed of the three following layers:

seeds

in

1.
An e p i d e r m i s c o n s i s t i n g of a layer of palisade cells (about 40 y by
10 y ) with a lumen that gradually tapers toward the cuticle; towards the upper
part the wall is thickened by nearly vertical bars, the sections of which are
distinctly seen in the surface view. The outer end of the cell is not flat but
shortly and bluntly conical.
2.
A layer of p a r e n c h y m a t o u s cells (about 15 y by 15 y ); these are
c o n t r a c t e d in the m i d d l e , and hence a s s u m e the shape of an h o u r - g l a s s ; they
differ from the hypodermal layer of the bean in not containing calcium oxalate
crystals.
3.
A layer of irregular parenchymatous cells with thin walls; this layer
varies very much in the extent to which it is developed.
The cotyledons are covered with an epidermis consisting of polygonal cells, all
of w h i c h are e l o n g a t e d in the same direction.
The cells of the c o t y l e d o n s
themselves are polygonal, their walls are thin and occasionally exhibit small
pits. They are filled w i t h starch and aleurone grains. The former occupy a
p o s i t i o n that is i n t e r m e d i a t e b e t w e e n bean and pea starch as regards their
shape and appearance.
Many are ovoid but les9 regularly so than bean starch;
many exhibit a fissured hilum and distinct concentric striae, but in others the
hilum is not to be seen nor are the striae distinct; size of the larger grains
30 y to 40 y .

210

The diagnostic characters

flour are (Figure 7.15):
(a) T h e
ends.

palisade

cells

(b) T h e h o u r - g l a s s
calcium oxalate.

of

with

cells

(c) T h e n e a r l y p a r a l l e l
c e l l s of the c o t y l e d o n s .
(d) T h e t h i n - w a l l e d
cotyledons.

lentil

conical

without

epidermal

cells

of

the

(e) T h e s t a r c h
intermediate
in
character b e t w e e n pea starch and
bean starch.

Figure 7.15.
16.

Haricot Bean

Lentil

Flour

Flour

T h e h a r i c o t b e a n , P h a s e o l u s v u l g a r i s , L i n n . , p o s s e s s e s an a n a t o m i c a l s t r u c t u r e
r e s e m b l i n g t h a t of o t h e r l e g u m i n o u s s e e d s .
In the s e e d - c o a t the f o l l o w i n g
l a y e r s c a n be d i s t i n g u i s h e d :
(1) A n e p i d e r m i s c o n s i s t i n g of p r i s m a t i c c e l l s ,
r a d i c a l l y a r r a n g e d , like p a l i s a d e c e l l s ; the w a l l s of these c e l l s are m u c h
t h i c k e n e d and p o s s e s s s l i t - l i k e p i t s .
(2) A l a y e r of n e a r l y s q u a r e , or
r e c t a n g u l a r c e l l s , e a c h c o n t a i n i n g a l a r g e p r i s m a t i c c r y s t a l of c a l c i u m
oxalate.
T h e s e c e l l s a r e o f t e n c a l l e d " b e a r e r c e l l s " , a n d a r e f o u n d in a l l
v a r i e t i e s of b e a n s , b u t in m a n y o t h e r l e g u m i n o u s s e e d s t h e y are c o n t r a c t e d in
the m i d d l e (in t r a n s v e r s e s e c t i o n ) and f r e e f r o m c a l c i u m o x a l a t e .
(3) A l a y e r
of p a r e n c h y m a t o u s t i s s u e .
The c e l l s of the c o t y l e d o n s are p o l y g o n a l and
e x h i b i t at t h e i r a n g l e s e i t h e r a c o l l e n c h y m a t o u s t h i c k e n i n g o r m o r e o r l e s s
conspicuous intercellular spaces.
T h e s t a r c h o f t h e h a r i c o t b e a n is in o v o i d g r a i n s , s e l d o m r o u n d e d , s o m e t i m e s
r e n i f o r m , or e x h i b i t i n g o n e or m o r e p r o t u b e r a n c e s .
T h e h i l u m , w h i c h is
u s u a l l y v e r y d i s t i n c t , is e l o n g a t e d or f i s s u r e d .
In s i z e the l a r g e g r a i n s v a r y
f r o m 30 y to 75 p ; h o w e v e r , m a n y s m a l l e r o n e s a r e to b e f o u n d .
T h e d i a g n o s t i c c h a r a c t e r s of the f l o u r
the h a r i c o t bean are (Figure 7.16):
(a) The r e m a r k a b l e
(b) The
calcium

palisade

bearer cells
oxalate.

( c ) T h e c e l l s of the

with

Figure 7.16.

Bean

Flour

211

cells.
crystals

cotyledons.

(d) The c h a r a c t e r i s t i c

of

starch.

of

17.

Buckwheat Flour

The fruit of the b u c k w h e a t (Fagopyrum e s c u l e n t u m , M o e n c h ) is an achene. The

p e r i c a r p is c o m p o s e d of (1) An e p i d e r m i s consisting of a single layer of
p r i s m a t i c c e l l s w i t h thickened walls. (2) A fibrous h y p o d e r m a consisting of
four or five layers of p o l y g o n a l cells w i t h thickened w a l l s .
(3) A layer of
brown cells.
(4) An inner epidermis of very long, flattened cells.
The seed is enveloped in three coats:
(1) An outer coat composed of cells with
v e r y sinuous w a l l s .
(2) A m i d d l e coat of cells w i t h lacunae.
(3) An inner
coat of elongated cells.
Within these coats is an aleurone layer consisting of
a single row of cubical cells; then the endosperm filled with starch.
The g r a i n s of s t a r c h are s i m p l e , and either isolated or a g g l o m e r a t e d into
masses. The isolated grains are bluntly or sometimes sharply angular, or often
rounded. They may attain 10 p or 12 p in diameter, but average about 4 y to 6p,
Buckwheat starch always contains a number of abnormal grains larger than the
o t h e r s ; they are i r r e g u l a r l y e n l a r g e d , often bearing some r e s e m b l a n c e to an
hour-glas s.
The diagnostic characters
are (Figure 7.17):
(a)

The characteristic

of buckwheat

flour

starch grains.

(b)
The e p i d e r m i s of the s e e d - c o a t ,
cells of which have very sinuous walls.
(c)
The m i d d l e layer,
exhibit lacunae.

the

cells

of

the

which

Figure 7.17.

18.

Buckwheat Flour

Curcuma Starch (East Indian Arrowroot)

C u r c u m a starch (Figure 7.18) is obtained from the r h i z o m e s of Cure um a

angustifolia, Roxb., C^ leucorrhiza, Roxb. and other species of Curcuma.
The grains of this starch are oval, elliptical, almost rectangular or rounded
in o u t l i n e .
At one of their e x t r e m i t i e s they usually t e r m i n a t e in a short
o b t u s e point in w h i c h the very e c c e n t r i c p u n c t i f o r m h i l u m is situated
s u r r o u n d e d by c o n c e n t r i c striae. The g r a i n s are so thin that w h e n v i e w e d on
their edges they appear to be e x t r e m e l y n a r r o w ; several m a y often be seen in
this position adhering together by their flat sides.
Mixed with these larger
grains are smaller ones of similar shape.
C u r c u m a starch g r a i n s average from 30 p to 60 p in length, 25 y to 35 p in
b r e a d t h , and 7 p to 8 P in thickness.
The length of the s m a l l e s t grains
s c a r c e l y e x c e e d s 15 p to 25 p but the largest grains from . leucorrhiza m a y
attain as much as 140 p .

212

Figure 7.18.

19.

T o u s les M o i s S t a r c h

(Queensland

Curcuma

Starch

Arrowroot)

T h i s s t a r c h ( F i g u r e 7.19) is o b t a i n e d f r o m the r h i z o m e s of C a n n a e d u l i s , L i n n . ,
a n d o t h e r s p e c i e s of C a n n a . T h e g r a i n s of w h i c h it c o n s i s t s are. so l a r g e as to
i m p a r t a s a t i n y - w h i t e a p p e a r a n c e to the s t a r c h .
T h e m a j o r i t y are s e l d o m l e s s
t h a n 6 0 y o r 7 0 M i n l e n g t h , w h i l s t t h e l a r g e s t o c c a s i o n a l l y r e a c h 1 1 0 p to
130 y .
T h e y are u s u a l l y s i m p l e and are e l l i p t i c a l , s l i g h t l y o v a l , c o n c h o i d a l ,
or s o m e t i m e s r e n i f o r m in o u t l i n e ; t h e y a r e f l a t t e n e d a n d o f t e n p r o l o n g e d at t h e
n a r r o w e r e n d to a s h o r t o b t u s e p o i n t in w h i c h t h e r o u n d e d h i l u m is s i t u a t e d ,
surrounded by concentric striae.

Figure 7.19.

Tous

les M o i s

213

Starch

20.

Y a m or D i o s c o r e a

Starch (British Guiana

Arrowroot)

T h i s v a r i e t y of s t a r c h ( F i g u r e 7.20) is o b t a i n e d f r o m D i o s c o r e a a l a t a , L i n n . ,
and
other
species
of D i o s c o r e a .
The
largest
of
the
grains
of
w h i c h i t is c o m p o s e d m e a s u r e 45 y t o 9 0 U in
l e n g t h a n d 2 5 y to 60y i n b r e a d t h , w h i l e t h e
s m a l l e r v a r y f r o m 15 p to 30 p in l e n g t h and
a b o u t h a l f t h a t i n b r e a d t h . In o u t l i n e t h e y
a r e v e r y v a r i a b l e , b e i n g o f t e n o v a l or
elliptical, three-sided, with rounded angles,
or s o m e t i m e s c u r v e d . T h e l a r g e r e x t r e m i t y is
o f t e n t r u n c a t e and in t h e o p p o s i t e n a r r o w e r
e x t r e m i t y t h e h i l u m is s i t u a t e d ; t h i s is
rounded,
eccentric
and
surrounded
by
concentric striae.

Figure

21.

7.20. D i o s c o r e a

Banana

Starch

(or P l a n t a i n )

Starch

T h i s is o b t a i n e d f r o m the u n r i p e f r u i t s o f M u s a s a p i e n t u m , L i n n . , and h a s a l s o
b e e n o f f e r e d in c o m m e r c e as G u i a n a a r r o w r o o t . T h e g r a i n s a r e s i m p l e and s h o w a
g r e a t v a r i a t i o n in o u t l i n e : s o m e a r e o v a l , e l l i p s o i d a l or e l o n g a t e d , w h i l s t
others are c u r v e d , b o t t l e - s h a p e d , bean-shaped, etc. They are always flattened,
a n d t h e r e f o r e a p p e a r n a r r o w a n d s a u s a g e - s h a p e d w h e n p r e s e n t i n g t h e i r e d g e s to
T h e h i l u m is r o u n d e d ,
the o b s e r v e r .
situated
near
one
extremity
and
surrounded by concentric striae.
The
l a r g e s t g r a i n s m e a s u r e 45 M t o 6 5 y ,
the s m a l l e s t about 7 y > i n t e r m e d i a t e
g r a i n s f r o m 22 y to 34 P ( F i g u r e 7.21).
T h e f r u i t s f r o m w h i c h t h e s t a r c h is
p r e p a r e d a r e o f t e n d i s t i n g u i s h e d as
p l a n t a i n s , and the p l a n t y i e l d i n g them
is s o m e t i m e s d i s t i n g u i s h e d b y the n a m e
Musa paradisiaca, Linn.

Figure
22.

Manihot

Starch

7.21.

Banana

Starch

(Cassava)

T h i s v a r i e t y o f s t a r c h ( F i g u r e 7 . 2 2 ) is o b t a i n e d in l a r g e q u a n t i t i e s f r o m t h e
t u b e r s of M a n i h o t u t i l i s s i m a , P o h l , and o t h e r s p e c i e s as M a n i h o t .
It is a l s o
k n o w n u n d e r the n a m e o f c a s s a v a s t a r c h , m a n d i o c s t a r c h , B r a z i l i a n , B a h i a , R i o
o r P a r a a r r o w r o o t , e t c . T h e m a j o r i t y o f i t is c o n v e r t e d i n t o t a p i o c a b e f o r e
exportation.
T h e g r a i n s a r e o r i g i n a l l y c o m p o u n d , c o n s i s t i n g of t w o , t h r e e or f o u r c o m p o n e n t
g r a i n s , and are o c c a s i o n a l l y f o u n d i n t a c t . M o s t of t h e m , h o w e v e r , h a v e b e e n
separated into their c o m p o n e n t grains.
T h e y are s e l d o m quite r o u n d .
M o s t of
t h e m e x h i b i t o n e or t w o flat s u r f a c e s w h e r e o t h e r of the c o n s t i t u e n t s of the
c o m p o u n d g r a i n h a v e b e e n a t t a c h e d , a n d a r e in c o n s e q u e n c e m u l 1 e r - s h a p e d , c a p s h a p e d , or s h o r t l y c o n i c a l , c u r v e d on one side and i r r e g u l a r on the o t h e r ,
etc., some are even p o l y g o n a l . The m a j o r i t y possess a distinct rounded linear
or s t e l l a t e h i l u m and d e l i c a t e c o n c e n t r i c s t r i a t i o n s .
T h e l a r g e s t m e a s u r e 25y
to 35 y in l e n g t h , the s m a l l e s t 5 y to 15 y ; m a n y r a n g e f r o m 15 y to 25 y .

214

Figure 7.22. Manihot

23.

Starch

Tapioca

T h i s s u b s t a n c e is p r e p a r e d f r o m m a n i h o t s t a r c h , by h e a t i n g and s t i r r i n g the
m o i s t s t a r c h u n t i l it a g g l o m e r a t e s i n t o the l i t t l e i r r e g u l a r , r u g g e d m a s s e s
that are k n o w n in c o m m e r c e as tapioca; it is usually exported in this form and
constitutes an important article of food.
The granules of tapioca soften w h e n
soaked in water for a few hours, and a small portion, taken preferably from the
w h i t e r and m o r e o p a q u e p a r t , c a n be
b r o k e n up w i t h a n e e d l e in a d r o p of
w a t e r and c o v e r e d w i t h a c o v e r s l i p , a
little
pressure
being
applied
if
necessary.
M a n y of the g r a i n s w i l l be
s e e n to h a v e p r e s e r v e d t h e i r o r i g i n a l
shape, and exhibit a distinct h i l u m ; in
m a n y the h i l u m is s t e l l a t e l y f i s s u r e d ;
in o t h e r s the c e n t r a l p a r t of the g r a i n
is a t r a n s l u c e n t m a s s , b u t the o u t l i n e
is s t i l l r e c o g n i z a b l e ; w h i l s t f i n a l l y
m a n y have s w o l l e n into a s h a p e l e s s ,
unrecognizable
mass.
These are
the
various stages in the gelatinisation of
the s t a r c h by h e a t in the p r e s e n c e of
m o i s t u r e ( F i g u r e 7.23).

Figure

24.

Tacca Starch (Tahiti

7.23.

Tapioca

Arrowroot)
It is o b t a i n e d f r o m the t u b e r s of T a c c a
pinnatifida, Linn.
The grains vary both
in size and s h a p e .
T y p i c a l o n e s are
r o u n d e d , o v a l or e v e n l e n t i c u l a r ; s o m e
are e l l i p t i c a l , a l m o s t t r i a n g u l a r , etc.
The hilum is generally fissured situated
near
the
centre
of t h e g r a i n ,
and
surrounded by c o n c e n t r i c striae.
The
l a r g e r g r a i n s m e a s u r e 38 y to 50 V , the
smaller 15 p to 25 y
(Figure 7.24).

Figure 7.24.

Tacca

Starch

215

25.

Sago

Starch

Sago starch is obtained from the stem of the sago palm, Metroxylon sagu, Rottb.
and a l l i e d trees.
T h e form of the g r a i n s of sago s t a r c h v a r i e s a c c o r d i n g as
t h e y are s i m p l e or c o m p o u n d .
Simple grains
a r e o v a l , r o u n d e d , etc. but the c o m p o u n d
grains have a very remarkable shape. Each of
t h e s e u s u a l l y c o n s i s t s of a l a r g e g r a i n to
which 1, 2 or 3 small ones are attached.
The
large grain is conical or muller-shaped, and
f r e q u e n t l y b e a r s one or t w o p r o j e c t i o n s , to
the flat e n d s of w h i c h the s m a l l e r g r a i n s
have been attached; sometimes these two flat
surfaces meet to form an angle.
The largest
g r a i n s m e a s u r e 50 p to 65 p in l e n g t h , the
s m a l l e s t 10 p to 20 p . The h i l u m is v e r y
distinct,
l i n e a r , t r a n s v e r s e or o b l i q u e ,
s o m e t i m e s stellate and usually surrounded by
d i s t i n c t s t r i a e ( F i g u r e 7.25).
Commercial
sago
starch
often
contains
debris
of
v e g e t a b l e t i s s u e , etc. left in it by the
imperfect
washing
it h a s
undergone.
S c 1 e r e n c h y m a t o u s c e l l s , h a i r s and c r y s t a l s
m a y thus be found in it.
Figure 7.25.
Sago

26,

Pearl Sago (East Indian

Sago)

Genuine or East Indian sago (pearl sago) is largely prepared in Singapore from
s a g o s t a r c h ( F i g u r e 7.26).
The s t a r c h is c o n v e r t e d into sago by h e a t i n g it
whilst moist, as described under tapioca. Several varieties occur in commerce,
d i f f e r i n g in s o u r c e and a p p e a r a n c e .
Pearl
s a g o , w h e n e x a m i n e d u n d e r the m i c r o s c o p e ,
e x h i b i t s s t a r c h g r a i n s in v a r i o u s s t a g e s of
transformation, induced by the heat to which
they have been subjected.
Some of them have
p r e s e r v e d their o r i g i n a l shape and can be
easily identified.
M a n y are m o r e or less
altered; in some the central portion has been
gelatinised
and
is
transparent
and
h o m o g e n e o u s ; o t h e r s h a v e s w o l l e n to an
unrecognizible gelatinous mass.

Figure
27.

Potato

7.26.

Pearl

Sago

Starch

Potato starch (Figure 7.27) is obtained from the tubers of Solanum tuberosum,
Linn.
It
is
composed
of g r a i n s
of v a r i a b l e
size,
some
being
Typical grains of this starch are
so large as to be visible to the naked eye.
f l a t t e n e d , and h a v e an o v a l , o v a t e , e l l i p s o i d a l or c o n c h o i d a l o u t l i n e .
The
hilum is punctiform, eccentric, and generally situated in the narrow end of the
grain; it is surrounded by numerous distinct concentric striations, some few of
which are much more conspicuous than the others.
In addition to these typical
g r a i n s t h e r e are a f e w o t h e r s , s m a l l e r in size and r o u n d e d in o u t l i n e , or
roundd on one side and flattened on the are sometimes attached by their flat
sides in twos or threes.
The largest grains vary in length from 75 p to llOp,
t h o s e of the m e d i u m size from 45 p to 65 p and the s m a l l e r ones from 15 P to
25p.

216

28.

Maranta Starch

M a r a n t a starch (Figure 7.28) is obtained from the r h i z o m e s of M a r a n t a

a r u n d i n a c e a , Linn, and other species of M a r a n t a .
It is c o m m o n l y k n o w n in
commerce as "arrowroot", a term however, which is also applied to the starches
of other and widely different plants.
The different v a r i e t i e s of a r r o w r o o t are distinguished by their geographical
sources. Maranta starch is known as Bermuda, St. Vincent, West Indian or Natal
arrowroot, according to the country in which it is prepared.
The grains of the M a r a n t a starch are s i m p l e
and rather large.
They are irregular in
shape, being r o u n d e d , ovoid, pear-shaped or
s o m e t i m e s a l m o s t triangular; the s m a l l e s t
ones are nearly spherical.
The largest bear
n u m e r o u s fine concentric striations, and a
c o n s p i c u o u s r o u n d e d , linear or stellate,
eccentric hilum. In some varieties of arrowroot (Natal), the rounded hilum predominates,
in o t h e r s (St. V i n c e n t ) the l i n e a r or
stellate; it often r e s e m b l e s the w i n g s of a
poised bird. They average about 30 y to 40 V
in length, but m a y attain to 45 M , 60 y , or
e v e n 75 y as, for i n s t a n c e , in B e r m u d a
arrowroot; the smaller grains vary from 7 y to
15 y .
Figure 7.28.

217

Maranta Starch

ACIDITY IR FLOUR
(Water Extract)
PRINCIPLE
The acidity of an aqueous extract prepared under standard
determined by titration and calculated as lactic acid.

conditions

is

APPARATUS
1.

Waterbath at 40C.

REAGERTS
1.

0.1 N NaOH.

PROCEDURE
W e i g h 18 g of flour into a 500 ml conical flask and add 200 ml of
carbon dioxide-free distilled water.
Stand the flask in a waterbath
at 40C for 1 hour so that the flask is covered to just about the
level of liquid.
Swirl occasionally to ensure complete mixing.
After 1 h o u r , filter and titrate 100 ml of the filtrate with 0.1 N
NaOH.
CALCULATION
Acidity, % lactic acid

ml of 0.1 N NaOH
1000

ml of 0.1 N NaOH

0il

90

100
9

10

(90 = equivalent weight of lactic acid).

IRTERPRETATIOR
Under tropical conditions, there have been adverse
white wheat flour as landed if the acidity was over
over about 0.45% almost invariably taste stale and
taste of w h i t e w h e a t flour correlates fairly well
extraction flours have a naturally higher acidity.

reports on consignments of
0.35%.
Samples containing
bitter and above 0.35% the
with the acidity.
Higher

REFERENCE
E g a n , H., Kirk, R.S. and S a w y e r , R., 1981. Pearson's
Food s, 8th Ed., Churchill Livingstone*

218

Chemical

Analysis

of

ACIDITY IB FLOOR
(Alcohol Extract)
PRINCIPLE
The s a m p l e
alkali.

is

shaken

with

ethanol

and

the

extract

titrated

with

standard

REAGENTS
1.

Ethanol, 90%, v/v, neutralized

to

phenolphthalein.

2.

Phenolphthalein, 1% in neutral

ethanol.

3.

0.05 N Sodium hydroxide

solution.

PROCEDURE
Weigh 5.0 g of sample into a conical flask, add 50 ml of 90% ethanol,
s t o p p e r the f l a s k , s h a k e and leave to stand o v e r n i g h t .
D e c a n t the
e x t r a c t t h r o u g h a f i l t e r , w a s h the r e s i d u e w i t h 20 m l of 90% v/v
n e u t r a l e t h a n o l and d e c a n t t h r o u g h the s a m e f i l t e r .
T i t r a t e the
c o m b i n e d f i l t r a t e w i t h 0.05 N s o d i u m h y d r o x i d e s o l u t i o n to the
phenolphthalein end-point.
CALCULATION
% acidity
(as sulphuric acid) =
3

ttre

1000

x .05 x 49 x 2 .
5

INTERPRETATION
Recently
sulphuric

milled
acid.

flours

have

alcohol

extract

acidity

of

0.03

- 0.04%

as

Analysis

of

REFERENCE
E g a n , H., K i r k , R.S. and S a w y e r , R., 1981.
Foods, 8th Ed., Churchill Livingstone.

219

Pearson's

Chemical

ASH IM FLOUR
PRINCIPLE
A portion of flour is ashed and the residue weighed.
APPARATUS
1.

Muffle

furnace.

2.

Analytical balance.

PROCEDURE
W e i g h 3-5 g flour in a tared silica dish.
furnace at 600C to constant weight.

Ignite in a muffle

CALCULATION
% ash =

wt

ash (
B> , x 100
wt flour (g)

INTERPRETATION
The outer portions of grains have a higher mineral content than the inner.
Therefore the ash of w h i t e flour will be less than that of w h o l e m e a l flour.
'Patent' flour gives
For wheat flours, the ash gives an indication of grade.
an ash of 0.3 - 0.4%, 'Straight Run' (72% extraction) gives about 0.45% and
' W h o l e m e a l ' gives 1.2 - 1.8%. H o w e v e r , if chalk has been added to the flour,
then ash figures are invalid.
REFERENCE
Official Methods of Analysis of the AOAC, 1984, 14.006.

220

IROH IR FLOUR
PRINCIPLE
I r o n is o f t e n a d d e d as a n u t r i e n t to e n r i c h e d f l o u r s , u s u a l l y as f e r r o u s
sulphate, ferric a m m o n i u m citrate or iron powder.
This method involves ashing
the f l o u r to r e m o v e o r g a n i c m a t e r i a l , r e d u c i n g f e r r i c ion to f e r r o u s w i t h
sulphur dioxide and reacting with o-phenanthroline. The resultant red compound
is determined spectrophotometrically.
APPARATUS
1.

Muffle

furnace.

2.

Steam bath.

3.

Hot

4.

Pipettes

5.

Spectrophotometer

plate.
and volumetric

flasks.

(visible

range).

REAGEHTS
1.

Glycerol-alcohol mixture

(1+1).

2.

Nitric acid,

concentrated.

3.

Hydrochloric

acid, 5 N.

4.

Hydrochloric

acid, dilute

(1 ml acid

5.
Sulphur dioxide solution,
bisulfite in 100 ml water.

2%.

to 100 ml with

Dissolve

6.

Sodium acetate solution, 2 N.

7.

Congo red

8.

o-Phenanthroline solution, 0.25% in water.

indicator

3.25g

water).

sodium

meta-

paper.

9.
Ferrous a m m o n i u m sulphate hexahydrate standard - dissolve 0.7024
g in w a t e r ,
add 2 d r o p s H C 1 and d i l u t e to 1 l i t r e .
D i l u t e 50 m l of
this to 1 l i t r e (1 m l = 0.005 mg Fe).
PROCEDURE
W e i g h an a m o u n t of flour c o n t a i n i n g up to 0.4 m g iron into a s i l i c a
ashing dish.
(Note t h a t i r o n is o f t e n a d d e d to f l o u r at 1.6 m g / 1 0 0
g).
Add 10 m l of the g l y c e r o l - a l c o h o l m i x t u r e .
A s h o v e r n i g h t at
600C (after initial heating w i t h an infrared lamp or at the m o u t h of
the open muffle).
C o o l and add 1 m l c o n c e n t r a t e d n i t r i c acid. E v a p o r a t e and r e - a s h 1
hour.
C o o l and a d d 5 m l 5 N H C 1 .
H e a t on a s t e a m b a t h 15 m i n u t e s
and f i l t e r t h r o u g h h a r d e n e d f i l t e r p a p e r into a 100 m l v o l u m e t r i c
flask.
Add 3 m l d i l u t e H C 1 to the s i l i c a d i s h , b r i n g i n g to b o i l on a h o t
p l a t e and f i l t e r u s i n g the s a m e f i l t e r and f l a s k as a b o v e .
Repeat
this process four more times.
Then wash the dish and filter with hot
water to m a k e the filtrate flask to the mark.

221

M i x and pipette 10 ml into a 25 ml volumetric flask. Add 1 ml 2%

sulphur dioxide solution. Add 2 N sodium acetate solution by burette
until the solution changes congo red paper from blue to pink.
Next,
add 2 ml 0.25% o-phenanthroline solution and make to the mark.
Let stand overnight to develop the red colour. Read the absorbance
in a 4 cm cell at 520 nm versus a blank prepared the same way as the
sample.
Prepare a standard curve by pipetting 0, 2, 5, 7 and 10 ml of the
dilute standard solution (0.005 mg Fe/ml) into five 25 ml volumetric
flasks. Add reagents as with the sample and read the absorbances.
Plot the absorbance versus the mg iron.
CALCULATION
Find the mg iron corresponding
standard curve.
This is 'w'.

mg iron/100 g flour =

to the sample absorbance using the

w x luuu
g sample taken

REFERENCE
This method
14.011-.013.

is adapted from Official Methods of Analysis of the AOAC, 1984,

It differs primarily in the reducing agent used.

222

7.3

BREAD

ROUTINE

ANALYSIS

B r e a d m a y be e n r i c h e d b y a d d i t i o n (to the f l o u r ) of t h i a m i n e , r i b o f l a v i n ,
n i a c i n , iron, v i t a m i n D and c a l c i u m .
P r e s e r v a t i v e s s u c h as p r o p i o n i c a c i d and
e n u l 8 i f i e r s a n d s t a b i l i z e r s a r e s o m e t i m e s u s e d in b r e a d m a k i n g .
Propionic,
s o r b i c and b e n z o i c a c i d s m a y be d e t e r m i n e d by the GLC p r o c e d u r e of G r a v e l a n d

(8).
T h e f i b r e c o n t e n t g i v e s an i n d i c a t i o n of w h e t h e r the b r e a d w a s m a d e f r o m
wholemeal
flour.
The residue
from
the fibre
test m a y be
examined
m i c r o s c o p i c a l l y to a s s e s s t h e a m o u n t o f f i l t h in t h e i n g r e d i e n t s . T h e f i b r e
m a y be d e t e r m i n e d by a n u m b e r of m e t h o d s , i n c l u d i n g the d i c h r o m a t e m e t h o d of
v a n de K a m e r a n d v a n G r i n k e l (9).
S t a r c h is d e t e r m i n e d b y t i t r a t i o n o f r e d u c i n g s u g a r s a f t e r h y d r o l y s i s w i t h a c i d
or b y a n e n z y m i c m e t h o d . T h e l a t t e r d e p e n d s o n c o n v e r s i o n o f a u t o c l a v e d s t a r c h
to g l u c o s e b y g l u c a m y l a s e , o x i d a t i o n o f t h e g l u c o s e to g l u c o n i c a c i d w i t h
l i b e r a t i o n of h y d r o g e n p e r o x i d e and r e a c t i o n of the l a t t e r w i t h o - d i a n i s i d i n e
to f o r m an o r a n g e - r e d c o l o u r .
M i l k s o l i d s in b r e a d a r e a s s e s s e d f r o m t h e
l e v e l s of l a c t o s e or o r o t i c a c i d .
The d e t e r m i n a t i o n of l a c t o s e in b r e a d
r e q u i r e s the p r i o r f e r m e n t a t i o n of o t h e r s u g a r s p r e s e n t .
N o r m a l l y , the b r e a d
is a i r d r i e d , a p o r t i o n t a k e n f o r m o i s t u r e a n d a n a l y t i c a l f i g u r e s a r e e x p r e s s e d
as a p e r c e n t a g e of the d r y m a t t e r s i n c e t h e m o i s t u r e c o n t e n t of f r e s h b r e a d
varies considerably. Recommended c o n d i t i o n s of t e m p e r a t u r e and p r e s s u r e for
d r y i n g v a r y (100C a n d 25 m m H g to c o n s t a n t w e i g h t o r 130C for 1 h o u r , o r 50C
a n d 1 0 - 2 0 m m H g t o c o n s t a n t w e i g h t o r 130C f o r 2 h o u r s , o r 105C t o c o n s t a n t
weight).

223

MOISTORE II BREAD
PRINCIPLE
The loaf is w e i g h e d , cut and air-dried, the air-dried product is weighed and
ground and the moisture determined on a small portion.
The total moisture is
calculated as a percentage of the whole loaf as received. The method is not
applicable to bread containing fruit.
PROCEDURE
Accurately weigh loaf of bread immediately upon receipt, using scales
sensitive to at least 0.2 g. If impossible to weigh accurately at
this time, seal sample in air-tight container and weigh accurately as
soon thereafter as is practicable. Preserve sample in such a manner
that no loss of bread solids can occur whereby loss would be
calculated as moisture.
Cut bread into slices 2-3 mm thick.
Spread slices on paper, let dry
in w a r m room (15-20 hours) and when apparently dry, break into
fragments.
If bread is not entirely crisp and brittle, let it dry
longer - until it is in equilibrium with moisture of air - so that no
moisture changes occur during grinding.
W e i g h , grind and weigh again to check absence of grinding losses.
Mix w e l l and d e t e r m i n e the moisture content on a small weighed
portion (about 2 g) by drying 1 hour at 130C.
CALCULATION
% moisture in bread = % moisture in dry ground sample x
weight of air-dried slices
weight of fresh loaf
REFERE1CE
Official Methods of Analysis of the AOAC, 1984, 14.087.

224

7.4

TEXT REFERENCES

1.

M A L O N E , B.
1969.
Journal
Chemists 52 (4) 800-805.

2.

PANEL ON FUMIGANT RESIDUES IN GRAIN, REPORT.

3.

M U T H U , M., K A S H I , K.P., &

Industry 4 18.2.78. 129-131.

4.

P E A R S O N , D.
Livingstone.

5.

GRIFFITHS, J.G.A.

6.

T I L L M A N S , J., HOLL, H. & J A N I V A L A , L.

mitteluntersuchung und Forschung 56 26.

7.

TILLMANS, J.

8.

G R A V E L A N D , A. 1972. Journal of the A s s o c i a t i o n of Official

Chemists 55 (5) 1024.

9.

VAN de KAMER, J.H. & Van GRINKEL, L.

1981.

MAJUMDER,

of Official

1974.

S.K.

Analytical

Analyst 99 570-576.

1 9 7 8.

Chemistry

The C h e m i c a l A n a l y s i s of Foods, 8th Ed.

1937.

1929.

of the A s s o c i a t i o n

and

Churchill

Analyst 62 510.
1928.

Zeitschrift

fur

Lebens-

Analyst 44 43.

1952.

Agricultural

Cereal Chemistry 29 239-51.

Further Reading
BUSHUK, W. 1976.
POMERANZ, Y.

1971.

HOUSTON, D.F.
TSEN, C.C.

Rye: Production, Chemistry and Technology AACC.

Wheat: Production, Chemistry and Technology AACC.

1972.

1974.

Rice: Production, Chemistry and Technology AACC.

Triticale, First Man-Made Cereal AACC.

K E N T - J O N E S , D.W. & A M O S ,
Northern Publishing Co.

A.J.

1957.

Modern

AACC Approved M e t h o d s , A m e r i c a n A s s o c i a t i o n
Minnesota, USA.
ICC-Standards, International Association
A-2320 Schwechat, Austria.

Cereal

Chemistry

of Cereal

5th

Chemists,

Ed.

The

St. Paul,

for Cereal Chemistry Schmidgasse 3-7,

225

8.
8.1

HERBS AND SPICES

HERBS

COMPOSITION
The following table is taken in part from Pearson's Chemical Analysis of Foods,
8th Ed., 1981, and r e p r e s e n t s a n a l y s e s of various herbs.
(Note that single
values represent one analysis. All other values are ranges.)

Herb
Basil
Bay
Marj oram
Mint
Parsley
Rosemary
Sage
Savory
Thyme

Total
Ash
Z

Acid
Insoluble
Ash Z

11.5
2.8 - 4.1
8.9 -12.0
10.0 -14.4
12.4 -18.4

0.2 - 0.8

5.4 -14.3
6.3 - 9.9
7.0 -19.2

0.3

0.4 - 2.7
0.3 - 2.2

1.0 - 6.0

0.5 - 3.5

0.1 - 0.8

0.8 - 9.8

226

Volatle
Oil
Z

1.8

0.7 - 2.3
1.2 - 2.5
0.5 - 3.0

6.6

0.9
0.6 - 1.5
0.9 - 1.5
0.4 - 2.5

Stalks
etc.
Z

10

2-12
4-15
2
4
5-15
2-

8.2

SPICES - SEEDS

COMPOSITION
Umbelliferous spices are called cremocarps and are a variety of splitting fruit
w h i c h d i v i d e s i n t o o n e - s e e d e d p a r t s k n o w n as m e r i c a r p s .
S o m e r a n g e s of
composition of these spice seeds are as follows:
Non-volatile
extract Z

Ash
Z

Spice
Anise
Caraway
Celery
Coriander
Cumm in
Dill
Fenne1

4.8 - 7.6
0.1

Cardamom
Fenugreek
Mace
Mus tard
Nutmeg

20
20
20
20

12

0.8

10
15
12

The composition of other spice seeds

Spice

8
8
15

Moisture
Z
<1.3
-

3.5 - 7.0
4.8 - 7.0
4.8

Ash
Z
<9.2
<6.0
1 .6 - 2. 5
3 .7 - 4. 5
1 .8 - 4. 5

14
18

20

Volatile
Oil Z
1.5
2.5
1.5
0.3
2.0

2.0

0.8

4.0
5.9
3.0

Crude
Fibre Z

17 - 22

1.0
4.0
4.0
4.0

are:
Non-volatile
extract Z
-

24 - 33
24 - 39
30 - 40

Volatile
Oil Z
> 4
-

4
-15
0.5- 1.0
-15
5

Crude
Fibre Z
< 8
-

4.7 - 8.0
1.4 - 4.2
2.0 - 3.7

ROUTINE A N A L Y S I S
Spices should be of normal pungency, usually assessed from the volatile oil and
the n o n - v o l a t i l e e t h e r e x t r a c t .
T h e y s h o u l d a l s o be free f r o m e x t r a n e o u s
material (assessed from the ash, acid-insoluble ash and examination for filth,
h a i r s , i n s e c t s and i n s e c t f r a g m e n t s and r o d e n t d r o p p i n g s ) .
T h e i d e n t i t y of
g r o u n d s p i c e s m u s t be c h e c k e d by a c a r e f u l m i c r o s c o p i c a l e x a m i n a t i o n .
It is
c o m m o n to d e t e r m i n e lead and arsenic in spices.
The I n t e r n a t i o n a l O r g a n i z a t i o n for S t a n d a r d i z a t i o n ( I S O ) has b e e n a c t i v e in
s t a n d a r d i z i n g m e t h o d s and c o m p o s i t i o n a l l i m i t s for s p i c e s .
ISO R 6 7 6 : 1 9 6 8
g i v e s the b o t a n i c a l , F r e n c h , R u s s i a n and E n g l i s h n a m e s of 68 s p i c e s and
condiments.
ISO 2825:1974 r e c o m m e n d s that in the preparation of samples for
analysis, they are gound to approximately 1 m m , avoiding undue heating and as
far as p o s s i b l e c o n t a c t w i t h the o u t s i d e air.
T h e y s h o u l d then be p l a c e d in
clean, dry, air-tight containers so as to nearly fill them.
The q u a l i t y of s p i c e s is a s s e s s e d f r o m a n u m b e r of v a l u e s in a d d i t i o n to the
v o l a t i l e and fixed oil c o n t e n t s .
Other values include crude fibre, w a t e r soluble ash, alcohol extract, cold water extract and total nitrogen.
Nitrogen
is determined by the routine Kjeldahl procedure.
Moisture is determined by the
D e a n and S t a r k e n t r a i n m e n t m e t h o d . e x c e p t in the c a s e of m u s t a r d .
F i l t h and
insect fragments can be determined by the AOAC procedures.
Added salt may be
found in powdered spices.
Whole spices m a y be faced with mineral oil or added
colour in an attempt to improve the appearance.
M i x t u r e s of s p i c e s a l s o m u s t be a n a l y z e d f r o m t i m e to t i m e . C u r r y p o w d e r is
one of the m o s t c o m m o n m i x t u r e s .
T h i s is u s u a l l y c o m p o s e d of a f e w of the
following - capsicum, cassia, cinnamon, coriander, ginger, turmeric, fenugreek,
f e n n e l , m u s t a r d , p e p p e r and p i m e n t o .
L e a v e s of B a y and M u r r a y a K o e n i g i i ,
cumin, dill, mace and c a r d a m o m are also possible though the last two are m o r e
expensive than the others.

227

IDENTIFICATION
Anise
Anise is the fruit of Pimpinella anisum, Linn. (Umbelliferae).
The fruits are
about 3 m m long, greenish-grey to b r o w n in colour, ovoid and somewhat laterally
compressed.
The volatile oil contains about 90% of anethole.
The transverse section of the fruit exhibits the following

structure:

a.
An outer epidermis composed of flattened cells and provided with stomata
as w e l l as w i t h n u m e r o u s s i n g l e h a i r s .
In s u r f a c e v i e w the e p i d e r m a l c e l l s
a p p e a r p o l y g o n a l and s t r o n g l y s t r i a t e d ; the h a i r s are s h o r t , c o n i c a l , t h i c k walled warty, and usually one-celled.
b.
The p a r e n c h y m a t o u s t i s s u e n e x t to the e p i d e r m i s is m a d e up of p o l y g o n a l
c e l l s , and is t r a v e r s e d by s e c r e t o r y d u c t s .
The n u m b e r of the l a t t e r is
v a r i a b l e , but a l w a y s c o n s i d e r a b l e , and they are p l a c e d close t o g e t h e r .
This
t i s s u e is a l s o t r a v e r s e d by a n u m b e r of f i b r o - v a s c u l a r b u n d l e s s u r r o u n d e d by
sclerenchymatous tissue of varying extent, the cells of which are polygonal and
have thickened pitted walls.
c.
An inner epidermis, consisting of a single row of cells, all of which are
elongated in the same direction.
d.
A s e e d - c o a t , w h i c h is r e p r e s e n t e d by a s i n g l e r o w of b r o w n
cells; in surface view these appear polygonal and isodiametric.

flattened

e. An endosperm, composed of polygonal cells

containing fixed oil and aleurone grains; in
the l a t t e r a g l o b o i d or a r o s e t t e of c a l c i u n
oxalate may be found.
The d i a g n o s t i c c h a r a c t e r s
f r u i t are ( F i g u r e 8.1):

of powdered

(1)

The short, stout, conical

(2)

The numerous, narrow, brown

(3)

The sclerenchyma of the

(4)

The contents of the endosperm

anise

hairs.
oil-ducts.

pericarp.
cells.
Figure 8.1.

Anise

Caraway
C a r a w a y is the f r u i t of C a r u m c arv i, L i n n . ( U m b e l l i f e r a e ) .
The fruits are
a b o u t 6 m m long w i t h a b r o w n , g l a b r o u s s u r f a c e and s l i g h t l y c u r v e d .
The
volatile oil contains about 50% carvone.
The slight brown Levant (or Mogador)
caraway and also Indian dill have been substituted for genuine caraway.
The fruit exhibits

the following

structure:

a.
An o u t e r e p i d e r m i s , c o m p o s e d of a x i a l l y e l o n g a t e d c e l l s w i t h s t r i a t e d
c u t i c l e and p i t t e d w a l l s ; h e r e and there a s t o m a is v i s i b l e , but it o f f e r s no
remarkable features.
b.
A narrow layer of parenchymatous tissue, consisting of irregular polygonal
cells;* this tissue is traversed by fibro-vascular bundles, which are situated
in the r i d g e s of the f r u i t , and are s u p p o r t e d by s t r a n d s of s c l e r e n c h y m a t o u s
c e l l s ; the l a t t e r p o s s e s s p i t t e d w a l l s , but v a r y g r e a t l y in size and shape.
Six large b r o w n vittae also occur in this tissue.

228

c. An inner epidermis, composed of polygonal, thin-walled cells, which are all

tangentially elongated and exhibit a very regular arrangement.
d. The s e e d - c o a t , c o n s i s t i n g of a s i n g l e layer of s m a l l p o l y g o n a l c e l l s of a
rather dark b r o w n colour.
e. The e n d o s p e r m , m a d e up of r a t h e r
grains and fixed oil.

thick-walled

cells containing

aleurone

The
diagnostic
characters
of
powdered caraway are (Figure 8.2):
(1) T h e a b u n d a n t
tis sue.

sc1erenchymatous

( 2 ) T h e a b s e n c e of
spiral and reticulate
(3) The striated

h a i r s and
cells.

of

epidermis.

(4) The l a r g e c e l l s of the i n n e r

epidermis
and
their
regular
arrangement.
(5) The small aleurone
Figure 8.2.

grains.

Caraway

Celery
Celery is the fruit of Apium graveolens.
The "seeds" are brown,
and very small.
The m o s t outstanding feature of the microscopic
the elongated crossing cells similar to fennel.

sub-spherical
structure are

C e l e r y salt is u s u a l l y a m i x t u r e of s a l t and g r o u n d c e l e r y seed.

r e q u i r e s a m i n i m u m of 20% c e l e r y s e e d , for e x a m p l e .
C e l e r y salt
contains m a g n e s i u m carbonate and may also contain calcium stearate.

Canada
usually

Coriander
This

is the fruit of Coriandrum

The dorsal portion exhibits

sativum, Linn.

in transverse

(Umbelliferae).

section:

a.
A n o u t e r e p i d e r m i s c o m p o s e d of t a b u l a r c e l l s w h i c h in s u r f a c e v i e w are
s e e n to be p o l y g o n a l , and h a v e s l i g h t l y t h i c k e n e d , p i t t e d w a l l s .
It is o f t e n
partially thrown off, especially from the intercostal regions; it is provided
with stomata, and in some of the cells a prismatic crystal of calcium oxalate
may be observed.
b.
A tissue, corresponding to the m e s o c a r p , which has undergone considerable
differentiation, and in which the following layers can be distinguished:
(1)
an o u t e r l a y e r of t a n g e n t i a l l y e l o n g a t e d p a r e n c h y m a t o u s c e l l s ; (2) a w e l l d e v e l o p e d l a y e r of s c 1 e r e n c h y m a , t r a v e r s e d by f i b r o - v a s c u 1 a r b u n d l e s , and
f o r m i n g a c o n t i n u o u s and v e r y t h i c k p r o t e c t i v e t i s s u e t h r o u g h o u t the e n t i r e
dorsal portion of the mericarp; the cells of which this layer is composed are
elongated, have thick, pitted w a l l s and cross in different directions; (c) one
or t w o r o w s of f l a t t e n e d t h i n - w a l l e d c e l l s ; (d) t w o or t h r e e r o w s of l a r g e ,
irregular polygonal cells with very thick, pitted walls.
c.
An i n n e r e p i d e r m i s of f l a t t e n e d , t a n g e n t i a l l y e l o n g a t e d c e l l s w h i c h in
surface view are seen to be rectangular, four or five times as long as they are
broad, and all elongated in the same direction.

229

d.
A seed-coat consisting
w i t h slightly w a v y walls.

of a s i n g l e layer of p a l e y e l l o w p o l y g o n a l

e.
An endosperm m a d e up of thick walled polygonal cells containing
grains, fixed oil, and small rosette-crystals of calcium oxalate.

cells

aleurone

T h e s t r u c t u r e of the c o m m i s s u r a l p o r t i o n of the f r u i t is s l i g h t l y d i f f e r e n t
from that of the dorsal portion; the sclerenchymatous layer is absent, and the
m e s o c a r p is traversed by two large secretory ducts (vittae).
The diagnostic c h a r a c t e r i s of p o w d e r e d
coriander fruit are (Figure 8.3):
(1) T h e e p i d e r m a l
crystals.

cells with

prismatic

(2) T h e f i b r o u s s c 1 e r e n c h y m a t o u s
of the p e r i c a r p .

layer

(3) The large sclerenchymatous cells in

the inner part of the pericarp, to which
the inner epidermis is often attached.
(4) The large secretory

ducts.

(5) The m i n u t e rosettes of

o x a l a t e in the e n d o s p e r m .

(The

last

two characters

calcium

are found

in other umbelliferous

fruits.)

Cunmin
The

fruit of Cuminum

The fruit exhibits

cyminum, Linn. (Umbel1 iferae).

the

following

structure:

a.
A n o u t e r e p i d e r m i s c o m p o s e d of p o l y g o n a l c e l l s and p r o v i d e d
secondary ridges w i t h conical, pluricellular, pluriserial hairs.

over

the

b.
A tissue, corresponding to the m e s o c a r p , traversed by five fibro-vascular
b u n d l e s s i t u a t e d b e l o w the p r i m a r y r i d g e s .
T h i s t i s s u e a l s o c o n t a i n s six
v i t t a e , four of w h i c h are placed b e l o w the secondary ridges and the remaining
t w o on the c o m m i s s u r a l surface.
In t h i s t i s s u e and n e a r the f i b r o - v a s c u l a r
b u n d l e s s c l e r e n c h y m a t o u s c e l l s of v a r y i n g s h a p e s are to be f o u n d ; s o m e are
p o l y g o n a l and e l o n g a t e d , o t h e r s s i n u o u s , etc., b u t a l l of t h e m h a v e t h i c k ,
pitted walls.
The bundles
t h e m s e l v e s are a c c o m p a n i e d by s c l e r e n c h y m a t o u s
fibres w i t h lignified walls.
c.
An inner e p i d e r m i s c o m p o s e d
e l o n g a t e in the same direction.
d.

A seed-coat

consisting

of

tolerably

of brown polygonal

regular

polygonal

cells

all

cells.

e. A n e n d o s p e r m w i t h t h i c k - w a l l e d c e l l s in w h i c h a l e u r o n e g r a i n s , fixed o i l
and small rosette crystals of calcium oxalate are contained.
The diagnostic
(1) The

characters of powdered

pluricellular,

pluriserial

(2) The s c l e r e n c h y m a t o u s

cells

from

cummin

fruit

hairs.
the

mesocarp.

230

are (Figure

8.4):

(3) The large

oil-ducts.

Dill
Dill is from Anethum graveolens.
The mericarps are about 4 mm x 2.5 m m , flat
and g l a b r o u s . The m a i n c o n s t i t u e n t of the v o l a t i l e oil is c a r v o n e as is a l s o
the case w i t h c a r a w a y .
T h e m a i n m i c r o s c o p i c a l f e a t u r e s are the s t r i a t e d
c u t i c l e of the e p i d e r m i s , the fixed o i l and the a l e u r o n e g r a i n s e n c l o s i n g a
microcrystal of calcium oxalate.
Fennel
The

fruit of Foeniculum

The transverse

capillaceum, Gilib.

(Umbelliferae).

section exhibits the following

a.
An outer epidermis,
furnished with stomata.

composed

characters:

of polygonal

cells

with

straight

walls

and

b.
P a r e n c h y m a t o u s t i s s u e ( m e s o c a r p ) c o m p o s e d of irregular polygonal cells;
m a n y of these are characterized by their reticulate or spiral thickening; they
are either isolated or form groups in the ridges of the fruit, near the fibrovascular bundles.
T h e r e a r e six l a r g e v i t t a e , e a s i l y d i s t i n g u i s h e d by the
b r o w n c o l o u r of t h e i r w a l l s ; four are s i t u a t e d on the d o r s a l s u r f a c e of the
fruit, and two on the c o m m i s s u r a l .
The bundles are composed of tracheids w i t h
a f e w b a s t c e l l s , s u p p o r t e d by a m a s s of s c 1 e r e n c h y m a t ous f i b r e s w i t h p i t t e d
wal1 s.
c.
An inner epidermis, composed of a single layer of n a r r o w , elongated cells;
t h e s e c e l l s a r e a r r a n g e d in g r o u p s of s o m e six or m o r e , w i t h t h e i r long a x e s
p a r a l l e l to one a n o t h e r , but at r i g h t a n g l e s or o b l i q u e l y to the long a x e s of
the cells of other groups.
d.

A seed coat;

this consists of a single

layer of b r o w n polygonal

cells.

e.
An endosperm m a d e up of rather thick walled polygonal cells, containing
a l e u r o n e g r a i n s , fixed o i l , and p r o t o p l a s m .
S o m e of the a l e u r o n e g r a i n s
contain a rounded globoid, others a small rosette of calcium oxalate.

231

The

d i a g n o s t i c c h a r a c t e r s of powdered

fenne

are (Figure 8.5):

(1) The spiral and r e t i c u l a t e cells of the

mesocarp.
(2) The narrow cells of the inner epidermis
and their charac teristic arrangement.
absence of hairs.

(3)

The

(4)

The thick-w ailed endosperm cells.

Figure 8.5.

Fennel

Cardamom
The fruits of Elettaria cardamomum, Maton

(Scitamineae).

The pericarp of the fruit presents the following

tissues:

a.
An outer epidermis consisting of a single row of irregular polygonal cells
with straight, smooth walls.
b.
A rather thick layer of p a r e n c h y m a traversed by numerous fibro-vascular
bundles, and containing scattered cells filled w i t h b r o w n i s h oleoresin.
The
f i b r o - v a s c u l a r b u n d l e s are supported by a m a s s of fibres, m o s t of w h i c h have
thickened, pitted walls.
c.
An inner epidermis,
less collapsed.

resembling

the outer in structure but usually more or

The arillus is very thin and composed of several rows of elongated, yellowish,
m o r e or less c o l l a p s e d , cells, c o n t a i n i n g small rounded or oval droplets of
oil.
The seed is composed of the following

tissues:

a.
An epidermis, consisting of cells which appear rectangular in transverse
s e c t i o n , but in surface v i e w are seen to be m u c h elongated and taper t o w a r d s
the ends; they are furnished with slightly thickened, undulating walls.
b.
A single r o w of s m a l l e r cells, also elongated in shape but crossing the
cells of the epidermis at right angles.
c.

A single row of large rectangular

d.
A narrow layer composed
is not distinctly visible.

oil-cells.

of several rows of cells,

the structure of which

e.
An inner e p i d e r m i s , consisting of a single row of b r o w n or y e l l o w i s h brown, radially elongated cells with very thick walls, the cavity being shallow
and almost entirely filled with a nodule of silica.
f.
A largely developed perisperm, the cells of which have thin walls, and are
packed w i t h m i n u t e starch grains; in the centre of each cell there is a
prismatic crystal of calcium oxalate.
g.

An endosperm

and embryo, the cells of which contain proteid matter.

232

The diagnostic

characters

of the powdered

pericarps

(1)

The parenchyma with e m p t y cells and scattered

(2)

The

Powdered

fibres

from

the

resin

cells.

bundles.

seeds are identified

(3)

The characteristic

(4)

The sclerenchymatous

(5) T h e f r a g m e n t s
small starch grains
crystals.

are (Figure 8.6):

by:

epidermis.
layer.

of p e r i s p e r m
with
and calcium oxalate

Figure 8.6.

Powdered

Cardasoa

Fenugreek
Fenugreek is from Trigonella foenum-graecum.
The seeds range in size up to 6.5
m m l o n g , 3 m m w i d e and 2.5 m m t h i c k .
They are hard, y e l l o w i s h b r o w n ,
irregularly rhomboidal and flattened.
T h e y h a v e a d e p r e s s i o n in one of the l o n g , n a r r o w s i d e s .
The s e e d s c o n t a i n
trigonelline and choline and are about 18% mucilage.
Microscopically there are
epidermal palisade cells which are about five times longer than they are w i d e ,
plus hour-glass cells with b a r - l i k e t h i c k e n i n g s , a l e u r o n e g r a i n s and s t a r c h .
Fenugreek is a principal constituent of m a n y curry powders.
Mace
Mace is the fleshy arillus surrounding the seeds of Myristica fragrans, Houtt.
(Myristicaceae).
It consists principally of p a r e n c h y m a t o u s t i s s u e c o n t a i n i n g
The cells of the
numerous oil cells, and traversed by fibro-vascular bundles.
p a r e n c h y m a (pa) are p o l y g o n a l and i s o d i a m e t r i c .
T h e y contai.i a r e m a r k a b l e
s u b s t a n c e , k n o w n as a m y l o d e x t r i n , e m b e d d e d in a f a t t y m a s s .
Am y lo-dextrin
o c c u r s in g r a i n s of v e r y i r r e g u l a r s h a p e .
S o m e t i m e s t h e y a r e d i s c o i d or
rounded but more often they are angular.
Solution
of
io d o - p o t a s s ium
iodide
colours them red.
The oil-cells contain
e i t h e r y e l l o w v o l a t i l e o i l or r e d d i s h b r o w n oleo-resin.
The e p i d e r m i s
of b o t h s u r f a c e s
is
covered with a thick cuticle.
In
s u r f a c e v i e w the e p i d e r m a l c e l l s (eps,
e p i ) are s t r o n g l y e l o n g a t e d
axially;
they are often fusiform, and have thick
walls.
Below the upper epidermis there
is a c o l l e n c h y m a t o u s h y p o d e r m a ; t h i s ,
h o w e v e r , is n o t c o n t i n u o u s or u n i f o r m ,
but disappears in some places, whilst in
o t h e r s it is c o m p o s e d of t w o r o w s of
cells .
Figure 8.7.

Powdered

Mace

233

The

diagnostic

characters

(1) T h e l a r g e , p o i n t e d ,
(2) T h e

of p o w d e r e d

thick-walled

mace

c e l l s of t h e

o i l - c e l l s , m a n y of w h i c h m a y be

(3) T h e g r a i n s of a m y l o - d e x t r i n

are ( F i g u r e

8.7):

epidermis.

broken.

in t h e p a r e n c h y m a t o u s

cells.

MasCard
ISO 1 2 3 7 - 1 9 7 4 s p e c i f i e s that m u s t a r d seed m u s t be the d r i e d c l e a n s e e d s of
S inap i s a l b a ( w h i t e or y e l l o w m u s t a r d ) , Bras s ica n i g r a ( b l a c k m u s t a r d ) or
B r a s s i c a j u n c e a (Indian" m u s t a r d ) .
T h e m u s t a r d s e e d s s h a l l b e w h o l e and m a t u r e
a n d s h a l l n o t c o n t a i n m o r e t h a n 2% o f e x t r a n e o u s m a t t e r o r o t h e r v e g e t a b l e
material.
E x t r a n e o u s seeds include charlock (Sinapis arvensis Linnaeus), rape
( B r a s s i c a n a p u s L i n n a e u s ) and M e l i l o t u s s p e c i e s .
T h e p r o p o r t i o n of d a m a g e d or
shrivelled m u s t a r d s seeds shall not exceed 2%.

the
a.

Black Mustard Seeds:

T h e s e e d s of B r a s s i c a n i g r a , K o c h ( C r u c i f e r a e ) .
s e e d - c o a t s the f o l l o w i n g layers can be d i s t i n g u i s h e d :
An e p i d e r m i s (am) c o m p o s e d

of l a r g e

thin-walled

cells containing

In

mucilage.

b.
A s i n g l e l a y e r of l a r g e p a r e n c h y m a t o u s c e l l s , the w a l l s of w h i c h a r e n o t
c o l l e n c h y m a t o u s , as t h o s e of w h i t e m u s t a r d s e e d s a r e .
These cells
are
g e n e r a l l y c o l l a p s e d , and lie c l o s e l y p r e s s e d on to the n e x t l a y e r of c e l l s .
In
t h e p o w d e r e d f o r m t h e y a r e n o t e a s y to s e e .
c.
A s i n g l e l a y e r o f d a r k b r o w n s c l e r e n c h y m a t o u s c e l l s ( s c ) w h i c h , in
t r a n s v e r s e s e c t i o n , e x h i b i t t h e v e r y c h a r a c t e r i s t i c t h i c k e n i n g ( s ' c ' ) s h o w n in
the i l l u s t r a t i o n .
S o m e of t h e s e c e l l s at r e g u l a r i n t e r v a l s a r e l o n g e r t h a n t h e
o t h e r s , a n d t h u s p r o d u c e t h e p i t t e d a p p e a r a n c e of the s e e d as w e l l as the
p e c u l i a r , p o l y g o n a l n e t w o r k s e e n in t h e s u r f a c e v i e w of the s e e d - c o a t s .
d.
A t h i n m e m b r a n o u s l a y e r ( c m ) c o n s i s t i n g of l a r g e , p o l y g o n a l , f l a t t e n e d
cells, containing a brownish amorphous substance.
T h i s l a y e r is c l o s e l y
a p p l i e d to t h e s c l e r e n c h y m a t o u s l a y e r , a n d in t h e p o w d e r g e n e r a l l y r e m a i n s
f i r m l y a d h e r e d to i t , p r o d u c i n g , i n p a r t , t h e c h a r a c t e r i s t i c c o l o u r o f t h e
seed .
e.
W i t h i n the s e e d - c o a t is an a l e u r o n e l a y e r (ap) c o n s i s t i n g o f r a t h e r t h i c k w a l l e d , p o l y g o n a l c e l l s , c o n t a i n i n g a l e u r o n e g r a i n s . N e x t to t h i s r o w of c e l l s
is a l a y e r c o m p o s e d o f s e v e r a l r o w s o f c o l l a p s e d p a r e n c h y m a t o u s c e l l s , t h e
c a v i t i e s o f w h i c h a r e o n l y i n d i s t i n c t l y v i s i b l e as f a i n t l i n e s .
f.
T h e c o t y l e d o n s (co) are c o v e r e d b y a t r a n s p a r e n t e p i d e r m i s , c o n s i s t i n g
polygonal cells.
T h e y c o n t a i n s m a l l i r r e g l u l a r a l e u r o n e g r a i n s , in e a c h
w h i c h n u m e r o u s m i n u t e g l o b o i d s can be d e t e c t e d .

of
of

T h e p o w d e r e d s e e d s c o n t a i n v e r y n u m e r o u s f r a g m e n t s of t h e d e l i c a t e t i s s u e of
t h e c o t y l e d o n s and r a d i c l e ; in g l y c e r i n t h e s e e x h i b i t t h e c h a r a c t e r i s t i c
a l e u r o n e g r a i n s , w h i c h m a y also be found s c a t t e r e d over the p r e p a r a t i o n .
E x a m i n e d in c h l o r a l h y d r a t e g l o b u l e s of f i x e d o i l are v e r y
conspicuous.
F r a g m e n t s of the s e e d - c o a t are e a s i l y r e c o g n i z e d by their b r o w n c o l o u r ; they
u s u a l l y p r e s e n t t h e i r s u r f a c e v i e w and e x h i b i t the p o l y g o n a l n e t w o r k a l l u d e d to
a b o v e . C o l o u r l e s s , t r a n s p a r e n t f r a g m e n t s of the e p i d e r m i s m a y a l s o be f o u n d as
w e l l as p o r t i o n s of the a l e u r o n e l a y e r .
The d i a g n o s t i c

characters

of p o w d e r e d

mustard

234

seeds

are

(Figure

8.8):

(1)
The
chymatous

dark,
layer.

yellowish-brown

scleren-

(2) The p o l y g o n a l n e t w o r k e x h i b i t e d by the

upper surface of that layer.
(3)
The small
numerous minute
(4)

aleurone grains,
globoids.

The mucilaginous

cells of the

each

with

epidermis.

Povdered

Figure 8.8.
Black Mustard

Seeds

tfhite M u s t a r d S e e d s :
The seeds of Brassica alba, Linn. (Cruciferae).
The
seeds are yellow in colour, and so minutely pitted that they appear smooth to
the naked eye.
In the seed-coats the following layers can be distinguished:
(a) An epidermis (am) made up of large cells containing mucilage
very rapidly and very considerably in contact w i t h water.

which

swells

(b) A collenchymatous layer (col) composed of two rows of polygonal cells, the
walls of which are thickened, particularly in the angles.
(c) A s c l e r e n c h y m a t o u s l a y e r (sc) c o n s i s t i n g of a s i n g l e r o w of c e l l s ,
lateral and inner walls of which are thickened and pale yellow in colour.
cells are tolerably uniform in size and arrangement.
The above three layers represent

the outer

the
The

seed-coat.

(d) A m e m b r a n e layer (cm) composed of two or three rows of strongly flattened

T h e s e are free f r o m the d a r k b r o w n p i g m e n t w h i c h is c o n t a i n e d in the
cells.
corresponding cells of black mustard seed.
Within the seed-coats is the e m b r y o surrounded by the aleurone layer (ap); the
l a t t e r c o n s i s t s of a s i n g l e r o w of i s o d i a m e t r i c p o l y g o n a l c e l l s w h i c h h a v e
u n i f o r m l y t h i c k e n e d w a l l s and g r a n u l a r c o n t e n t s .
The e p i d e r m i s of the
cotyledons (cc) is composed of irregular empty cells amongst which groups of
t w o or t h r e e s m a l l e r o n e s m a y be o b s e r v e d ; t h e s e are s t o m a t a in p r o c e s s of
f o r m a t ion.
The c e l l s of the c o t y l e d o n s t h e m s e l v e s (co) are filled w i t h
aleurone grains and fixed oil.
The aleurone grains are small and irregular in
shape; they contain numerous minute globoids but no crystalloids.
The p o w d e r c o n s i s t s , like that of
black m u s t a r d , largely of fragments
of the cotyledons and radicle.
The
portions of the seed-coat are pale
yellow in colour, and hence easily
d i s t i n g u i s h e d f r o m the r e d - b r o w n
f r a g m e n t s of b l a c k m u s t a r d .
The
polygonal n e t w o r k that is so easily
s e e n on the u p p e r s u r f a c e of the
sclerenchymatous
layer of b l a c k
mustard is m u c h less conspicuous.

Figure 8.9.
Powdered White Mustard

Seeds

235

The

diagnostic

characters

(1) The pale yellow

(2) T h e

epidermal

small

powdered

sclerenchymatous

cells

(3) The collenchymatouB

(4) T h e

of

irregular,

with

striated

hypodermal
aleurone

white

mustard

are

(Figure

8.9):

layer.
mucilage.

layer.
grains

containing

numerous

minute

globoid.

Mutmeg

N u t m e g is from M y r i s t ic a fragrans and consists of the kernel of the

(Mace is from the same, but is the dried extra seed coat or aril).
N u t m e g s are ovoid w i t h the B a n d a n u t m e g s m e a s u r i n g about
light b r o w n , f i n e l y pitted and h a v e r e t i c u l a t e m a r k i n g .

2 x

2.5

cm.

Bombay nutmegs (M_. malabaria), and Macassar nutmegs (M. argentea) are
narrower and do not have the aroma of the Banda (M. fragrans).

They

seed.

are

longer,

Microscopically, powdered nutmeg shows solid fat, starch grains and brown cells
in the perisperm.

236

UMBELLIFEROUS SEEDS
(TLC Identification)
PRINCIPLE
P e t r o l e u m ether e x t r a c t s
dinitrophenylhydrazine.

are

developed

by

TLC

and

visualized

with

2,

APPARATUS
1.

TLC

equipment.

2.

UV light source, 365 nm.

3.
P r e p a r e p l a t e s 0.25 m m thick from 25 g silica gel G in 50 ml of
0.05% a q u e o u s f l u o r e s c e i n s o d i u m .
He at at 105C for 30 m i n u t e s
b e f o r e use.
REAGENTS
1.

Petroleum ether BR

60-80C.

2.

Chloroform - benzene

3.

Bromine.

4.

2,4-dinitrophenylhydrazine,
chloric acid.

5.

Sulphuric acid containing

(1:1) developing

solvent.

saturated

solution

in

IN

hydro-

1% vanillin.

PROCEDURE
Shake 0.5 g powdered sample with 5 ml of petroleum ether.
After the
s u s p e n d e d m a t t e r h a s s e t t l e d , apply 0.03 m l of e x t r a c t to the TLC
p l a t e , a l l o w i n g the spot to b e c o m e up to 1 cm in d i a m e t e r .
Apply
e x t r a c t s of a u t h e n t i c s p e c i m e n s f o r c o m p a r i s o n .
Develop
the
c h r o m a t o g r a m for about 15 cm.
E x a m i n e u n d e r UV light and l i g h t l y
o u t l i n e any q u e n c h e d areas.
Treat b r i e f l y w i t h b r o m i n e v a p o u r
( c o n v e r t i n g f l u o r e s c e i n to e o s i n ) and e x a m i n e under UV light for
persisting fluorescein f l u o r e s c e n c e due to u n s a t u r a t e d s u b s t a n c e s .
Spray w i t h the 2 , 4 - d ini t r o p h e n y lhy dr a z ine solution. Aldehydes and
k e t o n e s a p p e a r as o r a n g e spots. Air dry and spray w i t h 1% v a n i l l i n
in s u l p h u r i c acid. K h e l l i n , t h y m o l and s i m i l a r s u b s t a n c e s give
c o l o u r e d spots r a p i d l y , f e n c h o n e and s o m e o t h e r s m a y take s e v e r a l
hours.
INTERPRETATION
Betts

found

the following R^ values

Anise
Caraway
Coriander
Cummin
Dill
Fennel

for Umbelliferous

seeds:

0.17, 0. 39
0.43
0.26
0.58, 0. 55
0.42
0.72, 0. 57,

REFERENCE
B e t t s , T.J., Q u a r t e r l y
16 131T.

Journal

of P h a r m a c y an.d Pharmacology

237

(Supplement)

4-

HOI-VOLATILE

EXTRACT

PRINCIPLE
The spice material is extracted with diethyl ether, the ether is distilled
off and the non-volatile residue is weighed.
APPARATUS
1.

Soxhlet or similar extraction

2.

Oven at 110 +_ 1C.

apparatus.

REAGENT
1.

Diethyl ether,

anhydrous.

PROCEDURE
Extract a weighed 2 g sample portion (ground to pass a 1 m m sieve) in
a continuous extraction apparatus with anhydrous diethyl ether for 18
hours.
R e m o v e the ether from the extract by distillation, followed
by b l o w i n g w i t h a s t r e a m of air, w i t h the flask kept on a b o i l i n g water bath, and dry the flask in the oven at 110 _+ 1C until the loss
in w e i g h t b e t w e e n t w o c o n s e c u t i v e w e i g h i n g s is less than 0.005g.
S h a k e the r e s i d u e in the flask w i t h 2 to 3 m l of a n h y d r o u s d i e t h y l
e t h e r at l a b o r a t o r y t e m p e r a t u r e s , a l l o w to s e t t l e and d e c a n t the
ether.
Repeat the extraction until no more of the residue dissolves.
Dry the f l a s k a g a i n u n t i l the loss in m a s s b e t w e e n t w o s u c c e s s i v e
weighings is less than 0.005g.
CALCULATION
T h e p e r c e n t a g e of n o n - v o l a t i l e e t h e r e x t r a c t in the s a m p l e , on the
dry basis, is equal to

(M

2>

100
"M;

100
100 - H

Where :
MQ

= g of sample

portion

Mj

= g of the residue obtained

M j = g of the final
H

after drying at 110C

residue.

= moisture content, as a percentage by weight, of the

sample as received.

INTERPRETATION
This determination is also referred to as the fixed oil or fixed ether extract.
The condenser water m a y be too hot for the refluxing of diethyl ether, in which
c a s e the m e t h o d as w r i t t e n c a n n o t be c a r r i e d out.
D i - i s o p r o p y l ether is
readily available and of higher boiling-point.
The author is not aware of any
r e p o r t c o m p a r i n g r e s u l t s u s i n g the t w o s o l v e n t s but it s e e m s l i k e l y that any
difference would be small.
REFERENCE
ISO 1108 - 1980.

238

VOLATILE OIL
PRINCIPLE
The d e t e r m i n a t i o n of v o l a t i l e oil in a s p i c e is m a d e by d i s t i l l i n g the s p i c e
with water, collecting the distillate in a graduated tube in which the aqueous
p o r t i o n of the d i s t i l l a t e is a u t o m a t i c a l l y s e p a r a t e d and r e t u r n e d to the
distilling flask, and measuring the v o l u m e of the oil.
The content of volatile
oil is expressed as a percentage v/w.

APPARATUS
1.
F l a s k , d i s t i l l i n g , 1 litre c a p a c i t y , p r e f e r a b l y w i t h
stirrer.
2.

Volatile

oil traps, Clevenger

LIGHTER

THAN

magnetic

type.

WATER

HEAVIER

THAN

WATER

Dimensions in m m .
It is essential to wash the apparatus with acetone
and water and then leave to stand in chromic-sulphuric acid mixture
with complete rinsing prior to use.

PROCEDURE
Grind the spice to pass a number 20 (850 m i c r o n ) sieve.
Weigh 20g of
s p i c e or e n o u g h to y i e l d 2 - 4 m l of oil if p o s s i b l e and p l a c e in the
f l a s k w i t h g l a s s b e a d s or p o r o u s e a r t h e n w a r e p i e c e s if a m a g n e t i c
s t i r r e r is n o t u s e d .
Add a b o u t 3 0 0 m l of w a t e r and a d r o p of
antifoam if necessary.
Fill the trap with water.
Place an efficient
w a t e r - c o o l e d c o n d e n s e r on top of the t r a p and h e a t the f l a s k w i t h
good stirring or agitation until boiling starts and continue boiling
m o d e r a t e l y b r i s k l y b u t so t h a t the l o w e r p a r t of the c o n d e n s e r
remains cold. Set the apparatus so that the condensate will not drop
d i r e c t l y on the s u r f a c e of the l i q u i d in the t r a p b u t run d o w n the
side walls.
Rotate the flask occasionally to wash d o w n any m a t e r i a l
adhering to the upper part of the walls.
Distil until 2 consecutive
readings taken at 1 hour intervals show no change in oil content ( > 6
hr).
R e m o v e the s o u r c e of h e a t and r e a d the v o l u m e of o i l ten
minutes or so later.
Calculate as v/w.
If the oil s e p a r a t e s in the g r a d u a t e d p o r t i o n of the trap or c l i n g s
to the w a l l s , add s e v e r a l d r o p s of a s a t u r a t e d a q u e o u s d e t e r g e n t
s o l u t i o n t h r o u g h the top of the c o n d e n s e r .
R e p e a t if n e c e s s a r y
although once is usually sufficient.
Distil for at least 10 minutes
after adding detergent in order to wash it out of the trap.

239

S o m e oils (e.g. cassia) have a density close to 1 or separate into 2

fractions in the trap (allspice, nutmeg).
For these, prior to adding
the s a m p l e to the flask, add 1.0ml xylene to the trap and distil
w i t h o u t s a m p l e for at least half an hour.
Cool and m o r e than ten
minutes later read the volume of xylene.
Add the sample and distil
for up to 6 hours as described above. Subtract the volume of xylene
from the total volume of the organic layer in the trap. Calculate as
before.
The oil obtained (without use of xylene) may be recovered, dried with
a very small amount of sodium sulphate and its characteristics, such
as density, RI etc. determined.

REFERENCES
Sage,

C.E. and

Fleck,

H.R.,

1934.

The Analyst

Official Methods of Analysis of the AOAC,

1984,

59., 614.
30.020-30.027.

Note: The a p p a r a t u s of Sage and Fleck can be constructed from ordinary

laboratory glassware.
Results by either method may be a little lower than
the true v a l u e , due to the slight solubility of some oils in water. The
results are also affected by the particle size of the sample.

240

8.3

S P I C E S - P O D S AOT) F R U I T S

COMPOSITIOH
A l l red pod p e p p e r s are s p e c i e s of C a p s i c u m a n d are f o u n d t h r o u g h o u t the w o r l d .
T h e w h o l e p o d s a r e u s u a l l y c a l l e d ' c h i l i e s ' a n d v a r y d r a m a t i c a l l y in s i z e ,
c o l o u r and p u n g e n c y .
D r i e d a n d g r o u n d C a p s i c u m o f m a n y s p e c i e s a r e u s e d as a s p i c e . T h e t w o m o r e
c o m m o n are cayenne and p a p r i k a .
C a y e n n e c a n b e m a d e f r o m C. f r u t e s c e n s L., C.
b a c c a t u m L . or o t h e r s m a l 1 - f r u i t e d s p e c i e s .
P a p r i k a is m u c h l e s s p u n g e n t and
is u s u a l l y m a d e f r o m
annuum.
T h e p u n g e n c y (or h o t n e s s ) of c a p i s c u m s is d u e to c a p s a i c i n ( C H ^ O C j 7 H 2 4 . N O 2 H )
the pepper oil.
A b o u t 0.05% of c a y e n n e p e p p e r is c a p s a i c i n , for e x a m p l e .
Some composition data

for c a y e n n e and p a p r i k a a r e as

Spice

Moisture
Z

Total
Ash Z

follows:

Ion-Volatile
Extract Z

Volatile
Oil Z

Cayenne

3.7 - 9 . 0

5.1 - 6.4

15 - 22

0.7 -

2.6

Paprika

7.0 - 9.5

5.6 - 7.6

7 - 1 2

0.3 -

1.5

Other spice fruits include b l a c k and w h i t e

c o m p o s i t i o n a l d a t a a r e as f o l l o w s :

Spice

Moisture
Z

pepper and

Total
Ash Z

allspice (pimento).

Non-Volatile
Extract Z

Volatile
Oil Z

Allspice

9.4

-10.5

4.1 - 4.8

4 . 3 - 7.7

3.0 - 5.2

Black

Pepper

8.7

-13.0

3.1 -

6.1

-10.7

0.5 -

2.5

White

Pepper

9.5

-14.5

0.6 - 2.5

6.2 - 9.7

0.5 -

1.8

6.4

in

IDENTIFICATION
Capsicums
T h e f r u i t of C a p s i c u m
following tissues:

annuum

L. (Paprika)

has

a pericarp

a.
An e p i d e r m i s of t a b u l a r c e l l s , w h i c h are s e e n
p o l y g o n a l and to h a v e t h i c k e n e d , p i t t e d , y e l l o w w a l l s .

in

composed

surface

view

of

to

the

be

b.
N e x t to t h e e p i d e r m i s is a h y p o d e r m a c o n s i s t i n g o f f o u r o r f i v e r o w s o f
tangentially elongated cells with c o l l e n c h y m a t o u s and s u b e r i z e d w a l l s ; these
c e l l s c o n t a i n red c h r o m o p l a s t s and d r o p l e t s of o i l .
T h e h y p o d e r m a is f o l l o w e d
b y p a r e n c h y m a t o u s t i s s u e m a d e u p of t h i n - w a l l e d , p o l y g o n a l c e l l s , a n d t r a v e r s e d
by numerous bicollateral bundles.
c.
L a s t l y , an i n n e r e p i d e r m i s c o m p o s e d of c e l l s w i t h t h i c k e n e d and p i t t e d
w a l l s , w h i c h in s u r f a c e v i e w a r e s e e n to b e i r r e g u l a r l y s i n u o u s .
These thickw a l l e d c e l l s a r e i n t e r r u p t e d at i n t e r v a l s b y b a n d s o f p o l y g o n a l t h i n - w a l l e d
c e l l s , t h e w h o l e f o r m i n g an e x t r e m e l y c h a r a c t e r i s t i c t i s s u e .
d.
T h e c a l y x p o s s e s s e s on its l o w e r s u r f a c e an e p i d e r m i s b e a r i n g s t o m a t a , and
c o m p o s e d of r e c t a n g u l a r c e l l s , w h i c h in s u r f a c e v i e w a r e p o l y g o n a l
and
e l o n g a t e d . T h e e p i d e r m i s of the u p p e r s u r f a c e is f o r m e d of i r r e g u l a r p o l y g o n a l

241

c e l l s w i t h p i t t e d w a l l s , and b e a r s s h o r t u n i c e l l u l a r c o n i c a l h a i r s
b i c e l l u l a r and p l u r i c e l l u l a r s t a l k e d g l a n d s of v a r y i n g s i z e .

as w e l l

as

e.
T h e e p i d e r m i s o f t h e s e e d - c o a t is v e r y c h a r a c t e r i s t i c . In s u r f a c e v i e w
t h e c e l l s o f w h i c h i t is c o m p o s e d a r e s e e n to b e v e r y l a r g e a n d p r o v i d e d w i t h
v e r y t h i c k s i n u o u s w a l l s , b u t in t r a n s v e r s e s e c t i o n t h e o u t e r w a l l is t h i n ,
w h i l s t the r a d i a l and inner w a l l s are t h i c k e n e d ; i m m e d i a t e l y b e l o w the
e p i d e r m i s is a l a y e r of p a r e n c h y m a t o u s t i s s u e m a d e up of p o l y g o n a l c e l l s w i t h
t h i n , p i t t e d w a l l s , n e x t to w h i c h t h e r e is a t h i c k e r l a y e r o f p o l y g o n a l ,
8 o d i a m e t r i c c e l l s . T h e c e l l s of the e n d o s p e r m are p o l y g o n a l and c o n t a i n s m a l l
aleurone grains.
f.
T h e p l a c e n t a is c o v e r e d w i t h an e p i d e r m i s of p o l y g o n a l c e l l s w i t h p i t t e d
walls.
B e l o w t h e c u t i c l e of t h e s e c e l l s o i l y d r o p s a r e s e c r e t e d in w h i c h the
a c t i v e c o n s t i t u e n t , c a p s a i c i n , is c o n t a i n e d ; t h e l a t t e r m a y s o m e t i m e s b e
N e x t to t h e e p i d e r m i s is a p a r e n c h y m a t o u s
o b s e r v e d in l a m e l l a r c r y s t a l s .
t i s s u e c o m p o s e d of s m a l l e r i r r e g u l a r c e l l s and t r a v e r s e d b y f i b r o - v a s c u l a r
bundles.
The
diagnostic
characters
c a p s i c u m s a r e ( F i g u r e 8.10):

of

powdered

(1) T h e i n n e r e p i d e r m i s of the p e r i c a r p w i t h
t h i c k - w a l l e d c e l l s i n t e r r u p t e d by b a n d s of
thin-walled cells;
(2) T h e e p i d e r m i s
of the s e e d - c o a t
with
large, thick-walled, sinuous pitted cells;
(3) T h e d r o p l e t s
fixed oil;

of y e l l o w or

(4) The
thickened
cells
e p i d e r m i s of t h e p e r i c a r p .

orange-coloured
of

the

outer

Figure 8.10
Powdered Capsicum Fruits
Black

Pepper

B l a c k p e p p e r is t h e u n r i p e f r u i t o f P i p e r n i g r u m , n o t to b e c o n f u s e d w i t h
p i m e n t o ( a l l s p i c e ) , t h e f r u i t of P i m e n t a o f f i c i n a l i s (an o b v i o u s d i f f e r e n c e is
t h a t the c a l y x and s t y l e s t i l l r e m a i n in the l a t t e r ) or p i m i e n t o , a v a r i e t y of
C a p s i c u m or r e d p e p p e r .
A transverse

s e c t i o n of b l a c k p e p p e r e x h i b i t s

the f o l l o w i n g

structure:

a.
A n o u t e r e p i d e r m i s c o n s i s t i n g of s m a l l c e l l s w i t h b r o w n c o n t e n t s and a
rather thick cuticle.
In s u r f a c e v i e w t h e s e c e l l s a p p e a r p o l y g o n a l , and h e r e
a n d t h e r e a s t o m a m a y b e s e e n ; m a n y of t h e m c o n t a i n s m a l l p r i s m a t i c c r y s t a l s of
calcium oxalate.
b.
A n o u t e r s c l e r e n c h y m a t o u s l a y e r a b u t t i n g u p o n the e p i d e r m i s or s e p a r a t e d
f r o m it b y a s i n g l e r o w of p a r e n c h y m a t o u s c e l l s . T h i s l a y e r is n o t c o n t i n u o u s ,
b u t is i n t e r r u p t e d a t i n t e r v a l s b y t h i n - w a l l e d p a r e n c h y m a t o u s c e l l s .
The
B C l e r e n c h y m a t o u 8 e l l s v a r y s o m e w h a t in s h a p e , b u t m o s t of t h e m are r a d i a l l y
e l o n g a t e d , and c o n t a i n a b r o w n s u b s t a n c e ; t h e i r w a l l s are t h i c k and p i t t e d .

242

c.
P a r e n c h y m a t o u s t i s s u e c o r r e s p o n d i n g to the m e s o c a r p , and c o n s t i t u t i n g the
b u l k of the p e r i c a r p .
The o u t e r l a y e r s of t h i s t i s s u e c o n s i s t of l a r g e
p o l y g o n a l c e l l s , a m o n g s t w h i c h an o c c a s i o n a l s t i l l l a r g e r o i l - c e l l m a y b e s e e n ;
t h e f o r m e r c o n t a i n a f e w s m a l l s t a r c h g r a i n s , t h e l a t t e r g l o b u l e s of v o l a t i l e
oil.
T h e i n n e r l a y e r s of p a r e n c h y m a t o u s c e l l s h a v e l i g n i f i e d w a l l s and are
m o r e s t r o n g l y t a n g e n t i a l l y e l o n g a t e d or e v e n f l a t t e n e d so as to p r e s e n t a w e l l
m a r k e d l i n e o f d e m a r c a t i o n , w h i c h is a c c e n t u a t e d b y t h e p r e s e n c e o f f i b r o vascular bundles.
O i l - c e l l s a r e m o r e n u m e r o u s in t h i s i n n e r p a r t of t h e
p a r e n c h y m a t o u s t i s s u e t h a n t h e y a r e in the o u t e r .
d.
A n i n n e r s c l e r e n c h y m a t o u s l a y e r c o n s i s t i n g of a s i n g l e r o w of c e l l s
t h i c k e n e d o n t h e i r r a d i a l a n d i n n e r t a n g e n t i a l w a l l s ; in s u r f a c e v i e w t h e s e
c e l l s a r e s e e n to b e i s o d i a m e t r i c , p o l y g o n a l , a n d to h a v e m o d e r a t e l y t h i c k ,
p i t t e d w a l l s ; t h e i r c a v i t i e s are c o l o u r l e s s and l a r g e r t h a n t h o s e of the o u t e r
l a y e r of s c l e r e n c h y m a t o u s c e l l s .
T h i s l a y e r of c e l l s is g e n e r a l l y a d h e r e n t to
the b r o w n s e e d - c o a t .
e.
A brown
a colourless
seed-coat.

and a y e l l o w l a y e r of c o l l a p s e d c e l l s to w h i c h is f i r m l y a t t a c h e d
l a y e r o f c o l l a p s e d c e l l s ; t h e s e l a s t t h r e e l a y e r s c o n s t i t u t e the

T h e k e r n e l of the seed c o n s i s t s a l m o s t e n t i r e l y of p e r i s p e r m . T h e o u t e r t w o or
t h r e e r o w s of c e l l s a r e p o l y g o n a l a n d c o n t a i n a l e u r o n e g r a i n s , b u t the o t h e r s
are e l o n g a t e d and are p a c k e d w i t h m i n u t e g r a i n s of s t a r c h .
Scattered
t h r o u g h o u t the p e r i s p e r m a r e c e l l s c o n t a i n i n g y e l l o w i s h v o l a t i l e o i l .
The diagnostic

characters

of p o w d e r e d

black pepper

are

(Figure

8.11):

(1) T h e o u t e r e p i d e r m i s , t o g e t h e r
with
the s u b j a c e n t
interrupted
sclerenchymatous layer.
(2) The
layer.

inner

(3) The s t a r c h
compact masses.

sclerenchymatous

grains,

often

in

(4) T h e o i l - c e l l s , the c o n t e n t s of
w h i c h are c o l o u r e d red by s u l p h u r i c
acid .

Figure 8.11.

White

Powdered Black

Pepper

Pepper

T h i s c o n s i s t s of the d r i e d w h o l e r i p e b e r r i e s of P i p e r n i g r u m f r o m w h i c h the
outer part of the p e r i c a r p has been r e m o v e d by a b r a s i o n after
previously
f e r m e n t i n g or s o a k i n g in w a t e r .
T h e q u a l i t y m a y b e a s s e s s e d f r o m the l e v e l of
c r u d e f i b r e , (0.5 - 5 . 0 % ) .

243

CAPSAICIN
PRINCIPLE
T h e p o w d e r e d s p i c e is e x t r a c t e d w i t h m e t h a n o l a n d t h e c a p s a i c i n s e p a r a t e d b y a n
ether-alkali partition extraction procedure.
T h e c a p s a i c i n is d e t e r m i n e d b y a
spectrophotometric difference method.
APPARATUS
1.

Soxhlet

type

continuous

2.

Separatory

3.

Spectrophotometer

extraction

apparatus.

funnels.
a n d 1 cm c u v e t t e s .

REAGENTS
1.
Carbon, purified.
S h a k e 10 g o f a c t i v a t e d c a r b o n w i t h 1 0 0 m l o f
d e h y d r a t e d m e t h a n o l , filter through a s i n t e r e d - g l a s s f u n n e l , and dry
t h e r e s i d u e a t 105C.
2.

Ether.

Diethyl

3.

Light

4.

Methanol,

5.

Sodium

petroleum.

ether, anhydrous
Boiling

peroxide-free.

r a n g e 8 0 t o 100C.

anhydrous.

chloride.

6.
H y d r o c h l o r i c a c i d , 0.1 N . R e a g e n t s o l u t i o n .
Dilute
c o n c e n t r a t e d h y d r o c h l o r i c acid, to 1 litre w i t h w a t e r .
7.
Hydrochloric
a c i d , 0.05 N.
hydrochloric acid, to 1 litre with
8.
Methanol,
water.
9.

Sodium

60%.

hydroxide

Dilute

Dilute
water.

3 volumes

solution,

0.1 N .

4.5 m l

of methanol

Prepare

of

with

freshly

9 m l of

concentrated

2 volumes

as

of

required.

10.
Phenol red indicator solution.
W a r m 20 m g o f p h e n o l r e d w i t h
1.1 m l o f 0 . 0 5 N s o d i u m h y d r o x i d e a n d 2 m l o f a l c o h o l ( 9 0 % v / v ) a n d
w h e n s o l u t i o n 3 c o m p l e t e , d i l u t e t h e s o l u t i o n t o 1 0 0 m l w i t h a l c o h o l
(20% v / v ) .
PROCEDURE
T r a n s f e r a b o u t 5 g ( a c c u r a t e l y w e i g h e d ) of the w e l l m i x e d s a m p l e ,
g r o u n d to p a s s a 3 0 - m e s h s i e v e , to a c o n t i n u o u s e x t r a c t i o n a p p a r a t u s ,
e x t r a c t w i t h d e h y d r a t e d m e t h a n o l for n o t less than 6 h o u r s , or until
the s a m p l e is e x h a u s t e d , and d i l u t e t h e e x t r a c t to 100 m l w i t h
dehydrated methanol.

T o 10 m l o f t h i s s o l u t i o n a d d 15 m l o f d e h y d r a t e d m e t h a n o l , 15 m l o f
w a t e r , 2 g o f s o d i u m c h l o r i d e a n d 5 m l o f 0.1 N s o d i u m h y d r o x i d e ,
m i x , a n d e x t r a c t w i t h t h r e e s u c c e s s i v e 10 m l p o r t i o n s o f l i g h t
p e t r o l e u m ( b o i l i n g r a n g e 8 0 t o 100C). W a s h t h e c o m b i n e d e x t r a c t s
w i t h t w o s u c c e s s i v e 5 m l p o r t i o n s of 60% m e t h a n o l and discard the
light petroleum extract; filter the combined aqueous solution and
w a s h i n g s t h r o u g h c o t t o n w o o l , w a s h i n g t h e f i l t e r w i t h 10 m l o f 5 0 %
methanol.

244

Evaporate the combined filtrate and washings on a water-bath until

the volume is reduced to about 5 m l , dilute the solution to about 50
m l w i t h w a t e r , a d j u s t the pH to 7.0 to 7.5 w i t h 0.1 N h y d r o c h l o r i c
acid, using either a pH meter or phenol red solution as indicator and
e x t r a c t w i t h six s u c c e s s i v e 20 m l p o r t i o n s of e t h e r , e n s u r i n g that
the c o r r e c t pH is m a i n t a i n e d d u r i n g the e x t r a c t i o n .
W a s h the
c o m b i n e d e x t r a c t s w i t h 10 ml of w a t e r , and d i s c a r d the a q u e o u s
s o l u t i o n and w a s h i n g s .
Add 20 m l of d e h y d r a t e d m e t h a n o l and
e v a p o r a t e on a w a t e r - b a t h in a f u m e c u p b o a r d u n t i l the v o l u m e is
reduced to about 1 ml.
Dilute the residue to 100 ml with dehydrated methanol, add 0.05 g of
decolourising charcoal, shake and filter through a fine grade filterp a p e r , d i s c a r d i n g the first 20 m l of f i l t r a t e .
To 10 ml of this
s o l u t i o n add 5 m l , a c c u r a t e l y m e a s u r e d , of 0.1 N s o d i u m h y d r o x i d e ,
cool and d i l u t e to 25 m l w i t h d e h y d r a t e d m e t h a n o l . To a f u r t h e r 10
m l add 5 ml, accurately measured, of 0.05 N hydrochloric acid, cool,
and dilute to 25 ml with dehydrated methanol.
M e a s u r e the e x t i n c t i o n of the a l k a l i n e s o l u t i o n
solution at the m a x i m a at 248 and 296 nm.

against

the

acid

U s i n g d e h y d r a t e d m e t h a n o l as s o l v e n t , d i l u t e 5 m l , a c c u r a t e l y
m e a s u r e d , of 0.1 N s o d i u m h y d r o x i d e to 25 ml and d i l u t e 5 m l ,
a c c u r a t e l y m e a s u r e d , of 0.05 N h y d r o c h l o r i c acid to 25 m l ; m e a s u r e
the extinction of the alkaline solution against the acid solution at
the m a x i m a again at 248 and 296 nm.
CALCULATION
F r o m the e x t i n c t i o n s at the t w o m a x i m a g i v e n by the s o l u t i o n s
containing the sample, deduct the corresponding extinctions given by
the blank solutions.
For purposes of calculation, use a value of 313
1 X
for the E
of c a p s a i c i n at 248 nm u n d e r t h e s e c o n d i t i o n s and of
1 cm
127 for that at 296 nm; calculate the proportion of capsaicin in the
sample from the extinction at each wavelength.
If the two results so
o b t a i n e d d i f f e r by not m o r e than 5%, the c o n t e n t of c a p s a i c i n is
given by the mean of the two results.
INTERPRETATION
Capsaicin is the substance which gives capsicums their pungency.
cayenne pepper powder should contain about 0.5% capsaicin.

As

example,

REFERENCES
British Pharmaceutical
Hartman, K.J., 1970.

Codex, 1973, 119.

Journal of Food Science j5_5, 543.

M a s a d a , Y., H a s h i m o t o ,
Science _36, 858.

K., I n o u e , T. and

S u z u k i , M.,

1971. J o u r n a l

of

Food

Note:
H a r t m a n and M a s a d a et al d e s c r i b e gas c h r o m a t o g r a p h i c m e t h o d s .
The
Scoville Index, defined as the greatest dilution expressed as the denomination
of the d i l u t i o n f r a c t i o n , at w h i c h a p u n g e n t s e n s a t i o n from c h i l l i e s is
p e r c e i v e d , under c o n d i t i o n s of the m e t h o d as d e s c r i b e d , m a y be d e t e r m i n e d by
ISO 3513-1977.

245

8.4

SPICES - ROOTS, BARKS AND FLOWERS

COMPOSITION

Roots
Ginger
Tumeric

8.0 -10.0

3.2
6.5

9.3
7.5

7.8 -10.5

4.1

5.7

5.0 -11.0

4.7
5.0

7.0
8.0

8.4 -13.9

Barks
Cassia
Cinnamon
Flowers
Cloves
Saffron

8.0

Non-Volatile
Extract X

Total
Ash X

Moisture
Spice

2 . 8 - 8.1

Volatile
Oil X

3.1
5.0

7.0 - 9.0

0.9
3.0

1.3 - 1.7

0.7 - 1.4

6.2 -10.1

14 - 20

Cassia is the bark of Cinnamomum cassia and the usual cinnamon of commerce is
the b a r k of C i n n a m o m u m z e y l a n i c u m .
They differ from each other and other
Cinnamomum species as follows:

Cinnamomum
cassia
Cinnamaldehyde

Cinnamomum
zeylanicum
(Cinnamon)

Cinnamomum
Burmanni

> 30

>30

Cinnamomum
pedatinervium
Cimmamomum
& Cinnamomum
s intok
loureirii

Eugenol
Diameter of
fibres (microns)
Calcium
oxalate

>40
minute
prisms

Acicular
crys tal s

Mucilage

9.5-10.9

1.9-2.1

Ash of
mucilage

6.1- 6.4

20.6-24.7

cork
present

thin,
no cork

Appearance

smal 1
prisms

> 40
Acicular
crystal s

IDENTIFICATION
Ginger
G i n g e r is the scraped and dried r h i z o m e of Zing iber officinale. There are a
n u m b e r of d i f f e r e n t types of ginger used c o m m e r c i a l l y throughout the world.
All are scraped rhizomes of a different species such as
m ioga used in Japan.
L i m e d g i n g e r is ginger treated w i t h lime to soften the skins before peeling.
The calcium content of limed ginger usually exceeds 1% (as CaO) while unlimed
is less than 0.5 %.
*

Crude fiber is a useful means to determine if the degree of scraping has been
sufficient.
The crude fiber of normal ginger is usually in the range of 1.7 6.5%. Excessive crude fiber would indicate possible insufficient scraping.

246

Microscopically, ginger

shows:

a.
S t a r c h g r a i n s w h i c h are s a c k - s h a p e d , ovoid or s i m p l e , are f a i n t l y
s t r i a t e d , h a v e an e c c e n t r i c h i l u m (length 1 2 - 5 0 m i c r o n s ) , and e x h i b i t an
asymmetric cross when examined using polarized light.
b.

Thin-walled

parenchymatous

cells.

Turmeric
T u r m e r i c is the r h i z o m e of Curc um a d o m e s t i c a .
The p r i m a r y r h i z o m e is pear
shaped and is called 'bulb' turmeric. The secondary rhizome is cylindrical and
is usually known as 'finger' turmeric.
The turmeric rhizomes are usually boiled or steamed
yellow starch usually appears gelantinized.

during preparation,

so the

Turmeric is most often used for its colour only and is considered inferior as a
flavouring agent.
It is used in m o s t curry p o w d e r p r e p a r a t i o n s and in s o m e
ground mixed spices.
Cassia Bark
C a s s i a is the b a r k of C i n n a m o m um
presents the following structure:

cassia,

a.
Cork, in which layers of thin-walled,
of cells with thickened, b r o w n walls.

Blume.

(Laurineae).

The

tabular cells alternate with

bark

layers

b.
C o r t e x , w h i c h is m o d e r a t e l y w i d e and c h a r a c t e r i z e d by the a b u n d a n c e of
s c l e r e n c h y m a t o u s c e l l s c o n t a i n e d in it. S o m e of these c e l l s h a v e v e r y t h i c k
walls with branching pits; others have comparatively large cavities and walls
that e x h i b i t a m o r e or less c o n s p i c u o u s o n e - s i d e d t h i c k e n i n g .
They o c c u r
e i t h e r i s o l a t e d or in s m a l l g r o u p s in the p r i m a r y c o r t e x , and also form a
s c l e r e n c h y m a t o u s r i n g , w h i c h is i n t e r r u p t e d at i n t e r v a l s by s m a l l g r o u p s of
p a r e n c h y m a t o u s c e l l s , and b e a r s on the outer m a r g i n s c a t t e r e d b u n d l e s of
pericyclic fibres.
c.
Bast ring, constituting the greater part of the bark.
It is traversed by
medullary rays two cells wide, and contains numerous secretion cells as well as
bast-fibres and sclerenchymatous cells.
The secretion cells are mostly larger
than the c e l l s of the b a s t p a r e n c h y m a , and are a x i a l l y e l o n g a t e d ; they m a y
contain either mucilage or volatile oil, or a mixture of both. The bast fibres
are e i t h e r i s o l a t e d , or o c c u r in g r o u p s of t w o or t h r e e ; they are l a r g e r b u t
less numerous than those of cinnamon bark.
The sclerenchymatous cells are also
either isolated or in small groups.
The cells of the bast parenchyma contain
starch grains which are considerably larger than those of cinnamon bark.
Many
c e l l s , e s p e c i a l l y t h o s e of the m e d u l l a r y r a y s , c o n t a i n n u m e r o u s m i n u t e
prismatic crystals of calcium oxalate.
The sieve-tubes are narrow, and have
small, transverse sieve-plates.
Typical specimens of cassia bark may be distinguished from typical specimens of
cinnamon bark by the presence of cork, by the larger, thicker, bast fibres, and
by the larger s t a r c h - g r a i n s , but the l o w e r g r a d e s of c i n n a m o n b a r k are o f t e n
difficult to distinguish from cassia.

247

The diagnostic

characters

of cassia bark are (Figure 8.12):

(1) The cork, some of the cells of which

are thick walled.
(2) The isolated bast

fibres.

(3) The sclerenchymatous cells, many of

which are more strongly thickened on one
side than on the other.
(4) The secretion
or mucilage.

cells,

containing

(5) The m i n u t e p r i s m a t i c
calcium oxalate.

oil

crystals

of

Figure 8.12. Powdered Cassia Bark

Cinnamon Bark
Cinnamon is usually the bark of Cinnamomum zeylanicum, Breyne (Laurineae). The
bark, which is deprived of the cork and of the majority of the cortex, presents
the following structure:
a.
Cortex, represented
polygonal cells.

by

two

or

three

rows

of

tangentially

elongated

b.
Bast ring, separated from the remains of the cortex by a continuous ring
of sclerenchymatous cells, with thickened pitted walls, the thickening being
o f t e n m o r e s t r o n g l y developed on one side than on the others.
On the outer
margin of this ring bundles of pericyclic fibres may be detected at intervals,
as in the case of cassia bark, but the s c l e r e n c h y m a t o u s ring of c i n n a m o n
differs from that of cassia in being continuous instead of interrupted.
The
bast ring is traversed by m e d u l l a r y rays, w h i c h are very n a r r o w near the
It contains s e c r e t i o n cells
c a n b i u m , but e n l a r g e t o w a r d s the periphery.
similar to those of cassia bark, and bast fibres that have very thick walls and
are most isolated. The sieve-tubes are arranged in tangential groups, which in
the outer p o r t i o n s of the bast ring are collapsed and exhibit traces only of
cavities; they are narrow and have transverse sieve-plates.
Many of the cells
of the cortical parenchyma and medullary rays contain small starch grains or
numerous minute crystals of calcium oxalate.
The diagnostic characters
bark are (Figure 8.13):

of

cinnamon

(1) The absence of cork.

(2) The sclerenchymatous cells, sometimes
thickened on one side m o r e than on the
others.
(3) The secretion cells containing oil or
mucilage.
(4) T h e
oxalate.

minute

crystals

of

(5) The small sieve-tubes with

plates.
Figure 8.13

Cinnamon

248

calcium

transverse

Cloves
Cloves are the dried flower-buds of Eugenia caryophyllata, Thunb. (Myrtaceae).
E a c h c l o v e c o n s i s t s of a s o m e w h a t q u a d r a n g u l a r s t e m - l i k e p o r t i o n s l i g h t l y
c o n t r a c t e d at the b a s e ; this part is s o m e t i m e s i n t e r r u p t e d as a c a l y x - t u b e ,
s o m e t i m e s as the solid l o w e r part of the ovary.
It is c r o w n e d by four t h i c k ,
divergent, suboval calyx-teeth in the centre of which there is a small rounded
b o d y c o n s i s t i n g of four i m b r i c a t e d p e t a l s e n c l o s i n g the dried s t a m e n s and
style.
The transverse section of the lower part of the stem-like portion exhibits the
following characters:
The e p i d e r m i s is c o m p o s e d of p o l y g o n a l c e l l s c o v e r e d
with a rather thick cuticle, and provided with stomata. Below the epidermis is
parenchymatous tissue well differentiated into three layers, viz. an outer one
w i t h r a d i a l l y e l o n g a t e d c e l l s , and n u m e r o u s f i b r o - v a s c u l a r b u n d l e s in w h i c h
thick 8clerenchymatous fibres are conspicuous elements, and an inner, lacunous
layer.
In the centre there is a moderately large fibro-vascular bundle.
Both
the teeth of the c a l y x and of the p e t a l s c o n s i s t c h i e f l y of p a r e n c h y m a t o u s
t i s s u e in w h i c h there are n u m e r o u s o i l - g l a n d s .
The a n t h e r s are c o m p o s e d of
small cells with pitted walls, and larger cells with spiral thickenings.
The
p o w d e r also c o n t a i n s n u m e r o u s p o l l e n g r a i n s as w e l l as p e r i c y c l i c fibres
derived from the bundles in the lower part of the clove.
P o w d e r e d c l o v e s p o s s e s s no w e l l - m a r k e d d i a g n o s t i c characters; the
features, may, however, be mentioned (Figure 8.14):

(1) E p i d e r m i s of l o w e r
thick cuticle.

part of o v a r y ,

(2) Epidermis of calyx-teeth

(3) Fragments of the

and

following

with

corolla.

oil-glands.

(4) P a r e n c h y m a of the c o r o l l a w i t h n u m e r o u s
rosettes of calcium oxalate. Powdered cloves
should not c o n t a i n se 1 e r e n c h y m a t o u s c e l l s
(clove stalks) or starch (mother-of-cloves).

Figure 8.14. Powdered

Cloves

Saffron
S a f f r o n c o n s i s t s of the s t i g m a t a and upper p a r t s of the s t y l e s of C r o c u s
It forms a loosely matted mass of dark, reddish-brown
sativus Linn. (Irideae).
f l a t t e n e d t h r e a d s , a m o n g s t w h i c h a f e w n a r r o w e r y e l l o w o n e s c a n be
distinguished.
The upper, enlarged part of the flattened threads is the stigma
of the flower, the lower narrower portion is the style.
The s t i g m a is c o m p o s e d of p o l y g o n a l or r o u n d e d , t h i n - w a l l e d , p a r e n c h y m a t o u s
cells containing mucilage and an orange-red colouring matter composed of crocin
and crocetin.
This tissue is traversed by small fibro-vascular bundles, which
appear rounded in transverse section.
It is covered by a slightly thickened,
easily detached cuticle, which on the upper surface of the stigma bears small
p r o t u b e r a n c e s (pr.).
Near the apex the s t i g m a is f u r n i s h e d w i t h large

249

papillae.
The y e l l o w i s h l o w e r p a r t o f the t h r e a d s o f s a f f r o n i s p r o v i d e d w i t h
an e p i d e r m i s c o n s i s t i n g o f s l i g h t l y s i n u o u s c e l l s , and i s t r a v e r s e d by a s m a l l
f i b r o - v a 8 c u l a r bundle.
The d i a g n o s t i c
characters
of
powdered s a f f r o n a r e ( F i g u r e 8 . 1 5 ) :
(1)
The o r a n g e - r e d
colouring
m a t t e r in the c e l l s ; i t i s s o l u b l e
in w a t e r , but i n s o l u b l e in f i x e d
oil.
(2)
The u p p e r
stigmata with
protuberances .
(3)

The l a r g e

epidermis of
the
small
papillose

pollen

grains.

Figure 8.15.

250

Powdered

Saffron

COLOURING POWER OP SAPPRON

PRINCIPLE
Dilute a cold water soluble extract, so as to bring the concentration to about
4 mg of saffron per 100 ml of solution.
Measure the absorbance at 440 nm in a
1 cm cell.
APPARATUS
1.

Pipette, 1 ml.

2.

Volumetric

flask, 500 ml.

3.
Spectrophotometer (or
absorbance at 440 nm).

any

other

apparatus

capable

4.
Q u a r t z or g l a s s cells for s p e c t r o p h o t o m e t r y ,
light at 440 nm, with optical path length of 1 cm.

of

measuring

transparent

to

PROCEDURE
Extract 2 g of saffron with 100 ml cold water.
Pipette 1 ml of the
s u p e r n a t a n t into a 500 m l v o l u m e t r i c flask and d i l u t e to the m a r k
with distilled water so as to obtain a solution containing about 4 mg
of saffron per 100 ml of solution.
M e a s u r e the a b s o r b a n c e of the final d i l u t i o n
using distilled water as reference.

at 440 nm

in a c e l l ,

CALCULATION
Note the absorbance measured
F

at 440 nm and calculate the absorptivity

1%

^ 1 cm
INTERPRETATION
The c o l o u r i n g p o w e r
extract at 440 nm.

of s a f f r o n is d e f i n e d as the a b s o r p t i v i t y of a 1%
1 %
1%
E
should be 110 for filaments and 150 for powder.
1 cm

REFERENCE
ISO

3632:1975.

251

8.5

TEXT

REFERENCES

None

Further Reading
HART, F.L.
Verlag.
PARRY,
Press,
April

&

FISjlER,

J.W.
1962.
London.
1956

- March

JACKSON & SNOWDON,

H.J.

1971.

Spices,

1957

their

inc.

1974.

Modern

Food

Morphology,

Articles

Powdered

Histology

entitled

Vegetable

Analysis

Food,

Drugs

Chapter

&

Food

parts

Trade

4-15.

Thornes.

AMERICAN SPICE TRADE A S S O C I A T I O N .

1968. O f f i c i a l Analytical
A v e n u e , PO Box 1 2 6 7 , E n g l e w o o d C l i f f s , N.J. 0 7 6 3 2 .
PRUTHI, J.S.
1 9 7 6 . S p i c e s and C o n d i m e n t s
G r e e n P a r k , New D e h l i ,
110016.

Springer-

Chemistry

Microscopy

S.

14

National

Methods

Book T r u s t ,

580

Sylvan

New D e l h i ,

A5

ROBERTS, L.A.
1 9 6 8 . GLC of C h l o r i n a t e d Hydrocarbon C o n t a m i n a n t s in O l e o r e s i n s ,
J o u r n a l of the A s s o c i a t i o n of O f f i c i a l A n a l y t i c a l C h e m i s t s ( 4 ) 8 2 5 - 2 8 .
STRAUSS,
121-22.

D.

1969.

Microscopy

STAHL,
W.H.,
SKARZYNSKI,
J o u r n a l of the A s s o c i a t i o n

of

Tamarind,

J.N. & VOELKER, W.A.

of O f f i c i a l A n a l y t i c a l

GERHARDT, .
1969. Typical Analytical
O n i o n , F l e i s c h w i r t s c h a f t (10) 1356-8.
GERHARDT,
U.
1969.
Fleischwirtschaft
(9)
GERHARDT,
197.

U.

1970.

Typical
1191-3.

Typical

Deutsche

Values

Analytical

Analyses

for

for

Lebensmittel-Rundschau

1 9 6 9 . GLC & TLC o f O r g a n o ,

C h e m i s t s ( 6 ) 1184-9.
Ginger,

Values

Herbs,

(4)

for

Cinnamon,

Nutmeg

Garlic

and

Fleischwirtschaft

and

Mustard,

(2)

192-4

and

S H A N K A R A N A R A Y A N A , M . L . , N A G A L A K S H M I , S. & N A T A R A J A N , C . P .
1970. Colorimetric
D e t e r m i n a t i o n of Pungent C o n s t i t u e n t s of P e p p e r , F l a v o u r I n d u s t r y ( 3 ) 173-5.
ANON.
1970. C u l t i v a t i o n , Histology,
Flavour Industry (8) 524-26.

Constituents

and

Adulterants

ANON.
1970. C u l t i v a t i o n ,
Mustard, Flavour Industry

Constituents

and

Uses

Histology,
( 9 ) 596-8.

ANON.
1970. Cultivation, Histology,
Flavour Industry (7) 452-3.
ANON.
Anise,

1970. C u l t i v a t i o n , Histology,
Flavour I n d u s t r y (7) 446-8.

Constituents

Constituents

SHANKARACHARYA, N.B. & NATARAJAN, C.P.

and U s e s , I n d i a n Food Packer ( 5 ) 28-36.

and

Uses

and U s e s

1971. Cardamon,

of

of

White

of

and

C.P.

1972. Fenugreek,

252

and

Star

Technology

F O R R E S T , J . E . , HEARCOCK, R.A. & FORREST, T.P.

1972. I d e n t i f i c a t i o n
Comporients of E s s e n t i a l O i l of M a c e , J o u r n a l of Chromatography 69 ( 1 )
SHANKARACHARYA, N.B. & NATARAJAN,
I n d i a n Spices (1) 2-12.

Black

Peppermint,

of A n i s e

Chemistry,

Caraway,

Composition

of M a j o r
115-21.
and

Use,

S H A N K A R A C H A R Y A , N.B. & N A T A R A J A N , C.P.

I n d i a n F o o d P a c k e r (6) 2 2 - 8 .

1971.

Cummin, Composition

S H A N K A R A C H A R Y A , M.L., R A G H A V A N , B. & N A T A R A J A N , C.P.

V a r i e t i e s , C h e m i s t r y and A n a l y s i s , L e b e n s m i t t e l - W i s s e n s c h a f t
191-7.

and

Use,

1972.
Mustard,
+ T e c h n o l o g i e (6)

P E Y R O N , L., S E N A U X , S., A C C H I A R D I , M . J . & B R O U A , M .

1972. A d u l t e r a t i o n of
B a s i l E s s e n c e w i t h Y l a n g - Y l a n g , A n n a l e s des F a l s i f i c a t i o n et de l ' E x p e r t i s e
C h i m i q u e (701) 2 4 7 - 5 4 .
ANON.
1972. Production, Composition
(3/4) 1 - 1 3 .

and Use of I n d i a n S p i c e s , I n d i a n

Spices

H A R T M A N , C.P., D I V A K A R , N.G. & R A O , U.N.N.

1973. A d u l t e r a t i o n of P e p p e r
P a p a y a S e e d s , J o u r n a l o f F o o d S c i e n c e a n d T e c h n o l o g y I n d i a 10 4 3 .
P R U T H I , J.S. & K U L K A R N I , B.M. 1 9 6 9 . D e t e c t i o n
B e r r i e s , I n d i a n Food P a c k e r 23 5 1 .

of P a p a y a

S e e d s in B l a c k

with

Pepper

K A R A S Z , A.B., C O C C O , F de & B O K U S , L .
1973.
F o o d s , J o u r n a l of the A s s o c i a t i o n of O f f i c i a l

R a p i d D e t e c t i o n of T u r m e r i c in
A n a l y t i c a l C h e m i s t s (3) _56_ 6 2 6 - 8 .

OSISIOGU,
I.U.W.
C h r o m a t o g r a p h y (1)

Ginger

1973.
200-3.

Adulteration

in

Galenicals,

Journal

of.

S E N , A.R., S A R D A R , P.K., S I L , S. & M A T H E W , T.V.

1973. Coriander, Composition
a n d A n a l y s i s , J o u r n a l o f F o o d S c i e n c e a n d T e c h n o l o g y , I n d i a 10 1 2 6 - 7 .
S E N , A.R., G U P T A , P.S. & D A S T I D A R , N.G.
1974. Detection
a n d C u r c u m a A r o m a t i c a in C u r c u m a L o n g a , A n a l y s t 99 ( 1 1 7 6 )

of C u r c u m a
153-5.

SUD'EVA,
N.G., S E N T I C H ,
V.Ya., P O P O V A ,
S.A. & P O L Y A K O V ,
C o m p o s i t i o n of A n i s e e d , I z v e s t i y a V y s s h i k h U c h e b n y k h Z a v e d e n i i ,
Tekhnologiya No. 2 131-2.

Zedoaria

A.F.
1 973.
Pischchevaya

S E N , A.R., S A R D A R , P.K., S I L , S. & M A T H E W , T.V.

19 73 .
Analysis
and
C o m p o s i t i o n of C e l e r y S e e d , J o u r n a l of F o o d S c i e n c e a n d T e c h n o l o g y I n d i a 10
187-8.
B H A L L A , K. & P U N E K A R , B.D.
1975. Adulteration
and A d u l t e r a t i o n of B l a c k P e p p e r , I n d i a n J o u r n a l
216-22.

of A s a f o e t i d a w i t h C o l o p h o n y
o f N u t r i t i o n a n d D i e t e t i c s (7)

H U M P H R E Y , A.M.
1 9 7 3 . TLC and GLC of S p i c e O i l s , T r o p i c a l
C o n f e r e n c e P a p e r s 12 3 - 6 .

Products

Institute

M I T R A , S.N., G U P T A , P.S. & S E N , A.R.

1971.
S t u d i e s on the C o m p o s i t i o n
Curry Powder.
J o u r n a l o f t h e I n s t i t u t e o f C h e m i s t s , I n d i a 4 3 (2) 8 1 - 2 .

of

S E N , A.R., S A R D A R , P.K. & S E N G U P T A , P.

1 97 7 .
J o u r n a l of the A s s o c i a t i o n of
O f f i c i a l A n a l y t i c a l C h e m i s t s (1) 6 0 2 3 5 d e s c r i b e a s i m p l e T L C m e t h o d
for
d i s t i n g u i s h i n g C a r a w a y S e e d s f r o m those of B l a c k C a r a w a y ( C a r u m b u l b o c a s t a n u m
Koch).
S E N , A.R., S E N G U P T A , P., S A R D A R , P.K. & R O Y , B.R.
197 7. D e s c r i b e a T L C M e t h o d
to d i s t i n g u i s h t r u e c a r d a m o m f r o m g r e a t e r c a r d a m o m .
J o u r n a l of t h e A s s o c i a t i o n
o f O f f i c i a l A n a l y t i c a l C h e m i s t s (1)
307-8.
H E A T H , H.
1974.
Tumeric.
Flavour

Herbs and Spices a B i b l i o g r a p h y ,

I n d u s t r y _5_ ( 5 / 6 ) 1 2 3 - 4 .

S E N G U P T A , P., S E N , A.R., B O S E , A . &

J o u r n a l o f the A s s o c i a t i o n o f O f f i c i a l

IX,

Tarragon,

M A T H E W , T.V.
197 3. A j o w a n
A n a l t y i c a l C h e m i s t s 56 1 5 1 0 .

253

&

Thyme,

Celery.

S E N , A.R.,
Coriander.

S E N G U P T A , P., M O N D A L , A . & R O Y , B . R .
1974.
Cumin, Caraway &
J o u r n a l o f t h e A s s o c i a t i o n of O f f i c i a l A n a l y t i c a l C h e m i s t s 57 7 6 3 .

BALLARIN, C. & BALLARIN, J.

ISO

1237:1981.

1972.

Determination

Fennel & Aniseed.

of Allyl

Pharmazie

Isothiocyanates

in

27

544.

Mustard.

R A G H A V A N , B., S H A N K A R A N A N Y A M A , M.L., N A G A L A K S H M I , S. & N A T A R A J A N , C.P.

1972.
C o l o r i m e t r i'c M e t h o d for A l l y l I s o t h i o c y c a n a t e s .
M i k r o c h i m i c a Acta No. 6 81822.
S H A N K A R A N A R A Y A N A , M . L . , N A G A L A K S H M I , S., R A G H A V A N , B . & N A T A R A J A N , C . P .
1972.
M e t h o d for A l l y l I s o t h i o c y a n a t e s using C h l o r a m i n e - T .
F l a v o u r I n d u s t r y (2) 7577 .
C H I K K S P U Y Y A I A H , K.S., S H A N K A R A N A R A Y A N A , M . L . & N A T A R A J A N , C.P.
Method.
F l a v o u r I n d u s t r y (10) 5 9 1 - 3 .
ISO 5 5 6 7 : 1 9 8 2 .
compounds.

Dehydrated

Garlic

ISO

Thin-layer

chromatography

3632:1975.

P A R V A N E H , V. 1 9 7 2 .
Saffron
P u b l i c A n a l y s t s 10 3 1 .
D H A R , D.N. & S U R I , S.C.
of C h e m i s t s ( I n d i a ) 4 6

and

- Determination

of

of s a f f r o n

Safflower.

volatile

A Rapid

organic

sulphur

pigments.

Journal

1974. Saffron Adulterants.

(4) 1 3 0 - 2 .

1971.

of

the

Association

J o u r n a l of the

of

Institute

H O H M A N N , B.
1969.
M i c r o s c o p i c a l I d e n t i f i c a t i o n of C i n n a m o n L e a v e s in C u r r y
P o w d e r and P o i n t s of D i f f e r e n c e f r o m L a u r e l L e a v e s .
Zeitschrift
fur
L e b e n s m i t t e l u n t e r s u c h u n g und F o r s c h u n g (3) 1 5 2 - 7 .
L A W R E N C E , B.M.
Food Technology

1 9 6 9 . B o t a n i c a l O r i g i n s of C i n n a m o n .
J o u r n a l (4) 1 7 8 - 8 0 .

L A W R E N C E , B.M. & H O G G , J.W.

M e d i c a (1) 1 - 5 .

1974.

Safrole

Canadian

in t w o C i n n a m o n u m

S T A H L , W.H., S K A R Z Y N S K I , N.N. & V O E L K E R , W.A.

D i s t i n g u i s h C i n n a m o n and C a s s i a .
Journal
Analytical Chemists 741.

Institute

spices.

of

Planta

1969.
M u c i l a g e D e t e r m i n a t i o n to
of the A s s o c i a t i o n of O f f i c i a l

D U T T A , A.B.
1961.
D i s t i n g u i s h i n g C i n n a m o n from Cassia by C h e m i c a l A n a l y s i s .
J o u r n a l of t h e A s s o c i a t i o n o f O f f i c i a l A g r i c u l t u r a l C h e m i s t s 44 (4) 6 3 9 - 4 0 .
M A R T I N D A L E , The Extra
54-5 (Asa f o e t i d a).

Pharmacopoeia,

1955.

The P h a r m a c e u t i c a l

C O U N C I L OF S C I E N T I F I C & I N D U S T R I A L R E S E A R C H , N E W D E H L I .
P h a r m a c e u t i c a l C o d e x , V o l . 1. ( A s a f o e t i d a ) .
UNITED

STATES PHARMACOPOIEA,

1936.

(Asafoetida).

254

1953.

Press

London,

The

Indian

9.
9.1

OILS & FATS

VEGETABLE OILS

COMPOSITIOH
Codex Alimentariu8 Commission recommended

standards for vegetable oils:

DnsaponiSaponififiable
cation
Iodine matter
Value
(maximum)
Value

Acid
value
(mg KOH/g
oil)(max)

Peroxide
value
(meq 0,/
kg oil;
(max)

0.6

10

Vegetable
Oil

Density
20/20

Refractive
Index

Soya Bean

0.9190.925

1.4661.470

189195

120143

15

Arachis

0.9140.917

1.4601.465

187196

80106

10

Cottonseed

0.9180.926

1.4581.466

189198

99119

15

Sunflower

0.9180.923

1.4671.469

188194

110143

15

virgin 4
not " 0 . 6

10

Rapeseed

0.9100.920

1.4651.469

168181

94120

20

virgin 4
not " 0 . 6

10

Maize

0.9170.925

1.4651.468

187195

103128

28

virgin 4
not " 0 . 6

10

Sesame(*)

0.9150.923

1.4651.469

187195

104120

20

virgin 4
not " 0 . 6

10

Safflower

0.9220.927

1.4671.470

186198

135150

15

Mustardseed

0.9100.921

1.4611.469

170184

92125

15

(*)

virgin 4
not " 0 . 6
0.6

0.6

virgin 4
not " 0 . 6

10

10

10

10

Also known as Gingelly, Benne, Ben, Till and Tillie oil.

The Codex has also recommended maximum levels

commonly used with vegetable oils. These are:
Antioxidants

for various

antioxidants

Maximum level of use

Propyl, octyl and dodecyl gallates

100 mg/kg individually or in

combinat ion

Butylated hydroxyanisole
Butylated hydroxytoluene

200 mg/kg individually or in

combination

(BHA)
(BHT)

Any combination of gallates with BHA

or BHT, or both

200 mg/kg but gallates not to

exceed 100 mg/kg

Ascorbyl palmitate
Ascorbyl stearate

200 mg/kg individually or in

combination

Dilauryl thiodipropionate

2Q0 mg/kg

255

The Codex p e r m i t s v e g e t a b l e oils also to contain b e t a - c a r o t e n e , a n n a t t o ,

curcumin, canthaxanthin, beta-apo-8'-carotenal, and methyl and ethyl esters of
beta-apo-8'-carotenoic acid and flavours of no known toxic hazard which may be
added for the purpose of restoring or standardizing colour or flavour as long
as the c o n s u m e r is not thereby m i s l e d .
0 i m e t h y 1 p o 1 ys i 1oxane, singly or
c o m b i n e d w i t h s i l i c o n d i o x i d e , m a y be added as a n t i f o a m up to a limit of 10
mg/kg and oxystearin as crystallization inhibitor up to 1250 mg/kg.
ROOTIHE ANALYSIS
Note that the analytical values for most oils are broadly similar, a reflection
of similar triglyceride compositions.
It is important to check that the values
for a s a m p l e lie w i t h i n the accepted range, since if they do not, it is
r e a s o n a b l e to a s s u m e a d u l t e r a t i o n until the contrary is proved.
However,
c l i m a t i c and other factors do influence these values.
For e x a m p l e , it is
c o n s i d e r e d in Italy that olive oil has an iodine value b e t w e e n 79 and 88,
r a t h e r than the range of 75-94 given in the Codex standard, w h i l e in North
Africa even higher values may be found and Tunisia has discussed introduction
of an upper limit of 97. Thus, although these determinations are valuable they
are not adequate by themselves.
Further tests to establish identity and detect
a d u l t e r a t i o n include the classical tests for specific oils (e.g. F i t e l s o n ' s
test for teaseed oil), examination of the unsaponifiable matter (e.g. tests for
p h y t o s t e r o l s ) , and detailed analysis of the t r i g l y c e r i d e s t h e m s e l v e s or the
component fatty acids.
The latter technique is often refined by measuring the
r a t i o of the p r o p o r t i o n of one acid to a n o t h e r , as this only varies w i t h i n a
small range in some cases. The Codex Committee on Fats & Oils has decided that
the inclusion of fatty acid composition in Codex standards is not advisable at
the present time.
Nevertheless, determination of this composition by GLC and
the c o m p a r i s o n w i t h the n o r m a l range m a y be of c o n s i d e r a b l e assistance in
establishing the identity of a sample.
W a t e r in oil is g e n e r a l l y d e t e r m i n e d by the method of Dean and Stark using
x y l e n e . The s t a b i l i t y of fats and oils is usually assessed by the S w i f t test
(IUPAC m e t h o d 2.506 (1979)).
T h e A m e r i c a n O i l C h e m i s t s S o c i e t y ( A O C S ) , D e u t s c h e G e s e l l s c h a f t fur
F e t t w i s s e n s c h a f t and the I n t e r n a t i o n a l Union of Pure and Applied C h e m i s t r y
(IUPAC) p u b l i s h s t a n d a r d i z e d m e t h o d s for the analysis of oils and fats (1,2).
The b o o k s by C h r i s t i e (3) and B o c k e n o o g e n (4) in p a r t i c u l a r are useful
i n t r o d u c t i o n s to s o m e of the m o r e recent techniques in oil and fat analysis
such as argentation chromatography and GLC.
The paper by Parodi (5) describes
the use of these techniques with some margarines, cooking oils and fat.
The squalene content of olive oil is higher than that of other oils. Hart and
Fisher (6) report values of 1360-7080 mg/kg in olive oil.
Cottonseed, peanut,
corn, soy, sunflower seed, teaseed, sesame seed and rapeseed oils showed values
in the range 3 0 - 4 9 0 m g / k g .
Rapeseed oil n a t u r a l l y contains high levels of
erucic acid glycerides. Varieties have now been bred which give oil containing
only a few percent of erucic acid g l y c e r i d e s (canbra oil).
A m e t h o d for
monitoring erucic acid levels may therefore be required.
It may be convenient
to carry out a general screening test for docosenoic acids (Conacher and Chadha
(7), C o n a c h e r (8)) and if n e c e s s a r y follow a procedure such as that of A c k m a n
et al (9) for the quantitative determination of erucic acid.
There is sometimes a need to assess the degree of hydrognation to which a oil
has been subjected.
The iodine value and refractive index fall and the melting
and solidification points rise as a result of hydrognation.
This is of little
assistance if the untreated oil is not available for comparison and advantage
w a s t h e r e f o r e taken of the e f f e c t s of the i s o m e r i z a t i o n w h i c h occurs during
hydrognation.
For example, part of the naturally occurring cis-oleic acid is
c o n v e r t e d to the tran s-i s o m e r , e l a i d i c a c i d .
This problem was first
i n v e s t i g a t e d s e v e r a l d e c a d e s ago b e f o r e the introduction of infra-red
s p e c t r o p h o t o m e t r y and g a s - l i q u i d c h r o m a t o g r a p h y .
The p r o c e d u r e of Cocks,
C h r i s t i a n and H a r d i n g (10) m e a s u r e d the so-called iso-oleic acids, taken to

256

m e a n those isomers of oleic acid with a melting-point of 20C or over.

Since
t h e s e h a v e lead s a l t s a l m o s t i n s o l u b l e in a l c o h o l and e t h e r , it w a s u s u a l l y
assumed that all unsaturated acids precipitated as insoluble lead salts, other
t h a n a s m a l l a m o u n t d u e to i n c o m p l e t e s e p a r a t i o n of s o l u b l e lead s a l t s f r o m
insoluble ones, were isomers of oleic acid having the same iodine value as that
acid.
The i s o - o l e i c acid v a l u e s o b t a i n e d a r e g e n e r a l l y in the r a n g e 5 - 3 0
p e r c e n t d e p e n d i n g on the i o d i n e v a l u e of the o r i g i n a l fat and the e x t e n t and
conditions of hydrognation.
T h e a d v e n t of i n f r a - r e d s p e c t r o p h o t o m e t r y p r o v i d e d a m u c h e a s i e r m e a n s of
a s s e s s i n g the e x t e n t of h y d r o g n a t i o n f r o m the p r o p o r t i o n of t r a n s - i s o m e r s
present.
Elaidic acid is the standard n o r m a l l y used.
Both sample and standard
are methylated to remove interference by the free carboxyl group. The standard
I.R. p r o c e d u r e is g i v e n in A O C S Cd 1 4 - 6 1 and in C o c k s and V a n R e d e (11).
The
p r o c e d u r e of A l l e n (12) m a y a l s o be u s e d b u t in that c a s e the s t a n d a r d c u r v e
must be prepared with material very similar to that to be analyzed (Huang and
F i r e s t o n e (13)). I U P A C m e t h o d 2.208. d e s c r i b e s the s e p a r a t i o n of
transoctadecenoic acids by TLC and quantitation by GLC.
The test of Synodines and Kovitas (14) appears to reliably distinguish virgin
oils from refined oils.
The section by Gracian in Boekenoogen (4) or the paper
by Matarese (15) are r e c o m m e n d e d reading before relying on the results of the
test.
The r e f r a c t i v e i n d e x is t a k e n at 20C for o i l s l i q u i d at that t e m p e r a t u r e and
at 40C, 60C or 80C for t h o s e t h a t are s o l i d at 20C.
The D l i n e s of s o d i u m
are the u s u a l l i g h t s o u r c e .
The t e m p e r a t u r e of m e a s u r e m e n t s h o u l d be +_ 2C
w i t h i n the s e l e c t e d t e m p e r a t u r e .
If a c o r r e c t i o n is n e c e s s a r y , 0.00035/ is
added to r e a d i n g s t a k e n a b o v e 20C to c o r r e c t to 20C and the s a m e a m o u n t
s u b t r a c t e d if r e a d i n g s are t a k e n b e l o w 20C. T h e f a c t o r b e c o m e s 0.00036/ at
40 and above.
If condenser water of a suitable temperature is not available
for circulating around the prism, a cold water supply m a y be fed into and out
of a closed conical flask by tubes that reach nearly to the bottom of the flask
and then connected to the instrument.
The conical flask is heated and suitable
a d j u s t m e n t of the tap and the h e a t i n g m a k e s it p o s s i b l e to pass w a t e r of the
desired temperature through the refractometer.
T h e r e l a t i v e d e n s i t y of o i l s and fats is d e t e r m i n e d by g r a v i t y b o t t l e or
pycnometer according to a general procedure.
It is conventional to record the
r e l a t i v e d e n s i t y of o i l s at 20C o r , if t h e y are not e n t i r e l y l i q u i d at that
t e m p e r a t u r e , at 40C, 60C or h i g h e r as a p p r o p r i a t e .
If the t e m p e r a t u r e of
m e a s u r e m e n t differs from the standard temperature by up to 5C, IUPAC method
2.101 ( 1 9 7 6 ) r e c o m m e n d s that c o r r e c t i o n s be m a d e a c c o r d i n g to the f o l l o w i n g
formulae :

pt =

ptj

(tj - t) x 0.00068

if tj > t

pt =

ptj

(t - tj) x 0.00068 if tj < t

If the correct coefficient of expansion of the

this figure should be substituted for 0.00068.

257

fat under examination

is k n o w n ,

SAPONIFICATION VALUE
PRINCIPLE
The saponification value denotes the weight
required to saponify one gram of oil.

in mg of potassium hydroxide (KOH)

APPARATUS
G l a s s flask, r e s i s t a n t to a l k a l i s , of about 200 ml capacity, which
can be fitted to a reflux condenser.
REAGENTS
1.

1% w/v solution of phenolphthalein in 95% ethanol.

2.
Approximately 0.5 N ethanolic KOH solution.
This is stored in a
b r o w n or y e l l o w glass bottle having a rubber stopper and then
d e c a n t e d b e f o r e use.
The solution should be c o l o u r l e s s or straw
yellow.
3.

0.5 N hydrochloric acid solution

(standardized).

PROCEDURE
W e i g h into the flask about 2 g of oil to w i t h i n 0.001 g and add
e x a c t l y 25 m l of 0.5 N e t h a n o l i c KOH solution. A t t a c h the flask to
the condenser. Boil gently, mixing from time to time.
A f t e r 60 m i n u t e s , stop h e a t i n g . (Note: For certain fats w h i c h are
d i f f i c u l t to saponify it is n e c e s s a r y to heat for longer than 60
minutes.) Add 4 to 5 drops of phenolphthalein solution.
Titrate the
hot soap solution with the 0.5 N hydrochloric acid solution.
Carry out a reagent blank
solution.

test on the ethanolic

potassium

hydroxide

CALCULATION

Saponification value
Where :
a
b
N
p

=
=
=
=

56.1 N (a - b)
P

ml of hydrochloric acid solution used in blank test

ml of hydrochloric acid solution used for the sample
exact normality of the hydrochloric acid solution used
weight in g of sample

REFERENCE
IUPAC

2.202.

258

IODINE VALUE
(Hanus M e t h o d )
PRINCIPLE
The u n s a t u r a t e d g l y c e r i d e s of an oil h a v e the a b i l i t y to a b s o r b a d e f i n i t e
amount of iodine, especially when aided by a carrier such as bromine, and thus
form saturated compounds.
The quantity of iodine absorbed is a measure of the
unsaturation of an oil or fat.
The iodine value is generally expressed as the
number of grams of iodine absorbed by 100 g of the oil.
The i o d i n e v a l u e is o f t e n the m o s t u s e f u l and e a s i l y d e t e r m i n e d f i g u r e for
i d e n t i f y i n g an oil or at l e a s t p l a c i n g it into a p a r t i c u l a r g r o u p .
It s h o u l d
also be noted that for natural oils and fats the less unsaturated fats with low
iodine values are solid at room temperature, or conversely, oils that are m o r e
h i g h l y u n s a t u r a t e d are l i q u i d s ( s h o w i n g t h e r e is a r e l a t i o n s h i p b e t w e e n the
m e l t i n g p o i n t s and the i o d i n e v a l u e s ) .
In g e n e r a l , the g r e a t e r the d e g r e e of
unsaturation (i.e., the higher the iodine value), the greater is the liability
of the oil or fat to b e c o m e rancid by oxidation.
REAGENTS
1.
H a n u s I o d i n e S o l u t i o n - M e a s u r e 825 ml a c e t i c acid and d i s s o l v e
13.615 g I in it w i t h the aid of h e a t .
C o o l , and t i t r a t e 25 m l w i t h 0.1N
s o d i u m thi o su 1 p h a t e . M e a s u r e a n o t h e r p o r t i o n of 200 m l a c e t i c acid and
add 3 m l Br.
To 5 m l of this s o l u t i o n add 10 m l 15% K I s o l u t i o n and
titrate with 0.1 N sodium thiosulphate.
Calculate volume of Br solution
r e q u i r e d to d o u b l e h a l o g e n c o n t e n t of r e m a i n i n g 8 0 0 m l I s o l u t i o n as
follows :
A

Where :
X = ml of Br solution to be added to 800 ml I solution.
A = 800 x thiosulphate equivalent of 1 ml Br solution.
B = thiosulphate equivalent of 1 ml Br solution.
If n e c e s s a r y , r e d u c e m i x e d
dilution with acetic acid.

solution

2.

Chloroform

3.

15% potassium

4.

Standard 0.1 N sodium

thiosulphate

5.

1% starch solution as

indicator.

iodide

solution

to

(freshly

proper

concentration

by

prepared)

solution.

PROCEDURE
W e i g h a c c u r a t e l y a b o u t 0.1 g of oil into a 500 m l s t o p p e r e d c o n i c a l
flask.
D i s s o l v e oil in 10 m l c h l o r o f o r m .
Add 25 m l H a n u s i o d i n e
solution from bulb pipette.
Keep in the dark for 30 minutes, shaking
occasionally.
Add 10 m l 15% p o t a s s i u m i o d i d e and 100 m l w a t e r ( w a s h i n g d o w n any
free iodine on stopper).
Titrate with 0.1N N a 2 S 2 0 3 , using 1% starch
s o l u t i o n as i n d i c a t o r .
(Note:
t o w a r d end of t i t r a t i o n , s t o p p e r
bottle and shake vigorously, so that any iodine remaining in solution
in C H C l j m a y b e t a k e n up b y K I s o l u t i o n . )
Conduct 2 blank
determinations along with determination on sample.

259

CALCULATION
Iodine number [(B - C) x N x 12.69]/g sample

(oil)

Where:
B average blank titration volume in ml
C sample titration volume in ml
N " normality of ^ 2 8 2 0 3

solution

12.69 = equivalent of I,
L

(126.9
1000

x 100)

INTERPRETATION
The

following

are typical iodine value ranges for selected oils and fats.
Product

Range (Iodine Value)

Beef tallow
Butter fat
Coconut oil
Corn oil
Lard oil
Lard
Mutton tallow
Palm oil
Peanut oil
Sesame oil

35
26
6
111
62
47
32
49
85
103

- 42
- 28
- 10
- 128
- 82
- 66
- 61
- 59
- 100
- 117

REFERENCE
Official Methods of Analysis of the AOAC, 1984, 28.021-.022.

260

UNSAPOIIFIABLE MATTER
PRINCIPLE
The unsaponifable matter is defined as the substances soluble in an oil which
after saponification are insoluble in water but soluble in the solvent used for
the d e t e r m i n a t i o n .
It includes lipids of natural origin such as sterols,
alcohols and hydrocarbons as well as any foreign organic matter non-volatile at
100C (mineral oils) which may be present. Light petroleum or diethyl ether is
used as a solvent but in m o s t cases the results w i l l differ according to the
solvent selected and, g e n e r a l l y , the use of diethyl ether will give a higher
result.
The method is applicable to all oils.
It is, h o w e v e r , only a p p r o x i m a t e for
certain oils having a high content of unsaponifiable matter. The percentage of
unsaponifiable matter is usually determined on the oil as received since, for
trade p u r p o s e s , it often has to be added to the percentage m o i s t u r e and
impurities to determine the net amount of oil.
APPARATUS
1.

150 ml flask fitted with a reflux condenser;

2.

500 ml separating

3.

Oven regulated at 103C (+_ 2C).

funnels;

REAGENTS
1.
2 N potassium hydroxide solution in ethanol (dissolve 120 g KOH
in 95% ethanol and m a k e up to 1 litre).
The reagent m u s t not be
darker than straw yellow.
2.

Light petroleum ether (BR 40-60) free from residue.

PROCEDURE
Weigh about 5 g of
approximately 2 N
Boil gently for an
condenser 50 ml of

fat to within 0.01 g into the flask. Add 50 ml of

ethanolic KOH solution.
A t t a c h the condenser.
hour. Stop heating. Add through the top of the
distilled water and shake.

After c o o l i n g , transfer to a separating funnel and rinse the flask

s e v e r a l t i m e s u s i n g in a l l 50 m l of l i g h t p e t r o l e u m .
Shake
v i g o r o u s l y for a m i n u t e .
Let it stand until there is c o m p l e t e
s e p a r a t i o n of the two phases, and d r a w off the soap solution into a
second separating funnel. If an e m u l s i o n should form, break it by
adding small q u a n t i t i e s of ethanol or of concentrated p o t a s s i u m
hydroxide solution.
Extract the soap solution twice m o r e , using 50 ml light p e t r o l e u m
each time.
C o m b i n e the 3 p e t r o l e u m e x t r a c t s in one separating
funnel, wash 3 times with 50 ml portions of 50% ethanol.
Pour off the p e t r o l e u m extract q u a n t i t a t i v e l y , if n e c e s s a r y in
instalments through the top of the funnel into a tared 250 ml flask.
Rinse the funnel with small quantities of light petroleum.
Evaporate
the solvent by d i s t i l l a t i o n over a bo i 1 i n g - w a t e r bath.
Dry the
residue in an oven at 103C for 15 m i n u t e s , placing the flask in a
Weigh after cooling in a desiccator.
horizontal position.
Repeat the drying for successive 15-minute periods until the loss of
weight between two successive weighings is less than 0.1 percent. If
the
a c o n s t a n t w e i g h t is n o t o b t a i n e d a f t e r 3 o p e r a t i o n s ,

261

unsaponifiable matter is probably contaminated. To ascertain whether

u n s a p o n i f i a b l e m a t t e r is free from soap, ignite the residue and
titrate the ash w i t h an aqueous solution of 0.1 N hydrochloric acid
in the presence of methyl orange.
CALCULATION
Percentage of unsaponifiable matter =
P
Where:

a weight of residue in g
p = weight of sample in g

In the case where the residue has been ignited and titrated with the
aqueous solution of 0.1 N hydrochl oric acid:
Percentage of unsaponifiable matte r
Where:

a
p
b
N

=
=
=
=

100 (a - 0.32Nb)
P

weight of residue in g
weight of sample in g
number of ml of 0.1 N hydrochloric acid used
exact normality of the aqueous solution of HCl

REFERENCE
IUPAC 2.401. The use of diethyl ether as an alternative solvent is described
in the method.

262

ACID VALUE
PRINCIPLE
The acid value is defined as Che weight in milligrams of potassium hydroxide
required to neutralize one gram of oil.
It is a relative measure of rancidity
as free fatty acids are usually formed during decomposition of oil glycerides.
REAGENTS
1.
0.5 N or 0.1 N ethanolic solution of potassium hydroxide
(standardized). This solution is stored in a brown or yellow glass
bottle furnished with a rubber stopper and then decanted for use.
The solution should be colourless or straw yellow.
A stable
colourless solution can be prepared in the following manner: reflux 1
L of ethanol w i t h 8 g of potassium hydroxide and 5 g of a l u m i n i u m
pellets for one hour, then distil immediately. Dissolve the required
amount of potassium hydroxide in the distillate.
Allow the whole to
stand for several days and decant off the clear supernatant liquid
from the deposited potassium carbonate.
The solution can also be
prepared without distillation in the following manner:
add 4 ml of
aluminium butylate to 1 L ethanol and allow the mixture to stand for
several days. Decant the supernatant liquid and dissolve therein the
necessary amount of potassium hydroxide.
This solution is ready for
use.
2.

1% w/v solution of phenolphthalein in 95% ethanol.

95% ethanol containing 5 drops of phenolphthalein solution in

3.
100 ml.
Neutralize i m m e d i a t e l y before use by means of the 0.1 N
ethanolic KOH solution.
PROCEDURE
W e i g h 5 to 10 g of oil to within 0.01 g into a 250 ml Erlenmeyer
flask.
Dissolve in 50-150 m l of a 1:1 v/v mixture of ethanol and
diethyl ether neutralized to phenolphthalein.
Titrate, while shaking, with the 0.5 N ethanolic KOH-solution (or 0.1
N for acidities less than 2 percent by w e i g h t ) until the colour of
the indicator changes.
CALCULATION
Acid Value
Where:

(56.1)(a)(N)
P

a = number of ml of the ethanolic KOH solution used

N = exact normality of the ethanolic KOH solution used
p = weight of sample in g

INTERPRETATION
If mineral acids are present, special procedures may be necessary.
REFERENCES
IUPAC 2.201

(1979).

L o w r y , R.R. and Tinsley, I.J., 1976.

Journal of the A m e r i c a n Oil
Society, 3 (7), 470-472 (Describes a colourimetric method).

263

Chemists

PEROXIDE VALUE
PRINCIPLE
The sample is added to boiling chloroform-acetic acid containing dissolved
p o t a s s i u m iodide. After cooling, the mixture is diluted with water and the
liberated iodine titrated with sodium thiosu1phate.
The amount of iodine
liberated by the sample is a measure of the active oxygen present.
APPARATUS
1.
100 ml round-bottomed flask with a ground-glass joint connecting
to a tube about 750 x 9 m m , the upper 150 m m cooled by a water
condenser.
2.

Microburner.

REAGENTS
1.

Chloroform.

2.

Glacial acetic acid, high purity.

3.

Potassium iodide, low in iodate.

4.

Standard sodium thiosulphate solution 0.002 N.

5.

Starch solution, about 1 percent.

PROCEDURE
Boil 10 ml of chloroform and 10 ml acetic acid in the flask until the
mixture refluxes.
Pour a solution of 1 g of potassium iodide in 1.3
ml of water d o w n the column sufficiently slowly that refluxing
continues without interruption.
This ensures that absence of oxygen
in the flask is maintained. Redissolve any precipitated potassium
iodide by adding of not m o r e than about 6 drops of boiled distilled
cooled water. Add about 1 or 2 g of sample, accurately weighed, down
the c o l u m n w i t h o u t interrupting the steady refluxing.
It is
convenient to add the sample to the flask via the column and reweigh
the beaker plus any residual oil, obtaining the weight taken for the
determination by difference. Turn off the cooling water in order to
raise the condensation level and so ensure that all the sample is
washed into the flask.
Boil the solution with sample added for 3-5
m i n u t e s , rapidly cool, dilute with 50 ml of water and titrate the
liberated iodine with 0.002 N sodium thiosulphate solution using
starch as indicator.
CALCULATION
Peroxide Value

ml

0-0002 N thiosulphate used

g oil taken

INTERPRETATION
Herzinger and Pazlar (16) have compared iodometric and colorimetric methods of
peroxide determination.
The Kreis test may also be used (Mehlenbacher (17)).
W i l l i a m s (18) describes a method for ketonic rancidity. The thiobarbituric
acid value (TBA) is also favoured by some workers (Tarladgis et al (19)). This
m e t h o d is thought to give more consistent results than m a n y others.
Codex
standards for oils and fats require that IUPAC method II.D.13 be used.
Results
from different methods should not be regarded as necessarily equivalent.

264

unincKs
Sully, B.D., 1954.

The Analyst 79, 86-90.

Lea, C.H. 1945. ..Journal of the Society of Chemical

Industry 64, 106.

Lea, C.H. 1946. Journal of the Society of Chemical Industry 65., 286.
Stuffins, C.B. and Weatherall, H., 1945. The Analyst 70, 403.

265

TITRE
PRIHCIPLE
The titre is the s o l i d i f i c a t i o n point of the w a t e r - i n s o l u b l e fatty acids,
p r e p a r e d as d e s c r i b e d and left to c r y s t a l l i z e in a d e s i c c a t o r at laboratory
temperature for about 24 hours before completing the determination. The method
is that of Dalican.
W a t e r - i n s o l u b l e fatty acids are those obtained after
saponification of a fat and decomposition of the soap formed according to the
m e t h o d d e s c r i b e d . B e c a u s e of the possible presence of fatty acids w h i c h are
p a r t i a l l y soluble in w a t e r , the details of this method m u s t be strictly
followed.
APPARATUS
1.

Round-bottomed basin of about 1500 ml capacity.

2.

Sand-bath.

3.
cm.

Glass test tube, length 12 cm, internal d i a m e t e r exactly 2.75

4.
Flat c i r c u l a r
support the tube.

cork having

a central

hole

just big enough

5.
W i d e - n e c k e d jar, height 13 c m , external d i a m e t e r
which the cork and the tube are fitted.

to

10 c m , into

6.
Thermometer accurately calibrated, graduated in 0.1 or 0.2C up
to 70C. The bulb is 2 cm long and 0.6 cm in d i a m e t e r .
REAGENTS
1.
Ethanolic potassium hydroxide solution:, dissolve 18 g KOH in 20
ml distilled water and dilute with 50 ml of 95% ethanol.
2.
Dilute sulphuric acid:
volume of distilled water.
3.

10% w/v sodium chloride

add conc. acid (SG = 1.84) to 4 times its

solution.

PROCEDURE
W e i g h 50 g of the fat into a 1500 ml basin.
Heat s l o w l y and
p r o g r e s s i v e l y up to 115-118C.
Add in one single o p e r a t i o n the
r e q u i s i t e q u a n t i t y of the ethanolic KOH solution. Stir v i g o r o u s l y
w i t h a spatula. Continue to heat gently w h i l e c o n s t a n t l y stirring
and scraping the mass with the spatula until the soap forms fragments
which no longer adhere to the spatula when lightly pressed under it.
Pour 1 litre b o i l i n g distilled w a t e r onto the soap. M a i n t a i n the
soap s o l u t i o n at b o i l i n g point for 45 m i n u t e s in order to drive off
the ethanol and obtain a clear soap solution.
Stop heating.
Replace
the evaporated water by cold water approximately weight for weight,
then c a r e f u l l y pour in 70 ml of the dilute sulphuric acid. Do not
allow undecomposed soap particles to adhere to the surface or rim of
the basin.
Bring to the boil and m a i n t a i n at the boiling point until the free
fatty acids float to the surface in a clear layer (in the special
case where the fatty material contains luric acid glycerides, heat
over a b o i l i n g - w a t e r bath and not by boiling the aqueous layer).

266

Wash the fatty acids twice, each time using 500 ml of boiling aqueous
s o d i u m c h l o r i d e s o l u t i o n . A f t e r e a c h w a s h i n g d r a w off the a q u e o u s
layer as completely as possible.
T r a n s f e r the fatty a c i d s to a flask. Add a n h y d r o u s s o d i u m s u l p h a t e
and filter t h r o u g h a dry f i l t e r paper.
A l l o w the fatty a c i d s to
c r y s t a l l i z e in a d e s i c c a t o r at l a b o r a t o r y t e m p e r a t u r e for about 24
hours before determining the titre.
B r i n g the t e m p e r a t u r e i n s i d e the jar to 20-25C b e l o w that of the
expected titre by immersing the jar in a heated or cooled water bath.
M e l t the fatty a c i d s at a t e m p e r a t u r e of about 10C above the titre
expected.
Pour the fat into the tube to a h e i g h t of about 5.5 cm.
Support the tube in the cork so that a length of about 3 cm projects
a b o v e it. S u s p e n d the t h e r m o m e t e r c a r e f u l l y in the c e n t r e of the
tube so that the b a s e of the bulb is 1 cm from the b o t t o m of the
tube.
The m e r c u r y c o l u m n falls r a p i d l y at first and then m o r e s l o w l y .
Simultaneously, the fatty acids crystallize firstly at the bottom of
the tube, gradually covering the base of the thermometer bulb.
When
the mercury column appears to be stationary during four observations
m a d e at 5 second i n t e r v a l s , stir the fatty a c i d s w i t h a r a p i d ,
c i r c u l a r m o v e m e n t of the t h e r m o m e t e r three t i m e s to the r i g h t and
three t i m e s to the left, thus b r e a k i n g up the c r y s t a l s f o r m e d .
R e p l a c e the t h e r m o m e t e r i m m e d i a t e l y in the c e n t r e of the tube and
take further readings.
The m e r c u r y c o l u m n , w h i c h fell s h a r p l y d u r i n g the a g i t a t i o n , n o w
rises again and attains a m a x i m u m before falling again.
This m a x i m u m
is the titre.
Carry out two determinations and if the difference between them does
not e x c e e d 0.2C, take the m e a n .
If a d i f f e r e n c e e x c e e d s 0.2C,
r e p e a t the d e t e r m i n a t i o n s until the r e a d i n g s agree to w i t h i n 0.2C.
Take the mean of two acceptable values.
INTERPRETATION
S o m e t y p i c a l titre f i g u r e s for v a r i o u s o i l s are:
c o t t o n s e e d 33, o l i v e
s e s a m e 23, t e a s e e d 14, a r a c h i s 30.
T h e s e f i g u r e s are g u i d e s only.
accurate comparison, authentic oils should be tested using this method.
REFERENCE
IUPAC 2.121

(1979).

267

23,
For

SOAP TEST IH EDIBLE OILS

PRINCIPLE
Detection of alkalinity using bromophenol blue as indicator.
APPARATUS
1.

150 mm x 15 mm

test tube.

REAGEHTS
1.

Solution of 0.1% of bromophenol blue in 95% v/v ethanol.

2.

Acetone, analytical grade containing 2% v/v water.

(A few drops of the solution of bromophenol blue should give a yellow

to yellow-green colour to the 2% water in acetone.)
PROCEDURE
Place 10 ml of the acetone and 1 drop of the bromophenol blue
solution in a test tube. The solution should have a yellow colour.
(If not, rinse the test tube with acetone until the blue colour
disappears). Add 10 g of the oil to the test tube, stopper with a
clean stopper, shake and allow to settle.
The presence of blue
colour in the upper acetone layer indicates the presence of soap.
IHTERPRETATIOH
The result is expressed as positive or negative.
edible oil.

The test is applicable to any

REFEREHCE
British Standard
(Alkalinity).

684:Section

2.5:1977.

268

Determination

of

Dissolved

Soap

ASACHIS (GROUNDNUT) OIL TEST

PRINCIPLE
T h i s test is d e s i g n e d for use w i t h a r a c h i s o i l , a l o n e or in a d m i x t u r e w i t h
liquid fatty oils.
It is not applicable to m i x t u r e s of arachis oil with solid
fats.
It i s not suitable for the evaluation of the purity of arachis oil.
A r a c h i d i c acid is p r e c i p i t a t e d f r o m the s a p o n i f i e d o i l s a m p l e , c r y s t a l l i z e d
f r o m 9 0 % e t h a n o l and d i s s o l v e d in d i e t h y l e t h e r .
T h e s o l u t i o n is t h e n
e v a p o r a t e d and the r e s i d u e d r i e d and w e i g h e d , a f t e r the c o n f i r m a t i o n of its
i d e n t i t y by the d e t e r m i n a t i o n of its m e l t i n g p o i n t w h i c h s h o u l d be 71C or
over.

REAGENTS
1.

Potassium hydroxide, 1.5 N solution

2.

Acetic acid, 33% (m/m)

3.

Ethanol, 70%

4.

Ethanol 90%

(v/v).

5.

Hydrochloric

acid,

6.

Diethyl

in 95%

(v/v) ethanol.

solution.

(v/v).

concentrated.

ether.

PROCEDURE
B o i l 5 g of the oil in a 250 m l c o n i c a l flask w i t h 25 m l of the
potassium hydroxide solution for 10 m i n u t e s under a reflux condenser.
Add to the hot solution 7.5 ml of the acetic acid solution and 100 ml
of the 70% ethanol to which 1 m l of HC1 has been added.
Maintain the
t e m p e r a t u r e of t h e m i x t u r e f o r 1 h at 12C to 14C.
If no
precipitate forms arachis oil is absent.
If t h e r e is a p r e c i p i t a t e , f i l t e r t h e s o l u t i o n
p r e c i p i t a t e w i t h the s a m e m i x t u r e of 70% e t h a n o l and
19C, breaking up the precipitate occasionally by means
wire bent into a loop.
Continue the washing until the
only a faint turbidity with water.

and w a s h the
H C 1 at 17C to
of a platinum
washings give

Note the total v o l u m e of the 70% ethanol used for the crystallization
D i s s o l v e t h e p r e c i p i t a t e in the s m a l l e s t p o s s i b l e
and w a s h i n g .
q u a n t i t y of the 9 0 % e t h a n o l (25 to 70 m l ) and cool the s o l u t i o n to
15C for 3 hours.
If no precipitate forms arachis oil is absent.
If any crystals appear, filter the solution and wash with about half
the v o l u m e o f 90% e t h a n o l used for c r y s t a l l i z a t i o n at the s a m e
t e m p e r a t u r e and t h e n w i t h 50 m l of the 70% e t h a n o l .
D i s s o l v e the
crystals in w a r m diethyl ether and filter into a tared flask, washing
the f i l t e r w e l l w i t h the e t h e r and a d d i n g the w a s h i n g s to the
s o l u t i o n in the f l a s k .
E v a p o r a t e the e t h e r and d r y the r e s i d u e at
100C and w e i g h .
D e t e r m i n e the m e l t i n g p o i n t of the c r y s t a l s ,
p r e f e r a b l y in a c l o s e d c a p i l l a r y m e l t i n g p o i n t t u b e a n d , if it is
found to be lower than 71C, recrystal1ize from 90% ethanol, transfer
to a t a r e d f l a s k , d r y , w e i g h as b e f o r e , and d e t e r m i n e the m e l t i n g
point.
If the m e l t i n g p o i n t is b e l o w 71C a r a c h i s oil s h o u l d be a s s u m e d to
be a b s e n t .
If the m e l t i n g p o i n t is 71C or o v e r add to the w e i g h t
thus found ( f r o m T a b l e s 1 and 2 r e s p e c t i v e l y ) , c o r r e c t i o n s for the
solubility of the crystals (a) in 90% ethanol and (b) in 70% ethanol,

269

in the latter case calculating the correction from the total quantity
of 70% ethanol used in precipitating and washing - including the 100
ml used in the first instance.

Table 1
Correction to be added per
70 ml of 90% (v/v) ethanol
at 15C

Weight of
crystals obtained
g

0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50

0.021
0.025
0.029
0.033
0.036
0.038
0.040
0.042
0.044
0.045

7
2
5
6
4
5
7
7
0
7

Table 2

Melting point
of crystals

Correction to be
added per 100 ml
70% (v/v) ethanol
at 50C
g

71.0
71.5
72.0
72.5
73.0

Factor for converting

corrected proportion
of arachidic acid to
arachis oil (*)

0.013 0
0.009 9

17.0

18.6

20.0

0.008 0

21.1

0.006 7
0.006 0

(*) Not needed

for

22 .0

calculation

CALCULATION
R e s u l t s are c a l c u l a t e d a p p l y i n g the a p p r o p r i a t e
expressed as g arachidic acid/kg oil.
g arachidic

acid/kg oil = corrected mass of dried

obtained (g) x 200

corrections,

and

crystals

Note:
If d e s i r e d , the a p p a r e n t % m / m of a r a c h i s oil p r e s e n t can be
e v a l u a t e d by a p p l y i n g the a p p r o p r i a t e f a c t o r in T a b l e 2, C o l u m n 3.
The r e s u l t s so o b t a i n e d are not s u i t a b l e for the e v a l u a t i o n of the
purity of arachis oil.
REFEREHCE
C A C / R M - 1 9 6 9 , w h i c h is based on BS 6 8 4 : 1 9 5 8 , M e t h o d s of A n a l y s i s of O i l s and
Fats, p. 97 and updated as BS 684:Section 2.31:1978, Arachis Oil Test (Evers).

270

COTTONSEED OIL TEST

PRINCIPLE
Based on the red c o l o u r d e v e l o p e d by c y c l o p r o p e n o i c a c i d s (such as s t e r c u l i c
and m a l v a l i c a c i d s ) u n d e r the c o n d i t i o n s of test in the p r e s e n c e of s u l p h u r .
Hydrognation and deodorisation wholly or partly destroy the chromogens and a
positive reaction is not given by an oil heated to 225C or above.
APPARATUS
1.
30 ml screw-capped bacteriological media bottle or other strong
g l a s s c o n t a i n e r w h i c h can be s t o p p e r e d .
A l t e r n a t i v e l y , use t e s t t u b e s , 150 x 25 m m .
2.
Water-bath, boiling or heating bath at 110-120 o C such as an oil
bath, heating block or bath of saturated salt solution.
REAGEHTS
1.
Sulphur reagent.
Mix equal
solution of 1% precipitated sulphur

v o l u m e s of a m y l a l c o h o l
in carbon disulphide.

and

PROCEDURE
Add 2.5 m l oil and 2.5 m l r e a g e n t to the b o t t l e or t e s t - t u b e .
If a
bottle is used, lightly screw on the cap. Sensitivity is improved by
r e t a r d i n g e v a p o r a t i o n of the c a r b o n d i s u l p h i d e and by c a r r y i n g out
under m o d e r a t e pressure.
If a t e s t - t u b e is u s e d , the cork m a y be
inserted firmly and kept in place by tying a piece of linen over it.
Although this enhances sensitivity, it may be omitted if it appears
to be hazardous.
If a bottle is used, keep in the boiling water-bath
for t w o h o u r s .
If a t e s t - t u b e is u s e d , k e e p at 70 - 80C for 30
minutes, loosen the stopper and transfer to a boiling water-bath or
preferably a heating apparatus at 110C for one and a half hours.
INTERPRETATION
The presence of 2% or more of cottonseed oil is indicated by a rose-red colour.
K'apok seed oil (Er i o d e n d r on an f r ac tuo s um ), b a o b a b or fony o i l , the oil of
S t e r c u l i a foe t id a and s o m e other o i l s not n o r m a l l y found in c o m m e r c e also
react.
The b u t t e r , lard, etc. of a n i m a l s fed on c o t t o n s e e d m a y give a faint
p o s i t i v e reaction.
REFERENCES
Halphen, G., 1897.
CAC/RM

Analyst 22, 326.

23-1970.

O f f i c i a l M e t h o d s of A n a l y s i s of the A O A C ,
1984, 28.120-.123, o b t a i n s a
quantitative result by using a standard preparation of cyclopropene fatty acids
in cottonseed oil methyl esters.

271

SESAME OIL TEST

PRINCIPLE
A colour is produced

from a reaction with sesamin in the oil.

APPARATUS
1.

Separator, cylindrical type, 25 ml.

Mark at 10 ml and 15 ml.

2.

Porcelain dish, flat-bottomed, about 60 mm diameter.

REAGEITS
1.

Concentrated

sulphuric acid.

2.
Furfural, 0.035Z in acetic anhydride. Use freshly distilled
furfural. Store in the refrigerator in a dark bottle with a w e l l fitting ground-glass stopper.
The solution keeps several months.
PROCEDURE
Pour 10 ml of oil followed by 5 ml of furfural reagent into the
separator. Stopper and shake vigorously approximately one minute.
Leave to separate.
Run 1-2 ml of the lower layer into the porcelain
dish, add 5-6 drops of sulphuric acid and mix by swirling.
IHTERPRETATIOH
For quantities of sesame seed oil higher than 1 percent, a dark blue coloration
with a slight greenish tint, develops immediately. For lesser amounts the blue
colour is p r o g r e s s i v e l y paler and the period required for the complete
d e v e l o p m e n t of the colour is increased.
0.25 percent in olive oil gives a
bluish grey colour which takes 5-10 minutes to develop. The test is generally
applicable.
REFERENCES
P a v o l i n i , L., 1934.
Olii M i n e r a l i , Grasse Saponi, Colori Vernici
41.
A l s o , see the s e c t i o n by G r a c i a n in B o c k e n o o g e n (4).
The B a n d o u i n Villavecchia-Fabris test is less dependable.

272

TEASEED OIL TKST

PRINCIPLE
Based on the Fitelson (modified L i e b e r m a n - B u r c h a r d ) test, i.e. red colour
developed by acetic anhydride in the presence of sulphuric acid in chloroform
solution of the oil.
APPARATUS
1.

150 mm x 15 mm test tube.

2.

2 ml pipette, graduated to 0.1 ml.

3.
Dropper so calibrated
0.22g.
4.

that 7 drops of oil w e i g h a p p r o x i m a t e l y

Water bath at 5C.

REAGENTS
1.

Chloroform.

2.

Concentrated

3.

Acetic

4.

Diethyl ether, anhydrous, stored over sodium and

sulphuric

acid.

anhydride.
peroxide-free.

PROCEDURE
Pipette into a test-tube 0.8 ml of acetic a n h y d r i d e , 1.5 ml of
c h l o r o f o r m and 0.2 m l of sulphuric acid.
Cool to 5C, then add
approximately 0.22 g (7 drops) of oil.
If any turbidity appears, add
acetic anhydride drop by drop with shaking until the solution becomes
clear.
Keep at 5C for 5 m i n u t e s .
Add 10 ml of diethyl ether
previously cooled to 5C. Stopper the test tube and mix immediately
by inverting it twice. Return the test tube to the bath at 5C. An
intense red colour which develops about a minute after the addition
of the ether, reaches a m a x i m u m and d i s a p p e a r s , indicates pure
teaseed oil.
A less intense colour suggests the presence of teaseed
oil, but caution m u s t be exercised in interpreting results in the
presence of olive oil. The test is g e n e r a l l y a p p l i c a b l e , but some
olive oils yield a pink colour and the test is therefore not reliable
for the detection of less than about 15% of teaseed oil in olive oil.
REFERENCES
F i t e l s o n , J., 1936.
Journal
Agricultural Chemists 19. 493.

of

the

Association

CAC/RM 24-1970.

273

of

Official

IDENTIFICATION OF OILS AND FATS

(GLC of Fatty Acid Methyl Esters)
PRINCIPLE
The methyl esters are formed using boron trifluoride or methanol and alkali and
separated by gas-liquid chromatography using a flame ionization detector.
The
m e t h y l esters are also suitable for analysis by TLC and IR. The pattern of
methyl esters can be compared to authentic oils for identification.
APPARATUS
1.

Gas liquid chromatograph with the following

characteristics:

a.
Injection system heated to a higher temperature (20 to 50C
than that of the column.
b.
Oven:
the oven should be capable of heating the column to
at least 220C and of maintaining the desired temperature to within
1<>C. If temperature programming is to be employed, an apparatus with
a twin column is recommended.
c.
Packed column: columns may be glass or stainless. However,
glass is preferred as the steel may decompose polyunsaturated fatty
a c i d s h a v i n g m o r e than 3 double bonds.
S o m e successful c o l u m n
packings are given below with the column length, internal diameter
and operating temperature indicated:
(1) 12-15% ethylene
C h r o m P (2 m x 4 m m , 180C).

glycol

succinate on 100/120 mesh Gas-

(2) 2-10% Apiezon-L on 80/100 mesh Chromosorb W or Celite

(2 m x 4 m m , 220C).
(3) 10% butan-l-4-diol
W or C e l i t e (2 m x 4 m m , 175C).

succinate on 80/100 mesh chromosorb

(4) 3% SE-30 on 100/120 mesh Chromosorb G, silanised (2 m

x 3 m m , 190C).
(5) 10% Silar-10C on 100/120 mesh Gas-Chrom Q (2 m x 4 m m ,
from 130-220C at 4C/min.).
(Note that column
chain, C20, fatty

lengths may have to be shortened to determine long

acids.)

Condition a newly prepared column by disconnecting the detector and

heating the column in the oven at the normal operating temperature,
while running the carrier gas at a rate of 20-60 ml/min.
Condition
for about 16 hours.
d.
Detector:
F l a m e - i o n i z a t i o n detector capable of being
heated to a t e m p e r a t u r e above that of the c o l u m n .
H y d r o g e n flow
should be about h a l f of the carrier gas and oxygen flow should be 5
to 10 times the hydrogen.
2.
Syringe:
microlitre.

Maximum

capacity

10 y 1, graduated

in tenths

of a

3.
Recorder:
If the recorded curve is to be used to calculate the
composition of the mixture analyzed, an electronic recorder of high
precision is required.
The characteritics of the recorder should be:

274

a.
second.

Rate

of

response

below

1.5

second,

b.

Width of the paper: 25 cm minimum.

c.

Paper speed:

preferably

below

25 to 150 cm/hr.

4.
Integrator (optional): Rapid and accurate calculation can be
performed with the help of an electronic integrator.
This must give
a linear response with adequate sensitivity, and the correction for
deviation of the base-line must be satisfactory.
5.

50 and 100 ml boiling flasks with ground

joints.

6.
Reflux c o n d e n s e r , 20 to 30 cm e f f e c t i v e
joint.
7.

Boiling chips

8.

Inlet tube for nitrogen.

length,

with

ground

(fat-free).

9.
Graduated pipette, capacity at least 10 ml, fitted with a rubber
bulb, or automatic pipette.
10.

Test tubes with ground

11.

250 ml separating

stoppers.

funnels.

REAGEHTS
1.
Carrier gas:
inert gas (nitrogen, h e l i u m , argon) t h o r o u g h l y
dried and containing less than 10 mg/kg of oxygen.
(Use at about 4060 ml/min for 4 mm ID columns).
2.
A u x i l i a r y gas: h y d r o g e n (99.9 percent min. purity) free from
organic impurities; air or oxygen, free from organic impurities.
3.
Reference standards:
a mixture of methyl esters, or the methyl
esters of an oil of known composition, preferably similar to that of
the fatty matter to be analyzed.
4.
M e t h a n o l i c sodium h y d r o x i d e solution, a p p r o x i m a t e l y 0.5 N:
Dissolve 2 g of sodium h y d r o x i d e in 100 m l m e t h a n o l containing not
m o r e than 0.5 percent m / m of water.
W h e n the solution has to be
stored for a considerable time, a small amount of white precipitate
of sodium carbonate m a y be formed; this has no effect on the
preparation of the methyl esters.
5.
Methanolic solution of boron trifluoride, 12 to 15 percent m/m.
14 and 50 percent solutions are a v a i l a b l e c o m m e r c i a l l y .
CAOTIOH:
B o r o n t r i f l u o r i d e is p o i s o n o u s .
F o r this r e a s o n , it is not
recommended that the analyst prepare the methanolic solution of boron
trifluoride from methanol and boron trifluoride.
However, if it is
quite u n a v o i d a b l e to prepare a solution of boron t r i f l u o r i d e from
gaseous boron t r i f l u o r i d e , the r e c o m m e n d e d method is:
Weigh 2 L
flask c o n t a i n i n g 1 L of m e t h a n o l . Cool in ice bath, and w i t h flask
still in bath, bubble BF^ from cylinder through a glass tube into the
methanol until 125g BF3 is absorbed. Perform operation in fume hood.
BF3 must be flowing through the glass tube before it is placed in and
until it is removed from methanol to prevent liquid from being drawn
into gas cylinder valve system.
Gas should not flow so fast that
white fumes emerge from flask. Reagent is stable two years.

275

Also note that m e t h a n o l i c boron trifluoride solution m a y produce

a d v e n t i t i o u s p e a k s on the graph in the r e g i o n of C20 ~ ^22 a c i d s .
Consequently, any new batch of reagent should be checked by preparing
the m e t h y l ester of pure oleic acid, and c h r o m a t o g r a p h i n g ; if an
extraneous peak appears, the reagent should be rejected.
The various
reagents must not give peaks interfering with those of methyl esters
of fatty acids during the gas liquid chromatography.
The methanolic
solutions of boron trifluoride must be stored in a refrigerator.
6.
H e p t a n e , c h r o m a t o g r a p h i c quality (Note that if C20 or higher
fatty acids are absent, hexane may be substituted.)
7.
R e d i s t i l l e d light p e t r o l e u m
than 1, residue-free: or hexane.
sodium

(BR 40-60C), b r o m i n e value

8.

Anhydrous

9.

Saturated aqueous solution of sodium chloride.

10.

Methyl red, 1 g/L solution in 60% v/v ethanol.

less

sulphate.

PROCEDURE
Prepare the
trifluoride
preferable
trifluoride

methyl esters of the fatty acids. The method using boron

g i v e s good r e s u l t s on a w i d e range of s a m p l e s and is
to a l t e r n a t i v e m e t h o d s w h i c h m a y be used in case boron
is not available.

B e c a u s e of the toxic character of boron t r i f l u o r i d e , the following

operations are best performed under a ventilated hood. All glassware
must be washed with water immediately after use.
If the oil or fatty acids include fatty acids containing more than 2
double bonds, it is advisable to purge the air from the methanol and
the flask by passing a stream of nitrogen into the methanol for a few
minutes.
Transfer about 350 mg of clear oil (filtered if necessary) to a 50 ml
c o n i c a l flask, add 6 ml of the 0.5 N m e t h a n o l i c sodium h y d r o x i d e
solution, 7 ml of methanolic boron trifluoride solution and a boiling
chip. Fit the c o n d e n s e r to the flask. Boil under reflux until the
d r o p l e t s of fat d i s a p p e a r (this m a y take 5 to 10 m i n u t e s , but in
e x c e p t i o n a l cases it m a y require m o r e than 10 minutes).
Add the
appropriate amount of methanolic boron trifluoride solution from the
b u l b or a u t o m a t i c pipette through the top of the condenser to the
boiling liquid.
Continue boiling for 2 minutes.
Add 2 to 5 m l of h e p t a n e (the precise a m o u n t does not affect the
r e a c t i o n ) to the boiling m i x t u r e through the top of the condenser.
Continue boiling for 1 minute. Withdraw the source of heat, and then
r e m o v e the c o n d e n s e r .
Add a small portion of saturated sodium
chloride solution to the flask in order to bring the level of liquid
into the n e c k of the flask. Transfer about 1 ml of the upper layer
(heptane solution) into a test-tube with a ground-glass neck and add
a little a n h y d r o u s sodium sulphate to r e m o v e any trace of water.
This s o l u t i o n w i l l c o n t a i n about 5 - 1 0 percent of m e t h y l esters and
may
be i n j e c t e d
directly
i n t o the c o l u m n
for gas
liquid
chromatography.
If the sample consists of fatty acids, not triglycerides, the former
may be methylated directly by proceeding as above, but omitting the
methanolic sodium hydroxide solution.

276

The amount of sample methyl esters injected should be adjusted to fit

the o p e r a t i n g c o n d i t i o n s and to be w i t h i n the linear r a n g e of the
detector and electrometer.
Carry out the analysis of a mixture of methyl stearate and oleate in
about equivalent proportions (e.g. methyl esters from cocoa butter).
Choose the size of the sample, the temperature of the column and the
carrier gas flow so that the m a x i m u m of the methyl stearate peak is
recorded about 15 minutes after the solvent peak, and reaches about
3/4 of full scale deflection.
The s a m p l e for e x a m i n a t i o n should be 0.1 to 1 p 1 of the h e p t a n e
s o l u t i o n of m e t h y l e s t e r s .
In the case of e s t e r s not in s o l u t i o n ,
prepare an approx. 10 percent solution in heptane and inject 0.1 to 2
yl of this.
If the search is for constituents present only in trace
amounts, the sample size may be increased (up to tenfold).
As a r u l e , the o p e r a t i n g c o n d i t i o n s w i l l be those d e f i n e d above.
Nevertheless, it is possible to work with a lower column temperature
where the determination of acids below C ^ i required, or at higher
temperature when determining fatty acids above C2g.
If possible, it
is r e c o m m e n d e d that a n a l y s i s be m a d e on t w o c o l u m n s of d i f f e r e n t
polarity to check for the absence of coincident peaks, for example in
the case of fish oils, or in the case of the simultaneous presence of
^18 - 3 an< * ^20-0 o r
^18-3 ant * conjugated C ^ g ^ .
On occasion, it is
possible to employ temperature programming.
If the sample contains
methyl esters of fatty acids below Cj^, it is necessary to inject the
s a m p l e at 100C (or at 50-60C if b u t y r i c acid is p r e s e n t ) , and to
raise the temperature at 4-8C/min. In some cases the two procedures
can be combined:
after the programmed heating, continue the elution
at a constant temperature until all the components have been eluted.
If a t e m p e r a t u r e p r o g r a m m i n g f a c i l i t y is not p r o v i d e d , w o r k at t w o
fixed temperatures between 100C and 195C.
Note that it is p r e f e r a b l e to a n a l y z e the m e t h y l e s t e r s as soon as
possible.
If necessary, the heptane solution containing the methyl
e s t e r s m a y be stored u n d e r an inert gas in a r e f r i g e r a t o r .
In the
case of p r o l o n g e d s t o r a g e , it is d e s i r a b l e to p r o t e c t the m e t h y l
esters from autoxidation by adding to the solution an antioxidant in
such c o n c e n t r a t i o n as w i l l not i n t e r f e r e w i t h the
subsequent
analysis, e.g. 0.005 percent m / v of BHT ( 2 , 6 - d i-1 er t - b u t y 1-t o 1 u e n e ) .
If necessary, the dry and solvent-free methyl esters may be stored 24
h o u r s u n d e r inert gas in a r e f r i g e r a t o r , or l o n g e r in a sealed tube
under vacuum in a freezer.
EXPRESSION OF RESULTS
Analyse a reference standard mixture of known composition under the
s a m e o p e r a t i n g c o n d i t i o n s as those e m p l o y e d for the s a m p l e , and
measure
the r e t e n t i o n d i s t a n c e s (or r e t e n t i o n t i m e s ) for the
constituent fatty acids. Using semi-logarithmic paper, construct the
graphs showing the logarithm of the retention distance (or retention
t i m e ) as a f u n c t i o n of the n u m b e r of c a r b o n a t o m s ; in i s o t h e r m a l
conditions, the graphs for straight chain acids of the same degree of
u n s a t u r a t i o n s h o u l d be s t r a i g h t l i n e s , and lines for h o m o l o g o u s
series of different degrees of unsaturation should be approximately
I d e n t i f y the p e a k s in the s a m p l e from these g r a p h s , if
parallel.
necessary by interpolation.
It is necessary to avoid conditions such
that "masked peaks" exist, i.e., where the resolution is insufficient
to separate two acids.

277

The fatty acid c o m p o s i t i o n of c o m m o n oils and fats is given in the

tables below.
The values are in percent and single values represent
m a x i m a (that value or less).
Maximum values of less than 1% are not
shown.
(Table i n f o r m a t i o n is from P e a r s o n ' s C h e m i c a l Analysis of
F o o d s , 8th Ed.).

Fatty
Acid

Coconut

C 6:0
C 8:0
C10 :0
C12 : 0
C14:0
Cl 6:0
C16 : 1
Cl 8:0
C18: 1
Cl 8:2
C18:3
C20:0
C20:1

Pain

1.2
3.4-15
3.2-15
41-56
13-23
4.2-12

1.2
0.5-5.9
32-59

Pala
Kernel
-

2.4-6.2
2.6-7.0
41-55
14-20
6.5-11

1.0-4.7
3.4-12
0.9-3.7

1.5-8.0
27-52
5-14
1.5
1.0

Ground
Nut

Cl 6:0
C16:1
Cl 8:0
C18: 1
Cl 8:2
C18: 3
C20:0
C20:1
C22:0
C22: 1
C24:0

6.0-16
1.0
1.3-6.5
35-72
13-45
1.0
1.0-3.0
0.5-2.1
1.0-5.0
2.0
0.5-3.0

Fatty
Acid
C14:0
C14:1
C15:0
C15 : i s o
C16:0
Cl 6 :1
C16:2
Cl 7 : 0
Cl 7: 1
Cl 7 :iso
C18:0
C18: 1
C18:2
Cl 8:3
C20: ail

Safflower

Se s ame

0.4-2.0
17-31
0.5-2.0
1.0-4.0
13-44
33-59
0.1-2.1

3.5-6.0
35-50
35-50
1.0
1.0

1.3-3.5
10-23
0.7-5.4

Fatty
Acid

Cottonseed

1.0
2.0-10

Soyabean

7.0-12

7.0-14

1.0-10
7-42
55-81
1.0

Maize

Sunflower

Olive

Rapeseed

8.0-19

3.0-10
1.0
1.0-10
14-65
20-75

7.5-20
0.3-3.5
0.5-3.5
56-83
3.5-20
1.5

2.5-6.0

0.5-4.0
19-50
34-62
2.0
1.0

1.5

1.0

0.9-2.1
50-66
18-30
6.0-14
0.1-1.2
0.1-4.3

5.0

Lard and Rendered

Pork Fat

Edible Tallow
and Premier Jus

0.5-1.5
20-32
1.7-5.0
5.0-24
35-62
3.0-16
1.5
4.0

1.4-7.8
0.5-1.5
0.5-1.0
1.5
17-37
0.7-8.8
1.0
0.5-2.0
1.0
1.5
6.0-40
26-50
0.5-5 .0
2.5

278

1.4-5.5
19-30
44-62
4.0-11
1.0
1.0

IHTERPRETATIOH
It is possible to quantitate from the m e t h y l ester c h r o m a t o g r a m s by comparison
to authentics.
This m e t h o d , h o w e v e r , is best used for identification of pure
o i l s or d e t e c t i o n of a d u l t e r a t i o n of an e x p e n s i v e oil by a c h e a p e r one.
For
e x a m p l e , s o y a b e a n oil c a n be d e t e c t e d as an a d u l t e r a n t of s e s a m e o i l by the
presence of C18:3 fatty acid.
Also, animal fat adulterants in vegetable oils
can be detected by C14:0, C16:l and the large amount of C18:0.
Note that methylation with boron trifluoride m a y lead to erroneous results
the following compounds:
a.
Compounds
epoxy);

having

secondary

b.

Compounds containing

c.

Conjugated

d.

Waxes.

oxygen

cyclopropane

polyunsaturated

groupings (hydroxy, hydroperoxy, keto,

and cyclopropene

compounds

and acetylenic

groups;
compounds;

For t h e s e it is p r e f e r a b l e to use one of the a l t e r n a t i v e m e t h o d s u n l e s s

amounts present are small (e.g. as in cottonseed oil).

REFERENCE
IUPAC 2.301, 2.302

(1979).

279

with

the

ALTERNATIVE METHYL ESTER PREPARATION METHOD

(Neutral Oils and Fats)
PRINCIPLE
This method is alternate to the boron trifluoride esterification procedure, for
use w i t h neutral oils and fats (i.e., those having an acid value of less than
2).
It involves m e t h y l ester ification of the fatty acids in an alkaline
medium.
APPARATUS
1.
High speed
plate.

stirrer, with heater (e.g. magnetic stirrer and hot-

2.

100 ml ground-necked conical or round-bottomed

3.

Inlet tube for passing nitrogen.

4.

Reflux condenser to fit the flask.

5.

Boiling chips

6.

125 ml separating

7.

50 ml narrow-mouthed conical flask.

flask.

(fat-free).
funnels.

REAGENTS
1.

Methanol, containing not more than 0.5% of water.

2.
Methanolic potassium hydroxide solution, approx. IN. Dissolve
5.6 g of p o t a s s i u m hydroxide in 100 ml of methanol conta ining not
more than 0.5 % m/m of water.
3.

Heptane, chromatographic

quality.

4.

Anhydrous sodium sulphate.

5.

Nitrogen, containing less than 5. mg/kg of oxygen.

PROCEDURE
If the oil includes fatty acids containing more than 2 double bonds,
it is advisable to purge the air from the methanol and the flask by
passing a stream of nitrogen into the methanol for a few minutes.
Transfer about 4 g of clear sample oil into a 100 ml round-bottomed
or conical flask. Add about 40 ml of m e t h a n o l , 0.5 ml of potassium
h y d r o x i d e solution and a boiling chip.
Fit under the reflux
c o n d e n s e r , stir and bring to the boil. The solution should b e c o m e
clear.
The reaction is usually complete after 5 to 10 minutes.
(Oils such as castor oil may not become completely clear).
Cool under running water and transfer the contents, to a 125 ml
separating funnel, rinsing the flask with 20 ml heptane. Add about
40 ml of w a t e r , shake and allow to separate. The esters pass into
the upper heptane layer.
Separate.
Extract the aqueous layer again
w i t h 20 ml heptane.
Combine the two extracts and wash them with
several 20 ml portions of water. Separate and dry the ester solution
over anhydrous sodium sulphate. Filter through cotton wool into a 50
ml conical flask and evaporate the solution down to approximately 20
ml over a water-bath, while passing a stream of nitrogen.
Note that

280

there is some risk of losing part of the most volatile methyl esters
if the solvent evaporation is prolonged or if the current of nitrogen
i s too vigourous.
REFERENCE
IUPAC

2.301,

2.302

(1979).

281

ALTERNATIVE METHYL ESTER PREPARATION METHOD

(Acidic Oils and Fats)
PRINCIPLE
This method
is an a l t e r n a t i v e to the b o r o n t r i f l u o r i d e
esterification
procedure, for use w i t h acidic oils and fats (i.e., those having an acid value
g r e a t e r than 2). It i n v o l v e s n e u t r a l i z a t i o n of p r e v i o u s l y f o r m e d free fatty
acids, alkaline methanolysis of the glycerides and esterification of the fatty
acids in acid m e d i u m .
APPARATUS
1.
High
plate) .

speed

stirrer

and

heater

2.

250 ml ground-necked

3.

Inlet tube for passing

4.

Reflux condenser to fit the

5.

Boiling chips

6.

250 ml separating

7.

100 ml narrow-necked

(e.g. a magnetic

conical or round-bottomed

stirrer

and

hot-

flask.

nitrogen.
flask.

(fat-free).
funnels.
conical

flask.

REAGENTS
1.
Sodium methoxide solution.
Dissolve lg of sodium
methanol containing not more than 0.5% m / m of water.

in 100 ml

of

2.
M e t h a n o l i c s o l u t i o n of a n h y d r o u s hydrochloric acid, approx. N:
Gaseous hydrochloric acid can easily be prepared in the laboratory by
simple displacement
from the c o m m e r c i a l s o l u t i o n by d r i p p i n g
c o n c e n t r a t e d s u l p h u r i c acid on to the h y d r o c h l o r i c acid s o l u t i o n .
The liberated gas is dried by bubbling through concentrated sulphuric
acid.
Since hydrochloric acid is very rapidly absorbed by methanol,
it is advisable to take the usual precautions in dissolving it, i.e.,
introduce the gas through a small inverted funnel with the rim just
touching the surface of the liquid.
Large quantities of methanolic
h y d r o c h l o r i c a c i d s o l u t i o n can be p r e p a r e d in a d v a n c e , as it k e e p s
well in glass-stoppered bottles stored in the dark.
3.

Heptane, chromatographic

4.

Anhydrous

5.

Nitrogen, containing less than 0.5 mg/kg of oxygen.

sodium

quality.

sulphate.

PROCEDURE
If the acid oil i n c l u d e s fatty a c i d s c o n t a i n i n g m o r e than 2 d o u b l e
bonds, it is advisable to purge the methanol and the flask by passing
a stream of nitrogen into the methanol for a few minutes.
Transfer
about 4 g of clear sample oil into a 250 ml conical or round-bottomed
f l a s k . Add 40 m l of the s o d i u m m e t h o x i d e s o l u t i o n . Fit the r e f l u x
c o n d e n s e r , stir and b r i n g to the b o i l .
The s o l u t i o n s h o u l d b e c o m e
C l e a r , w h i c h u s u a l l y o c c u r s in about 10 m i n u t e s .
The r e a c t i o n is
n o r m a l l y c o m p l e t e after 15 m i n u t e s .
Note that in the case of v e r y
acid oils and fats precipitation of sodium chloride occurs, owing to
the relatively high quantity of sodium methoxide.
This may lead to

282

b u m p i n g d u r i n g the s u b s e q u e n t b o i l i n g .
T h e p r e c i p i t a t e may be
f i l t e r e d o f f , b u t t h i s i s u s u a l l y u n n e c e s s a r y o w i n g to the s h o r t
p e r i o d of b o i l i n g p r e s c r i b e d .
Add at l e a s t 50 ml o f the h y d r o c h l o r i c a c i d s o l u t i o n to the f l a s k ,
and b o i l a g a i n for 10 m i n u t e s .
Cool under r u n n i n g w a t e r , add 100 ml
of w a t e r
to t h e f l a s k ,
t h e n p o u r t h e c o n t e n t s i n t o a 2 5 0 ml
s e p a r a t i n g f u n n e l and add 30 ml of h e p t a n e .
Shake v i g o r o u s l y and l e t
settle until
the 2 p h a s e s h a v e s e p a r a t e d .
C o l l e c t the h e p t a n e
e x t r a c t s and w a s h them s e v e r a l t i m e s w i t h w a t e r u n t i l
neutral.
S e p a r a t e and d r y o v e r a n h y d r o u s s o d i u m s u l p h a t e .
F i l t e r through
c o t t o n w o o l i n t o a 1 0 0 ml f l a s k and f i n a l l y e v a p o r a t e the s o l u t i o n
d o w n to 2 0 ml o v e r a b o i l i n g w a t e r - b a t h , w h i l e p a s s i n g a s t r e a m o f
nitrogen.
REFERENCE
IUPAC

2.301,

2.302

(1979).

283

9.2

MAKGAKIRE

COMPOSITION
The Codex Standard CAC/RS 32-1969 recommends
than 80% fat and not more than 16% water.

that margarine

contain not

less

V i t a m i n s A and e s t e r s , D, E and esters and other v i t a m i n s m a y be added and it

is recommended that maximum and minimum levels be established under national
legislation.
Sodium chloride, sugars and suitable edible proteins may also be
added and milk or milk products may be used in manufacture.
The following may
be added, the amount not being limited, otherwise than by good manufacturing
practice:
beta-carotene, annatto, curcumin, canthaxanthin, beta-apo-81c a r o t e n a l , m e t h y l and ethyl esters of b e t a - a p o - 8 1 - c a r o t e n o i c acid, natural
f l a v o u r s and their identical synthetic e q u i v a l e n t s (except those w h i c h are
known to represent a toxic hazard), other synthetic flavours approved by Codex
A l i m e n t a r i u s C o m m i s s i o n , m o n o - and d i g l y c e r i d e s of fatty acids, citric
d i g l y c e r i d e s of fatty acids, citric and lactic acids and their p o t a s s i u m and
sodium salts, L-tartaric acid and its sodium and sodium-potassium salts, sodium
h y d r o g e n c a r b o n a t e , sodium carbonate and sodium h y d r o x i d e , natural and
synthetic tocopherols.
Recommended

limits have been set for the following added

Additives

substances:

Maxiaim level of use

Mono- and diglycerides of fatty acids esterified

with the following acids: acetic, acetyltartaric,
citric, lactic, tartaric and their sodium and
calcium salts
Polyglycerol esters of fatty acids

10 g/kg
5 g/kg

1,2-propylene glycol esters of fatty acids

20 g/kg

Esters of fatty acids with polyalcohols other

than glycerol, such as sorbitan monopalmitate,
sorbitan monostearate, sorbitan tristearate

10 g/kg

Sucrose esters of fatty acids

10 g/kg

Preservatives
Sorbic acid and its sodium, potassium and
calcium salts
Benzoic acid and its sodium and potassium salts

1,000 mg/kg individually or in combination .

Antioxidants
Propyl, octyl, and dodecyl gallates
Butylated hydroxytoluene (BHT)
Butylated hydroxyanisole (BHA)

100 mg/kg individually or in combination

Ascorbyl

palmitate

Ascorbyl

stearate

200 mg/kg individually or in combination

Antioxidant

Synergists

Isopropyl citrate mixture

100 mg/kg

284

MaxBUIB

Metal Contaminants

Level

Iron (Fe)

1.5 mg/kg

Copper

0.1 mg/kg

Lead

(Cu)

0.1 mg/kg

(Pb)

Arsenic

0.1 mg/kg

(As)

ROUTINE ANALYSIS
Margarine may be analyzed by the methods used for butter. Determination of the
RPK values will enable assessment to be made of the amount of butterfat added,
if any. (See methods under 2.4 of this Manual).
CAC/RS 32-1969 details the AM C (1959) m e t h o d for v i t a m i n E, using paper
chromatography to separate the different tocopherols.
The method of Christie
et al (20) is easier to use, although the c h r o m a t o g r a p h y m a t e r i a l s specified
must be used.
Interference by dimers is unusual in practice.
Rancidity can be assessed by determining the free fatty acids (FFA) in the fat
portion of the m a r g a r i n e .
Freshly m a n u f a c t u r e d m a r g a r i n e should have FFA
values of about 0.16% calculated as oleic acid.

285

MOHOGLTCERISES IN MARGARINE
PRINCIPLE
This method determines the amount of 1-monoglycerides, conventionally expressed
as g l y c e r y l m o n o s t e a r a t e , p r e s e n t in a fatty m a t e r i a l such as m a r g a r i n e .
O x i d a t i o n of the m o n o g 1 y c e r i d e s by p e r i o d i c acid is the b a s i s of the m e t h o d ,
w h i c h is v a l i d in the p r e s e n c e of free g l y c e r o l .
Dry f i l t e r e d fat should be
taken for the examination.
APPARATUS
1.

Graduated

flasks, 100 ml

2.

Stoppered

flasks, 500 ml

3.

Stoppered

flask, 2 L.

REAGENTS
1.
Periodic acid solution:
weigh out 5.4 g of periodic acid into a
2 L glass-stoppered flask and dissolve in 100 m l of distilled water;
add to this a q u e o u s s o l u t i o n 1900 m l of g l a c i a l acetic acid.
Mix
thoroughly and keep away from the light.
2.

Aqueous

dissolved

in

citric

acid

solution:

Chloroform.

4.

Aqueous solution of potassium

6.

Aqueous

w/v

citric

acid

crystals

water.

3.

5.
Aqueous
standardized.

2%

sodium

starch

iodide, 15% w/v.

thiosulphate

indicator

solution

0.1

solution, 1% w/v.

PROCEDURE
The s a m p l e m u s t be h o m o g e n e o u s and to e n s u r e h o m o g e n e i t y the fat
s h o u l d be m e l t e d and v i g o r o u s l y s h a k e n . If the s a m p l e is solid but
a p p a r e n t l y h o m o g e n e o u s it should be l i q u i f i e d at a t e m p e r a t u r e not
e x c e e d i n g 10C a b o v e its m e l t i n g p o i n t .
Do not take a p o r t i o n for
a n a l y s i s u n t i l the w h o l e of the s a m p l e has b e e n l i q u i f i e d and w e l l
shaken.
The amount to be taken for analysis can be determined in the
light of its probable 1-monoglyceride content as shown in the table
below :

Presumed content of
1-monoglyceride present
in the sample (Z w/w)

Weight of the portion

to be taken for assay
(g)

100
75
50
40
30
20

0.3
0.4

0.6
0.7

1.0
1.5
3.0
6.0

10
5
3 or less

10.0

286

Weigh out the quantity for analysis with an accuracy of 0.1 percent,
and t r a n s f e r it q u a n t i t a t i v e l y i n t o a 100 m l g r a d u a t e d f l a s k u s i n g
successive small quantities of chloroform to aid the transfer until
the v o l u m e r e a c h e s a b o u t 50 m l .
Add 25 m l of a q u e o u s c i t r i c acid
s o l u t i o n in o r d e r to d e c o m p o s e any soap that m a y be p r e s e n t , s h a k e
vigorously for 1 m i n u t e and allow it to stand.
A f t e r the m i x t u r e has s e p a r a t e d into t w o p h a s e s r e m o v e the
supernatant aqueous phase as completely as possible and transfer it
to a n o t h e r 100 m l g r a d u a t e d flask.
(The w a s h i n g of the c h l o r o f o r m
solution m a y be effected by a suitable contrivance which will allow
the w a s h i n g to be c a r r i e d out w i t h e a s e and w i t h o u t any r i s k of
losing any of the aqueous or chloroform phases.)
Repeat the washing
of the c h l o r o f o r m p h a s e t w i c e m o r e , e i t h e r w i t h 25 m l of w a t e r or
w i t h 25 m l of the a q u e o u s c i t r i c a c i d s o l u t i o n w h e n e m u l s i o n s are
formed.
Combine the aqueous solutions in the second 100 ml flask and
fill up to the 100 m l m a r k with distilled water.
Make up a chloroform solution remaining in the first graduated flask
to t h e 100 m l m a r k w i t h c h l o r o f o r m .
P i p e t t e e x a c t l y 50 m l of
p e r i o d i c acid s o l u t i o n and 50 m l of the c h l o r o f o r m s o l u t i o n into a
500 m l glass-stoppered flask.
Shake for a few m i n u t e s , stopper the
flask and allow to stand away from light at room temperature for 30
minutes.
C a r r y o u t a b l a n k test u n d e r the s a m e c o n d i t i o n s u s i n g 50
ml of the periodic acid solution and 50 ml of chloroform.
A f t e r the 30 m i n u t e p e r i o d add a b o u t 20 m l of a q u e o u s p o t a s s i u m
i o d i d e s o l u t i o n b o t h to the s a m p l e and to the b l a n k . A l l o w t h e m to
s t a n d for a f u r t h e r m i n u t e .
Add a b o u t 100 m l of d i s t i l l e d w a t e r to
each.
T i t r a t e w i t h the 0.1 N s o d i u m t h i o s u l p h a t e s o l u t i o n w h i l e
shaking continuously in order to ensure thorough mixing.
Towards the
end of titration, add about 2 ml of starch solution as indicator and
t h e n c o n t i n u e t i t r a t i n g d r o p by d r o p w i t h v i g o r o u s a g i t a t i o n u n t i l
the end-point is reached.
The n u m b e r of m l used to t i t r a t e the s a m p l e s h o u l d not be less than
80 p e r c e n t of the n u m b e r of m l r e q u i r e d for the t i t r a t i o n of the
blank.
If this condition is not fulfilled, repeat the determination
using a smaller quantity for analysis, to ensure that the oxidation
of the 1-monoglycerides is complete.
CALCULATION
T h e p e r c e n t a g e of 1 - m o n o g 1 y c e r i d e s
monostearate, M W 358)
=

35.8

(calculated

as

glyceryl

(a-b) N
P

Where :
a =

n u m b e r of m l of 0.1 N s o d i u m
for the blank test

thiosulphate

solution

utilized

b =

n u m b e r of m l of 0.1 N s o d i u m
for the test sample

thiosulphate

solution

utilized

N =

the e x a c t
solution

p =

weight

normality

of

the

in g taken for the. determination.

REFERENCE
IUPAC 2.322

aqueous

(1979).

287

sodium

thiosulphate

VITAMIN A II MARGARINE
PRINCIPLE
The margarine is saponified and the unsaponifiable material is extracted and
eluted t h r o u g h an a d s o r p t i o n c o l u m n .
This separates the V i t a m i n A and
carotenes which are determined spectrophotometrically.
APPARATUS
1.

Spectrophotometer capable of readings at 325 nm and 450 nm.

2.
Chromatographic tubes 12 m m OD x 90 m m long with stem on lower
end. The tube can be fitted with a medium porosity glass frit at the
bottom, or a pledget of glass wool can be used.
3.

Vacuum source

(a water aspirator vacuum would

suffice.)

4.
Long w a v e l e n g t h (300 n m ) u l t r a v i o l e t light source for v i e w i n g
bands (use with caution).
REAGENTS
1.

Potassium hydroxide solution, 50% w/w (780 g/L).

2.
Ethanol, absolute and 95%. Check spectral purity by determining
a b s o r b a n c e at 300 nm in a 1 cm cell against water. It should not
e x c e e d 0.05.
3.
Ethyl ether, peroxide-free.
last 10% of distillate.

Distil fresh and discard

first and

4.
Petroleum ether (BR 30-60).
Check transmittance (T) at 300 nm
in a 1 cm cell against air. T should be greater than 85%.
5.

Eluting solution I - 16% ethyl ether in petroleum ether.

6.
E l u t i n g s o l u t i o n II - 25% ethyl ether in p e t r o l e u m ether.
(Note: dry both I and II w i t h anhydrous sodium sulphate and store
over bright copper metal turnings to prevent peroxide formation).
7.

Eluting

solution III - 10% absolute ethanol in petroleum

ether.

8.
Sodium sulphate, anhydrous.
Check acidity by dissolving 10% in
water. The solution must not be acidic to methyl red indicator.
9.
Alumina. Check mesh size as follows: All should pass a 60 mesh
s i e v e , up to 20% can be retained on a 100 m e s h , about 50% should be
retained by a 160 mesh and the rest should pass through. Activate the
a l u m i n a by h e a t i n g for 3 hours at 600C. After cooling add w a t e r
dropwise to a weighed amount until the alumina contains 3% m/m water.
Keep in a tightly closed container.
10. A l k a l i n e a l u m i n a - mix a portion of the dry activated a l u m i n a
w i t h an equal w e i g h t of 10% w / w KOH solution in a dish. Decant and
discard the e x c e s s liquid.
Dry overnight at 100C.
Crush dried
material and pass through a 60 mesh sieve. Take a weighed amount and
add w a t e r d r o p w i s e until 3 % m / m water has been added. Store in a
tightly capped bottle.

288

PROCEDURE
Weigh 10 +_ 0.1 g margarine. Add 75 ml absolute ethanol and 15 ml 50%
KOH solution.
S a p o n i f y by b o i l i n g 5 m i n u t e s .
Let cool for 20
minutes.
Transfer the cooled solution to a separatory funnel using about 100
m l total of w a t e r , for w a s h i n g s .
Add 100 e t h y l ether and shake to
extract.
Transfer the aqueous phase to another separatory funnel and
re-extract with four 50 ml portions of ethyl ether.
Combine all of
the ether e x t r a c t s and w a s h w i t h t w o 100 ml p o r t i o n s of w a t e r .
Discard washings.
Dry the c o m b i n e d e t h e r e x t r a c t s w i t h a n h y d r o u s
sodium sulphate.
Evaporate the combined dried ether extract on a steam bath to about
25 m l . T r a n s f e r to a 50 m l b e a k e r and c o n t i n u e e v a p o r a t i o n u n t i l a
viscous, oily material remains.
Remove from the steam bath and evaporate the last traces of solvent
u s i n g n i t r o g e n gas.
D i s s o l v e the o i l y m a t e r i a l in 5 m l p e t r o l e u m
ether and t r a n s f e r to a 10 ml v o l u m e t r i c flask.
M a k e to the m a r k
with petroleum ether.
This is the sample solution.
P r e p a r e a c h r o m a t o g r a p h i c c o l u m n by p a c k i n g (gravity and s l i g h t
tapping on the sides) 1 cm of the damp alumina, then 2 cm of alkaline
a l u m ina and f i n a l l y a n o t h e r 4 cm of the d a m p a l u m i n a *
P l a c e the
column exit stem through a stopper into a vacuum flask with side arm.
Attach to a vacuum source and control using a pinchcock.
Apply vacuum to the column and add 5 ml petroleum ether.
Next add by
p i p e t t e 5 m l s a m p l e s o l u t i o n and a n o t h e r 5 m l p e t r o l e u m ether.
As
the last of the p e t r o l e u m e t h e r e n t e r s the c o l u m n top, add 5 m l of
eluting solution I. Continue adding 5 ml portions of I until all of
the carotene has eluted from the column.
This can be noted when the
e l u a t e is no longer y e l l o w i s h .
C o m b i n e all of the c a r o t e n e e l u a t e
and save for further analysis.
Next, continue adding 5 ml portions of eluting solution I and monitor
the band of Vitamin A as it progresses down the column, using the UV
l a m p ( V i t a m i n A is f l u o r e s c e n t under UV). D i s c a r d the e l u a t e after
the carotene and before the Vitamin A begins to move.
The Vitamin A
should elute in less than 20 minutes.
C o n t i n u e e l u t i o n u n t i l no f l u o r e s c e n c e
eluate.
Combine all Vitamin A eluates.

is

noted

in

1 ml

of

the

E v a p o r a t e the c a r o t e n e and V i t a m i n A e l u a t e s s e p a r a t e l y on a s t e a m
bath to about 2 ml each.
Cool and remove the remaining solvent from
e a c h u s i n g n i t r o g e n or a v a c u u m .
D i s s o l v e the c a r o t e n e in 5 m l
p e t r o l e u m ether and t r a n s f e r to a 10 m l v o l u m e t r i c flask. M a k e to
the m a r k w i t h p e t r o l e u m ether.
D i s s o l v e the V i t a m i n A in 5 m l
absolute ethanol.
Transfer to a 10 ml volumetric flask and make to
the mark with absolute ethanol.
D e t e r m i n e the a b s o r b a n c e of the c a r o t e n e s o l u t i o n in a 1 cm cell at
450 nm using petroleum ether as the reference.
Similarly, determine the absorbance of the Vitamin A solution at 310
nm and 325 nm using absolute ethanol as the reference.
The ratio of
the absorbances at 310 and 325 should be 1 or less.

289

CALCULATION

Vitamin A m

,
\i / g

A,oc x 5.5
_JL2
g sample

Vitamin A in Intnl. Units/g

Carotene in

y/g

A-i 9 C x 18.3
-1
g s am p 1 e

A/, cn x 4.17
"
g sample

,
Carotene in Intnl. Units/g

Acn x 6.95
,
g sample

REFERENCES
Analtyical Methods Committee, 1964.

The Analyst 89_, 7.

Official Methods of Analysis of the AOAC, 1984. 43.001

- .007.

290

9.3

A N I M A L FAT

COMPOSITION
The recommended International General Standard for Edible Fats and Oils (CAC/RS
19-1969) defines edible oils and fats as:
foodstuffs composed of glycerides of
fatty acids of vegetable, animal or marine origin.
Fats of animal origin m u s t
be produced from animals in good health at the time of slaughter and be fit for
h u m a n consumption as determined by a competent authority recognized in national
legislation.
T h e y m a y c o n t a i n s m a l l a m o u n t s of o t h e r
l i p i d s such as
phosphatides, of unsaponifiable constituents and of free fatty acids naturally
present in the fat or oil.
The Codex recommended

Animal Pat

standards

for animal

fats

are:

Unsapon- Acid
Peroxide
ifiable
Value
value
SaponDensity Refractive ification Iodine m a t t e r
mg KOH/g meq 0/kg
(max)
(max )
(max)
Index
Value
Value
20/20 o

Lard
(400-200 )

0.8960.904

1.4481.460

192203

4570

10

1.3

10

Rende red
pork fat
(40-20 )

0.8940.906

1.4481.461

192203

4570

12

2.5

16

Edible
tallow
(40-20)

0.8930.904

1.4481.460

190202

3250

12

2.5

16

1.4481.460

190200

3247

10

10

Premier Jus . 0.8930.898

(40-20)
The

titres of animal fats must be within the

Pat

following ranges :
Titre

Lard
Rendered Pork Fat
Edible Tallow
Premier Jus

32 - 45C
32 - 45C
40 - 49C
42.5 - 47C

L a r d m a y c o n t a i n r e f i n e d l a r d , lard s t e a r i n e and h y d r o g e n a t e d lard p r o v i d e d

this is declared on the label, but the r e c o m m e n d e d standard does not apply to
refined lard.
Rendered pork fat m a y contain fat from bones, ears, tails, etc.
w h i c h lard m a y not.
The r e c o m m e n d e d s t a n d a r d d o e s n o t a p p l y to r e f i n e d
r e n d e r e d p o r k f a t , but the a r t i c l e m a y c o n t a i n r e f i n e d lard or r e n d e r e d p o r k
fat, hydrogenated or not, lard stearine and rendered pork fat stearine as long
as t h e s e are d e c l a r e d on the l a b e l . E d i b l e t a l l o w ( s y n o n y m : d r i p p i n g ) is the
p r o d u c t o b t a i n e d by r e n d e r i n g the c l e a n , s o u n d f a t t y t i s s u e s
(including
t r i m m i n g and cutting fats), attendant muscles and bones of bovine animals (Bos
t a u r u s ) a n d / o r s h e e p (Ovis ar ie s) in g o o d h e a l t h at t i m e of s l a u g h t e r and fit
for h u m a n consumption as determined by an authority recognized as competent in
national legislation.
P r e m i e r Jus ( S y n o n y m : O l e o S t o c k ) is the p r o d u c t
o b t a i n e d by r e n d e r i n g at l o w h e a t the f r e s h fat ( k i l l i n g f a t ) or h e a r t , c a u l ,
k i d n e y and m e s e n t e r y c o l l e c t e d at t i m e of s l a u g h t e r of b o v i n e a n i m a l s (Bos
t a u r u s ) in good h e a l t h at t i m e of s l a u g h t e r and fit for h u m a n c o n s u m p t i o n as
d e t e r m i n e d b y an a u t h o r i t y r e c o g n i z e d as c o m p e t e n t in n a t i o n a l l e g i s l a t i o n .
The raw material does not include cutting fats.

291

FREE FATTY ACIDS

PRINCIPLE
Animal tissues contain lipase enzymes which have the ability to hydrolyze fats,
s p l i t t i n g fatty a c i d s from the g l y c e r o l m o l e c u l e s . The extent to w h i c h this
has o c c u r r e d can be d e t e r m i n e d by the free fatty acid (FFA) content of the
animal fat by placing in solution and titration.
APPARATUS
1.

Steam bath.

2.

Drying

3.

Burette.

oven.

REAGENTS
1.

Chloroform.

2.

Ethanol, 95%.

3.

0.02 N sodium hydroxide solution, accurately

4.

Phenolphthalein solution 1%.

5.

Anhydrous sodium

standardized.

sulphate.

PROCEDURE
W e i g h 50 g of fat and m a c e r a t e with 200 ml c h l o r o f o r m . (Do not use
heat).
Filter through a paper containing anhydrous sodium sulphate.
Collect the filtrate in a stoppered flask.
Pipette 20 ml of the filtrate and evaporate the chloroform on a steam
bath. Dry in an oven at 100C for 3 hours. Cool in a d e s i c c a t o r and
(This d e t e r m i n e s the fat content of the filtrate
w e i g h the fat.
solution).
P i p e t t e 20 ml of the filtrate into a 250 ml flask.
Add 20 ml of
neutralized ethanol.
Titrate with 0.02 N sodium hydroxide solution
using phenolphthalein indicator.
CALCULATION
FFA (as % oleic acid in the fat)
Where:

V x N x 28.2
W

V = ml of NaOH used
N = normality of NaOH
W = g fat per 20 ml filtrate

INTERPRETATION
In good

quality animal

fat the FFA should not exceed 0.5% as'oleic acid.

REFERENCE
Official and Tentative Methods of the American Oil Chemists' Society, Ca 5a-40.

292

THIOBARBITURIC CID VALUE

PRINCIPLE
O x i d i z e d l i p i d s are f o r m e d as fats b e c o m e r a n c i d . T h i o b a r b i t u r i c (TBA) acid
w i l l r e a c t w i t h t h e s e l i p i d s to f o r m a r e d - c o l o u r e d c o m p l e x w h i c h can be
determined spectrophotometrically.
Malonaldehyde is one of the end products of
oxidative rancidity, and is believed to be involved in the reaction with TBA.
Therefore, the TBA value is expressed as rag malonaldehyde per kg sample.
The
TBA test is applicable to fatty foods (e.g. meat) as well as fats and oils.
APPARATUS
1.

Distillation

apparatus

2.

Glassbeads.

3.

Electric

4.

Pipette.

5.

Glass stoppered

6.

Spectrophotometer.

(flask, condenser, receiver).

mantle.

test

tube.

REAGENTS
1.

Hydrochloric

2.

Antifoam

acid, 4N.

liquid.

3.
Thiobarbituric acid reagent:
glacial acetic acid.

dissolve 0.2883 g in 100 ml of 90%

PROCEDURE
Macerate 10 g sample with 50 ml water for 2 minutes
to the distillation flask, using 47.5 ml water for
m l 4N HC1. (pH should be 1.5). Add a n t i f o a m and a
D i s t i l at a r a t e so that 50 m l of d i s t i l l a t e is
minutes from the time boiling commences.

and then transfer

washing.
Add 2.5
few glass b e a d s .
c o l l e c t e d in 10

Pipette 5 ml of the distillate into a glass-stoppered tube. Add 5 ml

TBA reagent.
Shake and heat in boiling water for 35 minutes.
Prepare a blank similarly, using 5 ml water, for 35 minutes.
Cool the s a m p l e and b l a n k tubes and m e a s u r e the a b s o r b a n c e of the
sample against the blank at 538 nm using 1 cm cells.
CALCULATION
TBA value

(as mg malonaldehyde per kg sample) = 7.8 x A

(A = absorbance of sample against

(Caution:
The method
be valid.)

must be

followed

blank)
exactly

for the 7.8 factor

to

REFERENCE
T a r l a d g i s , B.G., W a t t s , B.M., Y o u n a t h a n , M.T. and
the American Oil Chemists' Society _3_7, 44.

293

D u g a n , L., 1960. J o u r n a l

of

9.4

TEXT REFERENCES

1.

A M E R I C A N OIL C H E M I S T S SOCIETY.
Official & Tentative Methods of the
A m e r i c a n Oil C h e m i s t s ' Society 508 S. 6th Street, Champaign, Illinois
61820.

2.

IUPAC.
1979.
Standard Methods of the Analysis
Derivatives 6th Edition, Butterworths, London.

3.

CHRISTIE, W.W.

4.

B O C K E N O O G E N , H.A. 1964. Analysis & Characterisation of Oils, Fats and

Fat Products Vol I and II and 1968 Interscience.

5.

PARODI, P.W.
534.

6.

HART, F.L. & FISHER, H.J.

7.

CONACHER, H.B.S. & CHADHA, R.K.

Chemists Society 57 (5) 1161.

8.

CONACHER, H.B.S.
(3) 488.

9.

A C K M A N , R.G. et al 1977. Technical Report No. 57?, Fisheries and Marine

Service Halifax Laboratory, Halifax, Nova Scotia.

10.

COCKS, L.V., CHRISTIAN, B.C. & HARDING, G.

11.

COCKS, L.V. & Van REDE, C.

1966. Laboratory
Analyses Academic Press 1966.

12.

ALLEN, R.R. 1969. Journal of the American Oil Chemists '.Society 46 552.

13.

H U A N G , A. & F I R E S T O N E , D.
Official Analytical Chemists.

14.

SYNODINES, E. & KOVITAS, 1958. 408 Chim Chronia 23 21.

15.

M A T A R E S E , L. 1962.
429 1963.

16.

HERZINGER, V. & PAZLAR, M. 1975.

17.

M E H L E N B A C H E R , V.C. 1960. The

Press, Champaign, Illinois.

18.

WILLIAMS, K.A. 1966. Oils, Fats and Fatty Foods Churchill, London.

19.

T A R L A D G I S , B.G., W A T T S , B.M. & Y O U N A T H A N , M.T.

Journal of the Amrican Oil Chemists' Society 37 44.

20.

CHRISTIE, A.A., DEAN, A.C. & MILBURN, B.A. 1973. Analyst' 98 1164.

1973.

1976.

of

Oils,

Fats

and

Lipid Analysis Pergamon Press.

Journal of the American Oil Chemists Society 53 530-

1975.

1971.

Modern Food Analysis,

1974.

Journal

of

Springer-Verlag.
the

American

Oil

Journal of the American Oil Chemists Society 58

1971.

1931.

Journal

Analyst 56 368.

Handbook

of

the

for Oil

and

Association

Fat

of

Rivista Italianna delle Sostanze Grasse 39 74 and 40

Prumysl Potravin 26 (8) 473-474.

Analysis

of

Fats

and

Oils

The

& DUGAN,

Garrard

L.

1960.

Further Reading
D i e t a r y Fats and Oils

in Human Nutrition, FAO Food & Nutrition Paper No. 3

1977.
PATTERSON, H.B.W. 1983. Hydrognation of Fats & Oils. Applied

Science.

HScience
A M I L T O.N , R.J. & BHATI, A. 1980. Fats & Oils: Chemistry & Technology, Applied
294

ALLEN, J.C. & HAMILTON, R.J. 1983. Rancidity in Foods, Applied

PERKINS, E.G.

(Ed)

1978.

Analysis of Lipids & Lipoproteins.

WITTING, L.A.

(Ed)

1978.

Glycolipid Methodology

Official & Tentative Methods of Analysis, AOCS,

Illinois 61820
U.S.A.

295

Science.

AOCS.

508 South 6th

St.,

Champaign,

10.
10.1

BEVERAGES

ALCOHOLIC - DISTILLED

COMPOSITION
The five most common distilled spirits are brandy, gin, rum, vodka and whisky.
Brandy is distilled from the fermented juice of grapes or other fruits. Gin is
a diluted spirit flavored with juniper berries or other plant extracts. The
basic spirit is usually made from maize and rye with malted barley. Vodka is a
diluted spirit usually made from potato, wheat or other grains, with no added
flavouring.
Rum is a spirit distilled from f e r m e n t e d sugar cane juice or
molasses.
Whisky is generally meant to be Scotch Whisky but does include the
U. S. b o u r b o n w h i s k i e s .
C o m p o s i t i o n data for some distilled spirits are as f o l l o w s (note that these
date are ranges or approximations only, as composition can vary widely):
(Data
compiled from Pearson's Chemical Analysis of Food, 8th Ed.)

Ash I

Acidity Z
(as acetic)

Total
Esters
(*)

Furfural
(*)

Aidehydes
(*)

Higher
Alcohol
(*)

0.1-1.9

.004-.012

.041-.072

1.1-5.8

.02-.06

.24-.76

1.5-7.8

Gin

.01-.09

.008-.009

.015-.020

Rum

.29-.42

.025-.041

.082-.102

1.9-2.7

.07-.10

.24-.35

1.6-1.9

.030-.070

0.1-0.3

0.1-0.2

1.0-2.7

0.7-4.5

Spirit

Total
Solids!

Brandy

Whisky**

* expressed as g/L of ethanol

** Scotch whisky.

ROUTIHE ANALYSIS
D i s t i l l e d s p i r i t s should be e x a m i n e d for ethanol and total solids (extract).
Ethanol is normally over 37 percent v/v in spirits.
It may also be necessary
to e x a m i n e for ash, acidity, t a n n i n s , esters, furfural and other a l d e h y d e s ,
ketones, higher alcohols, methanol and isopropanol.
Hart and Fisher (1) give
s o m e a n a l y s i s for A m e r i c a n w h i s k i e s .
See also "Standard M e t h o d s of W h i s k y
A n a l y s i s " p u b l i s h e d by Scotch W h i s k y A s s o c i a t i o n , U.K. and the Reports of the
Research Committee on the analysis of Potable Spirits.
Liqueurs are usually sweetened and flavoured distilled spirits. The flavouring
components and other materials may interfere in the estimation of ethanol, in
w h i c h case one of the m e t h o d s of the BP. 1968, p. 1278 m a y be used.
The
p r e s e n c e of i n t e r f e r i n g substances is inferred w h e n the refractive index
reading of the distillate does not correspond to the specific gravity according
to the following table:

Specific Gravity
20/20
0.9710
0.9720
0.9730
0.9740
0.9750
0.9760
0.9770

Refractive
Index 20

Specific Gravity
20*/20*

1.34661
1.34605
1.34549
1.34493
1.34437
1.34380
1.34324

0.9860
0.9870
0.9880
0.9890
0.9900
0.9910
0.9920

296

Refractive
Index 20
1.33842
1.33796
1.33751
1.33705
1.33663
1.33620
1.33578

Specific Gravity
20/20
0.9780
0.9790
0.9800
0.9810
0.9820
0.9830
0.9840
0.9850

Refractive
Index 20 s

Specific Gravity
20/20*
0.9930
0.9940
0.9950
0.9960
0.9970
0.9980
0.9990
1.0000

1.34267
1.34211
1.34154
1.34098
1.34044
1.33991
1.33942
1.33892

Refractive
Index 20*
1.33540
1.33501
1.33466
1.33432
1.33397
1.33362
1.33331
1 .33300

The BP methods require that the sample be distilled and diluted to four times
its v o l u m e ( q u a d r u p l e bulk), so the use of the table w i l l r e q u i r e d i l u t i o n of
most liqueur distillates.
Both determination of SG and RI must be done at 20C
(or c o r r e c t e d t h e r e t o ) and the RI should not d i f f e r by m o r e than 0.00007 from
the v a l u e c o r r e s p o n d i n g to the s p e c i f i c g r a v i t y .
If n e c e s s a r y , the RI at
temperatures other than 20C can be related to the SG via AOAC tables.
Liqueur
chocolates should be steam-distilled prior to the determination of ethanol.

297

ETHANOL
PRINCIPLE
T h e s a m p l e is d i s t i l l e d and the s p e c i f i c g r a v i t y of the d i s t i l l a t e
the p r o p o r t i o n of alcohol being calculated from tables.

measured,

APPARATUS
1.
Distillation
flask with
'revenue' or West condenser.
2.

Specific

gravity

bottle or

an

efficient

condenser,

such

as

pyconometer.

PROCEDURE
General note:
The distillation m u s t be carried out under conditions
that do not incur loss of alcohol.
For example, the A m e r i c a n Society
of B r e w i n g C h e m i s t s method specifies that the condenser water must be
25C and the receiving flask should be surrounded w i t h ice or ice and
water.
A l l d e t e r m i n a t i o n s of g r a v i t y are m o s t c o n v e n i e n t l y and
a c c u r a t e l y c a r r i e d out at 20C and t h e r e f o r e the v o l u m e of s a m p l e
taken
initially
s h o u l d be at 20C but if this is
impossible,
temperature c o r r e c t i o n s m u s t be m a d e .
It is e s s e n t i a l
that
d i s t i l l a t e s be d i l u t e d to v o l u m e (or a m u l t i p l e of it) at the s a m e
t e m p e r a t u r e as that at w h i c h the sample was measured initially.
50 ml of sample is washed into a 100 ml distillation
Spirits;
flask w i t h not m o r e than 5 m l of w a t e r , distilled and the distillate
d i l u t e d to 50 m l .
T h e s p e c i f i c g r a v i t y is d e t e r m i n e d .
(See R C A P S
(2)).
The specific gravity d e t e r m i n e d without distillation does not
give seriously erroneous results for products such as vodka in which
the t o t a l s o l i d m a t t e r is s m a l l .
T h e solid m a t t e r i n c r e a s e s the
g r a v i t y and t h e r e f o r e the a l c o h o l p e r c e n t a g e c a l c u l a t e d from the
specific gravity without distillation is lower than the true value.
T h i s d i f f e r e n c e b e t w e e n the a p p a r e n t and true a l c o h o l v a l u e s is
referred to as the 'obscuration'.
This m a y be determined by dilution
of the a l c o h o l - f r e e r e s i d u e r e m a i n i n g f r o m d i s t i l l a t i o n to the
o r i g i n a l s a m p l e v o l u m e and d e t e r m i n a t i o n of the s p e c i f i c g r a v i t y .
S u b t r a c t 1 f r o m the S.G. and s u b t r a c t the r e s u l t f r o m the s p e c i f i c
gravity of the s a m p l e (obtained w i t h o u t distillation).
This gives a
result corresponding
to the t r u e a l c o h o l c o n t e n t .
I n s t e a d of
carrying out the d e t e r m i n a t i o n , it is s o m e t i m e s assumed that each 1
percent m / v of extract (total solids by drying to constant weight at
100C) increases the specific gravity by 0.0041.
In c o n t r a s t to p u r e s p i r i t s , s o m e l i q u e u r s m a y c o n t a i n s u f f i c i e n t
v o l a t i l e m a t t e r o t h e r t h a n a l c o h o l to g i v e an i n c o r r e c t v a l u e .
In
c a s e s of d o u b t , the s p e c i f i c g r a v i t y and r e f r a c t i v e i n d e x are
c o m p a r e d and the d i s t i l l a t e a c c e p t e d as of a d e q u a t e p u r i t y if the
values correspond to the same percentage of alcohol.
If not, proceed
by the method of the British P h a r m a c o p o e i a (1968).
Wines:
The OIV reference method requires that a 1 L flask and a
20 cm condenser should be used.
The equipment chosen m u s t be capable
of giving 99.8 percent recovery of alcohol distilled from it.
De-gas
the w i n e if necessary by shaking in a flask of about twice the v o l u m e
of l i q u i d .
M e a s u r e 2 0 0 m l or o t h e r s u i t a b l e v o l u m e into the
d i s t i l l a t i o n flask, rinsing the graduated flask with 4 x 5 ml water.
Add 10 ml of a 12% suspension of calcium oxide.
For very acid wines
add extra l i m e suspension until the w i n e becomes alkaline as shown by
p h e n o 1 ph tha 1 e in u s e d as an e x t e r n a l i n d i c a t o r .
Add p o r o u s pot and
distil into the flask in w h i c h the sample w a s measured after placing
a b o u t 10 m l of w a t e r in the f l a s k .
E n s u r e that the o u t l e t to the

298

c o n d e n s e r is i n s e r t e d w e l l into the f l a s k .
Distil about threequarters of the initial volume.
Wash out the distillation flask, and
r e t u r n the d i s t i l l a t e to it, r i n s i n g w i t h 4 x 5 m l of w a t e r as
before.
Add porous pot and 1 m l of 10 % sulphuric acid and re-distil
into the s a m e v o l u m e t r i c flask c o n t a i n i n g a b o u t 10 m l of w a t e r .
Dilute the distillate to the m a r k , ensuring that the temperature is
within 2C of that at which the wine was measured initially.
Beer:
D e - c a r b o n a t e by p o u r i n g f r o m o n e b e a k e r to a n o t h e r
several times.
Measure 100 ml of beer and place in the distillation
f l a s k , r i n s i n g the g r a d u a t e d f l a s k w i t h a t o t a l of 50 m l of w a t e r
used in several portions.
Distil 96 ml at the uniform rate in 30-60
minutes.
D i l u t e to v o l u m e , m i x w e l l and d e t e r m i n e the s p e c i f i c
gravity.
Determination of Specific Gravity of the

Distillate

W e i g h an e m p t y s p e c i f i c g r a v i t y b o t t l e or p y c n o m e t e r , fill to the
m a r k with distillate avoiding inclusion of air and carefully wiping
the o u t s i d e of the b o t t l e if n e c e s s a r y , and w e i g h .
A d j u s t the
t e m p e r a t u r e to 20C and e n s u r e that the b o t t l e or p y c n o m e t e r is
exactly full or at the graduation mark at that temperature, or adjust
to the m a r k and d e t e r m i n e the t e m p e r a t u r e .
R e p l a c e the d i s t i l l a t e
with water and weigh, adjusting the temperature or determining it as
for the distillate.
If both
then :

distillate

Apparent

SG

and

water

were

measured

at

the

same

temperature,

weight of volume of distillate

weight of equal volume of water

The percentage of alcohol v/v, m / m or m/v at 20C from determinations

carried out at 20C m a y be calculated from tables such as that of the
A m e r i c a n S o c i e t y of B r e w i n g C h e m i s t s or by A O A C .
U.K C u s t o m s and
E x c i s e T a b l e s r e l a t e v a l u e s at 20C to t h o s e at 15.5C.. The O I V
tables enable determinations at other temperatures to be calculated
to 20C and t h o s e of the A O A C e n a b l e d e t e r m i n a t i o n s
at
other
temperatures to be calculated to 15.56C.
It i 3 g e n e r a l l y d e s i r a b l e to c a r r y o u t the d e t e r m i n a t i o n s w i t h the
t e m p e r a t u r e o f t h e d i s t i l l a t e a n d of t h e w a t e r at t h e
same
temperature.
If t h i s is n o t d o n e , the a p p a r e n t s p e c i f i c g r a v i t y of
the water m a y be adjusted to the temperature at which the distillate
was weighed, according to the water density table b e l o w :
e.g. weight of gravity bottle = 49.0000 g
weight of gravity bottle + distillate at 18C = 100.4320 g
weight of distillate at 18C = 51.4320 g
weight of gravity bottle + water at 22C = 101.3330 g
weight of water at 22C = 52.3330 g
density of water at 22C = 0.997801
density of water at 18C = 0.998625
water required
52.3330

apparent

to fill gravity bottle at 18C would

x

0.998625
0.997801

specific
=

52.3762

gravity of distillate

at 18C = 51.4320
5 2.3762

0.9810

alcohol % v/v at 20C

weigh

(OIV tables) = 12.70

299

W a t e r density table
Physics, 49th Ed.)

(from

Density

G.S.

Kell,

(g/l)

of

Chemistry

Density

23
24
25
26
27
28
29
30
31
32
33
34
35

0.999728
0.999634
0.999526
0.999406
0.999273
0.999129
0.998972
0.998804
0.998625
0.998435
0.998234
0.998022
0.997801

10
11
12
1-3
14
15
16
17
18
19
20
21
22

Handbook

and

(g/

0.997569
0.997327
0.997075
0.996814
0.996544
0.996264
0.995976
0.995678
0.995372
0.995057
0.994734
0.994403
0.994063

One d e g r e e G a y - L u s s a c c o r r e s p o n d s to o n e - l i t r e of e t h a n o l in 100
l i t r e s of w i n e , b o t h m e a s u r e d at 15C.
The I n t e r n a t i o n a l d e g r e e
(Convention Internationale,
1 9 5 7 ) is the p e r c e n t a g e by v o l u m e
expressed at 20C.
Jaulmes and Marignan (3) showed that there was no
s i m p l e r e l a t i o n b e t w e e n the t w o and s u g g e s t e d use of the f o l l o w i n g
table :
20*72020/2015/15
20/20"
15/15
15/15
20/20
15"/15
6
7
8
9
10
11
12
13
14
15
16
17

6.03
7 .03
8.04
9.04
10.04
11.05
12.05
13.05
14.06
15.06
16 .06
17.07

18
19
20
21
25
30
40
50
60
70
80
90

0.03
0.03
0.04
0.04
0.04
0.05
0.05
0.05
0.06
0.06
0.06
0.07

18.07
19.07
20.07
21.08
25.08
30.09
40.08
50.07
60.07
70.06
80.05
90.03

0.07
0.07
0.07
0.08
0.08
0.09
0.08
0.07
0.07
0.06
0.05
0.03

REFERENCES
O I V ( O f f i c e I n t e r n a t i o n a l de la V i g n e et du V i n ) R e c e u i l des M e t h o d e s
d'Analyse, T978. (Note: When methods are stated to follow OIV procedures, the
description of the details of the procedure is not to be considered an official
t r a n s l a t i o n f r o m the F r e n c h . The o r i g i n a l F r e n c h text should be r e f e r r e d to
for the dfinitive text of OIV recommended methods).
Methods of Analysis of the American Society of Brewing Chemists,
Bee,

H.M.,

Customs

1970. Journal

and

Excise

of the Association

Spirit

of Public

Tables, U.K., current

300

Analysts_8,

edition.

1976.
97.

METHANOL
PRINCIPLE
Methanol is oxidised to formaldehyde and the colour developed by reaction
chronotropic acid is measured at 570 nm.
APPARATUS
1.

Spectrophotometer.

REAGENTS
1.
C h r o m o t r o p i c a c i d , 4 , 5 - d i h y d r o x y - n a p h t h a 1 e n e - 2 , 7 - d i s u l phonic
a c i d , or the d i s o d i u m salt.
If it is n e c e s s a r y to p u r i f y it,
d i s s o l v e 10 g of acid or salt in 25 ml w a t e r , adding 2 ml of
c o n c e n t r a t e d s u l p h u r i c acid in the latter case, add 50 m l of
methanol, boil and filter. Add 100 ml isopropanol, allow to cool and
filter off the c r y s t a l s .
D i s s o l v e 0.05 g c h r o m o t r o p i c acid or salt
in 35 ml w a t e r , cool to 0C and s l o w l y w i t h s t i r r i n g add 75 ml of
Prepare fresh.
sulphuric acid.
2.

Standard methanol, 0.05% in 5% v/v ethanol. in water.

3.

Phosphoric acid, 50% m/v.

4.

Potassium permanganate 5% m/v.

5.
Sod ium su 1 phi te 2% m / v .
standard iodine solution.

C h e c k the s t r e n g t h by t i t r a t i o n

with

PROCEDURE
D i l u t e the d i s t i l l a t e from the d e t e r m i n a t i o n of e t h a n o l so that it
contains 5 percent ethanol.
To 0.5 m l of d i l u t e d d i s t i l l a t e in a
t e s t - t u b e add 1 drop of 50% p h o s p h o r i c acid and
2 drops of 5%
potassium
permanganate.
S h a k e a n d l e a v e to s t a n d
10 rain.
Decolourize the potassium permanganate with sodium sulphite solution,
avoiding an excess.
Add 5 ml of 0.05% chromotropic acid solution and
leave 20 minutes at 70C.
In a s e r i e s of 50 m l f l a s k s add 2.5, 5, 10, 15, 20 and 25 ml of the
standard 0.05% methanol solution.
Dilute to 50 ml with 5% ethanol in
water.
T h e s e s o l u t i o n s c o n t a i n 0.025, 0.05 etc. g m e t h a n o l per
litre.
T r e a t 0.5 m l of e a c h as for the s a m p l e .
D e t e r m i n e the
a b s o r b a n c e at 570 nm u s i n g the 0.5 ml of each as for the s a m p l e .
Determine the absorbance at 570 nm using the solution prepared from
0.5 ml of 5% e t h a n o l in w a t e r as a blank.
CALCULATION
From a standard curve, calculate
distillate diluted to 5 percent.

the

g of m e t h a n o l

g/L of methanol = g/L in 5% distillate x (% ethanol

per

litre

of

in sample)/5

REFERENCE
O f f i c i a l M e t h o d s of A n a l y s i s of the A O A C , 1984, 9.102-.105.
OIV (4) routine method.
The reference method uses GLC.

301

This is the

with

HIGHER ALCOHOLS
PRINCIPLE
T h e s a m p l e is d i s t i l l e d a f t e r a d j u s t m e n t of the e t h a n o l l e v e l to 40 p e r c e n t .
The higher alcohols are reacted w i y h 4-hydroxybenzaldehyde-3-sulphonic acid in
the p r e s e n c e of s u l p h u r i c a c i d (the K o m a r o w s k y r e a c t i o n ) and the c o l o u r
c o m p a r e d w i t h standards.

APPARATUS
1.
2.

Distillation

apparatus.

Spectrophotometer.

REAGEHTS
1.

Sodium 4 - h y d r o x y b e n z a l d e h y d e - 3 - s u l p h o n a t e , 4% m/v

2.
Mixed pentanols.
methylbutan-l-ol) .
3.

Standard

higher

4.

Prepare the

in water.

I s o a m y 1 a 1 c o h o 1 , A R or e q u i v a l e n t ( m a i n l y 3 -

alcohol

following

solutions.

mixtures:

7.5 ml of 2-methylpropan-l-ol
5.0 ml of 2-methylpropan-l-ol
2.5 ml of 2-methylpropan-l-ol

and 2.5 ml of mixed

and 5.0 ml of mixed
and 7.5 ml of mixed

pentanols
pentanols
pentanols

From each of these foregoing m i x t u r e s , and also from 2 - m e t h y l p r o p a n l - o l and f r o m m i x e d p e n t a n o l s p r e p a r e s o l u t i o n s of 5 g of ( t o t a l )

higher alcohols in 100 m l of 40% v/v aqueous ethanol.
Further dilute
each of the five solutions so obtained with 40% v/v aqueous ethanol
to g i v e f i v e s e t s of d i l u t e s t a n d a r d s o l u t i o n s of ( t o t a l ) h i g h e r
a l c o h o l s , e a c h set c o n t a i n i n g .25, .50, .75, 1.25 and 2.50 g of
(total) higher alcohols per litre of solution.

PROCEDURE
If necessary, dilute the sample with water or ethanol to an ethanol
c o n c e n t r a t i o n of 4 0 percent +_ 1% v/v. To 50 ml of the diluted sample
add about 20 ml of water and distil.
Collect at least 47 ml in a 50
m l f l a s k , m i x , d i l u t e to v o l u m e w i t h w a t e r and m i x w e l l .
Transfer
0.10 m l of e a c h d i l u t e s t a n d a r d of a s e t , and a l s o of a 4 0 % v/v
a q u e o u s e t h a n o l s o l u t i o n as a b l a n k , to a s e r i e s of six 10 m l
v o l u m e t r i c f l a s k s and t h e n add in o r d e r :
0.20 m l of s o d i u m 4 h y d r o x y b e n z a l d e h y d e - 3 - s u l p h o n a t e solution, and 2.0 m l of concentrated
sulphuric acid.
M i x by swirling.
Place the flasks in a cold water bath and heat the water to boiling.
Boil for 30 m i n u t e s .
Remove the flasks and allow them to cool.
Make
the v o l u m e to 10 m l w i t h c o n c e n t r a t e d s u l p h u r i c a c i d .
Mix well.
M e a s u r e the absorbance at 445 nm and at 560 n m , using the blank as a
r e f e r e n c e in a 1 cm cell. R e p e a t for each of the o t h e r four sets of
dilute standards.
P l o t a b s o r b a n c e at 4 4 5 nm a g a i n s t the h i g h e r
alcohol c o n c e n t r a t i o n for each set, to give a group of five straight
calibration lines.
M e a s u r e the s l o p e s of the l i n e s as g r a m s of
h i g h e r a l c o h o l s per l i t r e of s o l u t i o n per u n i t of a b s o r b a n c e .
D e t e r m i n e the average ratio of absorbance at 560 nm to absorbance at
4 4 5 nm for e a c h set of s t a n d a r d s .
P l o t the s l o p e s a g a i n s t the
absorbance ratios.
(Note t h a t the a b s o r b a n c e at 5 6 0 n m m a y be m u c h
higher. If so, dilute and re-read, and correct the final calculation
for this dilution).

302

To e s t i m a t e the total h i g h e r a l c o h o l s in the s a m p l e , p r o c e e d as in

the 'blank test' using duplicate 0.10 ml aliquots of prepared sample
and of any one of the 25 d i l u t e s t a n d a r d h i g h e r a l c o h o l m i x t u r e s .
Use 0.10 m l of 4 0 % v/v a q u e o u s e t h a n o l s o l u t i o n as the b l a n k .
Calculate the average of the results of each pair of measurements.
CALCULATION
Let A = absorbance at 445 nm of the prepared

sample

B = absorbance at 560 nm of the prepared

sample

P = a b s o r b a n c e at 445 nm of the s t a n d a r d
the same time as the prepared sample

when measured

at

R = a b s o r b a n c e at 445 nm of the s t a n d a r d w h e n m e a s u r e d
the time the standard lines were established.

at

Calculate the ratio B/A and from the plot of the slopes in the 'blank
test' find the slope corresponding.
Let this slope be S.
The higher
a l c o h o l c o n t e n t of the s a m p l e , H, is g i v e n by H = .025 (SAR/P) g r a m s
per litre of ethanol in the sample.
If the o r i g i n a l s a m p l e c o n t a i n s V% v/v of e t h a n o l , w h e r e V is less
t h a n 40, and e t h a n o l has b e e n added to r a i s e the s t r e n g t h , then H =
(SAR/VP) grams per litre of ethanol in the sample.
For some products the proportion of 2-methylpropan-l-ol in the higher
a l c o h o l s is f a i r l y c o n s t a n t and can be m a t c h e d by a single set of
s t a n d a r d s , i.e. the s l o p e , S, can be t a k e n a l w a y s to be the s a m e .
Typical ratios of pentanols to 2-methylpropan-l-ol are: malt whisky,
1.7:1;
blended whisky,
1:1;
rum 3:1; cognac brandy, 2.6:1.
REFERENCE
Research Committee on the Analysis of Potable Spirits (RCAPS),' 1970.
Journal
of the A s s o c i a t i o n of P u b l i c A n a l y s t s _ 8 , 81. The R C A P S r e p o r t s g i v e four GLC
methods.
The 1984 A O A C d e t e r m i n e s "fusel oil" c o 1 o r i m e t r i c a 11 y and " h i g h e r
alcohols" by GLC, although the two terms are essentially synonymous.

303

ACIDITY
PRINCIPLE
Titration of acidity with alkali, detecting the end-point
or using phenolphthalein or bromothymol blue.

potentio-metrically

APPARATUS
1.

Steam-distillation

apparatus.

REAGENTS
1.

Sodium hydroxide, 0.1N.

2.

Bromothymol blue, 0.1% in neutral

3.

Phenolphthalein, 0.1% in 60% neutral

ethanol.
ethanol.

PROCEDURE
Total Acidity
Spirits :
Dilute six-fold with water adjusted to pH 7.8 and
C a l c u l a t e as b o t h a c e t i c and t a r t a r i c acids.
to pH 7.8.
preferable to use a pH meter, otherwise bromothymol blue.

titrate
It is

Wines:
B o i l 25 m l u n d e r r e f l u x for 20 m i n u t e s to e x p e l C02Wash
d o w n the c o n d e n s e r w i t h w a t e r and t i t r a t e to pH 7.8. C a l c u l a t e as
milliequivalents/L or as tartaric acid.
Beer :
Dilute with
phenolphthalein.
Fixed

water

and

titrate

potentiometrica11 y

or

to

Acidity

Spirits :
E v a p o r a t e 50 m l on a w a t e r - b a t h for 30 m i n u t e s after
e v a p o r a t i o n is c o m p l e t e .
Add 50 m l w a t e r (at pH 7-8), stir and
titrate.
Calculate as tartaric acid.
Wines : Calculate by difference,
as milliequivalents/L.

Total-Volatile,

as tartaric

acid

or

B e e r : E v a p o r a t e 20 ml to d r y n e s s , add w a t e r , e v a p o r a t e and r e p e a t
this process 3 or 4 times.
Add water and titrate with 0.1N alkali to
phenolphthalein.
Calculate as lactic and as acetic acids.
Volatile
Spirits :

Acidity
Calculate

as g acetic acid/100 L of alcohol in sample

1200

from

(V1-V2)

w h e r e S = % v/v e t h a n o l in the s a m p l e , V^ and V 2 are m l of 0.1N

alkali required to neutralize total or fixed acidity respectively.
Wines : Boil 50 ml of sample under reflux for 20 minutes, wash down
the condenser with water and steam distil.
Adjust the heat so that
the v o l u m e in the d i s t i l l a t i o n flask is about 25 m l and c o l l e c t at
least 200 m l of distillate.
Titrate to phenolphthalein and calculate
as milliequivalents per litre or as acetic and tartaric acid.
Beer :

Calculate

as acetic acid

from

304

(Total-Fixed).

REFERENCES
Official Methods of Analysis of the AOAC, 1984,
EEC Regulation 1539/71,
OIV Methods

Annex.

(4).

305

9.062-.064.

10.2

ALCOHOLIC-FERMEBTED

COMPOSITION
F e r m e n t e d b e v e r a g e s include both w i n e s and beers. W i n e s are the fermented
p r o d u c t s of g r a p e s or fruit juices.
Table w i n e s usually c o n t a i n 9 to 14%
ethanol derived from the original fermentation only. Fortified wines (such as
sherry, port, marsala, etc.) have added distilled spirits usually to about 20%
total ethan-ol.
S p a r k l i n g w i n e s (such as C h a m p a g n e ) have c a r b o n a t i o n from a
second in-bottle fermentation or from added pressurized carbon dioxide.
The deep colour of red wines is due to pigments extracted from the grape skins
d u r i n g the f e r m e n t a t i o n .
Red w i n e s also contain higher a m o u n t s of grape
tannins which give them their characteristic astringency.
White wines, on the
o t h e r h a n d , are m a d e from the expressed juice alone w i t h little or no grape
skin contact during fermentation.
A c o r r e c t acid b a l a n c e is very i m p o r t a n t for wines. Too little acid and the
w i n e w i l l taste flabby and not age well. Too m u c h acid and the w i n e w i l l be
unpalatable.
M o s t w i n e s have a fixed acidity of 0.3 to 0.55% calculated as
tartaric acid, and a volatile acidity of 0.03 to 0.35% as acetic acid.
If this
latter exceeds 0.14%, the wine may be unacceptable.
Often crystals are found
in a b o t t l e of w i n e , or adhering to the b o t t o m of the cork. These are m e r e l y
tartrate crystals and do not detract from the wine.
Residual sugar is another important feature of a wine.
Dry wines usually have
from 0 to 0.3% r e s i d u a l sugar, a l t h o u g h m o s t persons cannot o r g a n o l e p t i c a 1 1 y
detect sugar until it is about 1% or greater.
The amount of residual sugar in
a w i n e d e p e n d s on the sugar content of the original juice and w h e t h e r or not
the fermentation was stopped before completion.
The traditional manufacture of beer is from barley, which is allowed to sprout,
e n z y m e s being p r e s e n t w h i c h convert starch to sugars. Yeast is added to the
i n f u s i o n Dbtained from the sprouted barley.
The f e r m e n t a t i o n converts a
considerable proportion of the sugars to ethanol.
Bitters such as those from
hops are added at some stage and the ferment is cleared and filtered.
The specific gravity of the infusion before fermentation is greater than unity
due to the presence of dissolved solids, mainly sugars.
Fermentation lowers
the g r a v i t y b o t h b e c a u s e sugars are r e m o v e d and because e t h a n o l , w h i c h has a
l o w e r g r a v i t y than w a t e r , is produced.
The higher the specific gravity
("original gravity") of this unfermented infusion or beer wort the stronger the
beer from it if the fermentation is allowed to proceed to its completion.
For
this r e a s o n the o r i g i n a l gravity has b e e n used t r a d i t i o n a l l y for taxation
purposes.
It is determined experimentally by removal of the alcohol from the
f i n i s h e d a r t i c l e by d i s t i l l a t i o n and d e t e r m i n a t i o n of the alcohol from the
g r a v i t y of the d i s t i l l a t e . From this m a y be calculated the c o n c e n t r a t i o n of
sugar from w h i c h this alcohol was derived and w h a t c o n t r i b u t i o n that sugar
w o u l d m a k e to the gravity of the beer wort. This is added to the gravity of
the residue adjusted for volume, the result being an estimation of the original
gravity of the beer wort prior to fermentation.
B e e r s v a r y w i d e l y in c o m p o s i t i o n , d e p e n d i n g on ingredients and type of
fermentation.
Some composition data examples are given in the following table
(from Pearson's Chemical Analysis of Foods, 8th Ed.):
Type of
Beer
Pale Ale
Mild Ale
Bock
Lager
Stout

Alcohol
Z
4.30
3.15
4.50
3.20
4.30

Total
Solids Z
4.95
3.55
6.80
5.38
5.70

306

Acidity Z
(as Acetic)
0.15
0.08
0.12
0.17
0.17

Ash
Z
0.26
0.20
0.29
0.20
0.23

ROUTIHE ANALYSIS
The m o r e i m p o r t a n t d e t e r m i n a t i o n s on w i n e i n c l u d e e t h a n o l , s u l p h u r d i o x i d e ,
other p r e s e r v a t i v e s , g l y c e r o l , m e t h a n o l and t o t a l s o l i d s .
It m a y also be
n e c e s s a r y to d e t e r m i n e ash, a c i d i t y ( t o t a l , fixed and v o l a t i l e ) , h i g h e r
alcohols, sugar, added colour, isopropanol, artificial
sweeteners,
acetaldehyde, tartaric acid and diethyl pyrocarbonate.
O I V o f f i c i a l m e t h o d s (4) i n c l u d e p r o c e d u r e s for s a l i c y l i c ,
sorbic,
ph y d r o x yb e n zo i c (and e s t e r s ) , p - c h 1 o r o b e n z o ic and b e n z o i c a c i d s and
m o n o c h l o r o a c e t i c and monobrorno a c e t i c a c i d s a l l of w h i c h a r e
possible
preservatives.
All e x c e p t sorbic and p o s s i b l y h y d r o x y b e n z o i c a c i d s are
generally considered undesirable.
Both the OIV and AOAC give GLC methods for
d i e t h y l p y r o c a r b o n a t e (DEPC).
The OIV also d e s c r i b e s m e t h o d s for s u l p h u r
d i o x i d e , a c e t a l d e h y d e , s u g a r s , g l y c e r o l and 2 , 3 , - b u t a n e d i o 1 , s o r b i t o l and
mannitol and tartaric acid, among others.
B e e r s h o u l d be a n a l y z e d for a l c o h o l and o r i g i n a l g r a v i t y ,
acidity,
preservatives and toxic elements such as arsenic, lead and tin in canned beer.
C o b a l t has b e e n added as a froth s t a b i l i z e r and one i n s t a n c e of t o x i c i t y
a r i s i n g from this has b e e n r e p o r t e d .
In a d d i t i o n , beer m a y be e x a m i n e d for
carbon dioxide, extract, ash, chloride, glycerol, tannin, bitter substances and
d i e t h y l pyrocarbonate.
S o u r c e s of a n a l y t i c a l m e t h o d s for beer i n c l u d e m e t h o d s of a n a l y s i s of the
A m e r i c a n S o c i e t y of B r e w i n g C h e m i s t s , the E u r o p e a n B r e w e r y C o n v e n t i o n , the
Institute of Brewing Analysis Committee (5) and Hudson (6).
The ASBC methods
i n c l u d e q u a l i t y c r i t e r i a such as c o l o u r , t a s t e , foam c o l l a p s e rate and
turbidity.
For excise purposes, the specific gravity of the beer wort before fermentation
is of importance, the complete fermentation of worts of higher specific gravity
leading to stronger beers.
The gravity before fermentation is referred to as
the e x t r a c t of the o r i g i n a l w o r t or the o r i g i n a l g r a v i t y .
The A S B C g i v e s
formulae
from w h i c h the e x t r a c t and the real and a p p a r e n t d e g r e e s of
f e r m e n t a t i o n m a y be c a l c u l a t e d .
E s s e n t i a l l y the s a m e c a l c u l a t i o n but u s i n g
different terminology may be carried out as follows.
The alcohol is distilled
and the s p e c i f i c g r a v i t y of the d i s t i l l a t e at 15.5C d e t e r m i n e d .
1000 (1-SG)
is called the spirit i n d i c a t i o n .
The r e s i d u e in the flask is d i l u t e d to the
original volume of sample taken and the gravity of the solution determined at
15.5C or corrected to that temperature (specific gravity of the extract SGj).
Suppose

SG = 0.9946, SGj = 1.0197, then 1000(1-SG) = 5.4

If the total a c i d i t y e x c e e d s 0.1 p e r c e n t as a c e t i c a c i d , a c o r r e c t i o n

must be made to the spirit indication according to the following table:

CORRECTIONS TO BE MADE TO SPIRIT INDICATION OF BEER FOR EXCESS ACID

Acetic
Acid
0. 00

0.01

Corresponding degrees of spirit indication

0.08
0.02
0.03
0.04
0.06
0.07
0.05

0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0

0.02
0.15
0.28
0.40
0.53
0.66
0.78
0.91
1.04
1.16
1.31

0.04
0.17
0.29
0.42
0.55
0.67
0.80
0.93
1.06
1.18
1.33

0. 14
0. 27
0. 39
0. 52
0. 65
0. 77
0. 90
1. 03
1. 15
1. 29

0.06
0.18
0.31
0.43
0.56
0.69
0.81
0.94
1.07
1.19
1.35

0.07
0.19
0.32
0.44
0.57
0. 70
0.82
0.95
1.08
1.21
1.36

307

0.08
0.21
0.33
0.46
0.59
0.71
0.84
0.97
1.09
1.22
1.37

0.09
0.22
0.34
0.47
0.60
0. 72
0.85
0.98
1.10
1.23
1.38

0.11
0.23
0.35
0.48
0.61
0.73
0.86
0.99
1.11
1.25
1.40

0.12
0.24
0.36
0.49
0.62
0. 75
0.87
1.00
1.13
1.26
1.41

0.09
0.13
0.26
0.37
0.51
0.64
0.76
0.89
1.02
1.14
1.28
1.42

Suppose the acidity = 0.35%, the excess = 0.25 and therefore 0.33 must be added
to the spirit indication, which becomes 5.4 + 0.33 * 5.73.
From the following
table, this is equivalent to gravity of 25.18 lost by fermentation.

TABLE OF GRAVITY LOST FOR DETERMINIHG THE ORIGINAL GRAVITY OF WORTS OF BEER
Degrees
of
Spirit
Indication
0 0
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16

0 .00
4 .25
8 .50
12 .90
17 .30
21 .85
26 .40
31 .00
35 .65
40 .30
45 .00
49 .85
54 .85
59 .95
65 .10
70 .30
75 .60

0 .2

0 .42
4 .67
8 .94
13 .34
17 .75
22 .30
26 .86
31 .46
36 .11
40 .77
45 .48
50 .35
55 .36
60 .46
65 .62
70 .83

0 .85
5 .10
9 .38
13 .78
18 .21
22 .76
27 .32
31 .93
36 .58
41 .24
45 .97
50 .85
55 .87
60 .97
66 .14
71 .36

(The a b o v e t a b l e
2 2 3 2 ) U.K.)

_4

0
1 .27
5 .52
9 .82
14 .22
18 .66
23 .21
27 .78
32 .39
37 .04
41 . 71
46 .45
51 .35
5 6 .38
61 .48
66 .66
71 .89

0 ^7

0 zl

0 9.

3 .40
7 .65
12 .02
16 .42
20 .94
25 .49
30 .08
34 .72
39 .37
44 .06
48 .88
53 .85
58 .93
64 .03
69 .26
74 .54

3 .82
8 .07
12 .40
16 .8
21 .39
25 .94
30 .54
35 .18
39 .83
44 .53
49 .36
54 .35
59 .44
64 .54
69 .78
75 .07

1 .70
5 .95
10 .26
14 .66
19 .12
23 .67
28 .24
32 .86
37 .51
42 .18
46 .94
51 .85
56 .89
61 .99
67 .18
72 .42

2 .12
6 .37
10 .70
15 .10
19 .57
24 .12
28 .70
33 .32
37 .97
42 .65
47 .42
52 .35
57 .40
62 .51
67 .70
72 .95

2 .55
6 .80
11 .14
15 .54
20 .03
24 .58
29 .16
33 .79
38 .44
43 .12
47 .91
52 .85
57 .91
63 .01
68 .22
73 .48

2 .97
7 .22
11 .58
15 .98
20 .48
25 .03
29 .62
34 .25
38 .90
43 .59
48 .39
53 .35
58 .42
63 .52
68 .74
74 .01

is r e p r o d u c e d

from

The Beer R e g u l a t i o n s ,

1952 (SI 1952 No.

T h i s f i g u r e is a d d e d to the g r a v i t y of the e x t r a c t (x 1 0 0 0 ) giving 1019.7 +

25.18 = 1044.9 ( u s u a l l y e x p r e s s e d in this w a y but m a y a l s o be e x p r e s s e d as a
s p e c i f i c g r a v i t y of 1.0449).
E s s e r y and H a l l d e s c r i b e a q u i c k m e a n s of c a l c u l a t i o n .
Light beers may have
o r i g i n a l g r a v i t i e s of a r o u n d 1 0 3 0 - 1 0 4 0 and very s t r o n g b e e r s up to 1070 or
1080.
Total

solids may be calculated

TS(% m / v ) =

or determined

from the

formula:

1 0 0 0

< S G - I? - 1.1 xZ ash

3.86

by drying 25 ml at 100C to constant

308

weight.

10.3

TEA

COMPOSITION
T e a is a b e v e r a g e m a d e by i n f u s i n g a v e g e t a b l e s u b s t a n c e w i t h b o i l i n g w a t e r .
Herbs and m a n y other substances can be used to m a k e "teas", but traditional tea
is the p r e p a r e d l e a v e s of v a r i o u s s p e c i e s of C a m e 11 i a the a, i n c l u d i n g _T_.
a s s a m i c a , T. b o h e a , T_. s i n e n s i s and T_. v i r id i s .
T h e r e are t h r e e g e n e r a l t y p e s of t e a s , b l a c k , g r e e n and o o l o n g .
B l a c k tea is
f u l l y f e r m e n t e d , g r e e n is u n f e r m e n t e d and o o l o n g is p a r t i a l l y
fermented.
F e r m e n t a t i o n is a step in the process of tea manufacture which produces changes
in the tannin and other constituents.
Some legal standards

for tea from a few countries

Canada
Total

ash (%)

Water-soluble
Extractives

Australia

follows:

Switzerland

8 (max)
ash (%)

(%)

2.75

Caffeine

(%)

USA

Iraq

4-7

5-7.5

(min)

30 (min)

Stalks (%)

30

(min)
22

C o m p o s i t i o n d a t a for b l a c k and g r e e n tea are

Pearson's Chemical Analysis of Foods, 8th Ed.)

Constituent

as

(max)

follows:

Green Tea

Moisture %

3.9 -

9.5

6.1 - 9.2

Total ash %

4.9 -

6.5

5.2 -

Water

3.0 - 4.2

2.6 - 4.1

1.2 -

1.2 -

soluble ash %

Extractives %

2.0

30 - 50

Caffeine %

1.9 -

Tannin %

7.3 -15. 1

Crude

fibre %

3.6

14 - 18

(min)

30

(min)

10

(max)

1.0

(min)

(compiled

Black Tea

Alkalinity % (as K 2 0 )

ROUTINE

are as

from

7.2

1.6

33 - 45
1.5 - 4.3
-

9 - 15

ANALYSIS

T h e m o s t i m p o r t a n t d e t e r m i n a t i o n s on tea are a m i c r o s c o p i c a l e x a m i n a t i o n ,
moisture, water-soluble e x t r a c t i v e , f i l t h t e s t and the p r o p o r t i o n of s t a l k s .
Samples containing over 11% moisture may have attracted mould growth.
If an
infusion of the tea smells m u s t y it m a y be examined for mould with a hand-lens
or b i n o c u l a r m i c r o s c o p e and t e s t e d for m y c o t o x i n s if t e n t a t i v e or p o s i t i v e
i d e n t i f i c a t i o n of the m o u l d s u g g e s t s t h i s is w a r r a n t e d .
T h e t o t a l ash and
a c i d - i n s o 1 u b 1 e ash are u s e f u l i n d i c a t i o n s of the p r e s e n c e of any e x t r a n e o u s
mineral matter.
This is confirmed by the test for heavy filth.
A low w a t e r soluble ash and alkalinity of it indicate the presence of spent leaves.
These
also yield low water-soluble extractive and low caffeine figures.
Unless the

309

p r e s e n c e o f s p e n t l e a v e s is s u g g e s t e d b y t h e e x t r a c t i v e a n d a s h v a l u e s ,
d e t e r m i n a t i o n o f c a f f e i n e is n o t p a r t i c u l a r l y v a l u a b l e in r o u t i n e t e s t i n g .

the

A l t h o u g h t e a - c h e s t s a r e n o w l i n e d w i t h a l u m i n i u m , n o t l e a d , the lead c o n t e n t of
tea can o c c a s i o n a l l y be higher than desirable.
It is t h e r e f o r e i m p o r t a n t t o
t e s t for t h i s e l e m e n t , and a l s o w o r t h w h i l e to e x a m i n e for c o p p e r and a r s e n i c .
A t o n e t i m e P r u s s i a n b l u e w a s a d d e d to g r e e n tea to i m p r o v e its a p p e a r a n c e .
T h e p r e s e n c e of o t h e r a d d e d c o l o u r w o u l d p r o b a b l y be s u s p e c t e d by the
a p p e a r a n c e ' of a c o l d - w a t e r i n f u s i o n .
T e a m a y b'e e x a m i n e d f o r f i l t h b y t h e
g e n e r a l t e s t s for h e r b s and s p i c e s .
T h e a s h o f t e a s h o u l d b e d e t e r m i n e d b y h e a t i n g to as l o w a t e m p e r a t u r e a s
p o s s i b l e f o r a s h i n g s o t h a t t h e c o l o u r o f i t is g r e y o r b r o w n , n o t g r e e n
b e c a u s e of e x c e s s i v e o x i d a t i o n .
A g r e e n a s h u s u a l l y g i v e s r i s e to a p i n k
f i l t r a t e o n e x t r a c t i n g w i t h w a t e r , and m a k e s it d i f f i c u l t to see the e n d - p o i n t
w h e n t i t r a t i n g the a l k a l i n i t y .
E i t h e r t i t r a t e to pH 3.7 u s i n g a pH m e t e r , use
s c r e e n e d i n d i c a t o r , or e v a p o r a t e t h e e x t r a c t / a s h m i x t u r e u n t i l the p i n k c o l o u r
( d u e to m a n g a n e s e c o m p o u n d s ) is n o l o n g e r p r o d u c e d , b e f o r e d i l u t i n g to a b o u t 25
m l and f i l t e r i n g .

310

MICROSCOPIC EXAMINATION OF TEA

PRINCIPLE
T h e t e a is c l e a r e d w i t h
prepare for e x a m i n a t i o n .

chloral

hydrate

and bleached

with

hypochlorite

APPARATUS
1.

Microscope

and lamp.

REAGENTS
1.

Chloral

hydrate.

Dissolve

7 g in 5 m l w a t e r .

2.
S o d i u m h y p o c h l o r i t e s o l u t i o n , 5 % . If o n l y b l e a c h i n g p o w d e r is
a v a i l a b l e p r e p a r e as f o l l o w s :
D i s s o l v e 75g of c r y s t a l l i n e s o d i u m
c a r b o n a t e in 125 m l of d i s t i l l e d w a t e r ; r u b d o w n 5 0 g of c h l o r i n a t e d
l i m e in a m o r t a r w i t h 3 7 5 m l o f d i s t i l l e d w a t e r , a d d e d a l i t t l e a t a
time.
M i x the t w o fluids and shake o c c a s i o n a l l y d u r i n g three or four
hours;
filter.
3.

Phloroglucinol.

4.

Concentrated

1 % in 9 0 %

hydrochloric

ethanol.

acid.

PROCEDURE
A l l o w t h e l e a v e s to r e m a i n in c h l o r a l h y d r a t e s o l u t i o n for s e v e r a l
hours. Pour off the chloral hydrate and replace with hypochlorite
solution.
Once the leaves are adequately bleached, replace the
bleaching solution with phloroglucinol solution.
A l l o w this to
b e c o m e n e a r l y d r y b y e v a p o r a t i o n , then add a drop of c o n c e n t r a t e d
hydrochloric acid. The s c l e r e n c h y m a t o u s m a t t e r of w h i c h the h a i r s
a n d t h e i d i o b l a s t s l a r g e l y c o n s i s t is s t a i n e d p i n k .
Mount and
examine under low power. Apart from the t w o elements m e n t i o n e d , note
the s t o m a t a and c a l c i u m o x a l a t e .
Check that there are no e l e m e n t s
present that are n o t characteristic of tea.
A microscopic

description

o f t e a is a s

follows:

The u p p e r e p i d e r m i s is c o m p o s e d o f c e l l s w i t h u n d u l a t i n g w a l l s a n d
covered with a rather thick cuticle.
The lower e p i d e r m i s consists of
s m a l l e r c e l l s a n d is a l o n e p r o v i d e d w i t h s t o m a t a ; t h e l a t t e r a r e
s u r r o u n d e d b y three or four t a n g e n t i a l l y e l o n g a t e d c e l l s .
Simple
hairs occur on both surfaces of the leaf, b u t they are m o r e abundant
on the l o w e r ; t h e n u m b e r , h o w e v e r , v a r i e s w i t h t h e v a r i e t y of t e a ,
and w i t h the a g e of the leaf; they are u n i c e l lular, t a p e r i n g and
r a t h e r t h i c k w a l l e d , v a r y i n g v e r y m u c h in l e n g t h , b u t o f t e n a t t a i n i n g
500-700 y .
T h e m e s o p h y l l is h e t e r o g e n e o u s a n d a s y m m e t r i c a l .
I t is c h a r a c t e r i z e d
by the presence of a large n u m b e r of s c l e r e n c h y m a t o u s
idioblasts.
T h e s e are m o r e or less b r a n c h e d and w a r t y and o f t e n
extend
t r a n s v e r s e l y f r o m the u p p e r to the l o w e r e p i d e r m i s .
They vary very
m u c h in s h a p e a n d in t h e t h i c k n e s s o f t h e w a l l s .
The cells of the
spongy p a r e n c h y m a contain cluster crystals of c a l c i u m oxalate.
T h e m i d r i b is b i c o n v e x .
U n d e r e a c h e p i d e r m i s t h e r e is a l a y e r o f
c o l l e n c h y m a of varying thickness.
T h e w o o d is a r c h e d a n d t h e b a s t
c o n t a i n s c r y s t a l s o f c a l c i u m o x a l a t e . T h e m e r i s t e l e is s u r r o u n d e d b y
a p e r i c y c l e c o n s i s t i n g of s l i g h t l y l i g n i f i e d c e l l s a r r a n g e d in a
circle.
The cortical tissue contains idioblasts which are usually
rather larger and m o r e branched than those of the m e s o p h y l l .

311

T h e l i t t l e f r a g m e n t s of the s t e m s , w h i c h are o f t e n to be found in

ordinary tea, have a slightly different structure.
The wood in them
f o r m s a c i r c l e w i t h i n w h i c h t h e r e is a p i t h c o n t a i n i n g b r a n c h e d
idioblasts; these have c o m p a r a t i v e l y thin, pitted walls.

312

CAFFEINE IB TEA
PRINCIPLE
T h e c a f f e i n e is e x t r a c t e d w i t h e t h a n o l , t h e e t h a n o l r e m o v e d , t h e r e s i d u e t a k e n
up i n w a t e r a n d e x t r a c t e d i n t o c h l o r o f o r m . T h e c h l o r o f o r m is e v a p o r a t e d a n d
the r e s i d u e w e i g h e d .
APPARATUS
1.

Soxhlet

extraction

2.

Separator, 500 m l .

apparatus

or e q u i v a l e n t .

REAGENTS
1.

Ethanol,

absolute.

2.

Magnesium

oxide,

3.

Sulphuric

a c i d , 10% v / v .

4.

Sulphuric

acid, 0.5% v / v .

5.

Chloroform.

6.

Potassium

heavy.

hydroxide

solution, 1%.

PROCEDURE
M o i s t e n 10g of s a m p l e , g r o u n d fine e n o u g h to p a s s a 30 m e s h ( 0 . 5 m m )
s i e v e , w i t h e t h a n o l in an e x t r a c t i o n t h i m b l e .
T r a n s f e r to a S o x h l e t
and extract w i t h ethanol eight h o u r s . C o m p l e t e n e s s of e x t r a c t i o n m a y
be c h e c k e d b y u s i n g a f r e s h f l a s k and fresh e t h a n o l , e x t r a c t i n g a
f u r t h e r t w o h o u r s , e v a p o r a t i n g a n d c h e c k i n g t h a t t h e r e s i d u e is
negligible.
T r a n s f e r the e t h a n o l i c e x t r a c t w i t h h o t w a t e r to a
p o r c e l a i n d i s h c o n t a i n i n g 1 0 g h e a v y m a g n e s i u m o x i d e s u s p e n d e d in 1 0 0
ml water.
Evaporate slowly on the steam bath with frequent stirring
to a d r y , p o w d e r y m a s s .
Rub the residue with a pestle into a paste
w i t h b o i l i n g w a t e r a n d t r a n s f e r w i t h h o t w a t e r to a f i l t e r , c l e a n i n g
t h e d i s h w i t h a p o l i c e m a n . C o l l e c t t h e f i l t r a t e in a 1 l i t r e c o n i c a l
flask m a r k e d at 250 m l and wash with boiling water until the filtrate
reaches the mark.
A d d 2 0 m l o f 10 % s u l p h u r i c a c i d a n d b o i l g e n t l y
30 m i n u t e s , w i t h a f u n n e l in t h e n e c k o f t h e f l a s k to r e d u c e
evaporation.
C o o l , filter through a m o i s t e n e d double paper into the
s e p a r a t o r a n d w a s h w i t h s m a l l p o r t i o n s o f 0.5 % v / v s u l p h u r i c a c i d .
E x t r a c t w i t h 6 x 25 m l o f c h l o r o f o r m .
W a s h the combined c h l o r o f o r m
extracts with 5 m l of 1 % potassium hydroxide solution.
Filter the
chloroform
i n t o a t a r e d f l a s k ( 1 0 0 m l is s u i t a b l e ) .
Wash the
p o t a s s i u m h y d r o x i d e s o l u t i o n w i t h 2 x 10 m l c h l o r o f o r m , p o u r t h e s e
through the filter paper and w a s h the filter paper with c h l o r o f o r m ,
filtering all of these washings into the flask containing the
filtered c h l o r o f o r m e x t r a c t . E v a p o r a t e or d i s t i l the c h l o r o f o r m on a
s t e a m - b a t h , e v a p o r a t i n g t h e l a s t 1 0 - 1 5 m l c a r e f u l l y to a v o i d l o s s o f
r e s i d u e , d r y 3 0 m i n u t e s a t 100C a n d w e i g h .
CALCULATION
W e i g h t o f c a f f e i n e x 1 0 0 / 1 0 = % c a f f e i n e in s a m p l e .
D e t e r m i n e the
n i t r o g e n o n t h e r e s i d u e b y K j e l d a h l p r o c e d u r e , N x 3.464 = c a f f e i n e .
The

caffeine

absorbance

residue

measured

can

also

at 2 7 4 n m ,

be

taking

313

dissolved
in w a t e r , a n d t h e
1 2 =
E
525 for pure c a f f e i n e .
1 cm

REFERENCES
O f f i c i a l M e t h o d s of A n a l y s i s of the AOAC, 1 9 6 5 , 1 4 . 0 1 9 (Power-Chestnut
The 1 9 8 4 AOAC method g i v e s the m o d i f i e d B a i l e y - A n d r e w Method.

Method).

T L C h a s a l s o b e e n e m p l o y e d f o r d e t e c t i o n and q u a n t i t a t i v e e s t i m a t i o n of
caffeine.
See S e n g u p t a ,
P.,
Mondai,
A.,
Sen,
A.R.
and R o y ,
B.R.,
1975.
I n t e r n a t i o n a l F l a v o u r and Food A d d i t i v e s
340.

314

EXTRACTIVES FROM TEA

PRINCIPLE
A known weight of dried tea is boiled with water and an aliquot of the extract
is evaporated and the residue weighed.
The result is expressed as a percentage
of the dried tea.
APPARATUS
1.

Metal dish,

flat-bottomed.

2.

250 ml round-bottomed

3.

250 ml volumetric

4.

Metal evaporating dish, round

5.

Waterbath.

6.

Oven.

flask and reflux

condenser.

flask.
bottom.

PROCEDURE
Dry approximately 2g tea, accurately weighed, in the oven in a tared
f l a t - b o t t o m d i s h for 5 h o u r s at 100C.
R e m o v e the dish from the
o v e n , cool and w e i g h .
C a l c u l a t e the w e i g h t of tea t a k e n for the
test.
T r a n s f e r the dried tea to a 250 ml r o u n d - b o t t o m flask, add 1 0 0 m l
d i s t i l l e d w a t e r , and r e f l u x for one hour.
F i l t e r into a 250 m l
g r a d u a t e d flask. R e t u r n the filter paper w i t h the r e s i d u e in it to
the reflux flask, add a further 100 ml water and reflux for a further
Filter into the volumetric flask, rinse the reflux flask with
hour.
hot water and pass the rinsings through the filter.
Wash the filter
w i t h hot w a t e r u n t i l the v o l u m e t r i c flask is filled n e a r l y to the
mark.
C o o l , d i l u t e to 250 m l , m i x and p i p e t t e 50 m l into a w e i g h e d
metal evaporating dish.
Evaporate the solution on a waterbath, and
finally dry in the oven.
Cool and weigh.
CALCULATION
% water-soluble

extractives

weight of residue

250
50

100
weight of dried

tea

INTERPRETATION
M a n l e y found b l a c k tea to c o n t a i n 25.5-46.4% e x t r a c t i v e s and g r e e n teas 29.641.4%.
S e v e r a l c o u n t r i e s h a v e a legal l i m i t of 30%.
Spent tea l e a v e s , i.e.,
those that have been infused, dried and re-used give values of less than 10 %.
REFERENCE
Manley,

1965. Journal of the American Pharmaceutical

315

Association^,

101.

STALK IR TEA
PRINCIPLE
The sample is boiled with water, spread out and the stalk separated by use of
forceps.
The stalks and leaf fractions are dried separately and weighed'.
PROCEDURE
B o i l 5 g of the s a m p l e w i t h 500 m l of w a t e r for 15 m i n u t e s .
Pour
into a basin or other suitable receptacle with a white interior and
p i c k out the s t a l k s w i t h the aid of t w e e z e r s or f o r c e p s . T a k e care
not to c o n f u s e s t a l k w i t h leaf m i d r i b , or leaf f r a g m e n t s that h a v e
remained rolled.
Use of a hand-lens will enable foreign material to
be d e t e c t e d and e x a m i n e d s e p a r a t e l y .
Dry s t a l k and leaf p o r t i o n s
separately for 5 hours at 100C and weigh.
CALCULATION

Percent

stalk

weight of dried stalk x 100

wt. of dried stalk + wt. of dried leaf

INTERPRETATION
O v e r 25 % m a y be r e g a r d e d as e x c e s s i v e but s o m e c o u n t r i e s
lower limits. Crude fibre is also an index of stalk. While
n o r m a l q u o t a of s t a l k s has a fibre c o n t e n t of a r o u n d 10 %,
l i m i t is u s u a l l y 15 % ( m a x ) on a dry w e i g h t b a s i s , s t a l k y
m o r e and hard stalks are up to 60 % of crude fibre.

316

have considerably
usual tea with its
and the r e g u l a t o r y
tea c o n t a i n s m u c h

10.4

COFFEE

COMPOSITION
Coffee is sold as the raw green bean or after roasting and grinding.
Roasting
produces carbon dioxide which m a y continue to be released after packaging so a
swollen h e r m e t i c a l l y sealed container is usually not an indication of spoilage.
" F r e n c h c o f f e e " is a m i x t u r e of c o f f e e and c h i c o r y .
" V i e n n e s e c o f f e e " is a
mixture of coffee and fig seasoning or flavouring.
No other substances should
be present in either product.
Some compositional data for raw and roasted coffee, roasted chicory and
coffee are as follows:
(all figures are percents)

Rav Coffee
m in
max
aver
Moisture
8.2
Total Ash
3.0
Water-soluble
ash
Ditto (as % of
total ash
Alky, of sol.
ash as K 2 C O 3 )
Acid insoluble
ash (as % of
total ash)
Crude fibre
Tannin
Total nitrogen
Caffeine
1.1
Starch
Ether Extract
11.4
Extractives of
dry sample
25
Dextrose

Mal tose

ROUTINE

Roasted Coffee
m in
max
aver

Roasted Chicory
m in
max
aver

instant

Instant
Coffee
aver

10.3
4.0

0.3
3.4

5.6
4.9

2.2
4.3

2.5
4.0

12.0
6.7

5.5
5.0

3.98
11.21

2.9

3.0

3.6

3.2

1.6

3.3

2.8

65

85

75

5.5

1.9

3.2

2.4

20
6.9

0.0

13.8
4.5

1.8
-

13.7
34

9.0
2.7
1.3
-

12.2
-

10.5
-

2.1
0.9
0.9
8.0
23

15.3
-

3.3
1.8
3.5
14.2
33

13.0
4.6
2.6
1.2
2.3
13.5
-

10

35

0.9

3.9

70

78

1.4
Nil
2.1
2.1
-

2.24
3.4
-

100
2.13
4.44

ANALYSIS

Materials likely to be mixed with coffee generally have a higher proportion of

soluble m a t t e r ("extractives").
(One e x c e p t i o n is e x h a u s t e d c o f f e e f r o m the
preparation
of i n s t a n t p o w d e r s ) .
This d e t e r m i n a t i o n ,
together with a
microscopical examination, are the most important tests.
The moisture content
of g r o u n d c o f f e e s h o u l d be less t h a n 5 %, d e t e r m i n e d by d r y i n g 5g at 100C to
constant weight.
Caffeine content usually lies b e t w e e n 0.9 and 1.8 %, and m a y
be determined by the t w o - c o l u m n method.
In addition to microscopical examination, the presence of chicory may be shown
s i m p l y by s p r i n k l i n g the p o w d e r o n t o w a t e r in a m e a s u r i n g c y l i n d e r .
Coffee
floats while chicory particles start sinking within a few seconds, and leave
b e h i n d a b r o w n t r a i l of c a r a m e l .
The p r o p o r t i o n of c h i c o r y is u s u a l l y
d e t e r m i n e d by the a m o u n t of e x t r a c t i v e s p r e s e n t .
It 3 b e s t to a d h e r e to an
exact procedure as the results vary s o m e w h a t with the method used.
That of the
European Decaffeination Association is as follows:
Weigh 10g sample into 500 m l erlenmeyer.
Place a 20 cm glass stirring rod into
the f l a s k , add 200 m l w a t e r and w e i g h . B r i n g to b o i l w h i l e s t i r r i n g and b o i l
exactly 5 minutes.
C o o l , r e w e i g h , and add w a t e r to b r i n g w e i g h t b a c k to
original.
F i l t e r , e v a p o r a t e 25 m l .
D r y in air o v e n at 105C and w e i g h the
residue.
Continue the drying until constant weight is obtained.

317

C h i c o r y c o n t a i n s i n u l i n , w h i c h h y d r o l y s e s to l a e v u l o s e .
C o f f e e c o n t a i n s no
inulin.
T h e p r e s e n c e o f c h i c o r y is s h o w n by a p o s i t i v e r e a c t i o n
with
S e l i w a n o f f ' s reagent (0.05 % resorcinol in 33% V/V hydrochloric acid).
Clarify
a 2 % aqueous extract of coffee with a little neutral lead acetate and filter.
To 5 m l of the c l e a r f i l t r a t e add 5 m l of S e l i w a n o f f r e a g e n t f o l l o w e d hy 1 m l
of concentrated hydrochloric acid.
Boil for one minute.
A distinct red colour
produced on standing shows the presence of chicory in coffee.
Roasted cereals such as barley, oats and wheat and also soya and other products
Careful
m a y be mixed w i t h coffee, or coffee and chicory as coffee substitutes.
m i c r o s c o p i c a l e x a m i n a t i o n will reveal the presence of these products.
S a m p l e s of ground coffee have been e x a m i n e d , with coats, stems,
sugar, soil, sand and spent coffee the c o m m o n e s t adulterants.
L o w ash, nitrogen and caffeine in instant coffee could
added dextrin, m a l t o s e or dextrose.

indicate

roasted

maize,

the presence

of

Coffee extracts should be analyzed for m o i s t u r e , caffeine, insoluble residue,

s u l p h u r d i o x i d e and s o l v e n t r e s i d u e s .
M o i s t u r e of d r y c o f f e e e x t r a c t s is
d e t e r m i n e d by d r y i n g at 70C u n d e r a p r e s s u r e of a b o u t 25 m m of Hg.
Liquid
coffee extracts are dried at 70C and about 100 m m pressure of Hg for 16 hours.
E E C d i s c u s s i o n d o c u m e n t s s u g g e s t the S c h i 1 1 i n g - G a 1 1 GLC m e t h o d for s o l v e n t
residues.
I n s o l u b l e m a t t e r m a y be d e t e r m i n e d by U.S. F e d e r a l S p e c i f i c a t i o n
method HHH-C-57CC of 10/7/74.

318

MICROSCOPIC EXAMINATION OF COFFEE

PRINCIPLE
Ground coffee is boiled

with water, cleared and

stained

for

examination.

APPARATUS
M i c r o s c o p e , w i t h low p o w e r o b j e c t i v e ( h i g h - p o w e r o b j e c t i v e m a y be
needed occasionally).
PROCEDDRE
Boil about 0.5g of s a m p l e w i t h w a t e r so that m o s t of the c o l o u r is
extracted.
Drain and replace the volume of water used with chloral
hydrate.
Heat until the material is sufficiently cleared.
Wash out
the chloral hydrate and stain with phloroglucinol/hydrochloric acid.
Coffee is characterized by longitudinal
tous fibres (from the pericarp).

and

transverse

sclerenchyma-

C h i c o r y has v e r y large v e s s e l s up to 115 m i c r o n s across w h i c h h a v e

r a t h e r short pits.
Clearing with boiling 5 % sodium hydroxide
solution may also be used.

319

CAFFEINE IN COFFEE
PRINCIPLE

The m e t h o d d e s c r i b e d f o l l o w s the L e v i n e p r o c e d u r e .
It has b e e n c h o s e n from
a m o n g s t n u m e r o u s m e t h o d s , s t u d i e d c o m p a r a t i v e l y for g e n e r a l a p p l i c a b i l i t y ,
reproducibility, specificity, ease of application and rapidity.
The method is
s e n s i t i v e to d i s t u r b a n c e s a n d s m a l l v a r i a t i o n s w h i l e t h e a n a l y s i s is
proceeding.
It a p p l i e s to g r e e n c o f f e e , d e c a f f e i n a t e d g r e e n c o f f e e , roasted
coffee, decaffeinated roasted coffee, extracts of coffee, both dried and liquid
and d e c a f f e i n a t e d e x t r a c t s . The l o w e r l i m i t of d e t e c t i o n is 0.02% c a f f e i n e .
The c a f f e i n e is e x t r a c t e d f r o m the s a m p l e , in an a m m o n i a c a l m e d i u m .
After
successive purifications with diethyl ether on two chromatographic columns, the
caffeine is estimated spectrophoto-metrically in the ultra-violet range.
APPARATUS
1.
Chromatographic
columns,
diameter, with stopcocks.

250

2.

Ultra-violet

3.

Beaker, 100 ml.

4.

Boiling water

5.

One-mark volumetric

6.

One-mark pipettes, 2 and 5 ml.

7.

Analytical

8.

Coffee mill

mm

spectrophotometer with

long,

21-25

1 cm quartz

mm

interior

cells.

bath.
flasks 50 ml, 100 ml and 1000 ml.

balance.
(e.g. domestic) for roasted coffee

beans.

9.
T o o t h e d d i s k m i l l w i t h c o o l i n g j a c k e t or a n a l y t i c a l m i l l w i t h
s p a r e c u t t e r and c o o l i n g j a c k e t or s i m i l a r m i l l for g r e e n c o f f e e
beans.
10.
Sieve, woven wire cloth or perforated
microns or 630 microns.

plates, aperture

size

600

REAGENTS
1.

Sulphuric

2.

Sodium hydroxide, 2N

3.

Celite

4.
Ammonia
of water).

acid

solution.
solution.

545.
solution

(1 volume

of concentrated

ammonia + 2 volumes

5.
Diethyl ether, repurified by chromatography on a column of basic
a l u m i n u m o x i d e of a c t i v i t y g r a d e 1. P a s s 800 m l of d i e t h y l e t h e r
t h r o u g h a c o l u m n filled w i t h 100 g of a l u m i n u m o x i d e . The d i e t h y l
ether thus purified should be kept in a dark bottle until used.
6.

Caffeine, pure, anhydrous,

CgH^QN^02.

7.
Chloroform pure, repurified by
method described for diethyl ether.

320

chromatography

according

to

the

PROCEDURE
If necessary, mill the sample until it passes a sieve of 600 or 630y
aperture size.
(See ISO R e c o m m e n d a t i o n R 565 - Woven wire cloth and
p e r f o r a t e d p l a t e s in test s i e v e s .
N o m i n a l size of a p e r t u r e s ) .
A
p a r t of the s a m p l e
thus prepared
s h o u l d be t a k e n for
the
determination of dry matter.
For g r e e n c o f f e e and r o a s t e d c o f f e e , w e i g h , to the n e a r e s t 0.1 m g ,
a b o u t 1 g of the p r e p a r e d s a m p l e .
T r a n s f e r it to a 100 m l b e a k e r ,
add 5 m l of a m m o n i a s o l u t i o n and w a r m for t w o m i n u t e s on a b o i l i n g
w a t e r bath.
A l l o w to c o o l , t h e n t r a n s f e r to a 100 m l v o l u m e t r i c
flask and adjust to v o l u m e with distilled water. Take 5.0 ml of this
(turbid) solution, add 6 g of Celite and m i x carefully.
For d e c a f f e i n a t e d g r e e n c o f f e e and r o a s t e d c o f f e e w e i g h , to the
n e a r e s t 0.1 m g , a b o u t 1 g of the p r e p a r e d s a m p l e .
T r a n s f e r to a 100
m l b e a k e r , add 5 m l of a m m o n i a s o l u t i o n and w a r m for 2 m i n u t e s on a
boiling water bath.
Add 6g of Celite and m i x carefully.
For dried and liquid coffee extract, proceed as for green and roasted
coffee, above, except weigh portions of 0.5 g and 20 g respectively.
For the liquid extract, also use only 2 ml of the turbid solution and
3 g of C e l i t e .
For d e c a f f e i n a t e d c o f f e e e x t r a c t f o l l o w the s a m e p r o c e d u r e
decaffeinated coffee, above, except use a 0.5 g portion.
Prepare

a two layer alkaline column as

as

follows:

Layer A:

Mix carefully, by kneading with a flexible spatula blade,

3g of C e l i t e and 2 m l of s o d i u m h y d r o x i d e s o l u t i o n u n t i l
homogeneous.
A slightly w e t powder is obtained.
Transfer
this p o w d e r , in p o r t i o n s , into a c h r o m a t o g r a p h i c c o l u m n ,
the l o w e r p a r t of w h i c h is p a c k e d w i t h a w a d of c o t t o n or
glass wool.
T a m p d o w n the m i x t u r e , without force, with a
glass rod, one end of which is flattened to the d i a m e t e r of
the column until a perfectly homogeneous and compact layer
is obtained.
A small wad of cotton or glass wool can then
be p l a c e d on the top of l a y e r A.

Layer B:

Transfer the Celite sample mixture to the column on top of

layer A. D r y the b e a k e r w i t h a b o u t 1 g of C e l i t e u s i n g it
twice.
Tamp d o w n to obtain a h o m o g e n e o u s m a s s and place a
wad of cotton or glass wool on the top.

Prepare an acid column as

follows:

P l a c e in a s e c o n d c o l u m n , the l o w e r p a r t of w h i c h is p a c k e d w i t h a
small wad of cotton or glass wool, 2g of Celite and 2 m l of sulphuric
acid solution carefully mixed.
Place a wad of cotton or glass wool
on the top of the l a y e r to k e e p it in p l a c e .
M o u n t the c o l u m n s one a b o v e the o t h e r so that the e f f l u e n t of the
alkaline c o l u m n can drip directly on the acid column.
Pass 150 m l of
d i e t h y l e t h e r t h r o u g h the t w o c o l u m n s .
K e e p the s t o p c o c k in the
alkaline c o l u m n open. Adjust the stopcock of the acid column so that
a quantity of supernatant liquid remains above the layer. Pass 50 ml
of diethyl ether through the acid column, using the initial portion
to w a s h the top of the a l k a l i n e c o l u m n .
U s e this p o r t i o n a l s o for
the acid column.
Discard the effluent from the acid column.
Remove
alkaline column.
P a s s a s t r e a m of a i r , f r o m the top to the l o w e r
p a r t of the a c i d c o l u m n (e.g. u s i n g an i n f l a t e d r u b b e r b u l b ) , u n t i l
no more diethyl ether drips from the c o l u m n and the air flow from the

321

stopcock carries only a weak smell of diethyl ether. Elute the acid
c o l u m n with 45-50 m l of chloroform.
Collect the eluate in a 50 ml
volumetric flask, adjust to volume with chloroform and mix carefully.
The flow rate of diethyl ether and chloroform should not exceed 1.5-2
ml per minute.
Measure the absorption of the solution of caffeine in chloroform in
quartz- cells against chloroform at 276 nm (absorption m a x i m u m ) .
Measure also the absorption at 246 nm (absorption minimum) and at 306
nm in order to verify the purity of the caffeine obtained.
If the
absorption at 276 nm exceeds 1.3, repeat the m e a s u r e m e n t with the
diluted chloroform solution.
In this case the dilution factor must
be included in the calculation.
Prepare a reference solution of caffeine in the following manner:
Weigh to the nearest 0.1 mg, about 100 mg of pure anhydrous caffeine.
Place in a 1000 ml graduated flask.
Dissolve in c h l o r o f o r m , and
With a graduated pipette, take 5 ml of this
adjust to v o l u m e .
solution and adjust to a v o l u m e of 50 ml with chloroform.
Measure
the absorption of this solution.
The corrected absorption of the
reference solution should be in the region of 0.4.
Carry out at
least two determinations on the same prepared sample.
CALCULATION
For green or roasted coffee, the caffeine content, in grams per 100
grams of dry matter of the sample is equal to:
50 x 10 6 x C x X
5 x A x m x ms
Where :
C

the concentration of caffeine and the reference solution in

g/ml.

the corrected absorption of the purified extract, which is

absorption at 276 nm - 1/2 (absorption at 246 nm + absorption
at 306 nm).

the corrected absorption of the reference solution of caffeine,

w h i c h is: absorption at 176 nm - 1/2 (absorption at 246 nm absorption at 306 nm).

the mass, in grams, of the test portion.

ms =

the content of dry matter, percentage by mass, of the sample.

For all decaffeinated products, the caffeine content, in grams per

100 g of dry matter of the sample, is equal to:
5 x 10 5 x C x X
A x m x ms
Where:

C, X, A, m, ms are as above.

For dried and liquid coffee extracts, the caffeine content, in grams
per 100 g of dry matter of the sample, is equal to:
25 x 10 6 x C x X
A x m x ms
where:

C, X, A, m, ms are as above.

322

REFERENCE
ISO 4052:1983.

323

10.5 TEXT REFERENCES

1.

HART,

F.L. & FISHER, H.J. 1971. Modern Food Analysis,

2.

R E S E A R C H C O M M I T T E E ON THE A N A L Y S I S OF P O T A B L E SPIRITS (RCAPS), 1970.

J o u r n a l of the A s s o c i a t i o n of Public Analysts 8 81. 1972. H) 49. 1974. 1_2
40,45.

3.

J A U L M E S , P. &
Fraudes 46 336.

4.

OIV Receuil des Methodes Internationales d'Analyse des Vins.

5.

INSTITUTE OF BREWING ANALYSIS COMMITTEE.

of Brewing 77 181-226.

6.

HUDSON, J.R. 1960. Development

London, Institute of Brewing.

MARIGNAN,

R.

1953.

Annales

des

Springer-Verlag.

Falsifications

1971. Journal of the

et

Institute

of Brewing Analysis - A Historical

Review,

Further Reading
RIBEREAN-GAYON, J. & PEYNAUD, E. 1982.
B I R C H , G.G.
Science.

&

LINDLEY,

M.G.

1985.

Analyse et Controle des Vins.

Alcoholic

Beverages,

Elsevier

Dunod.
Applied

M e t h o d s of A n a l y s i s of the A m e r i c a n Society of B r e w i n g C h e m i s t s , 1976, 3340

Pilot Knob Road, St. Paul, MN 55121, USA.
JOHNSON, H. 1977. The World Atlas of Wine, Mitchell Beazley.
AMERINE, M.A. & OUGH, G.S.
GLENISTER, P.R.

1980. Methods for Analysis of Musts & Wines,

1975. Beer Deposits, Miles Laboratories

Wiley.

Inc.

Food Standards Committee Report on Beer, 1977, HMSO, London.

INTERNATIONAL STANDARDS ORGANIZATION
1572
1573
1574
1575
1576
1577
1578
1839
3103
3720
6078
6770

1980
1980
1980
1980
1975
1980
1975
1980
1980
1981
1982
1982

(ISO), Methods for analysis of tea:

Preparation of ground sample of known dry matter content

Loss in mass at 103C
Water extract
Total ash
Water-soluble ash and water-insoluble ash
Acid-insoluble ash
Alkalinity of water-soluble ash
Sampling
Preparation of liquor for use in sensory tests
Black tea - Specifications
Black tea - Vocabulary
Instant tea - Determination of free-flow and compacted
bulk derivatives.

324

APPENDIX
Abrviations Dsed in the Manual
Dnlts of Measure
absorbance

ng

gram
3
kilogram (10 g )
milligram U 0 _ g g )
microgram (10<j g )
nanogram (10
g)

L
ml
Ml

litre
millilitre
microlitre

m
cm
mm
in

metre
,-2
centimetre (10_.m)
millimetre (10
m)
i n c h ( e s ) (25 m m )

hr
min
sec

hour(s)
minute(s)
second(s )

rpm
o

revolutions per minute

g
kg
mg

(10~^L)
(10
L)

degrees Celsius (centigrade)

C
..
_
ra tion of:

distance spot moved

^
distance solvent moved

R,
Descriptive Units
AR

analytical reagent

BR

boiling

EDTA

ethylene diamine

G/G

ground

IIJ

International

MW

molecular weight

parts
parts
parts

maximum
minimum
average

%
ppm
ppb
max
min
ave

M
N

ID
OD

rs

3S

(grade)

range
tetracetic

acid

glass
Units

per hundred
per million
per billion

(percent)

molar
normal
interior diameter
outside diameter

325

mass in mass
mass in volume
volume in volume

m/m
m/v
v/v

Analytical Techniques
AAS
GLC
HPLC
PC
TLC
RI
SG
IR
UV
vis

=
m
-

=
=
=

atomic absorption spectrophotometry

gas-liquid chromatography
high performance liquid chromatography
paper chromatography
thin-layer chromatography
refractive index
specific gravity
infrared
ultraviolet
visible

Organizations and Agencies

AACC
AOAC
AOCS
BP
BS
CAC
EEC
ICC
ICUMSA
ISO
IUPAC
01V

=
=
=
=
=
=
=
=
=
=
=
=

American Association of Cereal Chemists

Association of Official Analytical Chemists
American Oil Chemists Society
British Pharmacopoeia
British Standards
Codex Alimentarius Commission
European Economic Community
International Association for Cereal Science and Technology
International Commission for Uniform Methods of Sugar Analysis
International Standardization Organization
International Union of Pure and Applied Chemistry
Office International de la Vigne et du Vin

326

ISBN 92-5-102412-X

9
M-87

789251

ISSN 0254-4725

024126
W6530E/2/11.97/200