J. A. Radley-Examination and Analysis of Starch and Starch Products-Springer (1976)

EXAMINATION AND ANALYSIS
OF STARCH AND STARCH PRODUCTS

EXAMINATION AND
ANALYSIS OF STARCH
AND STARCH PRODUCTS
Edited by
J. A. RADLEY
MoSco, e.Chemo, FoSoDoCo, FoRoI.e.
APPLIED SCIENCE PUBLISHERS LTD

LONDON
APPLIED SCIENCE PUBLISHERS LTD
RIPPLE ROAD, BARKING, ESSEX, ENGLAND
ISBN-13: 978-94-010-1334-5 e-ISBN-13: 978-94-010-1332-1

DOl: 10.1007/978-94-010-1332-1
WITH 7 TABLES AND 54 ILLUSTRATIONS
© APPLIED SCIENCE PUBLISHERS LTD 1976

Softcover reprint of the hardcover 1st edition 1976
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, recording, or otherwise,
without the prior written permission of the publishers, Applied Science
Publishers Ltd, Ripple Road, Barking, Essex, England
Galliard (Printers) Ltd Great Yarmouth

Preface
The literature of starch has proliferated in the last ten years at an almost
geometric rate and a number of important changes and developments in the
technology of starch and its derivatives have taken place which make it
highly desirable to review these in some depth.
The immensity of the subject determined the writer to seek the assistance
of a number of prominent workers throughout the world.
Where older work contains factual information of present value it has
been retained, generally in the form of Additional References. These are
brief abstracts which will help specialised searches in a branch of the
subject to complete the information given in the text. Inclusion of dis-
jointed information can often lead to the loss of coherence and clarity,
and the device of the Additional References, whilst allowing smooth
presentation, also allows the inclusion of up-to-the-minute material
appearing after the main text has been written.
The rewarding techniques of transmission and scanning electron
microscopy have been dealt with for the first time in a book of this nature.
Apart from the immense amount of important practical and theoretical
detail required to produce and use starch for many applications in a
number of important industries, a thorough knowledge is also required of
a number of aspects for the successful buying and selling of starch. This
book was written and published contemporaneously with two others
entitled Starch Production Technology and Industrial Uses of Starch and
Its Derivatives. The three books together provide a wide coverage of
starch technology and chemistry with the self-contained individual
volumes providing precise information for specialist readers.
My most sincere thanks are due to the contributors for their most
helpful and ready co-operation in getting out a volume that is as up-to-date
v
vi PREFACE
as humanly possible and to my secretary, Mrs R. M. Russell, for her

valuable help and care in producing the manuscript. I should also like to
record my thanks for the constructive criticism of many practical details
through the book in its early stages that were made by Mr Jack Seaman
before his tragic and untimely death. I have also to thank Dr G. Graefe,
the editor of Die Starke, for permission to reproduce a number of diagrams
from this journal and to various firms for supplying photographs of
equipment, etc., with permission to publish.
Finally, my thanks are also due to the publishers for their part in the
production of this book.
Contents
Preface v
1. The Microscopy of Starch by G. E. Moss 1
2. Electron Microscopy of Starch and Starch Products by

D. J. GALLANT and C. STERLING 33
3. The Rheology of Starch by A. H. A. DE WILLIGEN 61
4. Physical Methods of Characterising Starch by J. A. RADLEY 91
5. Chemical Analysis of Raw and Modified Starches by F. A. LYNE 133
6. Determination of Starch in Various Products by F. A. LYNE. 167
7. The Analysis of Starch Derivatives by J. VAN DER BI] 189
Index 215
vii
CHAPTER 1
The Microscopy of Starch

G. E. Moss
Commercial and Forensic Laboratories, 220-222 Elgar Road,
Reading, Berks. RG2 ODG, Great Britain
1.1 THE MICROSCOPE
The microscope is an extremely versatile instrumentl and it is the most

widely used and useful of the various instruments that have been applied
to the study of starches. It seems appropriate therefore, to briefly mention
some forms and adaptations that are of particular value to the starch
chemist.
The light microscope. In its simplest form, the light microscope is used
in conjunction with a tungsten filament lamp which can provide either top
or transmitted illumination. The latter is generally the more useful. Low
power magnification ( < x 100) is useful in assessing such items as 'extent
of aggregation' or 'purity'. Medium power ( x 100 to x 400) is suitable for
the identification and study of individual and small arrays of granules.
Higher powers (up to x 1500) may be required for studying granule
surface details and contaminants in gels. The modern microscope generally
has a turret carrying several objectives, permitting easy change of mag-
nifying power. Other features that have become fairly standard are:
Mechanical stage. This permits controlled movement of the specimen.
Sub-stage filter carrier. A blue filter is often used to balance the yellow-
ness of the tungsten filament lamp. A green filter is normally used for
photomicrography.
Binocular head. This is considered to be more restful to use, making

longer periods of use possible than with the monocular instrument. Its
1
J. A. Radley (ed.), Examination and Analysis of Starch and Starch Products

© Applied Science Publishers Ltd 1976
2 EXAMINATION AND ANALYSIS OF STARCH
use introduces an extra magnification factor, usually x 1·2 and marked on

the head.
These features are available for many modestly priced, as well as the
more expensive, standard instruments.
More specialised adaptions of the light microscope include:
The polarising microscope. In its least sophisticated (but still very useful)
form, this is an ordinary microscope having a polarising filter in its sub-
stage carrier and another polarising filter (analyser) held just above the
eyepiece (or in the eyepiece assembly) such that it can be rotated. Such a
simple form of polarising microscope is adequate for the observation of the
starch granule polarisation 'crosses'.
Measuring microscope. A microscope having an eyepiece graticule and

mechanical stage is suitable for determining the size distribution of
granules. The graticule should be calibrated against a stage micrometer,
and it is convenient to keep with the microscope, a table of calibrations
for the various eyepiece-objective combinations.
Hot-stage microscope. The Kofler hot-stage is an electrically heated box

with temperature control (up to 350°C) and thermometer well. Some
microscopes are specifically designed to receive a hot-stage. Others having
a circular stage with centering screws, a x 8 to x 10 objective having
sufficient working distance may with a little adaption, be fitted with a
hot-stage. Such an instrument provides an excellent means of measuring
gelatinisation temperatures by direct observation of the expanding
granule or its loss of birefringence.
Phase contrast. Instruments having special phase condensers and

matched phase objectives are useful for examining subjects that have such
small differences in refractive index that they would otherwise be invisible.
The technique is especially valuable in situations where staining of the
subject to improve visibility might produce artefacts. The author has
successfully used phase contrast illumination to study the form of
dispersions of gels prepared from modified starches.
Fluorescence microscope. Certain light microscopes having quartz iodine

light sources, may be equipped with appropriate filters, and so utilise the
near ultra violet wavelengths. Fluorochrome stains may then be employed
and their excited colours observed.
THE MICROSCOPY OF STARCH 3
Stereo microscope. The stereo or 3D microscope can be useful when

studying granule shape and aggregation. The relationship between these
factors and bulk density may well become clear from study of the 3-
dimensional image.
The electron microscope. The transmission electron microscope (used in
the study of structure) and the scanning electron microscope (used in the
study of surface topography) are essentially research instruments. They
are both currently playing an important role in advancing our knowledge
of the structural details of the starch granule. Clearly their application
differs markedly from that of the light microscope. Recent studies involving
the electron microscope are described in Chapter 2 of this book.
1.2 MICROSCOPICAL EXAMINATION

1.2.1 General considerations
Although starches from various plants may have similar analytical data
the form of the individual granules varies from sample to sample; and a
microscopical examination is a valuable diagnostic procedure for any
laboratory dealing with starches and starch products. Microscopical
examination may include the study of characteristic shape, granule size
distribution, the response to various stains and reagents, or measurement of
gelatinisation temperature.
1.2.2 Gelatinisation temperatures
In order to determine gelatinisation range, a starch is slurried in water
to make a 0·1 to 0·2 %dispersion. A small drop of the dispersion is placed
on a microscope slide, and a ring of viscous mineral oil is made around the
drop. A cover glass is then placed on the system which should be effectively
sealed by the oil and should contain no air bubbles. The slide is placed on
the stage of a hot-stage microscope and the temperature increased at 2°C
per minute. Since loss of polarisation crosses immediately precedes
swelling of the granules, observation of the disappearance of the crosses
affords the best means of observing the phenomenon. Not all the granules
swell or lose their birefringence at the same time, and it is appropriate to
record the temperatures at which first losses and 10, 25, 50, 75 and 90 %
birefringence losses are observed. 2
1.2.3 Mountants
Starches and starch-containing materials can often be mounted in water.
This mountant offers the advantage of subsequent staining by irrigation
(drawing a drop of stain solution under the cover slip by means of a piece
of dry tissue held against the opposite edge). Dextrins and pregelatinised
starches require non-aqueous mountants. Glycerol and Neutral Mountant
are both suitable and the latter may be used for making semi-permanent
slides (Fig. 1.1). It is possible to irrigate and partly displace these mountants
with iodine/potassium iodide for the purpose of proving identity of, for
example, pregelatinised starch fragments in a food mix. Other mountants
that have found use include dioxane-sandarac gum, and clove oil. The
latter is said to make the fissures in diastatically corroded granules very
prominent. 3
In general, for the examination of starches, it is highly desirable to use a
microscope fitted with polarising and analysing filters, and a calibrated
FIG. 1.1. Yellow potato dextrin swelling in aqueous glycerine (x 300). As the granules
swell, the peeling-off of layers may be observed.
eyepiece graticule. A mechanical stage is also a great advantage. When

examining granular starch, the size and shape of granules should be
observed; also the position of the hilum and any concentric rings around it.
The appearance of the granules in polarised light may also provide valuable
information, the granules of some starches showing a dark cross or V
character. The polarisation characters are very strong in some starches,
e.g. potato and less apparent in others, e.g. wheat. The characters may
disappear as the result of fracture by grinding or heat treatment. When the
dark cross is observed, the hilum is located at its intersection but as the
hilum becomes more eccentric the cross changes to the V character.
Over-dry or geiatinised granules have their internal forces relieved and the
refraction disappears. Remoistening extensively dried starch restores the
refraction.
The size of the granule is generally expressed as the length of its longest
axis in micro metres (1 f1m = 10- 6 m). The size of the largest and smallest
granules should be noted, as well as the average size of the sample.
The granules may be variously shaped according to source and may be
polygonal, round, oval or elliptical.
The shape of the granules from anyone plant may vary according to the
environment of growth. Maize starch granules which are angular and
highly refractive result from growth in a glutinous matrix which later
dehydrates and resists deformation by the growing starch granule. Those
granules from the crown of the maize kernel, however, are loosely packed
and are essentially round outline granules, being not constrained by
adjacent tissues during growth. Such granules are the fragile type, being
readily injured in grinding operations. Potato and arrowroot starches are
formed in moist conditions free from physical constraint and are rounded
or elliptical granules. Millet and rice starches grow in constrained con-
ditions and are tough, highly-angular granules.
The hilum varies in position, size and prominence for the various
starches. When a starch such as maize is dried to a low moisture-content,
the hilum often appears as a star-shaped crack. The form of such cracks
varies with the type of starch. Striations, if present in a granule can be
made more pronounced by treatment with dilute chromic acid solution.
When examining mixtures of starches and commercial products con-
taining starches, in addition to visual examination, two analytical
procedures may be employed:
(1) differential staining of components; or

(2) differential swelling in various reagents.
1.2.4 Staining techniques

Starches vary in their affinity for organic dyestuffs, and staining under
controlled conditions provides a useful means of obtaining information.
There are a number of quite different reasons for employing staining
procedures. The reasons for staining may be:
(1) To reveal starch granules in situ, for example, in the plant tissues or
in a food product; and staining may indicate the approximate
proportion of starch present in admixture with other ingredients.
(2) To differentiate between the various kinds of starch.
(3) To differentiate between various modified starches.
(4) To reveal damage in granules.
Several procedures for the staining of plant tissues are described in

detail by E. Gurr. 4 Kull's stain (acid fuchsin-aurantia-toluidine blue)
stains starch granules blue and mitochondria red. Schiiltze solution (chlor
zinc iodine) colours starch blue, proteins brown and cellulose walls violet.
Gram's iodine stains starch navy blue, proteins brown, cytoplasm light
brown, nuclei dark brown, chloroplasts blue or brown, cellulose walls
pale yellow and lignified walls deep yellow.
Successive staining by irrigation may help to differentiate such mixtures
as starch, protein and fats or oils in such items as food mixes and flours.
Dilute iodine/potassium iodide, Fast green and Sudan Blue are useful in
this context.
Since the starches of different plants react differently towards organic
dyestuffs, staining provides a means of distinguishing two or more different
starches in a mixture. F. D. Armitage 5 has pointed out, however, that
dyestuffs themselves may be of somewhat variable composition and it is
advisable to test samples prior to their regular application. This applies
particularly to soluble blue, orcein and safranine mixture used in Unna's
method. Armitage points out that anilin blue W.S. (C.L No. 707) which
has a slightly different formula from soluble blue, is the only one giving
the positive reaction with the Unna technique.
Suitable stains for permanent mounts are: methylene blue, safranine 0,
methyl violet and chlorazol fast red K. If the staining is carried out under
strictly comparable conditions, potato starch appears to stain more
heavily than wheat, maize and rice, in that order.
Metachrome red G agfa does not colour cereal starches,6 but stains
potato starch a bright golden yellow. It is important that the sample is
exactly pH 7 since in the presence of acid, wheat starch is also stained.
E. Unna 7 has proposed a differential staining procedure to distinguish

between potato, rye and wheat starches. The sample is suspended in a
mixture of 3 % phenol solution for 24 h. A drop is transferred to a slide
and allowed to dry. The slide is immersed in a solution containing soluble
blue, orcein and eosin in aqueous alcohol for 10 min, washed and immersed
in safranine solution for 15 min. It is then washed, immersed in 0·5 %
potassium dichromate for 30 min, washed with water, alcohol and xylene
and finally mounted in Canada balsam. Potato starch is stained dark red,
wheat starch pink, rye starch yellow to brown and gluten blue.
Potato starch is generally more heavily stained and more character-
istically stained than other starches. It behaves characteristically when
treated with Lamer's solution of methylene blue and examined in polarised
light. 8 According to A. P. Schulz and G. S. Steinhoff 9 it is identified by
staining blue with a mixture of methyl orange, fuchsin and methyl green.
Neutral red stains potato starch pink. Thionine has been used by G. Schutz
and L. Wein 10 to detect potato starch in bread. The potato starch is
stained lilac while remaining potato substance becomes reddish-violet or
blue. Both wheat and rye remain unstained. W. Neuwohner ll and Klauss 12
have used iodine/potassium iodide solution for the detection of potato
meal in wheat meal. The potato starch granules stain with a greater
intensity than the wheat starch. It is desirable when using iodine/potassium
iodide for staining starches not to exceed 0·3 % to avoid over-staining.
Iodine staining also distinguishes between red-staining waxy maize
varieties and blue-staining normal starch, the irrigation procedure
described by MacMasters 13 being applicable.
When examining instant food mixes containing pregelatinised starch and
sugar, it is of course necessary to use a non-aqueous medium such as
Neutral mountant. After mounting the particles thought to be starch, it is
then a simple matter to check the identity of the fragments by irrigating
with aqueous iodine/potassium iodide solution. This results in the staining
and slow swelling of the starch fragments, leaving no doubt as to their
identity. This procedure can be useful when a rapid outline analysis of a
powder product is required.
The adsorption of dyes by modified starches and starch derivatives
varies according to their ionic character. 14 Anionic character may be
imparted to starches by such means as oxidation, carboxymethylation and
phosphorylation. Cationic character may be imparted by the introduction
of substituted ammonia groups. Positively charged dyes such as methylene
blue (C.1.922), crystal violet (C.1.681), safranine 0 (C.1.841) and neutral
red (C.1.825), stain starches having anionic character. Negatively charged
dyes such as light green SF yellowish (C.1.670), acid fuchsin (C.1.691)

and Orange G (C.1.27), stain starches having cationic character.
When testing for anionic or cationic character in the above manner, it
is important to first wash the sample free of any acidity, alkalinity or
associated salts. Disperse approximately 50 mg of the starch in 20 to 25 ml
of the 0·1 % dye solution for 5 to 10 min with occasional agitation. The
starch should then be thoroughly washed with several aliquots of water
until the washings are clear and examined. The presence or absence of
staining indicates the ionic character of the starch. The intensity of
staining indicates the extent of modification.
Any starches that have been treated with anionic or cationic surfactants,
will possess ionic character due to the absorbed surfactant. Such starches
will also respond to positively and negatively charged dyes respectively.
The ionic character of surfactant-treated starches is however removed by
soxhlet extraction using 95 %ethanol.
Methylene blue has been used to differentially stain a dry blend of
pregelatinised corn starch and pregelatinised carboxymethyl starch. 14
The corn starch stains faint blue while the carboxymethyl starch stains
deep blue.
Reactive dyes, such as Levafix E-type, have been used by R. Stute 15
and H. U. W oelk 16 for the detection of structurally altered starch granules.
Structural alterations occurring from mechanical damage, heat-moisture
treatment or freeze drying can be deduced from the dye binding capacity,
which correlates with changes in X-ray pattern. The staining of the
granules by the reactive dyes is permanent.
Aqueous Congo Red (0·1 to 0·35 %) only stains starch granules that are
damaged. C. R. Jones 17 has referred to the damaged granules of wheat as
'ghosts', after their feint outline appearance. When mounted in congo red
solution, the whole of each damaged granule is uniformly stained orange-
pink. Any gluten is coloured bright brown, and it should be noted that
endosperm cell-wall tissues are stained a vivid pink.
1.2.5 Swelling methods

The starches of different plants may be distinguished by observing the
effect of various swelling agents. W. H. Symons 18 measured the concen-
tration of sodium hydroxide solution required to gelatinise the majority
of the granules of a sample. He found that when 0·1 g starch was stirred
with 1 ml sodium hydroxide solution and examined under the microscope,
the percentage concentrations of sodium hydroxide required were:
potato, 0'7; oat, 0·8; tous les mois, wheat and sago, 0'9; maize, rice and
cassava, 1·0. K. Baumann 19 used the fact that maize starch is less readily
swollen by 1·8 %potassium hydroxide solution than wheat or rye starches
and that the latter is ruptured more readily than wheat. Following
gelatinisation, the maize starch can be made more apparent by staining
with iodine. 20 Rye starch21 ,22 is also distinguished from other starches in
that the granules gelatinise faster in 9 % sodium salicylate solution. The
larger granules swell within one hour and lose birefringence. J. A. Radley23
has observed that 38 % formaldehyde solution swells potato, tapioca,
wheat and maize starches in that order, the swelling of potato and tapioca
being much more rapid.
1.3 MICROSCOPICAL APPEARANCE AND CHARACTER
Several textbooks have been written that provide very full descriptive data
of starches from a large number of sources. 24 - 29 The following descrip-
tions relate to a selection of starches of commercial importance, and the
reader is referred to the more specific literature for descriptive data
concerning those starches not mentioned.
1.3.1 Potato starch

The granules of potato starch vary greatly in size and shape; the largest
are often egg-shaped and are visible to the unaided eye. The majority are
flattened ellipsoids and the smallest may be perfectly spherical (Fig. 1.2).
The granules generally occur singly, although compound granules con-
sisting of two or three units may be seen on rare occasions. Granule size
ranges from 15 /lm to 100 /lm but O. Hoyer,30 who has examined samples
from many sources, considers the upper size limit to be 121 /lm. In the
trade, the starch having the largest average granule size is considered best
for some purposes. Average size for samples may range from 35·5 /lm to
12·5/lm.
The excentric hilum towards the narrow end of the grain is normally
well marked and surrounded by numerous concentric rings; these being
very distinct on some grains. The cross observed with crossed polariser and
analyser is well-defined, of irregular shape and centred at the hilum.
O. A. Sjostrom31 noted that during gelatinisation, the hydration develops
units of rounded form, arranged in an irregular manner. These show
individual striations that become obliterated as swelling proceeds.
The granules of thin-boiling starches made from potato starch, tend to
swell more in relation to original size than do those made from tapioca
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and maize starches. The hydration phenomenon shown by the unmodified

granules is also observed in the thin-boiling type. If potato dextrin is
mounted in glycerol-water mixture, the peeling off of layers can be
observed. 23 The difference between the mode of disintegration of potato
dextrin and thin-boiling potato starch is greater than with cereal starches.
According to V. Vilikovski,32 very dry seasons diminish the size of
granules formed in potatoes, and to obtain large granules, potash fertilisers
should be used. M. Dudkin and L. Serdyuk 33 have studied granule size in
relation to potato variety.
1.3.2 Wheat starch

Wheat starch granules are biconvex discs with a fairly regular circular
outline. Perfectly circular grains are more rare than in rye starch. They do
.,.,
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FIG. 1.3. Rye flour (approximltely 60 % starch) ( x 500) .

not show distinct striations or polarisation crosses, and the hilum is only
observed in a small number of grains. Granule size ranges from 35 JIm to
2 JIm and they are generally referred to as 'large' and 'small' granules.
The large granules are usually 25 to 35 JIm while the small ones are usually
2 to 8 JIm. The upper limit for size, according to O. Hoyer, 3 0 is 55 JIm.
When heated in water the granules swell, and at near boiling point,
they assume a characteristic curved shape, which has been used by
o. A. Sjostrom 31 to identify wheat starch in technical products. Rye
starch behaves similarly.
1.3.3 Rye starch

Except that they are larger and thicker, the starch granules of rye appear
similar to wheat (Fig. 1.3). The majority of granules are spheriodal, but
FIG. 1.4. Barley starch (x 500).

among the smaller granules bell-shaped ones are sometimes seen. Greenish
and Collin 25 state that, hat-shaped and bell-shaped ones are to be found
among the medium and smaller grains. Some grains have a very distinct
stellate central hilum and some show fine concentric rings. The granules
may be as large as 50 11m. J. G. A. Griffiths 34 has found that 5 to 11 %of
granules of diameter greater than 10 11m have stellate hila.
1.3.4 Barley starch

The granules of barley starch may be bulb-shaped elliptical or circular
in outline (Fig. 1.4). The larger granules are usually in the range 20 11m to
35 11m and the smallest 2 11m to 6 11m. The hilum is even less often seen than
in wheat. Some of the larger grains show striae. Barley has a larger pro-
portion of very small granules than wheat. The polarisation crosses are
indistinct. When barley and wheat starches are mixed, it is difficult to pick
them out. It is an easier matter to distinguish between the fragments of
chaff or bran of the two cereals.
1.3.5 Maize starch

Maize exhibits the polygonal type of starch granule (Fig. 1.5). The
granules from the more flinty part of the maize grain are angular in
outline, of fairly regular polyhedral shape and with well marked central
hilum. Those from the mealy part vary more in shape, some being almost
spherical. A striking point is the greater uniformity in size of the maize
starch granules, generally 10 11m to 25 11m. If the corn is of a soft mealy
variety the starch contains a preponderance of rounded granules having
an average diameter of 13 11m to 15 11m. Pickens and Englis35 find that
granules of hard corn starches are smaller and less rounded. The hilum is
well marked and starred with fissures, but striae are not normally observed.
Striae may, however, be observed in a few granules when viewed in
highly favourable conditions. According to Galt,24 concentric rings are
found only in granules of rounded form. The polarisation crosses are
distinct.
1.3.6 Oat starch

Oat has smaller granules than maize, being mostly in the range 5 11m to
811m. The grains are more or less polygonal or angular. They are often
united into characteristic rounded aggregates of variable size. When the
aggregates are broken apart, the central grains are generally seen to be
polygonal, while those from the periphery may be curved on one face.
Several small grains will sometimes be seen united end to end resembling a
FIG. 1.5. An oxidised maize starch (a thin boiling maize starch made by oxidation)
( x 400): (top) normal light; (bottom) polarised light.
rod-like structure. Occasionally among separated grains, lemon or

spindle-shaped grains will be observed. Hilum and striae are rarely visible.
1.3.7 Rice starch

The granules of rice starch are similar to those of oat, but smaller,
being mostly 3 pm to 5 pm diameter (Fig. 1.6). The central hilum is
difficult to observe and striae are visible only after treatment with dilute
chromic acid. Compound grains comprising several granules are some-
times observed, which present an overall angular outline. M. Wagenaar 36
has detected particles of rice in wheat, rye, oat, barley and buckwheat
flours by staining with fuchsin S. The acid dye is adsorbed by the protein
grains of the rice, and the revealed distribution of these protein grains
gives a characteristic appearance to the fragments of rice flour.
FIG. 1.6. Rice starch (x 500).

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n' n o ·'
1.3.8 Pea starch

This starch has features that are typical of the leguminous starches
(Fig. 1.7). The granules are translucent and rounded in outline, some being
elliptical. Each has a plane of symmetry such that both ends of the
granule appear the same. The granules are of very uniform size and the
hilum is a dark slit lying in a shallow depression running along the long
axis. The granules never show a stellate hilum unless fractured, and when
the slit is absent, the depression can still be seen. Concentric striae are
visible, and with polarised light, a dark V is seen at either end, the apex
of each touching either end of the hilum.
1.3.9 Sago starch

The granules of this starch are similar to medium-sized potato starch
granules (Fig. 1.8). The size is in the range 15 p,m to 65 p,m and mostly
20 p,m to 60 p,m. The granules are oval or egg-shaped, and some are of
truncated oval form. Striae are seen as faint concentric rings on the larger
granules. The hilum is excentric and the polarisation lines cross it in an
irregular manner. When gelatinised by heating in water, the swollen sago
starch granules appear as smooth bag-shaped forms that are not destroyed
by boiling. The swollen granules nearly always show a well-defined crater-
like opening at the end. This is a distinguishing feature by which sago
starch differs from arrowroot; which swells to a more irregular form having
a larger opening.
1.3.10 Arrowroot starches

The arrowroot starches (Fig. 1.9) really comprise a group of starches
prepared from Maranta arundinacea grown in widely separated localities.
They are found in the trade as St. Vincent, Natal, West Indian, Jamaican
and Bermuda arrowroots. The granules are similar to potato but differ in
size, being 13 p,m to 70 p,m and mostly 27 p,m to 54 p,m. The hilum is
usually excentric but may be central, and is sometimes fissured in the
form of a double curve. Concentric striae are always visible. The above
arrowroot starches all appear the same under the microscope, although
some physical properties may differ. Tous-Ies-mois is a variant having
much larger granules.
1.3.11 Cassava starch

The granules of cassava, manioc or tapioca starch are similar in size to
maize, and range from 5 p,m to 35 p,m (Fig. 1.10). The largest are usually
.-. \
\ .. ~
,} ~: .,
'l ,. ...,
. p ,,, ::ctIl
",
.. ~
.. 61 n
:;:0
" ~) , ~ '",
....... o
'-. "
,~ 8'""d
- , ii ><
'-It, o.."
... , '"
~ " ~
:;:0
()
,. ~,
::c
~~
~) C)".
FIG. 1.9. St Vincent arrowroot starch (x400): (left) normal light; (right) polarised light.
\0
-
TABLE 1.1
Microscopical characteristics of various starches
Name Source Granule size Granule shape Hilum Striae Fissures Comments
1. Acorn 5-20 pm Hemispherical or Simple and

'kettledrum' compound granules
2. Canna Canna edulis, 10--130 pm Elliptical, oval, Round and Distinct

coccinea most frequent- oyster or excentric
60 pm reniform
3. Curcuma Curcuma 30--60/lm Oval, elliptical, Punctiform Very distinct

species almost and very
(East rectangular or excentric
Indian rounded, thin
arrowroot)
4. Curcuma C. Leucorrhiz Generally

105/lm;
up to 145/lm
5. Batata Ipomoea Components- Components- Distinct and Well marked Some compound
batatas 10--50/lm often hemi- excentric granules 2-12, but
spherical generally 4-5
Brazilan Simple 8-25 /lm Centric or sI. Distinct components. Mostly
arrowroot excentric single. Some simple
6. Banana Musa Longest diam. Round to rod Distinct, very Well marked
paradisiaca 7-58/lm shaped excentric
generally
24-48/lm
7. Buckwheat Polygonum 4--15.um Simple polygonal Contains character-
/agopyrum chiefly 9 .urn similar to rice istic compound
granules of 2-9
components which
are somewhat
rounded
8. Arum Arum 3-27.um chiefly WeIl marked Indistinct or Radial from Compound granules
esculentum 13-20.um absent hilem 2-10 components
of unequal size
9. Broad bean Vida/abia 20-40.um Simple reniform, Centric Visible but OccasionaIly,
oval or rounded not marked from hilum
10. BuIlrush Pennisetum 8-25 .um Irregular or Centric Not visible OccasionaIly
MiIIet typhoideum round,
(Nigeria) polygonal
11. Chestnut Castanea 1·5-3O .um Variously shaped. Not Not visible Occasional twin and
vulgaris Only few of Large-three observable triplet granules
intermediate or four-sided,
sizes heart, kidney
or club shaped.
SmaIl-
rounded, egg
or pear shaped
12. FritiIIaria Mussel and bean Various Observable at Occasional compound

shaped Mussel shaped pointed end granules,
granules Oval triangular Observable at 2-3 components
32-71 .um Bean shaped pointed end
Breadth Observable on
27-55 .um concave side
Spherical SmaIl spherical
9-17.um concentricaIly
built (continues)
TABLE l.l-contd.
13. French bean Phaseolus 30-50 11m Rounded or oval Centric Fairly General, from
vulgaris marked hilum
14. Bean, Scarlet Phaseolus 20-35 Ilffi Rounded or oval Centric Not distinct General, Size differentiates
runner multif/orus radial from from French bean
hilum
15. Colocasia 10-55 11m Regular and oval Fairly Only on

esculenta distinct, inner part
at narrow of granule,
end then wide
zone with
none
16. Millet, Sorghum

Dhurra vulgare
(Durra, Dura) (Sudanese)
17. Millet, Sorghum I
Rounded
Nigerian, vulgare ~ Fissured, freq.
15-35 11m polygonal Centric Not visible
White Guinea (Nigerian) r stellate
shape
corn
18. Millet, Indian Sorghum
Jowar vulgare
(Intama)
19. Millet, Panicum 8 to 15 11m Polygonal or Centric and Not visible Sometimes sl.
Shama colonum Some up to rounded punctiform fissured
(India) 20 11m polygonal
20. Millet, Setaria 8-15 11m Rounded or Centric, Not visible Sometimes sl.
Italian italica polygonal fairly fissured
(India) distinct
21. Millet, Ragi Eleusine 6-20 pm Polygonal Centric, Not visible Sometimes sl.
coracana sometimes fissured
(India) faintly
fissured
22. Maranta Maranta 13-70 pm Similar to potato Sometimes Always Hilum often Always simple
(original arundinacea Chiefly Note size centric, visible, fissured in
Arrowroot) (West 27-54 pm difference more but not form of
Indies, often strong double
Jamaica, excentric curve like
Bermuda, flattened OJ
StVincent,
and Natal
23. Maranta Maranta 11-34 pm Simple Centric Only distinct Radial after Compound granules
nobilis after chromic observed very
chromic acid rarely, 2-5 small
acid treatment components
treatment
24. Mango Mangi/era 5-25 pm Mostly compound Very distinct Easily visible
indica very few simple
granules
25. Horse Aesculus Small 3-8 pm Round or Distinct and Concentrically Both simple and
Chestnut hippo- elliptical. often striated compound in small
castaneum Simple and fissured
some compound
Large 14-35 pm Simple and Distinct and Both simple and

compound. excentric compound in large.
Pear-shaped or Compound consists
conical, irregular of 1 large and 1 or
2 small
(continues)
TABLE l.l-contd.
26. Tacca (Tahiti Tacca 25-45 J.lfD. Simple---elliptical, Centric or Distinct

or Williams pinnatijidia egg or pear- only sl.
Arrowroot) and T. shaped excentric.
Oceanica Distinct
27. Waxy 6-30 pm Similar to maize Centric and Indistinct Distinct and Red iodine
Sorghum Chiefly about distinct radial from coloration
(Red Leoti) 15 J.lfD. hilum
28. Waxy Maize 5-25 pm Similar to Centric and Red brown

common maize distinct coloration with
iodine
29. Sweet potato 10-25 pm Polygonal Centric and Faint if any

predominate distinct
30. White Dioscorea Small- Simple, irregular, Distinct Distinct Greyish yellow colour
Dioscorea alata 15-30 J.lfD. long, oval or elliptical excentricity
7-15 pm wide, or rounded of 1/5-1/7
large-45-90 J.lfD. triangular.
by 25-60 pm Larger
wide extremity of
granule often
truncated
31. Yellow Igname 8-55 11m Much tissue Not readily Fairly Intense greyish
Dioscorea indien present. visible distinct yellow to brown
jaune Elliptical, egg, except in and very colour
pear- or heart- glycerol, numerous
shaped, but do is then
not appear distinct
truncated
32. Red Igname 17-119 11m Parenchymatous Distinct. Distinct Dirty red powder.
Dioscorea indien tissue generally Excentric Colour of granules
rouge present. turns bright red
Granules have with acid, blue
red colour. with alkali
Like White
variety but
narrower
33. Zamia 5-75 11m Hemispherical or Not well Not well Radial Mostly compound
(Florida dome shaped. marked marked fissures granules, 2-8
arrowroot) Some with when when components, often
large flat faces fresh. In fresh. In 3 components in a
and sl. rounded commer- commer- row. Clusters of
ends (middle cial cial calcium oxalate
components of prepara- prepara- crystals 14-60 11m
rows of 3) tions- tions- in diameter,
distinct distinct valuable aid to
i den tifica ti on
tv
00
ss::
~...,
(5
Z
>
Z
t:I
>
~r
><
'"t;J
o.."
'"
~
::c
("l
::r:
FIG. 1.12. Edible canna starch (x 375): (le/t) normal light; (right) polarised light.
• ', . L. G •
~
~o·b 00 (,. ,..-,

. D ,/ ...
.. .~ . " .
Q •
~
"' o. · ') s:::
~. ~ · D r
' h ~.) "''''~~';'.'' (~,
" .. . . ~. ../' ~
o
(")
'"
o
'" J~:I ~ / / ~/ .' .
-" ~
\....."11
.,. ...... ' • ..., ~, ~',' . ,
~
. . . ."') .. ," r: ' .
~ ~~oa
GJ ~. o ---~ Ti .. 0 '. \ , . g~
' '.
. c:)( ~ - .- . , .,J
r. .,.........-
· I .'
~' .•. v." ,"
/
." ' ' , '
.' "
FIG, 1.13. Green plantain starch (Musa pardisiaca L.) (x 188): (left) normal light; (right) polarised light.
tv
'C>
w
o
o 0
tl1
o C0 0
o 0 0 ~
~o . 0 ~
::l
.0 ()0'Oo a o
o z
0 0 "), ~()
(, rQO:n:) flcJ.cpO ~
QQ- ,~ (T oO
,PO 0 0 o 0 ~
~
~
en
\J o'rl
Q o~ en
>-3
>
::0
f, \ 0 0.0 ("l
0~ I II:
o
FIG. 1.14. White sweet potato starch (Ipomoea batatas) (x 375): (left) normal light; (right) polarised light.
25/tm to 35/tm and the smallest 5/tm to 15/tm. The granules appear round
with a flat surface on one side containing a conical pit which extends to a
well defined excentric hilum. Some granules are very nearly circular. In
polarised light, a well defined dark cross is observed.
Table 1.1 lists brief microscopical details for a number of the less
common starches. Photomicrographs of some of these are shown in
Figs. 1.11-1.14.
REFERENCES
1. Lyne, F. A. and Moss, G. E., Chem. & Ind., 1971, 388-392.

2. Schoch, T. J. and Maywald, E. c., Analyt. Chem., 1956,28 (3), 382-387.
3. Berliner, E., Miihlenlab., 1939,9, 13 and 87.
4. Gurr, E., The Rational Use of Dyes in Biology, Leonard Hill, London, 1965.
5. Armitage, F. D., Industrial Chemist, 1943, 18, 583.
6. Blunck, G., Zeit. Nahr. Genussm., 1918,36,49.
7. Unna, E., Zeit. Nahr. Genussm., 1918,36,49.
8. Bengen, F., Zeit. Nahr. Genussm., 1915,29,247.
9. Schulz, A. P. and Steinhoff, G. S., Zeit. Spiritus. Ind., 1932,55, 162.
10. Schutz, G. and Wein, L., Chem. Zeit., 1915,29, 143.
11. Neuwohner, W., Zeit. Tierernahr. Futtermitt., 1939,3, 1.
12. Klauss, Vorratspflege Lebensmitt, 1938, 1 (6--7).
13. MacMasters, M. M., in R. L. Whistler (ed.), Methods of Carbohydrate Chemistry,
Vol. IV, Academic Press, New York, 1964.
14. Schoch, T. J. and Maywald, E. C., Analyt. Chem., 1956,28 (3), 382-387.
15. Stute, R., Die Starke, 1973,25 (12), 409.
16. Stute, R. and Woelk, H. D., Die Starke, 1974,26 (1), 2.
17. Jones, C. R., Cereal Chem., 1940, 17, 135.
18. Symons, W. H., Pharm. J., 1882, 13, 237.
19. Baumann, K., Zeit. Nahr. Genussm., 1899,2,27.
20. Embrey, G., Analyst, 1900,25,315.
21. Lenz, W., Zeit. OjJentl. Chem., 1910, 15,224.
22. Greenish, H. G., The Microscopical Examination of Food and Drugs, Churchill,
London, 1923.
23. Radley, J. A., Starch and its Derivatives, Chapman & Hall, London, 1943.
24. Galt, H. G., The Microscopy of the Starches, Bailliere, London, 1900.
25. Greenish, H. G. and Collin, E., An Anatomical Atlas of Vegetable Powders, Churchill,
London, 1904.
26. Morris, T. N., Microscopic Analysis of Cattle Foods, Cambridge Univ. Press,
London, 1917.
27. Winton, A. L. and Winton, K. B., The Structure and Composition of Foods, 4 Vols.,
Wiley, New York, 1932-39.
28. Czaja, A. T., Handbuch der Starke, Die Mikroskopie der Starkekorner, P. Parey,
Berlin, 1969.
29. Seidemann, J., Starke-Atlas: Principles of Starch Microscopy and Description of
Varieties of Starch, P. Parey, Berlin, 1966.
30. Hoyer, 0., Chem. Zentralbl., 1911,2, 305.
31. Sjostrom, O. A., Ind. Eng. Chem., 1936,28, 72.
32. Vilikovski, V., Chem. Listy, 1911,5,412.

33. Dudkin, M. and Serdyuk, L., Fiziol. Biokhim. Kul't. Rast, 1974,6 (2), 163-5.
34. Griffiths, J. G. A., Analyst, 1937, 62, 510.
35. Pickens, L. and Englis, D. T., Food Res., 1940, 5, 563.
36. Wagenaar, M., Zeit. Unters. Lebensm., 1937, 54, 357.
CHAPTER 2
Electron Microscopy of Starch and Starch Products

D. J. GALLANT
Research Officer, Ministry of Agriculture, Station of Cereal Bio- and
Physical Chemistry, National Institute of Agronomical Research,
CERDIA, 91305 Massy, France
and
C. STERLING
Department of Food Science and Technology, University of California,
Davis, California 95616, USA
INTRODUCTION
For many years our knowledge of the physical structure and behaviour
of starch grains was based on evidence from study with the light micro-
scope. However, with that microscope it is not possible to explore the
so-called submicroscopic or 'fine' structure of starch. The smallest
resolution possible with the light microscope is about 0·2 pm.* Structures
with diameters smaller than this value cannot be seen because points
closer together than this distance cannot reflect light rays so that all will be
in the same phase. Cancellation of rays will occur and there will be no
discrete image.
• The meanings of the infra-submultiple unit prefixes adopted by the International

Committee on Weights and Measures are as follows:
Prefix Symbol Submultiples
milli m 10- 3
micro p 10- 6
nano n 10- 9
pico p 10- 12
Thus 400 nm (400 nanometers) = 0·4 pm = 4000 A (1 A = 0·1 nm).
33

Resolution, p, is a function of the wavelength of light, according to the

relationship of Abbe, 1
p oc 0·6U/A (1)
where A. is the wavelength of light in air, and A is the numerical aperture

of the objective lens. A is given by n sin oc, n being the refractive index of
the medium between object and objective lens and oc the aperture angle,
i.e. one half the cone angle of the light beam between object and objective.
Therefore, in the light microscope the wavelength of visible light is a
major limiting factor in the resolution of fine structure. It is in this respect
that the electron microscope has given the possibility of resolving details
of many orders of magnitude less than the light microscope. Where the
smallest wavelength of visible light is about 400 nm, the usual wavelength
of an electron beam is about 0·004 nm (4 pm). If the numerical aperture
for an electron beam were of the same magnitude as for the light micro-
scope, the potential resolution would be even less than the diameter of an
atom. It is this potentiality which has conferred a very great interest on
the use of electrons in microscopy. Thus, thanks to pioneering studies
around 1930 and later developments after 1950, there has been a spectacular
development of equipment for electron microscopy and a correspondingly
widely extended use of this equipment, resulting in completely new insights
into the fine structure of organic and inorganic bodies, often down to
molecular dimensions. The ultimate resolution of present-day electron
microscopes is of the order of 0·2-1·0 nm.
Today two principal techniques hold the most promise for the study of
fine structure: transmission electron microscopy (TEM) and scanning
electron microscopy (SEM). Although both use a primary electron beam
and an electromagnetic or electrostatic field to focus that beam, the
fundamental principles of image formation in each are quite different.
In TEM, observations are made on a fluorescent screen which is directly
bombarded by the electrons of the primary beam after the beam passes
through the sample. The sample must be extremely thin and must present
differences in electron absorption in its morphologically different parts.
In SEM, on the other hand, the irradiated specimen may be of any thick-
ness because a secondary radiation of electrons from the surface of the
specimen produces an apparent three-dimensional image on a remote
screen via photomultiplication. The sample must therefore have good
electrical conductivity lest the secondary beam be excessively strong
(because of a charging effect) and efface the image.
ELECTRON MICROSCOPY OF STARCH AND STARCH PRODUCTS 35
2.1 THEORETICAL ASPECTS OF THE ELECTRON

MICROSCOPE
The mathematical development of the principle of focalisation of an

electron beam was accomplished by H. Busch as a direct application of the
Hamiltonian theory.2 Busch created an analogy between the dynamic
behaviour of a point situated in a field of forces and the focalisation of a
luminous beam passing through environments of variable refractive
indices as developed by Hamilton. The 'point', of course, is the electron,
conceived of as a dual corpuscle-wave entity.3
It will be useful to digress slightly at this point to consider the manner
of production of a beam of continuously emitted electrons and how these
may be influenced in an electromagnetic field. In an enlarged type of
cathode ray tube a tungsten filament, the cathode, is heated to incandescence
(ca. 5000°C). If a difference of potential of many thousands of volts be
Filoment
Electron gun
Co thode and Wehnelt
--+---'- Anode
....
Electro - : : : : Object ~
Object
__ - 1,:,_ /
Condenser lens
....
". :<r- ~
< :'
...- ........
mognet ic •••• I" .>
lens •••• '
[ .
••••
Electrons
Cathode ray tube
Image Fluorescent screen Image 1 Screen

(b)
(a)
FIG.2.1. (a) Focusing of the electron beam by an electromagnetic field created around
wire coils. (b) Focusing of a beam of light by a vitreous condenser lens.
established between this cathode and an annular anode, electrons will be

emitted in a stream from cathode to anode, particularly if all other
particles have been removed under high vacuum so that there is no physical
barrier to the passage of the electrons. The electrons are accelerated by
the potential difference and pass in a straight line through the hole in the
anode. In TEM, the primary electrons strike a fluorescent screen at the
base of the tube. If an object is situated in the path of the beam, it will give
an image on the screen, a sort of shadow, because of its absorption and
deflection of those electrons which strike it. However its outline on the
screen will not be sharp because the beam is divergent and the edges of the
object scatter electrons out of phase. Now if an annular coil be placed
about the tube, and a current be passed through it, the magnetic field
created about the coil will interact with the electrons so that there is a
deflection of their trajectory, in a focusing effect. With the correct adjust-
ment of the coil current, it is possible to obtain a clear image of the object
on the screen (Fig. 2.1(a)).
It is immediately apparent that there is a physical parallel between the
focusing of light by a vitreous lens and the bending of a beam of electrons
by an electromagnetic field (Fig. 2.l(a) and (b)). Indeed the electro-
magnetic field, for a charged particle like an electron, can be regarded as
equivalent to a medium with a fictitious refractive index, and it is common
to speak of the annular coils which provide these fields in an electron
microscope as 'lenses'.
The amount of convergence brought about by the electromagnetic lens
is proportional to the so-called excitation factor, K:
(2)
where N is the number of turns in the coil, I the current passing through it,
and V the voltage, i.e. the difference in potential between cathode and
anode. Thus, by varying the accelerating voltage, it is possible-with the
use of a simple potentiometer-to vary continuously the excitation factor
of the electromagnetic lens. Equation 2 also shows the need for stability in
voltage and current for the focusing of the electromagnetic lens; the
slightest variation in either will change the angle of convergence of the
electron beam-and this angle, being normally quite small, is extremely
sensitive for correct focusing. This problem of sensitivity is related to the
very short wavelength of electrons and to the small diameter of the
electron beam.
The wavelength, A, of the electromagnetic vibration associated with the

displacement of the electron is given by the de Broglie equation: 3
A=~ (3)
mv
where h is Planck's constant and m the mass of the electron, corrected for
the velocity v. An approximate value of A may be found from the relation-
ship, (150/V)t, V being the accelerating voltage. Thus A at 50 kV is 5·4 pm;
at 100 kV, 3·7 pm and at 1000 kV, 0·9 pm. 4
Recalling that the general relationship for resolving power in the light
microscope is that it varies as a direct function of the wavelength and as
an inverse function of the numerical aperture (eqn. 1), it is to be stressed
that the numerical aperture of an electron beam is exceedingly small. The
conical aperture angle of the beam at the electromagnetic lens amounts to
no more than 10- 3 to 10- 4 radians. This limiting angle is imposed by
several considerations, the most important of which are the angular
aberrational effects. Thus, chromatic aberration is directly proportional to
the aperture angle, and spherical aberration is proportional to the cube of
that angle. The angular openings, and correspondingly the beam diameter,
must therefore be limited by the use of diaphragms with small pinholes in
the electron microscope. Additional collimation of the electron beam is
provided by the Wehnelt, a small cylinder that surrounds the cathode
(tungsten filament) and is negative to the cathode. Playing the role of an
electrostatic lens, the Wehnelt concentrates the electrons emitted by the
cathode into a fine-pencilled beam.
It should be pointed out that although the small aperture angle of the
electron microscope increases the resolution, it also increases the depth of
focus, which is a function of the resolution divided by the aperture angle.
For the light microscope, this depth is only 0·2 pm at a magnification of
1000, but for the electron microscope at the same magnification, the depth
of focus could be from 2·5 pm to 50 pm, depending on the aperture angle
used. 5 This depth of focus is extremely necessary in SEM and is occasion-
ally quite useful also in TEM.
The velocity of the electrons depends on the difference in potential
between cathode and anode. Thus the kinetic energy of the incident
electrons, which determines the power of penetration and scattering of
electrons in the specimen, is in large part dependent on the accelerating
voltage. For biological materials a voltage of 60-80 kV is adequate for
present-day observations with TEM, and the transmitted beam will be
satisfactory both for activating a fluorescent screen and for reducing the
silver grains in a photographic emulsion. In SEM, where minimal energies

of the incident beam are sufficient, the high voltage will generally range
from 1 to 25 kV.
2.2 SPECIAL FEATURES OF TEM
2.2.1 Apparatus
There is a close resemblance between the physical structure of a trans-
mission electron microscope and the classical light microscope (Fig. 2.2):
each has a system of three lenses (condenser, objective and ocular) which
are arranged in superposed fashion in the optical axis of the apparatus.
Object
on grid
Objective
1-~r~==::L Aperture
Intermediote
lens
Window
k----::;!;;:===;;iI-- Scree n
Projector
lectrons /
~
bserver
Pump
FIG. 2.2. Diagram of the transmission electron microscope.

The column in the former is usually much larger: often more than I m
high. Also, a vacuum is created in TEM, by the use of fore-pump and a
diffusion pump, amounting at least to 10- 5 mm Hg.
The formation of an image proceeds in a similar way. The light source
here is the 'electron gun', emitting the beam of electrons. This beam is
focused on the plane of the object by the condenser (C). The object absorbs
or scatters the incident electrons, and the transmitted beam is enlarged
several times by the succeeding lenses. These latter are the objective (0)
and then an ocular, composed of two lenses: the intermediate lens (LI),
of variable focus, permitting continuous magnification from 1 to 50
diameters, and the projector (P), of fixed magnification, which enlarges
the intermediate image to its final size by projection on a retractable
fluorescent screen or a photographic emulsion. The total magnification can
reach 100000 to 150000 diameters.
However, for a body to be seen with TEM, fine resolution and high
magnification are not sufficient. There must also be adequate contrast to
(a) (b) (c)
0ei eei
' ...
z+ e~
ei
e
~ ei
FIG. 2.3. Interaction of beam electrons (eJ -) with an atom: (a) elastic scattering;
(b) inelastic scattering; (c) no scattering. (ea - = atomic electron, Z + = atomic nucleus.)
the surrounding region in the image. Contrast is a function of the amount

of scattering (deflection) within the microscope, due to the interaction of
electrons with the atoms of the specimen. The electrons may be scattered
by interacting with the nucleus of the bombarded atom (elastic scattering,
Fig. 2.3(a)); by interacting with the electrons of the atom (inelastic
scattering, Fig. 2.3(b)); or they may pass through interatomic space
without deflection (Fig. 2.3(c)). The amount of scattering is directly
proportional to the size of the atoms in the specimen and inversely propor-
tional to the energy of the incident electrons. Because the atoms of
biological materials are generally rather small (C, H, 0, N), there will be
little scattering, and the trajectory of the electrons will be nearly linear.
When heavy atoms (Mn, Os, Au, Ag, Pb) are present, the incident electrons
interact strongly with them and thereby experience both energy loss and
considerable deflection.
These factors exercise the following requirements in specimen prepara-
tion: (1) the specimen must be very thin, usually of the order of 50 nm or
less, otherwise the electrons are scattered and lose energy to such an extent
that they cannot emerge from the specimen; (2) if the specimen is com-
posed only of elements of low atomic number, contrast must be enhanced
by introducing atoms of high atomic number. This is especially the case
with starch.
2.2.2 Specimen preparation

The usual techniques of TEM2, 5,6 are ultrathin sectioning (which may
be combined with heavy metal staining, autoradiography or shadowing)
and the production of replicas, almost always involving shadowing. These
methods, with various modifications, have been applied to starch, but
their success has been limited. Not only has the normal starch grain seemed
to be quite homogeneous, but it is also characterised by unstable behaviour
and variable reactivity.
In most cases the sample must be supported on an ultrathin membrane,
'transparent' to electrons, which is in turn supported on a screen-like
metallic grid, with from 100 to 400 meshes per inch. The specimen is
observed through the open meshes. The ultrathin membrane can be formed
by letting a drop of dilute solution of polymer (collodion or formvar)
spread on the surface of water; or the membrane may be a thin film of
carbon or silicon, produced by evaporation under vacuum. Usually
ultrathin sections, cut on a special microtome from material embedded in
a cross linked resin after chemical fixation and dehydration, are mounted
on the membrane covering the grid. 7 -11 However, some newer resins are
now tough enough so that sections may be put directly on a grid without a
supporting membrane.
Generally sections of the normal starch grain are devoid of identifiable
subunits, and it is necessary to bring about some sort of artificial differen-
tiation in the starch substance. Thus, more amorphous or looser regions
can be preferentially dissolved by acid hydrolysis 12 - 24 or by enzymatic
digestion 7.12.18.21.24-37 before the starch grain is embedded and
sectioned. Some workers have used amylase on the thin sections of the
starch grain as they lie on the grid. 7,32,38,39 Another approach to differen-
tiation is to disperse starch material, as by gelatinisation, 40- 42 placing a
droplet of the gelatinised suspension on the membrane of the grid.
Many efforts have been made to effect heavy atom staining in order to
achieve differentiation. Such efforts in recent years have been especially
fruitful, as they have been based on reactions with specific groups. It is
first of all worthy of note that the classic heavy atom fixatives of protein
materials have proved virtually useless in staining starch: OsO 4/ 2. 14 .43 - 47
glutaraldehyde plus OS04,31,48,49 OS04 + phosphotungstic acid,
uranium nitrate and chI oro platinic acid. 43 ,44,50,51 Potassium perman-
ganate also seems devoid of effect, except at pH 5 for a long time or with
lintnerised starch, 12,18,52,53 which indicates a reduction of the manganese
by the liberated aldehyde groups, with the probable formation of Mn0 2.
Similarly, reduction probably occurs when chromic acid is used. 17
Specific group reactions are those based on the use of periodic acid,
which yields a starch dialdehyde by oxidising the hydroxyl groups at C 2
and C 3. The dialdehyde can react with ammoniacal silver (pH 10-12) to
precipitate silver crystals. 21 ,24,54-56 This particular reaction has the
disadvantage of the treatments with chromic acid and potassium per-
manganate in that it produces large precipitates (i.e. larger than 10 nm).
However, if the starch dialdehyde is first reacted for a long time with
thiosemicarbazide (TSC) or thiocarbohydrazide (TCH) and then a silver
salt (silver nitrate or proteinate), the reaction is stoichiometric, with 1 atom
of silver fixed per molecule of TSC or TCH (Fig. 2.4). The reaction was
first studied chemically by Edel 57 in cellulose. Later Edel's process was
developed for TEM in starch by Gallant2 1,54,55 and in cellulose by
Kassenbeck and Hagege. 58 It was used in a similar way in glycogen and
animal mucopolysaccharides by Thiery. 59 It may be used for bulk material
before embedding 21 ,22,24,34,59-63 or on thin sections. 19 ,24,31,32,64 The
reaction does not occur with polysaccharides of {3,1-3 structure 65 but can
take place wherever 2,3-dialdehydes may be formed by periodic acid
treatment. In some conditions unsaturated lipids and amino acid terminal
groups may react. 24,31 A quicker oxidation occurs with lead tetraacetate,
followed by potassium permanganate, and the stain seems more
intense. 22 ,24 Metal alcoholates are produced by reacting an alkali metal
with the hydroxyl groups of the starch. 19 ,24,59,66
FIG. 2.4. TEM view of com starch (x 26 400), lightly oxidised by periodic acid, then
treated with thiosemicarbazide and silver nitrate. The darker layers contain more
reduced silver.
Some stammg reagents, which give good differentiation in starch

sections, have incompletely understood reactions. These include lead salts
such as plumbites, 19,24, 67 lead hydroxide and lead citrate. 19 ,24,29,47 ,48,68
Unfortunately, despite its being a heavy atom and having a natural
affinity for starch, iodine is not useful in staining: it is volatile and
evaporates in the column of the microscope; and it is itself a powerful
oxidant, damaging both starch and the microscope parts. However lead
iodide has been used successfully to stain starch, and the stain may be
specific. 2 4,69
The surface of the starch grain can also be studied by TEM. Recourse
is had to the technique of replica formation by shadowing (Fig. 2.5), in
which a small amount of metal (Ag, Au, Cr, Pd, Pt, U) is evaporated at a
Broken storch
Formvor
I Gloss
+ C)
f'u
(Shodow U
U C
c
- * , S t o r c h / uc
~.,
I Formvor
(+ ethylene dich loride) 1 Grid
U C
• .Sto~h/UC
1+ 30% ,h"m;, ood I \ u 'G,;d
~~
""'"'o;.=-........... C -::: u C
Grid
FIG. 2.5. Process of preparing a typical replica by shadowing with uranium (U) and
carbon (C) and then digesting the original material.
small angle to the plane of the starch surface to form a very thin layer,
with reliefs. That is, for any protuberance, the metal is deposited more
heavily on the exposed side and more lightly on the side facing away. In
the microscope, differences in relative absorption and scattering of the
electron beam by the different thicknesses of metal deposit give the optical
effect of a surface being lighted from one side. The metal layer is usually
reinforced by another, evaporated layer of carbon, silicon, germanium or
silica. Any thickness of starch substance can be used because the starch is
removed by digestion after the shadowing operation. 70, 72 Sometimes a
negative replica-a mould or impression with a plastic such as collodion 73

or cellulose acetate 74-is made first, and then the negative replica is
shadowed. Thin sections too have been shadowed, either directly21,24,31,
55,56,60 or after removal of some of the embedding resin. 7- 11 ,20,38,39
The shadow technique has also been used to examine internal fracture
faces of starch grains. 17 ,70 -72, 75, 76
The method of freeze-etching is another recent development in TEM.
In this method, material is frozen in water or glycerol-water solution, cut
with a sharp knife to create small fracture surfaces, and then, after
evaporation of some of the aqueous matrix, shadowed with heavy metal,
and finally coated with carbon. The original material is then dissolved or
digested. Internal fracture surfaces are made evident as well as external
'etch' surfaces. Fractures are promoted systematically in areas of weakness,
characterised by the presence oflipoidal materials, 7 7 and thus this method
has not been employed extensively with starch ll , 7 8,99 or its derivatives. 80
Cathode ray tubes

Polaroid
boxl
Q)
>
u
Q)
E
0 1'-----"
FIG. 2.6. Diagram of the scanning electron microscope.

2.3 SPECIAL FEATURES OF SEM
2.3.1 Apparatus
Like the microscope for TEM, that for SEM is composed of a column,
an electron gun and a focusing system of lenses for the primary electron
beam (Fig. 2.6). There is, in addition, a scanning device by which the very
fine primary electron beam ('probe') is caused to make successive sweeps
e., :""1
! :':""'-Electron probe
.j
Reflected electrons :"J Secondary electrons
detector detector
(cathodo-Iuminescence) ~/)s \ ' / /f?/ detector

~ V X - rays
--
1;>-;) Sample
Electromotive force
0'
measurement Electrons absorbed
by sample
Transmitted electrons
detector
FIG. 2.7. Possibilities of analysis with SEM (after Kimoto and Russ S1 ).
of the area of interest by electromagnetic deflection. These sweeps occur

along orthogonal co-ordinates, illuminating the area point by point. As
the beam strikes the object, various types of secondary phenomena are
induced, including emission of secondary electrons, reflection (back-
scatter) of primary electrons, cathodoluminescence and beam-induced
conductivity. The intensity of these phenomena depends on the energy of
the primary beam and the composition of the object. 81,82 They are
detected by appropriate receivers (Fig. 2.7), with image formation governed
( a) (b) I :
II
Scintilla tor
Det ecto r
Gol d s hell Gold s hell
10 nm F=====: ~==~!===J.tt:::;2=:t:::=;=:+:-Surfuce of scmpl e
E
::t.
Sample
Hol der
FIG. 2.S. Formation of image with secondary electrons (e;) and reflected primary
electrons (er -) in SEM. Beam in (a) is of low energy; beam in (b) is of high energy
(after Kimoto and Russ 81 and Philibert 82 ).
principally by electron emission (Fig. 2.8) which is detected by a scintillator-

photomultiplier receiver. After amplification, the signal from the latter
receiver is brought to two cathode ray tubes, each with a fluorescent screen
and a sweep mechanism, synchronised with that of the primary beam, for
image formation. One of these tubes, with finer definition, is used for
photographing the image; the other is for observation. The three-
dimensional illusion of SEM is enhanced by the depth of focus and by the
brighter highlighting of upper level surfaces due to their richer yield of
emitted secondary electrons.
Magnification is determined by the ratio between the sweep amplitude
of the beam and that of the cathode ray tube and can reach 140000 to
200 000. The resolution is approximately equal to the diameter of the
electron beam. In the best conditions, because of diffraction and diffusion
at the object surface, this resolution is not less than about 10 nm and can
often be up to 25 nm. Thus SEM is not suitable for study of very fine
structure.
2.3.2 Specimen preparation

There are special problems with biological materials: (1) they are
generally poor conductors and hence weak emitters, provoking coarse
electron discharges-i.e. a charging effect; (2) because these materials
permit deep electron penetration, secondary electrons are produced not
only at the surface but in subjacent regions, so that the clarity of the image
is impaired (Fig. 2.8); (3) often, as is the case with starch,83.84 they are
easily damaged by primary electrons of high energy. These problems can
be ameliorated by depositing (by vacuum evaporation) a layer of gold,
gold-palladium or carbon and gold (10-20 nm thick) on the surface of the
specimen. Gold is a good conductor and emitter and will supply the
secondary electrons for image formation.
Starch specimens have been examined with SEM in untreated
fiour 25 ,26,33,63,79,85-87 or after special treatments to reveal the
protein 87- 90 or lipid 91 components. Starch grains have also been studied
in the native state (Figs. 2.9_2.10).24,25,84-87,89,90,92-103 The use of
amylolytic enzymes has revealed the course of digestive action as it varies
with the enzyme 24 ,25,33-36,63,88,104,105 and with the origin of the starch
grain (Figs. 2.11, 2.12(c)_(d)).30,61,106 SEM has been employed in
examining the structure of fracture faces in native 107 ,109,129 and
lintnerised24 ,83 starch and in discerning the physical effects of ultra-
sound 84 ,103 (Fig. 2.12(a)); it has been used to explore the action of
dimethyl sulphoxide in swelling starch grains, 110 encapsulation or grafting
with other polymers, 111 - 116 and gelatinisation processing. 101,11 5,117,118
It has also been used to compare hard and soft endosperm in cereal
grains 89 ,90,l15,118 and the effects of other technological treatments 33 ,36,
37,63,86,98,115,118 on starch grains.
2.4 APPLICATIONS AND RESULTS
The state of our knowledge of the ultrastructure of starch has been

summarised in several recent publications. These include resumes of the
ontogeny, cytology, macromolecular structure and biosynthesis of
starch. 24 ,48,119-121 At this point we should present some results which
are relatively new and which have not been cited in the above-mentioned
resumes.
2.4.1 Cytological studies
Although certain reactions are specific for particular polysaccharides,
there is not uniform agreement on the significance of the still widely-used
.j::..
00
tT1
~
:>
s::
~
oz
~
~
><:
~
v.>
o'>"l
~:;tI
n
::t::
(a) (b)
FIG. 2.9. (a) SEM view of native normal maize starch (x 18(0). (b) SEM view of native amylomaize starch ( x 2880).
tl1
r
tl1
(")
...,
:;>:I
o
z
~
n
~
til
(")
~
><:
o.."
til
~
:;>:I
(")
II:
>
S
~
::c
(")
II:
"d
:;>:I
§
c::
(")
...,
til
(c) (d)
FIG . 2.9. (c) SEM view of native waxy maize starch ( x 1800). (d) SEM view of native rice starch (x2880). ,l::..
1.0
( d) VI
o
\, I
,l'
~
...,~
oz
~
"(b)
~
E
til
o
"l
en
~
~
(")
::t:
(a)
FIG. 2.10. SEM views of development of the native starch grain of wheat (after Evers 95 ). Scales illustrated: (a)-(d) 1 pm; (e) 2 pm;
(f) 5 pm.
(a)
(b)
FIG.2.11. SEM view of native wheat starch during digestion by pancreatic juice of hog:
(a) x 3740; (b) x 4680.
Vl
N
tTl
~
~
~
o
Z
~
~
~
'"
o>rj
;!'"
::c
g
(a) (b)
FIG. 2.12. (a) SEM view of potato starch sonicated in air (x 5810). (b) SEM view of native manioc starch (x 3360).
(c) (d)
FIG. 2.12. (c) and (d). SEM view of native manioc starch during digestion by pancreatic juice of hog ( x 8(00).
more general reactions, such as that of the lead salts. It has been
supposed122 that lead, as plumbite ion, forms hydrogen bonds with the
substrate. This would clearly be a non-specific kind of staining, regardless
of the minute and circumspect details suggested for its employment.
However, new techniques of staining permit the localisation of the cellular
polysaccharides and the tracing of the course of their biosynthesis and
growth in relation to other structures of the cell. 14 ,24,48,121
An elegant application of specific reagents in TEM concerns the
identification of the nature of the starch in parenchyma cells and in sieve
tubes of the bean. 29 The starch grains were stained with uranyl nitrate and
lead citrate after digestion of the ultrathin sections with bacterial a.-
amylase and with pullulanase. It could thus be shown that there was an
abundance of glycogen-like a.,1-6 branching in the sieve tube starch,
which differed from the more typical starch in the parenchyma cells.
2.4.2 Ultrastructural studies

The treatment of the starch grain for study of its fine structure is fraught
with difficulties. The starch grain is very delicate, physically, and it is
susceptible to heat and to many chemical reagents, so it is easily degraded.
It may be presumed, however, that the types of degradation will be
related to the macromolecular structure of the starch grain, and this thesis
underlies the studies of ultrastructure in electron microscopy.
The surface of the native starch grain is basically smooth (Figs. 2.9,
2,10, 2.12(b)), as shown by surface replicas,73,123 freeze-etching 78 ,79 and
SEM. 84 ,92-95,97,107-110 But sometimes it shows indentations of small
starch granules (Fig. 2.9(c)_(d))12,24,124 or protein bodies in hard
endosperm. 89 ,115,118,124 Internally, replicas of fracture faces usually
show a finely granular structure, 8-11,21,24,38,39,56, 78,129 butthere is also
evidence of a fibrillar constitution. 17, 75,76,78,11 7 A radially fibrillar con-
stitution is also revealed in TEM by gamma radiation, followed by silver
staining ofthe induced groups21 , 55 and in SEM by ultrasonic radiation. 84
Examination of fracture faces in SEM similarly provides evidence of a
radial disposition of the structural elements of the starch grain, often as
rope-like fibrillar strands. 83 ,108,109
The mild acid hydrolysis of starch (lintnerisation) permits the extraction
of a part of each of the superposed, concentric layers of the starch grain.
It is not yet certain whether the starch removed is less crystalline or simply
less dense than that remaining. New evidence, from silver reduction,
suggests that the latter possibility is more reasonable. 12 5 However the
more acid-resistant centre ('nucleus') of the cereal starch grain 12 ,15,18,21,

24,109 seems to be more crystalline than the outer region and is demon-
strable even in the native grain. 30 ,61,92,106,126
The hydrolysis of starch by salivary amylase, 38 by diastases during
germination, 12,18,25,31 by a-amylase of germinated wheat/ 04 by sprouted
wheat,33 by glucoamylase,35,l05 by bacterial a_amylase,24,32,62 by
pancreatic juice, 24,30,34,61,106 and by pancreatic a_amylase 24 ,34,37 ,63,109
has shown that the differences in the kinetics of amylolysis are related to
the structure of the starch grain (Figs. 2.11, 2.12(c)-(d)). Enzymic action
is manifested either by an exocorrosion alone or by exocorrosion accom-
panied by a more or less rapid endocorrosion. It is presumed that hydrolysis
proceeds most rapidly in the less crystalline or less dense regions of the
starch grain. Although differences have been found in the effects of
different amylases on wheat starch,l 04 other results on different types of
starch 24 ,30,37,106 suggest that the effects are related more significantly to
specific differences among the starches rather than to differences among
the amylases used.
2.5 EVALUATION AND PERSPECTIVES

It is possible to use other atomic particles to form images, notably protons,
neutrons and certain positive ions. However, the use of electrons has the
advantages of simplicity of production, ease of focusing by a magnetic
field, high capacity of excitation of a fluorescent screen, ready sensitisation
of a photographic emulsion, and small extent of scatter on traversing a
specimen. 12 7 When these are joined to a thousandfold finer resolution
(TEM) and a more than hundred-fold greater depth of focus than in the
light microscope, the extraordinary efficacy and power of the electron
microscope becomes evident. Ancillary devices have been developed to
heat, refrigerate, manipulate, lyophilise and even freeze-etch the specimen
while it is being observed.
Nevertheless, there are certain limitations on the use of the electron
microscope. Because the column must be kept under a very high vacuum,
it is not yet possible to observe biological processes or enzymatic reactions
or any event that requires the presence of liquid water. The extreme
thinness of the sample in TEM imposes similar restrictions where it is
desired to observe the progress, perhaps, of events such as fermentation or
baking of micro-samples. A further difficulty is the appearance of artefacts,
structures which were not present in the original material. Examination by
TEM requires impregnation with fixing solutions (which involve chemical
reactions such as oxidation, saponification, neutralisation or cross

linking), intensive dehydration, impregnation with resins, polymerisation
of the resins at high temperatures, removing ultrathin sections from the
surface of water, and often staining with heavy metal solutions in very
acid or alkaline conditions. These operations are bound to influence
profoundly the fine structure of the original material and give rise to
artefacts. The avidity of starch for water gives rise to dark band-like
artefacts as the thin section of the starch grain swells and folds. 12 ,2o,24,31,
50,51,60,79 These bands were mistakenly interpreted as being proteinaceous
inclusions. 43 The tendency to swell and fold is obviously directly related
to the surface area of the grain section, being more marked in larger than
smaller grains,31 and inversely related to its thickness. 24 ,6o Similarly, the
'halo', a clear space about the starch grain in sections of the plastidal
stroma, may be such an artifact.
Artefacts may also be produced when replicas are made. During the
many manipulations in the replica process it is possible to cause secondary
fissures, fractures, folding, flattening, etc. In some cases not all the original
material is removed after shadowing. In these cases, SEM-despite its
poorer resolution-is an advantageous substitute. Indeed, in spite of its
imperfections, SEM is much more practical than TEM. Specimen prepara-
tion and observation are far more rapid-to such a degree, indeed, that SEM
can be used in controlling industrial processes of non-biological nature.
The future of SEM may become more important as ways are found to
enhance its power of resolution. Certainly, efforts will be made also to use
SEM in a discriminatory way, to distinguish between different kinds of
compounds. Some recent devices for SEM (cryounit, freeze etching and
ultrafine microanalysis) are useful in giving great promise of the utility of
this new technique for technological food science. In 1942, Langeron128
wrote, a propos of the electron microscope, 'At the present time, the
biological applications of the electron microscope are richer in hope than
in reality .... The results hitherto obtained do not suggest the use of this
instrument in micrographic practice in the very near future.' The present
chapter contradicts this pessimistic perspective, and it is to be expected
that the future may be even more fertile than the past.
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92. Evers, A. D., Die Starke, 1969,21, 96.
93. Hall, D. M. and Sayre, J. G., Textile Res. J., 1969,39, 1044.
94. Hall, D. M. and Sayre, J. G., ibid., 1970,40,256.
95. Evers, A. D., Die Starke, 1971,23, 157.
96. Hall, D. M., Van Patten, E., Brown, J. L., Harmon, G. R. and Nix, G. H., Ind.
Eng. Chem. Prod. Res. Develop., 1971, 10,171.
98. Palmer, G. H., J. Inst. Brew., 1972,78,326.
99. Banks, W., Greenwood, C. T. and Muir, D. D., Die Starke, 1973, 25, 225.
100. Hall, D. M. and Sayre, J. G., ibid., 1973, 25, 119.
101. Hill, R. D. and Dronzek, B. L., ibid., 1973, 25, 367.
102. Banks, W., Greenwood, C. T. and Muir, D. D., ibid., 1974,26,45.
103. Degrois, M., Gallant, D., Baldo, P. and Guilbot, A., Ultrasonics, 1974, 129.
104. Evers, A. D. and McDermott, E. E., Die Starke, 1970, 12,23.
105. Evers, A. D., Gough, B. M. and Pybus, J. N., ibid., 1971,23, 16.
106. Derrien, A., Gallant, D. and Aumaitre, A., Ann. Bioi. Anim. Bioch. Biophys., 1971,
11,326.
108. Richter, I.-E., Die Starke, 1970,22,20.
109. Gallant, D. and Guilbot, A., ibid., 1975 (in press).
111. Gruber, E., Alloush, S., John, K. and Schurz, J., Die Starke, 1972,24,251.
112. Gruber, E., Alloush, S., John, K. and Schurz, J., ibid., 1973, 25, 325.
113. Chanzy, H. D. and Revol, J. F., ibid., 1973,25, 131.
114. Fanta, G. F., Baker, F. L., Burr, R. c., Doane, W. M. and Russell, C. R., ibid.,
1973,25, 157.
115. Farber, B., D.E.A. de Cytologie et morphogenese Vegetales, Universite de Paris VI,
Paris, 1971.
116. Chanzy, H. D. and Revol, J. F., Die Starke, 1974, 25, 197.
117. Sterling, C., ibid., 1974,26, 105.
118. Farber, B. and Gallant, D., Ann. Zootech., 1975 (in press).
119. Frey-Wyssling, A. and Miihlethaler, K., Ultrastructural Plant Cytology, Elsevier,
Amsterdam, 1965.
120. Sterling, c., 'The Structure of the Starch Grain', in J. A. Radley (ed.), Starch and
Its Derivatives, 4th ed., Chapman & Hall, London, 1968.
121. Badenhuizen, N. P., Struktur und Bildung des Starkekorns: Handbuch der Starke,
VI-2, Paul Parey, Berlin, 1971.
122. Karnovski, J., J. Bioi. Biochem, 1961,2, 729.
123. Kore-Eda, A. and Nikuni, Z., Mem. Inst. Sci. Ind. Res., Osaka Univ., 1955, 12, 141.
124. Gallant, D., Farber, B. and Guilbot, A., Die Starke, 1975 (in press).
125. Sterling, C., ibid., 1973, 25, 115.
126. Czaja, A. T., ibid., 1972,24, 77.
127. Selme, P., Le microscope electronique, Coll. 'Que Sais-je?', No. 1045, Press Univ.
France, Paris, 1963.
128. Langeron, M., Precis de Microscopie, Col. de Precis Medicaux, 6th ed., Masson,
Paris, 1942.
129. Ferri, S., J. Ultrastr. Res., 1974,47,420.
CHAPTER 3
The Rheology of Starch

A. H. A. DE WILLIGEN
Rijksweg 49, Glimmen (GR), The Netherlands
INTRODUCTION
Many rheologically interesting materials consist of a mixture of solid and

liquid phases as is the case with the starch-water systems known as pastes.
The discontinuous phase can be solid or semi-solid. Usually it consists of
swollen granules or of fragments of such granules, from 300 J1. down to
less than I J1. in diameter. In some cases the particles are preformed during
an industrial process, in others the solid phase is formed from a solution.
In many products the particles as such occupy a substantial proportion
of the volume. A paste may consist of swollen grains with only sufficient
liquid to fill a small volume of interstices. A starch gel may contain no
more than traces of free liquid. Even crystallising particles-although not
occupying an important volume themselves-may entrap large volumes
of liquid by the formation of networks, giving the entire system, or its
major parts, the properties of a rigid structure.
A characteristic of such starch-water systems is the gel character of the
particles themselves. They are able to swell and shrink, to lose their solid
structure and to regain it. A heavy stress disintegrates them or at least
remodels them in an irreversible way, but even then a new structure may
be reformed.
In the older literature there is a tendency to describe the flow of pastes
as that of water with addition of starch, influencing its viscosity. In that
case the first approximation is that of viscous flow with a viscosity
dependent on starch concentration according to one or several modifica-
tions of the Einstein law. 57 As a more intricate approximation for warm
pastes it is customary to describe its flow as that of a pseudoplastic
fluid. 5 8 Where possible these conventions will be followed. This chapter
61

deals mainly with starch as a discontinuous phase. Restricting the treat-

ment to viscous or pseudoplastic flow alone would allow insufficient scope
for the properties of concentrated pastes and gels, so important in practical
applications. Therefore the discussion will start-in an unusual way-at
the other end, viz. with air-dry starch as the most concentrated starch
suspension.
The difficulty of such a treatment is that no branch of science has
attempted to deal with this problem as a whole. For instance the know-
ledge of fundamental relations between particle size on the one hand and
fluid velocity on the other is still scanty. Nevertheless in our experience
this treatment of the phenomena opens up new vistas for practical
application.
3.1 AIR-DRY STARCH
Air-dry starch is seldom a free-flowing powder. It has 'crunch'. In not too

thin a layer it has a certain compactness and resistance against pressure,
but on squeezing it with the fingers it gives way suddenly, often emitting a
very distinct high pitched tone or 'squeak'. The feel and the sound depend
on the water content. In potato starch this phenomenon disappears after
drying below 15 % and again at 22 % or over leaving a slightly oily feeling.
In commercial potato starch, of about 20 %, 'crunch' can be eliminated by
the addition of a very small amount (0'1 % or less) of a solid. Any fine
powder will do: calcium phosphate, magnesium oxide, fine iron oxides,
they all give a free-flowing starch.
On the other hand, in a dry starch, free of crunch, this property can be
reconstituted by admixture of 0·2 % or sometimes less of a liquid im-
miscible with water, e.g. a hydrocarbon or chlorinated hydrocarbon makes
the grains stick together again. However the least surplus causes an oily
feeling, and it would be difficult to get the right sound in such a way.
The most likely hypothesis is therefore, that the grain surfaces stick
together at certain wet places, where the surface is covered by liquid in a
volume of 0·2 % or less. The fine powders mentioned above would fill up
these sticky places and in that way diminish the coherence of the starch
grains.
The feel is dependent on the grain diameter. In potato starch certain
fractions have a better crunch than others. Offal qualities ('seconds' and
'thirds') never have the right crunch, mostly because of a high content of
particles below 20 J1 diameter. Crunch is more pronounced in potato
THE RHEOLOGY OF STARCH 63
starch than in maize starch. Starches with very small grains such as rice
starch are almost free-flowing and have less crunch.
3.2 STARCH SUSPENSIONS
3.2.1 General discussion

This part will be restricted to rigid particles under such conditions of
stress that no disruption of the granules occurs.
Let us consider a suspension of starch in air or water, flowing through a
horizontal pipe or over a horizontal plane. Under laminar conditions of
fluid flow, the suspension is unstable: the granules settle. For particle
transport the velocity of the liquid must be chosen such that the vertical
component of a turbulent motion opposes the settling movement.
This is quite obvious in an air suspension. Even under conditions of
fully turbulent flow of the medium it is difficult to keep dry starch particles
uniformly spread over the cross-section of the tube. A substantial fraction
settles in the lower part where it goes into saltation, viz. the granules are
leaping from point to point over the lower surface. 1 From time to time
one settles down, but it is immediately raised again by collision with a
more rapid successor. The energy for such particle movements is with-
drawn from the energy of motion of the medium, so that a lower velocity
is found for the suspension than for the pure medium at the same stress.
A measurement with classical rheological apparatus under such con-
ditions would lead to the conclusion that the viscosity of the suspension
is higher than that of the medium.
Now consider a perpendicular plane parallel to the main direction of
flow. In a very dilute suspension in viscous flow all particles move parallel
to this plane, and no particle is passing through it. However, in a slightly
more concentrated suspension this is not the case. Owing to the settling
movement collisions occur, in which the energy is transferred in all
directions, and not only in that of the main stream. In the major proportion
of the collisions there is a component perpendicular to the plane. Thus a
turbulent component is generated in that direction, and again the energy
for it is withdrawn from that of the medium.
In conventional rheology the viscous flow of a liquid is treated as that of
a multiple collection of volume elements moving along parallel lines, in
the direction of the shear gradient. In starch suspensions on the contrary
the minimal element is the starch grain with its surrounding liquid. In
comparison to the usual apparatus this is a relatively big portion, and
moreover it does not satisfy the condition of parallel flow. Therefore a

discussion of its motion in terms of viscosity or pseudoplasticity will have
a limited value only.
Now consider a dilute suspension, in which the particles have settled on
the bottom. For a turbulent flow of a pure liquid the flow parameters are
correlated by the law of Prandtl: 2
U = KVlogz/k (1)
in which U is the mean velocity in the direction of the main stream, K is a
constant, z is the distance from wall and k a factor, depending on the
roughness of the wall. As z/k is dimensionless V obtains the dimension of
a velocity, characterising the mean value of velocity at any distance from
the wall.
For a wall covered with particles, k is of the order of 1/30 of the particle
diameter. The law of Prandtl therefore takes the following form:
U = KVlog 30z/D (2)
in which D is the mean particle diameter.
The drag on the wall of a rheometer is proportional to V 2 and therefore
under a given value for z/k to U 2 • Again in covering the wall with a
coating of particles their diameter takes the place of the wall roughness.
F or a given U, the drag is higher, the greater the diameter of the particles.
However these considerations apply for fully turbulent flow only.
A theoretical discussion of semi-turbulent flow of concentrated sus-
pensions of settling particles would be quite complicated. Measurement is
in practice interfered with by the sedimentation. Bagnold 3 and Mason 65
therefore studied a simpler case, viz uniform spherical particles in a
medium of equal specific gravity, using a Mac-Michael type rheometer.
In relatively concentrated suspensions an extra shear stress is found, over
and above that of the liquid, depending on volume concentration of the
particles, density P, particle diameter D, velocity U and slit width y. In
the turbulent region the extra shear stress is proportional to
(3)
In concentrated suspensions this extra shear stress can assume quite
important values, up to 100 times that of the medium. Bagnold demon-
strates that this is caused by the interaction of the rigid particles. He sub-
stitutes the wall of the inner bob by a rubber membrane. In that case a
pressure is found, perpendicular to the main direction of flow, and again
proportional to the eqn. (3) given above.
In concentrated suspensions therefore the collision effect is not neces-

sarily caused by the settling movement only, but also by the friction of one
layer of particles over the next one; in both cases a non-laminar motion
results.
Two aspects in these experiments are important in the case of a starch
suspension. The first is that the function (3) contains the diameter of the
granule D. We may transform it by dividing the slit width y by D,
substituting y and D by
y' = y/D
By that means eqn. (3) is transformed into
(4)
viz the diameter, y, of the slit should be measured in the particle diameter
D.
In this way the rheology of starch suspensions fits into the laws of
turbulent flow of liquids.
3.2.2 Dilatancy
Concentrated starch suspensions show the phenomenon of dilatancy. 4
Starch milks of 40 %dry matter (24 0 Be) or over are difficult to pump and
they flow irregularly even at small velocities. In smooth-walled pipes the
movement has a tendency to proceed with uniform velocity except for a
thin friction layer next to the wall. 5 The inner part moves as a cylinder of
solid material. On passing a nozzle such a flow tends to be broken up in
alternating pieces of semi-solid and more dilute suspension, giving a
varying profile on leaving an orifice. If in such a case a pressure can build
up (e.g. when a constant volume of suspension is brought into motion),
the flow will almost surely stop altogether.
In pressure-velocity studies of concentrated milks (from 34 %solids on)
an unusual relation is found. At very low pressure the velocity of move-
ment is roughly proportional to the pressure. At increased pressure the
velocity-pressure relation diminishes, and in some special cases a region
will be found where the velocity is almost independent of pressure.
However in most such cases after a further increase of pressure the flow
stops entirely.
In its most extreme form this dilatancy was well known in the starch
factories at the time when settling of the starch was done on tables or in
vats, followed by hand-discharge. Manipulation of a spade in the semi-
solid mass on the table was quite an art. The workers made a solid by the
pressure of the spade and kept it solid by a slight manipulation of the

spade up to the moment when it could be discharged elsewhere. But
the problem is still there, if in another form. The washing is now done by
settling in centrifuges or hydrocyclones, from which the starch is dis-
charged through nozzles in the form of a concentrated suspension. The
efficiency of this operation depends on the dry matter content obtained,
and therefore the concentration is chosen as high as dilatancy allows
(e.g. 23° Be).
3.2.3 Expansion and shrinking of unpasted starch

Up to this point the movement of starch milk has been described in
terms which could as well be applied to other suspensions, e.g. sand in
water. However there is a fundamental difference. Starch in contact with
water does not have a constant volume, but a variable one. In this respect
potato starch is the most extreme example. In water at 25°C an air-dry
starch absorbs water up to about 40 % of its dry matter, whereby its
volume expands.
This uptake of water is an exothermic reaction. Therefore the equilib-
rium state is dependent on temperature. The higher the temperature, the
less water is taken in (however this is valid only up to that temperature
where the first granules are pasted, viz. in potato starch up to 52°C). At
lower temperatures, more water is absorbed. As the volume increases with
water uptake, the paradoxical situation is prevailing that starch in water has
a negative coefficient of expansion. The following examples for potato
starch in water are given: 6
Equilibrium percentage Volume/mljg

Temperature
(% water in air-dry starch) abs. dry starch
o 33·5 1·099
25 29·5 1·029
45 26·2 0·973
As the occurrence of dilatancy depends on the volume percentage of the

solid phase and not on its dry matter content, the maximum amount of
dry matter tolerable for handling in a given machine is firmly dependent on
temperature. Hence the concentrating efficiency of hydrocyclones and
nozzle centrifuges is also affected by temperature. Around the freezing
temperature their achievements are rather poor. An optimal separation
will be obtained somewhat above room temperature, e.g. 25-30°C.
The volume of starch also depends on the composition of the con-

tinuous phase around it.7 As with all gels there is a Donnan equilibrium
between the internal ions (bound phosphorus in potato starch, fatty acids
in corn starch) and the ions ofthe immersion liquid. This has an important
influence in the manufacture of potato starch. The crude soup obtained by
milling of the tubers contains potassium salts to the order of 0·1 N, which
concentration makes the grain swell to almost its maximum volume. In
the course of the washing process these potassium salts are eliminated,
thereby making the grain shrink and improving the efficiency of the
separation. The same holds for corn starch manufacture, during the
elimination of the sulphurous acid used in steeping.
3.3 STARCH PASTES
The pasting of starch in water is described in Starch Production

Technology, to which we refer the reader for the rheologically non-
relevant part of the extremely vast literature. In this chapter the subject is
divided in two parts: the paste formed by swollen granules, and the liquid
dispersion obtained by their almost complete disintegration (see Section
3.4).
On pasting the granule swells to a multiple of its original volume, up to
30 times or more. An 8 %suspension, flowing like water at room tempera-
ture, changes into an almost solid substance at 60°C. In the first instance
this is not caused by the development of special bonds between the
particles or in the surrounding liquid, but simply by the absence of liquid.
In this case the granule absorbs almost all the water. The remaining fluid is
scarcely sufficient for acting as a lubricant between the moving particles.
The heated, dilute suspension behaves as a concentrated suspension
with one important exception. No dilatancy is shown. The particle is no
longer as solid as in the cold. Instead, after application of a high pressure
or a high stirring torque, the swollen particle is distorted or elongated to
give way to its neighbours and-in more extreme cases-it is disrupted.
Under steep shear gradients this causes rather abrupt changes in
rheological behaviour, so that special methods of measurements are
needed.
3.3.1 Methods of following gelatinisation

An elaborate description of these methods can be found in Starch
Production Technology, so that this section can be restricted to the
rheological methods, and to literature from 1965 on. In view of what has
been said about the complex nature of the pasting process it will be clear
that a thorough study is possible only when the heating is performed under
rigid standardisation of all factors concerned and with continuous record-
ing of the rapidly changing consistency.
A series of instruments has been designed to meet these requirements,
of which the Corn Industries Recording ViscometerlO.ll.12 and the
VI-Viscograph 13 were especially adapted to concentrated pastes of potato
starch and the Brabender Amylograph 8 to flour. Attention should be
drawn to the fact, that in principle the apparatus have all been developed
between 1938 and 1950 and that all later modifications have been on the
same general lines. For a more thorough comparison of the relative
merits of these instruments, see Reference 14.
For starch the only instrument now available is the Brabender Visco-
graph. 9 It works on the following principle (Fig. 3.1). A mixture of starch
and water is put into a beaker provided with a set of vertical pins. It is
coupled to a synchronous drive, which turns it around a vertical axis,
and a stirrer is put in also consisting of a set of vertical pins. The mixture
is heated by a set of heating spirals cylindrically arranged around the
FIG. 3.1. Measuring bowl and sensor for the Brabender Viscograph. (Courtesy
Brabender OHG, Duisburg.)
beaker. The rate of heating and cooling is controlled by a contact

thermometer with continuously changing adjustment. By this means the
temperature is raised at the pace of I-t°Cjmin until a temperature of 92 or
95°C is reached. From this point on the paste is kept at a constant tem-
perature (usually for 15 min) after which a cooling spiral (with a tap also
controlled by the thermometer) provides a constantly declining tempera-
ture until 30°C is reached. The torque of the stirring rod is registered by a
dynamometer, so that a curve is obtained in which consistency is plotted
against time (viz temperature).
The maintenance and calibration of this instrument is a constant source
of trouble. A set of instructions is provided by the makers and in Reference
69. As long as the instrument is in perfect order and the instructions for
preparing the paste are rigidly followed a reproduceability of 2 % can be
obtained. However no absolute data can be derived, as the flow of the
paste around the pins is quite turbulent. Attempts to calibrate the instru-
ment with the aid of Newton liquids 67 ,68 were not successful.
In addition to these viscograph-type instruments a number of other
apparatus has been used. A review 61 compares the Brabender viscograph
with five other instruments, viz, the Brabender Plastograph, the Brookfield
viscometer, the Drageviscosimeter system Epprecht, the Rotavisko of
Haake and a capillary viscometer. Woldendorp62 tries to correlate the
results of another group of instruments with his subjective judgement of
'long' and 'short'. Curiously this proved to be well-correlated with the
results of the Brookfield viscometer, a rotation viscometer in which the
distance of the cylinders is relatively high, and not strictly defined (it can
be used in any vessel, however wide).
3.3.2 Interpretation of the viscogram

The influence of paste concentration on the stirring torque of the VI
viscograph is given in Fig. 3.2.18 In dilute suspensions (potato starch under
2 %) the torque is small. Swollen granules still find enough room for a free
motion, relatively unhampered by their neighbours. In somewhat higher
concentrations the stirring torque builds up very gradually, in proportion
with the swelling of the granules (which does not occur simultaneously,
or suddenly). In more concentrated suspensions however (over 6-7 %with
potato starch, over 12-14 %with corn starch) the water absorption proceeds
in such a rapid way, that the stirring energy increases, within 2-3 min up
to the maximum capacity of the apparatus. 19 - 22
Bechtel 21 and Anker and Geddes 8 have shown that there is a linear
relationship between the logarithm of the maximum stirring torque 't' and
the logarithm of paste concentration c of the form

log 1" = a + b log c (5)
in which formula a and b are coefficients, depending on the apparatus.
The same relationship is found in References 18 and 9. In all these
publications b is above 1, but below 2. Attempts to describe the influence
in terms of the Einstein law,15.16 with b = 1, make no allowance for the
>-
u
c
.!'!
<f)
<f)
c
o
U
Time
FIG . 3.2
rapid increase of 1" at higher paste concentration. Probably the point of

importance is the rapidly diminishing amount of free liquid acting as a
lubricant between the particles in motion, and the pliability of the particle
permitting its movement in the presence of so small an amount of con-
tinuous phase. Microphotographs 59 seem to confirm this. For the cooked
paste a value of b around unity is found,18 viz the Einstein law is valid.
With unmodified starch the stirring of so stiff a paste causes a rapid
breakage of the particles, and this is accompanied by a gradually diminish-
ing torque. This is quite obvious in potato starch,S 9 but it can be found in
other starches too, if a suitable (higher) concentration is chosen, e.g. 15 %
for corn starch.
The first explanation of the falling torque has been that a considerable
part of the starch out of the broken granules dissolves in water, leaving a
smaller volume of particles. The dissolution is a fact (see Starch Production
Technology. However the torque at first falls more rapidly than can
be explained by dissolution. Also in this case the clearance of the instru-
ment should be compared with the granule diameter. At the maximum of
the curve the main diameter is high, up to 3-4 times that of the unswollen
grain. In potato starch this amounts to an average diameter of over 10011,
and a maximum value of over 300 11. The factor dUjdy' in formula (4) is
high and therefore stirring resistance is high. The big grains however are
immediately broken, the factor dUjdy' goes down and the stirring resist-
ance diminishes proportionally. In many cases this change suffices for the
explanation of the diminishing torque independent of starch in solution.
For industrial users of starch the general shape of the viscogram has
proved to be of value, not only for the prediction of the cooking process.
Several groups of users have tried to correlate their results with the graphs
and have found certain relationships to hold. A high peak indicates the
ease with which the granules are disintegrated, an important property in
the use of potato starch on fibres, e.g. in the adhesive, paper and textile
industries. A prolonged period of high consistency is appreciated in the
food industry (see cross linked starches) in cases where the swollen starch
must be left as fully intact as possible.
In other applications the main requirement is a rapid breakdown to a
solution of very low consistency, which is indicated in the graph by a return
to the zero line of the scale. So for the regular user, the viscograph makes
possible a rapid judgement of any sample submitted by the manufacturers.
On the other hand we urge caution against an easy interpretation of the
visco graph curve in terms of industrial use. In many applications of
starch (e.g. paper, textiles), the flow of the paste must be regulated on two
completely different levels. On the one hand the user is interested in the
flow in pumps and through pipelines, or in the orifices of his equipment.
On the other hand there is the flow between fibres, or in the pores of other
materials, for which the starch paste is used as a binder. In the latter case
there are opposing requirements. In some applications the starchy material
must be able to pass into the pores, in other cases such as textile and paper
finishing the main part (or at least a certain fraction) should not go
through.
With macro-rheological methods there are two chances of error. In the
first place the pastes in the instruments are intensively stirred and particles
could be much more distorted than in industrial application. In the second
place a consistency measurement is a crude, overall yardstick for the
complex properties of the paste. The same high consistency can be found
with relatively large remnants of the starch granule structure dispersed in
a thin liquid as with few smaller parts in a medium of high viscosity.
We therefore urge the reader always to combine the examination by
viscograph with the microscopic methods described in Starch Production
Technology and eventually with the sedimentation methods described

in that work, after a suitable dilution of the paste.
3.3.3 Comparison of different types of starch

In general the various kinds of commercial starches show gradual
differences only. Potato starch however takes a rather extreme position, as
will be shown in an example.
In Fig. 3.3 the viscograms of 7 kinds of starch are given as recorded by
the VI-Viscograph, in 8 % paste. 23 • 64 Potato starch shows by far the
higher peak. But this is not a characteristic property of potato starch
exclusively. The same type of curve can be obtained with corn starch at a
concentration of 16 %. The difference is that the high peak viscosity of
potato starch is obtained at a much lower concentration, in a range used
commercially.
>-
u
C
<Il
~ 50
If)
c
o
<..)
o
Time (min)
FIG. 3.3.
The other starches differ from potato starch and also among themselves
on two points. The maximum consistency differs, and the curve leaves the
zero line after different times, viz at a different temperature.
In the concentration region up to 10 %of the cereal starches swell much
less than potato or cassava starch. Hence the granules stay more rigid and
are embedded in more fluid, so that less granule breakage occurs during
cooking. Accordingly the paste is built up primarily from entire granules
or well-organised parts of granules. Less starch is present in the inter-

stitial liquid. Therefore the particles are easier to separate (the paste is
called 'short') and on cooling more fragments control the nature of the
retrogradation process (see Section 3.5).
3.3.4 Influence of the double layer

This part is most important with respect to potato starch, less so for the
cereal starches. Potato starch contains up to 0·28 % of phosphorus
(calculated as P 20 5 in dry matter). This component is firmly bound to the
starch, mainly as a monoester, and for the major part to the branched
molecules, the amylopectin fraction. There is the equivalent of about
4 meq/lOO g of dry matter of negative groups, acting as an ion exchanger.
In the potato juice K and Mg are the most important compensating
cations. But after washing the starch with hard water K is exchanged
against Ca and Mg up to 3 meq or more divalent ions. In older, less well
equipped factories lactic acid bacteria lower the pH; in that case hydrogen
ion may displace all other ones.
Veselovskii 24 finds that the phosphorus content of potato starch depends
on potato variety. It also can be increased by high applications of phos-
phorus fertilisers on the soil before or during planting of the potato;2 5
several other agronomic factors contribute to it. 26 ,27 In every case a
correlation is found between this content and the rheological behaviour of
the starch. The maximum of the viscograph curve and-but to a lesser
extent-the viscosity of the cooked paste are increased by a higher
phosphorus content. 28,29 However, the causal relation is still far from
clear; in many cases phosphorus application and influence of potato
variety have a different regression equation. 25
Exchange of monovalent ions against divalent has an essential influence
on the pasting process (Fig. 3.4). The maximu~ of the viscograph curve is
depressed and shifted to a higher temperature. The influence on the
consistency of the cooked paste is not important. However more and
better organised granule fractions remain after cooking.
Fundamentally the influence of phosphorus must be considered as that
of a Donnan equilibrium. The influence of electrolytes in the cooking
water on the pasting process is in agreement with this conception. The
internal pressure in the swelling granule is thereby diminished and the
maximum of the viscograph curve is lowered. 3o ,31
The phosphorus bond in potato starch is not at all stable. Hydrolysis
takes place spontaneously,32 with a corresponding decline of the visco-
graph maximum. After a year's storage in a temperate climate the influence
i, noticeable in almost every application. Some users of unmodified starch

therefore have to change their formula as soon as the product from a new
potato crop becomes available. The process is most easily followed by a
determination of free phosphate. Its content goes up from almost zero in
a freshly prepared product to well over 30 % of total P after some years.
100
;>,
u
c
Q)
V>
V> 50
c
0
U
O~~--~----~-----L-----L----~-- __
25
Time (min)
FIG. 3.4.
In warm climates the hydrolysis goes on much more rapidly. After a

storage of 1-2 months at 50°C the usefulness of potato starch can be
seriously affected. For a purposely performed hydrolysis see under next
section.
3.3.5 Cross linked starches

Under the collective denotation 'cross linked starches' the industry
supplies a series of derivatives in which the swollen granule is reinforced
in such a way, that its breakdown is prevented or at least hampered. The
most simple case, with potato starch, is a heat moisture treatment 33 • 34
during which a hydrolysis of the phosphorus bond takes place.
In Fig. 3.5 some examples of visco graph curves are given. 35 • 36 A short
treatment shifts the maximum of the viscograph curve to the right, viz
to a longer time of heating of the paste and a higher temperature. A more
prolonged treatment retards the maximum to the second part (above the
cooking point of the VI-Viscograph, or in the 95°C-period of the CI
Recording Viscometer). A stilI longer treatment diminishes granule
swelling to such an extent, that a decidedly lower consistency is reached;
in principle it would be possible to reach a state where no swelling occurs,
viz the recording pen remains on the zero line.
With potato starch, such a reinforcement can also be obtained by the

addition of small amounts of aldehydes (not an industrial proposition
because the preparations are not stable), or with a number of divalent
reagents (e.g. epichlorhydrin or POCI 3 , 0·1 % or less).
Such starches are used as thickening agents. At temperatures above 60°C
they absorb an ample amount of water without much dissolution, giving a
short paste. A considerable amount of this product is used for canned
foods. 37 The amount of cross linking can be regulated in accordance with
temperature and time of sterilisation in such a way that the maximum of
the viscograph curve would occur under the exact conditions to which
the product is subjected.
100
>-
u
.,c:
.,., 50
c:
o
u
a
Time (min)
FIG. 3.5.
By this means a mainly liquid product can be changed to a suspension

of solids in only a small amount of liquid, but without much real coherence.
It therefore could combine a low yield value with a high consistency. The
particle diameter-an important factor in food acceptability tests-can be
regulated through the choice of the original starch and the swelling
capacity of the derivative. There is a range from under 10 J1 (rice starch)
to more than 200 J1 (potato starch).
3.4 COOKED PASTES

Owing to the rapid changes taking place at the beginning of the pasting
process, the study of the stress-strain relationship meets almost unsur-
mountable difficulties. The CI Recording Viscometer has been designed
for measurements to be performed at several speeds of stirring, only under

special experimental conditions can a more or less pseudoplastic flow be
demonstrated, from the curve maximum in Reference 11.
The determination of the rheological curve is much easier after a certain
heating and stirring period, viz when the initial, rapid change has declined.
Under these circumstances the rheological equation of the paste can be
written as that of a pseudoplastic liquid:
log, = a log D + log K (6)
in which the stirring torque, is correlated with the velocity gradient D by
factors a and K, dependent on the state of the paste. In Reference 11 it is
shown, that under the turbulent conditions of the CI Recording Viscometer
the factor a goes up from about 0'35, shortly after the pasting point, to
about 0·5 for the cooked paste after being stirred for one hour at a high
speed (10% potato starch paste).
In not too Iowa concentration (e.g. 5 %for potato starch) the paste does
not settle in an inconvenient way, so that measurements can be made with
any ordinary rheometer providing that the clearance in the apparatus is
greater than that of the maximum dimensions of the starch granule
remnants present in the paste. This indicates that the cone and plate
rheometer, with its varying clearance dimensions from very small to high,
is not suitable.
The measurement by concentric cylinder visco meters is thoroughly
discussed by Schutz et aI.38-40.70 They draw attention to the fact that
the usual formula for the calculation of the relation of velocity and stress
parameters are derived for Newtonian liquids only. For a pseudoplastic
liquid special corrections have to be made and this is readily demonstrated
by carrying out measurements using the same paste with cups and cylinders
of widely varying dimensions when it will be found that eqn. (6) still holds,
but with a slightly varying value of a and quite important deviations of K
depending on cup and bob dimensions.
Schutz et al. find however that deviations of this kind can be eliminated
by multiplication of D in formula (6) by a factor A, calculated from cup
radius R, bob radius r and coefficient a (from eqn. 6) in the following way:
A = 1 - (r/R)2
(7)
a[1 - (rj R)2/a]
This formula will hold for any pseudoplastic liquid, viz any liquid in
which the factor a from eqn. (6) deviates from unity. In this way measure-
ments in apparatus of varying dimensions can be brought into one
formula (6) by which method a and K truly represent the properties of the
paste.
On warm, well stirred pastes the value of the factor a do not deviate
much from 0·5, so that in practical applications one measurement would
suffice to establish K. In this way the flow of paste through apparatus,
pumps, pipe lines can be predicted.
However for the application of such pastes more information must be
known than K alone. Even in potato starch pastes it is very difficult to
destroy the last traces of micellar organisation. In industrial practice this
is almost never realised. 42 The fact that a is always so far from unity is
also significant. It tends to confirm the thesis of Klausner 43 that pseudo-
plastic flow itself is an indication of the presence of at least two phases.
'"
::>
~ 1600
(?
O~--~L----J ____ ~ ____- L_ _ _ _- L_ _ _

6 12 18 24 30
Time of heoting / stirr i ng (min)
FIG. 3.6.
The action of flow during practical application can cause separation of the
phases, so that it is not always certain that the paste will behave as a
liquid.
In this connection some comment should be made on the usual ways of
preparing starch pastes for industrial applications and its influence on
rheology. The paste should be stirred thoroughly in the process. The
influence of such a treatment can be seen in Fig. 3.6. 11 In this case stirring
causes a 30 %extra consistency loss. In the somewhat old fashioned open
steam method a similar result is obtained. The starch granule after swelling
is locally subjected to heavy vibrations caused by the collapse of steam
bubbles below 100°C and broken into pieces.
3.5 COLD PASTES
So much has been written on the subject of gel formation that a complete
survey seems to be almost impossible. For a review from the structural
point of view we refer to Chapter 3 of Starch Production Technology and
to Rees 45 and, for a more rheological treatment of gel properties, to
Hermans. 46 On cooling the more or less pseudoplastic starch paste
develops a structure by retrogradation (see Starch Production Technology,
Chapter 7). Not only is there an increase in the K of formula (6), com-
parable to the increasing viscosity in a cooling Newtonian liquid but there
is also a change in character of the flow taking place. A certain rigidity
develops in the system which also has a reversible component showing
elastic behaviour and reacting against stress, which component is for an
important part of an elastic nature.
The main problem at this stage is the structure of the paste. In many
cases the warm paste still contains a significant volume of granule frag-
ments, so that after cooling and in the course of several hours a volume of
elastic particles will be formed. Around them a starch solution is able to
retrograde giving a gel. Are the fragments glued together by this second
process, or is the elasticity mainly that of the fragments?
As an illustration of the problem we suggest the following model. A
vessel is filled with a number of dry rubber balls, each having its own
elasticity, but no force is needed to separate it from its neighbours. On the
balls is placed a lid somewhat smaller than the opening of the vessel, but
with a margin so small that no ball can escape. On putting pressure on the
lid the vessel and its contents react elastically. The same vessel and the
same balls are used again, but now the balls are glued together, layer by
layer. In this situation quite a force is needed to separate the balls. How-
ever the reaction to pressure on the lid remains about the same. Which of
those two situations is that of the cold paste?
For an answer to this question Hamer 47 immerses Saareplates (provided
with vertical stems) in a cooling corn starch paste. After cooling to room
temperature-and keeping it cool for some time-he applies vertical loads
to the plates via the stems. Under these conditions even very small loads
give a slow but sustained movement, whose velocity is dependent on the
load. No yield value is found. However it is known that in corn starch
paste the interstitial liquid is not very concentrated. A well-stirred and
heated potato starch would be a better object for such a study because so
much more starch has gone into solution. The experiment was therefore
repeated by de Willigen 48 in an 8 %potato starch paste, heated for 48 min
under a most rapid stirring and cooled during 20 h. In this thoroughly

disorganised paste no yield value was found . At every load, however
small, the movement of the plate was sustained until the paste broke away.
For these cases the model of the loose rubber balls is better suited than
that of the connected balls.
Meanwhile, from these examples, we do not want to conclude that no
bond between the retrograded fragments can be formed . On the contrary
a gelation of the interstitial liquid is often clearly shown. But under many
conditions the main forces in the gel structure are those in the fragments,
those in the continuous phase being the weaker ones. Anyhow for the
structure the gel properties are in principle such as described in 45 and,46
but with different parameters in the two phases.
Probably the main factor in the cooling phase is the remnant of a
structure in the granule fragments. This serves as a framework for crystal-
lisation. In a relatively short time, e.g. a few hours, a structure could be
rebuilt within this framework with a strength comparable to that of the
original grain. In the liquid phase on the contrary the network must be
built up almost from scratch. It may be presumed that also, for that
purpose, the granule remnants act as centres, viz the retrogradation
works from these centres outwards. In the section on derivatives some
examples are given of the initial weakness of the ensuing crystal pattern
during its growth. It could be supposed that in many cases a group of
needles is formed and no continuous network structure.
The strain-time relation of this kind of gel is given in Fig. 3.7. 49 At
time to a piece of gel is loaded with a plunger weighing about 13 gjcm 2 •
C, '
Cree
A I P recovery
C 2 :- - - - - - - - - - - - - - - - - - - 0
,
O ~____________~
C3~'____________________~E~___
~ ~ ~
Time
FIG. 3.7.
An instantaneous elastic deformation is obtained with a strain OA,

followed by a slow creep to B. When at the time tIthe load is taken away
there is an instantaneous elastic reaction BC l , almost equal to OA and a
creep recovery C l C 2 • The distance DE accounts for the irreversible part
of the deformation. In rheological terms these gels can be said to show
plasto-elastic deformation.
Creep and creep recovery are important after cooling of 'long' pastes
(well-cooked pastes of potato or cassava starch, also in waxy-maize
starch), viz in those pastes in which the original structure has been seriously
disturbed. They are much weaker in grain starch puddings, or in potato
starch pastes cooled immediately after pasting or pasted with a minimum
of stirring. In these pastes the influence of retrogradation in the interstitial
liquid can be at best very weak. In spite of that the gel behaves as an
elastic body.
The literature on cold paste rheology contains a large number of
experiments carried out on the cooling paste under rapid stirring. For this
purpose both the Corn Industries Recording Viscometer and the Brabender
Viscograph are provided with cooling circuits. From the foregoing it will
be clear that in such a proceeding the effect is mainly due to the recrystal-
lisation of the granule fragments and that the organisation of the inter-
stitial liquid is, to high degree, disturbed or broken by stirring. However
the fragments tend to congeal in an elongated form owing to the constant
forces of deformation applied in the apparatus. Nevertheless the stirring
torque as a rule increases very sharply during cooling, indicating a rapid
reinforcement of the particle structure. 9
In studying gel structure preference should be given to methods of
congelation in undisturbed pastes. In this wayan overall structure can be
formed, and moreover any particle keeps its original form. A review of
older methods is given in Reference 14. Practical methods for thin gels are
reviewed in Reference 60. Two systems prevail. The best defined value is
that of a fully reversible deformation obtained under a small load (rigidity,
elasticity). The other extreme is to use so heavy a load, that the gel is
ruptured (gel strength). As the results of both systems depend on the way
of pasting and cooking of the starch, it is impossible to correlate the data
in the literature into one rigid system. The following picture, cited from
References 14 and 49 might account for the greater part of the facts.
A certain minimum degree of swelling of the starch grains is necessary
for a coherent gel. When swelling is too low, a suspension of more or less
swollen particles will result and the available water is not fully immobilised.
In that state potato starch particles have a tendency to syneresis, viz to
exude some of the water absorbed on pasting during the cooling period.
In more advanced states of pasting a rigid gel with high elasticity will be
formed, evidently consisting of swollen starch grains. In potato starch, in
particular, but also in other starches, a continuation of heating and stirring
of the warm paste correlates with decreasing firmness and strength of
the cold paste. Cooked pastes of potato starch have, as a rule, passed the
optimal state of swelling, so that gel firmness and strength are less than the
maximal. Such pastes have a 'long' structure, viz on cutting with a spoon
no sharp cutting surface is obtained and a thread of jelly drags behind.
Also the gel tends to flow from the spoon.
High mechanical shear of the warm paste causes a reduction in gel
strength. Whittenberger 51 submitted an 8 % paste at 75°C to high
shear and succeeded in obtaining a product which did not gel on cooling.
The properties of the gels are highly dependent on starch concentration.
In maize starch milk puddings, Rutgers,60 found a rapid increase of gel
strength around 4 % starch. For example the time for the solution to flow
through a mobilometer increases tenfold when the concentration is
increased from 3·6 to 4·4 % starch.
The present day multiplicity of starch derivatives further complicates the
picture since a number of methods are available for the modification of the
gel formation in a paste. On one hand there is the cross linked series of
derivatives, which give a paste with swollen, but not broken granules
especially suited for gel formation. On the other hand retrogradation can
be hampered, or completely prevented, by esterification (e.g. acetylation,
phosphorylation), or etherification (e.g. with propylene oxide).52 Com-
binations of both methods of substitution are in use.
In such a combination of cross linking and esterification of potato starch
or waxy maize starch it is possible to obtain a clear gel, with low gel
strength, short texture and a high granule stability especially suitable for
use in frozen foods. For opaque gels equivalent derivatives are made from
grain starches,52 but as a rule these derivatives have a somewhat higher
gel strength.
Certain additions to the aqueous phase hamper the swelling and break-
age of the starch granules on cooking and exert an influence on the gel
properties of the cooked paste. This is especially the case with sugar in
concentrations of 10% and over. 51 A potato starch saturated with Ca-ions
will give a firmer gel than the corresponding K starch, and the Ca-starch
gel is the shorter. 48 A further complication with these systems is the
influence of the grain diameter or, in more thoroughly cooked and stirred
pastes, of the mean diameter of the fragments. Attention should be drawn
to the influence of the diameter according to formula (4), which is clearly

found in this kind of gel. In our view this is the explanation of the wide
variety of methods of gel testing. A method which is exactly right for a gel
with small particles might not work so well as one with granules of a
larger diameter (which might require an instrument with larger orifices or
clearances, or to vary the load, or both).
3.6 PASTES AND JELLIES OF PRE-COOKED STARCH
It has been shown above that the cooking of a starch paste is a process
depending on many variables. Considerable experience is needed to make
up a paste to a definite specification with regard to particle diameter and
overall consistency. Around 1930 there were already derivatives on the
market in a pre-cooked form and the first extensive application was a
powder for making a wall-paper adhesive by mixing with cold water.
The advantage of such preparations for the user of small quantities lies
in the easy reproducibility of the 'reconstituting' process. In the factory,
the pasting is carried out on an industrial scale under rigorously standard-
ised conditions to produce a paste of top quality which the small consumer
cannot achieve and large amounts of these preparations are marketed, as
adhesives, for paper making, textile sizing and many other applications.
In their rheological behaviour this kind of preparation has a dual
particle structure. In the first place there is the structure as in any warm
paste, containing smaller or larger fragments of starch granules. The paste
with this structure is dried, mostly on rotary drum dryers. 53 The layer is
scraped off, milled and sieved. These particles form the second structure
with particle diameters well above that of original grains. These fragments
undergo retrogradation, with a partial reversion to the structure of the
original grain. However in these products water absorption in the cold is
high in comparison with the natural starch.
During reconstitution the most simple dried starch paste swells, takes up
water, but does not disintegrate and the starch molecules do not readily
go into solution. The paste formed is a short one consisting of swollen
layer particles without much coherence. In the early days the users of dry
wall-paper adhesive complained that the paste was much 'shorter' than the
freshly cooked flour pastes they were accustomed to. This was corrected
by the addition of alkali or salts. 54
Now, after about 40 years of industrial practice such complaints are
eliminated by the use of esters and ethers instead of simple starches, and
retrogradation, as well as granule breakdown are controlled as described

under Section 3.5. In this way the reconstituted paste can be formulated
to be as long or as short as desired.
3.7 HYDROLYSED AND OXIDISED STARCHES
These derivatives are made for the explicit purpose of making a Newtonian,
or almost Newtonian, solution of a much higher concentration than would
be possible with native starch. For this purpose the structure of the starch
granule must be destroyed, either immediately after pasting or at a some-
what higher temperature.
The classic example is the Lintner starch. This is prepared by treating
starch in a cold 2N hydrochloric acid. The degree of scission of the starch
molecules, catalysed by the hydrogen ion, can be regulated by the time of
treatment, e.g. some days or weeks. After this immersion up to 25 % of the
starch has dissolved in the acid, mostly in the form of smaller oligo-
saccharides, e.g. DP 20 or less. The residual powder still shows the starch
structure, but after neutralisation it is readily soluble in hot water, giving
a clear Newtonian solution. A concentration of up to 30% is possible (a
30 % paste of potato starch, if possible to make, is a stiff mass).
Practical methods of scission are by acid (cold or warm), by enzymatic
hydrolysis and by oxidation, mostly with sodium hypochlorite, 5 6 at pH
8-9. The choice of method depends on the kind of starch and the purpose
in application. Solutions are Newtonian, or nearly so, and concentrations
can be very high. Such solutions are almost free of structure and are,
therefore, able to penetrate fibres, e.g. paper, textiles and can act as a glue.
On cooling, a structure is formed by retrogradation. The rheological
change in a paste of enzymatically hydrolysed starch has been measured by
Schutz 5 5 (Fig. 3.8). The stress-strain relation at different holding times is
given as that of a pseudoplastic fluid, without yield value, and is described
by formulae which are in principle similar to formula (6). However the
exponent a, almost unity immediately after cooking, goes down regularly
until after 80 min it is about 0'5, viz a value is obtained about similar to
that of a well-stirred cooked potato starch paste. Also in this case no sign
of a coherent network structure is found.
The velocity of retrogradation and its effect on liquid rheology is
dependent on the degree of scission (see R. Collison 66). However a some-
what more severe treatment causes a decline in retrogradation. In the
manufacture of these products there is a possibility of making a product
with extra heavy retrogradation as well as one which does not crystallise at
all on cooling.
Newer research on oxidation of starch has proved the possibility of
making products with more carboxyl groups and less hydrolysis than
before. Such derivatives give more viscous solutions and retrogradation is
hampered or completely prevented by the carboxyl groups.
In all these cases the warm solutions show no abnormalities and their
viscosity can be measured in absolute units in any suitable viscometer.
log 0
FIG. 3.8.
3.8 DEXTRINS AND SYRUPS
The object of manufacture of these products is to prepare a Newtonian

solution of high viscosity. Retrogradation, however small, is undesirable,
e.g. a solution of a yellow dextrin as sold as an adhesive for desk use
should retain its initial viscosity after months or years of storage. It should
neither go up or down. Poor products develop a structure (often a visible
turbidity) which in the long run tends to solidify the liquid. Such a develop-
ment is fundamentally a retrogradation, and can be reversed by heating
well above 60°C.
The white dextrins, on the contrary, are manufactured with the intention
to provide a backsetting solution. Theoretically they can be compared
with the retrograding solutions obtained by oxidation or hydrolytic
scission of starch.
Syrups and solutions of dextrose are Newtonian liquids up to high
concentrations. As a rule some solid matter does not alter the rheological
behaviour of such viscous fluids. Tregubov and collaborators 63 find that

in fondant masses composed of crystallised dextrose and syrup or hydrol
20-25 % of dextrose crystals can be present before the behaviour of the
mixture will deviate. Higher concentrations of solids are pseudoplastic.
REFERENCES
1. Bagnold, R. A., The Physics 0/ Blown Sand and Desert Dunes, Methuen, London,
1954.
2. PrandtI, L., Z. Ver. Deut. Ing., 1933, 77, 105; see also 1, and Townsend, A. A.,
The Structure o/Turbulent Shear Flow, Cambridge Univ. Press, London, 1956.
3. Bagnold, R. A., Proc. Roy. Soc. (London), 1954, A225, 49.
4. Roscoe, R., in Flow Properties 0/ Disperse Systems (by J. J. Hermans), North-
Holland Publishing Company, Amsterdam, 1953.
5. Karnis, A., Goldsmith, H. L. and Mason, S. E., J. Colloid Inter! Sci., 1966,22,531.
6. de Willigen, A. H. A. and de Groot, P. W., Die Starke, 1967,19,368.
7. de Willigen, A. H. A., Die Starke, 1951,3,75.
8. Anker, C. A. and Geddes, W. F., Cereal Chem., 1944, 335.
9. Pagenstedt, B., Die Starke, 1951,3,202.
10. Kesler, C. C. and Bechtel, W. G., Ind. Eng. Chem., 1947, 19, 16.
11. de Willigen, A. H. A., Analytical Chemistry, 1953,25, 314.
12. de Willigen, A. H. A., Die Starke, 1953, 5, 36.
13. Selling, H. J. and van Lamoen, F. L. J., Chem. Weekblad, 1947,43, 602.
14. Scott Blair, G. W., Foodstuffs, Their Plasticity, Fluidity and Consistency, North-
Holland Publishing Company, Amsterdam, 1953.
15. Brimhall, B. and Hixon, R. M., Ind. Eng. Chem. (Anal. Ed.), 1941, 13, 193.
16. Gallay, W., Can. J. Research, 1936, 14,360, 391,409.
17. Leach, H. W., Cereal Chemistry, 1963,40,593.
18. Hofstee, J., Chem. Weekblad, 1950,46, 515.
19. Hofstee, J., Die Starke, 1962, 14, 318.
20. Schoch, T. J., Cereal Chem., 1957, 34, 141.
21. Bechtel, W. G., Cereal Chem., 1947,24,200.
22. Bechtel, W. G. and Fischer, E. K., J. Coli. Chem., 1949,4,265.
23. de Willigen, A. H. A., Hofstee, J. and de Groot, P. W., Chem. Weekblad, 1948,44,
422.
Hofstee, J., Die Starke, 1953,4, 83.
24. Veselovskii, I. A., Am. Potato J., 1940, 17, 330.
25. de Willigen, A. H. A., Die Starke, 1951,3, 147.
26. de Willigen, A. H. A., Die Starke, 1952,4,213.
27. de Willigen, A. H. A., Proc. 0/ 9th Congresso Internazionale Industrie Agrarie,
Rome, 1952, c.s. al T.P. 1.
28. de Willigen, A. H. A., Vestnik slovenskega kemijskega drustva, 1954, I, 131.
29. Goerlitz, H., Z. Landwirtsch. Versuchs-Untersuchungsw., 1960,7, 121.
30. Ganz, A. J., Cereal Chem., 1965,42,431.
31. de Willigen, A. H. A., Kort Bericht no 28 van het Proe/station voor Aardappel-
verwerking, Groningen, 1950.
32. de Willigen, A. H. A., Chemisch Weekblad, 1947,43, 153.
33. Wiegel, E., Kolloid Z., 1933,62, 310.
34. Parlov, A., Germ. Pat. 629,798, 11 Mar. 1931.
35. Hofstee, J., Vestnik slovenskega kemijskega drustva, 1954, 1, 125.

36. de Willigen, A. H. A., Chemisch Weekblad, 1950,46,529; 1954, 1, 125.
37. Vogel, W. F., Conserva, 1969, 18, 1.
38. Schutz, R. A., Die Starke, 1963, 15, 394.
39. Schutz, R. A. and Nedonchelle, Y., Comt. Rend., 1965,261,5111.
40. Schutz, R. A., Die Starke, 1966, 18, 180.
41. Nedonchelle, Y., Sur la rheologie des solutions concentn!es de carbohydrates macro-
moleculaires, Diss. Strasbourg, 1968.
42. Caesar, G. V., in Chemistry and Industry of Starch (by R. W. Kerr), Academic Press,
New York, 1944.
43. Klausner, Y., Kolloid-Z., 1961, 174, 109.
45. Rees, D. A., Polysaccharide gels and network, in Advances in Carbohydrate Chemistry
and Biochemistry, 1969,24,267.
46. Hermans, P. H., Flow Properties of Disperse Systems, North-Holland Publishing
Co., Amsterdam, 1953, p. 61.
47. Hamer, W. J., J. Res. Nat. Bureau Standards, 1947,39,29.
49. Hofstee, J. and de Willigen, A. H. A., Chem. Weekblad, 1950,46,649.
50. Rutgers, R., J. Sci. Food Agric., 1958,2, 61, 69.
51. Whittenberger, R. T. and Nutting, G. C., Ind. Eng. Chem., 1948,40, 1407.
52. Hester, E. E., c.s., Cereal Chem., 1956,33,91.
53. Kantorowicz, J., DRP 224,663, 14 Feb. 1909.
54. Stadlinger, H., Klebstoffe aus Starke erzeugnissen, Elsner, Berlin, 1938.
55. Schutz, R. A., Rheologica Acta, 1969,8, 349.
56. Potze, J. and Hiemstra, P., Die Starke, 1963, 15, 217.
57. Rutgers, R., Rheologica Acta, 1962,2,202.
58. Scott Blair, G. W., A Survey of General and Applied Rheology, Pitman, London,
1949, p. 54.
59. Hofstee, J., Die Starke, 1962, 14, 320, (Abb. 8 and 9); 322 (Abb. 15).
60. Rutgers, R., J. Sci. Food Agric., 1958,9, 61, 69.
61. Grun, H. H., Die Starke, 1963, 15, 60.
62. Woldendorp, P. and de Noord, K. G., Die Starke, 1966, 18,293.
63. Chvorova, L. S. and Tregubov, N. N., Sakharnaja Prom., 1969,43,61.
64. Hofstee, J., Die Starke, 1953, 4, 83.
65. Mason, J., Colloid Sci., 1957,12,243; 1959, 14,457; 1961,16,238.
66. Collison, R., Starch and Its Derivatives (Ed. J. A. Radley) 4th edit., Part I, p. 198,
Fig. 6.4, Chapman & Hall, London, 1968.
67. Johnson, J. A., Trans. Am. Assoc. Cereal Chem., 1954, 12, 292.
68. Wood, F. W. and Goff, T. C., Die Starke, 1973,25,89.
69. Corn Industries Research Foundation, Standard Analytical Methods of the Member
Companies, Washington, D. c., 1972.
70. Schutz, R. A., Die Rheologie auf dem Starkegebiet, Paul Parey, Berlin, 1974.
ADDITIONAL REFERENCES
Major reference works:

Scott Blair, G. W., Foodstuffs, Their Plasticity, Fluidity and Consistency, North-Holland
Publishing Company, Amsterdam, 1953. Chapter 1, 'Starch' (by J. Hofstee and
A. H. A. de Willigen) contains the literature on rheology to 1951).
Aehnelt, W. R., Die Starke, Starkesirup, Starkezucker, Steinkopff, Dresden, 1951.

(8087 references.)
Samec, M., Kolloidchemie der Starke, Steinkopff, Dresden, 1927.
Schutz, R. A., Die Rheologie auf dem Starkegebiet, Paul Parey, Berlin, 1974.
Ulmann, M., Die Starke, Lieferung 11, Akademie-Verlag, Dresden, 1968.
Other references:
Ansart, M., Ind. Aliment. Agric. (Paris), 1962, 79, 821; Die Starke, 1955, 7, 136. (Test
methods for starches.)
Bean, M. L., Food Research, 1959,24,665. (Effect of 10 different sugars on the hot-paste
viscosity curves and gel of 5% corn starch paste.)
BeIche, J. R., Tappi, 1957,40,94. (Urea will lower the viscosity and retard the gelation
of modified starches but not of unmodified.)
Blinc, M., Die Starke, 1970,22,181. (Waxy corn starch and high amylose starch modified
by the influence of temperature.)
Bock, W. et al., Die Starke, 1967, 19, 87. (Investigations on starch gels using the
ridgelimeter.)
Brimhall, B. and Hixon, R. M., Cereal Chem., 1942, 19, 425. (Interpretation of starch
pastes.)
Chichester, C. O. and Sterling, C., Cereal Chem., 1957, 34, 233. (Stress relaxation in
starch gels.)
Chikubu, S., Chem. Abstr., 1960, 54, 10357. (Viscosity of non-glutinuous rice starch
pastes in the Brabender amylograph.)
De, H. N. et al., Pakistan J. Sci. Ind. Res., 1966, 9, 239. (Starch granule diameter
positively correlated with tuber diameter.)
Daum, U. and Benninga, H., Tappi, 1970,53, 1710. (Interaction between clay and starch
in paper coating colours, rheology of mixture.)
Djakovic, L. and Dokic, P., Die Starke, 1972,24, 195. (Rheological characterisation of
starch gels.)
Erdi, N. Z., et al., J. Colloid Interface Sci., 1965, 28, 36. (Rheological correlated with
particle diameter.)
Farrow, F. D. et al., J. Text. Inst., 1923, 14,414; 1928,19, 18. (The flow of starch paste
through a capillary.)
Fetzer, W. R. and Kirst, L. c., Cereal Chem., 1959, 36, 108. (The estimation of starch
paste fluidities.)
Goering, K. J., Cereal Chem., 1970,47, 592. (Cooking viscosity curves of barley starch.)
Ghose, V., Chem. Abstr., 1956,50, 7435i. (Viscous properties of starch pastes in alkaline
media.)
Goto, F., Die Starke, 1969, 21, 128. (Studies in the Brabender Plastograph.)
Goto, F., Die Starke, 1972, 21, 267. (Gelatinisation properties of highly concentrated
starch suspension by Brabender plastograph.)
Gupta, S. L., Indian J. Chem., 1970, 8, 536. (Influence of certain modifications of starch
on its sorption of water.)
Hersiczky, A., Die Starke, 1965, 17, 1. (Effect of the concentration of the starch paste on
the accuracy of Hoppler viscosity measurements.)
Higginbotham, R. S., Shirley Institute Memoirs, 1946,20, 1. (The flow of starch pastes.)
Hollo, J., Nahrung, 1959,3, 617, 877, 1051. (Studies on paste formation in starch.)
Hollo, J., Nahrung, 1961,5, 506. (Paste formation in heat-treated potato starch.)
Honsch, W. M., Die Starke, 1956, 8, 277. (Brabender diagrams of modified wheat
starches.)
Hsieh, P. T., Chemistry (Tapei), 1961, 231. (Viscosity of cassave and sweet potato
starches.)
van Hallie, T. B., Bijdrage tot de kennis der verstijfseling en retrogradatie van zetmeel
door middel van de Rontgenspectrografie, Diss, Amsterdam, 1930.
Kawakami, K., Chern. Abstr., 1959, 53, 22601. (Brabender viscosity of gelatinised
starches.)
Kawamura, Y., Chern. Abstr., 1962,57,2479. (Viscosity curves of wheat starch pastes.)
Kihara, Y., Chern. Abstr., 1965, 62, 12006. (Viscosity of starches in rheometer UR-1.)
Kite, F. E., Die Starke, 1963, 15, 131. (Functional properties of food starches.)
Kite, F. E., Bakers Dig., 1957,21,42. (Properties of thick-boiling starches.)
Knyaginichev, M. I., Kolloid Zhur., 1956, 18, 38. (Viscosity of gelatinised potato starch
not reproducible. Reproducible value was obtained by treating with N NaOH at
room temperature.)
Koehler, R., Die Starke, 1963, 15, 56. (Basic concepts in rheology.)
Kopriva, B., Chern. Abstr., 1965, 63, 15083. (Sizes prepared from potato starch show
light thixotropy.)
Kopriva, B., Listy Cvkrovar., 1963,79, 141,224; 1963,80, 156. (Determination using a
Hoeppler viscometer.)
Kornienkoy, P. A. and Pvgin, T. S., Porosch. Met., 1968, 8, 101. (The physicochem.
state of starch paste changed continuously with time.)
Kovalevskaya, E. E. and Kurilenko, O. D., Chern. Abstr., 1965,62,5428. (Rheological
properties of starch pastes with admixtures.)
Kretovich, V. L. et al., Dokl. Akad. Nauk SSSR, 1970, 190, 1480. (Effect of heavy water
on the viscosity of starch solutions.)
Kurilenko, O. D., Chern. Abstr., 1961,55, 16842. (The rheological properties of solutions
of starch paste, amylose and amylopectin.)
Kuwajima, S., Chern. Abstr., 1958,52, 1660. (Variation of viscosity of starch paste.)
Lancaster, E. B. et al., Cereal Chern., 1966,43, 637. (Rheological properties of alkaline
starch pastes.)
Lancaster, E. B., Cereal Chern. Today, 1968, 13, 248. (Alkalisorbtion and swelling of
starch.)
Leach, H. W., Cereal Chern., 1959, 36, 534. (Structure of the starch granule.)
Lobanov, D. I., Chern. Abstr., 1961,22877. (Starch gelatinisation.)
Maslova, G. M., Die Starke, 1967,7,26 (referat). (The rheological properties of potato
starch pastes.)
Mazurs, E. G. et al., Cereal Chern., 1957,34, 141. (Brabender viscosity curves.)
McDonald, J. W., USP 3,103,451, 10 Sept. 1963. (Non-congealing cereal starch pastes.)
Mellies, R. L., J. Chern. Eng. Data, 1960,5, 169; Die Starke, 1961, 13, 114. (The Corn
Industries Recording Viscometer.)
Meyer, K. H. and Fuld, M., Helv. Chirn. Acta, 1942,25,391. (On the viscosity of starch
pastes.)
Meis, P. E., Treadway, R. H. and Smith, L. F., Ind. Eng. Chern., 1944, 36, 159. (The
influence of electrolytes on starch paste.)
Miller, B. S. and Mench, J. W., 'The viscosity of starch sirups', Purdue University,
Lafayette, Ind., 1946.
Milyutin, A. A., Chern. Abstr., 1958,52,2437. (The dependence of viscosity on the rate
of treating of starch during the drying process.)
Morsi, M. K. E. S., Univ. Microfilrns, 1965, Order 65-14548. (physical-chemical proper-
ties of starch-water systems.)
Nakagaki, M., Bull. Chern. Soc. Japan, 1961, 34, 316; Chern. Abstr., 1961, 55, 16079.
(Dynamic viscosity and dynamic rigidity of starch solutions.)
Nakagaki, M., Chern. Abstr., 1962, 57, 7683. (Viscoelasticity and structure change of
starch solutions.)
Nakamura, Z., Chern. Abstr., 1964, 61, 9624. (Viscosity of wheat starch, gel strength
wheat starch. Effect of the addition of salts, acids, alkalis.)
Nara, S. et al., Chern. Abstr., 1965,63,5881. (Effect of monoglycerides on the viscosity
and swelling of starch.)
Nishiuchi, T., Chern. Abstr., 1965,63,778. (Decrease of viscosity of an aqueous solution
of carboxymethyl starch.)
Osman, E. M., Cereal Chern., 1960, 37, 464. (Starch paste and oils in Brabender
amylograph.)
Ott, M., Cereal Chern., 1965, 42, 476. (Gel formation as related to concentration of
amylose and degree of starch swelling.)
Ozasa, H. et al., Chern. Abstr., 1959, 53, 12733. (Correlation of viscosity and freezing-
point depression in dilute starch-water systems.)
Patel, C., USP 3,152,925, 18 Aug. 1961. (High viscosity starch derivatives by treating in
unmodified starch with dichlorobutene.)
Patel, C. and Pyle, R. E., USP 3,271,387, 6 Sept. 1966. (High viscosity starch derivatives
with alkyl chlorothioformiates.)
Popov, I. D., Chern. Abstr., 1960,54, 10457. (The influence of salts on the viscosity of
starch solutions.)
Ramaszeder, K., Die Starke, 1971, 23, 176. (Rheological examination of textile starch
pastes.)
Rankin, J. C., et al., Die Stiirke, 1972, 187. (Acid-modified wheat flours, pasting and
dispersion properties.)
Reinders, M. A. and Gotlieb, K. F., Neth. Pat. 6,707,287, 1968. (Reaction of starch
with 90% amylopectin and POCI 3 .)
Richardson, W. A., Shirley Institute Mernoirs, 1933, 12, 101. (The flow of starch pastes.)
Robinson, J. V., Tappi, 1966,49,505. (The effect on urea on the viscosity of starch and
casein solutions.)
Samec, M., Die Stiirke, 1956, 8, 107. (Ageing of solutions of starch fractions.)
Schaffer, W. c., Cereal Chern., 1962,39,304. (Intrinsic viscosity of dehaldehyde starch.)
Schierbaum, F., Die Starke, 1966, 18, 110. (Heat-moisture treatment.)
Schierbaum, F., Germ. Pat. 38,076, 29 June 1964. (Edible swollen starch.)
Schoch, T. J., Cereal Chern. Today, 1959,4,202; Die Stiirke, 1959, 11, 156. (Brabender
viscosity curves and Brookfield viscosity at different rate of shear for various thick-
boiling starches: corn, tapioca, waxy sorghum, cross-bonded waxy sorghum.)
van Schoonneveldt, J. V. et al., Landbouwk. Tijdschr., 1968, 80, 63. (Effect of super-
phosphate manuring on yield of starch potato varieties and on the quality of the
starch.)
Schutz, R. A., Bull. Inst. Text., 1959, 81, 87. (Description of a viscograph-type of
apparatus for cooking starch paste.)
Schutz, R. A., Contribution it I'etude de I'amidon, Diss., Strasbourg, 1962.
Schutz, R. A., Die Stiirke, 1971, 23, 359. (Characterisation of starches with respect to
their applications in the textile and paper industry.)
Seves, A., Ric. Doc. Tessile, 1965, 2, 15. (Gelation of oxydised and esterified starch on
prolonged standing.)
Seves, A. and Croce, A., Ind. Carta (Milan), 1966, 4, 209. (Mechanical treatment of
starch paste caused disappearance of thixotropy, converting it in a pseudoplastic
substance.)
Shimizv, T., Chern. Abstr., 1962, 57, 10908. (Swelling of starches cross-linked by
epichlorohydrin.)
Shimizu, Y., Chern. Abstr., 1956,50, 8092. (Jelly strength.)
Smidsroed, O. et al., Carbohydrate Research, 1967,5,582. (Degradation rates measured
by viscosity measurements.)
Sterling, c., Food Research, 1956,21, 680. (Relation of age with strain retardation in a
starch gel.)
Sterling, c., Chern. Abstr., 1958, 52, 6664. (Crystallisation is the predominant factor
conferring rigidity on the gel.)
Sugimoto, K. et at., Chern. Abstr., 1966, 65, 20345. (Viscosity of potato starch as
measured by a B-type viscometer and an amylograph.)
Suzuki, H., Chern. Abstr., 1956,50, 16147. (Viscosity in 5 N KOH or NaOH.)
Suzuki, H., Chern. Abstr., 1959, 53, 3747. (Viscograph curves of acid-treated starches.)
Suzuki, S., Chern. Abstr., 1965, 63, 5882. (Amylograph cooling curves of potato, sweet
potato, wheat and corn starch.)
Takahashi, S. et al., Chern. Abstr., 1958, 52, 21185. (Viscoelastic properties of starch
paste.)
Takahashi, S. Chern. Abstr., 1959,53, 15610. (Effects of metal ions on potato starch.)
Tani, T., Chern. Abstr., 1957,51,9190. (Flow curves of rice-starch pastes in the Stormer
viscometer.)
Tegge, G., Die Starke, 1961, 13, 292. (Viscograph curves, influence of hard water, salts
and of P-content.)
Uematsu, T., Chern. Abstr., 1959, 53, 20932. (A method of measurement of viscoelastic
constants of pastelike materials at very low frequencies by electrical equipment.)
Tolmasquim, S., Chern. Abstr., 1966, 64, 19953. (Starch of Cicer arietinum is of the
cross-linked type, caused by the presence of fat.)
Ulmann, M., Die Starke, 1953, 5, 307. (Hoppler viscometer insufficient for the evalua-
tion of potato starch paste.)
Vasileva, E. G. and Veksler, B. A., Sakh. Prorn., 1967,41,51. (Changes in plasticity of
the sirup during crystallisation of dextrose.)
Wataribe, H., Jap. Pat. 14,488, 5 Nov. 1960. (Starch paste having sufficient stability.)
Wegner, H. and Winkler, S., Die Starke, 1954,6, 187. (Requirements for and measure-
ments of potato starch pastes.)
de Willigen, A. H. A., Vestnik Slovenskega Kerniiskega Drustva, 1954, 1, 131 (Samec
number). (Raising phosphorus content and viscosity of potato starch by agricultural
measures.)
de Willigen, A. H. A., Kali, 1957,33. (Viscosity of potato starch and manuring of the
potato.)
Winkler, S., Die Starke, 1957, 9, 213. (Measurements with the Viskowaage.)
Winkler, S., Die Starke, 1961,13,319. (Viscograph curves of H- and Kation-starches.)
Winkler, S., Die Starke, 1965, 17, 381. (Comparison of two viscometers.)
Winkler, S., Die Starke, 1966, 18, 316. (Comparative measurements of structural
viscosity of starch solutions.)
Winkler, S., Luckow, G. and Donie, H., Die Starke, 1971,23,325; 1972,24,58.
Wollerman, L. A., Cereal Sci-Today, 1958,3,244. (Properties ofpregelatinised starches.)
Yasumatsu, K., J. Food Sci., 1964, 29 (2), 198. (Changes of characteristics of starch
during gelatinisation in the presence or absence of fatty acid.)
CHAPTER 4
Physical Methods of Characterising Starch
INTRODUCTION
The industrial uses of starch are so numerous and the requirements of the
product are so varied, that a large number of techniques are necessary for
its complete characterisation. The problems encountered are further com-
plicated because of the many varieties of starch which are important in
industry and the numerous modifications produced by oxidation, acid
modification, dextrinisation and other methods. Some tests are made by
the starch manufacturer to control the quality of his products during
processing or to ensure that they meet required specifications. The same
or other tests may be made by the consumer to check the specifications or
to compare competitive products. Still other investigative methods are
used in research on fundamental problems of starch chemistry or in the
development of new or improved products. Standardised procedures for
the analysis and examination of maize starch have been laid down by the
Com Industries Research Association and these include the determination
of inherent viscosity and colour.
In the consideration of the problem of investigating starch it is important
to note that the literature on the subject contains almost innumerable
references to instruments and techniques for the purpose, of which some
are only of historical interest at present. It would be impossible to consider
all of them, and in this chapter attention will be centred on those of
importance at the present time, and especially on those which proved
valuable in the industrial evaluation of starch products. It occasionally
happens that methods which are reported in the literature have not proved
of general value for this purpose, and on the other hand some methods of
great value in industry have not been published and are therefore not
widely known.
91

Starch products find their greatest use in the textile, paper, adhesive and
food industries. They are usually cooked with water and used as hot or
cold fluids or as gels. It is customary to refer to dispersions of cooked
starch in water which are fluids as starch pastes, and this term is used to
indicate even those which are very dilute and which do not seem pasty at
all. While in many theoretical studies quite dilute pastes are often used in
order to avoid complicating effects which occur at higher concentrations,
many of the industrial tests must be made on pastes of a great enough
concentration so that the properties determined permit an adequate
prediction to be made of the industrial performance of the product. The
number of tests required and their nature depend on the purpose of the
examination and the circumstances under which it is made. They will be
enumerated and classified below and the description of the individual
tests will be given later in the chapter.
It is apparent that the starch manufacturer, knowing the variety of
starch and the treatment to which it has been subjected, need only make a
limited number of tests when the purpose is quality control and standardisa-
tion of the product. Those usually made are:
(a) Viscosity or fluidity of hot starch paste
(b) Alkali fluidity, especially for acid modified starches
(c) Colour
(d) Odour
(e) Hydrogen ion activity or pH
(f) Acidity or alkalinity
(g) Moisture
In addition, some supplementary tests are made of starches intended for

certain uses. These include:
(a) Cold paste viscosity if the starch is to be used cold as in the case of
certain adhesives; or at relatively low temperature, 120° to 135°F
(50° to 57°C), for paper coating.
(b) Gel properties if it is intended for use as a gel or in products such
as salad dressing, pudding and some candies where its use depends
on its gelling tendency in combination with other materials.
(c) Foreign matter.
(d) Bacterial tests if it is to be used in food.
(e) Flow properties or mobility if it is to be used for certain purposes
such as a dusting powder for rubber where a highly mobile product
is necessary, or in condiments where it is often used with the ground
PHYSICAL METHODS OF CHARACTERISING STARCH 93
materials to cause them to sift easily. On the other hand there are
uses for which a mobile starch is unsuited. An example is its use in
pharmaceutical tablets such as aspirin where a mobile starch will
not form a firm, strong tablet.
For the examination of an unknown starch or for the comparison of
competing starches some of the following tests must usually be made in
addition to those listed above:
(a) Microscopic examination (see Chapter 1).
(b) Analysis for mineral matter. This may be simply a determination of
total ash, or a qualitative or quantitative analysis may be required.
(c) Analysis for nitrogen. This may be necessary to determine the
presence of added nitrogenous matter.
(d) Analysis for fat, to determine whether fats, oils, soaps or similar
products may have been added.
(e) Determination of water soluble material, which may show the
extent of modification of the starch or the presence of added
substances.
(f) Examination of opacity of the paste, which is of assistance in
determining the variety of starch.
(g) Determination of the gelatinisation range, which is of value in
establishing the variety of starch and the treatment it has been given.
All of the methods given above are also useful in theoretical research or
in the development of new or improved products, and in addition
numerous other techniques may be employed. Important among these are:
(a) Alkali liability number 2 - 4
(b) Copper reducing number 5 ,6
(c) Iodine titration 7,8
(d) Fractionation of starch by complex formation 9 -1 0
(e) Action of enzymes 11
It is the function of this chapter to present only the physical methods of
testing starch. Tests of a chemical nature have been discussed elsewhere
(see Chapter 5).
4.1 VISCOSITY
The most important of all industrial tests used to characterise starches is

that of paste viscosity. It is generally recognised that to obtain a measure
of the true viscosity of a hot starch paste it must be determined at very low
concentration. 12 At higher concentrations pastes deviate from true

Newtonian properties of viscous flow. 13 While much research has been
done on very dilute pastes, examinations made for industrial purposes
must be made on those which are more concentrated and which have
anomalous viscosity. Such terms as 'viscosity', apparant viscosity and
consistency have been proposed 14 for this property. However within the
starch industry it is generally referred to simply as viscosity and this term
will be used in this chapter to correspond to general usage.
The anomalous viscosity of starch pastes has been investigated by
Farrow and co-workers/ 8,19 Alsberg,z° GallaY,21,22 Katz,z3 Brimhall
and Hixon,12 Taylor and Beckmann,24 Schoch25 and many others. These
studies show that the properties of pastes of unmodified starches, even
after considerable cooking, are not primarily colloidal phenomena but are
due to the presence oflarger aggregates including undisintegrated granules.
The work of McDowell and Usher 26 indicates that the swollen granules
form a structure which encloses portions of the liquid phase, thus increasing
the viscosity. The presence of such structural viscosity can be demonstrated
by making a viscosity determination of a hot paste of unmodified corn
starch immediately after agitating it. A second test made after the paste
has stood a short time without agitation will show an increase in viscosity.
For the determination of starch viscosity, any good viscometer can be
used, and an examination of the literature shows that practically all have
been, by one investigator or another. A review of some of the methods has
been given by Blinc and Samec, 27 while excellent discussions of visco meters
and the problems of viscometry are given by Barr, 15 Hatschek 28 and
Bingham,z9 whilst A. H. de Willigen has discussed the rheology of starch
pastes and suspension in connection with various designs of instruments
in Chapter 3.
Because the observed viscosity of starch depends on the technique
employed in preparing the paste and in making the test,31 the entire
procedure must be standardised with the greatest care in order to obtain
satisfactory results. To be specific, the factors which must be controlled
with precision are given below.
(a) The initial temperature of the water used for the paste should not
vary greatly
(b) The rate of increase of temperature must be the same in all tests
(c) The highest temperature to which the paste is cooked should be
closely controlled, and should not vary by more than a few tenths
degree C
(d) The rate of stirring must be uniform

(e) The type of stirring and the dimensions of the stirrer must be
accurately duplicated. It is essential that the entire quantity of paste
be heated uniformly. This requires that the paste which forms on the
sides and bottom be stirred into the main body of material. If this
is not done an insulating layer forms on the walls of the vessel and
retards the cooking of the remainder
(f) It is necessary that the time of cooking and the total elapsed time
from the beginning of the cooking process until the viscosity test is
started be closely duplicated
(g) Evaporation of water from the paste must be kept uniform and
should be minimised by use of a close-fitting cover with a condenser
(h) Differences in pH produce changes in viscosity, which must be
considered in testing
(i) Extraneous materials of many kinds both organic and inorganic
including salts, acids, alkalis,32-35,37,38 fats 32 and proteins,36
affect viscosity. This, of course, includes minerals which may be
found in natural waters (see also Industrial Uses of Starch and Its
Derivatives, Chapter 2).
It follows that for viscosity tests the paste should be made with distilled
water, it should be cooked in a water bath which is thermostatically
controlled, stirring should be done mechanically and the stirrer should be
powered by a motor which will maintain constant speed under varying
load. Furthermore, when the paste is transferred to the viscometer it is
necessary to perform this operation quickly to minimise the drop in
temperature which will occur. The viscometer must be preheated to the
temperature of the test to avoid heat loss from the paste to the instrument,
and great care must be exercised to ensure that it is clean.
4.1.1 Orifice flow

The commonest method used for the industrial determination of starch
viscosity is the measurement of the rate of flow of a definite volume of
paste through an orifice. In some cases the time in seconds required for a
definite volume to flow is measured, and this is taken as the relative
viscosity without changing it into absolute viscosity units. In other cases
the volume which flows in a definite time is measured. This is called the
fluidity of the paste. It will be seen that these quantities are the inverse of
each other, so that a starch of low viscosity has a high fluidity.
Among the instruments used are the Scott,39,42 Engler,41 ,42

Saybolt 40 ,42 and Redwood 42 ,44 viscometers. The Scott appears to be the
most widely used industrially, and although there are many techniques 43 , 74
employed for using this instrument, one typical procedure will be described
here. This description is given in considerable detail as an example of the
care that must be taken to standardise the technique of cooking and testing
by any method, and is applicable to other instruments than the Scott.
4.1.2 Scott test

Weigh the starch accurately on an analytical balance, transfer it to a
German silver beaker of 600 ml capacity and add 280 ml of distilled water
at room temperature. Stir the mixture to suspend the starch, then place
the beaker in a rapidly boiling water bath of large size and maintain it at
the boiling point during the cooking. Start a stopwatch at the moment the
beaker is placed in the water bath and stir the paste vigorously for exactly
five minutes. Then cover the beaker with a watch glass and allow it to cook
for 5 min without stirring. Next remove the cover, allow the condensate to
drain into the paste and stir it for 15 s. Replace the cover and continue
cooking until 14 min 45 s total cooking time. Remove the cover, drain as
before and stir it for 15 s. Rates of stirring in different laboratories vary
from 120 to 250 rpm. Whatever rate is used must be accurately reproduced
in all tests.
While the paste is cooking, heat the Scott cup in its water bath which
contains also a conical flask of 250 ml capacity graduated at 200 ml.
At the end of the IS-min cooking period quickly measure 200 ml of paste
in the flask and pour it immediately into the Scott cup. Release the plunger
at once and at the same time start a stopwatch. The time for a definite
volume of paste to flow is known as the Scott viscosity.
Because of the great range of viscosity of starches, the weight of starch
used and the volume of flow which is timed vary, depending on the
variety of starch and its modification. The same volume of water is used for
all tests. For thick starches such as tapioca 10 g may be used, for un-
modified corn starch 15 g; for moderately thin-boiling starches 28·5 g
and for those of greatest modification 100 g. The flow of either 50 or 100 ml
of paste is timed, the volume being chosen so that the time is adequate for
accurate measurement and yet is not unduly long. The rate of shear
decreases during the test. Therefore the head of liquid in the Scott cup
should not be allowed to become low, and volumes greater than 100 ml are
not timed. In order to obtain uniform cooking conditions it is necessary
that the liquid level in the water bath be maintained constant. Care must
be taken to prevent paste being stirred into the beaker above the paste
level. If a ridge forms, it may harden and such material transferred to the
viscometer cup may partly or completely plug the orifice.
In an effort to overcome variations in cooking procedure, most labora-
tories now use motor-driven stirrers. Direct introduction of steam into the
paste for heating and stirring has been applied. 45 This simulates the
cooking method employed by many starch users. It requires careful
adjustment of steam to produce a uniform rate of heating the starch and a
constant rate of boiling. The Scott cup is frequently provided with an
overflow at the 200 ml mark so that the paste can be introduced quickly
without first measuring it in another vessel.
4.1.3 Knife-edge orifice

Another type of orifice viscometer frequently used for both starches and
dextrins is one which employs as an orifice a small hole in a thin plate of
metal. This is known as a knife-edge orifice. An instrument of this type
was developed at the Penick & Ford laboratory for control and sales
service testing. It consists of a metal plate 0·8 mm thick through which
the orifice is drilled, a glass cylinder of 33 mm inside diameter and 305 mm
length with a section marked by two etched lines to contain 100 ml. The
lower of these lines is 120 mm from the bottom of the tube. The glass tube
is connected to the metal plate by means of a simple frame, with a rubber
gasket to prevent leakage. For various ranges of viscosity, tubes with
orifices of 1, 2 and 3·5 mm diameter are used. The apparatus may be
enclosed in a constant temperature water bath. Each instrument is
standardised by comparison with a master instrument which is kept for
this purpose. The cooked paste is poured into the tube until it is filled
above the upper etched line, and the time of flow of the paste contained
between the lines is noted. This instrument has proved excellent for rapid
testing.
4.1.4 Correction for moisture

Commercial starches contain varying amounts of moisture depending
on the source and the conditions in drying and storing them. This will
affect the viscosity, for if a standard weight of starch is used in testing, the
greater the moisture-content the lower will be the weight of dry starch it
contains. For this reason a correction is often made for moisture. This may
be done by first determining the moisture in the starch and weighing
amounts which will contain the same quantity of dry substance. Where
viscosity tests are made frequently on a routine basis a correction chart is
usually made on which the viscosity on a dry basis is graphed against

viscosity at various moisture levels. The advantages are that the viscosity
test may be made without waiting for the results of the moisture analysis,
and the routine worker will always weigh the same amount of starch.
4.1.5 Pipettes
Where a rapid approximate determination is to be made on a paste of
rather low viscosity, the Dudley47 or other pipette is often used. The paste
is drawn into the pipette and the flow of the measured volume is timed.
The pipette may be equipped with a constant-temperature jacket as in the
case of the pipette used by Chrzaszcz and Piorozek. 51 In industrial testing,
pipettes are often used without any means of maintaining a constant
temperature. Such determinations are subject to considerable error but
have the advantage of greater speed and convenience.
4.1.6 Standardising orifice and pipette viscometers

A frequently used method of standardising or comparing orifice and
pipette viscometers is to determine the time of flow of a definite volume of
water at some chosen temperature. It has been shown by Bue1 46 and by
Balderston 47 that this method is inadequate. One reason is that the water
time is often very low so that errors in measuring time will be relatively
high. Another reason of great significance is that the relationship between
time of flow and viscosity for this type of instrument is given by the
formula nip = At - Bit where n is viscosity, p is density, t is time of flow
and A and B are instrument constants.16.30.48 It is obvious that it is not
possible to evaluate the two constants by means of a single determination.
At least two determinations must be made using materials which differ
considerably in viscosity, and which preferably lie in the range of those to
be tested. Errors are minimised where an instrument is made according to
rigid specifications of orifice diameter and length, as is the case with the
Redwood, Engler and Saybolt. They are serious where viscometers are
made of a funnel with a glass orifice 49 such as are often used for the
alkali fluidity test, and with pipettes, 47.50 for it is practically impossible
to draw out a number of glass orifices which will be alike.
4.1.7 Capillary viscometers

Many investigators have used capillary viscometers for starch research.
With dilute pastes capillaries without applied pressure have been used, 27
while with more concentrated pastes pressure is applied, as by Richardson
and Waite 32 and Brimhall and Hixon. 12 The use of applied pressure has
proved advantageous for studying starch at various rates of shear.

Capillary viscometers are not used very frequently for industrial examina-
tion of starch because of the greater convenience and ruggedness of other
types. They have proved to be excellent research tools.
There is, however, one standardised test for inherent viscosity which is
recommended by the Corn Industries Research Foundation (tentative
standard 5-20-53) in which a weighed starch sample is dispersed in sodium
hydroxide solution using a standard technique. Relative viscosity of the
sample dispersion is determined by measuring the efflux time of the
dispersion and the solvent with a Cannon-Ubbelohde viscometer, Size 75,
Catalogue No. CUBU (Cannon Instrument Co., P.O. Box 812, State
College, Pa., USA). For convenience the viscometer is equipped with a
Cannon Instrument Co., Catalogue No. N120 Neoprene rubber holder.
The sample is ground to 20 mesh or finer, and an accurately weighed
sample representing one gram of dry substance of the sample is transferred
quantitatively to a 400 ml beaker to which is added 100 ml of distilled
water at 25°C in a constant temperature bath, to which 100 ml of 2·00 M
standard sodium hydroxide solution at 25°C is added whilst the solution
is stirred with a standard stirring device for 30 min. The liquid is then
filtered by gravity through a coarse porosity glass funnel. Three determina-
tions of viscosity are made, using the viscometer, which is then thoroughly
cleaned and dried, and the viscosity of a 1·00 M sodium hydroxide
solution determined.
The efflux time of the starch solution (dry substance concentration of
0·5 g/100 ml)(T) divided by the efflux time for the solvent (To) is the
relative viscosity. The inherent viscosity is defined as the natural logarithm
of the relative viscosity divided by the concentration, therefore:
.. 2·303 log (r
Inherent VISCOSIty «(0 dl/g = 0.5
4.1.8 Rotational viscometers

Commercial viscometers of the rotational type are widely used for
determinations of starch viscosity, though not as frequently as orifice
viscometers. They are often used for testing commercial products which
contain starch and are especially useful when it is desired to study viscosity
at several rates of shear.
4.1.9 Stormer viscometer

This instrument consists of a rotating cylinder within a sample cup
which is surrounded by a small water bath. The cylinder is rotated by
means of a falling weight, and an indicator shows the number of revolutions

made by the cylinder. In the usual procedure the time for 100 revolutions
is taken as the Stormer viscosity, using a weight which will enable the
test to be made in a reasonable time. It was used by Rask and Alsberg, 52
Glarum,53 MacMasters and Hilbert 54 and others. Geddes and Dawson
have derived an equation for transforming Stormer times into absolute
viscosity units. 55 More recently Fischer and Lindsley have modified the
Stormer viscometer to make it suitable for measurements in absolute
viscosity units, 123 and have applied it to the rheological study of starch. 124
Their work shows that starch pastes are pseudoplastic, for the curves of
torque versus rate of shear have no linearity, and no intercept on the torque
axis corresponding to a yield value.
In a typical routine industrial procedure from 7 to 15 g of starch,
depending on the degree of modification, is dispersed in 100 ml of distilled
water. The slurry is placed in a boiling water bath and stirred until it
reaches 90°C. It is then covered and is allowed to cook undisturbed until
30 min after heating was begun. The water bath on the viscometer has
meanwhile been heated. The sample is poured into the Stormer cup and
the weight is released. The cylinder is allowed to make 10 to 15 revolutions
in order to avoid the error due to inertia when it is started, and the next
100 revolutions are timed. Another procedure is described in detail by
Barham. 74 Searle's viscometer 56 is very similar in principle to the Stormer
and can be used equally well.
4.1.10 Macmichael viscometer 5 7

Gallay and Bell 21 reported the use of this viscometer in their studies of
starch viscosity. A critical study of the instrument has been made by
Herschel. 58,59 It is used regularly in the food industries for flour,60 as
well as for prepared products which contain starch, such as puddings and
salad dressings. In the paper industry it is often used for studying the
properties of paper coatings. 125 ,126 It has a sample cup surrounded by a
small water bath. Suspended centrally in the cup by means of a torsion
wire is a disc. The cup is rotated at constant speed by an electric motor
and the twist imparted to the wire is measured in arbitrary units by means
of a pointer and dial. There are several interchangeable wires of different
sizes so that a wide range of viscosities can be covered. The speed of
rotation can be changed readily so that it is possible to study a sample at
various rates of shear. This is especially useful in the study of thixotropy,
dilatency and plasticity, as in the preparation of paper coatings.
When used with hot starch pastes two difficulties appear. One is that
with thick starch pastes the wire does not reach an equilibrium rapidly.
Instead the reading slowly decreases over a period of several minutes due
to breakdown of paste structure. In tests with 5 % unmodified corn starch
Bechtel found that the reading fell 16 units over a period of 6 min. This
makes it difficult to decide what reading to take as the viscosity and makes
any figure purely arbitrary. The other difficulty is that the water bath,
like that of the Stormer, is very small. Even by use of the supplementary
heater provided, it is difficult to control paste temperature accurately
because of loss of heat to the rather massive disc and the supporting
column which surrounds the torsion wire.
4.1.11 Brookfield viscometer

Another rotational instrument of somewhat different principle is the
Brookfield viscometer. 61 It consists of a spindle which is turned at constant
speed by an electric motor. Viscosity is indicated by a pointer on a dial
and it is very simple to express the results in absolute viscosity units. There
are several easily interchangeable spindles so that it can be used over a
wide range of viscosities. One model permits operation at several speeds
so that it can be used to determine the effect of different rates of shear.
It may be held in the hand during a test, and a guard is provided so that it
will be centred in a 600 ml beaker.
To evaluate a starch sample, an aqueous suspension containing as low
as 2-3 % (dry substance) of an unmodified starch, or lower for a cross-
bonded product, or as high as 50-60 %for a highly modified starch product
such as dextrin, is stirred in a metal beaker which is then placed in boiling
water or steam bath, and the suspension cooked whilst stirring in the
prescribed manner. The time of heating is generally about 30 min, depend-
ing on the volume of suspension and the type of sample, and the paste
temperature should reach about 95°C. In limiting the sample evaluation,
type and vigour of stirring, the rate of temperature rise, and the cooking
time, should be reproduced from sample to sample.
Since most starch pastes are non-Newtonian, viscosities may be deter-
mined with several spindles at different speeds to obtain data indicating
the shear dependence. The same speed and spindle should be employed in
all sample comparisons involving a single observation, and the spindle
number and speed should be reported with all calculated viscosities.
From the foregoing it will be seen that it has many of the same advan-
tages and disadvantages as the MacMichael when used for hot starch paste
determinations. One advantage is that it can be used without the necessity
of transferring the paste to a special viscometer cup with the resulting
lowering of temperature and loss of time. However, it is observed that

during tests with this instrument the temperature of the paste slowly falls,
due to the large surface exposed to the air. As in the case of the
MacMichael, continued rotation of the spindle in a thick boiling starch
brings about a steady fall in the reading, making it difficult to select a
value for the viscosity of the paste. Its greatest use with starch is in the
evaluation of comparatively cool mixtures of starch and other materials,
such as paper coatings. The complete list of the rotational devices that
have been used in starch paste work would include the Rotovisco, the
Ferranti-Shirley cone plate viscometer, the Fann V-G meter, the Hercules
Hi-Shear instrument and a number of others.
4.1.12 Falling sphere viscometer

The faIling sphere viscometer has been applied to the testing of starch
products. An excellent discussion of the theory of this method and the
possible errors and their correction has been given by Barr. 1 7 The instru-
ment required according to theory must use a sphere of small diameter
compared to that of the fall tube. Such a viscometer was used by Gibson
and Jacobs. 62 One possible error is due to the fact that the faIling sphere
may tend to wander from a straight path. 63 A difficulty in applying this
instrument to starch pastes is that many pastes, such as those of cereal
starches, are rather opaque and it is difficult to observe and time the fall
of the sphere. H6ppler 64 developed a relative viscometer using the falling
sphere principle in which these difficulties have been overcome. It has been
described by Wobser and Miiller. 65 In this instrument a glass fall tube of
about 1·6 cm diameter and 20 cm length is enclosed in a water bath
equipped with a means for maintaining constant temperature. The fall
tube is inclined at an angle of 10° from vertical. It has a series of spheres
of varying diameter and material to cover a wide range of viscosities.
The inclined tube acts as a guide, causing the sphere to follow a straight
path. The spheres are only slightly smaller than the diameter of the tube
so that they can be observed easily in starch pastes. However, this type of
instrument is very sensitive to structural viscosity of the pastes, as reported
by Morgan and Vaughn,49 so that its application would seem to be limited
either to very dilute pastes or to those of highly modified starches. It has
in fact been used for the purpose of determining structure in pastes by
Komm and Martin 66 who measured the time of fall in pastes of starches
and dextrins at various intervals of time and extrapolated to zero time.
The time of fall was found to increase with each passage to a final value
which they called the stable structural viscosity.
It was found by Bechtel that a falling sphere viscometer or theoretical

design gives erratic results with thick starch pastes, that with the HappIer
type the time of fall in 5 %pastes of unmodified corn starch increases with
successive passages of the ball if the recommended 3 min is allowed to
elapse between passage, and that results with different cooks of the same
starch do not agree well. To obtain agreement it was necessary to pass the
ball through the paste several times without allowing time between
passages, before timing a fall. This makes the method a slow one.
The falling or rolling ball and the falling plunger principles have been
employed with some success and these and other commercial instruments
have been described by Van Wazer and co-workers. 139
In every case it must be remembered viscometric and/or rheological
properties of starch pastes are functions of the method of preparation as
well as of sample origin and history, and consequently it is imperative that
adequate and reproducible pasting procedures be employed prior to
viscosity determination or paste characterisation by any method.
4.2 COLD PASTE VISCOSITY
One cannot derive the viscosity of a cold paste from the hot paste viscosity
determination. Starches from different sources and those given different
treatment in manufacture differ widely in the extent to which their pastes
thicken on cooling. While unmodified tapioca, potato and waxy maize
starches have high hot paste viscosities, their cold pastes thicken or 'set
back' much less than those of the common cereal starches. When a cereal
starch such as corn starch is modified by acid to a moderate extent, the
tendency of the cold paste to congeal is much greater than would be
expected from its hot paste viscosity. On the other hand, when such a
starch is oxidised by hypochlorite, the tendency of the cold paste to
thicken is much reduced. Therefore, whenever a starch is to be used in the
form of a cold paste, it is necessary to determine the paste viscosity under
approximately the conditions of temperature and concentration at which
it will be used.
For the determination of cold paste viscosity orifice or capillary
viscometers can be used if the concentration of starch is low, for example
in the order of 2 %or less for unmodified corn starch, or if the starch is highly
modified so that the cold paste is thin. Katz,l with 1 % corn and potato
starch, used an Ostwald viscometer. Where it is necessary to test pastes of
higher concentration and of low degree of modification so that the cold
pastes are rather thick, they tend to clog the orifice. In such cases a
rotational instrument such as the Stormer or MacMichael can be used to
advantage. The paste should be prepared with all the precautions required
for a hot paste, and in the concentration range in which results will be
significant in terms of the intended use of the starch. When the paste has
been cooked it is placed in a constant-temperature bath to cool to the
temperature desired for testing. The cooling period may be about 3 h, but
whatever time is allowed, must be kept uniform so that all pastes will cool
at the same rate and to the same temperature. If a skin forms on the
surface it must be removed prior to testing.
Since the extent of viscosity increase depends on the time that the paste
stands, it is frequently necessary to make further tests after longer periods
of time. In commercial practice two tests are often made, one after 3 h
and one after 24 h. From these data reasonably accurate information is
gained about the rate and extent of thickening. It should be observed that
the above procedure refers to fluid pastes. If a gel has formed on cooling,
it is necessary to use a lower starch concentration.
4.3 VISCOSITY CURVES
There are numerous different pieces of apparatus for viscosity measure-

ments which are used in industry and the academic fields to obtain
rheological data by the scale and measuring systems of the instruments
used. Unfortunately the data so obtained cannot be compared because of
the different units and systems associated with each instrument. No
suitable standardised method seems, so far, to have been devised. The
standardisation of methods depends on the ability of the apparatus to
produce reproducible results when uniform working conditions are
observed.
W. Kempf and G. Kalender 142 have determined the viscosities of
various starches under different conditions using the Brabender visco-
graph, Rotovisco instrument and the HappIer falling-ball viscosimeter to
establish optimum conditions for the reproducibility of results. It seems
to be possible to review comparatively the rheological properties and
behaviour of starches with regard to their importance in the field of
technical applications, if methods are available which enable test data to be
obtained reproducibly.
The viscosity of a paste changes continuously with heating and stirring.
When starch gelatinises the viscosity begins to increase with the swelling
of the granules and this continues until a maximum is reached, after which
it decreases more or less depending on the variety of starch and the
modification it has been given during manufacture. The instruments and
methods previously described are best suited for making single determina-
tions of paste viscosity. They have been used to make a number of deter-
minations from which a viscosity curve has been derived, but the process
is rather laborious because of the necessity of cooking large samples and
of cleaning the viscometer between tests.
Instruments have been developed by which a series of viscosity curves
can be recorded on a chart. Such instruments are now widely used and
are of great value because they completely characterise a starch and give
the temperature of initial viscosity rise, gelatinisation range, maximum
viscosity, time of cooking and temperature at the maximum, and the
viscosity at any time during the cooking period, from which the rate and
extent of decrease in viscosity after the maximum can be obtained. When
it is desired, a cooling curve can also be made.
Another advantage of these instruments is that the entire cooking and
testing procedure can be controlled automatically so that the results are
entirely free from the effects of variations in technique due to different
operators. The value and importance of this feature is made apparent by
reference to the detailed cooking and testing procedure given under the
Scott Test, in which a slight deviation from the described technique alters
the result of the test.
The consistometer of Caesar 6 7.68 has a beaker for the paste, surrounded
by an electrically heated water bath. The paste is stirred by a streamlined
agitator driven by a constant-speed electric motor, and the changes in
viscosity are followed by observing the power input in watts required to
drive the agitator through the paste at uniform speed. These readings are
then graphed against paste temperature. By passing a stream of cooling
water through the water bath the changes in viscosity during cooling can
be observed. Its function is to measure viscous and plastic effects in
concentrated pastes, with a working range of 15 to 30 % concentration of
starches. Caesar has stated that the lower limit of concentration which
can be used is 10%.
In their study of the gelatinisation of starch, Mullen and Pacsu 116
developed a consistometer of the same principles but somewhat different
design. Radley 69 has designed a similar instrument, only that the power
input into the motor is maintained constant and the speed of the stirrer
is allowed to vary. In this case motor speed is graphed against paste
temperature. The curves are the inverse of those obtained by Caesar. A
FIG.4.1. Barham, Wagoner and Reed Viscometer. (1) Oil bath. (3) Electric heaters.
(9) Cooling coil. (11) Rotating solution cup. (13) Suspended torque cylinder. (Courtesy
H. N. Barham.)
recording viscometer designed for concentrated pastes by Bauer has been

described by Glabe. 70 It measures the torque against two paddles which
are driven at constant speed, and the results are graphed by means of a
recording dynamometer. Houtz 71 reported the use of a consistometer
similar in principle to Caesar's, in which the power input required to
drive a stirrer through the paste is measured.
Barham, Wagoner and Reed 72 have designed a continuous reading
concentric cylinder viscometer for starch (Fig. 4.1). The outer cylinder is
driven at constant speed in an electrically heated oil bath. The torque on
both internal and external walls of the inner cylinder is measured by
adjusting weights on a balance beam until the resistance of the paste is
equalled. This instrument is sensitive in a much lower range of viscosities
than those previously described. It has been designed for pastes of between
5 and 10% concentration. They have recently applied their instrument to
the study of potato, sweet potato and sorghum starch. 73, 74 From the
results of many tests they find that their precision is within ± 2 % (see
Fig. 4.1).
Higginbotham, 117 also, developed a consistometer for continuous
measurement of starch viscosity, and has applied it to problems arising
in the evaluation of starches for use in the textile industry. It is sufficiently
sensitive for testing concentrations as low as 5 % of unmodified starches
and results have been found reproducible within 1 %. A paddle is driven
at constant speed through a sample of 170 ml and the torque against the
paddle is transmitted through a differential to a quadrant balance.
Readings may be made at any time by means of a pointer and scale. In
order to reproduce industrial conditions of preparing sizing pastes, they
are usually cooked at 99°C.
The VI-Viscograph of Selling and Lamoen 122 employs a stirrer
driven by a synchronous motor in a housing which rotates on a ball
bearing. Attached to the motor housing is a coil spring. When the stirrer
operates in a starch paste the torque against it causes the motor housing
to rotate until the torque is balanced by the spring. The coil spring is
calibrated in gram-centimetres and results are graphed by means of a
recorder.
4.3.1 Brabender amylograpb

A widely used instrument is the Brabender Amylograph, which is
available commercially. It has been described by Brabender,75 and by
Muller (see Fig. 4.2).76 The vessel in which the starch is pasted is rotated
at a constant speed of 75 rpm in an electrically heated air bath by means
, r--.;,
.
FIG. 4.2. The Brabender AmyJograph. (Courtesy Brabender OHG, Duisburg.)

of a synchronous motor. The cover, which does not touch the rotating
vessel, has several small cylindrical rods which extend down into the paste.
It is fastened to a vertical shaft which is connected to a coiled torsion
spring. The rods stir the paste when the vessel is rotated, and the torque
of the paste against them turns the shaft until it is balanced by the torsion
spring. Attached to the spring is a pen by means of which the changes in
viscosity of the paste are recorded on a chart of the strip type. An interesting
feature is a means for controlling the temperature rise so that it occurs at
the constant rate of about I·5°C/min. The thermoregulator can be pre-set
so that at any desired temperature no further rise will occur. A means for
controlling the rate of cooling so that it too can be made constant, is
provided. The method of Schoch and co-workers 135 is recommended but
alternative procedures, e.g. that of Anker and Geddes, 3 6 who have made a
critical evaluation of the instrument, can be used. The latter found that
duplicate curves agree closely both in the temperature at which significant
changes occur and in the values of viscosity. They tested the effect of
adding wheat gluten, buffers, enzymes and cold gelatinising agents. They
also showed results obtained with a series of modified corn starches. Meiss,
Treadway and Smith 61 used this instrument for measurements with potato
starch and studied the effects of drying methods and of water soluble
materials.
When using the Brabender instrument for evaluating starches from
different sources, or with different histories of chemical and physical
treatment, or both, complete cooking and cooling curves can be determined
for each sample at 5 to 8 concentrations chosen over the range of the
instrument. When these curves are plotted and superimposed on rect-
angular co-ordinates, a family of curves is obtained. These contain five
successive points of interest.
1. The highest viscosity that the user may encounter during the prepara-
tion of a usable paste, is indicated irrespective of the temperature
(peak viscosity).
2. The viscosity of the paste, when it reaches the temperature of 95°C
in relation to the peak viscosity, reflects the ease of cooking starch.
3. After cooking for one hour at 95°C, the viscosity curve indicates the
stability or breakdown of the paste.
4. The viscosity of the cooked paste after cooling to 50°C is a measure
of the thickening produced by cooling.
5. The final viscosity after stirring for one hour at 50°C indicates the
stability of the cooked paste to mechanical treatment.
Thus the viscosities corresponding to each significant point may be

taken from the resulting family of curves and plotted on linear co-
ordinates against logarithm of sample concentration, and this plotting
technique facilitates direct comparison of samples of extreme variation
in viscosity because 100% change in concentration on the logarithmic
concentration ordinate is represented by the same linear distance at all
concentrations.
4.3.2 Corn Industries viscometer

This viscometer, developed under a fellowship of the Corn Industries
Research Foundation, has been described by Kesler and Bechtel. 118 It is
being manufactured by the Gaertner Scientific Corporation of Chicago
and is available for general use. The purpose was to design a continuous
automatic recording viscometer suitable for general industrial testing in
the range of concentrations from 5 to 10 % of unmodified starch. The
viscometer has been patented127 and has been adopted as the official
testing instrument of the corn starch industry of the USA. It has been
used by Bechtel and Fischer 128 for the study of the flow of properties of
starch pastes. Another operating technique, e.g. the suggested technique
of TAPPI 140 will be apparent to those familiar with starch pastes
characteristics.
Starch, suspended in water, is heated in a metal beaker equipped with a
close fitting cover and condenser which prevents loss of water by evapora-
tion. Heat is supplied by an electrically heated, thermostatically controlled
water bath. Provision has been made for circulating cold water through
the water bath so that viscosity may be measured during cooling of the
paste.
For any viscosity test, the starch must be stirred before it gelatinises, to
prevent lumping. After gelatinisation begins, stirring should be of such a
nature that the entire quantity of paste is cooked uniformly at the same
temperature. The importance of adequate stirring is readily apparent,
since the viscosity depends on the temperature to which the starch is
cooked as well as the time that it is maintained there. Uniform cooking is
not easy to accomplish, for convection currents cease in all but the most
dilute pastes, and the starch thickens on the walls of the vessel to form an
insulating layer which, if it is not efficiently removed, results in rather
large differences in temperature between the paste at the walls and that at
the centre. Proper stirring is accomplished in this viscometer by use of a
mechanical stirrer consisting of two parts, one of which scrapes the wall
and bottom of the beaker to remove the adhering layer. The scraper is
turned directly by a synchronous motor. Through the hollow shaft of the

scraper is a shaft on which is a four-bladed propeller. The propeller has
the double function of stirring the body of the paste and of providing the
means of measuring viscosity. Power is transmitted to it through a
differential. The resistance which the propeller encounters is transmitted by
means of this differential to a dynamometer which actuates the pen of the
recorder.
To balance the torque a pendulum balance is used. Sensitivity over the
wide range of viscosities covered by starches is achieved by use of a series
of easily interchangeable weights. This method has advantages over the
use of torsion wires because of the ease of changing weights and because
they can be interchanged without requiring re-standardisation of the
viscometer.
Application has been made to the study of a number of starch properties
including the effect of rate of heating, final paste temperature, concentra-
tion, pH and defatting of corn starch. 119 Use of the viscometer for studying
enzyme conversions and cooling curves has also been reported. 12 0
4.4 COLD GELATISING AGENTS
Not only does hot water gelatinise starch to form pastes, but many
inorganic and organic chemicals cause starch to gelatinise in cold water.
These have been studied extensively by many investigators and their
effects are described elsewhere in this book. Several methods of evaluating
starches based on the use of added chemicals have been proposed. Among
these are the thiocyanate viscosity method of Richardson 77 and of
Jambuserwala. 78 Methods based on the use of soap have been proposed by
Houtz,71 Heald,7 9 and Kesler and Black. 8 0 However, the only method of
this type which has come into general use is the alkali fluidity procedure
first described by Buel 46 which is used regularly in the corn starch industry
for characterising certain kinds of starch products.
4.4.1 Alkali fluidity test

It has been found that for starches of one variety and of one method of
modification the alkali fluidities increase regularly with the extent of
modification and can be used to predict the properties of the pastes cooked
in the usual manner. For example, acid-modified corn starches are often
sold on the basis of their fluidity and this usually refers to their alkali
fluidity number. It should be noted that where starches of different kinds
are compared, the alkali fluidities do not give the same relative values as
the pastes prepared by cooking the starches with water. If, for example,
corn starch and tapioca starch of the same alkali fluidity number are
cooked, it is found that their viscosities are quite different. Similarly if
two starches are manufactured by different processes they may have the
same alkali fluidity number but will give cooked pastes which differ
widely. Nevertheless, when its limitations are understood, the method is of
great value and is frequently used both for factory control of the modifying
process and as a specification for the finished product.
Various procedures and funnels are in use 49 but the following directions
are typical. The fluidity funnel is an ordinary glass funnel of 4 in (100 mm)
diameter with the stem cut short. A glass tube of the same diameter as the
stem is drawn out to form an orifice about 1~ in (1·6 mm) in diameter.
Funnel stem and tube are joined by a short piece of rubber tubing, so that
the total length of stem and tip is about 3 in (75 mm). The tip is made so
that when 110 ml of distilled water at 75°F (24°C) is in the funnel, 100 ml
will flow out in 70 s. (As stated above, any such funnel should be further
standardised by use of other liquids of higher viscosity.)
5 g of starch is placed in a 250 ml beaker to which 10 ml of distilled
water is added, and a smooth slurry made. Then 90 ml of 1 % sodium
hydroxide at 75°F (24°C) is added and the paste is stirred for 3 min at
about 70 rpm. It is then placed in a water bath maintained at 75°F (24°C)
where it remains for 30 min without stirring. The fluidity funnel is also
immersed in the bath until 3 min before the test is made, when it is removed
and is allowed to drain. A finger is held under the tip, the funnel is tilted
somewhat, then the paste is poured in carefully so that the stem is filled
and free from bubbles. A 100 ml graduated cylinder is placed under the
tip and the paste is allowed to flow. The volume of paste which flows in
70 s, timed with a stopwatch, is the alkali fluidity.
4.4.2 Gel testing

When the more concentrated pastes of certain starches stand at room
temperature for a few hours, they set to rigid gels. Because of this property
they are used in the food industry for some candies, puddings and other
products,81 and for other industrial purposes. Evaluation of the gel
properties of starches intended for such uses is essential to ensure suitability
of the product.
Properties of starch gels can be determined with accuracy only if the
same precautions are followed in cooking the starch that were given for
viscosity determinations. In addition, the tests must be made under
carefully standardised conditions. The strength of gels depends on the time

of setting, the temperature at which they are stored and tested, the
dimensions of the mould, and in some testing methods on the nature of the
surface. In an unpublished work of Bechtel, it has been found that starch
gels increase in strength rapidly during the first 10 h and that during the
period from 18 to 24 h there is little change. With increase in temperature
the strength decreases rather rapidly at about 12°e and the decrease acceler-
ates as the temperature rises. Furthermore, if tests are made with a plunger
without removing gels from their moulds there will be effects due to the
walls and bottom unless the mould has a diameter several times that of the
plunger, and the gel has a depth of at least 1-!- in (37·5 mm). If a starch gel
is allowed to set with the surface exposed to air, even in a well-filled
stopper jar, a tough skin may form which destroys the value of any test
made through it. It is not advisable to remove the skin before testing, for
this alters the properties of the gel. Formation of the skin is often prevented
by covering the surface with kerosene immediately after pouring the hot
paste into the mould. A method of avoiding this effect is to remove the
gel from the mould and test it through the bottom. 82 In addition, results of
tests depend on the rate at which the load is added to the gel, so pre-
cautions must be observed to keep this reasonably constant. One such
instrument used in this method is shown in Fig. 4.3.
A number of methods have been proposed for testing gel properties.
Descriptions of the older ones are given by Sheppard 83 and Alexander. 84
In general, the methods employed measure either the force required to
break the gel, or the deformation of the gel caused by applying a definite
load. Since different properties are measured in the two types of tests
there is no general relationship between them. 85 • 88 This has led to the
development of instruments which make both measurements.
The term 'gel strength' or 'jelly strength' are often used loosely by
various investigators for either of the properties given above. Studies by
Brimhall and Hixon 85 show that the resistance of starch gels to deforma-
tion is true rigidity 8 6 and that results can be expressed in absolute units if
measurements are made in an apparatus capable of such standardisation.
Rough determinations of relative rigidity may be made by pressing the
surfaces of a series of gels with the finger tips or by removing them from
the mould and observing the deformation from the shape of the mould. 8 7
Such determinations are not adequate for work of high accuracy, and
because no numerical value can be given to the result they are not suitable
for comparisons of gels prepared at different times.
Many gel testers have been described in the literature, especially for
FIG. 4.3. Schematic drawing of embedded-disc gelometer. A is a platform adjustable

in height. B is a frame attached to the pan of a triple-beam laboratory balance. C
represents a means of applying a continuously increasing load to the disc.
testing glue, gelatine and pectin gels. Most of these have not been success-
fully applied to starch testing, because as a rule they are not sufficiently
sensitive and precise for measuring the properties of starch gels, which are
generally far less strong.
4.4.3 Rigidity
The rigidometer of Brimhall and Hixon 8 5 consists of a tall cylinder
with a long narrow tube suspended in it from a torsion wire. The head of
the wire is equipped with a pointer and the top of the jar has a graduated
scale. A paste is poured into the cylinder, the tube is centred, and the paste
is allowed to form a gel. The wire is twisted an arbitrary amount and the
resulting turn of the tube is measured by the deflection of a beam of light
reflected from a mirror on the tube to a large scale. Care must be exercised
that the wire is not twisted through an angle great enough to shear the
gel, as this would destroy the value of the measurements. Torsion wires may
be calibrated in absolute units so that the modulus of rigidity can be
calculated.
The Bloom gelometer 89 which is used in the glue and gelatine industry
is sometimes used for starch gels of high concentration. This instrument
measures the weight required to produce a depression of 4 mm depth in
the gel by a cylindrical plunger 12·5 mm in diameter. Weight is added to

the plunger in the form of lead shot, the addition of which is controlled
automatically by electrical contacts. The result of the test is known as the
'Bloom number', which is a measure of relative rigidity.
4.4.4 Breaking strength

The Tarr-Baker jelly tester,90.91 is frequently used 87 for evaluating
breaking strength. A plunger is slowly forced into a gel by an increasing
head of water. By means of a manometer the pressure is read at which the
plunger breaks the gel.
The method of Saare and Martens 92 is also used. 93 In this procedure a
disc of standard size is suspended horizontally by a rod attached to the
centre of one side at a definite depth in a paste which is then allowed to gel.
The jar containing the gel is placed on a bridge above the pan of a balance
and the rod is attached to the beam. Weight is added to the opposite pan
at constant rate, usually by pouring lead shot, until the disc breaks the gel.
Recently an improvement of this method was reported by Hamer 121 who
places the jar with the gel on an adjustable platform above the balance pan.
He adds mercury at constant rate to the other pan and adjusts the platform
so that the balance beam is maintained horizontal. In this way he obtains
the breaking strength with greater precision than heretofore, and from the
change in height of the platform he gets a measure of the deformability of
the gel under load before it breaks. Hamer has reported the effect of size
of the disc, its depth, gel temperature, and the rate of adding load, and
demonstrates the necessity of a standardised method for precise results.
The embedded-disc principle has been used recently by Bechtel 129 in a
gelometer suitable for rapid testing of both relative rigidity and breaking
strength of starch gels. This gelometer has been found to be very sensitive
to slight differences in gel properties, and to give results of high precision.
The tube penetrometer of Fuchs 93 consists of a sharpened metal tube
similar to a cork borer attached to a metal shaft which is held in a vertical
position by two bearings. A weight is placed on a small platform on the
top of the shaft, the penetrometer is released, and the time is noted for the
instrument to cut a measured distance into the gel. The usual commercial
penetrometers have not been found satisfactory, in general, for testing
starch gels because they lack the requisite sensitivity.
4.4.5 Rigidity and breaking strength

Hixon and Brimhall 82 have developed a gelometer for measuring both
relative rigidity and breaking strength of starch gels. It is designed
especially for gels of 6 to 9 % concentration. The paste is allowed to gel

in a 400 ml beaker from which it is removed and placed bottom down on a
level platform which has a circular hole at the centre. This procedure avoids
the effect of skin and ensures testing a smooth level surface. Suction is
applied at a slow constant rate through the hole in the platform. Readings
of suction are made on a manometer, while corresponding deformation is
measured by the height of a column of water. The test may be continued
until the gel breaks, and thus in a single test both relative rigidity and
breaking strength are found. They have shown that relative rigidities
measured with the gelometer correspond well with measurements made
with their rigidometer:
Another instrument suggested for this purpose is the gelometer of
Saxl. 9 5 It consists of a sensitive balance, on the platform on which the
gel is placed. By means of a counterpoise on a beam the balance is adjusted
to zero. A plunger is then moved by a rack and pinion until it is just in
contact with the gel. The plunger is lowered a measured distance and
weights are added to the opposite side of the balance until it is again at
zero. A series of readings may be obtained which give the relative rigidity
and finally the 'yield point' at which, without further addition of load, the
plunger penetrates the gel.
Moreover, compressive loads can also be systematically decreased so
that it is possible to use the instrument to measure quantitatively an entire
hysteresis loop, and what Saxl terms the dynamic characteristics of plastic
materials. By this means the elastic recovery of a gel can be determined,
and the line of demarcation found between permanent plastic deformation
and elastic recovery, before the material has been stressed beyond its
yield point. It has been found in this laboratory that the Saxl gelometer is
sufficiently precise and sensitive to show marked differences between
commercial grades of acid-modified corn starch.
4.4.6 Syneresis
On being allowed to stand, starch gels, like others, tend to lose water
leaving a more concentrated gel. This process of ageing is called syneresis.
A study of the syneresis of starch gels was made by Chapman and
Buchanan, 9 6 who found that the amount of syneresis increases with the
surface exposed, and with age. With increase in starch concentration it
decreases, while the length of cooking the paste has little effect. Acetates,
sulphates, oxalates and citrates were found to increase syneresis, while
many other salts retard it. Of the salts tested, those most effective in
retarding it were chlorides of calcium, strontium and barium, sodium
iodide, trisodium phosphate and potassium sulphocyanate. No relation-

ship was found between granule size and the extent of syneresis. It is an
undesirable property, and the best starches for gels are those with this
property at a minimum.
A method of evaluating syneresis is as follows. Gels are made under
identical conditions of cooking and storage. When the gel is firm it is
carefully removed from the mould so that the structure is not damaged,
and is placed on a sheet of filter paper in a place free from drafts and at a
uniform temperature. After definite intervals the distance the water has
travelled in the filter paper is measured. The best starches produce the
smallest water rings.
4.5 GELATINISATION TEMPERATURE

When a starch is heated with water the granules lose their birefringence,
then as the temperature rises they swell to several times their original size,
the viscosity begins to increase, and the appearance changes from that of a
cloudy suspension to a translucent paste. These changes occur in tem-
perature ranges which are characteristic of the type of starch and the
nature and extent of its modification. This makes observations of the
temperatures at which they occur of value in the differentiation and
identification of starches.
Much research has been conducted in this field, in which all of the kinds
of change given above have been used for the determination of gelatinisa-
tion range, the range of temperature in which the observed change occurs.
Since the changes do not occur simultaneously,2s it is apparent that the
gelatinisation range found depends on the property observed. In the older
literature a gelatinisation point was often given. This is misleading,
because some granules of a given starch will undergo any of the above
changes at a lower temperature than others. The gelatinisation point is
thus specific for each granule, but of doubtful significance when applied
to a sample of starch. For this reason it is now more usual to determine
the gelatinisation range.
A factor which must not be overlooked is that gelatinisation range is
influenced by the conditions of the test. Alsberg and Rask 97 showed that
the pH of the sample, the rate at which it is heated, and the extent to
which the starch has been dried previous to the test all affect the result.
The presence of added chemicals may also have an effect, and at least in
most methods the result depends on the concentration at which the starch
is tested.
4.5.1 Loss of birefringence

A small quantity of starch may be suspended in water in a test tube and
slowly heated in a water bath. Samples are taken at regular temperature
intervals and are examined under a polarising microscope. Or the sus-
pension may be heated in a microscope hot stage using electricity,98 or
hot water. 99 Various points have been taken as the gelatinisation tempera-
ture. Nyman lOO reported the temperature at which birefringence dis-
appeared in the larger granules, Reichert lOl reported the temperature of
disappearance in most granules, while that of complete disappearance
was taken by Francis and Smith. 99 Dox and Roark 98 chose the tempera-
ture of disappearance of birefringence in all granules large enough to show
the characteristic shape and markings.
4.5.2 Translucency
Samec l02 observed that if an incandescent light bulb is immersed in a
glass vessel containing a starch suspension which is being heated, a sudden
change in translucency occurs at a definite temperature. Cook and
Axtmeyer l03 devised a procedure by which a beam of light is passed
through a starch suspension and actuates a photoelectric cell connected to
a micro-ammeter. Readings of current and temperature are made and the
results when graphed give curves showing the beginning of gelatinisation
and the range. Cook and Axtmeyer stated that the method is capable of
accurate duplicability and that it is suitable for the identification of
starches and for determining the treatment given them during manufacture.
The method of Cook and Axtmeyer was refined by Morgan l04 who
made a study of the gelatinisation range of a large number of commercial
starches. Characteristic curves were obtained for each kind of starch, and
modification of the starch could be followed by the changes in the curves,
for with increased modification gelatinisation was found to occur at
progressively lower temperatures. Morgan also showed the application of
his method to the quantitative analysis of mixtures of starches. An
interesting feature of the method is that only 0·33 g of starch is needed.
The suspension is heated at a constant rate of 2·SoCfmin so that the test
can be completed in half an hour. Kuntzel and Doehner l 0 5 used a similar
method but included a recorder to obtain the transmission-temperature
curve automatically.
4.5.3 Refractometric method

A rapid optical method of great convenience which has been developed
in the laboratory of Penick & Ford is based on the use of an Abbe
refractometer. The starch suspension is heated slowly, and at regular

intervals a drop is transferred to the refractometer and the refractive index
is read. During gelatinisation the refractive index rises from zero to a
maximum. It is graphed against temperature, giving a curve which is
characteristic of the kind of starch and the degree of modification. The
method has proved to be sensitive and capable of accurate duplication.
It is also used to measure the state and concentration of sizes and dressings
in the textile industry and was used by Radley to examine the state of
gelatinisation of steam heated and modified starches. 141
4.5.4 Viscosity
Ostwald10 6 used the increase in viscosity of starch paste as a means of
determining gelatinisation points, making a series of viscometric tests at
increasing temperatures. He regarded as the ge1atinisation point the
temperature at which viscosity showed a sudden large increase. Alsberg
and Rask 97 studied the ge1atinisation of wheat and corn starches by
determining viscosity at various temperatures using the Stormer viscometer.
Their results show that the increase in viscosity with rising temperature is a
gradual process taking place over a range of 25 to 30 degrees. They there-
fore concluded that there was no definite temperature of gelatinisation, but
rather a gelatinisation range, a conclusion also drawn by Radley141 from
his work. The newer visco meters by which a viscosity-temperature curve is
recorded, or by means of which a series of readings can be made readily,
are well suited for the determination of ge1atinisation range. A great
advantage of these instruments for this purpose is that the gelatinisation
range is found in the same test that gives the viscosity curve. This method
appears to be equally as sensitive as any other.
4.5.5 Moisture
A comprehensive study of the problems involved in the accurate
determination of moisture in starch has been made by Sair and Fetzer.t07
They found that moisture can be removed completely without causing
decomposition of the starch, either by distillation with toluene using a
modification of the Bidwell-Sterling method/os or by heating it in a
vacuum oven to constant weight at 100°C. At an oven temperature of
135°C it was found that dextrins and highly modified starches lost volatile
matter other than water. Another finding of great importance was that
starch heated for as long as 40 h at 100qC in an air oven gave a moisture
analysis about 1%below that obtained in the vacuum oven. Their methods
with the precautions given in their original article, are suitable for reference
methods of great accuracy.
It is often desirable in practice to use rapid oven methods. One fre-
quently used routine procedure is to heat 5 g of starch in a metal moisture
dish for 4 h in a vacuum oven at 105°C. The official German method
requires that 5 g of starch be heated for 1 h at 50°C then 3 h at 120°C in
an air oven. 1 0 9 There are available various rapid moisture testers by
means of which a moisture value can be obtained in a few minutes. Such
arbitrary methods should be used only after comparison of the results
with one of the reference methods. A correction chart may then be made
for obtaining true moisture-content from the results of the rapid test.
4.6 FOREIGN MATTER
When starch is to be used for food or f01 paper coating, the presence of
foreign matter is very undesirable, and the determination of the amount
and its nature is essential.
Several methods are available for the separation of foreign matter from
starch. One rapid method which is frequently used is to sieve a definite
weight of dry powdered starch through a 200-mesh screen or through
No. 17 bolting silk until the starch has passed the screen leaving upon it
the foreign matter. Another is to suspend 50 g of starch by stirring in
500 ml of distilled water. The suspension is then passed through a layer
of No. 16 or No. 17 bolting silk. Other methods are presented in a hand-
book prepared by the US Food and Drug Administration,l1o including
procedures applicable to food products which contain starch.
If the starch is oily, as in the case of confectioners' moulding starch, it is
advisable to remove the oil by washing it with acetone. This greatly
improves its sieving properties. Acetone is to be preferred to ether or
petroleum ether because if the starch is to be suspended in water prior to
sieving, the acetone will dissolve readily.
When the foreign matter has been separated from the starch its amount
may be estimated. The nature of the particles may be determined by
microscopic examination. The handbook referred to above 11 0 has proved
of great value for the identification of foreign matter as it gives a discussion
of methods of microanalysis which may be employed, and descriptions
and illustrations of various kinds of contamination which may be present.
For starches and dextrins needed in first class work, the absence of
specks due to bran, dirt and particles of metallic origin is often essential,
and is a criterion of cleanliness. To measure specks, a sheet of glass with a

10 cm (100 mm) square marked on it, is laid flat on top of a layer of starch
or dextrin spread out on paper, and the number of specks enclosed in the
square is counted. Many firms have their own specifications of the number
of specks which will be tolerated. Particles of mineral or metallic origin
are separated quite readily by adding a weighed amount of starch to an
organic solvent with a specific gravity of about 1·5, and after suspension
has been accomplished in an Erlenmeyer flat-bottomed flask, allowing the
mixture to settle for some 5 min and then syphoning off the suspension,
the metallic or mineral particles will be found on the bottom of the flask.
Instead of complete removal of the starch in this method, water can be
used as a suspending liquid, and the starch allowed to settle as a hard
cake on the bottom before pouring off the supernatant liquor when the
foreign particles will be clearly seen through the bottom of the flask.
K. Takeichi and S. Ikeda 136 find that sweet potato and wheat starches
can be well graded for dirt, colour and protein content by mixing 1 g of
the dried starch in a white porcelain crucible with 3 ml of water, followed
by 5 ml Nil sodium hydroxide solution. The specks can be very easily
seen and counted.
On squeezing some starch samples between the fingers, they give way
suddenly, often emitting a distinct high-pitched 'squeak' or 'crunch'. At
one time, some attention was paid to this, since it was considered that the
better the 'squeak' or 'crunch' the better the quality. But this belief has
now been put in proper perspective by A. H. de Willigen in Chapter 3,
to which the reader is referred.
4.6.1 Odour
Examination for odour is made to determine possible rancidity of the
fat present in cereal starches, and mouldiness which may be due to poor
storage conditions. One method is to place a quantity of starch in a flask
or beaker, then add water at 160 to 170°F, stir quickly and note the odour.
Another and less rapid method is to place the starch in a jar with a tight-
fitting top and heat it for 16 h at 130°F. As soon as the jar is opened the
odour is noted. Unpleasant odours can be detected readily by either
procedure.
4.7 HYDROGEN ION ACTIVITY
The problems encountered in measuring pH suspension of starch in water

were investigated by Ripperton. 111 His work shows that the observed pH
changes in starch concentrations up to 5 % above which it is constant. He

found that if the starch is allowed to settle and the test made on the
supernatant liquid, the observed pH is somewhat different from that of
the suspension, and that it is not constant. To reach equilibrium it was
found that the suspension must be stirred for 10 to 30 min. In the standard
method of the Corn Industries Research Foundation (tentative standard
5-23-55) the stirring is standardised at 30 min.
Two kinds of acidity are measured: (a) the extractable, and (b) the paste
acidity. In the first the sample is ground and 10 g of it (plus or minus 0·1 g)
stirred in 100 ml of distilled water for 30 min, gravity filtered and titrated
with 0·1 N sodium hydroxide against phenolphthalein indicator.
In the determination of paste acidity, the weight taken is the same, but
the amount of water taken is 300 ml and the suspension brought to the
boil on a hot plate or over an open flame for approximately 15 min,
stirring occasionally, and allowed to boil for 10 min. Again the sample is
titrated immediately with standard 0·1 N sodium hydroxide against
phenolphthalein.
Since many acids contribute to the acidity of the sample, the value is
reported as milli-equivalents of acid per unit sample weight.
The pH can be determined electro metrically before the starch settles
out in the determination of destructible acidity, and it has been found in
some cases when the glass electrode is used, that starch coats the electrode,
with the result that determinations are erratic. This can be prevented by
stirring the suspension during the test, and by making it as rapidly as
possible.
4.8 COLOUR
Several methods are in use for the determination of colour in starch

products, ranging from visual inspection to the use of a photometer. One
very simple method is to place a small pile of starch on a porcelain plate
and place adjacent to it similar piles of starches which have been selected
as colour standards for matching. By means of a clear glass plate the piles
are carefully flattened so that they touch each other to form a continuous
layer. The sample is compared with the standards by observing them in
diffused daylight. It matches the standard when no boundary can be seen.
Another method consists in extracting the coloured material by sus-
pending 10 g of starch in 200 ml of 2 % alcoholic sodium hydroxide. The
suspension is heated to boiling, then is cooled for 30 min with continuous
shaking. After filtering, the liquid is compared with standards by use of a

colorimeter. This method is based on the assumption that the colour is
due to alcohol soluble materials, which is not always true.
The Lovibond tintometer is often used. The colour reflected from a
smooth surface of powdered starch is matched by interposing standard
Lovibond series 200 red and series 510 yellow colour glasses in the light
reflected from the smooth white surface of a magnesia block placed beside
the sample.
Various visual or photoelectric spectrophotometers of the reflectance
type are also used. The chief precaution in any measurement is to use
powdered starch to ensure the observation of a smooth surface.
The Corn Industries Research Foundation extensively examined and
standardised the various procedures for the analysis of products of the
corn industry, and with the regard to the colour of corn starch the methods
developed have been laid down in the standard analytical methods of this
body. The Tentative Standard Method 11-18-57 is specifically devoted to
the detailed procedure for the determination of colour by spectrophoto-
metric means in which the instrument specified is a Beckman Model B
spectrophotometer equipped with the integrating sphere diffuse reflectant
attachment, with beam-expanding lenses and a blue sensitive photo-tube,
or equivalent equipment, and the analyst is advised to calibrate and
operate the instrument according to the manufacturer's instructions, as
the reflectant standard, Vitrolite glass block secondary standard, calibrated
against freshly prepared magnesium oxide is used. The reflectance (%R)
at 450 mJ1, 550 mJ1 and 600 mJ1 is measured with this instrument, and the
calculation involved is:
Colour = log %R at 600 mJ1 - log %R at 450 mJ1
Brightness = %R at 550 mJ1
Greyness = 2 - log %R at 550 mJ1
C.LE. colour specifications, purity and luminous reflectants for starch
and other near white products derived from corn, can be estimated from
colour and brightness values by use of the following relationship:
C.I.E. % purity (estimated) = (l07 x colour) + 0·1
C.I.E. % luminous reflectance (estimated) = brightness - 0·3
In Japan, great importance is attached to the colour in the sweet potato
starch industry, in view of the content of yellow coloured compounds liable
to be found in the final starch. In the USA the Brice-Keene photometer13 0
is used to determine the photoelectric reflectance of the sample, and
express it as a percentage of the whiteness of a standard plate calibrated

against pure precipitated magnesium oxide. In Japan, however, the
Hunter reflectometer, which is used in the paper trade, attached to a
photoelectric colorimeter AKA No. 50 of the Kotaki Manufacturing Co.,
and the integrating sphere of the same Company, has been used, but any
other good commercial instrument will serve. 131 ,132 T. Fukada and
T. Maezawa 133 measure the reflectance at 452 m{l. S. Suzuki et al. 134
have also made studies on the measurement of whiteness of sweet potato
starch.
4.9 FLOW PROPERTIES OR MOBILITY OF DRY STARCH
The readiness with which powdered starch flows can be measured by

shaking a definite amount of the dry starch in a covered sieve, using a
mechanical shaking device to ensure uniformity for all samples. The
amount of starch which passes through the screen in a definite time is a
measure of its mobility.94 Another and very easily performed test is to
place a small sample of powdered starch of standardised size and conical
shape on a horizontal glass plate which can be inclined and behind which
is a scale showing the angle of inclination. The plate is slowly inclined at a
constant rate and the angle is noted at which the starch begins to slide.
The more mobile starches flow at a smaller inclination.
4.10 WATER SOLUBLE MATERIAL
Determination of the amount of water soluble matter in starch is of value

because it gives an indication of the presence of added materials. In a
dextrinised product it is also useful as a means of determining roughly the
degree of conversion, for the more highly converted dextrins and gums
contain larger amounts of soluble constituents.
Methods for determining the amount of water soluble material all
depend on suspending starch in water at a definite temperature and
concentration. The suspension is maintained by agitation, then is filtered
and the filtrate analysed.
One typical procedure is to suspend 10 g of starch in distilled water to a
total volume of 200 ml. The flask is shaken vigorously each half-hour for
3 h. This must be done at constant temperature duplicated for all tests.
The suspension is filtered and an aliquot of20 ml of the filtrate is evaporated

to dryness on a steam bath. It is then dried to constant weight in an oven
at 105°C and the residue is weighed.
4.11 OTHER PROPERTIES
Certain other characteristics of pastes of different starches are used in

comparing or differentiating them, and although the tests are qualitative
in nature, the observations are frequently of great value. One of these is
the relative opacity of pastes. When pastes at the same concentration are
compared it is found that those of ordinary cereal starches are more
opaque than those of root or waxy cereal starches.
Differences in texture may likewise be observed. If paste is allowed to
flow from a stirring rod, some pastes break abruptly. These are termed
'short' pastes. Others are more cohesive and form a long string. They are
called 'long' pastes. Unmodified tapioca and potato starches form 'long'
pastes while those of unmodified corn and wheat are 'short'. Gallay112
has discussed long and short pastes and believes that differences are due
to the effective volumes and deformability of the swollen granules.
A. H. de Willigen further discusses this matter in Chapter 3.
4.11.1 Dustiness
In one patent 137 for making dustless starch, by blending in 0·005-5·0%
of glyceryl acetates or similar polyhydric alcohol esters, a test is given to
measure the 'dustiness' of powders wherein 10 g of the product under
test is allowed to fall freely through a glass column open at both ends.
After 4 s the bottom of the tube is closed with a tared card and any air
suspended starch in the column allowed to settle on the closure which is
then re-weighed.
Another test is also described 138 wherein a pre-determined amount of
material is agitated under pre-determined conditions, e.g. by rolling in a
small drum for a given time and speed of rotation and passing air through
it at a given rate of flow and collecting and weighing the extrained starch
dust.
4.12 DEXTRINS AND GUMS
In general, the methods already given for starches are also applied to the
examination of dextrins and gums. An excellent example of the use of
several of these techniques is given by Brimhall,113 who used oxidation

with periodate, amylose content, solubility, the action of enzymes, end-
group analysis, copper-reducing number and alkali liability in her study
of pyrodextrins.
4.12.1 Viscosity
Because the viscosity of these products usually is so much lower than
that of starches, tests are made using much more concentrated pastes.
From 30 to 50 % or even higher concentrations may be used, depending
on the product tested. An orifice or pipette viscometer is used industrially
and it may be jacketed with a water bath to maintain constant temperature.
The dextrin is made into a smooth suspension with cold water and is then
cooked to 85°C after which it is cooled to some standard temperature and
the viscosity test is made. The temperature chosen is usually near room
temperature. If the dextrin is to be used in an adhesive which contains
borax, the same proportion of borax is added to it before cooking it for
the viscosity test. This is necessary because the effect of borax in altering
viscosity differs with the type of product. It should be noted that the
addition of 0·5 to 1 %of sodium hydroxide, based on the weight of dextrin,
increases the effect of the borax and this must be considered in testing.
Some dextrins and gums, when prepared as pastes, thicken or 'set back'
and turn cloudy on standing. For some purposes this is a disadvantage.
The extent of set-back may be determined by making a viscosity test after
24 h and comparing the result with that found immediately after cooling
the paste. With insufficiently roasted dextrins the development of cloudiness
is very marked and such dextrins do not behave normally in adhesive
formulations.
4.12.2 Alkali fluidity test

The alkali fluidity test described above has been modified in the Penick &
Ford laboratory so that it can be applied to the evaluation of dextrins and
gums. For this purpose either 20 or 40 g of the dextrin is used, depending
on the degree of modification. This is suspended in 50 ml of distilled water
at 75°F (24°C), then 50 ml of 4 %sodium hydroxide at the same tempera-
ture is added. The other details of procedure are the same as those pre-
viously given. The method is subject to the same limitations as when
applied to starches, i.e. it cannot be used to predict the paste properties of
dextrins from different varieties of starch, nor those products prepared by
different processes. It is a valuable means of factory control of the extent
of dextrinisation, of specification of finished product, and of comparing

different dextrins and gums.
4.12.3 Water soluble material

The amount of water soluble material present is used to obtain informa-
tion about the extent of conversion of the dextrin and its suitability for
certain purposes. A method for this determination is to suspend 20 g of
dextrin in 200 ml of distilled water, at a definite temperature such as 20°C.
The suspension is shaken mechanically for 30 min at this temperature and
is filtered. Since in a pure dextrin the soluble matter consists entirely of
starch conversion products, the concentration can be determined in the
filtrate by a refractometer, as shown by Tolman and SmithY4 If the
refractometer has a sugar scale, the concentration is given directly, other-
wise it may be obtained from refractive index-percentage sucrose tables
which may be found in any handbook on sugar, such as that of Browne
and Zerban. 115 Dipping refractometers are very convenient and rapid,
and pocket refractometers have proved of great value in the factory.
Concentrations may also be measured directly by means of a Brix hydro-
meter. The concentration read must be multiplied by 10 to obtain the
percentage of soluble carbohydrate since the dextrin was made in a 1 to 10
solution for the test.
4.12.4 Other tests

It is frequently necessary to ensure uniformity in the colour of dextrins
to be used for certain purposes such as in paper coating. Flavour and
odour are important when dextrins are used in foods. In addition, pH,
acidity and alkalinity and moisture-content must often be controlled.
Reducing sugars are generally determined to ascertain the extent of
conversion and the suitability of a dextrin for an intended purpose. For
this purpose any standard procedure may be used, although the ferricyanide
method is widely preferred.
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37. Wiegel, E., Zeit. Spiritusind., 1933,56,62.
38. Wiedmer, C., Tiba, 1936,4, 103, 107.
39. MacNider, G. M., J. Ind. Eng. Chem., 1912,4,417.
40. Pierson, G. G., Ind. Eng. Chem. Anal. Ed., 1934,6, 183.
41. Chrzaszcz, T. and Janicki, J., Biochem. Zeit., 1932,256,252.
42. Nivling, W. A., Starches, Their Fluidity and Viscosity in Relation to Sizing Value/or
Textiles, New York, 1912, pp. 2-12.
43. U.S. Federal Standard Stock Catalog. JJJ-S-701, Sec. IV, Part 5,5 Dec. 1933, p. 3.
44. Ermen, W. F. A., J. Soc. Chem. Ind., 1907,26, 501.
45. MacNider, G. M., Ind. Eng. Chem., 1917, 9, 597.
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York, Orign. Comm., 1912, 13, 63.
47. Balderston, L., J. American Leather Chemists Assn., 1913, 8, 47.
48. Sheely, M. L., Ind. Eng. Chem., 1923, 15, 1109.
49. Morgan, W. L. and Vaughn, N. L., Ind. Eng. Chem., 1943,35,233.
50. Briggs, C. A. and McCarthy, J. L., Paper Trade J., 1942, 114 (4), 37.
51. Chrzaszcz, T. and Piorozek, S., Zeit. Spiritusind, 1910,33,66.

52. Rask, O. S. and Alsberg, C. L., Cereal Chem., 1924, 1, 7.
53. Glarum, S. N., American Dyestuff Reporter, 1934,23, 175.
54. MacMasters, M. M. and Hilbert, G. E., Ind. Eng. Chem., 1944,36, 958.
55. Geddes, J. A. and Dawson, D. H., Ind. Eng. Chem., 1942,34, 163.
56. Searle, G. F. c., Proc. Cambridge Phil. Soc., 1912, 16, 600.
57. MacMichael, R. F., Ind. Eng. Chem., 1915,7,961.
58. Herschel, W. H., Ind. Eng. Chem., 1920, 12,282.
59. Herschel, W. H., J. Optical Soc. America, 1923,7,335.
60. Bayfield, E. G., Cereal Chem., 1934, 11, 121.
61. Meiss, P. E., Treadway, R. H. and Smith, L. T., Ind. Eng. Chem., 1944, 36, 159.
62. Gibson, W. H. and Jacobs, L. M., J. Chem. Soc., 1920, 117,473.
63. Valenta, E., Chem. Ztg, 1906,30, 583.
64. HappIer, F., Zeit. Techn. Physik, 1933,4, 165.
65. Wobser, R. and Muller, F., Kolloid-Beihefte, 1941,52, 165.
66. Komm, I. E. and Martin, U., Vorratspflege u. Lebensmittelforsch, 1939,2,635,650.
67. Caesar, G. V., Ind. Eng. Chem., 1932,24, 1432.
68. Caesar, G. V. and Moore, E., Ind. Eng. Chem., 1935,27, 1447.
69. Radley, J. A., Starch and Its Derivatives, Chapman & Hall, London, 1940, p. 43.
70. Glabe, E. F., Cereal Chem., 1939, 16, 661.
71. Houtz, H., Paper Trade J., 1941, 113 (6), 32.
72. Barham, H. N., Wagoner, J. A. and Reed, G. N., Ind. Eng. Chem., 1942,34, 1490.
73. Barham, H. N., Wagoner, J. A., Williams, B. M. and Reed, G. N., J. Agricultural
Research, 1944,68, 331.
74. Barham, H. N., Wagoner, J. A., Harclerode, E. H. and Campbell, C. L., Kansas
Agricultural Experiment Station Bull., 1946,61.
75. Brabender, C. W., Muhlelab., 1937,7, 121.
76. Muller, G. J., Intern. Tech. Chim. Ind. Agri., 6th Congr., Budapest, 1939, 2, 529.
77. Richardson, W. A., Chemistry and Industry, 1939, 17, 464.
78. Jambuserwala, C. B., J. Textile Inst., 1941,32, T201.
79. Heald, A. M., Paper Trade J., 1941, 113 (2), 39.
80. Kesler, C. C. and Black, W. c., Paper Trade J., 1942, 114 (21), 57.
81. Woodruff, S. and Nicoli, L., Cereal Chem., 1931,8,243.
82. Hixon, R. M. and Brimhall, B., Ind. Eng. Chem. Anal. Ed., 1941, 13, 193.
83. Sheppard, S. E., Gelatin in Photography, Vol. 1, New York, 1923.
84. Alexander, J., Glue and Gelatin, New York, 1923.
85. Brimhall, B. and Hixon, R. M., Ind. Eng. Chem. Anal. Ed., 1939, 11, 358.
86. Bingham, E. C.,J. Rheol., 1930, 1, 511.
87. Woodruff, S. and MacMasters, M. M., Illinois Agricultural Experiment Station
Bull., 1938, 445.
88. Saxl, I. J., Physics, 1936, 7, 62.
89. de Beukelaer, F. L., Ind. Eng. Chem. Anal. Ed., 1930,2, 348.
90. Tarr, L. W., Delaware Agricultural Experiment Station Bull., 1926, 142.
91. Baker, G., Ind. Eng. Chem., 1926, 18, 89.
92. Saare, O. and Martens, P., Zeit. Spiritusind, 1903,26,436.
93. Kerr, R. W., Chemistry and Industry of Starch, Academic Press, New York, 1944,
pp.97-98.
94. Kerr, R. W., loco cit., p. 109.
95. Saxl, E. J., Ind. Eng. Chem. Anal. Ed., 1938, 10, 82.
96. Chapman, O. W. and Buchanan, J. H., Iowa State College J. Science, 1930,4,441.
97. Alsberg, C. L. and Rask, O. S., Cereal Chem., 1924, 1, 107.
98. Dox, A. W. and Roark, G. W.,J. Am. Chem. Soc., 1917,39,742.
99. Francis, C. K. and Smith, O. C., J. Ind. Eng. Chem., 1916, 8, 509.
100. Nyman, M., Zeit. Nahr., Genussm., 1912, 24, 673.

101. Reichert, E. T., Carnegie Inst. 0/ Washington, Publ. 173, Pt. 1, 1913.
102. Samec, M., Kolloid-Beihe/te, 1912,3,126; Kolloidchemie der Stiirke, Dresden, 1927,
pp. 169-170.
103. Cook, D. H. and Axtmeyer, A. J., Ind. Eng. Chem. Anal. Ed., 1937, 9, 226.
104. Morgan, W. L., Ind. Eng. Chem. Anal. Ed., 1940, 12, 313.
105. Kuntzel, A. and Doehner, K., Kolloid Zeit., 1939, 86, 124.
106. Ostwald, W., Trans. Faraday Soc., 1913, 9, 34.
107. Sair, L. and Fetzer, W. R., Ind. Eng. Chem. Anal. Ed., 1942, 14, 843.
108. Cleland, J. E. and Fetzer, W. R., Ind. Eng. Chem. Anal. Ed., 1941, 13, 858; 1942,
14,27,124.
109. Sprockhoff, M., Zeit. Spiritusind, 1929,52,27.
110. Microanalysis 0/ Food and Drug Products, Food and Drug Circular No.1, Federal
Security Agency, Washington, 1944.
111. Ripperton, J. c., Hawaii Agricultural Experiment Station Bull., 63, 1931.
112. Gallay, W., Can. J. Research, 1936, B14, 409.
113. Brimhall, B., Ind. Eng. Chem., 1944,36,72.
114. Tolman, L. M. and Smith, W. B., J. Am. Chem. Soc., 1906,28, 1476.
115. Browne, C. A. and Zerban, F. W., Sugar Analysis, New York, 1941, pp. 1206-13.
116. Mullen, J. W. and Pacsu, E., Ind. Eng. Chem., 1942,34,807.
117. Higginbotham, R. S., J. Textile Inst., 1947,38, T131; Shirley Inst. Memoirs, 1946,
20 (26), 1.
118. Kesler, C. C. and Bechtel, W. G., Analytical Chem., 1947, 19, 16.
119. Bechtel, W. G., Cereal Chem., 1947,24,200.
120. Bechtel, W. G. and Kesler, C. C., Paper Trade J., 1947, 125 (16), 35.
121. Hamer, W. J., J. Research Nat!. Bur. Standards, 1947,39 (1), 29.
122. Selling, H. J. and van Lamoen, F. L. J., Chem. Weekblad, 1947,43, 602.
123. Lindsley, C. H. and Fischer, E. K., J. Applied Phys., 1947, 18, 988.
124. Fischer, E. K. and Lindsley, C. H., J. Colloid Sci., 1948, 3, Ill.
125. Bechtel, W. G., Kesler, C. C. and Stinchfield, J., Starch/or Paper Coating, TAPPI
Monograph Series No.3, New York, 1947, Ch. 11.
126. Frost, F. H., ibid., Ch. 21.
127. Bechtel, W. G. and Kesler, C. c., USP 2,491,639.
128. Bechtel, W. G. and Fischer, E. K., J. Colloid Sci., 1949, 4, 265.
129. Bechtel, W. G., J. Colloid Sci., 1950,5,260.
130. Keene, J. C. and Brice, B. A., Ind. Eng. Chem. Anal. Ed., 1937, 9, 258.
131. Suzuki, S. and Nemoto, Y., Denpun Kogyo Gakkaishi, 1954-5, 2, 61, 118.
132. Suzuki, S. and Nemoto, Y., ibid., 1955-6,3,66, 111.
133. Fukuda, T. and Maezawa, T., ibid., 1957,5,9,11.
134. Suzuki, S., Yoshikawa, S. and Arai, K., J. Technol. Soc. Starch (Japan), 1956,
4 (2), No. 12.
135. Mazurs, E. G., Schoch, T. J. and Kite, F. E., Cereal Chem., 1957,34, 141.
136. Takeichi, K. and Ikeda, S., Denpun Kogyo Gakkaishi, 1956,3, 128.
137. Marotta, N. G. and Ryan, F. F., USP 3,173,807, 16 Mar. 1965.
138. Hamilton, R. M., USP 3,087,839, 30 Apr. 1963.
139. van Wazer, J. R., Lyons, J. W., Khim, K. Y. and Colwell, R. E., Viscosity and Flow
Measurement: A Laboratory Handbook 0/ Rheology, Interscience, New York, 1963.
140. Suggested Method T637 sm-53, Apr. 1953, Tentative and Official Methods-
Recommended Practices-Specifications, Technical Association of the Pulp and
Paper Industry, 155 East 44th Street, New York, N.Y.
141. Radley, J. A., Die Stiirke, 1960, 12 (8), 232.
142. Kempf, W. and Kalender, G., ibid., 1972,24 (7), 270.
Ramaszeder, K., Die Starke, 1971, 22, 176. (Discusses rheological examination of
textiles pastes and concluded rotational viscometers are best for viscosity determina-
tion.)
Shibukawa, S. and Fukuba, H., Kaseigaku Zasshi, 1973,24 (1), 45. (Convenient method
to measure starch gelatinisation rate in foodstuffs. Gelatinised starches dehydrated
with alcohol, dried, ground and sieved and resuspended in H20. Rates of A to B of
sample to fully gelatinised sample gave values comparable with amperometric
titration-A is initial turbidity, B turbidity after 100 min.)
CHAPTER 5
Chemical Analysis of Raw and Modified Starches

F. A. LYNE
Public Analyst, 220 Elgar Road, Reading, Berks. RG20DG, Great Britain
INTRODUCTION
The examination of starch for quality control purposes to establish its

origin, or to assess its suitability for a given purpose may be divided into
three categories: (a) microscopical examination (Chapter 1), (b) Physical
examination (Chapter 4) and (c) Chemical analysis.
The dividing line between the three types of examination is not sharp.
Thus selective staining for microscopical examination is a chemical process
which may reveal differences in molecular structure due to modification by
chemical treatment. Physical methods may be employed to estimate
chemical constituents, e.g. the determination of moisture content by
dielectric measurement and changes in physical properties due to retro-
gradation may be measured by chemical means, e.g. by iodine titration.
The literature on the analysis of starch is voluminous, comprising many
hundreds of references. An attempt has been made to select those methods
which have become standard practice or which may be of practical value
for routine control or research. The most comprehensive source on the
analysis of corn starch is contained in Corn Industries Research Founda-
tion, Standard Methods of Analysis 7 to which reference is made in this
chapter and de Willigen and Gerritsen's82 valuable account of the Analysis
of Potato Starch. The methods given in these two works are usually
applicable to other starches.
5.1 MOISTURE
The moisture content of a starch is of economic importance as a purchaser
does not wish to pay for an excessive amount of water and also the storage
133

properties of the starch will be dependent upon moisture content. A high

moisture content will permit the growth of moulds and other micro-
organisms and will affect its free-flowing properties by 'balling'. The
normal moisture content varies from starch to starch (see Table 5.3).
In air-dry wheat starch the moisture content is usually around 13 %whilst
that of potato is 18 to 22 %. Furlong! suggests a maximum limit of 12·5 %
for moisture in superfine tapioca. The British Pharmacopoeia2 requires
that maize, rice and wheat starch shall contain not more than 14 %
moisture and that potato starch shall contain not more than 20 %.
The determination of the moisture content of any natural organic
substance is fraught with the basic difficulty that there is no absolute
definition of 'moisture content' or fundamental reference method for
determining it. Moisture may be present as free water, it may be loosely
held in a mechanical structure of varying degrees of stability as in a gel.
Some may be loosely attached to the molecules as in hydrated salts and
the strength of the molecular attachment will vary from unstable hydrates
to substances which form anhydrides by greater or lesser amounts of
heating. All methods for the determination of the moisture content of
natural organic substances are therefore empirical and 'moisture content'
must be related to the method of determination.
Moisture determinations have been made by L. Sair 3 on 22 commercial
starches and modified starches using distillation, vacuum-oven and air-
oven methods and reasons for differences in the results given by the
different methods are suggested. He concludes that the toluene distillation
procedure and the vacuum-oyen method at 100°C are suitable reference
methods. Rapid oven procedures involving temperatures as high as 140°C
he considers reliable with most starch products but not with acid-modified
or hypochlorite-modified products which should not be taken above 100°C.
It is interesting that this worker found that the sorptive power of modified
starches decreases with increasing modification but that the sorbed water
is held with equal, if not greater, tenacity than the water held by the
original unmodified starch. This the author can confirm from his own
experience on the subject.
Methods of determination include:
(a) Oven methods, including vacuum oven, high temperature and other
modifications
(b) Azeotropic distillation
(c) Karl Fischer
(d) Electrical properties
CHEMICAL ANALYSIS OF RAW AND MODIFIED STARCHES 135
(e) Gas evolution

(f) Miscellaneous.
5.1.1 Oven methods

When determining the moisture content of starch there is a further
complication due to the fact that starches gel between 57°C and 67°C so
that it is advisable to keep the temperature at 40°C for some hours until
the greater proportion of the water has been driven off and then to raise
the temperature to 120°C for 4-6 h. The author has found that the drying
time can be reduced by the addition of 5 ml of absolute alcohol to every
10 g of starch.
Sprockhoff4 gives the official German method in which 5 g of starch
are heated for 1 h at 50°C and then for 3 h at 120°C.
The British Pharmacopoeia 2 directs that the starch shall be dried to
constant weight at 105°C.
The International Association for Cereal Chemistry has adopted a basic
reference method for the determination of the moisture content of cereals
and cereal products. This has been in turn accepted by Sub-Committee 4-
Cereals and Pulses of ISOjTC34-Agricultural Food Products and has
been published as a British Standard Method. 5
The principle of the method is the determination of the loss in mass
when the product is brought into equilibrium with a dry atmosphere at a
temperature between 45 and 50°C and a pressure of 13 millibar (10 mm Hg
to 20 mm Hg).
3 g of the sample of specified particle size (less than 1·7 mm of which
less than 10 % by mass are over 1 mm and more than 50 % are less than
1 mm) are placed in a steel dish with a tightly fitting lid. The dish (with lid
removed) is placed in a drying tube which also contains a boat containing
a layer of phosphorus pentoxide. The drying tube is evacuated to about
13-26 millibar (10-20 mm Hg) and the portion of the drying tube con-
taining the dish is placed in an oven at 50°C. After about 100 h the tube
is removed from the oven, allowed to cool and air admitted through a
drying train. The lid is placed on the dish and it is quickly weighed. The
process is repeated, renewing the phosphorus pentoxide when it shows
signs of agglomeration, until the product reaches constant mass (i.e. less
than 0·000 6 g difference between successive weighings made at intervals
of 48 h). At least two determinations should be done on each sample
which should agree within 0·1 %.
Such methods are slow and unsuitable for large numbers of samples.
For control purposes it is necessary to devise methods which will give
results which are comparable with standard reference methods in the

minimum time. A later British Standard Specification 71 conforms with
methods prepared by Working Group 2-Moisture Content of Starch of
ISO/TC 93-Starch (including Derivatives and By Products) and published
as ISO Recommendation 1666. This cautiously avoids any controversy
over the definition of 'moisture' by referring to the 'loss in mass on drying'
and gives two methods, one involving oven-drying to constant weight at
130°C at atmospheric pressure and the other oven-drying at 100°C or
73°C at reduced pressure. The first is a routine procedure and the other is
designed to avoid any chemical alteration in the substance particularly
oxidation and loss of volatile organic substances. A number of moisture
testers have been devised and put on the market which employ higher
temperatures, forced circulation, built-in balances and similar devices,
e.g. Brabender, Carter Simon. In the former 'semi-automatic' type, the
heating time is one hour at 130°C with warm air flow and a built-in
balance which weighs the dishes whilst still hot. The moisture content is
shown on an illuminated scale. The Carter Simon oven is operated at
155°C and the time is 15 min. Dishes are placed in the oven at 5 min
intervals, pass through the oven and emerge at 5 min intervals.
A critical examination of a number of methods used to determine the
moisture in potato starch has been carried out by W. L. Porter and
C. O. Willits. 6 They used two types of mechanical convection oven, viz. a
Brabender moisture tester (an air oven with forced circulation) has a
balance incorporated in such a manner that the sample can be weighed
without removing it from the oven. The oven was regulated to different
temperatures and the loss in weight was noted at definite time intervals
until constant. With the Precision floor model several samples for each
particular drying temperature were placed in the oven, one being removed
after each time interval, so that repeated heating and re-cooling was
avoided. A similar procedure was adopted with a table· model, gravity
convection oven. Carter Simon tests were made with a number of samples
weighed at the same time, but passed through the oven at different rates
so that the time of drying was varied. Tests in a Weber vacuum oven
«5 mm of mercury) were made in the same manner as with the mechanical
convection oven. The loss of weight of potato starch recorded by the
Brabender moisture tester becomes greater with successively higher
temperatures, the rate of loss at each temperature being higher at the
beginning and falling off rapidly until the weight is constant. The curves
show that the total loss of volatile matter is dependent upon the tempera-
ture. At a high drying temperature a light brown discoloration appears in
the time necessary to establish constancy of weight. This alteration does

not affect the weight and determinations of the spectral reflectance and
estimation of the colour given with iodine by aqueous extracts showed,
respectively, that no change in composition and solubility occurred before
the constant weight period. With drying at 180°C appreciable decom-
position, indicated by further loss in weight and formation of erthro-
dextrins, occurred only when heating was continued for 4 h after the
attaining of constant weight. Any method for the determination of
moisture based on the steep portion of the isothermal curves showing the
relation between loss of weight (ordinate) and time (abscissa) is subject to
considerable error produced by relatively small time differences. This may
account for the inconclusive data reported in the literature of moisture-
content of potato starch, since most of the standard methods specify
conditions represented by points on the steep portions of the curves. A
more closely reproducible method would consist in the use of a temperature
indicated on the flattest position of the curve and a time long enough to
ensure constancy of weight. With the Brabender moisture tester the
method would involve heating at 135°-145°C for 30--60 min to the constant
weight. Now that this basic method has been established it should be
possible to employ any other method that will duplicate these moisture
values within the limit of error of the specified procedure. With potato
starch dried in a gravity convection oven at 100°C for 24 h the loss in
weight never quite reached the value obtained in the mechanical con-
vection oven at 100°C nor was the drying so rapid. In neither form of oven
did the loss reach that established by the basic method. Vacuum drying
at 5 mm of mercury at 80°C gave a value in close agreement with that of
the basic method, constant weight being reached in 22-24 h. Vacuum
drying at 100°C gave a slightly higher value with constancy in 5 h. Drying
at 100°C by any of the other methods gave lower values. At 135°C the air
drying methods used, with the exception of the Carter Simon method, gave
results comparable with those of the basic method. At a higher temperature
(150°C) slight decomposition occurred. Quick approximations can be
made, however, by heating at such a temperature for a time that will give
a loss of weight comparable with that of the basic method (e.g. the Carter
Simon moisture test which is operated at 155°C for 15 min), but heating
must be stopped at the end of the specified time.
The Standard oven method of the Corn Industries Research Founda-
tion 7 uses vacuum drying. Starch samples containing hard granular pellets
should be ground, taking precautions to prevent significant loss of moisture.
However, in most cases, grinding is not necessary.
Weigh accurately about 5 g of sample into a predried, cooled and tared

moisture dish. Place dish and coYer (cover removed) in vacuum oven
operating at 120°C and maintain at a pressure not in excess of 100 mm
(Hg) for 4 h. While sample is drying, bleed a small stream of air through
the drying train and oven.
Shut off the vacuum line and slowly fill the oven with air drawn through
the drying train. Open oven, quickly close dish with cover, place in
desiccator until cool (30 min usually sufficient) and weigh. The author has
found that the cooling time can be substantially reduced by placing the
dish on a block of aluminium.
This procedure is not applicable to highly modified starches which show
evidence of decomposition (usually discoloration) under the conditions
specified.
5.1.2 Azeotropic distillation (Dean and Stark)

Azetropic distillation with a suitable liquid enables the moisture content
of starch to be determined with speed and accuracy. T. H. Fairbrother
and R. J. Wood 8 found that distillation with tetrachlorethane or carbon
tetrachloride allows the moisture in flour to be estimated in 20 min with
an accuracy of ±0·5%.
Suitable liquids are given in Table 5.1.
TABLE 5.1
Boiling pt.
Added liquid A Boiling pt. A 01 azeotrope Wt %olA
with water
Benzene 80'2°C 69'25°C 8·83

Cyc10hexane 80'75°C 68'26°C 8·33
Toluene llO'7°C 84'I°C 19-6
Toluene has become the standard distillation liquid. The Com Industries
Research Foundation 9 regard azeotropic distillation to be the most
accurate method for all starches and employ it as a referee technique and
to establish conditions for the estimation of moisture by simplified
methods for control analysis. The standard apparatus consists of a 250 ml
squat type distillation flask connected by ground glass joint to a graduated
trap or receiver, capacity 5 ml graduated in tenths of a millilitre, which is
connected to a drip-tip-water jacketed condenser 40 cm long.
Starch samples containing hard granular pellets should be ground,
taking precautions to prevent significant loss of moisture. However, in
most cases, grinding is not necessary.
Place 5 to 8 g of prepared asbestos in a distillation flask and dry the

flask and its contents by heating overnight in an air oven at 100°C. Weigh
accurately about 30 g of sample and transfer to the distillation flask con-
taining asbestos. Add approximately 150 ml of toluene to the distillation
flask. Place the flask and its contents in the bath on the distillation rack
and attach the flask and condenser. Place a loose-fitting test tube over the
upper end of the condenser. Start the distillation at such a rate that
approximately 2 drops of condensate per second fall from the tip of the
condenser. After 1 h increase the distillation rate to approximately 4 drops
per second.
Continue the distillation until the volume of water in the trap ceases to
increase (8 h usually sufficient). Wash down the condenser with about
10 ml of toluene and continue the distillation for 30 min.
Disconnect the trap and immerse the graduated portion containing the
distillate in a water bath at 20°C until the contents of the trap assume the
temperature of the bath. Read the volume of water in the trap, estimating
to the closest hundredth of a millilitre. Determine a blank by the above
procedure substituting about 3 g of water, weighed accurately, for the
sample.
The official A.0.A.c. method 10 is similar in principle but the entire
process is said to be complete within 1 h. For removal of any drops of
water adhering to the condcnser the A.O.A.C. suggests a tube brush
attached to a piece of copper wire. A spiral of wire which fits loosely
inside the condenser tube has been found to be very satisfactory for this
purpose. The apparatus for determination of water by azeotropic (or
entrainment) distillation is the subject of British Standard Specification
756:1952. Five sizes of ordinary type receiver for light liquids (e.g.
petroleum spirit, toluene) and two sizes for heavier liquids (e.g. trichloro-
ethylene, perchlorethylene) are specified.
5.1.3 Karl Fischer method

In 1935 Karl Fischer 60 - 62 introduced a complex reagent consisting of
pyridine, methanol, sulphur dioxide and iodine which has a specificity
for water.
The reaction may be represented by the equations:
C sH sN·I 2 + CsHsN· S02 + CsHsN + H 20 ~

2C sH sN· HI + CsHsNO . S02
CsHsNO· S02 + CH 3 0H --> CsHsNH . S04CH3
When the Karl Fischer reagent is added to a methanol solution con-

taining a small amount of water, the free iodine disappears and the end-
point in a titration is marked by the disappearance of all free iodine.
This may be detected visually by the disappearance of the iodine colour or
electrometrically. Starch cannot be used as an indicator as the environment
is anhydrous. A typical formulation contains the following anhydrous
reagents:
2420 ml pyridine, 752 g iodine, 6000 ml methanol, 192 g sulphur dioxide
The Karl Fischer reagent can be standardised so that 1 ml is equivalent
to 5 mg of water. It can be purchased from laboratory suppliers already
standardised. The standardisation must be checked frequently as the
reagent is not stable. For this purpose solutions of water in methanol can
be obtained each ml of which contains 5 mg water. Alternatively the
Karl Fischer reagent can be standardised by using sodium tartarate
dihydrate which contains 15·66 % water. When this is dissolved in
anhydrous methanol the water of crystallisation reacts with Karl Fischer
reagent as if it were free moisture.
When starch is dispersed in anhydrous methanol, the moisture content
of the starch is extracted by the methanol and titrated with Karl Fischer
reagent. The particle size is clearly an important factor as is also the time
of contact and the type of starch. The differences in contact times required
are evidently associated with the relative firmness with which moisture is
held by the samplesY The Corn Industries Foundation does not specify
a contact time but directs that the sample shall, if necessary, be ground to
20 mesh or finer.
The apparatus for Karl Fischer determinations are designed to store
and measure the reagent without contact with moist air or any other
possible contamination by water and to carry out titrations (automatically
or manually) in a dry atmosphere. Traces of moisture must be removed
from the apparatus and flaming the titration flask has been found to be
desirable. The extraction of the moisture from the starch into the an-
hydrous methanol can be speeded up by warming the mixture. The end-
point is detected electrometrically by means of two platinum electrodes.
A small emf is applied to the electrodes which remain polarised in the
presence of water but are depolarised immediately an excess of iodine is
present thus allowing a current to flow. Stirring is effected by a magnetic
stirrer or by a stream of dry nitrogen.
In order to eliminate loss or gain of moisture during grinding, the
sample may be disintegrated and dispersed whilst submerged in dry
methanol. An apparatus which combines this operation with automatic

titration has been developed by ICI and marketed by laboratory suppliers
(e.g. Townson and Mercer (61A-400)).
5.1.4 Electrical properties

The electrical resistance or dielectric constant of many materials vary
according to their moisture content. 12 - 14 Provided the material to be
tested is of uniform granularity so that it can be packed reproducibly into
a cell the moisture content can be read off directly on a scale, once an
instrument has been calibrated for the material under test. In order to
obtain uniformity of packing the test material is compressed into the cells.
Such instruments are rapid in operation giving an 'on the spot' moisture
content within a few seconds and can claim an accuracy of about ± 0·05 %
on homogenous matcrials such as starch. Examples of such instruments
include the Marconi Moisture Meter 72 in which the material under test
is compressed into a special cell under a pressure of 1000 psi (6·89 N/mm2)
and the resistivity measured; the N.P.L. Moisture Meter 73 in which the
sample is placed in a cup or cell but not compressed, and the Tag-
Heppenstall 74 Moisture Meter in which the material is passed between
rotating cylindrical electrodes.
5.1.5 Gas evolution

T. Zerewitinoff 15 used a solution of methyl magnesium iodide in
pyridine to determine moisture in starch by measuring the amount of
methanol liberated and found close agreement with values obtained by
vacuum drying methods.
The reaction between water and calcium carbide to give acetylene is on
thc basis of the 'Speedy' moisture tester. A known weight of the starch
sample is shaken with an absorbent powder containing calcium carbide.
The pressure of acetylene produced is indicated on a gauge which may be
calibrated directly in percentage moisture.
The reaction between moisture and calcium carbide is also employed
in the Chopin moisture tester. The completion of the removal of moisture
when the sample is heated to 150°C is indicated by the cessation of
evolution of acetylene and the extinction of the flame with which it burns.
This method appears to be obsolete.
5.1.6 Miscellaneous methods

J. A. Radley has made use of the hygroscopic nature of absolute ethyl
alcohol to determine the moisture content of potato starch by finding the
specific gravity of the supernatant liquid obtained by shaking known

weights of alcohol and starch. The refractive index of the liquid can also be
used, particularly if a dipping refractometer is used.
Saare 16 gives a rapid method for estimating moisture in potato starch
which is claimed to be accurate to 0·5 %. 100 g of starch in a graduated
flask are made into a suspension with distilled water, the volume made up
to 250 ml at l7·5°C and then weighed. If S represents the weight of the
starch plus the water in the flask then the water-content of the starch is
giyen by the formula:
o 289·4 - S
%water = 0.394
The weighing should be done as accurately as possible, as a difference of
0·1 in S represents a final difference of 0·25 %water.
Infra red and near infra red methods have been used for the determina-
tion of moisture using dimethyl formamide and methyl sulphate as
solvents. 75 These are claimed to be more accurate than vacuum oven
methods and are sufficiently rapid for quality control.
According to Pande 76 a new technique called hygrophotography has
been developed by Sivdyian. This relies on the reversal of the photo-
chemical reaction on a photographic plate by water. It is claimed that the
plates can be easily calibrated and it is possible to make micro determina-
tions of water with good precision.
Results. The range of moisture contents of various starches is shown in

Table 5.3.
5.2 MINERAL MATTER
The determination of mineral matter may reveal the presence of sand or

dirt, give some indication of the grade of starch and may also show
whether the starch has been 'chemicalled'.
The presence of calcium in the ash may be ascribed to the hardness of
the washing water or may be due to the use of calcium bisulphite during
manufacture as suggested by H. Tryller 17 but the latter practice seems now
to be obsolete.
Ashing at too high a temperature may cause volatilisation of some of the
mineral constituents such as sodium chloride. The temperature specified
by the A.O.A.C. 1s is 525°C at which temperature sodium chloride does
not sublime the method being as follows:
Heat sample of appropriate weight for product being examined (usually

5-10 g) in 50-100 ml platinum dish at 100°C until H 2 0 is expelled; add
few drops pure olive oil and heat slowly over flame until swelling stops.
Place dish in muffle at ca. 525° and leave until white ash is obtained.
Moisten ash with H 2 0, dry on steam bath and then on hot plate, and
re-ash in muffle at 525°C to constant weight.
The Starch Industries Research Foundation 19 also employ a tempera-
ture of 525°C, the method recommended being:
Weigh 5 g (± 0·1 g) of corn starch into a preheated, cooled and
accurately-tared platinum or silica dish. In the case of starch containing
added inorganic materials, use sufficient sample to provide from 50 to
200 mg of ash. Heat gently over an open flame or on a hot plate until
sample is thoroughly carbonised. Ignite the sample during this charring
process. Special attention should be given to the preliminary carbonisation
since excessive foaming may cause loss of sample and ash. Place in muffle
furnace at 525°C and heat until ash is free from carbon (2 h usually
sufficient). Cool in desiccator and weigh.
W. V61ksen 20 suggests that the time ofashing can be materially reduced
by the addition of 'Ash Aids'. After a preliminary ashing the material is
cooled and moistened with 10 % ammonium nitrate solution or alcoholic
magnesium acetate which is carefully taken to dryness to avoid spitting
and the incineration completed by a further short period in the furnace.
A correction is necessary if the latter solution is used for the magnesium
oxide arising from the solution.
5.2.1 Calcium
Calcium may be determined on the ash by dissolving in hydrochloric
acid, neutralising with ammonia, precipitating the calcium as oxalate,
filtering and titrating the oxalate with standard permanganate solution.
The amount of calcium is so small in normal samples of starch that it
would be necessary to ash large quantities of sample which is time-
consuming and inconvenient. Ashing can be avoided by extracting the
calcium with 1·2 N hydrochloric acid as in the Corn Industries Research
method. 21 Weigh 100 g (± 1 g) of sample into a dry I-litre Erlenmeyer
flask. Add 400 ml of 1·2 N hydrochloric acid solution, stopper and agitate
continuously at a moderate rate for 5 min. Filter the slurry through a dry
Whatman No. 12 paper into a dry receiving flask. Transfer 200 ml of
filtrate to a 400 ml beaker. Place 200 ml of 1·2 N hydrochloric acid in a
second beaker to serve as a blank, and carry the sample and blank con-
currently through the procedure. With the aid of a pH meter, adjust
solution pH to 4·5-5·5 by addition of concentrated ammonia. Add 3 drops
of concentrated hydrochloric acid and place in a boiling-water bath.
To the hot solution, add 25 ml of saturated ammonium oxalate solution
and 30 g of urea. Stir to dissolve, cover with a watch glass, and heat in
the boiling-water bath for 2·5 h. Assurance of completion of the neutralisa-
tion reaction is best obtained by external use of bromo thymol blue
indicator (blue colour if neutralisation is complete). If the indicator is
used internally, no colour change is observed until the solution cools.
Allow reaction mixture to cool to room temperature. Filter by gravity
through Whatman No. 42 paper (since titration is carried out in the
precipitation beaker; quantitative transfer of the precipitate is unnecessary).
Wash the beaker and paper with four 25 ml portions of saturated calcium
oxalate solution followed by two 10 m1 portions of distilled water. Return
filter paper and precipitate to the precipitation beaker: add 100 ml of
distilled water and 10 ml of 12 N sulphuric acid while stirring. Heat to
70-80°C with stirring to macerate the paper and dissolve the precipitate.
Add three drops of manganese sulphate solution; titrate the hot mixture
with 0·1 N potassium permanganate solution to a faint pink end point
which persists for 30 s.
Calcium may also be determined by flame photometry or by atomic
absorption spectrophotometry the latter being the more precise.
The following method is that given in the Fertilisers and Feeding Stuffs
(Amendment) Regulations 1970: 22
Reagents: Calcium stock solution-Dry calcium carbonate at 105°C
for 1 h. Transfer 2·497 g into a 1 litre volumetric flask using approximately
100 ml water. Add slowly with swirling 60 ml N hydrochloric acid. When
all the calcium carbonate has dissolved, dilute to 1 I with water.
1 ml == 1 mg calcium
Calcium dilute solution-dilute 20 ml calcium stock solution to 200 ml.

1 ml == 100 jlg calcium
Calcium working standard solutions-add 10 ml releasing agent to each

of six 100 ml volumetric flasks. Measure 0, 3, 6, 9, 12, 15 ml dilute calcium
solution (1 ml == 100 jlg calcium) into the flasks and dilute to 100 m1 with
water. The flasks contain 0, 3, 6, 9, 12, 15 jlg Ca per ml respectively.
Lanthanum oxide solution (releasing agent)-wet 117·3 g lanthanum
oxide, La203, low in calcium with water. Add 350 ml concentrated

hydrochloric acid (d = 1·18) slowly, and shake until all the lanthanum
oxide is dissolved. Allow to cool and dilute to 1 I with water.
Set up the instrument using the line at 422·7 nm. Use a fuel-rich flame.
Add releasing agent and water to a suitable aliquot of the sample solution,
to produce a standard volume of solution to contain between 5 and 10 flg
of calcium per ml and 10 %v/v releasing agent. Prepare a blank solution
from which only the sample has been omitted. Spray water into the flame
and zero the instrument. Spray successively in triplicate, the standard
solutions, sample and blank, washing the instrument through with water
between each spraying. Plot the mean reading obtained for each standard
solution against its calcium content. Determine the calcium content of the
sample and blank solutions from the graph and from the difference
between them calculate the calcium content of the sample. If a number of
samples is being examined, one or more standard solutions must be
resprayed at intervals during the course of the analyses.
5.2.2 Phosphorus
Ashing may give low results due to the formation of fused glassy residues
which are difficultly soluble, these may be avoided if the sample is first
mixed with calcium oxide and the temperature of incineration not allowed
to exceed 500°C. Alternatively the organic matter may be destroyed by
wet combustion using nitric acid/sulphuric acid mixture or nitric acid and
permanganate.
The phosphorus may be precipitated as phosphomolybdate or, better, as
the quinolinium phosphomolybdate or, in view of the small amount of
phosphorus in samples of starch, spectrophotometrically as molyb-
divanado-phosphoric acid. This method is employed by Corn Industries
Research Foundation 23 as follows:
Standardisation Curve: pipette 5·0, 10·0 and 15·0 ml of standard
phosphorus solution into respective 100 ml volumetric flasks, and use
another flask for a blank. To each flask add in order, 10 ml of 29 %nitric
acid, 10 ml of 0·25 % ammonium vanadate, and 10 ml of 5 % ammonium
molybdate, mixing thoroughly after addition of each reagent. To avoid
interference from precipitation and side reactions, reagents must be added
in the order stated. Since the vanadate and molybdate are present in large
excess, volumes of reagents need not be controlled more closely than
± 1 ml. Dilute to volume with distilled water, mix thoroughly and allow
to stand for 10 min. Using the blank as a reference solution at 100 %
transmISSIOn, determine the transmission of each standard at 460 mJi.

Plot log % transmission versus mg of phosphorus per 100 ml. The
standardisation curve is reproducible and need be checked only when
fresh reagents are prepared.
Analysis of corn starch. Weigh accurately 10 g of corn starch into a
platinum or silica dish; add 10 ml of 2 %calcium acetate solution in a fine
stream, distributing the solution uniformly in the sample. If desired, 3 ml
of a saturated solution of magnesium nitrate and 7 ml of water may be
substituted for the calcium acetate. The two systems give comparable
results; calcium acetate is recommended principally because it yields a
higher-density ash and because an ignited magnesium nitrate blank must
be included when using that salt. Place the dish on a hot plate and carefully
evaporate to dryness, then increase heat and carbonise the sample on the
hot plate or over a gas flame. Place the dish in a muffle furnace at 600-
650°C until the ash is free of carbon (1-2 h). If difficulty is experienced in
obtaining a carbon-free ash, the dish may be removed from the muffle,
cooled, and the residue moistened with several drops of 29 % nitric acid.
Heating is then continued.
Cool to room temperature and wet the ash with 15 ml of water. Slowly
wash down the sides of the dish with 5 ml of 29 %nitric acid; quantitatively
transfer to a 200-ml volumetric flask, rinsing the dish with three 20-ml
portions of distilled water. Dilute to volume with distilled water and mix
thoroughly. (If not clear, gravity filter through a retentive paper.) Transfer
an aliquot selected to contain not more than 1·5 mg of phosphorus to a
100 ml volumetric flask, and add 50 ml of water to another flask to serve
as a blank. To each flask add, in order, 10 ml of 29 % nitric acid, 10 ml of
0·25 % ammonium vanadate, and 10 ml of 5 % ammonium molybdate,
mixing thoroughly after addition of each reagent. Dilute to volume with
water, mix thoroughly and allow to stand for 10 min. Determine %trans-
mission of the sample at 460 mJi, using the blank as a reference solution
at 100 % transmission. Read mg of phosphorus in the aliquot from the
standardisation curve.
This method is also employed in the Fertilisers and Feeding Stuffs
Regulations 1968 24 which directs as follows:
Weigh to the nearest mg about 5 g of the sample into a capsule or dish;
add 1 g of calcium oxide, mix well and thoroughly wet with a little water.
Dry the mixture and incinerate to a temperature not exceeding 500°C
until completely charred. Cool, transfer the contents of the capsule or dish
to a 250 ml beaker and add 10 ml of water; then slowly add 12 ml of

concentrated hydrochloric acid, taking suitable precautions to avoid loss
by effervescence and finally 5 ml of concentrated nitric acid. Heat to
incipient boiling and keep at this temperature for 10 min. Dilute with
about 10 ml of water, filter, transfer the insoluble matter to the filter paper
with a minimum amount of water and wash twice with small volumes of
water. Then transfer the filter paper and insoluble matter to the original
capsule or dish and incinerate until all the carbon is destroyed. Combine
the ash with the filtrate and heat to boiling point. Cool, transfer to a
250 ml volumetric flask, dilute to the mark, mix well and filter. Discard
the first 10 or 20 ml of the filtrate.
From a burette measure into a series of 100 ml volumetric flasks 25·0,
26·0,27·0,28·0,29·0,30·0 and 31·0 ml of the standard phosphate solution
(i.e. 5·0, 5·2, 5·4, 5·6, 5·8, 6·0 and 6·2 mg phosphoric acid). Add 25 ml of
the vanadium molybdate reagent to each flask and dilute to 100 ml with
water making sure that the temperature of the reagent and the dilution
water is 20°C. Shake and allow to stand for 10 min. Set the spectrophoto-
meter to the correct wavelength, say 420 nm, fill two 1 cm cells with the
5·0 mg solution and check the optical density of the cells. If there is a
small difference, select the cell with the smaller reading as the standard
reference cell.
Determine the apparent optical density at 20°C (corrected for cell
differences) of the 5·2, 5·4, 5·6, 5·8, 6·0 and 6·2 mg phosphoric acid
solutions referred to the 5·0 mg phosphoric acid solution as standard.
Plot a calibration graph of scale readings against known phosphoric acid
content.
Successively dilute a portion of the solution prepared as above so that
the final volume of about 25 ml contains between 5·5 and 6·2 mg phosphoric
acid, taking care that the dilution water is at a temperature of 20°C.
Transfer this final volume to a 100 ml volumetric flask, add 25 ml of
the vanadium molybdate reagent (at a temperature of 20°C), dilute to the
mark, mix and allow to stand for 10 min. At the same time transfer 25 ml
of the standard phosphate solution (at 20°C) into a second 100 ml
volumetric flask. Add 25 ml of the vanadium molybdate reagent (at 20°C)
dilute to the mark, mix and allow to stand for 10 min.
Measure the difference in optical density at 20°C between the two
solutions and estimate the phosphoric acid content of the volume of the
unknown solution from the calibration graph.
Calculate the phosphoric acid content of the sample from known
dilution factors and the weight of the sample.
Prepare a fresh reference standard for each series of readings on the

instrument.
5.2.3 Fat
T. C. Taylor and J. M. Nelson,25 considered that a small amount of the
fat contained in maize starch is in combination with the starch and not
removable by solvents, a conclusion which has since been proved wrong.
On hydrolysis this fat is set free and appears as palmitic acid, etc.
Further work by T. C. Taylor and L. Lehrman 63 has showed that the
fatty matter has approximately the following percentage composition:
palmitic acid 24, oleic acid 40, linoleic acid 36. The above workers find
that the percentage of fatty matter in maize starch is approximately 0·5,
in rice starch 0·83, in sago starch 0·11 and in cassava starch 0·12.
Thus, for some years a distinction was made between the so-called
'combined fatty acids' and the free fatty acids, the latter being readily
removable by extraction with ether or petroleum ether in a Soxhlet
apparatus, the former requiring acid hydrolysis of the starch before being
set free. It is now known that all the fat in the granules is but loosely held,
the major portion being removed from intact granules by a Soxhlet
extraction with methanol for 10 h. When the granules are disintegrated a
short extraction gives complete and easy removal. 26
K. A. Clendenning and D. E. Wright2 7 have carefully prepared pure
samples of starch according to the methods of various workers. 28 - 31
The fat content was determined by the acid-digestion method and it was
found that the fat content of the waxy starches was consistently lower than
that of the corresponding non-waxy cereal starches. Oat starches had by
far the highest fat content averaging 1·2 %. The fat content of legume,
bulb and tuber starches was low but measurable.
According to Schoch 32 defatting may be accomplished by hot extraction
with a suitable hydrophylic fat solvent which will remove the fat without
swelling or gelatinising the starch granules (e.g. methanol, ethanol, 80%
dioxane, 2 methoxyethanol (cellosolve)). Hydrocarbons, ethers or
chlorinated solvents do not extract fatty materials from starches.
5.2.4 Determination of total fat

The following method taken from the Standard Analytical Methods of
the Corn Industries Research Foundation 64 is similar in principle to the
method of Schoch: 33
Weigh 25 g (±0·1 g) of sample, transfer to a 600 ml beaker and suspend
in 100 ml of distilled water. Mix 100 ml of concentrated hydrochloric acid
with 200 ml of distilled water, heat to boiling, and add to the starch
suspension. Heat acidified starch sample to boiling and boil for 5 min or
until a negative starch test is obtained upon addition of a weak iodine
solution. Place in a cold-water bath (below 25°C) for 30 min to coagulate
fatty acids.
Gravity filter reaction mixture through Whatman No.1 paper and wash
residue with distilled water at room temperature until the filtrate is neutral
to methyl orange indicator. Wipe adhering fat from inside of beaker with a
clean filter paper and combine with main residue. Fold filter paper con-
taining the residue, place on a watch glass and dry for 3 h in air oven at 50°C
or overnight in a warm place. Place folded filter paper containing dried
residue in extraction shell. Plug top of shell with cotton extracted pre-
viously with carbon tetrachloride and place in extractor. Attach a previously
dried and weighed Erlenmeyer flask containing about 50 ml of carbon
tetrachloride. Attach water-cooled condenser and place assembly on
heater. Be sure all connections in the extraction assembly are tight to avoid
loss of solvent during extraction. Adjust heat to produce 150 to 200 drops
of condensed solvent per minute and extract for 3 h. Disconnect flask and
evaporate solvent on steam bath until no odour of solvent remains. Place
in vacuum oven for 1 h at 100°C. Prolonged drying of the extract at
elevated temperatures may cause high results due to fat oxidation. Cool
in desiccator and weigh.
5.2.5 Protein
The amount of protein in a sample of starch will depend upon the origin
of the starch and the degree of refinement. Good grades of wheat starch
contain less than 0·2 % protein, maize starch contains 0·1--0·2 %, protein,
tapioca shows a greater range of protein content ranging from 0·15 % in
the highest grades to 1·0 %, whilst potato starch contains only a negligible
amount of protein. Millet starch has the highest protein content-over
1 %; commercial rice starch may also be high in protein.
Direct determination of protein is seldom attempted although methods
involving the precipitation of protein have been devised and the advent
of automated amino acid analysers has made a more direct approach to
protein analysis practical. For routine purposes the protein content is
estimated by applying a factor to the nitrogen content as determined by the
kjeldahl method. The factor is empirical and is based on the average nitrogen
content ofthe proteins present. For all vegetable proteins other than wheat
the factor adopted is 6·25 but for wheat, in which the average nitrogen
content of the proteins is higher, the factor of 5·7 is usually employed.
In order to achieve inter-laboratory agreement, standard methods have

been laid down which specify the catalyst to be employed, time of digestion,
etc. 3 4 - 3 6 The catalysts commonly used are metallic mercury, mercuric
oxide, copper and selenium. Sodium or potassium sulphates are added
mainly to raise the temperature of digestion and hence cut down the time.
The following method which is based on the official method laid down
in the Fertilisers and Feeding Stuffs Regulations 196024 uses mercury or
mercuric oxide as catalyst. For convenience tablets containing the appro-
priate amount of potassium sulphate and catalyst may be obtained from
laboratory chemical suppliers. About 10 g of starch weighed to the nearest
mg (this assumes that the protein content is <0·5% as in the case of most
commercial starches, smaller amounts may be taken for starches with
higher nitrogen content) is placed in an 800 ml kjedahl flask with 10 g
potassium sulphate, 60 ml conc. H 2S0 4 and two small globules of mercury
or approximately 0·5 g of mercuric oxide. Heat gently over a small flame
until frothing ceases and the liquid is practically colourless. Continue to
heat for a further 2 h. Avoid local overheating. If frothing is excessive, add
about 0·5 g of paraffin wax.
Dissolve the cooled digest in water, and make up to a total volume of
about 250 ml. Taking precautions against loss of ammonia, add sufficient
50% sodium hydroxide solution to neutralise the acid and 10 ml in excess;
then add 5 g of sodium thiosulphate, mix well and connect immediately
to a distillation apparatus. Distil into an appropriate volume of 0·2 N acid,
controlling the rate of distillation so that not less than 150 ml distil in
30 min. Titrate the excess of acid with 0·2 N sodium hydroxide solution,
using methyl red solution as an indicator. Carry out a blank test on the
reagents using 2 g of sucrose in place of the sample. Express the result in
terms of nitrogen. 1 ml 0·2 N acid = 0·0028 g nitrogen.
5.3 ACIDITY
The acidity of starch is chiefly related to the amount of amylo-phosphoric

acid present as hydrolysable salts (H. Tryller37 ) but may be due in part
to the presence of sulphur dioxide used as a preservative (this must not
exceed 100 ppm in starch for food purposes vide Preservatives Regula-
tions 38) or, in the case of low grade starches, to the presence of propionic
and other organic acids produced by the controlled fermentation of
carbohydrate during steeping.
C. Scheele 39 considers the pH to be of more value than the titratable
DETERMINATION OF STARCH IN VARIOUS PRODUCTS 151
acidity. A complete picture would be obtained by determining the

electro metric titration curve.
A rough measure of the pH may be rapidly obtained by spreading a
small heap of starch on a plate and moistening it with a few drops of a
suitable indicator.
The Standard Analytical Methods of the Corn Industries Research
Foundation 4 0 include methods for the determination of the pH of starches
which can be gelatinised by heating with water on a water bath, in which
case the pH of a 5 %gelatinised paste is determined electrometrically whilst
in the case of starches which are substantially insoluble in water at room
temperature the determination is done on a 20 %slurry. The same standard
methods include the determination of extractable acidity which is a
measure of the acidity of a cold water filtered extract and the acidity of a
paste which has been prepared by gelatinisation. The latter will include
free acids which are only available after gelatinisation.
Thc methods are as follows:
If necessary grind sample completely through a laboratory cutting mill
to 20 mesh or finer, taking precautions to prevent significant loss of
moisture, and mix thoroughly.
Weigh 10 g (±0·1 g) of sample, transfer to a suitable container and add
100 ml of distilled water at room temperature. Distilled water for sample
extraction and filtrate dilution shall be of such quality that 200 ml will
require not more than 0·05 ml of 0·1 N acid or base to obtain the methyl
red or phenolphthalein end points, respectively. Cover container and
agitate continuously at a moderate rate for 30 min. Gravity filter through
a good quality filter paper into a clean and dry flask and discard first 25 m!.
In the case of pregelatinised starches and others having a substantial
concentration of solubles, reduce the sample weight to avoid an excessively
high viscosity, do not filter, titrate entire sample and adjust calculation
accordingly. Pipette 50·0 ml of filtrate into a clean Erlenmeyer flask,
dilute with 50 m1 of distilled water and add 1 ml of phenolphthalein
indicator. Titrate immediately with standard 0·1 N sodium hydroxide to
the first permanent pink colour. Alternatively the solution may be titrated
electrometrically in which case a pH value of 8·3 is taken as the end point.
For the acidity after gelatinisation:
If necessary grind sample completely through a laboratory cutting mill

to 20 mesh or finer, taking precautions to prevent significant loss of
moisture, and mix thoroughly.
Weigh 10 g (±0·1 g) of sample, add to 300 ml of distilled water in a

suitable container at room temperature and mix thoroughly. Distilled
water for sample preparation shall be of such quality that 200 ml will
require not more than 0·05 ml of 0·1 N acid or base to obtain the methyl
red or phenolphthalein end points, respectively. Bring to a boil on a hot
plate or over an open flame in approximately 15 min, stirring occasionally,
and boil for 10 min. Remove from heat source, add 1 ml of phenolphthalein
indicator and titrate immediately with standard 0·1 N sodium hydroxide
to the first permanent pink colour.
5.4 THE ALKALI-LABILE VALUE
A technique which may prove of value in routine and factory examination

of starch, modified starch and dextrin has been elaborated by T. C. Taylor
and his co-workers. 41- 43 It depends on the fact that these substances
contain a portion termed 'alkali-labile' which is readily acted upon by
alkali. Various pre-treatments of starch bring about changes in the amount
of this alkali-labile portion, and Taylor's method, whilst giving empiric
values, is precise, semi-micro and readily duplicated, providing the tech-
nique laid down is followed rigidly. The method detects changes which
cannot be determined by viscosity tests, iodine reaction, specific rotation
or initial reducing value determinations. Tests should be done in duplicate
at least and are carried out as follows: 43
50·0 mg ± 0·1 mg of starch are weighed into a Pyrex test tube 8 in x lin
and 10 ml of 0·1 N NaOH added. The tube, loosely stoppered, is floated
on a boiling water-bath for 1 h, after which it is cooled for 30 sunder
running cold water, 10 ml of 0·1 N HCI being added immediately it is
removed from the cold water and the contents of the tube well mixed by
thoroughly shaking. The mixture is transferred to a 250 ml Erlenmeyer
flask, the tube being washed twice with 10 ml distilled water and the
washings added to the flask.
Two drops of nitrazine yellow solution are added and the liquid is
neutralised with 0·1 N NaOH. Five ml of the alkali are then added,
followed immediately by 5·0 ml of 0·025 M standard iodine solution; the
last three operations should be carried out within 3 min. The flask is kept
at 25-30°C for 45 ± 1 min in the dark, and then 5·0 ml of conc. HCI
added, mixed well by shaking and immediately titrated with 0·025 M

thiosulphate solution. If the back titration is less than 3 ml the resulting
alkali-labile value will be low and 7 ml instead of 5·0 ml of iodine solution
should be added for the oxidation of a second sample, so that a reading
of at least 3 ml is obtained in the back titration with thiosulphate. One
hundred times the number of milligrams of iodine consumed divided by
the weight of the sample in mg gives the alkali-labile value.
Pastes may be precipitated with acetone-free dry methyl alcohol after
which the alkali-labile value of the carbohydrate material may be deter-
mined. If the paste contains no electrolyte the addition of a few drops of
0·1 N BaCl 2 greatly assists the precipitation; free chlorine, hypochlorous
acid, peroxides, borax or sulphur dioxide should be absent, as these
absorb iodine.
If the long chains of glucose units forming the starch molecule are
parallel and co-ordinate links exist between the R of the OR groups of
some chains and the 0 bridges of others as has been suggested the free
aldehyde groups at the end of the chain, although primarily chemically
free, might be protected by a dovetail-like fitting end to end of the bundles
of parallel-bound chains. Disassociation of the co-ordinately-linked
chains from one another would make the aldehyde groups available whilst
hydrolytic scission of glucosidic linkages would give shorter chains and
consequently new aldehydic groups. Taylor and Keresztesy 43 think the
great increase in alkali-labile value in making soluble starch by dry-
grinding (see Starch Production Technology), or by the Lintner acid-
process, is due to the terminal aldehyde exposed by disassociation of
the chains, whereas the slower hydrolytic breakdown of the glucosidic
links is responsible for the fairly steady rate of fall ultimately attained.
A good grade, commercial air-dry maize starch gives an alkali-labile
value of about 22, tapioca starch 14, a thin-boiling starch 60 and a yellow
dextrin 20. The method should be of value in examining oxidised and
acid-treated soluble starches.
M. Samec and B. Skerl 44 have studied the dependence of alkali-labile
value on the length of boiling and temperature to differentiate between
erythro- and amylo-substances. Native and partially attacked starch gave
quite different results. The fractionation methods previously employed by
these workers, e.g. pressure cooking, electro-dialysis, coagulation by
freezing or by ageing, caused no structural changes as judged by alkali-
labile value. They consider that a large number of starches can be
characterised by this value.
5.5 ALKALI NUMBER
T. J. Schoch and C. C. Jensen 45 have devised a simplified alkalimetric

method to estimate the relative hydrolytic degradation of starch. They
consider that Taylor's alkali-labile value is liable to variations at the hands
of different workers, especially where the starch has been precipitated by
solvents, as the latter are tenaciously retained even on prolonged drying.
Schoch and Jensen digest 0·5 g of starch (calculated on a dry basis)
with sodium hydroxide. The starch, which should pass a 60-mesh sieve, is
introduced into an eight-ounce flask and shaken with 10 ml of water until
suspended, 25 ml of 0·4 N sodium hydroxide are added, taking care to
agitate the contents of the flask so as to obtain uniform gelatinisation of
the starch, followed by 65 ml of hot distilled water. The flask is closed
with a bung carrying a bunsen valve and immediately placed in a vigorously
boiling water-bath. After heating for exactly 60 min the flask is placed in
cold water and 50 to 75 ml of cold distilled water are added to the contents
of the flask to halt the decomposition. The liquid is then titrated against
thymol blue to a yellow end-point with 0·2 N sulphuric acid. With highly
coloured compounds the titration is carried out to a pH value of 8, using a
glass electrode. The alkali number is calculated as (ml of acid to titrate
blank - ml of acid to titrate sample) x normality of acid x 10 -:- weight
of sample on dry basis.
Close adherence to the above procedure is recommended. Should the
starch product contain added acid or alkali, sufficient to affect the alkali
number, I g of the product is gelatinised and titrated with alkali or acid
against thymol blue, correcting the alkali number for this titre. With cold-
swelling products the sample should be introduced into a perfectly dry
digestion flask and wetted with 1-2 ml of benzene; 25 ml of 0·4 N sodium
hydroxide solution are then added followed by 75 ml of hot water. In this
way complete dispersion without the formation of lumps is obtained.
Commercial corn and wheat starches have consistently higher alkali
numbers than those of the common tuber starches. Schoch and Jensen
find the following values: tapioca, 5·9-6·9; potato, 5·7-6·9; rice, 6·7-7·5;
wheat, 9·7-11·5; maize, 9·8-12·1; maize (specially prepared by non-
aqueous steeping) 10·6, 11·4.
By leaching at temperatures just below the gelatinising point raw corn
starch gave a minor fraction of soluble carbohydrate possessing a high
alkali number (35·9). The insoluble residue had an alkali number, 8·8,
and the raw starch, 11·2.
By fractionation with cold water a typical acid modified thin-boiling
starch was found to be heterogeneous in character. The initial thin-boiling

starch gave a value of 44·1, the cold water soluble fraction 50·6 and the
insoluble residue 36·9 for the alkali number.
With increasing degree of acid conversion the alkali number rises
progressively in thin-boiling starches, e.g. 20-fluidity, 14·5; 40-fluidity,
15·0; 60-fluidity, 15·7; 75-fluidity, 20·7; 90-fluidity, 41·5; white dextrin,
25 %cold water soluble, 56· 3; white dextrin, 65 %cold water soluble, 62·6;
Lintner's soluble starch, 66·4.
British gums and yellow dextrins show alkali numbers only a little
higher than that of raw corn starch, e.g. British gum, 17·5 %soluble, 16·5;
British gum, 80 %soluble, 15·6; gum, 85 %soluble, 16· 3; dextrin, acetic acid
conversion, 98 %soluble, 16·4; yellow dextrin, nitric acid conversion, 85 %
soluble, 19·9; yellow dextrin, hydrochloric acid conversion, 97 % soluble,
23·0. The alkali numbers bear no relation to viscosity or solubility. With
oxidised starches the alkali number is generally lower than that of the
raw corn starch. Alkali lability, however determined, must not be construed
as a quantitative evaluation of aldehyde content, but merely as an empirical
index of hydrolysis.
5.6 IODIMETRIC DETERMINATION OF AMYLOSE
Starches normally contain two polysaccharide components, amylose which

consists of a straight (unbranched) chain which has a strong affinity for
iodine with which it gives a deep blue complex and amylo pectin which
consists of a branched chain having little affinity for iodine with which it
gives a red coloration. A full discussion by Hollo and Szeitli of the reaction
between starch and iodine is given in Chapter 7 Starch and Its Derivatives,
J. A. Radley, Chapman & Hall, London (1968).
The affinity between amylose and iodine has been used to determine the
amount of amylose in starches. The pioneer work by Bates, French and
Rundle,46 who used a potentiometric titration method has been modified
by Schoch et al. 47
Stock iodine solution. 83 mg KI + 2000 g iodine and 37 g potassium

chloride dissolved in small amount of water and then diluted to 1 1. Store
in brown bottle in dark.
Working iodine solution. Dilute stock solution x 10. Prepared fresh each
day as it is unstable.
Apparatus. Potentiometer sensitivity ± 0·1 mv or pH meter with

millivolt scale for less precise work. Bright Pt and Calomel electrodes.
Constant temperature bath 30°C ± 0·1°C.
Preparation of standard graph. A graph relating free iodine in solution

to emf is prepared as follows:
Dissolve 373 mg KCI and 830 mg KI in 100 ml of distilled water.
Insert in constant temperature bath at 30°C, insert calomel and platinum
electrodes and stir mechanically. Add working iodine solution in small
increments from burette and note emf for range corresponding to 230-
285 mv. Plot curve. From the curve a chart may be derived showing the
concentration of free iodine in solution against the potential in millivolts.
Determination. Defat starch sample by extraction with 95 % ethanol in

Soxhlet extractor for 24 h. Dry and grind to pass 60 mesh sieve. Weigh
out 100 mg (± 0·1 mg) of whole starch (half this amount iflinear fraction
only or double the amount if only branched fraction) into clean dry,
counterpoised 250 ml beaker. Add 1 ml water to suspend sample. Add
5 ml of 1·0 N potassium hydroxide and disperse by grinding with a stirring
rod. Place in refrigerator for 30 min stirring occasionally until clear
solution is obtained. Neutralise with 0·5 N HCI to methyl orange add
10 ml 0·5 N KI solution. Remove and rinse stirring rod. Add sufficient
water to bring total weight of solution to 100·9 g. If difficulty is experienced
in obtaining clear solution add 25-50 ml water and warm, cool to room
temperature and proceed as before. Titrate potentiometrically as in the
preparation of the standard graph at 30°C with constant mechanical
stirring, plotting about 15 points between 230 and 280 mv. Allow about
2 min for the solution to stabilise between each addition of iodine. From
the calibration chart or standard graph read off the amount of free iodine
in the solution against the observed emf and from the difference between
the total amount of iodine added and the amount of free iodine remaining
in solution, obtain the amount of bound iodine. Plot free iodine against
bound iodine and extrapolate the straight upper portion of the curve
back to the zero axis. The value of this intercept is used to calculate the
iodine affinity of the sample.
.
%Iodme . mg of bound iodine at zero intercept x 100
00 affinIty = .
mg of sample taken (on dry basIs)
Replicate determinations should agree with ±0·08 %.
Typical values for the purified lincar (amylose) fraction from various
starches are as follows:
Maize (corn) 19'0%, wheat 19·9%, potato 19·9%, tapioca 18·6%.
The calculation of the amylose contcnt of a given sample of starch from
these figures can only be approximate as there are possible intermediate
fractions between strictly linear chains (amylose) and highly branched
(amylo pectin) chains and long straight side chains may have some iodine
affinity.
Foster 48 has suggested that 'iodine affinity' should more properly be
designated 'iodine binding capacity' (I.B.C.).
Alternative amperometric methods of determining iodine absorption
are dealt with in Radley, J. A. Starch and Its Derivatives, Chapman &
Hall, London (1968).
The iodine absorption may also be measured by the 'Blue Value,49
based on the work of McCready and Hassid. 5 0
This is determined as follows:
Reagents. N· NaOH solution; N· HCI solution; iodine solution

containing 2 mg iodine/ml + 20 mg/KI per ml.
Apparatus. Spectrophotometer 1 cm cell.
Method. To 0·5 mg of sample dispersed in 1 ml water in a 50 ml

graduated flask add 0·5 ml of N . NaOH solution and warm for 3 min on
boiling water bath. Cool, neutralise by the addition of N . HCI solution
and add 0·07-0·1 gm of potassium hydrogen tartarate. Dilute with water
to about 45 ml then add 0·5 ml of Iodine solution (supra), mix and allow
to stand for 20 min at room temperature. Measure absorbance at 680 m,u.
Use iodine solution of equal concentration as reference solution.
Absorbance x 4
Blue value = C(mg/dl)
where C is the concentration of the carbohydrate solution. The blue colour

may not be fully developed due to retrogradation or incomplete solution.
If this is suspected more vigorous means of obtaining a solution must be
adopted and the method chosen must be the one which gives the maximum
blue value. If other substances are present which absorb iodine, additional
iodine solution must be added. Comparison of the 'Blue value' of the
sample with that of pure amylose will be a measure of the amylose content.
5.7 PENTOSAN CONTENT OF WHEAT STARCH
Baker, Parker and Mize,51 have separated the starch and gluten of wheat
flour by the ordinary gluten working technique, the starch being collected
by centrifuging. The top or 'amylodextrin' layer was separated from the
prime quality starch in the lower layer and purified by successive washing
and centrifuge treatments. Upon analysis the 'amylodextrin' fraction was
found to contain 14·0% of pentosans using the Schmidt-Nielson and
Hammer method. 59 The prime quality starch gave a content of 0·4 %
pentosans by this method. These authors concluded that the wheat starch
granules were all coated with insoluble pentosans, the higher pentosan
content of the 'amylodextrin' fraction being attributed to the smaller
average particle size and the concomitant greater surface area of this
fraction as compared with the prime quality starch fraction.
Clendenning and Wright27 find the same differences between the two
fractions. They found that the phloroglucide of the second distillate from
prime quality wheat starch was completely soluble in 95 % ethanol. Using
the Hughes-Acree bromine oxidation method 65 or phloroglucinol
precipitation 66 for the estimation of 'furfural', dextrose gave apparent
pentosan contents of the same order (0·4 %). Evidently with large samples
of starch redistillation from saturated sodium chloride does not ensure
complete removal of hydroxy-methyl-furfural as has hitherto been
assumed.
5.S THE EXAMINATION OF MODIFIED STARCHES
With these products it is generally desired to characterise them either for

use or for matching purposes. Oxidiscd starches are invariably whiter in
colour than acid-treated starches, and a microscopical examination will at
once determine the raw material used. In use the most important property
is the viscosity, but in endeavouring to characterise a starch for matching
purposes a number of drawbacks are encountered in viscometric deter-
minations. Some soluble starches made by acid treatment are not washed
free from acid or the acid is incompletely neutralised, and this leads to a
rapid fall in the initial viscosity on making a solution. Solutions of soluble
starch are also sensitive to the presence of impurities and electrolytes and
to mechanical treatment. Highly modified starch swells before dissolving
and must be heated to complete the dispersion, and such heating brings
about further modification.
In spite of these drawbacks, viscometric methods are employed for the

examination of these products, together with the reducing value by the
method ofW. A. Richardson, Higginbotham and Farrow. 53 With potato,
maize and sago starches which have been modified by hot acid treatment
followed by neutralisation, these workers find that the degree of modifica-
tion is accurately measured by the reducing value (R) which does not,
however, indicate the degree of modification of oxidised starches or those
treated in the granular state with acids. Starches in these two classes are
washed free from reagent after modification, and such washing removes the
water-soluble reducing substances.
F. F. Farley and R. M. Hixon 54 describe a much simpler method than
that of Richardson and co-workers. They noticed that when measuring
the reducing power of a series of starches by the Gore and Steele 55
modification of the Hagedorn and Jensen 56 ferrocyanide method the
apparent maltose equivalent of the more soluble products when converted
to milligrams of copper gave values equal to the copper numbers obtained
by the longer method of Richardson et al. For raw starch and very
slightly solubilised starch, high values were obtained by the Gore and
Steele method attributable to the visible entrapment in the starch of
iodine which was very difficultly released for measurement in the
thiosulphate solution.
Martin and Newton 57 avoided this difficulty when determining maltose
in maltose-starch mixtures by using Hassid's ceric sulphate titration. 58
Farley and Hixon, therefore, oxidise the sample with excess potassium
ferricyanide and the ferrous ion produced is titrated after 15 min at lOooe
with ceric sulphate. The method gives the same results in the starch and
dextrin range, but not in the range of sugars, as that of Richardson et al.
The reducing power increases with increasing conversion of starch products
except for the electrolytically oxidised starches, which, because of the
method of oxidation and washing, have values equal to or lower than
those of raw starches. The method can be used to follow the conversion,
by both acid and alkali, of starch to thin-boiling starches, Gore starches
or dextrins formed by acid or alkaline catalysts. Table 5.2 shows the
results obtained.
Interesting results in the examination of soluble starches have been
obtained by Richardson,67 using the following technique. A suspension
of starch in cold water is poured into sufficient boiling water to produce a
2·5 % solution which is heated for I min and then cooled. After passing
through a homogeniser the concentration is determined by the dichromate
method. 45 To 10 ml of this solution is added 15 ml of a 50% calcium
thiocyanate solution, a control solution of 30 g of this salt in 100 ml of

water also being prepared. 10 ml of each solution are used in separate
U-tube visco meters (cf. Higginbotham and Richardson 68) and n, the
viscosity of the starch solution relative to the control, is determined at
2S C ± SoC, and if c is the concentration of the starch in g 100/ml, the
O
'thiocyanate viscosity' (TV) is given by the quantity (log n)/c. Where c is

between 0·6 and 1·0 %, TV is independent of c, and so long as c is known
TABLE 5.2
Reducing power of starches and starch products
Product Rcu mg/g
Pearl starch (control) 6·8-7·9

Commercial maize starches 7'7,9'4,10'1,11'2,11'6
Waxy maize starch 9·0
Thin boiling starch, 40 fluidity 6·5
Thin boiling starch, 90 fluidity 27·2
Chlorinated starch, 2·5 % chlorine 14·9
Chlorinated starch, 5'0% chlorine 32·9
Electrolytically oxidised starches 6'8,3'0,6'3,4'5,7'5
Alkali dextrin A 10·5
Alkali dextrin B 33·0
Alkali dextrin C 71-0
Acid dextrin A 12·0
Acid dextrin B 25·5
Acid dextrin C 39·0
Gore starch, 5 h conversion 19-6
Gore starch 42 h conversion 61·6
Gore starch 96 h conversion 120·0
Maltose 1900
Glucose 2800
accurately it is unnecessary to adjust it to a given value. TV runs parallel

to R for acid-modified neutralised starches, and, like this value, may be
used as a satisfactory index of the degree of modification. TV is preferable
to R as the modification index for oxidised and acid-treated, washed
starches (vide supra).
With unmodified or lightly modified starches TV is very sensitive to
mechanical treatment, but this effect diminishes with increase in the
degree of modification until, when R exceeds 40, homogenisation is
superfluous. With oxidised starches, however, R may be lower than for an
acid-treated, neutralised starch of the same degree of modification and
may not, therefore, need homogenisation even when R is less than 40.
M. I. Knyaginichev 69 uses 30 or 50 % sodium salicylate solutions

containing 0·2 g of starch per 100 ml for the examination of starches.
He finds the viscosities for the different starches and modified products
are unaffected by time and often differ more than the corresponding
aqueous solutions. Incidentally, this worker finds that the larger granules
give more viscous pastes than do the smaller granules.
5.9 DETERMINATION OF CARBOXYL GROUPS

L. H. Elizer 70 has developed a method for the determination of carboxyl
groups in commercial starches modified by oxidation. Briefly a weighed
sample of the starch is left in contact with a solution of silver o-nitro-
phenolate for 18 h and the clear, supernatant liquid is then titrated with
ammonium thiocyanate. A more convenient method, which gives slightly
higher values, depends on the use of copper acetate and the clear super-
natant liquid is titrated, iodometrically. This method can be varied to give
a satisfactory routine procedure by following the copper acetate treatment
by the addition of potassium ferrocyanide solution to the supernatant
liquid and comparing the colour produced with standards.
E. K. Gladding and C. B. Purves 52 estimate the carboxyl groups
present in oxystarch and oxycellulose by treatment with hydroxylamine
hydrochloride to form the oxime and titrating the liberated hydrochloric
acid with standard alkali. Condensation occurs within 1·5 h with oxystarch,
the carboxyl groups being classed as a 'fast' type.
By the judicious use of the microscope and the determination of the
reducing value R, the thiocyanate viscosity and the alkali-labile value the
previous history and degree of modification of a sample of soluble starch
may be determined sufficiently accurately to enable it to be matched for
factory production.
Gas chromatography has been used for the determination of starch in
plant materials by C. W. Ford 77 by converting starch to the trimethyl silyl
derivative. This technique could probably be extended to investigate the
components of mixtures of starch and starch derivatives.
Gas chromatography has also been employed by Vomhof and Thomas 78
to determine the amount of dry matter in starch hydrolysates. They com-
pared various other instrumental methods with the Corn Industries
Research standard reference methods. They concluded that infra red was
of little use in this context. The various components of starch have been
separated by paper chromatography 7 9 using saturated baryta solution as
the mobile solvent.
TABLE 5.3
Specific
H 2O Ash Fat Protein %
Sample rotation
% % % (N X 6·25)
degrees
Wheat Marquis 9·21 0·18 0·57 0·21 202-3

Wheat Thatcher 9·33 0'15 0·51 0·21 202'7
Wheat Garnet 11-19 0·20 0·53 0·22 202'5
Wheat Dicklow 11·03 0·28 0·57 0·29 202-8
Wheat Blanca 11·93 0·24 0·61 0'18 203·2
Wheat Ontario
soft winter 9·07 0·14 0·48 0·17 202·3
Wheat Mindum 12·27 0·21 0'59 0·23 202·7
Wheat Kharkov 11·27 0·24 0·55 0·18 202-6
Wheat Jones Fife 11-06 0·27 0'58 0·19 203'1
Maize 11·36 0·05 0'62 0·29 202·3
Maize 9'12 0·05 0·64 0·37 202-9
Maize 10·44 0·05 0·71 0·37 202·9
Maize 11·29 0·07 0·72 0·39 203·2
Maize 9·08 0·07 0·77 0·27 20304
Waxy Maize 10'50 0·05 0·20 0·26 202·3
Barley 9·53 0·14 0'73 0'18 203·5
Waxy Barley 9·26 0·12 0·43 0·27 20H
Waxy Barley 13095 0·23 0·30 0·09 202·5
Rye 9·53 0·14 0·47 0·18 202-8
Oat 10·78 0·10 1·20 0'31 202·6
Oat 9·46 0·20 1-17 0·31 203·1
Rice 10·07 0'59 0·66 0·46 202-8
Rice 10·17 0·28 0·87 0·56 203·8
Rice 11·95 0·65 0·68 0·51 202·7
Rice 11·21 0·33 0·45 0·14 202-6
Waxy Rice 9·95 0·36 0·12 0·54 203·1
Waxy Rice 10·19 0·15 0·10 0·06 202-3
Grain Sorghum 12·91 0·41 0·72 0·14 202-3
Waxy Sorghum 10·23 0·32 0·39 0·34 202-3
Millet 10·46 0·53 0·91 1·28 201·4
Buckwheat 15-88 0·19 0·53 0·32 203·4
Pea 10·84 0·22 0·18 0·36 199·4
Bean 14·15 0·20 0·25 0·30 200·2
Lima Bean 9·73 0·12 0·17 0·19 200·5
Potato 12-88 0·30 0·18 0·17 204·8
Potato 14·80 0·32 0·12 0·11 203-8
Potato 14·84 0·34 0·11 0·08 203·7
Potato 12·18 0·32 0·11 0·08 20309
Potato 10·13 0·29 0·13 0·18 204'4
Potato 9·15 0·40 0·12 0·10 203'7
Potato IH2 0·33 0·15 0·08 20304
Sweet Potato 10·19 0·20 0·13 0·07 203·3
Sweet Potato 10·43 0·21 0·12 0·07 203·5
Arrowroot 8·94 0·12 0·15 0·08 203·6
Arrowroot 10·49 0·16 0·16 0·09 202·9
Tapioca 10·13 0·10 0·27 0·09 203·0
Tapioca 10·17 0·08 0·27 0·14 202-6
Easter Lily 8·74 0·37 0·27 0'04 20309
5.10 DAMAGED GRAINS
The proportion of damaged grains in a sample of starch may give some

indication of the treatment which it has received during manufacture.
Heavy grinding will increase the proportion of damaged grains which in
turn will affect the rheological properties of gels. Damaged grains will
swell with cold water, this may be an advantage for certain applications
but the viscosity curve will differ considerably from that of a sample
containing a lower proportion of damaged grains.
Besides being more readily swollen by cold water the damaged grains
are more readily attacked by enzymes. 8 0 This forms the basis of a method
for the estimation of damaged grains 81 in which the starch suspension is
digested for 30 min with e>:-amylase, the residual starch dissolved in calcium
chloride solution and determined polarimetrically. Comparison with a
sample which has not been treated with e>:-amylase gives the proportion of
damaged grains.
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24. Fertilisers and Feeding Stuffs Regulations, 1968, S.I.218/1968, HMSO, London.
25. Taylor, T. C. and Nelson, J. M.,J. Arn. Chern. Soc., 1920,42,1726.
26. Griffiths, J. G. A., Analyst, 1937,62, 510.
27. Clendenning, K. A. and Wright, D. E., Canad. J. Res., 1945, 23B, 131.
28. Shewfelt, A. L. and Adams, G. A., Can. Chem. Process Ind., 1944,28,502.
29. Ling, A. R., J. Inst. Brewing, 1922,28,843.
30. Sandstedt, R. M. et al., Cereal Chem., 1939, 16, 780.
31. Eynon, L. and Lane, J. H., Starch, W. Heffer & Sons Ltd, Cambridge, 1928.
32. Whistler, Methods in Carbohydrate Chemistry, Vol. IV, Academic Press, New York,
1963, p. 56.
33. Ibid., p. 59.
35. Official Methods of Analysis, A.O.A.C., 9th ed., 1960, 2.036.
36. Whistler, Carbohydrate Chemistry, Vol. IV, Academic Press, New York, 1964, p. 47.
37. Tryller, H., Zeit. Spiritusind, 1934, 57, 19.
38. Preservatives in Food Regulations, 1962, S.I. No. 1532/1962, HMSO, London.
39. Scheele, c., Afzelius, J. and Leander, K., Zeit. Spiritusind, 1937,60,163.
41. Taylor, T. C. and Salzmann, G. M., J. Amer. Chem. Soc., 1933,55,264.
42. Taylor, T. c., Fletcher, H. H. and Adams, M. H., Ind. Eng. Chem. (Amal. Ed.),
1935, 7, 321.
43. Taylor, T. C. and Keresztesy, J. c., Ind. Eng. Chem., 1936, 28, 502.
44. Samec, M. and Skerl, B., Kolloidchem. Bich., 1937, 47, 91.
45. Schoch, T. J. and Jensen, C. C., Ind. Eng. Chem. (Anal. Ed.), 1940, 12, 531.
46. Bates, F. L., French, D. and Rundle, R. E., J. Amer. Chem. Soc., 1943, 65, 142.
47. Wilson, A. J., Jr., Schoch, T. J. and Hudson, C. S., J. Am. Chem. Soc., 1943, 65,
1380.
48. Foster, J. F., in Starch Chemistry and Technology, ed. Whistler & Paschall, Vol. I,
Academic Press, New York, 1964, p. 371.
49. Gilbert, G. A. and Spragg, S. P., Methods in Carbohydrate Chemistry, Academic
Press, New York, 1964, p. 168.
50. McCready, R. M. and Hassid, W. Z., J. Am. Chem. Soc., 1943,65, 1154.
51. Baker, J. c., Parker, H. K. and Mize, M. D., Cereal Chem., 1943,20,267.
52. Gladding, E. K. and Purves, C. B., Paper Trade J., 1943, 116, Tappi 150.
53. Richardson, W. A., Higginbotham, R. S. and Farrow, F. D., J. Text. Inst., 1936,27,
131T.
54. Farley, F. F. and Hixon, R. M., Ind. Chem. (Anal. Ed.), 1941, 13, 616.
55. Gore, H. C. and Steele, H. K., ibid., 1935,7, 324.
56. Hagedorn, M. and Jensen, B. N., Biochem. Z., 1923, 135, 46.
57. Martin and Newton, J. N., Cereal Chem., 1938, 15, 456.
58. Hassid, W. Z., Ind. Eng. Chem. (Anal. Ed.), 1936, 8, 138; 1937,9,228; 1940,12,142.
59. Schmidt-Neilson, S. and Hammer, L., Kgl. Norske. Videnskab. Selskab. Forh.,
1932,5,84.
60. Fischer, K., Angew. Chem., 1935,48,394.
61. Smith et al., J. Am. Chem. Soc., 1939, 61, 2407.
62. Jones, A. G., Analyst., 1951,76, 5.
63. Taylor, T. C. and Lehrman, L., J. Am. Chem. Soc., 1920, 42, 1726.
65. Hughes, E. E. and Acree, S. S., Ind. Eng. Chem. (Anal. Ed.), 1934, 6, 123.
66. Official Methods of Analysis, A.O.A.C., 10th ed., 1965, 22.05()....22.051.
67. Richardson, W. A., Chem. and Ind., 1939,58,468.
68. Higginbotham, R. S. and Richardson, W. A., J. Soc. Chem. Ind., 1938, 57, 239.
69. Knyaginichev, M. I., Colloid J. (USSR), 1939,5,899.
70. Elizer, L. H., Ind. Eng. Chem. (Anal. Ed.), 1942, 14, 635.
71. BS 4628: Part 2, 1970. Methods of Test for Starch: Pt. I, Determination of Loss in
Mass on Drying.
72. Brocklesby, C. T., Cereal Chem., 1951,28, 83.

73. Kent-Jones, D. W. and Amos, A. J., Modern Cereal Chemistry, Northern Publishing
Co. Ltd, Liverpool, 1957, p. 535.
74. Ibid., p. 539.
75. Vomhof, D. W. and Thomas, J. H., Anal. Chem., 1970, 42 (11), 1230-3.
76. Pande, A., Laboratory Practice, 1971,20,117-120.
77. Ford, C. W., Anal. Biochem., 1974 (2), 564-568.
78. Vomhof, W. and Thomas, J. H., Amer. Soc. Brew. Chem. Proc., 1969, 139--41.
79. Patel, J. M. and Patel, N. B., Marathwada Univ. J. Sc., 1971, 10 (3), A.5-A.9 (Eng.).
80. Lelievre, J., Die Starke, 1974, 26 (3), 80.
81. Chiang, B. Y., Miller, G. and Johnson, J. A., Cereal Chem., 1973, 50 (1), 44--49.
82. de WiIIigen, A. H. A. and Patscheider-Gerritsen, G. A., The Analysis of Potato
Starch, Glimmen, The Netherlands, 1975.
CHAPTER 6
Determination of Starch in Various Products

F. A. LYNE
Public Analyst, 220 Elgar Road, Reading, Berks. RG2 ODG,
Great Britain
INTRODUCTION
Many methods are in use for the determination of starch, but most of
them are applicable to a limited type of work only. They may be roughly
classified under the following headings:
I. Non-hydrolytic methods. In these the starch is dispersed in a solvent,

and then (a) recovered and weighed, or (b) precipitated from the solvent
in the form of a derivative, or (c) determined polarimetrically.
2. Hydrolytic methods. In these the starch is hydrolysed to reducing

sugar and the sugar determined. The inversion may be carried out by
means of (a) acid, (b) enzymes or (c) enzymes, followed by acid treatment.
6.1 NON-HYDROLYTIC METHODS INVOLVING

DIRECT WEIGHING OF STARCH
Methods involving the dispersion of starch in various solvents to give

filterable solutions from which the starch may be recovered by precipita-
tion and weighed have been devised. Solvents have included hydrochloric
and trichloracetic2 acids, calcium chloride,3.4 potassium thiocyanate,
zinc chloride,l magnesium chloride,5 caustic alkalis, glycerol and form-
amide. Under specified conditions hydrolysis of the starch is minimal but
the physical properties of the recovered starch may have been radically
altered.
167

The best known of such methods is that due to O. s. Rask 6 in which the
starch is dispersed in cold concentrated hydrochloric acid from which the
starch is recovered by precipitation with alcohol. This method was at one
time adopted as a tentative method by the American Association of
Official Agricultural Chemists. 7 C. W. Herd and D. W. Kent-Jones 8
modified the method for application to mill feeds and mill products. Their
procedure was as follows:
1 g of the material is well mixed with 1 g of acid-washed sand and

covered with ethyl ether in a centrifuge tube, well stirred for about 1 min,
after which it is centrifuged and the liquid poured off. This is repeated
twice more, so reducing the filtration difficulties, due to impurities, which
would arise later in the determination. To the residue is added 2·5 ml
water and 0·25 ml Nil caustic soda solution, which is thoroughly stirred in.
Fifteen minutes later 5 ml of pure methyl alcohol are added and mixed in,
followed by 5 ml of dilute methyl alcohol (5 ml alcohol, 2·5 ml water),
and after mixing and centrifuging the alcoholic layer is removed. The
residue is washed twice with 10 ml of the diluted alcohol, and finally
given three washings with water.
The residue is mixed to a thick paste with a few ml of water, taking
care that no lumps are formed. A total of 20 ml of water is employed to
transfer the paste to a 100 ml flask, to which 20 ml of concentrated
hydrochloric acid are then added, and the total volume is made up to 100
ml with Rask's acid, using this first to rinse out the centrifuge tube. After
shaking the flask, the contents are filtered through a Gooch crucible having
a layer of acid-washed sand superimposed on the asbestos; 50 ml of the
filtrate are pipetted into a 200 ml beaker containing 110-115 ml of 96%
alcohol, and not until the pipette has drained is the liquid in the beaker
thoroughly stirred. A flocculent precipitate is formed; after it has partially
settlcd the contents of the beaker are centrifuged for 10 min and the residue
washed four times with 70% (by volume) alcohol and twice with 96%
alcohol to remove the last trace of acid, care being taken that the residue
and alcohol are thoroughly mixed each time.
It is essential that the time between the addition of the acid to the
sample and the precipitation of the starch with the alcohol does not exceed
35 min, otherwise hydrolysis of the starch may materially affect the
results. The final residue is transferred to a tared Gooch crucible, using
96 %alcohol, washed with ethyl ether and dried in the oven at 40 a C for 10
to 15 min; this is followed by heating to l30 a C until the weight is
constant.
Several workers 9-11 have compared the results obtained by Rask's

method and its modifications with those obtained by hydrolytic and
polarimetric methods.
Other possible solvents have been tried such as salicyclic and/or lactic
acid under pressure suggested by W. H. Krug and H. W. Wiley12 and
trichloroacetic acid by P. Biourge 2 but these were found to give high
results due to degradation of pentosans and hemicelluloses.
Caustic alkalis have also been used to extract starch from foodstuffs 13 ,14
followed by precipitation of the starch with alcohol.
The following method is that recommended by the Society of Public
Analysts for the determination of starch in Sausages: 15
Digest 20 g of sausages with 300 ml of 5 % alcoholic potash on a
water bath. Filter off the insoluble matter and wash with alcoholic
potash. Wash the insoluble matter into a beaker with 200 ml of
warm water, add 40 ml of N aqueous potassium hydroxide and warm
to dissolve the starch. Cool and make up to 250 ml with water in a
volumetric flask. Add a 50 ml aliquot to a beaker containing 300 ml of
90 % v /v alcohol acidified with acetic acid (equivalent to 8 ml of N
potassium hydroxide), stir thoroughly and allow to stand (preferably
overnight). Filter off the starch, wash with alcohol, then with ether
and dry at 100°C. After weighing, incinerate and deduct the ash from
the weight of the residue to give the starch in 4 g of original sample.
6.2 NON-HYDROLYTIC STARCH-IODINE METHODS
The formation of additive products between starch and iodine was used
by A. Kaiser 16 as a method for determining starch, and later Th. von
Fellenberg 17,18 used calcium chloride solution to dissolve starch, which
was then precipitated by the addition of iodine solution. The calcium
chloride acts as a salting-out agent for the starch-iodine complex, and this
is decomposed with alcohol to give starch. J. J. Chinoy and F. W.
Edwards, 19 other workers 20,21 and H. Weiss 22 have used similar methods.
J. C. Smalf 3 uses ammonium sulphate to salt out the starch iodide from
aqueous dispersions and to remove the dextrin-iodine complexes present.
W. Whale,24 using an iodometric method for food products, points out
that the presence of dextrin introduces an error not easily overcome, and
in such cases advocates one or other of the hydrolytic methods. He has
successfully applied the volumetric iodide method to the determination of
starch in cocoa and sweetened chocolate. 24
The method of Hling and Whittle 25 has been modified by Bagnall and
Smith26 for the determination of starch in lemon curd but could be used
for many foods:
Reflux 20 g of sample with 100 ml 95 % alcohol in a boiling-water
bath for 6 h. Filter on a Buchner funnel. Re-extract the residue as
before with more alcohol and filter. To the residue on the Buchner
funnel add 75 ml of 0·7 % aqueous potassium hydroxide solution and
gelatinise the starch by simmering gently for 30 min. Transfer the hot
liquid to a 200 ml volumetric flask, cool and make up to volume.
Filter. Neutralise 20 ml of filtrate with 5% acetic acid using phenol-
phthalein. Add 8 ml of 0·1 N iodine solution and 4 ml of either 10%
potassium acetate solution or alcohol and allow to stand until the
precipitate settles and centrifuge. Decant off the supernatant liquid.
Rub the residue with a rod and treat with 12 ml of a mixture con-
taining 10 ml of95% alcohol and 2 ml 0·1 N sodium thiosulphate by
addition in several small quantities. When the particles are thoroughly
broken up with the rod, add 25 ml of 80 % alcohol and filter through
a weighed Gooch crucible. Wash the residue with 95 % alcohol, dry
and weigh the starch.
The colour of the starch-iodine complex has also been used for the
determination of starch in flue-cured tobacco using an auto analyser112
with a precision of ± 3'9 % and for the determination of amylose
in starches and floursY3 The conditions must be standardised in
order to get reproducible results.
6.3 NON-HYDROLYTIC METHODS USING POLARIMETRY
In the third class of non-hydrolytic methods the starch is dispersed or

dissolved in a solvent and the amount of starch present in solution is
determined by means of the polarimeter. Methods depending on the use of
this instrument presuppose a constant specific rotation value for the
starches occurring in definable classes of plant materials. Unfortunately
there appears in the literature values for the specific rotation of starch
ranging from + 1800 to + 220 0 • To quote Kerr 27 ' ... one is tempted to
believe that each worker has his own method . . .'. A number of agents
have been used to prepare the starch solutions for polarimetric measure-
ment. A. Baudry28,29 refluxes the sample with benzoic or salicylic acid
and examines the filtrate polarimetrically, the polarimeter being graduated
for reading the starch-content directly. A modification of this method has
been used by L. Pellet. 3 0 Effrone 1 uses hydrochloric acid as the solubilising

agent, and after polarisation makes allowance for the small amount of
glucose present, which he determines by titrating with Fehling's solution.
D. Crispo 32 uses caustic potash solution for polarising, and C. Mannich
and K. Lenz 33 boil the starch with a concentrated solution of calcium
chloride, which is N/500 with respect to acetic acid, and thus obtain a
clear solution suitable for direct polarimetric observation. Calcium
chloride solutions are also favoured by Hopkins.
The Mannich-Lenz procedure or its modifications have been favoured
by a number of workers, but K. A. Clendenning 34 finds it unsuitable for
the determination of starch in gluten and describes an improved method.
The sample is boiled for 15 min with a calcium chloride solution (d 1'30)
of approximately pH 2·5. Frothing is controlled by the addition of one or
two drops of n-octyl alcohol, constant volume being maintained during
boiling by the addition of water. The dissolved proteins are precipitated
with 20 % stannic chloride solution prior to filtration and polarisation.
The procedure is rapid and the starch found is low but a correction can be
made so that the final, corrected figure for starch is within ±0·5 % of the
true value.
Earle and Milner 35 have determined the specific rotation of maize (2),
wheat (2), waxy maize, tapioca and potato starches, using calcium chloride
dispersions in an improved polarimetric method. The specific rotations
were 203'5, 202'6, 202'4, 201·2, 203'8, 203·5 and 203'7, respectively. These
results were in close agreement with those obtained by the diastase-acid
hydrolysis method with a conversion factor of 0·92 instead of 0·9. The
method was also applied to rye, barley and sorghums.
The solution of starch in calcium chloride solution has been used for the
determination of starch in sausage meat by Fraser and Holmes 36 after
precipitation of protein with Carrez's solutions. Other solvents for starch
using polarimetry include dimethyl sulphoxide suggested by Garcia and
Wolf 111 who compared it with calcium chloride.
6.3.1 Methods based on hydrochloric acid solutions

C. J. Lintner37 ,38 triturates 5 g of the sample with 20 ml water, then
mixed with 40 ml concentrated hydrochloric acid, and after standing 30
min transfers to a flask, using hydrochloric acid of s.g. 1·125. 10 ml of a
4 %phosphotungstic acid solution are added and the volume made up to
200 ml with hydrochloric acid (s.g. 1'125). After filtering the solution, the
rotation is determined in a 200 mm tube with sodium light. Lintner found
[(J(]~ = 200·3 for barley starch. The amount of starch in a sample can
be found from the formula:

Percentage = 4000 x observed
]20
rotation
L x [oc D
where L = length of tube in decimetres and [OC]D = specific rotatory

power of barley starch under above conditions.
O. Wenglein39 used sulphuric acid instead of hydrochloric acid and
obtained for barley starch a specific rotary power [OC]D = 191·7.
Lintner 40 ,41 has also used this method, but thinks that the sulphuric acid
used by Wenglein may cause decomposition of the starch and suggests
using a weaker acid of s.g. 1·4.
Although M. Canet and O. Durieux 42 found Lintner's method to be
satisfactory with starch and amylaceous materials they suggest the specific
rotary power [oc]~ = 202 be used in the above formula. The specific
rotary powers of the more important starches have been determined by
J. Konig and co-workers,43 using Lintner's hydrochloric acid method and
Ewers' method (see later). Their results show that the starch probably
undergoes hydrolysis during the precipitation of the solution by Ewers'
procedure, and that noticeable errors would be introduced into the
determination by this method if the average value of [oc]~ was taken, as
the rotations for different starches vary more widely than by Lintner's
method. With the latter method, the value [oc]~ = 202 is sufficiently
accurate for ordinary work. The two methods have also been compared by
S. Hals and S. Heggenhangen. 44
E. Ewers 45 treated the starch with glacial acetic acid, hydrochloric acid
and hot water, and used potassium ferrocyanide to clear the solution,
allowance being made for the rotation due to soluble carbohydrates.
Later 46 - 48 he used dilute hydrochloric acid (1·124 % by weight) with
which the starch was heated, clarified with sodium molybdate or phos-
photungstic acid, filtered and polarised. The details of Ewers' method are
as follows: 5 g of finely powdered material are washed into a 100 ml
graduated flask with 25 ml of 1·124 %hydrochloric acid, a further quantity
of the same acid being used to wash down the neck of the flask after it has
been thoroughly shaken with the first acid addition. After several more
shakings the flask is heated in a boiling-water bath for exactly 15 min,
during the first three of which it is constantly rotated. After making up,
with cold water, to about 90 ml 2 ml of sodium molybdate solution, made
by fusing 30 g of molybdic acid with 25 g dry sodium carbonate and
dissolving the product in water, making up to 250 ml and filtering,
is added.
Alternatively, 10 ml of a 4 % solution of phosphotungstic acid may be

added, the volume made up to the mark with water, the flask shaken, the
liquid then filtered and polarised in a 200 mm tube. If a saccharimeter
is used, the percentage of starch is obtained by mUltiplying the Ventske
reading by 1·912. The specific rotation for barley starch was found to be
181· 5 by this method. A similar method was employed by him for estimating
starch in potatoes. 4 9
Ewers' method has been very widely used for the determination of
starch and many detailed studies have been published all of which stress
the importance of standardising all the variables. Schaechter et al. 1 07
have pointed to the need to standardise the quantity of sample, the drying
time, the degree of agitation, etc. Dudas has investigated the time factor, 108
the influence of the various clearing agents 109 and the amount of
stirring. 11 0 He concluded that of the available clearing agents Carrez
solution was the best and that the other variables needed careful
standardisation.
V. Jahn,50 estimates the amount of starch in sausages, meat pastes or
mayonnaise by applying Ewers' method to the residue from extraction
with alcoholic potash in the following manner: 20 g of the material are
digested for several hours on the water-bath with 50 ml of 8 % alcoholic
potash, and, after filtering, the residue is washed with 96 % alcohol, water
being added to make a total weight of 25 g. The mixture is treated with
0·5 N hydrochloric acid until neutral to phenolphthalein, after which it is
heated with 25 ml dilute hydrochloric acid (80 ml of 25 % acid diluted to
1 1) at 100°C for 15 min to disperse the starch. After cooling 6 ml of a 4 %
phosphotungstic acid solution are added, the whole diluted to 100 ml
clarified with kieselguhr, and filtered. The filtrate is examined in the
polarimeter and the reading on the sugar scale x 0·475 represents the
percentage of pure starch present. M.1. Knyaginichev and Y. K. Palilova 51
have found that starch from legumes have a lower specific rotation
([ctng = 192·7) than that from wheat starch, and consider that the value
of the specific rotation runs parallel with the degree of evolutionary
development. They found, for example, that the starch from primitive
varieties of scaly grain wheat has a lower specific rotation ([ct]~ = 199·8)
than that from cultivated varieties with naked grain ([ctng = 204·0).
The significance of this in botanical and horticultural investigation will be
appreciated. J. Kavcic 52 has also found that the starch from four different
varieties of Solanum tuberosum (potato) showed different values for optical
rotation as well as for mean diameter of grains, ash, and nine other different
properties of the starches. The values of these properties were consistent
within anyone variety but differed from the corresponding values for
other varieties.
Table 6.1 shows the figures obtained by J. Konig and co-workers 53
using both Ewers' and Lintner's methods on the same starches. Ewers'
method gives considerably lower rotations and vary much more with
different starches. A mean value could not be used for all without serious
TABLE 6.1
[aID Lintner starch by [aID Ewers starch by

Starch
Difference Hydrolysis Difference Hydrolysis
Potato 201'2 204·5 191'8 195·0

Maize 201·2 205·2 182·5 186·2
Rice 200·8 203·2 185-8 188·0
Wheat 200·3 20H 182'7 185-9
Rye 200·4 205·8 182-9
Barley 198·2 205·5 180·0 186·7
Oat 193·2 201·8 180·0
Millet 183·7 201·2 165·5
Bean 204·6 208·3 169·4 172-4
Lentil 195-8 204·8 181·3 185-4
Pea 198·0 201·0 185·0 18708
Buckwheat 19% 201·6 170·0 171·7
Arrowroot 198'6 201·9 182·9 185·0
Maranta 204·7 212·7 177-4 184'3
Palm sago 202-6 20% 180·3 186'5
Cassava 203·9 210·9 181·5 187·7
Pepper 201·9 20708 179·2 184·5
Banana 209·8 196·4
error in some cases. The percentages of starch were obtained both by

difference, i.e. 100 minus the sum of the percentages of other constituents
and by hydrolysis to dextrose and estimation of the sugar which is con-
sidered the more accurate method of the two. 54
When examining substances containing optically active constituents in
addition to the starch, resort can be made to thorough washing with cold
water, alcohol and ether, to remove the interfering substances.
Thus it will be seen that there are widely different values obtained for the
specific rotation of starch and in the literature values ranging between
[a]D = + 180° and 220° may be found. This may arise partly from the
difficulty of obtaining clear and undegraded solutions of starch, for most
values reported for high molecular weight dextrins which are readily
soluble lie in the much shorter range of [a]D = 190 and 200°. Meyer 55
0
dissolved 'amylose' in alkali, neutralised and measured the optical rotation

immediately and obtained a value of [a]o = +220° ± 5°. This value
is much higher than the generally accepted values for whole starch.
Aqueous solutions of amylopectin are highly opalescent and somewhat
opaque and give a somewhat uncertain value of [a]o = +200°. Kerr
reports 28 that freshly prepared amylose, butanol precipitated, gives clear
solutions giving [a]o = 200° with an uncertainty of about 2°. With this
material there is usually a small amount which is undissolved even after
several minutes boiling and furthermore it contains butanol and water of
hydration so that solutions of known amylose strength are not easy to
prepare. The amount of amylose in solution, however, can be determined
by evaporating an aliquot portion of the liquor, used in the determination
to dryness.
Freudenberg's equation 5 6
[M]n ~ [Mh + (n - 2)[M]oojoo
(where n is the number of glucose residues in the amylose chain [M]n and
[M]2 the molecular rotations of an n-membered amylose chain and
maltose, respectively, and [M]oojoo is the molecular rotation per glucose
unit of an infinitely long amylose chain) allows one to calculate the value
of the specific rotation of an amylose chain of any given number of
glucose units after calculating that of an infinite chain of glucose residues.
The fact that amylodextrins give clear solutions is of great value here, as,
for example, with an amylodextrin of 22 glucose residues having a specific
rotation of [a]o = + 193°, maltose with [a]o = + 131 ° the respective
molecular rotation per glucose residue is [M]2 = 47200. The molecular
rotation per glucose residue is [M]oojoo = 32200, which corresponds to a
specific rotation for the infinitely long amylose chain of + 199°, agreeing
quite well with the figure obtained with butanol-precipitated amylose.
6.3.2 Methods employing calcium chloride solution

Etheridge 57 has found that the starch content that is reported by
different workers varied widely when the Mannich-Lenz-Hopkins pro-
cedure is applied to equal weights of the same sample of starch; for a
single sample of maize starch the various workers reported starch contents
ranging from 85·74 to 90·88 % while with wheat starch the variation was
from 85·75 to 89·45%. It has been reported 58 that starch concentration
has an important effect on the specific rotation value, causing it to decrease
from +207° at 0·9% to +199·4° at 5·0% starch under the conditions of
the Lintner method. Mannich and Lenz 3 presented data upon the effect of
various factors upon the optical rotatory powcr but their work was
confined to wheat starch and they did not indicate how they dctermined
the 'true starch content'. The multiplicity of optical rotatory values for
starch and of the methods and conditions for obtaining these values con-
stituted a most unsatisfactory state of affairs. Fortunately K. A.
Clendenning and D. E. Wright 59 have now investigated the effect of
solvent, pH, salt concentration, extraction temperature and time, filtration
technique, starch concentration and polarisation temperature upon the
optical rotatory power of wheat starch dissolved in aqueous calcium
chloride solutions. Clendenning 60 then extended this work to other
starches. He found that the addition of small amounts of 0·8 %acetic acid
to concentrated calcium chloride solutions as practised in the Mannich-
Lenz method causes a remarkable increase in the pH value. With 15 min
boiling at pH 2·1 to 3·0 little effect was exercised on the specific rotatory
power; above pH 4·0 the starch solutions are cloudy, difficult to filter and
gel on standing; at pH values below 2·0 the specific rotatory power is
depressed. The specific rotatory power is depressed by rising extraction
temperature to an extent varying with time and pH value but is unaffected
up to 1 h boiling period between pH values of 2·2-2·5. It is increased
quite remarkably, however, by rising salt concentration. Substituting
magnesium chloride for calcium chloride increased the specific rotation
value for wheat starch approximately 7°. The concentration of starch
appears to have a negligible effect but a rising polarisation temperature
causes a decrease in the value over the temperature range of 20°C to 35°C.
Clendenning considers a satisfactory solution of calcium chloride to be
one having a density of 1·30 adjusted to pH 2·2-2·5 by the addition of 2 cc
of 0·8 % acetic acid to 60 cc of the essentially unadjusted salt solution
(pH 5·5), or by acidifying the salt solutions en masse with glacial acetic
acid as suggested by Earle and Milner. 3 5 At this acidity level the specific
rotatory value is relatively insensitive to variations in the time of heating
provided alterations in salt concentration are avoided. A suitable boiling
time appears to be about 15 min. After heating the solution is cooled and
the volume adjusted with distilled water. With starches of low fat content
no cloudiness is occasioned by this step. The specific rotation is depressed,
however, because of the accompanying decrease in salt concentration. 61
Clendenning prefers to use calcium chloride solution of the same tempera-
ture. Filtration is generally required and the first fractions of filtrate
should be discarded as otherwise the sorptive properties of the filter paper
for water give high values.
The filtrate is transferred to water-jacketed 2 dm polarimeter tubes, the

temperature being maintained constant since a relationship exists between
the specific rotation value and polarisation temperature, the relationship
being expressed tentatively by the expression [oc]i, = [oc]~ - 0·12(t - 20).
A similar relationship exists in the case of sugars. 62,114 Rotating the tube
while viewing the field 115 reveals cover glasses rendered optically active,
by pressure from the screw-caps, or eccentricity of the tube. Handling the
tubes by the caps only is recommended by Browne and Zerban 62 in order
to avoid heating of the tubes by the hands thus leading to striations,
which may also arise if the tube is insufficiently rinsed out with the solution
before the final filling.
Clendenning and Wright59 have applied the above method to various
samples of starch from the same species or genus, and from different
varieties grown in different locations.
The specific rotation values for 48 samples of starch representing 20
different genera, or species, showed little divergence from 203°. Hard,
soft and durum wheats gave starches having values lying between 202·3°
to 203'2°. Average values for various starches were wheat, 202'7°;
maize, 202·9°; waxy maize, -202'3°; barley, 203'5°; waxy barley, 202'5°;
rye, 202.8°; oat, 202.9°; rice 203.0°; waxy rice, 202.7°; grain sorghum,
203.2°; waxy sorghum, 202.3°; buck-wheat, 203'4°; millet (impure), 201.4°;
sweet potato, 203.4°; arrowroot, 203'3°; tapioca, 202'8°; potato, 204.1 ° ;
lily (bulb), 203'9°; pea, 199'4°; bean, 200'2°; and lima bean, 200'5°. If
starch is stored for periods of time longer than ten years a decrease takes
place in the specific rotation value. Thus the root, bulb and tuber starches
show specific rotation values that, on the whole, corresponded closely
with those of the cereal starches, potato starches having the highest values.
The legume starches were characterised by low values averaging 200°.
There were probably some impurities present, but in view of the care
exercised by these workers in preparing these starches it appears advisable
from the present evidence to employ this lower value in analytical
applications of the calcium chloride polarimetric procedure to legume
products.
Thus a specific rotation value of + 203° is suitable for calculations of
starch content in most applications of the calcium chloride polarimetric
procedure, although potato and legume starches have higher and lower
values ( + 204'2, + 200°), respectively, which should be employed. It now
appears that the specific rotation value of starch from the same genus does
not vary widely with the conditions under which the starch is formed in
the plant. Very old starch samples are unsuitable for standardisation
purpose or for fundamental studies in this field since Clendenning and

Wright found wheat starch 10 years old to give a specific rotation of
+ 199·8° and one 18 years old to give a value of 197'6°.
The Corn Industries Research Foundation 63 solubilises the starch by
means of boiling aqueous calcium chloride after removal of interfering
substances by extraction with aqueous alcohol and precipitation of
protein by uranyl acetate. Mercuric chloride is added to the alcohol
solvent to inhibit enzyme action.
Reagents
Alcohol solvent-O'1 % mercuric chloride in 900 ml water plus 100 ml
of95% EtOH.
Calcium chloride solution-550 g of CaCl 2 • 2H 2 0 dissolved in 760 ml
water and adjusted to s.g. 1·30 and pH 2·0 (± 0·1) the pH being adjusted
by the addition of glacial acetic acid.
Uranyl acetate solution-1O g of uranyl acetate dihydrate dissolved in
80 ml of water and 20 ml of glacial acetic acid.
Heat to not over 60°C and add 100 mg of calcium chloride solution
(supra).
Method
Transfer about 2 g of sample, accurately weighed to a test tube and
extract by shaking vigorously for 2 min with 10 ml of alcohol solvent.
Filter through 9 cm hardened paper with suction and wash with about
25 ml of alcohol solvent.
Transfer filter paper and contents to 250 ml beaker. Macerate with 10 ml
water. Add 60 ml of calcium chloride solution and bring to boil in about
5 min whilst stirring. Boil vigorously for 30 min with occasional stirring
and maintain volume by addition of water. Cool to room temperature.
Add 10 ml of uranyl acetate solution and dilute to 100 ml in volumetric
flask by the addition of calcium chloride solution. Mix, allow to stand
for about 5 min. Filter through 18·5 cm folded filter paper rejecting first
portion of filtrate. Determine optical rotation of the filtrate in a 2-dcm
tube. A blank should be done on the reagents and observed rotation
corrected accordingly.
0/ h Degrees angular rotation x 100 x 100

/0 Starc = --------------
o 2 d cm x 203 x weight taken (g)
6.4 HYDROLYTIC METHODS
6.4.1 Acid hydrolysis

The amount of glucose obtained by acid hydrolysis of starch multiplied
by a factor of 0·90 should indicate the amount of starch present but
W. A. Noyes and co-workers 64 consider that a complete recovery cannot
be obtained unless the factor 0·93 is used. This is a completely empirical
figure, but it has been adopted by a number of workers, as losses certainly
do occur which may be due to the presence in the hydrolysate of di-
saccharides having a lower reducing power than glucose. Once it has been
formed, the glucose undergoes no change on heating with acid, unless the
concentration of the latter is abnormally high or the duration of the
heating is excessive. 49 Acid-hydrolysis methods are limited to starch
determinations in materials which are free from other cellulosic materials
which may also yield glucose on treatment with boiling acid. Cellulose is
less readily attacked by dilute acid than starch at about 60-80°C for a
short time, and G. S. Fraps 65 uses 0·02 N acid to separate the starch from
the insoluble matter, completing the hydrolysis with stronger acid in the
usual way and correcting the results for the pentosans present.
Ling 66 considers that none of the polarimetric methods in which acid
is used as a converting agent gives reliable results, at any rate, for starch
in cereals, because certain other substances pass into solution.
A typical method employing acid hydrolysis is used for the determina-
tion of starch in flour: 6 7
Reagents
Hydrochloric acid s.g. 1·125
Sodium hydroxide 5 N
Method
Mix about 2·5 g of flour with 50 ml of cold water to a smooth suspension
and allow to stand for 1 h. Having filtered to remove soluble material add
to the residue 20 ml of hydrochloric acid and 200 ml water and reflux for
2! h. Cool, make nearly neutral with 5 N sodium hydroxide and make up
to 250 m!. Determine sugar (as dextrose) by Lane and Eynon method.
Dextrose x 0·90 = starch.
6.4.2 Enzymic hydrolysis

As the products of enzyme action on starch consist of a mixture of
sugars and dextrins, numerous methods have been elaborated that embrace
both enzymatic and chemical or physical treatments. Enzymatic methods

are most useful where other carbohydrate material, capable of hydrolysis
to glucose with acid, is present besides the starch.
Several methods of an empirical kind which yield products giving
definite values for the reducing power or the rotation of polarised light
must be carried out under strictly controlled conditions. In other methods
the amount of each end-product present in the mixture is found by different
ways, and the amount of each product present calculated by the use of
simultaneous equations. A third group of methods comprises those in
which a preliminary enzymic reaction is used to separate the starch from
other bodies present; the separated degradation products are hydrolysed
to glucose, which is estimated and the amount of starch deduced.
Barley and malt diastase methods

So many diastatic methods have been proposed and employed that only
a few can be mentioned here.
Both IX- and fJ-amylase are present in malt, the former causing lique-
faction of the starch and the latter, the so-called saccharogenic enzyme,
coverting the amylose to maltose. E. Waldeschmidt-Leitz, M. Reichel and
A. Parr 68 have shown that, contrary to previous belief, both enzymes are
present in varying amounts in ungerminated barley, and G. Nordh and
E. Ohlsson 69 have found that both enzymes possess saccharogenic and
dextrinogenic activity. Such observations throw doubt on the accuracy of
the results obtained by the method of Ling, Nanji and Harper,70 in which
the precipitated, undried diastase from ungerminated barley 71 is used
on the assumption that only the amylose is attacked, and that the ratio of
amylose to amylopectin is 2:1. As previously stated the accuracy of this
ratio is disputed by several workers,72 - 74 although H. Luers and
F. Weininger 75 found the method gives concordant results.
Simultaneously with the barley or wheat conversion, Ling and his
co-workers make a blank estimation, using high-grade potato starch, and
determine the amount of maltose present, either iodometrically or by
Fehling's method, expressing the result as a percentage of the starch. The
percentage of starch in the barley or wheat is expressed as 100ml M, in
which m is the maltose obtained from 100 parts of dry cereal and M is the
maltose present in the potato-starch experiment expressed in 100 parts of
the dry starch. The accuracy of the method depends on the amylose-
maltose figure for potato starch and the extent to which this is applicable
to other starches and cereal products. If this relationship holds the method
is reliable and precise.
In a number of methods germinated barley is used, and maltose,

together with a small amount of dextrins of varying constitution is pro-
duced. Ling 7 6 has used malts with a diastatic activity of 20-100, as
measured by his scale, under strictly controlled conditions, and has
obtained maltose corresponding to 80-87'5 % of the weight of starch. In
determining the amount of starch in cereal products the amount of maltose
produced is compared with that theoretically obtainable by the same malt.
Ling and Price 7 7 propose to avoid limiting the method to the use of malt
of diastatic power of 80 Lintner by means of a simple formula.
H. T. Brown 78 • 79 extracts the malt or barley for 9 or 3 h, respectively,
with alcohol to remove sugars and certain nitrogenous compounds, boils
the sample under examination with water, and digests it at 57°C with an
active malt extract. After 1 h the liquid is boiled, cooled and filtered, and
the maltose-content is calculated on the assumption that 84-4 parts of
maltose correspond to 100 parts of starch. This assumption is justified
only if the malt from which the active extract is prepared has a diastatic
power of 80 Lintner, but malts with a lower diastatic activity give less, and
highly active malts give more maltose than the above figure. Considerable
error has been found to occur when starch in sweet potatoes is estimated
with the malt diastase method using the usual procedure. Pre-treatment
with calcium or barium hydroxide solution prevents, to a very great extent,
the action of malt diastase on certain non-starchy constituents usually
determined as starch, and for the most accurate determination of starch
in sweet potatoes such pre-treatment has been found to be essential. 80
C. F. Pce and B. J. Jukkola 81 have examined the effect of seven
commonly used preservatives on the diastase method for determining
starch. In most cases an increasing amount of preservative resulted in a
lower starch recovery this effect being chemical rather than due to change
in pH. The amount of the decrease in the percentage of starch recovered
is not significant inasmuch as the amounts of the preservative used were
far in excess of those ordinarily found in food products which are generally
examined for starch. Potassium nitrate, boric acid and borax have little
effect, sodium bisulphite or salicylate were detrimental in amounts over
certain limits, whilst sodium benzoate had the largest effect.
Takadiastase
Takadiastase preparations, which contain many different enzymes, 82
were introduced as a quantitative reagent for determining starch by
W. A. Davis and A. J. Daish 83 in 1914. 1. D. Collins 84 pointed out that,
at proper pH value and correct time, a high concentration of the enzyme
gives complete hydrolysis to glucose, basing her conclusions on the fact

that takadiastase, and also acid hydrolysis, gave a recovery of 93 % of the
dry weight of starch; if the factor 0·93 is used, she suggests that a recovery
of nearly 100% is obtained. Denny 85 and o. Lehman 86 report complete
recovery of starch by this method. The use of takadiastase is widespread
among workers on plant products, and thus deserves attention, but it
should be remembered that these preparations also contain enzymes which
act on materials other than starch, so that its use should be examined
critically before trying any new unproven departure from previous
work.
J. H. Van de Kamer 87 has suggested boiling the disintegrated, starch-
bearing material with water and treating the extract with pancreatic
amylase, after which the reaction mixture is centrifuged to remove
pentosans and hemicelluloses and the sugars determined. Too many
unknown and variable factors appear to enter into consideration in this
method to warrant its use before it has received critical attention.
Amyloglucosidase
Amyloglucosidase is an enzyme which is produced by fungi, particularly
Aspergillus, Rhizopus and Endomyces. Commercially, Aspergillus niger is
used for its production. It has the advantage over the other amylases that
it splits starch into glucose by splitting glucose units from the non-reducing
ends of starch chains. Pazur et al. 8 8 have shown that it acts preferentially
on longer chains. The enzyme also hydrolyses glucosides, although the
rate of reaction is often slow.
Amyloglucosidase has been employed for the determination of starch
by Salo and Salmi 8 9 as follows:
1 g of sample is mixed with about 10 g of sand and extracted with
80 % ethanol in a soxhlet apparatus for about 5 h. The residue is then
dried at room temperature and transferred to a conical flask containing a
few ml of water. After the addition of 25 ml of water the mixture is boiled
for 5 min. Any starch adhering to the sides of the flask is then rinsed down
with 25 ml of hot water and the boiling continued for a further 5 min.
The mixture is then cooled, 50 mg of amyloglucosidase, and 25 ml of
acetic acid/sodium acetate buffer pH 4·8 containing 0·01 % sodium
merthiolate added and the flask almost filled with water. After incubation
at 40°C for 20-22 h, shaking hourly for the first 3-4 h [alternatively the
mixture may be incubated at 60°C for 4-5 h shaking every 30 min],
the contents of the flask are filtered through a filter paper, washed and the
volume made up to 500 ml. The concentration of sugar in the filtrate may
be determined by one of the standard methods such as the Somogyi

method. 90
For materials with a low starch content the shorter incubation at the
higher temperature is said to give the more accurate result and a method
of removing interfering substances such as cysteine by shaking the solution
with ion exchange resins is described in the original paper but the
corrections are marginal.
The availability of glucoamylase in pure form has led to specific methods
for starch determination. P. Thivend et al.,t°l hydrolyse starch with
glucoamylase to give D-glucose which is then treated with glucose oxidase.
The method does not distinguish between starch and glycogen but with
this exception, it is specific for starch and is recommended for the deter-
mination of starch in complex media.
The same principle has been adopted by Dekker and Richards 102 for
the determination of starch in plant material.
6.4.3 Combined enzyme and acid hydrolysis

Methods embracing the acid hydrolysis of the products obtained by
enzyme action have found wide favour in America. Maerker 91 extracts
3 g of the finely ground material with ether, after which it is boiled with
100 mI of water, cooled to 65°C and treated for about 2 h with 10 ml of a
10 %infusion of malt. It is then heated on a boiling water-bath for 30 min.
On cooling to 65°C another 10 ml of malt infusion are added, and after
half an hour the contents of the flask are boiled, cooled, made up to
250 ml and filtered. Two hundred ml of the filtrate are heated on a boiling
water-bath for 2-!- h with 15 ml of 25 % hydrochloric acid. After cooling
and neutralising, the dextrose present is estimated by one of the standard
methods, the conversion factor 0·90 being used to calculate the weight of
starch present in the sample. A correction is made for the reducing power
of the malt extract, which is determined separately.
R. P. Walton and M. R. Coe 92 have worked out a method in which the
insoluble non-starch material present in the products of diastatic hydrolysis
are removed by filtration, the pectin being precipitated by 60 % alcohol,
which does not precipitate the dextrins present. The precautions to be
taken in this work are contained in the preliminary papers by these
workers. 93 - 95 Hartmann and HiIIig 96 suggest that starch products
containing much protein matter should be digested overnight with pepsin,
a process which would destroy the proteins that occlude starch and at
times render results unreliable. P. Fleury and G. Boyeldieu 97 determine
starch in bread prepared for diabetic patients, by hydrolysing with dilute
sulphuric acid and precipitating the proteins by the addition of an acid

solution of mercuric sulphate which they claim to be better for this
purpose than lead acetate. The dextrose remaining is then determined by
polarimetric or reduction methods. A. Hock 98 uses diastase followed by
acid treatment, but his chief modification is connected with the adequate
removal of fats, protein and water-soluble substances that may interfere.
Trop and Grossman 1 03 have published a method for the determination
of starch in foods which they claim to be simple and rapid. Acid hydrolysis
converts the starch to glucose which is treated with glucose oxidase. The
amount of oxygen required for the oxidation is measured polarographic-
ally. When starch hydrolysates contain dextrins the determination of
maltose by chemical methods may give high results. A. S. Schultz, R. A.
Fisher and co-workers 99 have determined the maltose in such products
by fermenting the sugar with yeast. During acid hydrolysis of starch the
highest maltose content they found was 25·2 %by the fermentation method
whereas chemical methods indicated 43-47 % which, they consider,
indicates a lack of specificity in the latter methods.
Herd and Kent-Jones,8 surveying the field of enzymes for use in the
determination of starch, point out that the difficulties arising from the
various methods proposed for the determination of starch in natural
materials may be summarised as follows:
1. Acid hydrolysis: presence of other hydrolysable carbohydrates.
2. Hydrolysis by prepared diastase: variable hydrolytic powers and the
unknown action on various components of the starch.
3. Hydrolysis by malt diastase: variable hydrolytic action on hemi-
celluloses.
4. Hydrolysis by barley diastase: as 3; and in addition, it is also
necessary to assume that the ratio amylose/maltose for other starches
is the same as for potato starch.
A survey of methods for the determination of starch has been carried
out by M. P. Etheridge. 10o
The investigation included the official American methods of hydrolysis
with hydrochloric acid and with diastase and acid, and the Hopkins
modification,4 of the Mannich-Lenz calcium chloride method. 3 Pure corn,
wheat and potato starches were used, their moisture, ash and protein-
contents being determined and a rough estimate of the starch obtained by
difference. For the determination of the moisture, the results obtained by
drying for 24 h in a Freas oven at 105-106°C compared favourably with
those given by drying in vacuo for a shorter period. Natural materials,
such as cake flour, whole wheat flour and whole Lima beans, were also
used. When the calcium chloride method was applied to these, the samples
were washed on filter paper, instead of being centrifuged with alcohol, as
recommended by Hopkins. Contrary to statements in the literature, this
worker found that all the starch in the reputed pure and commercial
starches was not obtained either by hydrolysis with hydrochloric acid or
by diastase treatment followed by acid hydrolysis. The results were even
lower than the 96 to 97 % of the total starch found by Noyes et al. 64 On
the other hand, with natural materials the diastase and hydrochloric acid
method (a) gave fairly concordant results comparing better with those
obtained by the calcium chloride method (b). Thus the following mean
percentages were found: cake flour, (a) 72·87, (b) 75·90; whole wheat
flour, (a) 66·95, (b) 67·29; rice bran, (a) 6·84, (b) 8·22; corn meal, (a) 62·44,
(b) 63·76; whole rice, (a) 71·75, (b) 77·31; Lima beans (a) 45·56, (b) 42·54.
In most instances the calcium chloride method was very satisfactory with
the pure starches. The Hopkins modification seemed to be the most
promising single method and, by careful control of heating and the use of
accelerated filtration, can be advantageously applied to natural materials.
Other chemicals were tried as dispersing agents in an effort to prevent
filtration difficulties. The use of calcium nitrate and sodium salicylate
showed possibilities, although the dispersions were still difficult to filter.
On the other hand, sulphosalicylic and formic acids gave promising
results, and the dispersions could be readily filtered.
Other comparative studies of the various methods of starch determina-
tion have been published by Saunders, Potter et al. 104 who compared
chemical, polarimetric and enzymatic methods for the determination of
starch in wheat milling fractions. They considered that enzymatic methods
were best for those products which had a high fibre content.
Mauser and Thomas 1 05 have worked on the standardisation of methods
for starch determination and consider that enzymatic methods are the
most accurate although extraction of the starch with alcohol posed some
problems. Banks, Greenwood and Muir 106 used a semi-micro method
which needed only 7·5-20 mg of starch. The starch was solubilised in hot
aqueous CaCl z and hydrolysed by a-amylase and glucose oxidase.
The following method using diastase is used for the determination of
starch in cocoa but could be adapted for many foods:
To 5 g of defatted material add 100 ml of 12 %alcohol, shake, filter and
wash with 20 ml LM.S. Heat the extracted material with 50 ml of distilled
water in a boiling water-bath for 20 min. Cover, add 0·1 g of diastase and
incubate at 50 -55°C for 2 h. Cool, make up to 250 ml and filter. Mix
0
200 ml of the filtrate with 20 ml hydrochloric acid (e.g. 1·125) and heat in
boiling water for 3 h. Cool, neutralise with NaOH and make up to 250 ml.
Determine reducing sugars in solution by Fehlings or other suitable
method. Dextrose x 0·90 = starch.
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88. Pazur, J. H. and Ando, T., J. Bioi. Chem., 1959,234, 1966-1970; ibid., 1960,235,
297-302.
89. Salo and Salmi, J. Sci. Agric. Soc., Finland, 1962,40 (i), 38-45.
90. Somogyi, M., J. Bioi. Chem., 1945, 160, 61-68.
91. Maerker, M., Chem. Ztg., 1885,9, 319.
92. Walton, R. P. and Coe, M. R., J. Agric. Res., 1923, 23, 995.
93. Coe, R. M., J.A.O.A.C., 1923-24,7, 341.
94. Coe, R. M., ibid., 1924-25,8, 358.
95. Walton, R. P. and Coe, R. M., ibid., 1923-24,7, 995.
96. Hartmann and Hillig, ibid., 1926,9,482.
97. Fleury, P. P. and Boye1dieu, G., Ann. Falsi/., 1928,21, 124.
98. Hock, A., Biochem. Zeit., 1937, 294, 336.
99. Schultz, A. S., Fisher, R. A. et al., Ind. Eng. Chem., Anal. Ed., 1943, 15, 496.
100. Etheridge, M. P., J.A.O.A.C., 1941,24, 113.
101. Thivend, P., Mercier, C. and Guilbot, A., Methods in Carbohydrate Chemistry,
1972,6, 100-105.
102. Dekker, R. F. H. and Richards, C. N., J. Sci. Fd and Agric., 1971,22,441.
103. Trop, M. and Grossman, S., J.A.O.A.C., 1972,55, 1191.
104. Saunders, R. M., Potter, A. L. et al., Cereal Chem., 1970,47 (2), 140-146.
105. Meuser, F. and Thomas, B., Ber. Getreide Chim-Tag., Detmold, 1972, 111-20.
(Ger.)
106. Banks, W., Greenwood, C. T. and Muir, D. D., Die Starke, 1970,23 (4), 105-108.
107. Schaechter, D., Copony, W. and Staniscu, R., An. Inst. Cercet Cult., Cartojului
Sfec1ei Zahar Bousov. Cartoful, 1969, 219-24.
108. Dudas, F., Acta Univ. Agr. Brno. Fac. Agron., 1971, 19 (4), 719-24 (Czech.).
109. Dudas, F., Die Starke, 1972,24 (11),367-369; 1973,25 (8), 263.
110. Dudas, F., ibid., 1971,23 (11), 390-393.
Ill. Garcia, Q. J. and Wolf, M. J., Cereal Chem., 1972,49,298-306.
112. Anon., Tobacco Science, 1970, 14, 164-166.
113. Williams, c., Kuzina, F. D. and Heynka, I., Cereal Chem., 1970,47 (4),411-442.
114. Bates, F. J. et al., Circular C 440, National Bureau of Standards, Washington,
1942.
115. Bronstead, J. N., Chem. Revs., 1928,5,231.
CHAPTER 7
The Analysis of Starch Derivatives

J. VAN DER Bu
Scholten-Honig NV, Foxhol (Gr), The Netherlands
INTRODUCTION
There exists in the literature some difference of opinion as to the definitions

of modified starch and starch derivatives. The I.s.o. (International
Organisation for Standardisation) Technical Committee 93 distinguishes
only between native starch and modified starch, the latter being defined as
native starch treated in such a way as to modify one or more of its original
physical or chemical properties. The term 'modified starch' thus includes:
pregelatinised starch, oxidised starch, thin boiling starch, dextrin,
dialdehyde starch, starch ester, starch ether, etc.
The term 'starch derivative' is not included in the I.s.o. terminology but
in this chapter we will consider as a starch derivative a modified starch in
which the hydroxyl groups of the starch have been totally or partially
substituted in a chemical reaction. The main products derived from starch
by substitution of hydroxy I groups are:
1. Starch esters
2. Starch ethers
In the case of an unknown starch product, the following analytical
questions arise:
(a) Is it a native starch or a modified starch and if the latter, is it a starch
derivative?
As we are dealing with derivatives only, the next questions are:
(b) Which groups have been introduced?
(c) What is the degree of substitution, i.e. how many groups have been
introduced?
189

Answering these questions is the main task for the analytical laboratory
of a factory that manufactures starch derivatives. Only in exceptional
cases a fourth question is posed:
(d) What is the substituent group distribution in the derivative?
In this chapter methods of analysis for answering these questions for
starch derivatives only will briefly be given.
7.1 STARCH ESTERS
The esterification of starch can be carried out with inorganic and organic
acids and their salts, anhydrides or acid chlorides, or with specific reagents
as for instance vinyl esters. The principal esters of commerce are: acetates
and phosphates; less known are the sulphates, maleates, succinates,
xanthates, etc.
7.1.1 Qualitative tests for starch esters

A general qualitative test for ester groups is the infrared spectrum from
which important conclusions can often be drawn. Aliphatic and aralkyl
esters are characterised by a specific absorption band at 1720 cm -1,
starch phosphates at 1240-1200 cm -1 and starch xanthates at 1320 and
970 cm -1. Starch sulphates have no specific band in the infrared spectrum.
Although it is usually possible to infer from the infrared spectrum which
type of ester group is present, it is often difficult to prove exactly what
group it is, especially with aliphatic esters.
Usually one tries to trans-esterify the ester with methanol and identify
the resulting methylesters by gaschromatography.1 Another method is to
saponify the starch ester and identify the isolated fatty acids as hydroxamic
acids by paper chromatography. 2 The presence of phosphates and
xanthates is normally derived from the infrared spectrum and checked
with a qualitative test for phosphorus or sulphur.
7.1.2 Quantitative determination of starch esters

If one knows which group is present, the next question normally is
what is the degree of substitution (DS), i.e. how many ester groups have
been introduced per anhydro glucose unit (AGU).
With starch esters prepared from aliphatic or aralkyl acids the acid
content is usually calculated from the saponification value. Due to the
THE ANALYSIS OF STARCH DERIVATIVES 191
alkali-lability of most of the polysaccharides the saponification has to be

performed under mild conditions, i.e. at low temperature.
A method specially devised for labile compounds is given by Freuden-
berg3 and consists in trans-esterification of the starch ester with an alcohol.
distillation of this ester and determination by saponification (see als0 4, S).
It is also possible to determine the acetyl content of a starch acetate from
its infrared spectrum. 6
With starch phosphates the DS is calculated from the phosphorus
content and with starch xanthates the DS can be determined as for the
cellulose xanthates, i.e. from the sulphur content of the xanthate benzyl
ester prepared according to
S
II
R· O· C-SNa + Br' CH 2 . C 6 Hs -+
S
II
R· 0 . C-S-CH2 . C 6 Hs + NaBr
In some cases the substituent content of aliphatic esters (e.g. cellulose
esters) can be determined by pyrolysis of the esters and gas-chromato-
graphy of the decomposition products. 7
The pyrolysis of esters normally follows the pattern:
o 0
/' /'
R· C-OCH2' CH 2R -+ R· C-OH + CH 2=CH' R
7.2 STARCH ETHERS
Starch ethers are as a rule prepared by reaction of sodium starch with

etherifying agents such as epoxides, chlorohydrins, halogen carbonic
acids, etc. The principal starch ethers at present are the alkyl and hydroxy-
alkyl starches and the carboxymethyl starches, while the interest in
cationic starch ethers is growing. The starch ethers can be divided into
three groups according to their properties:
(a) Non-ionic ethers, obtained by reacting starch with epoxides or
chlorohydrins, alkyl- or aralkylhalogens and sometimes with
unsaturated compounds as acetylene (-+ vinylstarch).
(b) Anionic starch ethers, from starch and monochloro acetic add,
propane sultone, etc.
(c) Cationic starches, from starch with nitrogen compounds as diethyl-
aminoethylchloroethane, epoxypropane amines, ethylene-imine,
etc.
For each of these groups there are special methods of analysis, utilising
existing methods for cellulose ethers.
7.2.1 Qualitative tests for starch ethers

(a) Non-ionic starch ethers
Due to the presence of many hydroxyl and ether groups in starch itself,
the infrared spectrum of an alkyl or hydroxyalkyl ether of starch gives us,
in general, little information and the detection of these ether groups is
therefore normally carried out by chemical means, using the same methods
as in the quantitative determination.
(b) Anionic starch ethers, e.g. reaction products of starch and monochloro
acetic acid, propane-sultone, etc.
Carboxymethyl starch. The carboxyl group is easily detected in the
infrared spectrum, but no differentiation can be made between starch
obtained by modification with an oxidising agent and carboxymethyl
starch obtained by derivatisation of starch with monochloroacetic acid.
A high carboxyl content may be assumed to originate from carboxymethyl
starch. If the product evolves carbon dioxide on heating with hydrochloric
acid (uronic acid) it is an oxidised starch, if not it is carboxymethyl
starch. 8 Another method for the detection and quantitative determination
of carboxylic groups is based on the binding affinity of methylene blue and
carboxylate ion. 9
Carboxymethyl starch can further be demonstrated by the formation of
glycollic acid on heating with 50 %sulphuric acid, which glycollic acid is
determined by its reaction with chromotropic acid. 1 0
The presence of sulphopropylether of starch, normally prepared by the
reaction of starch with propane-sultone:
St· OH + CH 2 • CH 2 • CH 2 --+ St· O' CH 2 • CH 2 • CH 2 • S03Na
I I
o S02
is indicated by the sulphur content of the sample and the fact that this
does not alter on saponification, as is the case with starch sulphates.
(c) Cationic starch ethers

These are the latest types of starch ethers of commercial importance.
Although their presence is easily shown from the nitrogen content of
these compounds, it is often very difficult to determine the type of
substituent.
7.2.2 Quantitative determination of starch ethers

(a) Non-ionic starch ethers
As mentioned under Non-ionic starch ethers the determination of ether
groups is normally carried out by chemical means. There are only a few
methods known to split ether groups, of which the main are:
(1) The splitting of alkyl or hydroxyalkyl groups with hydriodic acid.
The products obtained are alkyl iodides and, with hydroxyalkyl ethers,
sometimes olefines.
The hydriodic acid method is generally used and only the methods for
determining the end products differ. Zeisel,l1 in 1885, elaborated this
method for the determination of methoxyl groups in phenol ethers. The
principle is that phenol ethers are split in such a way that the halogen is
bound to the alkyl residue:
C6H5 . OCH 3 + HI ~ C 6H 50H + CH3I
Per mole phenol ether one mole of alkyliodide is formed and the quantita-
tive determination thereof can, therefore, serve for the quantitative
determination of the phenol ether. The CH3I formed is collected in an
alcoholic silver nitrate solution, forming a complex salt AgN0 3 . AgI,
from which, on acidification, AgI is liberated and determined gravi-
metrically. The original method has been extended to mixtures containing
OCH 3 and OC 2H 5-groups. The alkyl iodides are led into an alcoholic
(CH 3hN solution. The (CH3)4NI formed is practically insoluble in
absolute alcohol and can be determined gravimetrically. The filtrate is
diluted with water, nitric acid and silver nitrate added and the silver iodide
thus formed weighed.
The Zeisel method gives good results for the determination of methoxyl
groups but takes a lot of time, as is the case with most gravimetric methods.
Viebock and co-workers 12 ,13 have simplified it by introduction of a
volumetric method. These authors demonstrated that alkyl iodides react
with bromine and acetic acid in the following way:
RI + Br 2 ~ RBr + IBr
IBr + 2Br2 + 3H 20 ~ HI0 3 + 5HBr
The excess of bromine is decomposed with formic acid and the iodate
determined iodimetrically:
HI0 3 + 5HI ~ 31 2 + 3H 20
31 2 + 6Na 2S20 3 ~ 6NaI + 3Na2S406
Gran 14 later on replaced the bromine in acetic acid solution by a solution
of bromine in a phosphate buffer and obtained good results with this
aqueous solution.
The Zeisel and Viebock method can also be used for the higher alkyl
ethers but is not suitable for the determination of the hydroxyalkylethers,
which are usually manufactured from cellulose or starch with ethylene
oxide or propylene oxide according to the reaction:
""/
ROH + CH2-CH-R1 ~ R· OCH 2 . CHOH· R1
o
R1 = H or CH 3
Morgan 15 has investigated the reaction of HI with compounds of this
type with half ethers of ethylene glycol R· OCH 2 . CH 20H and found
that the sole end products are ethyl iodide and ethylene.
As to the course of the reaction Morgan considers ethylene iodide,
ICH 2CH 2I, is an intermediate product, preceded perhaps by the formation
of ethylene iodohydrin (ICH 2CH 20H). As soon as the ethylene iodide
has been formed, various new reactions may take place
ICH 2 . CH 2I ~ CH 2=CH 2 + 12
+ HI ~ CH 3 . CH 2I + 12
I· CH 2CH 2 . I
CH 2=CH 2 + HI ~ CH 3 . CH 2I
In any case the two end products are always ethyl iodide and ethylene and
the sum of these products is equivalent to the number of glycol ether
groups originally present in the ether. The final formulation is:
ROCH 2 . CH 20H + (3 + x)HJ ~
RJ + (X)CH3 . CH 2J + (1 - x)CH=CH 2 + J 2 + 2H 20
For the quantitative determination of hydroxylalkyl groups the two end
products of fission, alkyl iodide and olefine, have to be determined. The
determination cannot be performed by the Viebock method in this case,
but is carried out as follows:
After purification the gas mixture from the reaction with HI is bubbled
through a standardised alcoholic silver nitrate solution, in which after
acidification with HN0 3 the reaction:
RI + AgN0 3 ~ AgI + RON0 2
takes place. The excess of AgN0 3 is back titrated with ammonium
thiocyanate. The olefine which is not absorbed is thereafter collected in a
bromine solution in acetic acid and reacts with the bromine according to:
CH 2=CH 2 + Br2 ~ BrCH 2 · CH 2Br
The excess of bromine is determined iodimetrically after addition of
potassium iodide:
Br2 + 2KI ~ 2KBr + 12
12 + 2Na 2S 20 3 ~ 2NaI + Na2S406
Many variations have been applied to the Vieb6ck and the Morgan
methods with respect to apparatus, scrubber solution and absorbing
solutions.
The foregoing methods do not identify the particular olefine present
and hence the alkyl group. To solve this problem two general methods can
be applied:
1. Gas chromatography of the alkyl iodides either direct or after
absorption.
Many methods are in use for this. Kratzl 16 collects the alkyl iodides
prepared according to the Zeisel method in a cooled vessel, separates the
iodides by gas-liquid chromatography and determines the separate com-
pounds according to Vieb6ck. A somewhat analogous method is used by
Ehrenberger 17 who also determined the gas fractions (olefins) volumetric-
ally.
Other authors collect the iodides in a cold trap, or in a solvent, and then
determine the iodides by GLC with, or without, an internal standard. 18 ,19
A variant on this method is the extraction of the reaction mixture of the
ether and hydriodic acid with a solvent and GLC of an aliquot of the
extract. In this case the use of an internal standard is strongly recom-
mended. 2o ,21
2. Another method for determination of the nature of the ether groups
has been used by Japanese investigators and is based on combustion ofthe
alkyl iodides obtained by carrying out Zeisel's reaction.
CnH 2nO ~ CnH 2n + 1I ~ nC0 2 + tI2
The quantitatively formed iodine and carbon dioxide are determined.

The iodine is absorbed on silver gauze and the carbon dioxide in an
absorption tube.
The mole ratio I 2:C0 2 depends on the type of alkoxy groups, wherein
n must correlate with the number of carbon atoms in the alkoxy group.
When the chain length is known only the iodine formed has to be
determined. 22
(2) For the analysis of the aralkyl ethers obtained by the reaction of
starch with benzylchloride a different method is usually employed as the
reaction with HI forms mainly toluene and only very small amounts of
iodide. The methods of analysis here are hydrogenolysis or acetolysis of
the ethers. 23
Hydrogenolysis reaction:
C 6Hs . CH 20R + H2 --+ C6HS . CH 3 + HOR
The hydrogenolysis is carried out chemically with alcohol and sodium,
or catalytically, using platinum, palladium or Raney nickel in acetic acid.
Phenyl- and 2-phenylethyl ethers are not split by H 2 and Pd.
The debenzylation proceeds much more readily by acetolysis than by
hydrogenolysis according to
H 2 SO±
. . ) ROCOCH 3 + CH 3 . CO . OCH 2 . C6HS
+ acetIc anhydrIde acetylated benzylacetate
carbohydrate
The products formed in these reactions: toluene and benzyl acetate are
usually determined by gas chromatographic methods.
(3) Other reagents including acylhalides have been suggested to split
ether groups,24 but these reagents have found no use in the analysis of
starch ethers.
(4) Special methods of analysis for specific starch derivatives include:
(i) A spectrophotometric method for the methoxyl group which has
been described by Mathers 2s and consists of the hydrolytic cleavage of
methoxyl to methanol with sulphuric acid. Oxidation of the methanol
with permanganate yields formaldehyde, which is determined colori-
metrically with chromotropic acid. The method is specific for the methoxyl
group.
(ii) The determination of the hydroxy-ethyl group in starch ethers by
pyrolysis-GLC technique is mentioned by Tai. 26 Pyrolysis for 10 min at
400°C of HE starch gives a number of peaks in the chromatogram, of
which the height of the acetaldehyde peak correlates with hydroxyethyl
ether content. The problem with this method lies in the reproducibility of
the pyrolysis.
(iii) A quantitative determination of the ethyl group in O-ethyl and
O-ethyl hydroxyethyl cellulose is mentioned by Jacin 27 and is a modifica-
tion of a method of Lemieux. 2 8 Ethylcellulose is oxidised with CrO 3 and
the acetic acid formed determined with a gas chromatograph. This method
is specific for the ethoxyl group, the hydroxyethyl group is not determined.
(iv) For the spectrophotometric determination of the hydroxypropyl-
ether groups in hydroxypropyl starch ethers Johnson 29 describes a method
whereby the ether group is hydrolysed with sulphuric or phosphoric acid.
The propylene glycol formed is dehydrogenated with these acids to
propionaldehyde and the enolform of allyl alcohol, which products give a
purple colour with ninhydrin. This ninhydrin colour reaction is specific for
propionaldehyde, and does not react with other compounds which may be
formed. Large amounts offormaldehyde seem to inhibit the colour develop-
ment. Otherwise the method is specific for the hydroxypropyl group in this
group of substances.
(v) A special non-ionic starch ether is the cyanoethyl ether formed by
reacting starch with acrylonitrile. This ether is easily recognised in the
infra red spectrum and the degree of substitution is, in this case, calculated
from its nitrogen content.
(vi) The type and content of substituent of certain cellulose ethers can
be determined from the pyrolysis of these products. With ethers the usual
decomposition products are carbonyl compounds and hydrocarbons: 7
R· CH 2 • CH 2 0 . CH 2 CH 2 R ~ RCH 2 . CH=O + R· CH 2 . CH 3
(5) Some special methods of analysis for anionic starch ethers include:
(i) Determination of alkoxyl groups by quantitative vapour-phase infra-
red spectroscopy of alkyl iodides. This method 3 0 seems to give good
results in the determination of methoxyl and ethoxyl groups but
has found no wide application.
(ii) Recently 31 a method of identification has been described for
methyl, hydroxyethyl and hydroxypropyl cellulose derivatives by
thermal degradation of the ethers in a time-of-flight mass
spectrometer.
(b) Analysis of anionic starch ethers, e.g. the reaction products of starch and
monochloro acetic acid and propane sultone
(i) Carboxymethyl starch. The quantitative determination of the degree
of substitution of carboxymethyl starches with low degree of substitution
is usually done by means of potentiometric titration of the carboxylic acid

group. In the case of starches containing phosphate groups a correction
has to be made for this. 21 In highly substituted carboxymethyl starches the
carboxyl content may be determined by conductometric titration. 32 A
further method for the determination of carboxylic groups in oxidised and
carboxymethyl starches is based on determination of the soda content of
the ash of the purified derivative. 33
(ii) Sulphopropylethers of starch. The degree of substitution of these

ethers prepared from starch and propane sultone is calculated from the
sulphur content. 34
(c) Analysis of cationic starch ethers

As mentioned under Cationic starch ethers it is often difficult to determine
here the type of substituent and therefore the degree of substitution.
Sometimes conclusions can be drawn from the potentiometric titration
curves.
Further methods which may give clues to the constitution of cationic
starches include the Ralopont blue test 35 and turbidimetric titration
according to the principle described by Lambert,36 but it is not possible to
give a review at present of the methods of analysis of cationic starch
compounds.
(d) The definition of the degree of substitution (DS) and molar substitution
(MS)
The two names are defined by Bollenback 66 as follows: the degree of
substitution (DS) is a measure of the substitution of an anhydro glucose
unit without regard to the molecular size of the substituent. In fact it gives
the number of hydroxyl groups in an anhydroglucose unit (AGU) that
has been substituted.
The molar substitution (MS) however is a measure of the number of
moles of reagent which have been introduced per anhydroglucose unit
and are determined separately in the analysis. It follows that MS ;;::: DS.
In the preparation of derivatives with monofunctional reagents such as,
alkyl or aralkyl chlorides, monochloro acetic acid, propane sultone, etc.,
only one mole of reagent reacts with a hydroxyl-group, but in the prepara-
tion of hydroxylalkyl ethers, the hydroxyl group of the derivative side
chain acts in competition for the original OR-groups of the AGU and
thus polymerisation in the side chain may take place. In the Zeisel deter-
mination each alkoxy group is determined and thus for each OR group
substituted, more than one alkoxy group may be found. What normally is
described as a DS is in fact mostly a MS because the determination of the
DS in hydroxy alkyl derivatives is often a very tedious problem.
7.3 SUBSTITUENT GROUP DISTRIBUTION IN ESTERS AND

ETHERS OF CELLULOSE AND STARCH (Table 7.1)
After one has determined the type of substituent and the degree of sub-
stitution of starch derivatives, the next step may be the substituent groups
distribution (SGD) in these products. Much work has been done on SGD
of cellulose derivatives but until now very little on starch derivatives and
in both cases, for logical reasons, only on the ethers. The scheme normally
followed for the ethers is to hydrolyse the polysaccharide derivative in
question with sulphuric acid, neutralise with Ba(OHh or BaC0 3 and
separate the different constituents of the hydrolysate by means of selective
extraction, paper, thin layer, column or gas chromatography.
From the amount of isomer the distribution of groups can be calculated.
Because of the hydrolysis step normally required it is clear that this method
is unfit for esters and has only been used for ethers in this form. For esters
indirect methods for studying the distribution of substituents have to be
used which, as a rule, are much less accurate.
The following cellulose derivatives have been investigated for substituent
group distribution.
7.3.1 Cellulose ethers

1.Methylcellulose 37 - 3 9
2.Ethylcellulose 40
3.Hydroxyethylcellulose 41 - 44
4.Ethylhydroxyethylcellulose 45
5.Carboxymethylcellulose 46 - 48
6.Methylsulphonylethylcellulose 49 ,50 (cellulose + methyl vinyl sul-
phone)
7. Carbamoethyl cellulose 51 (cellulose + acrylamide)
8. Cyanoethyl cellulose 52 (cellulose + acrylonitrile)
9. O-(2-aminoethyI) cellulose 53
10. O-(2-diethylaminoethyl) cellulose 54 ,70
11. Allylcellulose 55
TABLE 7.1
Substituent group distribution in cellulose ethers
Ratio of substituents
Reagent Refer-
DS Remarks
ence
2-0 3-0 6-0
Methyl chloride 0'11; 0·16 2·50 0·50 1·00 39

Dimethyl sulphate 0·59 1·75 0·50 1·00 37b
Ethyl chloride 0'73; 1·29 2·25 0·50 1·00 40
Ethyl sulphate 1 0·80 1·00 ex 40
Ethylene oxide 0·60 0·30 0·10 1·00 41 Much sub-
stitution on
the new
OH-group
Sodium chloro acetate 0·44 0·64 0·36 1·00 46
Sodium chloro acetate 0·75-0'98 0·80 0·40 1·00 48
Methyl vinyl sulphone 0·11 0·20 0·03 1·00 49,50
Acrylamide 0·78 0·47 0·05 1·00 51
Acrylonitrile 0·09 52 2-0 ~ 6-0
Sodium 2-aminoethyl
sulphate 0·14 0·64 0·14 1·00 53
2-Diethylamino-
ethylchloride 0·048 1·27 0'35 1·00 54
Sodium aIIyl sulphate 0·07 0'70 0·20 1'00 55
Substituent group analysis in cellulose esters
Ratio of substituents
Refer-
Reagent DS Remarks
ence
2-0 3-0 6-0
Acetic anhydride 56
0·4-0·66 57 Substitution
mainly at C-6
Carbon disulphide+ KOH 58 No definite
conclusion
0'31-0·41 59 Substitution
mainly at C-6
60 Substitution
mainly at C-6
7.3.2 Cellulose esters

1. Cellulose acetate 56
2. Cellulose xanthate 5 7 - 60
Substituent group distribution in starch derivatives. Compared with

cellulose only little work has been done on the SGD of starch derivatives.
The main compounds are:
7.3.3 Starch ethers

1. Methyl starch 61- 63
2. Hydroxyethyl starch 64 - 67
3. Hydroxypropyl starch 68
4. Vinyl starch 69
5. O-(2-diethylaminoethyl) starch 70
7.3.4 Starch esters

1. Amylose acetate 71
2. Starch xanthate 72,73
7.4 SUMMARY
Although some progress has been made in the last two decades, as yet, in
most, cases it is not possible to predict the position of the hydroxyl group
that will mainly be substituted in preparing derivatives of cellulose and
starch. The reason for this is, that there are several factors which influence
the direction of the substitution, and some of these may work in the same
direction or contrary.
Croon found in 1957 that the distribution of substituents in cellulose is
governed by:
(a) The relative rate constant, and

(b) Differences in accessibility of the hydroxyl group.
In later years some rules for the interaction of specific groups in cellulose
were found, of which the main are:
1. Methylation at the 2- or 6-hydroxyl group, or both enhances the

reactivity of the 3-hydroxyl group. 61
2. Steric factors may exert a considerable influence on the distribution

of substituents, with heavier substituents there is a shift to the
6-hydroxyl group. 61 So for instance, there is a progressive decrease in
the relative amount of substitution at the 2-hydroxyl in proceeding
from methylchloride to ethylchloride, to 2-diethylaminoethylchloride.
The most complete explanation for the different reactivity of the
hydroxyl groups in cellulose and starch has been given by Roberts and
Rowland. 70
They state that, in general, reaction at the 2-hydroxyl group in cellulose
predominates in the Williamson (rate-controlled) reactions, whereas
reaction at the 6-hydroxyl group predominates in the Michael (equilibrium
controlled) reactions. They further found that in these reactions the
reactivity of the 2-hydroxyl group in starch is somewhat lower and more
nearly equal to that of the 6-hydroxyl group, than for the corresponding
reactions in cellulose.
The factors which influence the substituent group distribution are
summarised in the same paper on the reactivities of the 2-, 3- and 6-
hydroxyl groups of the glucopyranosyl residue. They are:
(a) The class and specific nature of the attacking agent
(b) The size of the reagent molecule
(c) The conditions of the reactions
(d) The glucosidic structure.
Points (a) and (b) have already been mentioned. With reference to (c)
most of the reactions have been performed in alkaline and aqueous medium
but they also compared the reaction of 2-(diethylamino)ethylchloride with
cellulose and starch in aqueous and dioxane media and found higher ratios
of substitution at the 2- and 3-hydroxyl group relative to that at the
6-hydroxyl group with cellulose in dioxane than in water, and in starch the
increase on the 2- and 3-hydroxyl groups was even more pronounced. In
all cases the degree of substitution in dioxane was much lower than in
water. As to (d), it has already been shown in the foregoing that the
glucosidic structure in cellulose and starch (amylose) influences the sub-
stituent group distribution.
Very little work has yet been carried out on the substituent group
distribution of cellulose and starch esters in the form of xanthate and
acetate.
The existing data for cellulose xanthate does not show which group is the
more reactive, the 2- or 6-hydroxyl. All authors agree that on ripening of
viscose the amount of 2- and 3-xanthate decrease.
In starch xanthate the 6-hydroxyl groups seems to be preferred, as is the

case with amylose acetate. Much work remains however, to be done
especially on the substituent group distribution of starch esters.
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VycudiIik, W. and Machata, G., Chromatographia, 1973,6, 384; C.A., 79, 147533r.
Ware, G. M., Mikrochemie, 1930,2,352; C.A., 25,896.
Waszeciak, P. and Nadeau, H. G., Anal. Chem., 1964,36,764; C.A., 61, 2960b.
White, E. P., Anal. Chem., 1944, 16, 207; C.A., 38, 1980 4 •
Yorston, F. H. and Pichette, A. H., Pulp Paper Mag. Can., 1949,50,114; C.A., 44, 4247i.
Zeisel, S., Sitzungsber. d.K.Akad.d. Wiss., 1885,92, 1431.
Zeisel, S., Monatsh., 1885, 6, 989.
Zeisel, S., Monatsh., 1886,7,406.
Carboxyl group determination

Achwal, W. B. and Shanker, G., Svensk Papperst., 1972, 75, 131; C.A., 77, 103504m.
Ant-Wuorinen, O. and Visapaa, A., Svensk Papperst., 1958,61,27; C.A., 52, 21063f.
Baucek, J., Textil, 1957, 12,464; C.A., 55, 4949d.
Bouttemy, M., Bull. Soc. chim. France, 1960,887; C.A., 55, 3054i.
Brissaud, L., Mem. poudres, 1938,28,43; C.A., 33, 7559 2 •
Cheung, H. C., Carroll, B. and Weill, C. E., Anal. Chem., 1960,32,818; C.A., 55, 243b.
Christofferson, K. and Samuelson, 0., Svensk Papperst., 1960,63, 749; C.A., 55, 4948k.
Conner, A Z. and Eyler, R. W., Anal. Chem., 1950,22,1129; C.A., 45, 985f.
Croessman, F., Klaus, W., Mergenthaler, E. and Souci, S. W., Z. Lebensm. Unters.,
1964, 125,413; C.A., 62, 4517f.
Davidson, G. F., J. Textile Inst., 1948,39, T59; C.A., 42, 4743g.
Davidson, G. F. and NeveIl, T. P., J. Textile Inst., 1948,39, Tl02; C.A., 42, 4743h.
Doering, H., das Papier, 1956, 10, 140; C.A., 50, 12467e.
Dujan, J., Ind. y. quim., 1959, 19, 52; C.A., 53, 15555h.
Elizer, L. H., Anal. Chem., 1942, 14, 635; C.A., 36,5112 6 •
Ellington, A. C. and Purves, C. B., Can. J. Chem., 1953, 31, 801; C.A., 48, 3051d.
Epstein, J. A. and Lewin, M., Textile Res. J., 1960,30,652; C.A., 54, 25789b.
Ermelenko, I. N., Trudi Inst. Fiz. i. Mat. Akad. Nauk Belorus S.S.R., 1956, 117; C.A.,
56,3691e.
Ermelenko, I. N. and Chirkova, G. N., Zh. Analit. Khirn., 1963, 18, 994; C.A., 59,
14580b.
Eyler, R. W., Klug, E. D. and Diephuis, F., Anal. Chern., 1947, 19,24; C.A., 41, 1837e.
Fahmy, Y. A. and Mansour, 0., Indian Pulp Paper, 1966,20,535; C.A., 65, 5646c.
Finkelstein, M. Z., Timokhin, I. M. and Mukhamedov, Kh. U., Izvest. Vysshikh. Asheb.
Zave denii. Neft i. Gaz., 1958,45; C.A., 53, 16809i.
Ibid., Khirn. Nauka i Prom., 1959,4,677; C.A., 54, 8654d.
Francis, C. Y., Anal. Chern., 1953,25,941; C.A., 47, 9865i.
Franzon, 0., Samuelson, 0., Bodforss, B. and Ehn, E., Svensk Papperst., 1957,60,706;
C.A., 52, 9589i.
Fujii, S. and Harada, M., Eisei Shikenjo Hokoku, 1958,75,471; C.A., 52, 18933g.
Fujii, S. and Harada, M., Eisei Shikenjo Hokoku, 1959,77,153; C.A., 55, 10878a.
Ghosh, K. G., Raman, M. R., Dey, A. N. and Balakrishna, K J., J. Sci. and Ind. Res.
(India), 1960, 19, B323; C.A., 55, 6858d.
Ghosh, K G. and Balakrishna, K. J., J. Sci. and Ind. Res. (India), 1962, 21, B194;
C.A., 57, 3663c.
Graham, H. D., J. Dairy Sci., 1972,55,42; C.A., 76, 57825w.
Green, J. W., Methods in Carbohyd. Chern., 1963,3,322; C.A., 58, 12755f.
Hansi, W., Klaus, W. and Mercator, K, Tenside, 1968, 5, 281; C.A., 70, 5295e.
Heymann, E. and Rabinov, G., Trans. Faraday Soc., 1942,38,209; C.A., 36,7311 6 •
Horovic, A. and Djordjevic, B., Glasnik. Khern. Drushtva Beograd, 1959, 22, 509;
C.A., 56, 3692i.
Hostomsky, J., Tolgyessy, J. and Krivan, Y., Chern. Zvesti., 1960, 14, 290; C.A., 54,
25787g.
Ikeda, S. and Takeichi, K., Denpun Kogyo Gakkaishi, 1961,9,45; C.A., 58, 1623d.
Ikeda, S., Denpun Kogyo Gakkaishi, 1963, 10, 74; C.A., 63, 5881h.
Khundkar, M. H. and Bhattacharjee, A. K, Chemist. Analyst, 1964,53,109; C.A., 62,
1835g.
Klug, E. D., Encycl. Polymer Sci. Techn., 1964,3,520; C.A., 65, 5648d.
Kunovits, G. and Hoffmann, F., Seifen, Dele, Fette, Wachse, 1972, 98,250; C.A., 77,
50394s.
Lejaren, A. H. and Pascu, E., Textile Res. J., 16, 390; C.A., 40,6814 3 •
Mattison, M. F. and Legendre, K. A., Anal. Chern., 1952,24, 1942; C.A., 47, 3598h.
Matyus, S., Magy Textiltechn., 1965, 17, 568; C.A., 64, 14333e.
McKillican, M. E. and Purves, C. B., Can. J. Chern., 1954,32,312; C.A., 48, 9094c.
Mukhopadhyay, S. and Mittra, B. Ch., Anal. Chern., 1973,45,1775; C.A., 79, 93631a.
Nabar, G. M. and Shenai, Y. A., J. Appl. Polyrn. Sci., 1970, 14, 1215; C.A., 73, 46841k.
Nemitz, G., Die Starke, 1962, 14,276; C.A., 58, 1623a.
Norstedt, I. and Samuelson, 0., Svensk Papperst., 1966, 69, 417; C.A., 65, 15655f.
Pacault, A. and Bouttemy, M., Bull. Soc. Chirn. France, 1950,663; C.A., 45, 1341i.
Philipp, B., Rheder, W. and Lang, H., das Papier, 1965, 19, 1; C.A., 62, 7991g.
Rebek, M., Baumgartner, H., Wagner, J., Beck, H. and Kirnbauer, A., Monatsh.,
1957,88,956; C.A., 52, 6781g.
Rebek, M. and Beck, H., das Papier, 1958, 12,201; C.A., 52, 14159g.
Rebek, M. and Stiibschen-Kirchner, H., das Papier, 1960, 14, 175; C.A., 54, 16821c.
Rebek, M., Kirnbauer, A. and Semlitsch, M. F. K, das Papier, 1960, 14, 510; C.A., 55,
5941d.
Samuelson, O. and Wennerblom, A., Svensk Papperst., 1955,58,713; C.A., 51, 18583f.
Samuelson, O. and Wikstrom, L. A., Svensk Papperst., 1960,63,543; C.A., 55, 981g.
Samuelson, O. and Tornell, B., Svensk Papperst., 1961,64,155; C.A., 55, 14904f.
Samuelson, O. and Tornell, B., Svensk Papperst., 1961,64,198; C.A., 55, 14904h.
Samuelson, 0., Methods in Carbohyd. Chern., 1963, 3, 31; C.A., 58, 12754g.
Schwenkedel, S., Textil Praxis, 1958, 13, 1268; C.A., 54, 11488e.
Simionescu, C. and Asandei, N., Chim. Anal., 1958,40,204; C.A., 52, 19121e.
Sjostrom, E. and Haglund, P., Svensk Papperst., 1961,64,438; C.A., 55, 21576h.
Slavik, I., Pasteka, M. and Kucerova, M., Svensk Papperst., 1967,70,229; C.A., 67,
33967g.
Ibid., Svensk Papperst., 1967,70,365; C.A., 67, 91827u.
Slavik, I. and Kucerova, M., Faser/orsch. Textiltech., 1966, 17, 26; C.A., 64, 17849g.
Sloan, J. W., Mehltretter, C. L. and Senti, F. R., J. Chem. Eng. Data, 1962, 7, 156;
C.A., 57, 2479d.
Sobue, H., Okubo, M. and Kaunami, S., J. Chem. Soc. Japan, 1954,57,247; C.A., 49,
2727i.
Tomita, S. and Terajima, K., Kogyo Kagaku Zasshi, 1969, 72, 532; C.A., 71, 81680n.
Unruh, C. c., McGee, P. A., Fowler, W. F. and Kenyon, W.O., J. Am. Chem. Soc.,
1947,69,349; C.A., 41, 2891b.
Usmanov, Kh. U. and Perlina, R. V., Uzbeksk. Khim. Zh., 1961,22; C.A., 56, 15700d.
Vink, H., Makromol. Chem., 1969, 122,271; C.A., 70, 107642h.
Weber, O. H., J. prakt. Chem., 1941,158,33; C.A., 36,653 5 •
Wilson, K., Svensk Papperst., 1948, 51, 45; C.A., 42, 6535f.
Wilson, K., Svensk Papperst., 1956,59,218; C.A., 51, 15118c.
Wilson, K., Svensk Papperst., 1960, 63, 714; C.A., 55, 5942c.
Wilson, K., Svensk Papperst., 1966,69, 386; C.A., 65, 12401b.
Wilson, K. and Mandel, J., Tappi, 1961,44, 131; C.A., 55, 14903i.
Zaalberg Van Zelst, E. F., Plastica, 1949,2, 360; C.A., 44, 3252h.
Zubrev, N. I., Lure, I. S. and Lukyanov, A. B., Sakh. Prom., 1973, 68; C.A., 80, 16734e.
Substituent group distribution in cellulose and starch
Adams, G. A. and Castagne, A. E., Can. J. Res., 1949,27, B924; C.A., 44, 3252g.
Berry, J. W., Deutschman, A. J. and Evans, J. P., J. Org. Chem., 1964,29,2619; C.A.,
61, 12069h.
Bines, B. J. and Whelan, W. J., J. Chem. Soc., 1962,4232; C.A., 58, 2496g.
Bjorndal, H., Lindberg, B. and Rosell, K. G., J. Polymer Science, 1972, 523; C.A., 76,
87375s.
Bollenback, G. N., Golik, R. S. and Parrish, F. W., Cereal Chem., 1969,46,304; C.A.,
71,40541z.
Bose, J. L., J. Appl. Polymer Sci., 1971, 15,2999; C.A., 76, 101421n.
Brownell, H. H. and Purves, C. B., Can. J. Chem., 1957,35,677; C.A., 52, 271f.
Bullock, A. L., Rowland, S. P. and Cirino, V. 0., Textile Res. J., 1970,40,313; C.A., 73,
36372u.
Croon, I. and Lindberg, B., Svensk Popperst., 1956,59, 794; C.A., 51, 17769i.
Croon, I. and Lindberg, B., Svensk Papperst., 1957,60, 82; C.A., 52, 719f.
Croon, I. and Lindberg, B., Svensk Papperst., 1957,60,843; C.A., 52, 9588g.
Croon, I., Lindberg, B. and Ros, A., Svensk Papperst., 1958,61,35; C.A., 52, 21066a.
Croon, I., Svensk Papperst., 1958,61,919; C.A., 53, 22910d.
Croon, I. and Flamm, E., Svensk Papperst., 1958, 61, 963; C.A., 54, 2735g.
Croon, I., Svensk Papperst., 1959,62,700; C.A., 54, 3941d.
Croon, I. and Purves, C. B., Svensk Popperst., 1959,62, 876; C.A., 54, 8071f.
Croon, I., Svensk Papperst., 1960, 63, 247; C.A., 54, 15925h.
Croon, I. and Lindberg, B., Acta Chem. Scond., 1957, 11, 192; C.A., 52, 9589b.
Croon, I., Acta Chem. Scand., 1959, 13, 1235; C.A., 55, 27077a.
Croon, I. and Manley, R. S. J., Methods in Carbohyd. Chem., 1963,3,280; C.A., 58,
12755e.
De Belder, A. N. and Norrman, B., Carbohyd. Res., 1968,8, 1; C.A., 69, 106988t.
De Belder, A. N. and Norrman, B., Carbohyd. Res., 1969, 10, 391; C.A., 71, 70846v.
Doane, W. M., Russell, C. R. and Rist, C. E., Die Stiirke, 1965,17,176; C.A., 63, 8595b.
Doane, W. M., Smith, N. L., Russell, C. R. and Rist, C. E., Ibid., 225; C.A., 63, 10157g.
Dunbrant, S. and Samuelson, 0., Tappi, 1963,46, 520; C.A., 59, 15474e.
Ezra, G. and Zilkha, A., J. Macromol. Sci. Chem., 1969,3, 1589; C.A., 71, 126269d.
Fink, A. L. and Hay, G. W., Can. J. Chem., 1969,47,845; C.A., 70, 68660r.
Gardner, T. S. and Purves, C. B., J. Am. Chem. Soc., 1942,64,1539; C.A., 36, 5343 6 •
Haworth, S., Roberts, J. G. and Sagar, B. F., Carbohyd. Res., 1969, 9, 491; C.A., 71,
13288r.
Haworth, S., Jones, D. M., Roberts, J. G. and Sagar, B. F., Carbohyd. Res., 1969, 10, 1;
C.A., 71, 40440r.
Hoiness, D. E., Wade, C. P. and Rowland, S. P., Can. J. Chem., 1968,46,667; C.A., 68,
11488d.
Husemann, E., Reinhardt, M. and Kafka, M., Makromol. Chem., 1960, 41, 184; C.A.,
55, 27929b.
Husemann, E. and Kafka, M., Makromol. Chem., 1960,41,208; C.A., 55, 27929f.
Jullander, I., das Papier, 1965, 19, 166.
Konishi, H., Sen. i. Gakkaishi, 1961, 17,1175; C.A., 56, 11836a.
Kubik, J. and Talian, I., Petrochimia, 1971, 11, 8.
Lenz, R. W., J. Am. Chem. Soc., 1960,82,182; C.A., 54, 18372g.
Lindberg, A., Svensk Papperst., 1974,77,286; C.A., 80, 137748s.
Lukanoff, T., Faser/orsch. Textiltech., 1965, 16, 540 ; C.A., 64, 9935t.
Mahoney, J. F. and Purves, C. B., J. Am. Chem. Soc., 1942,64,9; C.A., 36, 1175 6 •
Mahoney, J. F. and Purves, C. B., Ibid., 1942, 64, 15; C.A., 36,1176 1 .
Neely, W. Brock, Nott, J. and Roberts, C. B., Anal. Chem., 1962,34,1423; C.A., 57,
15392f.
Norrman, B., Acta Chem. Scand., 1968,22, 1381; C.A., 70, 4470w.
Philipp, B. and Chung-Hung, Chu, Faser/orsch. Texliltech., 1965, 16, 244; C.A., 63.
5886b.
Purves, C. B., Chem. Can., 1960, 12, 25; C.A., 59, 15474d.
Ramnas, O. and Samuelson, 0., Svensk Papperst., 1968,71, 674; C.A., 70, 59047u.
Roberts, E. J. and Rowland, S. P., Carbohyd. Res., 1967,5,1; C.A., 67, 117152a.
Can. J. Chem., 1967, 45, 261; C.A., 66, 55691 u.
Roberts, E. J. and Rowland, S. P., Can. J. Chem., 1969,47, 1571; C.A., 71, 4655f
Rowland, S. P., Chem. Eng. News., 1966, Jan. 31, 30.
Rowland, S. P., Cirino, V. O. and Bullock, A. L., Can. J. Chem., 1966,44, 1051; C. A.,
64,17850h.
Spurlin, H. M., J. Am. Chem. Soc., 1939,61,2222; C.A., 33, 8400 9 •
Srivastava, H. C. and Ramalingam, K. V., Die Starke, 1967, 19,295; C.A., 67, 101230p.
Stratta, J., Tappi, 1963, 46, 717; C.A., 60, 7018c.
Timell, T., Svensk Papperst., 1948,51, 52; C.A., 42, 8466g.
Timell, T., Svensk. Kem. Tid., 1950, 62, 49; C.A., 44, 6119g.
Timell, T., Ibid., 1950,62,129; C.A., 44, l1087d.
Timell, T. E., Svensk Papperst., 1952,55,649; C.A., 48, 10336h.
Timell, T. E. and Spurlin, H. M., Ibid., 1952,55, 700; C.A., 48, 10337c.
Treiber, E., das Papier, 1970, 24, 901.
Usov, A. I., Kurnetsova, Z. I. and Arkhipova, V. S., Vysokomol. Soedin. Ser. B., 1973,
15, 147; C.A., 79, 32804n.
Wirick, M. G., J. Polym. Sci. AI., 1968,6,1705; C.A., 69, 3818h.
Yoshimura, S., Sen. i. Gakkaishi., 1965,21,560; C.A., 64, 3828g.
Ester group determination in starch in cellulose esters

Awasthy, A. K., Belcher, R. and MacDonald, A. M. G., Anal. Chim. Acta, 1965,33,
311; C.A., 63, 10686c.
Balandina, V. A. and Novikova, E. M., Plasticheskie Massy., 1960, 53; C.A., 55,
19307i.
Bischoff, K. H. and Linow, K. J., Faser/orsch. Textiltech., 1960, 11, 245; C.A., 54,
18953e.
Bockman, C. D., Appl. Spectroscopy, 1961, 15, 84; C.A., 56, 6196g.
Bockman, C. D., Appl. Spectroscopy, 1961, 15, 85; C.A., 56, 10422d.
Budinsky, B., Chern. Listy, 1956,50,1936; C.A., 51, 4213d.
Budinsky, B., Collect. Czechoslov. Chern. Cornrnuns., 1957,22, 1440; C.A., 52, 7945c.
Chun, Ku, Hua Hsueh Tung Pao, 1965, 509; C.A., 64, 9935a.
Cramer, F. B., Gardner, T. S. and Purves, C. B., Anal. Chern., 1943, 15, 319; C.A., 37,
40328.
Dumazert, C. and Senequier, R., Bull. Soc. Pharrn. Marseille, 1960,9,235; C.A., 56,
5403i.
Dyer, E. and Williams, H. D., Tappi, 1957, 40, 14; C.A., 51, 4703b.
Eberstadt, diss. Heidelberg, 1909.
Emelin, E. A. and Smyslova, N. F., Zavodsk. Lab., 1966, 32, 280; C.A., 64, 17849c.
Franz, J., Plaste u. Kautschuk, 1960,7,493; C.A., 56, 4999i.
Freudenberg, K., Ann., 1923, 433, 230.
Frohwein, Y. Z., Israel J. Chern., 1964,2,57; C.A., 61, 9657b.
Garetto, G. and Ruffoni, A., Anal. Chern., 1955,27,400; C.A., 49, 8596c.
Genung, L. B. and Mallatt, R. C., Anal. Chern., 1941, 13, 369; C.A., 35,5308 8.
Genung, L. B., Anal. Chern., 1950, 22, 401; C.A., 44, 6121i.
Genung, L. B. et al., Anal. Chern., 1952,24,400; C.A., 46, 4789i.
Gore, T. S. and Gupte, S. S., Mikrochirn. Acta, 1962,486; C.A., 57, 52d.
HekeIer, W., Kunststoffe, 1968,58,365; C.A., 69, 60115a.
Huchette, M., Die Starke, 1963,15,275; C.A., 60, 752f.
Hurtubise, F. G., Tappi, 1962, 45, 460; C.A., 60, 5731e.
Inglis, A. S., Mikrochirn. Acta, 1958,228; C.A., 53, 8944e.
Inglis, A. S., Treatise Anal. Chern., 1971, 14, 161; C.A., 75, 104932n.
Kainz, G., Z. Anal. Chern., 1959, 166, 32; C.A., 53, 16606i.
Lemieux, R. U. and Purves, C. B., Can. J. Res., 1947,25, B485; C.A., 42, 843g.
Lipparini, L. and Garutti, M. A., Quad. Merceol., 1967,51,35; C.A., 67, 65675b.
MaIm, C. J., Nadeau, G. F. and Genung, L. B., Anal. Chern., 1942, 14,292; C.A., 36,
2716 4 .
MaIm, C. J., Genung, L. B., Williams, R. F. and Pile, M. A., Anal. Chern., 1944, 16,
501; C.A., 38, 5400 9 •
McComb, D. A. and McCready, R. M., Anal. Chern., 1957,29,819; C.A., 51, 11178c.
Mironov, D. P., Grishin, E. P., Zharkov, V. V. and Pogosov, Yu. L., Plast. Massy.,
1970, 64; C.A., 72, 10l972k.
Muetgeert, J., Hiemstra, P. and Bus, W. C., Die Starke, 1958,10,303; C.A., 53, 9706g.
Murray, T. F., Staud, C. J. and Gray, H. Le B., Anal. Chern., 1931,3,269; C.A., 25,
4701.
Phillips, M., Anal. Chern., 1934, 6, 321; C.A., 28, 6394 2 •
Prey, V., SchindIbauer, H. and Maday, E., Die Starke, 1973, 25, 73; C.A., 79, 712Od.
Redfarn, C. A., Plastics (London), 1958,23,33; C.A., 52, 6781h.
Roberts, H. J., Starch Chern. Technol., 1967, 2, 293; C.A., 69, 28743t.
Roudier, A. and Nick, D., Assoc. Tech. Ind. Papetiere Bull., 1961,269; C.A., 59, 15470i.
Schoeniger, W., Lieb, H. and EI Din Ibrahim, M. G., Mikrochirn. Acta, 1954,96; C.A.,
48, 4369i.
Slavik, I., Pasteka, M. and Kucerova, M., Faser/orsch. Textiltech., 1967, 18, 584; C.A.,
68, 4114Ot.
Steyermark, A. and Loeschiiuer, E. E., J. Ass. Offic. Agr. Chern., 1954,37,433; C.A., 48,
9273e.
Subcommittee on Acyl analysis, Anal. Chern., 1952,24,400; C.A., 46, 4789i.

Tanghe, L. J., Genung, L. B. and Warren, J., Methods Carbohyd. Chern., 1963,3,201;
C.A., 58, 12755e.
Taniguchi, M., J. Soc. Chern. Ind. Japan, 1941,44,956; C.A., 43, 1977i.
Wandel, M. and Tengler, H., Gurnrni, Asbest, Kunststoffe, 1966, 19, 141; C.A., 65,
2459h.
Warren Mench, J., Methods in Carbohyd. Chern., 1963,3,201; C.A., 58, 12755c.
Wernimont, G., Anal. Chern., 1951,23, 1572; C.A., 46, 1382f.
Whistler, R. L., Adv. in Carbohyd. Chern., 1945, 1,279; C.A., 40, 4679 2 .
Whistler, R. L. and Jeanes, A., Anal. Chern., 1943, 15, 317; C.A., 37, 4032 7 •
Wiesenberger, E., Mikrochern. Ver. Mikrochirn. Acta, 1947, 33, 51; C.A., 41, 3017d.
Wiesenberger, E., Mikrochirn. Acta, 1954, 127; C.A., 48, 4262h.
Zemplen, G., Gerecs, A. and Hadacsy, I., Berichte, 1936,69,1827; C.A., 30, 67121.
Index
Acid hydrolysis, 179 Beckman Model B spectrophotometer,

enzyme hydrolysis, and, 183-6 123
Acidity Birefringence loss and gelatinisation
determination of, 150-2 temperature, 118
measurement of, 122 Bloom number, 115
Adhesives, 82 Blue value, 157
Air-dry starch, rheology, 62-3 Brabender
Aldehydes, 75 Amylograph, 68, 107-10
Alkali fluidity test, 111, 126 Plastograph, 69
Alkali-labile value, 152-3 Viscograph, 68, 69, 80
Alkali number, 154-5 Breaking strength, 115-16
Alkoxyl groups, 196, 197 Brix hydrometer, 127
Alkyl iodides, 193, 195 Bromine, 194
Amylodextrin fraction, 158 Brookfield Viscometer, 69
Amyloglucosidase, 182
Amylose, iodimetric determination
of, 155-7 Calcium, 142
Apio starch, 21 bisulphite, 142
Aralkyl ethers, 196 chloride solution, 169, 175-9
Arrowroot starch, 19 determination of, 143
granules of, 18 Canna starch, 28
microscopy, 18 Carboxyl groups, determination of,
Ash content, 162 161
Azeotropic distillation for moisture Carboxymethyl starch, 192, 197
content, 138-9 Cassava starch, 20
granules of, 18
microscopy, 18
Balling, 134 Cellulose
Barley derivatives, substituent group
diastase methods, 180 distribution, 199
starch,12 ethers, 197
granules of, 13 substituent group distribution, 199
microscopy, 13 xanthate, 202
215
216 INDEX
Ceric sulphate titration, 159 Electron microscopy, 3, 33-59

Characterisation of starch, 91-131 apparatus, 38-40
Chemical analysis, raw and modified applications and results, 47-55
starches, 133-65 cytological studies, 47
CI Recording Viscometer, 75 evaluation and perspectives, 55-6
Cold pastes, rheology, 78-82 resolution, 37
Colour scanning, 34, 45-7
corn starch, of, 123 apparatus, 45-6
determination, 122-4 evaluation and perspectives, 56
specifications, 123 specimen preparation, 47
sweet potato, in, 123 theoretical aspects, 35
Consistometer, 105, 107 transmission, 34, 38-44
Cooked pastes, rheology, 75-82 apparatus, 38---40
Corn starch evaluation and perspectives, 55-6
colour, 123 specimen preparation, 40-4
phosphorus determination, 146 ultrastructural studies, 54-5
Creep and creep recovery, 80 Embedded-disc principle, 115
Cross linked starches, 74-75 Enzyme hydrolysis, 179-86
Cross linking, 81 Esterification, 81
Cytological studies, 47-54 Ethyl group, 197
Excitation factor, 36
Expansion of unpasted starch, 66-7
Damaged grains, 163
de Broglie equation, 37
Fat content, 162
Degree of substitution, 190-91, 198-9
determination of, 148-9
de Willigen, A. H. A., 61
Fatty acids, 148
Dextrins, 84 Flow properties, measurement of, 124
alkali fluidity test, 126
Foreign matter, separation and
examination of, 125-7
estimation of, 120-1
viscosity tests, 126 Freeze-etching, 44
water soluble material in, 127
Diastase methods, 180
Dilatancy, 65
Gallant, D. J., 33
Donnan equilibrium, 67, 73
Gas
Drageviscosimeter system Epprecht, chromatography, 161, 195, 196
69 evolution and moisture content, 141
Dustiness, 125 -liquid chromatography, 195
Dustless starch, 125
Gel
Dyes formation, 78-82
adsorption of, 7 strength, 113
reactive, 8 structure, 79, 80
testing, 82, 112-14
Gelatinisation, 105
Efflux time, 99 cold water, 111-17
Einstein law, 70 methods of following, 67
Electrical properties and moisture temperature, 3, 117-20
content, 141 birefringence loss, and, 118
INDEX 217
Gelatinisation-contd. Iodine, 169

temperature-contd. affinity, 156
moisture content, and, 119-20 solution, 155, 157
refractive index, and, 118-19 Ionic char <!cter, 8
translucency, and, 118
viscosity, and, 119
Gelometers, 114, 116 Karl Fischer method for moisture
Granular starch, examining, 5 content, 139-41
Granules Knife-edge orifice, 97
arrowroot starches, 18
barley starch, 13
cassava starch, 18 Lead citrate, 54
damaged, 8 Lintner starch, 83
maize starch, 13 Lovibond tintometer, 123
oat starch, 13 Luminous reflectance, 123
pea starch, 18 Lyne, F. A., 133, 167
potato starch, 9
rice starch, 15
rye starch, 12 Maize starch, 14,48,49
sago starch, 18 analysis and examination, 91
size and shape of, 5 granules, 13
wheat starch, 11 microscopy, 13
Green plantain starch, 29 Malt diastase methods, 180
Gums Maltose-starch mixtures, 159
alkali fluidity test, 126 Manioc starch, 53
examination of, 125-7 Mannich-Lenz-Hopkins procedure,
viscosity tests, 126 175
Mannich-Lenz procedure, 171
Methoxyl group, 193, 196
Microscope, 1-3
Hilum, 5, 9,12,18 electron, see Electron microscopy
Hughes-Acree bromine oxidation features of, 1
method, 158 fluorescence, 2
Hydrochloric acid solutions, 171-5 hot-stage, 2
Hydrogen ion activity, 121-2 light, 1, 33, 34
Hydrogenolysis reaction, 196 measuring, 2
Hydrolysed starches, 83 polarising, 2
Hydrolysis, 55, 74 stereo, 3
Hydroxyethyl group, 196, 197 Microscopical examination, 3-9
Hydroxyl groups, 189, 198,201,202, Microscopy, 1-32
203 arrowroot starches, 18
Hydroxylalkyl groups, 194 barley starch, 13
Hydroxypropylether groups, 197 cassava starch, 18
characteristics of starches, 22-7
electron, see Electron microscopy
maize starch, 13
Iodimetric determination of amylose, oat starch, 13
155-7 pea starch, 18
218 INDEX
Microscopy-contd. Parenchyma cells, 54

potato starch, 9-11 Pea starch
rice starch, 15 granules, 18
rye starch, 12 microscopy, 18
sago starch, 18 Pentosan content of wheat starch, 158
wheat starch, 11 pH determination, 121-2, 150-2
Mineral matter, determination of, Phase
142-50 condensers, 2
Modified starch objectives, 2
definition, 189 Phosphorus
examination of, 158 content of potato starch, 73
Moisture determination, 145, 146
content, 162 Photoelectric colorimeter, 124
determination of, 133-42 Photometer, 123
azeotropic distillation, 138-9 Pipettes, 98
electrical properties, by, 141 Polarimetry, 170--78
gas evolution, by, 141 Potassium
Karl Fischer method, 139-41 ferrocyanide, 161
miscellaneous methods, 141-2 hydroxide, 9
oven methods, 135-8 Potato starch, 10, 52, 72, 75
economic importance of, 133-4 granules, 9
electrical properties, and, 141 influence of double layer, 73
gas evolution, and, 141 microscopy, 9-11
gelatinisation temperature, and, moisture, 136, 142
119-20 pastes, 77, 81
normal,134 phosphorus
specifications, 136 content, 73
correction for, 97 determination, 146
tester, 137 viscogram, 72
Molar substitution, 198-9 Prandtl, law of, 64
Monochloroacetic acid, 192, 197 Precooked starch, pastes and jellies
Moss, G. E., 1 of,82-3
Mountants, 3-5, 7 Propane sultone, 192, 197
Protein
content, 162
determination of, 149-50
Newtonian solution, 83, 84
Purity, 123
Oat starch
granules, 13 Quality control, 92
microscopy, 13
Odour, examination for, 121
Opacity of pastes, 125
Orifice-flow viscosity test, 95 Reconstitution, 82
Oven methods for moisture content, Recording Viscometer, 68, 74, 80
135-8 Reflectance, 123
Oxidised starches, 83 Reflectometer, 124
INDEX 219
Refractive index and gelatinisation Starch

temperature, 118-19 derivatives, 81
Refractometers, 127 analysis, 189-213
Retrogradation, 83, 84 definition, 189
Rheological curve, 76 substituent group distribution,
Rheology, 61-90 201
air-dry starch, 62-3 determination of
cold pastes, 78-82 hydrolytic methods, 179-86
comparison of different types of non-hydrolytic methods involving
starch, 72-3 direct weighing, 167-9
cooked pastes, 75-82 non-hydrolytic methods using
starch polarimetry, 170--8
pastes, 67-75 non-hydrolytic starch-iodine
suspensions, 63-7 methods, 169-70
Rice starch, 15 starch products, in, 167-88
granules, 15 esters, 82, 189, 190-1
microscopy, 15 pyrolysis, 191
Rigidity testing, 114, 115 qualitative tests, 190
Rigidometer,114 quantitative determination, 190
Rotavisko of Haake, 69 substituent group distribution,
Rye 201
flour, 11 ethers, 82, 189, 191-9
starch anionic, 192, 197
granules, 12 cationic, 193, 198
microscopy, 12 non-ionic, 192, 193, 197
qualitative tests, 192
quantitative determination, 193-9
Saareplates, 78 substituent group distribution, 201
Sago starch, 17 pastes, 92
granules, 18 cold, viscosity, 103
microscopy, 18 cooking, 82
Scott test, 96 rheology, 67-75
Shrinking of unpasted starch, 66-7 viscosity tests, 93-103
Silver products, starch determination in,
nitrate solution, 195 167-88
o-nitrophenolate, 161 suspensions, rheology, 63-7
Sodium xanthate, 203
hydroxide, 8 Steric factors, 202
salicylate solutions, 161 Sterling, c., 33
Specific rotation, 162, 171, 176, 177 Stress-strain relationship, 75
Specifications, moisture content, 136 Substituent group distribution
Spectrometer, 197 cellulose
Spectrophotometers, 123 derivatives, 199
Spectrophotometry, 196 ethers, 199
Staining starch
reagents, 42 derivatives, 201
techniques, 6-8 esters, 201
Standardisation, 92, 104 ethers, 201
220 INDEX
Sulphopropylether of starch, 192, 198 Viscometers-contd.

Surfactants, 8 corn industries, 110--11
Sweet potato falling sphere, 102
colour in, 123 Macmichael, 100
whiteness of, 124 recording, 107
Swelling, 80 rotational, 99,101,102
agents, 8-9 standardising, 98
Syneresis, 116-17 Stormer, 99
Syrups, 84 Viscosity
cold paste, 103-4
curves, 104-11
Takadiastase, 181 determination of, 94, 95
Tarr-Baker jelly tester, 115 dextrins and gums, 126
Tests, physical, 91-3 gelatinisation temperature, and, 119
Texture, differences in, 125 knife-edge orifice, 97
Thickening agents, 75 orifice flow method, 95-6
Thiocyanate viscosity, 160, 161 pipette method, 98
Thiocyanate viscosity method, 111 tests
Translucency and gelatinisation correction for moisture, 97
temperature, 118 starch paste, 93-103
Tube penetrometer, 115 VI-Viscograph, 68, 72, 74, 107
Water soluble material

Uranyl nitrate, 54 determination, 124-5
dextrins, in, 127
Wheatstarch,50,51
van der Bij, J., 189 granules, 11
Velocity, Scott test, 96 microscopy, 11
Viscograms, 69-72 pent os an content, 158
Viscometers, 94, 95, 119, 126 White lentil flour, 16
Brookfield, 101 White sweet potato starch, 30
Cannon-Ubbelohde,99
capillary, 69, 98
continuous automatic recording, Yellow potato dextrin, 4
110
continuous reading concentric
cylinder, 107 Zeisel's reaction, 195

J. A. Radley-Examination and Analysis of Starch and Starch Products-Springer (1976)

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J. A. Radley-Examination and Analysis of Starch and Starch Products-Springer (1976)

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EXAMINATION AND ANALYSIS

OF STARCH AND STARCH PRODUCTS

APPLIED SCIENCE PUBLISHERS LTD

ISBN-13: 978-94-010-1334-5 e-ISBN-13: 978-94-010-1332-1

WITH 7 TABLES AND 54 ILLUSTRATIONS

© APPLIED SCIENCE PUBLISHERS LTD 1976

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

Galliard (Printers) Ltd Great Yarmouth

as humanly possible and to my secretary, Mrs R. M. Russell, for her

1. The Microscopy of Starch by G. E. Moss 1

2. Electron Microscopy of Starch and Starch Products by

3. The Rheology of Starch by A. H. A. DE WILLIGEN 61

4. Physical Methods of Characterising Starch by J. A. RADLEY 91

5. Chemical Analysis of Raw and Modified Starches by F. A. LYNE 133

6. Determination of Starch in Various Products by F. A. LYNE. 167

7. The Analysis of Starch Derivatives by J. VAN DER BI] 189

The Microscopy of Starch

1.1 THE MICROSCOPE

The microscope is an extremely versatile instrumentl and it is the most

Mechanical stage. This permits controlled movement of the specimen.

Binocular head. This is considered to be more restful to use, making

J. A. Radley (ed.), Examination and Analysis of Starch and Starch Products

use introduces an extra magnification factor, usually x 1·2 and marked on

Measuring microscope. A microscope having an eyepiece graticule and

Hot-stage microscope. The Kofler hot-stage is an electrically heated box

Phase contrast. Instruments having special phase condensers and

Fluorescence microscope. Certain light microscopes having quartz iodine

Stereo microscope. The stereo or 3D microscope can be useful when

1.2 MICROSCOPICAL EXAMINATION

eyepiece graticule. A mechanical stage is also a great advantage. When

(1) differential staining of components; or

1.2.4 Staining techniques

Several procedures for the staining of plant tissues are described in

E. Unna 7 has proposed a differential staining procedure to distinguish

dyes such as light green SF yellowish (C.1.670), acid fuchsin (C.1.691)

1.2.5 Swelling methods

1.3 MICROSCOPICAL APPEARANCE AND CHARACTER

1.3.1 Potato starch

and maize starches. The hydration phenomenon shown by the unmodified

1.3.2 Wheat starch

FIG. 1.3. Rye flour (approximltely 60 % starch) ( x 500) .

1.3.3 Rye starch

FIG. 1.4. Barley starch (x 500).

1.3.4 Barley starch

1.3.5 Maize starch

1.3.6 Oat starch

rod-like structure. Occasionally among separated grains, lemon or

1.3.7 Rice starch

FIG. 1.6. Rice starch (x 500).

1.3.8 Pea starch

1.3.9 Sago starch

1.3.10 Arrowroot starches

1.3.11 Cassava starch

1. Acorn 5-20 pm Hemispherical or Simple and

2. Canna Canna edulis, 10--130 pm Elliptical, oval, Round and Distinct

3. Curcuma Curcuma 30--60/lm Oval, elliptical, Punctiform Very distinct

4. Curcuma C. Leucorrhiz Generally

12. FritiIIaria Mussel and bean Various Observable at Occasional compound

15. Colocasia 10-55 11m Regular and oval Fairly Only on

16. Millet, Sorghum

Large 14-35 pm Simple and Distinct and Both simple and

26. Tacca (Tahiti Tacca 25-45 J.lfD. Simple---elliptical, Centric or Distinct

28. Waxy Maize 5-25 pm Similar to Centric and Red brown