31.10.

2017

Color Reactions And Reducing Sugar

Reactions of Carbohydrates
Experiment 6
Ebru AKHARMAN, Gebze Technical University, Turkey

AIM:
Carbohydrates are carbon compounds that contain great amounts of hydroxyl groups. The
simplest carbohydrates also comprise either an aldehyde moiety ( polyhydroxyaldehydes ) or a
ketone moiety (polyhydroxyketones). All carbohydrates can be categorized as monosacchrides,
oligosaccharides or polysaccharides. Somewhere from two to ten monosaccharide units, linked
by glycosidic bonds, make up an oligosaccharide. Polysaccharides are much bigger, having
hundreds of monosaccharide units. The existence of the hydroxyl groups permits
carbohydrates to interrelate with the aqueous nature and to participate in hydrogen bonding,
both inside and between chains. Derivatives of carbohydrates can contain nitrogen, phosphate
and sulfur compounds. Carbohydrates also can unite with lipids to form glycolipids or with
proteins to form glycoproteins. In this experiment,the carbohydrates were detected with color
reactions and reducing sugar reactions.
fertilization, preventing pathogenesis, blood
INTRODUCTION: clotting and development.
A carbohydrate is an organic compound
with the general formula Cm(H2O)n, that is, Color Reactions of Carbohydrates:
consists only of carbon, hydrogen and Concentrated non oxidizing acids can
oxygen, with the last two in the 2:1 atom convert carbahydrates into furfural or
ratio. Carbohydrates make up the bulk of furfural derivatives. These in turn can react
organic substances on earth and perform with phenolic compounds or aromatic
numerous roles in living things.
The carbohydrates (saccharides) are amines to produce colored compounds.
divided into four chemical groups: Accordingly, carbohydrate chemistry
monosaccharides, disaccharides, includes a number of so called color
oligosaccharides and polysaccharides. reactions. Some of these are general,
Polysaccharides serve for the storage of reacting with any carbohydrate; others,
energy and as structural components. however, are more specific, reacting with
Structural polysaccharides are frequently specific carbohydrates; others are used in
found in combination wit proteins quantitative analysis of carbohydrates.
Reaction with anthrone and sulfuric
(glycoproteins or mucoproteins) or lipids
acid:
(lipopolysaccharides). The 5-carbon
The Anthrone method is an example of a
monosaccharide ribose is an important
colorimetric method of determining the
component of and the backbone of the
concentration of the total sugars in a
genetic molecule known as RNA. The related
sample.Sugars react with the anthrone
deoxyribose is a component of DNA.
reagent under acidic conditions to yield a
Saccharides and their derivatives include
blue - green color. The sample is mixed with
many other important biomolecules that
sulfuric acid and the anthrone reagent and
play key roles in the immune system,

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then boiled until the reaction is completed.

The solution is then allowed to cool and its You must move seliwanoff test to
absorbance is measured at 620 nm. There is distinguish sucrose from fructose.
a linear relationship between the
absorbance and the amount of sugar that
was present in the original sample. This
method determines both reducing and non-
reducing sugars because of the presence of
the strongly oxidizing sulfuric acid. Like the
other methods it is non-stoichemetric and
therefore it is necessary to prepare a
calibration curve using a series of standards
of known carbohydrate concentration.

Image 2: Seliwanoff Test Image

Image 1: Anthron Test Image
Reaction with Resorcinol and
Hydrochloric acid:
Seliwanoffs test is a compound test which
separates aldose and ketose sugars. Ketoses
are differentiated from aldoses by means of
their ketone/aldehyde usefulness. If the
sugar contains a ketone bunch, it is a ketose.
If the sugar contains an aldehyde bunch, it is
an aldose. This test is much like Bials test.
This test depends on the rule that, when
heated, ketones are more quickly dried out
than aldoses. It is named after Theodor
Seliwanoff, the scientific expert, who
performed the test. At the point when it is
added to a solution containing ketones, a
red shading is framed quickly giving a
positive test. At the point when added to an
element containing aldoses, a slower
framing pink light is seen.
Reaction with Orcinol and
Hydrochloric Acid:
The reagents are the chemical compounds
which are necessary to perform some
chemical test. Just like any other test, there
are few reagents that are used in this test.
The compounds being used in this test acid
are Ferric acid, hydrochloric acid, and
orcinol. The reagent is prepared by
combining these compounds in a balanced
manner. The reaction works in a way that
pentose is dehydrated by the reagent and a
furfural form is formed. Orcinol then reacts
with this furfural which then reacts with
iron to give a bluish colored product and the
presence of pentose is detected. A positive
result in indicated when a bluish color
appears in the solution. Remember that only
bluish color indicates a positive test. If some
other color appears then the result is
negative. And this was an easy experiment
to perform and check the presence of
pentose.
Image 3: Bial s Test Image

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Quantitative application of a color detection of reducing sugars are

reactions: Dinitrosalicylic acid Test, and Nelson
The carbohydrate content of unknown method.
biologic products can be determined by a Reaction with Benedict s Reagent:
very simple and fairly reliable procedure. It is an equal-volume mixture of aqueous
This method does not include previous solution of Copper Sulfate (CuSO4) and
hydrolysis of the sample and liberation of sodium citrate. Here sodium citrate is the
the constituting monosaccharides, but uses complexing agent. Benedict's solution is a
concentrated sulfuric acid in the presence of deep-blue alkaline solution used to test for
phenol. Carbohydrates form either the presence of the aldehyde functional
furfuraldehyde or its homologs with strong group- CHO. Formation of red coloured
acid. These derivatives of the carbohydrates copper(I) oxide indicates the formation of a
produce colored compounds by precipitate. This precipitate is insoluble in
polymerization or condensation with water. As Benedicts test continues, the
aromatic phenols. This is the basis of the concentration of reducing sugar increases.
Molisch reaction and also of Bial's orcinol Following this condition, high amount of
assay which was originally designed for
brick-red colour precipitate will be formed
pentoses and hexuronic acids. The anthrone
at the end of the test tube. Sometimes you
reaction is perhaps the best known version.
will find small amounts of copper oxide
Certain SH-containing molecules also react
along with brick-red precipitate. Sodium
with the furfural derivatives. The phenol -
carbonate of Benedicts reagent facilitates
sulfuric acid procedur helps to detecting
the alkaline conditions which are required
concentration of carbohydrates.
for the redox reaction. Another compound
Reducing Sugar Reactions of
Sodium citrate complexes with the copper
Carbohydrates:
(II) ions to avoid degradation into copper(I)
A reducing sugar is any sugar that, in a
ions during storage. Some complex type of
solution, has an aldehyde or a ketone group.
carbohydrates like starch or amylum
The enolization of sugars under alkaline
consisting of a large number of glucose
conditions is an important consideration in
monomer units joined by glycosidic bonds.
reduction tests. The ability of a sugar to
Starch or amylum dont react or react very
reduce alkaline test reagents depends on
poorly with Benedict's reagent, due to the
the availability of an aldehyde or keto group
relatively small number of reducing sugar
for reduction reactions. A number of sugars
units. Inositol is another complex
especially disaccharides or polysaccharides
carbohydrate which produces a negative
have glycosidic linkages which involve
result with Benedicts test.
bonding a carbohydrate (sugar) molecule to
another one, and hence there is no reducing
group on the sugar; like in the case of
sucrose, glycogen, starch and dextrin. In the
case of reducing sugars, the presence of
alkali causes extensive enolization
especially at high pH and temperature. This
leads to a higher susceptibility to oxidation
reactions than at neutral or acidic pH. These
sugars, therefore, become potential agents
capable of reducing Cu+2 to Cu+, Ag+ to Ag
and so fort. Most commonly used tests for Image 4: Benedict Test Image

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Reaction with Dinitrosalicylic Acid: The blue color developed is compared with
3,5-Dinitrosalicylic acid is an aromatic a set of standards in a colorimeter at
compound that reacts with reducing sugars 620nm.
and other reducing molecules to form 3-
amino-5-nitrosalicylic acid, which absorbs
light strongly at 540 nm. It was first
introduced as a method to detect reducing
substances in urine and has since been
widely used, for example, for quantifying
carbohydrates levels in blood. It is mainly
used in assay of alpha-amylase. However,
enzymatic methods are usually preferred
due to DNS lack of specificity. An assay for
determination of galacturonic acid with 3,5-
dinitrosalicylic acid was developed that
substantially extends the linear range of Image 6: Nelson Method Image
detection compared to a previously
published method with this reagent. MATERIAL AND METHODS:

For reaction with anthrone and sulfuric acid

Image 5: DNS Test Image
Quantitative Application of Reducing
Sugar Reaction(Nelson Method):
Sugars with reducing property (arising out
of the presence of a potential aldehyde or
keto groups) are called reducing sugars.
Some of the reducing sugards are glucose,
galactose, lactose and maltose. The Nelson-
Somogyi method is one of the classical and
widely used methods for the quantitative
determination of reducing sugars.
The reducing sugars when heated with
alkaline copper tartrate reduce the copper
from the cupric to cuprous state and thus
cuprous oxide is formed. When cuprous
oxide is treated with arsenomolybdic acid,
the reduction of molybdic acid to
molybdenum blue takes place.
reactions:
Solutions of ribose, glucose, fructose,
lactose, sucrose and starch
Anthrone reagent
Sulfuric acid
Tubes.
Heater and marker
The names of carbonhydrates solutions
are writed on tubes.
Carbonhydrates solutions are added 1
drop to tubes.
10 drops anthrone reagent are added to
every carbohydrate solutions.
Then, every solutions are mixed and
heated at 5 minute.
The heating mixtures are cold. Changes
are observed and recorded
For reaction with Benedict s reactions:
Solutions of ribose, glucose, fructose,
lactose, sucrose and starch
Benedict s reagent
Tubes
Heater and marker
The names of carbonhydrates solutions
are writed on tubes.
Carbonhydrates solutions are added 1
drop to tubes.

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10 drops Benedict s reagent are added to The concentration of unknown is

every carbohydrate solutions calculated.
Then, every solutions are mixed and
heated at 5 minute. ml ml Water ml
The heating mixtures are cold. Changes Standart Unknown
are observed and recorded. A 0 1 0
For reaction with Bial s test reactions: B 0,2 0,8 0
Solutions of ribose, glucose, fructose, C 0,4 0,6 0
lactose, sucrose and starch D 0,6 0,4 0
Orcinol reagent E 0,8 0,2 0
Tubes F 1 0 0
Heater and marker
G 0 0 1
The names of carbonhydrates solutions
Table 1: Concentration Table
are writed on tubes.
Carbonhydrates solutions are added 1
drop to tubes. RESULTS:
2 drops Orcinol reagent are added to every 1: Anthrone when mixed with sulfuric acid
carbohydrate solutions and carbohydrate, gives a blue or gren color.
Then, every solutions are mixed and
heated at 5 minute.
The heating mixtures are cold. Changes
are observed and recorded.
For reaction with Dinitrosalicylic acid
reactions:
Solution of maltose
Dinitrosalicylic acid
Tubes
96 well plate
Heater and marker
The names of carbonhydrates solutions
are writed on tubes.
Image 6: Anthron reagent Image
Carbonhydrates solutions are added 1
Different carbohydrates give different color
drop to tubes.
with anthrone reagent.
2 drops Dinitrosalicylic acid are added to
every carbohydrate solutions
The prepairing mixed solutions are diluted
according to detected amounts.
Then, every tubes are mixed and heated at
5 minute.
The heating mixtures are cold. Changes are
observed and recorded.
DNS reagent is 500 l added to every
tubes.
200 l sample is added to 96 well plate.
The absorbance of samples are measured
540 nm.
Image 7: Anthron Reagent Image

5

2: Only bluish color indicates a positive
test. If some other color appears then the
result is negative. And this was an easy
experiment to perform and check the
presence of pentose and ribose.
Image 8: Orcinol Reagent Image
Different carbohydrates give different color
with orcinol reagent.
Image 9: Orcinol Reagent Image
3. The Benedict s reagent contains cupric
ions, which in an alkaline environment,
oxidize the aldehyde group to carboxylic
acid. Cupric ions are reduced to oxide, which
forms a red precipitate.
This test is a test to determine whether or
not the carbohydrate contains a free
aldehyde or ketone group. In Benedict s
reaction, copper is reduced and the product
forms a red precipitate.
31.10.2017
Image 10: Benedict reagent Image
4: DNS (Dinitrosalicylic acid) is one of the
reagents used to estimate reducing sugars
in the solution. It is a spectrophotometric
method. 3,5-Dinitrosalicylic acid is reduced
to 3 amino 5 nitro salicylic acid while
oxidizing the reducing sugars. The color
change can be quantified
spectrophotometrically at a wavelength of
540 nm. The intensity of the color observed
will be proportional to the concentration of
glucose present in the solution.
Image 11: DNS reagent Image
Different reducing sugars generallyyield
different color intensities; thus, it is
necessary to calibrate for each sugar. In
addition to theoxidation of the carbonyl
groups in the sugar, other side reactions
such as the decomposition of sugar also
competes for the availability of 3,5-
dinitrosalicylic acid. As a
consequence,carboxymethyl cellulose can
affect the calibration curve by enhancing the
intensity of the developed color.
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REP REP AVG-Blank

REP 1 REP 2 REP 3 AVG. AVG. Concentration
A(blank) 0,121 0,092 0,226 0,1065 0,0000 0 g / ml
B 0,146 0,143 0,139 0,1445 0,0380 80 g / ml
C 0,239 0,247 0,250 0,2485 0,1420 160 g / ml
D 0,330 0,343 0,318 0,3365 0,2300 240 g / ml
E 0,567 0,578 0,629 0,5725 0,4660 320 g / ml
F 0,632 0,670 0,695 0,6825 0,5760 400 g / ml
G(unknown) 0,578 0,584 0,634 0,5810 0,4745 X g / ml
Table 2: Absorbance Table

This table is obtained from different concentration values. The absorbance of these
concentrations values are measured with colorimetry. The absorbance of blank is removed from
other absorbance values. Thus, effect of buffer is removed. These datas are used to create
absorbance concentration graph.

Y = 0, 0015X 0,0617 ( y = 0,4745)

0,4745= 0, 0015X 0,0617
0,4745+ 0,0617 = 0, 0015 X
0, 4807 = 0, 0015X
0, 5362
= 320,44
0, 0015
The concentration of carbohydrate is 320,44 / .

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DISCUSSION:
Carbohydrates are important biological RESOURCES:
molecules for living things. The different 1)http://www.worldofchemicals.com/557/
chemical groups involved in carbohydrates chemistry-articles/benedicts-reagent-test-
add different qualities to them. These for-monosaccharides.html
properties play a crucial role in the 2)http://www.cloudclone.com/items/K697
reactions of carbonhydrates. Carbohydrates .html
can be identified or differentiated through
3)http://www.biocyclopedia.com/index/pl
different tests. Concentrated non- oxidizing
ant_protocols/carbohydrates/reducing_sug
acids can convert carbohydrates into
ars_by_Nelson-Somogyi_method.php
furfural or furfural derivatives. These in
turn can react with phenolic compounds or
aromatic amines to produce colored
compounds. The resulting colors tell us
whether the test is negative or positive. By
interpreting the resulting color, we may
have an idea of the type of carbohydrate. A
carbohydrate that has either a free aldehyde
or ketone group will exhiit a number of
characteristic reactions. One of these is the
ability to reduce the cupric ion in alkaline
solution. In a case such as this eventhough
the carbohydrate undergoes oxidation
historically attention has been called to the
reduction aspect of the reaction and
carbohydrates that can undergo this tpe of
reaction have been called reducing sugars.
The reducing sugar reactions and color
reactions are used in chromatographic
detection of carbohydrates and quantitative
analysis of carbohydrates. The diluted
maltose solution was placed in a 96-well
plate and the absorbance was measured
with a colorimeter. As a result of this
measurement we have found that each
concentration has different absorbance
values. Repeated three times, we found that
the solutions we placed in the 96 well plate
had different absorbance values. This
difference may be due to carelessness of the
person doing the experiment. The two
closest values from the absorbances
obtained were used for the standard curve.