COLOR REACTIONS AND REDUCING SUGAR REACTIONS OF CARBOHYDRATES

Rveyda AKN, Gebze Technical University, Turkey

Experiment 6
AIM
To characterize carbohydrates present in an unknown solution on the basis of various chemical assays.

INTRODUCTION
Most of the chemical compounds present in aldehydes and ketones. Aldehydes (CHO) and
living organisms contain skeletons of ketones ( = CO) constitute the major groups in
covalently bonded carbon atoms (C-C-C-C). carbohydrates.
These compounds are known as organic
compounds, because most of these are either
present in, or produced by living things.
Organic compounds are the major components
of cells and tissues. They provide energy for life
processes, participate in and regulate
metabolic reactions, and transmit information.
Organic macromolecules in living organisms
can be classified as either carbohydrates, (A carbohydrates)
proteins, lipids, or nucleic acids, among others.
These macromolecules are always made of Heating of monosaccharides under acidic
smaller subunits. The subunits of
conditions (concentrated sulphuric,
macromolecules are held together with
hydrochloric acids) leads to their dehydration.
covalent bonds, and have different structures
From pentoses the furfural is generated, and in
and properties. For example, lipids made of case of hexoses 5-hydroxymethylfurfural.
fatty acids have many C-H bonds and relatively Pentoses more easily undergo dehydration
little oxygen, while proteins made of amino compering to hexoses and ketoses undergoes
acids have amino groups (-NH2) and carboxyl (- dehydration more easliy than aldoses.
COOH) groups. These characteristic groups Disaccharides react more slowly in comparison
impart different chemical properties to to all monosaccharides. This phenomenon is
macromolecules for example, due to the fact that oligo- and polysaccharides
monosaccharides such as glucose are polar and have to be hydrolysed first and then undergo
soluble in water, while lipids are nonpolar and dehydration.
insoluble in water. Carbohydrates are
compounds that contain carbon, hydrogen and
oxygen. Carbohydrates include a variety of
compounds, such as sugars, starches, and
celluloses. While sugars and starches serve as
energy sources for cells; celluloses are
structural components of the walls that
surround plant cells. The term carbohydrate
literally means "hydrated (H20) carbon"
Carbohydrates may contain one sugar
molecule (monosaccharides), two sugar
molecules (disaccharides), or many sugar units
(polysaccharides). Carbohydrates are
polyhydroxy aldehydes and ketones or
substances that hydrolyze to yield polyhydroxy
(Heating of saccharides with concentrated
acids leads to the loss of one water molecule
in the first phase and then two water
molecules. As a result furfural is formed a
heterocyclic aldehyde or its derivatives.)
Furfural and 5-hydroxymethylfurfural are
colourless substances. They form colour
products of the condensation with phenols: -
naphthol, resorcin and orcin.
Reducing Sugar:
Sugars that contain aldehyde groups that are
oxidised to carboxylic acids are classified as
reducing sugars. They are classified as reducing
sugars since they reduce the Cu2+ to Cu+ which
forms as a red precipitate, copper (I) oxide.
aldehydes (and hence aldoses) are readily
oxidised. In order for oxidation to occur, the
cyclic form must first ring-open to give the
reactive aldehyde. So any sugar that contains a
hemi-acetal will be a reducing sugar. But
glycosides which are acetals are not reducing
sugars. Ketoses can also be reducing sugars
because they can isomerise (a tautomerisation)
to aldoses via an enediol.
Molischs Test:
This is a common test for all carbohydrates
larger than tetroses. The test is on the basis
that pentoses and hexoses are dehydrated by
conc. Sulphuric acid to form furfural or
hydroxymethylfurfural, respectively. These
products condense with -naphthol to form
purple condensation product. Disaccharides
react more slowly in comparison to all
monosaccharides. This phenomenon is due to
the fact that oligo- and polysaccharides have to
be hydrolysed first and then undergo
dehydration.
Fufural -Naphthol
Fehlings Test:
This forms the reduction test of carbohydrates.
Fehlings solution contains blue alkaline cupric
hydroxide solution, heated with reducing
sugars gets reduced to yellow or red cuprous
oxide and is precipitated. Hence, formation of
the yellow or brownish-red colored precipitate
helps in the detection of reducing sugars in the
test solution.
Benedicts Test:
As in Fehlings test, free aldehyde or keto group
in the reducing sugars reduce cupric hydroxide
in alkaline medium to red colored cuprous
oxide. Depending on the concentration of
sugars, yellow to green color is developed. All
monosaccharides are reducing sugars as they
all have a free reactive carbonyl group. Some
disaccharides, like maltose, have exposed
carbonyl groups and are also reducing sugars,
but less reactive than monosaccharides.
Barfoeds Test:
Barfoed's test is used to detect the presence of
monosaccharide (reducing) sugars in solution.
Barfoed's reagent, a mixture of ethanoic
(acetic) acid and copper(II) acetate, is
combined with the test solution and boiled. A
red copper(II) oxide precipitate is formed will
indicates the presence of reducing sugar. The
reaction will be negative in the presence of
disaccharide sugars because they are weaker
reducing agents. This test is specific for
monosaccharides. Due to the weakly acidic
nature of Barfoed's reagent, it is reduced only
by monosaccharide.
Seliwanoffs Test:
It is a color reaction specific for ketoses. When
conce: HCl is added. ketoses undergo
dehydration to yield furfural derivatives more
rapidly than aldoses. These derivatives form
complexes with resorcinol to yield deep red
color. The test reagent causes the dehydration
of ketohexoses to form 5-
hydroxymethylfurfural.5-
hydroxymethylfurfural reacts with resorcinol
present in the test reagent to produce a red
product within two minutes (reaction not
shown). Aldohexoses reacts so more slowly to
form the same product.

Bials Test:
Bials test is used to distinguish between
pentoses and hexoses. They react with Bials
reagent and are converted to furfural. Orcinol
and furfural condense in the presence of ferric
ion to form a colored product. Appearance of
green colour or precipitate indicates the
presence of pentoses and formation of muddy
brown precipitate shows the presence of
hexoses.
Iodine Test:
This test is used for the detection of starch in
the solution. The blue-black colour is due to the
formation of starch-iodine complex. Starch
contain polymer of -amylose and amylopectin
which forms a complex with iodine to give the
blue black colour.
Osazone Test:
The ketoses and aldoses react with
phenylhydrazine to produce a
phenylhydrazone which further reacts with
another two molecules of phenylhydrazine to
yield osazone. Needle-shaped yellow osazone
crystals are produced by glucose, fructose and
mannose, whereas lactosazone produces
mushroom shaped crystals. Crystals of
different shapes will be shown by different
osazones. Flower-shaped crystals are produced
by maltose.
MATERALS AND METHODS
96 well plate, 0.5% solutions (of ribose,
glucose, fructose, lactose, sucrose and
starch), anthrone, H2SO4, orcinol, sodium
bicarbonate, DNS
Parts A-B are qualitative tests.Perform
parts A and B on 0.5% solutions of ribose,
glicose, fructoose, lactose, sucrose and
starch.
A. Reaction with anthrone and sulfuric acid
Place 1 drop of carbohydrates solution on
the spotting plate and add 10 drops of
anthrone reagent (0.2% anthrone in
concentrated sulfuric acid). Mix well and
observe the color.
B. Reaction with orcinol and hydrochloric
acid
Place about 1 drop of carbohydrate
solution on the spotting plate and add 2
drops of orcinol reagent (2 g orcinol and 0.2
ferric chloride per liter of concertrated
HCI). Mix well and then heat on a hot-plate
for 1 min. Cool. Observe the color.
Reducing Sugar Reactions (Experimental):
A. Reaction with Benedicts Reagent
Place 1 drop of carbohydrate on the
spotting plate and add 10 drops of
Benedicts reagent (an alkaline solution
containing the cupric ion). Mix well and
then heat on a hot late for about 5 minutes.
Observe the results.
B. Reaction with dinitrosalicylic acid
Dilute 1 drop of carbohydrate solution with
5 drops of water. Then add 2 drops of
dinitrosalicylic acid (DNS) reagent (an
alkaline solution containing dinitrosalicylic
acid). Mix well and then heat on a hot plate
for about 5 minutes. Observe the results.
C. Quantitative application of a reducing
sugar reaction
Reducing carbohydrates can be
determined by a variety of methods, one of
which is the socalled Nelson method. In
this method, an alkaline solution
containing the cupric ion is first used to
oxidize the carbohydrate. The cuprous
 oxide formed as a result of this is in turn Color Change
used to reduce the arsenomolybdate ion Lactose -
and give rise to a deep bluegreen Ribose light green
chromophore that can be determined
Starch -
colorimetrically. The purpose of this
experiment is to acquaint you with the Sucrose blue
Nelson method as an example of a Glucose -
quantitative application of a reducing Fructose blue-green
sugar reaction. Over the years, methods
such as this have been used widely used in
both industrial and clinical laboratories.
While they are losing popularity in
laboratories concerned with clinical
chemistry, they continue to be popular in
laboratories concerned with starch
chemistry. You will make use of this
method in a subsequent experiment Figure 1
involving the action of amylase on starch.
Before starting the experiment outlined
below, be sure all of the glassware you
intend to use is thoroughly clean since the
experiments you performed in parts A and
B used %0,5 solutions of carbohydrates
and your pipettes and test tubes might still
contain enough carbohydrate to ruin your
assay.
Figure 2

Tube No ml Standard ml water ml Unknown DNS Reaction with orcinol and hydrochloric acid:
0 0 1.0 0 0.5
1 drop of carbohydrate solution on the spotting
1 0.2 0.8 0 0.5
plate and 2 drops of orcinol reagent.
2 0.4 0.6 0 0.5
3 0.6 0.4 0 0.5 Color Change
4 0.8 0.2 0 0.5 Lactose -
5 1.0 0 0 0.5 Ribose -
6 0 0 1.0 0.5 Starch -
Sucrose -
Add to each tube 500 L DNS. Place the tubes Glucose -
in an actively boiling water bath for 5 minutes. Fructose -
Cool the tubes in a beaker of cold water (1-2
min).Dilute to the graduation mark and mix
well. Determine the absorbance of each tube at Reducing Sugar Reactions (Experimental):
540 nm. Set tube 0 at zero absorbance.
Reaction with Benedicts Reagent:

RESULT Color change graphics;

Reaction with anthrone and sulfuric acid: Lactose Ribose Starch Sucrose Glucose Fructose
Benedicts (10 drops) blue-green red red - blue-green -
We dropped a drop ribose, glucose, fructose, DNS (2 drops) red red - - red red
lactose, sucrose, starch and 10 drops anthrone
in each tubes.
 Tube No ml Standard ml water ml Unknown DNS
0 0 1.0 0 0.5
1 0.2 0.8 0 0.5
2 0.4 0.6 0 0.5
3 0.6 0.4 0 0.5
4 0.8 0.2 0 0.5
5 1.0 0 0 0.5
6 0 0 1.0 0.5

Figure 3

Reaction with dinitrosalicylic acid:

1 drop of carbohydrate solution with 5 drops of

water then added 2 drops of DNS.
Figure 6 (First Situation)

Figure 4 (First Situation)

Figure 7 (boiling water bath for 5 minutes)

Figure 5

Quantitative application of a reducing sugar

reaction: Figure 8 (place 200 l in 96 well plate)

Determine the absorbance of each tube at 540

nm;
 vlab.amrita.edu/?sub=3&brch=63&sim=631&c
nt=1

http://www.chem.ucalgary.ca/courses/351/C
arey5th/Ch25/ch25-2-5.html

R. L. Whistler and M. L. Wolform, Methods in

Carbohydrate Chemistry, Vol I, Academic
Press, New York, 1962. This is the classic fully
detailed description of these reactions.

J. F. Robyt and B. J. White, Biochemical

Techniques: Theory and Practice, 2nd Edition.
Calculations: Waveland Press, Prospect Hts. IL, 1992. This
book by two professors at Iowa State provides
y=0.001x-0.0433 a clear description of the color reactions for
unknown absorbance= 0.3294 sugars.
0.3294=0.001x-0.0433
x=372.7

DISCUSSION
The most important thing in this experiment is
to determine the carbohydrate
(monosaccharide, disaccharide..). We can use
chromatography when determining
carbohydrates, even chromatography is
better. However we used color reactions in
experiment. One of the most important
considerations in the experiment is the
conversion of carbohydrates to furfural. (first
stage) After that, it combines with the
necessary chemicals to give color.
Carbohydrates arent given color section
experiment in reaction with orcinol and
hydrochloric acid. Probably 2 g orcinol and 0.2
ferric chloride per liter of concertrated HCI
condition may not have been made. On the
other hand, lactose and glucose are blue-green
color (Benedicts 10 drop) in reaction with
Benedicts Reagent. Great posibillity they are
not well mixed so they are blue- green. Actually
they had to be red.

REFERENCES
msu.edu/course/lbs/145/luckie/Lab1.html

katedrachemii.sum.edu.pl/uploaded/Carbohy
drates%20reactions_students.pdf