QUALITATIVE COLOR REACTIONS FOR CARBOHYDRATES

Tiffany Espiritu, Lorenz Rey A. Esteban, Kathleen Francisco, Casey Garcia, Aimee Hermoso

Group IV, 2E- Pharmacy, Faculty of Pharmacy, University of Santo Tomas

ABSTRACT

Carbohydrates refer to compounds of the general formula Cn(H2O)n (Campbell & Farrel,
2012). They are of different types, namely: monosaccharides, oligosaccharides, and
polysaccharides. In the experiment, a polysaccharide was used, which is glycogen. The glycogen
was extracted from chicken liver and was subjected to acid hydrolysis. The hydrolysate obtained
was then used for the color reactions. In this experiment, the qualitative tests for carbohydrates
were Benedicts test, Barfoeds test, Seliwanoffs test, Bials Orcinol test, Mucic acid test, and
Phenylhydrazone test. Aside from the hydrolysate, other carbohydrate solutions were also tested.
These were glucose, fructose, xylose, lactose, sucrose, and starch. For the Benedicts test,
glucose, fructose, xylose, lactose, starch, and the hydrolysate yielded positive results; for
Barfoeds test the solutions that showed positive results were glucose, fructose, and xylose;
fructose and sucrose yielded positive results for Seliwanoffs test; and for Bials orcinol test, only
xylose showed a positive result. For the Mucic acid test, galactose was the one that produced
colorless crystals. And lastly, for Phenylhydrazone test, fructose, xylose, lactose, and glucose
were the carbohydrate solutions that formed osazones.

INTRODUCTION The different qualitative tests are the

Carbohydrates are polyhydroxy
aldehydes (aldoses) and polyhydroxy
ketones (ketoses) with the general formula
(CH2O)n (Crisostomo, et al., 2010). The
three classifications of carbohydrates are
monosaccharides, oligosaccharides, and
polysaccharides. According to Campbell and
Farrel (2012), monosaccharides are the
simple sugars which are the building blocks
of carbohydrates. A monosaccharide can be
a polyhydroxy aldehyde (aldose) or a
polyhydroxy ketone (ketose).
Oligosaccharides are formed when a few
monosaccharides are linked with each other,
and polysaccharides are formed when many
monosaccharides are bonded together.
following: Benedicts test, Barfoeds test,
Seliwanoffs test, Bial Orcinols test, Mucic
acid test, and Phenylhydrazone test.
Benedicts test is a test to detect
reducing sugars. The reagent used is
Benedicts reagent which contains copper
(II) ions in alkaline solution with sodium
citrate. The positive visible result of this test
is a formation of brick-red precipitate.
Barfoeds test shows a positive
result for reducing monosaccharides. This is
done by treating a sample with Barfoeds
reagent (a solution of cupric acetate and
acetic acid). The result is a formation of
brick-red precipitate.

In performing the qualitative color A test to distinguish between

reactions, carbohydrate solutions (glucose,
fructose, xylose, lactose, sucrose, and
starch) and a hydrolyzed polysaccharide
were teste. In this experiment, glycogen,
which was obtained from chicken liver was
the one used.
hexoses with a ketone group and hexoses
that are aldehydes is called Seliwanoffs
test. To perform this, Seliwanoffs reagent is
added to the samples. Seliwanoffs reagent
is resorcinol in six molars (6M) hydrochloric
acid (HCl). The positive result is a formation
of cherry-red precipitate. Other sugars
appear as yellow to faint pink colors.
Bials Orcinol test is a test for
pentoses. The reagent used contains orcinol
(5-methylresorcinol) in concentrated
hydrochloric acid (HCl) with a small amount
of ferric chloride (FeCl3). A positive test is
indicated by a formation of a blue-green
solution. All other colors formed are
negative results since hexoses also generally
reacts in this test.
Mucic acid test uses strong
oxidizing agents like concentrated nitric acid
(HNO3). In this test, colorless crystals are
produced as a result of the oxidation of
galactose. The crystals are called the
dicarboxylic acid or the mucic acid.
By their formation of osazones with
a phenylhydrazine reagent, reducing sugars
may be differentiated from each other. This
can be done by performing the
phenylhydrazone test. The reducing sugars
may be differentiated by microscopic
examination of their formed osazones.
PROCEDURE
Benedicts, Barfoeds, Seliwanoffs, and bath for thirty (30) minutes. The time yellow
Bials Orcinol Tests
Seven test tubes were prepared for
each test. In these separate test tubes, five
(5) drops of the hydrolysate and the
carbohydrate solutions (glucose, fructose,
xylose, lactose, sucrose, and starch) were
placed. One (1) milliliter of the required
reagent was then added to each tube. After
the reagents had been added, all test tubes
were placed, at the same time, into a boiling
water bath. Test tubes that gave positive
visible results were removed and the time it
took for them to appear was noted.
Mucic Acid Test
Three (3) drops each of the
carbohydrate solutions (galactose and
lactose) and three (3) drops of nitric acid
(HNO3) were mixed on a glass slide. After
which, the glass slide was passed over a
small flame until it was almost dry and was
cooled to room temperature. The last
procedure was to examine the crystals
formed under the microscope.
Phenylhydrazone Test
The first step performed for this test
was to prepare the phenylhydrazine reagent.
This was done by mixing two (2) grams of
phenylhydrazine hydrochloride, three (3)
grams sodium acetate (CH3COONa), and ten
(10) milliliters of distilled water. The
reagent was placed in a warm water bath and
was stirred until it became clear.
In different test tubes, two (2) drops
of the carbohydrate solutions (glucose,
fructose, xylose, lactose, sucrose, and
starch) were mixed with four (4) drops of
the freshly prepared phenylhydrazine
reagent. The test tubes were covered with
cotton and were heated in a boiling water
crystals first appeared was recorded.
After the required time for heating
was done, the tubes were cooled. Samples of
crystals that appeared in the test tubes were
smeared in glass slides and were observed
under the microscope.
RESULTS AND DISCUSSION
From the experiment, the following results
were obtained:
 Table 1. Results of the color reactions hydroxymethylfurfural that
condenses with resorcinol.
Carbohydrate Benedicts Barfoeds Seliwanoffs Bials
Solution A blue-green solution is the
Glucose Brick- red Brick-red No No positive result for Bials orcinol test,
ppt. ppt. change change which is a test for pentoses. From the
(1 min., 17 (2 mins.) experiment, only xylose showed this
secs.) result. Therefore, it can be said that
Fructose Brick- red Brick-red Cherry-red Brown xylose is a pentose. The production of a
ppt. ppt. (2 ppt. (2 mins.) soln blue-green solution is due to the
(1 min., 45 mins.) dehydration of pentoses to form
secs.) furfural that condenses with orcinol.
Xylose Brick- red Brick-red No Blue-
ppt. ppt. (2 change green
(1 min., 11 mins., 30 soln Figure 1. Mucic acid test under the
secs.) secs.) microscope
Lactose No No No No
change change change change
Sucrose Brick- red No Cherry-red Brown
ppt. change ppt. soln
(1 min., 7 (2 mins., 15
secs.) secs.)
Starch No No No No
change change change change
Hydrolysate: Brick- red No No Green
Glycogen ppt. change change Soln
(10 mins.,
22 secs.) For the Mucic acid test,
galactose is the carbohydrate that
Based on Benedicts test, the formed crystals. These are called the
reducing sugars are glucose, fructose, dicarboxylic acid or the mucic acid.
xylose, and sucrose. It can also be said that a These crystals were formed due to
reducing sugar is present in glycogen since the oxidation of galactose.
it yielded a positive result. The brick-red
precipitate was formed due to the reduction Results for the
of blue cupric ion to cuprous oxide (Cu2O). phenylhydrazone test showed that
fructose, xylose, lactose, and
The monosaccharides in the samples glucose were the carbohydrate
tested are glucose, fructose, and xylose solutions that formed osazones.
because they all formed brick-red precipitate
with Barfoeds test. The precipitate was Figure 2. Osazone formed by fructose
produced because of the reduction of the
monosaccharides to cuprous oxide (Cu2O).

Fructose and sucrose showed

positive results for Seliwanoffs test. These
indicate that both are ketohexoses. The
cherry-red precipitate is due to the
dehydration of ketohexoses to form
Figure 3. Osazone formed by xylose
Figure 4. Osazone formed by lactose
Figure 5. Osazone formed by glucose
CONCLUSION BASED ON
OBJECTIVES
From the various qualitative color
reactions performed in the experiment, it can
be concluded that glucose, fructose, xylose,
and sucrose are all reducing sugars since
they showed positive results for Benedicts
test.
Basing from Barfoeds test, the
monosaccharides are the following: glucose,
fructose, and xylose. For Seliwanoffs test,
the ketohexoses are fructose and sucrose.
Formation of a blue-green solution
in Bials orcinol test indicates that a
carbohydrate solution is a pentose. In this
test, only xylose showed this result. Xylose
is therefore a pentose.
Through the Mucic acid test, it can
be said that galactose undergone oxidation
upon the addition of nitric acid (HNO3).
This can be seen when the slide is examined
under the microscope where colorless
crystals are formed.
Finally, in the phenylhydrazone test,
fructose, xylose, lactose, and glucose were
the carbohydrate solutions that formed
osazones. The osazones they produced can
be used as a means to distinguish them from
one another.
REFERENCES
Campbell, M.K., & Farrell, S.O. (2012).
Biochemistry (7th ed.). Brooks/Cole
Cengage Learning.
Crisostomo, A.C., et al. (2010).
Laboratory Manual in General
Biochemistry. C & E Publishing,
Inc.
Barfoeds Test (n.d.). Retrieved February
26, 2014 from
http://www.harpercollege.edu/tm-
ps/chm/100/dgodambe/thedisk/carb
o/barf/barfoed.htm
Test for Carbohydrates (n.d.) Retrieved
February 26, 2014 from
http://facstaff.gpc.edu/~msakuta/che
m1152L/lab8.pdf
Bials Test (n.d.) Retrieved February 26,
2014 from http://www.harpercollege
.edu/tmps/chm/100/dgodambe/
thedisk/carbo/bial/bials.h