QUALITATIVE TESTS FOR CARBOHYDRATES

Dianne I. Pascua, Francesca F. Perez, Romulo Vincent S. Perez,

Lia S. Priela and Zeth Raquedan
Group 8 2C Medical Technology Biochemistry Laboratory
ABSTRACT
The experiment involved five carbohydrates subjected to different qualitative tests. Glucose, fructose,
xylose, lactose and sucrose were used for Benedicts Test. All five sugars gave a positive result of brick
red precipitate. Barfoeds Test was used on glucose, fructose, xylose, lactose and sucrose. Xylose was the
first to produce a red precipitate, followed by fructose, glucose and sucrose. Lactose did now form a
precipitate. Seliwanoffs test was performed using glucose, fructose and xylose. Fructose was the only
carbohydrate that gave the positive result of cherry-red precipitate. Bials-Orcinol Test was performed
using glucose, fructose and xylose. Xylose gave the positive result of dark green solution. Mucic acid test
was performed using lactose then the crystals were observed using a microscope. The Phenylhydrazone
test was performed on lactose and sucrose. The crystals obtained were then observed under the
microscope.

INTRODUCTION
Carbohydrates
are
polyhydroxyaldehydes
(aldoses) and polyhydroxyketones (ketoses) with
the general formula of (CH2O)n, where n is equal
to 3 or more.
Carbohydrates are the simplest biomolecules
and are the most important source of energy for
the body. They are widely distributed in animal
and plant tissues. In plants, they are produced by
photosynthesis resulting to cellulose and starch.
In animals, they are in the form of glucose and
glycogen. They are sources of a large part of the
heat and mechanical energy requirements of the
body. Other functions of glucose include: for
structural integrity (cellulose); for transport of
nutrients (glycoproteins in plasma); for catalytic
activity
(ribonuclease
and
amylase);
for
regulatory function (FSH, LH); for immunity
(immunoglobulin, interferon and Rh factor); for
cell differentiation (ABO blood group); as a
constituent of the cell membrane; for blood
clotting; and as protective cellular coat proteins
(fibrinogen).
Carbohydrates can be classified into 3 general
classes
depending
on
the
number
of
carbohydrate
molecules
they
contain:
monosaccharides, which are the simplest;
oligosaccharides,
which
contain
2-10
monosaccharide units; and polysaccharides,
which contain more than 10 monosaccharide
units.
Monosaccharides are further classified by the
number of carbon atoms they contain. Mono-

saccharides having 5 carbons are called pentoses

while those with 6 carbon atoms are called
hexoses. Ribose and xylose are examples of
aldopentoses, while glucose and galactose are
examples of aldohexoses. Fructose is an example
of a ketohexose. Oligosaccharides are subdivided
into disaccharides (2 monosaccharide units),
trisaccharides (3 monosaccharide units) and so
forth. Sucrose, maltose and lactose are common
examples of disaccharides. Polysaccharides are
made up of hundreds of thousands of
monosaccharide units connected in different
patterns. Starch, cellulose and glycogen are the
most important examples of polysaccharides.

EXPERIMENTAL
A. Samples used
Glucose, fructose, xylose, lactose, sucrose
B. Procedure
Benedicts test: 5 test tubes were prepared
each separately containing 5 drops of glucose,
fructose, xylose, lactose and sucrose. 1 mL of
Benedicts reagent was added to each of the 5
test tubes. The test tubes were then subjected to
a boiling water bath for 5 minutes. The colors of
the results were then noted.
Barfoeds test: 5 test tubes were prepared each
separately containing 5 drops of glucose,
fructose, xylose, lactose and sucrose. 1 mL of
Barfoeds reagent was added to each of the 5 test
tubes. The test tubes were then subjected to a

boiling water bath. The time it took for a visible

result to appear was noted.
Seliwanoffs Test: 3 test tubes were prepared
each separately containing 5 drops of glucose,
fructose and xylose. 1 mL of Seliwanoffs reagent
was added to each of the 3 test tubes. The test
tubes were then subjected to a boiling water bath
for 5 minutes. The colors of the results were then
noted.
Bials-Orcinol Test: 3 test tubes were prepared
each separately containing 5 drops of glucose,
fructose and xylose. 1 mL of Bials-Orcinol
reagent was added to each of the test tubes. The
test tubes were then subjected to a boiling water
bath for 5 minutes. The colors of the results were
then noted.
Mucic Acid Test: 3 drops of the carbohydrate
solution and 3 drops of conc. HNO3 were mixed in
a test tube. 10 mL of distilled H2O was then
added. The test tube was then placed in a water
bath until the solution cleared. The crystals were
then isolated and observed under a microscope.
Phenylhydrazone Test: The phenylhydrazine
reagent
was
prepared
by
mixing
2 g
phenylhydrazine hydrochloride, 3 g CH3COONa
and 10 mL distilled H2O in a test tube. The
reagent was then placed in a water bath and
stirred until the solution cleared. In 2 different
test tubes each separately containing 2 drops of
lactose
and
sucrose,
4
drops
of
the
phenylhydrazine reagent was placed. The test
tubes were the covered with cotton and
subjected to a boiling water bath for 30 minutes.
The test tubes were then cooled and the crystals
isolated and observed under a microscope.

RESULTS AND DISCUSSION

Table 1. Results from Benedicts Test
Carbohydrate Solution
Glucose
Fructose
Xylose
Lactose
Sucrose

Benedicts Test
Brick-red precipitate
Brick-red precipitate
Brick-red precipitate
Brick-red precipitate
Brick-red precipitate

The Benedicts Test is a test for reducing

sugars. The Benedicts reagent is composed of
copper sulfate, sodium citrate and copper (II)
ions in alkaline solution. The alkaline condition
causes isomeric transformation of ketoses to

aldoses resulting in the reduction of blue cupric

ion to cuprous oxide (Cu2O), a brick-red
precipitate.
This
is
exhibited
by
all
monosaccharides and most disaccharides. All
carbohydrate samples yielded a brick-red
precipitate. Sucrose, which is a disaccharide,
should have been the only one that did not
produce a brick-red precipitate since it is a nonreducing sugar. This may be due to an error by
the experimenter.
Table 2. Results from Barfoed's Test
Carbohydrate
Solution
Glucose
Fructose
Xylose

Barfoeds Test
Brick-red precipitate (2:24
minutes)
Brick-red precipitate (2:00
minutes)
Brick-red precipitate (1:58
minutes)

Lactose

No precipitate

Sucrose

Brick-red precipitate (5:00

minutes)

The
Barfoeds
Test
detects
reducing
monosaccharides
in
the
presence
of
disaccharides. The reagent uses copper ions to
detect reducing sugars in acidic solution. Xylose
was the first carbohydrate to form a precipitate,
followed by fructose, glucose and sucrose.
Lactose did not form a precipitate. Lactose is a
reducing sugar.
Table 3. Results from Seliwanoff's Test
Carbohydrate Solution
Glucose
Fructose
Xylose

Seliwanoffs Test
Orange solution
Cherry-red condensate
Black solution

In Seliwanoffs Test, the reagent is resorcinol.

Ketohexoses undergo dehydration to form
hydroxymethylfurfural
that
condenses
with
resorcinol to form a cherry-red condensation
product. Since fructose yielded a cherry-red
condensate, it is a ketohexose.
Table 4. Results from Bial's Orcinol Test
Carbohydrate Solution
Glucose
Fructose
Xylose

Bials-Orcinol Test
Yellow solution
Brown solution
Dark green solution

In Bials-Orcinol Test, the ragent contains

orcinol in conc. HCl with a small amount of FeCl 3.
Pentoses undergo dehydration to form furfural
that condenses with orcinol to create a bluegreen solution. Since xylose yielded a dark green
solution, it is a pentose.
In the Mucic Acid Test, aldohexoses are
converted to their corresponding dicarboxylic
acids in the presence of strong oxidizing agents
like conc. HNO3. The dicarboxylic acid (mucic
acid) produced from the oxidation of galactose is
relatively insoluble and separates out as colorless
crystals.

Figure 2. Osazone crystals from lactose

CONCLUSION

Figure 1. Mucic acid crystals

In the Phenylhydrazone Test, reducing sugars
may be differentiated from each other by the
phenylhydrazones (osazones) they form with the
phenylhydrazine reagent. The osazones from
different reducing sugars have characteristic
crystalline forms. They also differ in their
solubility in hot water.

Based from the results of the qualitative tests,

the carbohydrates can be classified as simple or
complez; monosaccharide, oligosaccharide or
polysaccharide;
reducing
or
non-reducing;
pentose or hexose; and aldose or ketose.
The simple sugars are glucose, fructose,
xylose, lactose and sucrose. Starch is a complex
sugar.
The monosaccharides are glucose, fructose and
xylose. Lactose and sucrose are disaccharides.
Starch is a polysaccharide.
The reducing sugars are glucose, fructose,
xylose and lactose. The non-reducing sugar is
sucrose.
Sugars that are hexoses are glucose, fructose,
lactose and sucrose. Xylose is a pentose sugar.
Sugars that are aldoses are glucose and xylose.
Fructose is a ketose.

REFERENCES
[1] Crisostomo, A. C., Daya, M. L., De
Guia, R. M., Farrow, F. L., Gabona, M. G.,
Liu, M. I., . . . Ysrael, M. C.
(2010). Laboratory Manual in General

Biochemistry. Quezon City, Philippines: C

& E Publishing, Inc.

[2] Horton, H. R., Moran, L. A., Ochs, R. S.,

Rawn, D. J., Scrimgeour, K. G., Moser, R.
L., et al. (2008). Principles of
biochemistry (3rd ed.). New York:
Prentice Hall.

[3] Campbell, M. K., & Farrell, S. O.

(2012). Biochemistry (7th ed.). Belmont,
California: Brooks/Cole, Cangage
Learning.

[4] Qualitative Analysis of Carbohydrates

(Theory) : Biochemistry Virtual Lab I :
Biotechnology and Biomedical Engineering
: Amrita Vishwa Vidyapeetham Virtual
Lab. (n.d.). Retrieved February 19, 2014,
from
http://amrita.vlab.co.in/?sub=3&brch=63
&sim=631&cnt=1