Abstract

The experiment in reactions of carbohydrates is done to distinguish one carbohydrate from

another by conducting different quantitative tests, and to identify two unknown samples based
on the results. Nine carbohydrates were placed in separate test tubes, where two are
unknown samples, are subjected to different tests. The known carbohydrate samples are
glucose, galactose, ribose, fructose, lactose, sucrose, and starch. The different tests
conducted in the experiment are Molisch test, Iodine test, Benedicts test, Barfoeds test,
Seliwanoffs test and 2,4-DNP test. For Molisch and Iodine test, 1 ml of the reagent are added
to each of the samples and observed for reactions. For Benedict, Barfoed, Seliwanoff, and
2,4-DNP test, 1 ml of the reagent are added to the different samples and heated using water
bath. After observing and comparing the results, the unknown 1 is identified as fructose while
the unknown 2 is identified as the lactose.
Introduction
Carbohydrates are carbon compounds that contain great amounts of hydroxyl groups. All
carbohydrates can be categorized as monosacchrides, oligosaccharides or polysaccharides.
Monosaccharides consist of single carbohydrate molecule, containing between 3 to 7
carbons. Examples of monosaccharide are glucose and fructose, which is our primary energy
source. They are broken down quickly by the body and are building blocks for complex
carbohydrates. A disaccharide consists of two monosaccharaides that are linked together.
Starch from plants and glycogen from animals used to store glucose for readily accessible
form. They come from lactose, maltose and sucrose. Oligosaccharides are complex
carbohydrates as they are consist of three to ten sugars. They are rich in vitamins and
minerals; and, because they are fiber-rich, they are slower to digest than a simple
carbohydrate. On the other hand, polysaccharides are also complex carbohydrates and are
rich in vitamins, minerals and fiber; but they have larger numbers of sugars than
oligosaccharide.
Carbohydrates can be differentiated by their reactions with various reagents. Qualitative tests
can be done to yield the carbohydrates reactions by their change in color after reacting to
different reagents. Each test reagents have specific functional groups. These reactions can
identify the presence or absence of carbohydrates in the sample.
Materials and Method
For the identification of unknown carbohydrate samples, 1.00 ml of the known
carbohydrate samples and the two unknown samples were transferred on separate test
tubes. Six different tests are done and observed for their reactions. The different tests
performed are the following:
TEST

AMOUNT OF REAGENT

PROCEDURE

Iodine test

1.00 ml Molisch reagent +

1.00 ml concentrated H2SO4
1.00 ml iodine reagent

Benedicts test

1.00 ml Benedict reagent

Heated using water bath

Barfoeds test

1.00 ml Barfoed reagent

Heated using water bath

Seliwanoffs test

1.00 ml Seliwanoff reagent

Heated using water bath

2,4-DNP test

1.00 ml 2,4-DNP

Heated using water bath

Molisch test

Visible color changes are observed on each reaction. After the tests were performed, the
identities of the unknown samples were determined by comparing it to the known
carbohydrate samples.

Results and discussion

The table below summarizes the positive results for the carbohydrates tested with different
reagents.
Table 1. Positive Reactions for Carbohydrates Test
Test
Positive Color Change
Molisch
Violet
Iodine
Blue-black complex
Benedict
Rust-colored precipitates
Barfoed
Orange-red color
Seliwanoff
Deep red color
2,4-DNP
Yellow-black crystals
Carbohydrates can be identified through their reactions in different tests. Molisch test is a
general test for all carbohydrates which detects the presence of carbonyl in carbohydrates
that shows a deep purple color upon oxidation. In iodine test, a positive reaction shows a
blue-black complex which indicates the presence of starch. Benedicts test is used to
determine the presence of reducing sugars which gives a rust-colored precipitate. Barfoeds
test is used to distinguish the presence of reducing monosaccharides and disaccharides
which shows a orange-red color for positive reaction. Seliwanoff test distinguishes aldoses
from ketoses, wherein the formation of deep red color indicates a positive reaction for ketose.
The 2,4-DNP test is also a general test for carbohydrates except starch, which shows a
yellow-black color for its positive reaction.
Table 2. Reaction of Carbohydrates Samples
Samples
Molisch
Iodine
Benedict
Barfoed
Glucose
++

++
+
Galactose
++

++
+
Ribose
++

++
+
Fructose
++

++
+
Lactose
++

++

Sucrose
++

Starch
+
+

Unknown 1
++

++
+
Unknown 2
++

++

Identity of Unknown 1
Fructose
Identity of Unknown 2
Lactose
Legend: ++ positive, fast reaction / + positive, slow reaction /
negative, no reaction

Seliwanoff

++

++

2,4-DNP
+
+
+
+
+
++
+
+
+

In the experiment, all the samples gave a positive result in Molisch test because all of the
samples contain a carbonyl group. The iodine test is done to distinguish starch, so the starch
is the only sample that gave a positive reaction, while the others gave a negative or no
reaction.
In Benedicts test, only starch gave a negative reaction. In Barfoeds test, only lactose,
sucrose (both disaccharides), and starch (a polysaccharide) gave negative reactions because
Barfoeds test only give a positive result to monosaccharides. In Seliwanoff test, fructose and
starch are the only reagents which gave a positive result because fructose is basically a
ketose, and the sucrose is a polysaccharide formed from glucose and fructose. After
comparing all the results acquired from the experiment, the unknown 1 is identified as
fructose and unknown 2 is identified as lactose.

Reference:
(1) Legaspi. G.A. (2012). Essentials of Biochemistry Laboratory.
(2) Reactions of Carbohydrates | Biochemistry for Medics ... (n.d.). Retrieved October 22,
2016, from http://www.namrata.co/category/practical-biochemistry/reactions-of-carbohydrates/