SCIENCE 103: BIOCHEMISTRY

UNIVERSITY OF THE CORDILLERAS

COLLEGE OF ARTS AND SCIENCES
INSTRUCTOR: MRS. MA. CRISTINA P. BENTADAN

GROUP #: ___6____________________ SCORE: __________________

YEAR AND SECTION: __BSP2A_______________ DATE: __01-29-21___
(Source: Baliton, M.C., SLU Lab Manual)

ACTIVITY 6
TEST FOR CARBOHYDRATES

I. OBJECTIVES:
 This activity will enable students to:
o Understand the physical and chemical properties of carbohydrates
o Determine classifications of carbohydrates
o Determine the different tests for carbohydrates

II. PROCEDURE (Research Work)

 Please do a research work regarding the following:
(DO NOT perform these experiments at home)

A. GENERAL COLOR TEST (5 POINTS EACH ITEM)

1. Molisch Test
Definition: t is a sensitive chemical test for all carbohydrates, and some compounds containing
carbohydrates in a combined form, based on the dehydration of the carbohydrate by sulfuric
acid to produce an aldehyde (either furfural or a derivative), which then condenses with the
phenolic structure resulting in a red or purple-colored compound..
Purpose: It can be used to differentiate proteins and amino acids from carbohydrates.
Procedure:
(a) Place 2 mL of a known carbohydrate solution in a test tube, add 1 drop of Molisch’s
reagent (10% α-naphthol in ethanol).
(b) Pour 1-2 mL of conc. H2SO4 down the side of the test tube, so that it forms a layer at
the bottom of the tube.
(c) Observe the color at the interface between two layers and compare your result with a
control test
Result: It will then be resulted from the development of a purple ring at the layer formed by the
concentrated acid and is a positive indicator for Molisch’s test. If no purple or reddish-purple
color arises, the given analyte does not contain any carbohydrate.

2. Anthrone Test
Definition: Anthrone Test is a group test for carbohydrates that provides a rapid and convenient
method for quantification of carbohydrates that are either free or bound to any lipids or proteins.
Purpose: To detect the presence of carbohydrates in a given solution like blood, serum, milk,etc.
and to quantify the concentration of free and bound carbohydrates in a solution.
Procedure:

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(a) Prepare the sample in test tube and take distilled water in another tube as control.
(b) Then add 2ml of anthrone reagent to all the tubes and mix thoroughly all the content of
the tube.
(c) Observe for color change.
Result: The presence of a blue-green complex indicates the presence of carbohydrates in the
given solution.

B. TESTS FOR REDUCING SUGARS

1. Benedict’s Test
Definition: It is a chemical test that can be used to check for the presence of reducing sugars in a
given analyte.
Purpose: It is used to determine the presence or absence of reducing sugar in the solution.
Procedure:
(a) Taking one milliliter of the analyte solution in a test tube and mixing it with two
milliliters of Benedict’s reagent.
(b) Then, this mixture must be heated in a hot water bath for approximately 3 minutes (or
until a visible change in color occurs).
*Benedict’s solution can be prepared by complexing cupric ions (Cu2+ cations) from the copper
sulfate pentahydrate with citric acid molecules in a basic environment provided by sodium
carbonate.
Result: When exposed to reducing sugars, the reactions undergone by Benedict’s reagent result
in the formation of a brick-red precipitate, which indicates a positive Benedict’s test.

2. Nylander’s Test
Definition: It is a chemical test used for detecting the presence of reducing sugars. The solution
forms a black precipitate in a positive reaction.
Purpose: It is used to detect the presence of a reducing sugar.
Procedure:
(a) Take 5 ml sample solution in a test tube.
(b) Add 0-5 ml Nylander’s reagent.
(c) Boil for 3 minutes and cool.
Result: Whensugar is present; there is a brown to black colouration of the fluid where the
metallic bismuth settles down. If the dark coloration occurs as the fluid is cooling down, it does
not prove the presence of sugar. The reaction takes place when the proportion of sugar in the
urine is 0.1 percent. If the Nylander test gives a negative result, there is no need to do any further
test.

3. Barfoed’s Test
Definition: Barfoed’s test is a chemical test used for detecting the presence of monosaccharides.
It is based on the reduction of copper (II) acetate to copper (I) oxide, which forms a brick-red
precipitate.
Purpose: It is used to differentiate reducing monosaccharides from reducing disaccharides sugar.
Procedure: Three ml of Barfoed’s reagent (a solution of cupric acetate and acetic acid) is added.
The solution is then heated in a boiling water bath for three minutes.
(a) Take 1ml of test sample in dry test tube.
(b) Take 1ml of distilled water in another tube as control.
(c) Add 2ml of Barfoed’s reagent to all the tubes.

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(d) Keep in boiling water bath.
(e) Look for the developmental of brick red precipitate.
(f) Note the time taken to develop color.
Result: A red copper (II) oxide precipitate is formed that indicates the presence of reducing
sugar. The reaction was negative in the presence of disaccharide sugars because they are weaker
reducing agents.

4. Fehling’s Test
Definition: is a chemical test to detect reducing sugars and aldehydes in a solution, devised by
the German chemist Herman Christian von Fehling (1812-1885).
Purpose: It is used to differentiate between water-soluble carbohydrate and ketone functional
groups, and as a test for reducing sugars and non-reducing sugars, supplementary to the Tollen’s
reagent test.
Procedure:
(a) Add the sample in a dry test tube.
(b) Distilled water should be kept in another tube as control.
(c) Fehling’s solution to be added in the tubes.
(d) The tubes must be kept in water bath.
(e) Make observations and record if there is any developmental and red precipitate.
Result: Formation of red precipitate confirms the presence of reducing sugars. The copper ions
present in fehling’s solution in +3 state is reduced to +2 oxidation state and in alkaline medium it
is precipitated as red cuprous oxide.

5. Picric Acid Test

Definition: The name “picric” comes from the Greek word “pikros”, meaning bitter, due to its
bitter taste. It is one of the acidic phenols. Like other strongly nitrated organic compounds, picric
acid is an explosive, hence its primary use.

Purpose:  It is used for the presence of reducing sugars.

Procedure:
(a) Add 1ml of the above reagent to 1mL of the test solution followed by 0.5 mL of 10%
Sod. Carbonate solution.
(b) Heat the test tube in a boiling water bath.
(c) Observe the color of the solution.
Result: In the presence of reducing sugars, the solution stains red; a sodium salt of picric acid is
formed.

6. Tollen’s Test
Definition: It is a qualitative test also knows as mirror test.
Purpose: It used to differentiate reducing sugars from non-reducing sugars. It is very useful
method to distinguish between aldehydes and ketones.
Procedure:

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(a) Take two clean, dry test tubes and add 1ml of the test sample in one test tube and 1 ml of
distilled water in another as blank.
(b) Add 2 ml of Tollen’s reagent to both the test tubes.
(c) Keep both the test tubes in a water bath for 1 min.
(d) Observe the formation of color and note it down.
Result: Carbohydrates reacts with Tollens reagent forms a silver mirror on the inner walls of the
test tube. This confirms the presence of reducing sugars. Silver ions are reduced to metallic
silver.

C. MUCIC ACID TEST FOR GALACTOSE AND LACTOSE

Definition: Mucic acid test is a test that is highly specific and is used for the detection of the
presence of galactose and lactose. It is also termed galactaric acid that is named after the product
of the reaction.
Purpose: The mucic acid test is used to identify the presence of the sugar galactose in food or in
synthetics manufacture.
Procedures:
(a) Take 6 ml of each distilled water and test sugar solutions in four test tubes separately.
(b) Add 1 ml of Mucic acid reagent (concentrated nitric acid) to each tube.
(c) Heat the test tubes in the wa ter bath for 1-1/2 hours until the volume of the solution is
reduced to 2-3 ml.
(d) Let the test tube sit overnight before collecting the results.
Result: The formation of crystal at the bottom of the tube indicates a positive result which means
that the sample solution has galactose or its derivatives. The absence of such crystals indicates a
negative result and represents that the sample doesn’t have galactose or its derivative. The
solution might still have other carbohydrates.

D. SELIWANOFF’S TEST FOR KETOSES

Definition: Seliwanoff’s test is used to differentiate between sugars that have a ketone group
(ketose) and sugars that have an aldehyde group (aldoses). This test is a timed color reaction
specific to ketohexoses.
Purpose: To test which distinguishes between aldose and ketose sugars. If the sugar contains
a ketone group, it is a ketose.
Procedures
(a) Take two clean, dry test tubes and add 1 ml of the test sample in one test tube and 1 ml of
distilled water in another as blank.
(b) Add 2 ml of Seliwanoffs’ reagent to both the test tubes.
(c) Keep both the test tubes in a water bath for 1 min.
(d) Observe the formation of color and note it down.
Result: The formation of the cherry red-colored complex indicates a positive result which means
that the given sample contains ketoses. The absence of such color or the appearance of the color
after a prolonged period of time indicates a negative result which means that the test sample
doesn’t have ketoses.

E. TESTS FOR PENTOSES

1. ANILINE ACETATE TEST

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Definition: It is a chemical test for the presence of certain carbohydrates, where in they are
converted to furfural with hydrochloric acid that reacts with aniline acetate to produce a bright
pink color.
Purpose: To identify the presence of certain carbohydrates.
Procedure:
(a) A dry sample is dissolved in a small volume of hydrochloric acid and briefly heated.
(b) A piece of paper, previously impregnated with aniline acetate, is exposed to the vapor
from the sample solution.
Result: A bright pink color on the paper is positive for the presence of pentoses.

2. BIAL’S TEST
Definition: It is a chemical test performed to detect the presence of pentoses and pentosans. A
derivation of this test termed the Bial’s Orchintest is performed to detect the presence of RNA in
solutions. This test is based on the principle that pentosans are hydrolysed into pentoses under
hydrolysis.
Purpose: To detect the presence of carbohydrates and to distinguish the pentoses and pentosans
from other derivatives of carbohydrates like the hexoses.
Procedure:
(a) Pipette out different volumes (50 µl, 100 µl, and so on) of ribose solution from the
supplied stock solution (200µg /ml) into a series of test tubes and make up the volume to
1 mL with distilled water.
(b) Take a tube labeled as one as blank containing 1ml of just distilled water and the rest of
the tubes labeled 2 to 9 for construction of a standard curve. Tubes 10-15 are for the
unknown samples.
(c) Add 5 ml of the bial’s reagent to each tube and mix well by vortexing.
(d) Cool the tubes.
(e) Cover the tubes with caps on top and incubate at 90°C for 17 minutes or boiling water
bath for 10 minutes.
(f) Cool the tubes to room temperature and measure the optical density of the solutions at
620 nm against a blank.
(g) Prepare a standard curve of absorbance against ribose concentration.
(h) Determine the amount of ribose in the unknown sample by plotting a standard curve of
A620 on the Y-axis and concentration of Ribose on the X-axis.
Result: The presence of a blue-green complex indicates the presence of pentoses in the sample.

III. QUESTIONS FOR RESEARCH. (2 POINTS EACH)

1. Why do all sugars give a positive result for Molisch’s Test? Does positive Test confirm the
presence of sugar? Explain.
A positive reaction for Molisch's test is given by almost all carbohydrates (exceptions
include tetroses & trioses). All carbohydrates such as monosaccharides, disaccharides and
polysaccharides give a positive reaction for Molisch test. Monosaccharides give a rapid
positive test. Disaccharides and polysaccharides react slower. It is based on the dehydration
of the carbohydrate by Sulphuric acid to produce an aldehyde, which condenses with two
molecules of α-naphthol, resulting in appearance of a purple ring at the interface.
2. From what other sources are pentose obtained?
- Pentose sugars are can also be obtained from woody biomass and various crop residues.
3. How can Seliwanoff’s test be used to distinguish fructose from sucrose?

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- Seliwanoff's test is a chemical test which distinguishes between aldose and ketose sugars.
A mixture of concentrated HCl and resorcinol of 3ml is added to the sugar. Then the
solution is boiled for little time. If it is a ketose sugar like, it is fructose and sucrose if the
solution turns cherry red. Also, If the sugar contains a ketone group, it is a ketose. If a
sugar contains an aldehyde group, it is an aldose
4. What compounds other than napthanol can be used for Molisch’s Test?
- The other compounds that can be used as reagent for the test is the molisch's test for the
presence or determination of carbohydrates in a solution by the use of Molisch reagent or
alpha-napthol and concentrated sulfuric acid
5. Why is it important that Fehlings solution be freshly prepared?
- Fehling's solution must be freshly prepared and not beforehand, for like all other copper
test solutions, it becomes unreliable in a short time, thereafter indicating sugar where none
exists. Also, it decomposes with time and becomes non-effective for the tests
6. Differentiate the action of a strong base from a weak base on a reducing sugar.
- A weak base is one that only partially dissociates to give ions in solution, while a  strong
base is one that fully dissociates to give ions in solution. Weak bases only partially
dissociate in a solution, while the strong bases dissociate fully in a solution.
7. How are conditions of Barfoed’s Test different from those of Benedicts Test?
- These two tests (Barfoed’s Test and Benedicts Test) are extremely similar. Barfoed's test
detects monosaccharides through a reduction reaction that precipitates copper (I) oxide
(which is red in color), and Benedict's reagent detects reducing sugars by producing the
same precipitate. However, the only real difference is that Benedict's produces a greater
volume of precipitate and the precipitate can vary in color depending on the amount of
sugar present.
8. Account for the presence of a black precipitate in Nylanders Test.
- When Nylander's reagent, which consists of bismuth nitrate, potassium sodium tartrate
and potassium hydroxide, is added to a solution with reducing sugars, a black
precipitate of metallic bismuth is formed.

IV. GENERALIZATION: (Note: In the generalisation, do not use the “first person” in making
statements. Focus your topic on the title of the exercise. (10 POINTS)
Carbohydrates are the most abundant and diverse class of organic compounds occurring in
nature. There are several types of tests for Carbohydrates such as: Molisch’s Test,
Anthrone Test, Benedict’s Test, Nylander’s Test, Barfoed’s Test, Fehling’s Test, Picric
Acid Test, Tollen’s Test, Mucic Acid Test, Seliwanoff’s Test, Aniline Acetate Test, and
Bial’s Test. Understanding the different tests would help to know how different chemicals
would react to the solution – be it in reducing sugars or to confirm the presence of starch,
carbohydrates, and so on. In addition, since carbohydrates are the most abundant and
diverse class of organic compounds, having a knowledge in the physical and chemical
properties of carbohydrates would help to determine its function and classification not only
on conducting test experiments but also understand its components as humans consume
carbohydrates as the primary fuel source of the body.