1.

Molisch Test (Alpha-Naphthol Reaction) 

For each of the given tests for the biological molecules above, identify and illustrate in t erms
of : 
. 

a. Description (one or two sentences only) 

1.Molisch Test (Alpha-Naphthol Reaction) 

A. Description
Molisch’s test is a chemical test which is used to check for the presence of carbohydrates in
a given analyte.
B. Reagents used
Molisch’s test involves the addition of :
Molisch’s reagent (a solution of ∝-naphthol in 5% ethanol)
Test solution: 5 % Glucose, 5 % Sucrose, 5 % Starch and;
Concentrated H2SO4 (sulphuric acid).
C.Procedures
2-3 drops of Molisch’s reagent must be added to a small amount of the analyte in a test tube
and mixed well. Now, a few drops of concentrated sulphuric acid must be added drop-wise
along the walls of the test tube to facilitate the formation of a layer and avoid mixing.
D.Illustrative Reaction

E.Expected Results
In Molisch’s test, the carbohydrate (if present) undergoes
dehydration upon the introduction of concentrated sulphuric acid,
resulting in the formation of an aldehyde. This aldehyde undergoes
condensation along with two phenol-type molecules (such as ∝-
naphthol, resorcinol, and thymol), resulting in the formation of a
purple or reddish-purple coloured complex.The development of a
purple ring at the layer formed by the concentrated acid is a
positive indicator for Molisch’s test. If no purple or reddish-
purple colour arises, the given analyte does not contain any
carbohydrate.
2. Heller’s Ring Test 

A. Description
Heller’s test is a biochemical test performed to detect proteins in a sample by the denaturation
of those proteins by the addition of strong acids. Heller’s test usually uses concentrated nitric
acid for the denaturation of proteins.
B. Reagents used.
Nitric acid (HNO3)
Sample
Materials required:
Test tubes
Test tube stand
Pipettes
C.Procedures
 1. In a clean and dry test tube, 2 ml of concentrated nitric acid is taken.
2. To this, 2 ml of urine or other sample is added. The sample should be poured from the
sidewall of the test tube in an inclined position in order to form a layer of the sample above
the nitric acid.
3. The test tube is then observed for the formation of a white ring at the junction of the two
layers.
D.Illustrative Reaction

E.Expected Results
Positive result: A positive result is represented by the formation of a white ring
(precipitated protein) at the junction of the two distinct layers. This indicates the
presence of proteins in the given sample.
Negative result: A negative result is represented by the absence of a white ring. This
indicates the absence of protein in the sample.Concentrated nitric acid is added to a
protein solution from the side of the test tube to form two layers. A white ring appears
between the two layers if the test is positive.
3. Xanthoproteic Reaction
A.Description
Xanthoproteic test is a chemical test which is used to check for the presence of proteins
containing an aromatic nucleus.
B.Reagents used
Xanthoproteic test involves the addition of:
Test solution: 1 % tyrosine, 1 % tryptophan, 1 % phenylalanine, 1% any other amino
acid, protein solution (albumin)
Nitric acid
40% NaOH
C.Procedures
1 ml of test solution must be added to a small amount of the analyte in a test tube. Now,
add 1ml of concentrated nitric acid (HNO3) must be added drop-wise along the walls of
the test tube to facilitate the formation of a layer and avoid mixing. Mix and heat the
protein solution, initially white precipitate appears due to denaturation of protein, which
turns yellow on heating).Cool under tap water.Add 1 ml of 40% NaOH to make the
 solution alkaline.Observe whether the mixture turns orange red in colour.

D. Illustrative Reaction

E. Expected
Results
When the strong basic solution is added the colour of obtained products turns darker (from
yellow to orange). The positive result of xanthoproteic reaction gives: tyrosine, tryptophan
and phenylalanine.Colour changes to orange indicating the presence of aromatic amino
acids. The negative result of the test: colour does not changes indicating absence of aromatic
amino acids.
4. Fehling’s Test
A. Description
Fehling’s solution is a chemical reagent 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.
B. Reagents used
Test solution: 5 % Glucose, 5 % Sucrose, 5 % fructose, 5 % Lactose, 5 % Starc
Fehling’s reagent (solution A: CuSO4.5H2O
Fehling’s reagent ( solution B: Sodium potassium tartrate)
Water bath
Pipettes
Dry test tubes
C. Procedure/s
1.Take 1ml of sample in dry test tube.
2.Take 1ml of distilled water in another tube as control.
3.Add 1ml of Fehling’s reagent (A and B) to all the tubes.
 4.Keep in boiling water bath.
5.Look for the development of red precipitate.

D. Illustrative Reaction
Fehling’s test: A chemical reaction used to determine if a molecule contains an aldehyde
or a terminal α-hydroxy ketone. To perform the test one adds Fehling’s solution (which is
blue due to the presence of Cu2+) to the unknown. If the unknown contains an aldehyde
the Cu2+ is reduced to Cu+ which forms a red Cu2O precipitate. A terminal α-hydroxy
ketone also gives a positive test because the Fehling’s solution oxidizes a terminal α-
hydroxy ketone to an aldehyde.

Fehling's solution is blue due to dissolved Cu2+. The solution is

Add glucose

Addition of glucose causes reduction of Cu2+ to red Cu2O, forming a

E. Expected Result
The appearance of a reddish-brown precipitate indicates a positive result and the presence of
reducing sugars.The absence of the reddish precipitate or the appearance of deep blue color
indicates a negative result and lack of reducing sugars.

5. Biuret Reaction
A. Description
Biuret test is a chemical test used to determine the presence of peptide bond in a substance.
B. Reagent used
 Copper sulfate (CuSO40
Sodium hydroxide (NaOH)
Sodium potassium tartarate (commonly known as Rochelle salt)
C. Procedure/s
1. Take 3 clean and dry test tubes.
2. Add 1-2 ml of the test solution, egg albumin and deionized water in the
respective test tubes.
3. Add 1-2 ml of Biuret reagent to all the test tubes.
4. Shake well and allow the mixtures to stand for 5 minutes.
5. Observe for any color change.
D. Illustrative Reaction

E. Expected Results
The expected result is the solution remains blue means that the proteins are not present. And
if the solution turn from blue to deep purple means that the proteins are present.

6. Benedict’s Test
A. Description
Benedict's reagent is a chemical reagent and complex mixture of sodium carbonate, sodium
citrate and copper(II) sulfate pentahydrate.
B. Reagent used
mixing copper sulfate pentahydrate (CuSO4. 5H2O),
sodium citrate (Na3C6H5O7), and
sodium carbonate (Na2CO3) in distilled water

C. Procedure/s
1. Approximately 1 ml of sample is placed into a clean test tube.
2. 2 ml (10 drops) of Benedict’s reagent (CuSO4) is placed in the test tube.
3. The solution is then heated in a boiling water bath for 3-5 minutes.
4. Observe for color change in the solution of test tubes or precipitate
 formation.

D. Illustrative Reaction

Positive
Benedict’s Test: Formation of a reddish precipitate within three minutes. Reducing sugars
present. Example: Glucose
Negative Benedict’s Test: No color change (Remains Blue). Reducing sugars absent.
Example: Sucrose.
E. Expected Result
If the color upon boiling is changed into green, then there would be 0.1 to 0.5
percent sugar in solution.
If it changes color to yellow, then 0.5 to 1 percent sugar is present.
If it changes to orange, then it means that 1 to 1.5 percent sugar is present.
If color changes to red,then 1.5 to 2.0 percent sugar is present.
And if color changes to brick red,it means that more than 2 percent sugar is
present in solution.

References:
Nigam S. C. and Omkar (2003). Experimental Animal Physiology and Biochemistry. New Age
International Pvt. Limited. New Delhi.
D (012). Biochemistry. Fourteenth Edition. Academic Publishers. Kolkata.
Kyle, R. A., & Shampo, M. A. (1988). Johann Heller and the Nitric Acid Ring Test. Mayo Clinic
Proceedings, 63(9), 955.doi:10.1016/s0025-6196(12)62703-7
https://bioquochem.com/principals-of-various-protein-precipitation-methods
https://www.onlinebiologynotes.com/
http://www.chem.ucla.edu/~harding/IGOC/F/fehlings_test.html
https://www.chemistrylearner.com/biuret-test.html
https://microbiologyinfo.com/benedicts-test-principle-composition-preparation-procedure-and-result-
interpretation/#:~:text=Result%20Interpretation%20of%20Benedict's%20Test&text=If
%20it%20changes%20color%20to,2.0%20percent%20sugar%20is%20present.&text=Negative
%20Benedict's%20Test%3A%20No%20color%20change%20(Remains%20Bl