Roll No.

___________________________

D. Y. PATIL DEEMED TO BE UNIVERSITY, NAVI MUMBAI.

VIRTUAL LAB REPORT

2023 – 2024

SCHOOL OF BIOTECHNOLOGY AND BIOINFORMATICS,

PLOT NO 50, SECTOR 15, C.B.D. BELAPUR.
 Certificate
This is to certify that the virtual lab experiments entered in this

report have been performed and reported by Mr.

/Ms.______________________ studying in the School of

Biotechnology and Bioinformatics, D. Y. Patil Deemed to be

University, Navi Mumbai, BBT, Semester V, Batch ___ Roll

Number _________________ during the academic year 2023-

2024.

College Seal Date:

Internal Examiner External Examiner

D. Y. PATIL DEEMED TO BE UNIVERSITY, NAVI MUMBAI
SCHOOL OF BIOTECHNOLOGY AND BIOINFORMATICS

Virtual Lab Report

Program- BBT Semester- V

Practical Title- Qualitative analysis of amino acids

Experiment. No.- 8

Date and Time- 12 October 2024, 10 am

Approach or Amino acids are building blocks of all proteins, and are linked in series
principle by peptide bond (-CONH-) to form the primary structure of a protein.
Amino acids possess an amine group, a carboxylic acid group and a
varying side chain that differs between different amino acids.
There are 20 naturally occurring amino acids, which vary from one
another with respect to their side chains. Their melting points are
extremely high (usually exceeding 200°C), and at their pI, they exist as
zwitterions, rather than as unionized molecules.
Amino acids respond to all typical chemical reactions associated with
compounds that contain carboxylic acid and amino groups, usually
under conditions where the zwitter ions form is present in only small
quantities. All amino acids (except glycine) exhibit optical activity due
to the presence of an asymmetric α – Carbon atom. Amino acids with an
L – configuration are present in all naturally occurring proteins,
whereas those with D – forms are found in antibiotics and in bacterial
cell walls.

Details of 1. Ninhydrin Test:

Animation- To 1ml of amino acid solution taken in a test tube, add few drops of
ninhydrin reagent and vortex the contents. Place the test tube in a
boiling water bath for 5 minutes and cool to room temperature.

2. Xanthoproteic acid Test:

To 1ml of the amino acid solution taken in a test tube, add few drops of
nitric acid and vortex the contents. Boil the contents over a Bunsen
flame, using a test tube holder, for few minutes. Cool the test tube under
running tap water and add few drops of alkali.

3. Pauly's diazo Test:

Take 1ml of sulphanilic acid reagent in a test tube and chill the contents
in a small ice bucket. Add few drops of prechilled sodium nitrite
solution and vortex. Add immediately few drops of pre chilled amino
acid solution and vortex. This is followed by dropwise addition of
sodium carbonate solution until the color appears.

4. Millon's Test:
To 1ml of the amino acid solution in a test tube, add few drops of
millon’s reagent and vortex. Boil the contents over a Bunsen flame for 3
– 5 minutes. Cool the contents under running tap water and add few
drops of sodium nitrite solution.

5. Histidine Test:
To 1ml of amino acid solution, add 5% bromine in 33% acetic acid until
an yellow color was formed. After 10 minutes, add 2ml of 5%
ammonium carbonate solution and placed in a boiling water bath for 10
minutes.

6. Hopkins-Cole Test:
Mix 1 ml of the amino acid solution with 1 ml acetic acid – glyoxilic
acid reagent, in a test tube, vortex. Then carefully, add conc. Sulphuric
acid along the side of the test tube, keeping the tube in an inclined
 position (do not shake the test tube , while adding acid)

7. Sakaguchi Test:
To 1 ml of prechilled amino acid solution and few drops of NaOH is
mixed and 2 drops of alpha naphthol is added. Mix thoroughly and add
4-5 drops of hypobromite reagent and observe.

8. Lead sulphide Test:

To 1ml of the amino acid solution taken in a test tube, add few drops of
sodium hydroxide (40%) and boil the contents for 5 – 10min over a
bunsen burner. Cool the contents and add few drops of 10% Lead
acetate solution and observe.

9. Folin's McCarthy Sullivan Test:

To 1ml of the amino acid solution taken in a test tube, add few drops of
sodium hydroxide (5N), followed by addition of few drops of glycine
(1%) and 10% sodium nitroprusside solution and vortex. Place the test
tube in a hot water bath, maintained at 40°C, for 15 minutes. Cool the
test tube in ice cold water for 5 minutes and add 0.5ml of 6N HCl.
Vortex the contents and allow to stand for 15 minutes at room
temperature.

10. Isatin Test:

Apply a drop of imino acid solution on a filter paper strip and dry the
spot using a hot air gun / Hair dryer or hot air oven. Applay a drop of
isatin reagent on to the dried spot. Repeat the drying procedure with hot
air gun for few minutes and observe.

Self-Evaluation-
Principle:
Ninhydrin test
1 : In the pH range of 4-8, all α- amino acids react with ninhydrin (triketohydrindene
hydrate), a powerful oxidizing agent to give a purple colored product
(diketohydrin) termed Rhuemann’s purple. All primary amines and ammonia react
 similarly but without the liberation of carbon dioxide. The imino acids proline and
hydroxyproline also react with ninhydrin, but they give a yellow colored complex
instead of a purple one. Besides amino acids, other complex structures such as
peptides, peptones and proteins also react positively when subjected to the
ninhydrin reaction.

Xanthoproteic acid test

Aromatic amino acids, such as Phenyl alanine, tyrosine and tryptophan, respond to
this test. In the presence of concentrated nitric acid, the aromatic phenyl ring is
2 :
nitrated to give yellow colored nitro-derivatives. At alkaline pH, the color changes
to orange due to the ionization of the phenolic group

Materials Required:
1. Glass wares
2. Test tubes
3. Test tube holder
4. Water bath
5. Spatula
3 : 6. Dropper
7. Bunsen burner
8. Icebox
9. Vortex mixer
10. Filter paper strip
11. Hair drier

Procedure:

1. Ninhydrin Test:
To 1ml of amino acid solution taken in a test tube, add few drops of ninhydrin
reagent and vortex the contents. Place the test tube in a boiling water bath for 5
minutes and cool to room temperature.

2. Xanthoproteic acid Test:

To 1ml of the amino acid solution taken in a test tube, add few drops of nitric acid
and vortex the contents. Boil the contents over a Bunsen flame, using a test tube
holder, for few minutes. Cool the test tube under running tap water and add few
drops of alkali.
4 :
3. Pauly's diazo Test:
Take 1ml of sulphanilic acid reagent in a test tube and chill the contents in a small
ice bucket. Add few drops of prechilled sodium nitrite solution and vortex. Add
immediately few drops of pre chilled amino acid solution and vortex. This is
followed by dropwise addition of sodium carbonate solution until the color
appears.

4. Millon's Test:
To 1ml of the amino acid solution in a test tube, add few drops of millon’s reagent
and vortex. Boil the contents over a Bunsen flame for 3 – 5 minutes. Cool the
contents under running tap water and add few drops of sodium nitrite solution.
 5. Histidine Test:
To 1ml of amino acid solution, add 5% bromine in 33% acetic acid until an yellow
color was formed. After 10 minutes, add 2ml of 5% ammonium carbonate solution
and placed in a boiling water bath for 10 minutes.

6. Hopkins-Cole Test:
Mix 1 ml of the amino acid solution with 1 ml acetic acid – glyoxilic acid reagent,
in a test tube, vortex. Then carefully, add conc. Sulphuric acid along the side of the
test tube, keeping the tube in an inclined position (do not shake the test tube ,
while adding acid)

7. Sakaguchi Test:
To 1 ml of prechilled amino acid solution and few drops of NaOH is mixed and 2
drops of alpha naphthol is added. Mix thoroughly and add 4-5 drops of
hypobromite reagent and observe.

8. Lead sulphide Test:

To 1ml of the amino acid solution taken in a test tube, add few drops of sodium
hydroxide (40%) and boil the contents for 5 – 10min over a bunsen burner. Cool
the contents and add few drops of 10% Lead acetate solution and observe.

9. Folin's McCarthy Sullivan Test:

To 1ml of the amino acid solution taken in a test tube, add few drops of sodium
hydroxide (5N), followed by addition of few drops of glycine (1%) and 10%
sodium nitroprusside solution and vortex. Place the test tube in a hot water bath,
maintained at 40°C, for 15 minutes. Cool the test tube in ice cold water for 5
minutes and add 0.5ml of 6N HCl. Vortex the contents and allow to stand for 15
minutes at room temperature.

10. Isatin Test:

Apply a drop of imino acid solution on a filter paper strip and dry the spot using a
hot air gun / Hair dryer or hot air oven. Applay a drop of isatin reagent on to the
dried spot. Repeat the drying procedure with hot air gun for few minutes and
observe

Time spent- 25 mins

Level of
Understanding
achieved