Biochemistry

Biochemistry || lab 3
Amino Acids
Amino acids are molecules containing an amine group, a carboxylic acid group and a side chain that varies
betwen different amino acids. Amino acids of the general formula RCH(NH2)COOH are amphoteric, behaving
as amines in some reactions and as carboxylic acids in others.
Qualitative tests of Amino acids:-
1. Ninhydrin test : General test.
Objective:
to detect a-L-amino acids can be used also to detect free amino acids and carboxylic acid groups on proteins
and peptides
Method:
1-place 1ml of each of the solutions in a test tube and add 2ml of ninhydrin solution
2-Boil the mixture over a water bath
3-Allow to cool and observe the purple color formed
Result:
Free Amino Acid gives a purple color
Comment:
All amino acids that have a free amino group will give positive result (purple color), while not free amino
group-proline will give a yellow color
2- Xanthoproteic test: for aromatic group

(phenylalanine,Tyrosine,Tryptophan)
Objective :
This test is used to differentiate between aromatic amino acidswhich give positive Results and other amino
acids. Amino acids containing an aromatic nucleus form yellow nitro derivatives on heating with
concentrated HNO3. The salts of these derivatives are orange in color.
Method:
1 Label five tubes (1-5), then add 0.5 ml of each amino acidsolution and phenol solutions to those test tubes
each alone.
2 Add a few drops of concentrated HNO3.
3 Compare the color with that given by blank using waterinstead.
4 Now COOL THOROUGHLY under the tap and CAUTIOSLY add 5 drops of 10M NaOH to make the solution
strongly alkaline
Result:
-Tyrosine:light yellow
-Tryptophan: light orange
-Phenylalanine: colourless
Comment:
Amino acids containing an aromatic nucleus form yellow nitro derivatives on heating with concentrated
HNO3. The salts of these derivatives are orange in colour.
3-Millon’s test:(for phenol group tyr)

Objective:
This test is specific for tyrosine, the only amino acid containing aphenol group, hydroxyl group battached to
benzene ring.
Method:
1 Label 4 test tubes (1-5).
2 Add 1 ml of test solutions in separate tubes and the phenol solution in one tube.
3 Add to each tube 2 ml Millon's reagent and shake it well.
4 Place the test tubes in the boiling bath with care until the color of tyrosine appears.
Result:
Positive result (brick red color)
Comment :
the nitrated tyrosine complexes mercury ions in the Solution to form a brick-red solution or precipitate of
nitrated tyrosine, In all cases, appearance of red color is positive test.
4- Acree-Rosenheim test :(for indol group trp )
Objective:
It’s also known as aldehyde test. This is used for detecting the presence tryptophan.
Method:
1 -add 1 ml of formaldehyde, add 1 ml of 1% amino acid solution.
2 Pour 1 ml H2SO4 conc down the side of the sloping test tube.
3 Observe the formation of purple color at the interface between the
two layers.
Result :
Concentrated sulfuric acid is added to Form two layers violet zone in the junction point of two layers is a
positive result.
comment:
-Positive test: Appearance of a purple ring between the two layers due to the presence of tryptophan.
-Negative test: No appearance of a purple ring between the two layers due to the absence of tryptophan
5-Sakaguchi test : (for quanidine group)
Objective:
Sakaguchi test is a specific test for detection ofamino acid containing Qauanidium group [R-NH-C= (NH2)2+-
NH2]. In other words it's a test for Quanidines, i.e Arginine.
Method:
1-Add 1ml of Arginine sample.
2-Add 2ml of sodium hydroxide 10% and mix well
3-Add 3-5 drops of alpha naphthol and mix well
4-Add sodium hypobromide drop by drop until the deep red color appears
Result:
Positive result (deep red color)
Comment :
In alkaline solution, arginine react with a-Naphthol and sodium hypobromite / chlorite as an oxidize agent,
to form red complexes as a positive result.
6- Nitroprusside test :
Objective: it is specific for cystine and it’s commonly used to detect the ketones in the urine
Method:
1-Put 1ml amino acid solution into the test tube.
2-Add 0.25ml nitroprusside solution and shake thoroughly.
3 -Add 0.25ml ammonium hydroxide. Observe the color change.
Result :
Positive result (dark pink color)
Comment:
-Positive test: Formation of red color indicate presence of cysteine.
-Negative test: No formation of red color indicate absence of cysteine.
Uses of Nitroprusside Test

•Nitroprusside test is an analytical test for the detection of cysteine or cysteine-containing proteins in a
sample.
•The test can also be used in the diagnosis of cystinuria as the presence of cysteine in urine is a pathological
feature of the disease.
•The nitroprusside test also detects compounds like ketoacids and ketones in blood and urine that helps in
the determination of the degree of ketonuria and ketonemia.
7- pauly’s test: (for His & Tyr)

Principle:
Pauly’s test is used to detect histidine and tyrosine based on the coupling of diazonium salts with these
amino acids. The test involves sulphanilic acid, which undergoes diazotization to form a diazonium salt. This
salt reacts with histidine and tyrosine under alkaline conditions to produce colored azo dyes:
• Histidine: Dark red/cherry color
• Tyrosine: Cherry red/bronze-yellow color
The reaction relies on acidic diazotization followed by alkaline coupling to form the colored product.
Method:
1 To a test tube, add 1 ml of chilled 1% sulfanilic acid
2 added a few drops of pre-chilled 5% sodium nitrite and mixed in a vortex.
3 Add 1 ml of 1% amino acid sample immediately to the test tube and mixed in a vortex.
4 add Few drops of 10% sodium carbonate to the test tube drop by drop until color begins to appear.
Result:
-Histidine : gives dark red
-tyrosine : gives light red
Comment:
Pauly’s test is a sensitive method for detecting histidine and tyrosine based on diazo coupling. It requires
precise pH control, and potential interference from other compounds may affect accuracy.
1-Put 1ml amino acid solution into the test tube.

2-Add 0.25ml nitroprusside solution and shake thoroughly.
3 -Add 0.25ml ammonium hydroxide. Observe the color change.
Result :
Positive result (dark pink color)
Comment:
-Positive test: Formation of red color indicate presence of cysteine.
-Negative test: No formation of red color indicate absence of cysteine.
Uses of Nitroprusside Test

•Nitroprusside test is an analytical test for the detection of cysteine or cysteine-containing proteins in a
sample.
•The test can also be used in the diagnosis of cystinuria as the presence of cysteine in urine is a pathological
feature of the disease.
•The nitroprusside test also detects compounds like ketoacids and ketones in blood and urine that helps in
the determination of the degree of ketonuria and ketonemia.
P6
7- pauly’s test: (for His & Tyr)
Principle:
Pauly’s test is used to detect histidine and tyrosine based on the coupling of diazonium salts with these
amino acids. The test involves sulphanilic acid, which undergoes diazotization to form a diazonium salt. This
salt reacts with histidine and tyrosine under alkaline conditions to produce colored azo dyes:
• Histidine: Dark red/cherry color
• Tyrosine: Cherry red/bronze-yellow color
The reaction relies on acidic diazotization followed by alkaline coupling to form the colored product.
Method:
1 To a test tube, add 1 ml of chilled 1% sulfanilic acid
2 added a few drops of pre-chilled 5% sodium nitrite and mixed in a vortex.
3 Add 1 ml of 1% amino acid sample immediately to the test tube and mixed in a vortex.
4 add Few drops of 10% sodium carbonate to the test tube drop by drop until color begins to appear.
Result:
-Histidine : gives dark red
-tyrosine : gives light red
Comment:
Pauly’s test is a sensitive method for detecting histidine and tyrosine based on diazo coupling. It requires
precise pH control, and potential interference from other compounds may affect accuracy.
P7

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Biochemistry || lab 3

2- Xanthoproteic test: for aromatic group

3-Millon’s test:(for phenol group tyr)

4- Acree-Rosenheim test :(for indol group trp )

5-Sakaguchi test : (for quanidine group)

Uses of Nitroprusside Test

7- pauly’s test: (for His & Tyr)

1-Put 1ml amino acid solution into the test tube.

Uses of Nitroprusside Test

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