Ninhydrin Test

Amino acids contain a free amino group and a free carboxylic acid group that
react together with ninhydrin to produce a coloured product. When an amino
group is attached to the first, or alpha, carbon on the amino acid’s carbon chain,
the amino group’s nitrogen atom is part of a blue-purple product. Proteins also
contain free amino groups on the alpha carbon and can react with ninhydrin to
produce a blue-purple product.

Amino acids that have secondary amino group attachments also react with
ninhydrin. However, when the amino group is secondary, the condensation
product is yellow. For example, the amino acid proline, which contains a
secondary amino group, reacts with ninhydrin, as shown in Equation 3.
Blue-purple and yellow reaction products positively identify free amino groups
on amino acids and proteins.

Biuret Test: The Biuret Test is a general test for proteins. When a protein
reacts with copper(II) sulfate (blue), the positive test is the formation of a violet
colored complex.
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 nitrated to give yellow colored nitro-derivatives. At alkaline pH,
the color changes to orange due to the ionization of the phenolic group.

Millon-Nasse Test
Millon-Nasse test is a test specific for tyrosine, the only amino acid containing a
phenol group, a hydroxyl group attached to a benzene ring. In Millon’s test, the
phenol group of tyrosine is first nitrated by nitric acid in the test solution. Then
the nitrated tyrosine complexes mercury (I) and mercury (II) ions in the solution
to form a red precipitate or a red solution, both positive results. Proteins that
contain tyrosine will, therefore, yield a positive result.
However, some proteins containing tyrosine initially forms a white precipitate
that turns red when heated, while others form a red solution immediately. Both
results are considered positive.

Hopkins-Cole Test:
In the Hopkins-Cole reaction, the indole ring reacts with glyoxylic acid in the
presence of a strong acid (H2SO4in the experiment’s case) to form a violet
cyclic product. The mechanism for this reaction is shown in the equation
below.

The Hopkins-Cole test confirms the presence of tryptophan in Albumin. The

protein solution is hydrolyzed by the concentrated H2SO4 at the solution
interface. Once the tryptophan is free, it reacts with the glyoxylic acid to form
the violet product.

However, the results for the two qualitative tests were questionable. In the
case of Gelatin, according to Cole (2000), the protein lacks tryptophan which
does not agree to the results obtained in Hopkins-Cole test. In addition,
albumin contains tryptophan but showed negative results towards bromine
water test.
Conclusion
Amino acids are building blocks of proteins. They possess an amine
group, a carboxylic acid group and a varying side chain that differs between
different amino acids. These side chains are essential in determining the
presence of amino acids in proteins through qualitative test. The qualitative
tests conducted in this experiment make use of various color-producing
reagents which is dependent upon the side chains present in the test samples.
Ninhydrin reaction was used to confirm the sample as an amino acid or protein.
Biuret test was significant in distinguishing proteins from amino acids.
Xanthoproteic reaction is for classifying amino acids with benzene derivative.
Millon’s test was used to check the presence of tyrosine in the test samples.
Both Hopkins-Cole and Bromine water test are important for tryptophan
determination. Pauly reaction confirms the existence of tyrosine and histidine
in the sample. Reduced Sulfur test makes use of the sulphur in determining the
presence of cysteine and cysteine. And Sakaguchi’s reaction is useful in
arginine determination.
Albumin was confirmed to have the presence of tryptophan, tyrosine,
histidine, cysteine, and arginine. While for gelatin, only the existence of
tyrosine, histidine, and arginine were observed. Hair was also included in the
Lead Acetate Reaction to check the presence of cysteine.

References
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on Biochemistry. 38(1): 88–95.
Cole, CGB. Francis, F.J., editor. (2000). Gelatin.. Encyclopedia of Food
Science and Technology, 2nd ed. 4 Vols. New York: John Wiley & Sons,
pp. 1183-1188.
Galewska Z., Gogiel T., Małkowski A., Romanowicz L., Sobolewski K.,
Wolańska M. (2013). Biochemistry Workbook. Medical University of Białystok.
Milio, F. & Loffredo, W. (1995). Qualitative testing for amino acids and proteins.
USA: Chemical Education Resources, Inc.
Nigam, A & Ayyagari, A. (2007). Lab Manual in Biochemistry: Immunology and
Biotechnology. New Delhi: Tata McGraw-Hill Education. ISBN:
9780070617674
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