VI.

Analysis
Amino acids are key components in living organisms since they
perform functions that are important such as being the building blocks of
proteins and being the intermediates in metabolism. Amino acids contain an
amine group, a carboxylic acid group and a side chain that varies among
different amino acids. This side chain is the one that determines the class of
amino acid: polar, nonpolar, neutral, basic or acidic. It is also this side chain
that determines that characteristic of the protein it forms. As these
functional groups in proteins and amino acids react, it produces products
that vary in color and characteristic. In this experiment, various color-
producing reagents are used in order to detect the presence of certain
functional group.
In the ninhydrin reaction, the ninhydrin must be neutral in order for it
to react with with α- amino acids found in proteins. This is due to the fact
that when the pH of the solution is in the range of 4-8 (close to neutral), all
α- amino acids react with ninhydrin which is a powerful oxidizing agent,
which then results in a purple colored product named Rhuemann’s purple. If
the pH is not close to neutral, for example, a pH of 1-2, the reaction will give
off ammonia but there will be no change in color.
In the ninhydrin reaction, marbles were also used while heating the
solutions in the water bath. The marbles are used in order to trap the heat
inside the test tubes to prevent heat escape. This enables the heat to
circulate in the test tubes in order for the reaction to proceed.
In the biuret test, a very dilute solution of CuSO 4 is used. This is in
order to introduce copper ion(Cu2+) to the solution. When peptide bonds are
present in the alkaline solution, the Cu 2+ will form a coordination complex
with 4 nitrogen atoms coming from the peptide bonds. This complex with
Cu2+ and nitrogen make the color of the CuSO 4 solution change from violet to
blue. This change is dependent on the number of peptide bonds present in
the solution. Therefore, the more proteins there are in a solution, the more
intense is the color formed.
The Xanthoproteic and Millon-Nasse tests may be used in urine
examination for proteins but not ideal. In urinary examinations, the presence
of proteins such as albumins may signify an unhealthy kidney. Xanthoproteic
and Million-Nasse tests can detect certain proteins such as albumin. But it is
not recommended since it can only detect proteins that have certain
characteristics. In Xanthoproteic test, it is sensitive to proteins with aromatic
amino acids, such as tyrosine and tryptophan. In Millon-Nasse test, it is
specific to phenolic hydroxy group. Therefore, these tests are limited to
detect a short range of proteins only, making it not an ideal in urinary
examination for proteins.
Bromine water interacts with free tryptophan and n-amyl alcohol to
form a pinkish lavender complex. However, if there is excess bromine water,
the pink color may be masked by the color of the reagent. The colored
complex is soluble at the alcohol layer.
The ninhydrin reaction can be used to show up to what stage the
hydrolysis of a protein proceeds. Ninhydrin reacts with ammonia and primary
amines. Since amino acids can react with ninhydrin to yield CO 2, ammonia,
and an aldehyde of one carbon atom fewer than the original amino acid.
Therefore, we can then determine the stage of the hydrolysis with the use of
the aldehyde that has one carbon fewer than the original amino acid.

Discussion for Each Test

Ninhydrin Test
The ninhydrin is a powerful oxidizing agent that gives a purple colored
product which is also known as Rhuemann’s purple to indicate that an amino
group is attached to the alpha carbon on the amino acid, the amino group’s
nitrogen atom is part of the colored product. Amino acids that have
secondary amino group attached to them can also react with ninhydrin.

However, when the amino group is secondary, the resulting product is yellow.
Biuret Test
The Biuret Test identifies the presence of proteins with more than two
peptide bonds. The reaction in this test involves the complex formation of
the proteins with Cu2+ ions in a strongly alkaline solution.
Ex:
Amino acid(albumin) +10% NaOH + 0.5% CuSO4  dark violet
Xanthoproteic Test
Xanthoproteic is sensitive to proteins with aromatic amino acids, such
as tyrosine and tryptophan. The aromatic groups present in the amino acid
are acid derivatives of benzene and undergoes reactions the same as
benzene and benzene derivatives. Thus, the yellow product formed in the
reaction is due to the nitration of the benzene ring with nitric acid.

Millon-Nasse
This test is specific to proteins with phenolic hydroxy group thus,
specific to tyrosine since tyrosine is the only amino acid containing a phenol
group. The phenol group is first nitrated by nitric acid. Then the nitrated
tyrosine complexes mercury (I) and mercury (II) ions in the solution to form a
red precipitate or solution.
tyrosine + millon’s reagent +heat  red flocculent precipitate
Hopskin’s Cole
Hopkins-Cole test is a specific test for amino acid containing an indole
group thus, specific to tryptophan–the only amino acid containing an indole
group. The solution is hydrolyzed by the concentrated sulfuric acid. When the
tryptophan is free, it reacts with the glyoxylic acid to form the violet product.

Bromine Water
The Bromine water tests free tryptophan in solutions. Free tryptophan
interacts with bromine water and n-amyl alcohol to form a pinkish lavender
complex. However, the pink color may be masked by the color of the reagent
if excess reagent is added. The colored complex is soluble at the alcohol
layer.
Pauly
Reaction
 The principle behind the Pauly’s reaction is diazotized. Sulfanilic acid
will be diazotized with the addition of NaNO2 and Na2CO3 and formed
diazotized component. The diazonium component reacts with the imidazole
ring of histidine and a phenol group of tyrosine to form dark red compound.
Lead Acetate
This test is specific for sulfur containing amino acids. Sulfur containing
amino acids, upon boiling with NaOH, yields Na 2S. This reaction is due to the
partial conversion of the organic sulfur into inorganic sulfide, which can be
detected by precipitating it using the Pb(OAc)2 solution.

Sakaguchi Reaction
Sakaguchi reaction tests generally for the presence of proteins with the
amino acid arginine. It will give positive results in proteins with arginine
amino acids because of the functional group guanidine. The guanidine group
reacts with alpha napthol and sodium hypobromite to form deep red color.
VII. CONCLUSION
Color reactions yield products that vary in color intensity due to the
presence of certain functional groups in proteins. In the Ninhydrin test,
casein and albumin gave a positive result indicated by the solution with a
color violet. The Biuret test identified the presence of proteins in solutions
that gave off a violet color in casein and albumin. The presence of an
aromatic ring in amino acids was identified by the Xanthoproteic test which
gave off a yellow color of the solution, it was positive when tested with,
tryptophan, tyrosine and. Millon-Nasse gave a red solution to indicate the
presence of a phenol group, which is specific to the protein tyrosine. The
Hopkins-Cole reaction was used to identify tryptophan in the sample by the
presence of a violet ring, but it. Free tryptophan was tested and found to be
present in the amino acid sample using bromine water test. The Pauly
reaction yielded a red solution to indicate the presence of histidine and
tyrosine. Sulfur was present in the samples by testing it using the lead
acetate reaction confirming that cystine contains sulfur. Lastly, in the
Sakaguchi reaction, there was a presence of arginine which was indicated by
a color red solution.

Reference:
Agaceta, C. (2015). Laboratory manual in pharmaceutical biochemistry [PDF File]. Available
from http://s3.amazonaws.com/academia.edu.documents/47188098/Biochemistry_
Laboratory_Manual.pdf

Experiment 2 – Qualitative analysis of amino acids and proteins [PDF File]. (2003).
Available from http://www.chem.boun.edu.tr/wpcontent/uploads/2014/04/Chem-415-
Experiment-2.pdf

Milio, F., & Loffredo, W. (2001). Qualitative testing for amino acids and proteins [PDF file].
Available from http://labopslton.wikispaces.com/file/view/qualitative+testing+for+
amino+acids+%26+proteins.pdf