EXPT 6.

ANALYSIS

1.) Why must the solution to be tested with ninhydrin be neutral?

- In the Ninhydrin test, the samples were neutralized with NaOAc and was
added with Ninhydrin solution. The samples must be neutral because
Ninhydrin is a chemical used to detect ammoniaor primary and
secondary amines. Amino acids also react with the Ninhydrin solution at
pH=4. That is why the samples should be neutralized, so that all α- amino
acids that react with ninhydrin (triketohydrindene hydrate) can be
detected.

2.) What is the use of the marble in the ninhydrin reaction?

- The test tube was stoppered with a marble to prevent evaporation during
the heating period of the solution since it was heat to a boiling water bath
for 1-2 minutes.

3.) A very dilute solution of CuSo4 is used in the biuret test. Why?

- The Biuret Test is done to show the presence of peptide bonds, which
are the basis for the formation of proteins. These bonds will make the
blue Biuret reagent turn purple. CuSO4 reacts with compounds
containing two or more peptide bonds to give a violet colored product
which is due to formation of co-ordination complex of cupric ions with
unshared electron pairs of peptide nitrogen and O2 of water.

4.) Are the Xanthoproteic and Millon-Nasse tests satisfactory for use in the
urinary examination for protein? Why?

5.) Why does the bromine water be avoided in the test for free tryptophan?

- 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.
6.) Which test can be used to show up to what stage the hydrolysis of a protein
proceeds?

7.) Discuss each test to detect groups in proteins and amino acids and write
equations to support results.

Ninhydrin Test:

Ninhydrin is a strong oxidizing agent used in amino acid analysis for the
precise determination of protein quantities. It is mainly used as a detector in
liquid chromatography methods coupled with ninhydrin post-column
derivatization systems. The reagent which is initially yellow reacts with free
alpha amino groups present in all amino acids, proteins, or peptides and forms
a deep blue or purple colored complex known as Ruhemann’s purple.

The samples that were used for the test were neutralized with solid NaOAc
with 2-3 drops of ninhydrin solution. It was then covered with marbles and was
heated in a boiling water bath for 2-3 minutes. Colors deep blue and purple
were observed in the solution.

Biuret Test:

The Biuret Test positively identifies the presence of proteins (not less than two
peptides). The reaction in this test involves the complex formation of the
proteins with Cu2+ ions in a strongly alkaline solution. The samples were
mixed with 0.5 mL of 10% NaOH with 1-2 drops of 0.5% CuSO4. The reason
why the samples were mixed with NaOH because the biuret test for proteins
solution reacts in a basic solution to form a deep violet complex.
Xanthoproteic Test:

The Xanthoproteic test uses a nitration reaction to determine the presence of

proteins in a solution. When the sample is treated with a hot, concentrated nitric
acid it reacts with aromatic amino acids such as phenylalanine, tyrosine and
tryptophan and forms a yellow colored product known as Xantho protein. With
the addition of strong base such as NH3 or NaOH, it further changes to deep-
orange color. So, this test gives a positive result in those proteins which contain
amino acids that have aromatic rings in their side chains.

Each of the samples were added with 0.5mL of concentrated HNO3, it was then
observed if a white precipitated was formed, if present, the solution was then
heated and observed if the precipitate turned yellow. The solutions were cooled
and were added with 10% NaOH but there was no change in the color of the
precipitate observed. The intensity of the yellow color deepens when the
reaction occurs in basic solution. This reaction is one of the reactions that occur
if you spill a concentrated solution of nitric acid onto your skin. The proteins in
skin contain tyrosine and tryptophan, which become nitrated and turn yellow.
Hopkins-Cole Reaction:

This chemical test is used to identify the presence of tryptophan, the only amino
acid containing in dole group. When the protein solution is mixed with Hopkins-
Cole reagent the in-dole ring reacts with the glyoxylic acid in the presence of
concentrated sulfuric acid and forms a violet or purple colored product,
signifying a positive result. The protein solution is hydrolyzed by the
concentrated H2SO4 at the solution interface. Once the tryp-tophan is free, it
reacts with the glyoxylic acid to form the violet product.

Bromine Water Test

It is used to detect the presence of unsaturated compounds of alkene and

alkyne. Such a test for alkenes works via the mechanism of making alkenes or
hydrocarbons, having a minimum of one double bond that undergoes addition
reactions. The alkenes and hydrocarbons combine with bromine to impart a
colorless appearance to this element. As for the change in bromine's color,
alkenes are colorless and therefore, their combination with bromine causes the
latter to lose color, as well as it gets consumed in the reaction process. The
color of the alcohol layer that was observed was colorless. The equation for this
reaction is:

H2 = CH2 ---> H2BrC - CbrH2

Pauly Reaction:

In five test tubes, casein, albumin, histidine, tyrosine and a blank were treated
with sulfanilic acid with NaNO2and was cooled in an ice bath for 3 minutes and
made to alkaline with Na2CO3. The principle behind the Pauly’s reaction is
diazotized. Sulfanilic acid will bediazotized with the addition of NaNO2and
Na2CO3and formed diazotized component.7Thediazonium component reacts
with the imidazole ring of histidine and a phenol group of tyrosine to form dark
red compound. However, in the experiment, instead of forming a red compound,
yellow product was obtained. Thus, a negative result was observed.

Lead Acetate Reaction:

Four test tubes were obtained with casein, albumin, a blank, and an untreated
piece of hair were added with NaOH and Pb (OAc)2. Covered with marble, the
samples were heated to boiling under a water bath for a few minutes. The
albumin and the test tube which contains the hair gave a positive result. Sulphur
containing amino acids upon boiling with NaOH, yields Na2S. This reaction is
due to the partial conversion of the organic Sulphur into inorganic.

Sakaguchi Reaction:

The Sakaguchi reagent is used to test for a certain amino acid and proteins.
The amino acid that is detected in this test is arginine. Since arginine has a
guanidine group in its side chain, it gives a red color with α-naphthol in the
presence of an oxidizing agent like bromine solution Based on our
observation, the casein solution turned color red after it was mix thoroughly
with 0.5mL bromine water.
References:

(Kumar, P.) Qualitative and Quantitative Tests for Amino Acids and Proteins
https://vlab.amrita.edu/?sub=3&brch=63&sim=1094&cnt=1
(Kamineni, S., Manepally, M., Kamineni P.) (2016.) Musculoskeletal Protein
Analysis Techniques - A Review