Procedures

The gluten hydrolysate was tested with

different
characterization
reagents
accessible in the laboratory specifically:
Biuret, Ninhydrin, Xanthoproteic, Millons
Hopkins-Cole, Sakaguchi, Nitroprusside,
Fohls, Paulys and Test for Amides. 10
test tubes were prepared for all of the test
reactions. Each test tube consists of 1 mL
of distilled water added to 0.5 mL of
hydrolyzed samples.
Biuret Test
The group added 2-3 drops of 0.1 M
CuSO4 solution to a mixture of the sample
and 20 drops of 2.5M NaOH. The test tube
was shaken and the color changes in the
solution were observed.
Ninhydrin Test
For this test, 6-10 drops of 0.1% ninhydrin
solution was placed into the sample and
then it was heated in a boiling water bath.
After that, appearance of blue-violet
coloration was taken note of.
Xanthoproteic Test
10 drops of concentrated nitric acid and
concentrated sodium hydroxide was
slowly added to the diluted sample and
then mixed. Color changes after each
addition was observed. After that, 10 drops
of concentrated NaOH was added, then it
was to be observed whether there is any
color change.
Millons Test
5 drops of Millons reagent was added to
the diluted sample, and the color changes
were noted.
Hopkins-Cole Test
20 drops of Hopkins-Cole reagent was
slowly added to the sample, and then
mixed. While the test tube was inclined,
concentrated sulfuric acid of about 20
drops was slowly added, without shaking,
and the color at the interface was taken
note of.
Sakaguchi Test
10 drops of 10% NaOH and 10 drops of
0.02% naphthol solution was added to the

sample. The solution was left to stand for 3

minutes. Subsequently, 3 drops of 2%
NaOBr was added then the color produced
was noted.
Nitroprusside Test
0.5 mL of 3M NaOH was added to the 0.5
mL of the sample. Next, 0.25 ml of 2%
nitroprusside solution was added and th
formation of a red solution was observed.
Fohls Test
5 drops of 30% NaOH and 2 drops of 5%
(CH3COO)2Pb was added to the sample.
It was then place in a boiling water bath.
Then, the appearance of a dark (black or
brown) sediment was noted.
Pauly Test
The diazo reagent was prepared by mixing
3-5 drops of 1% sulfanilic acid with 3
drops of 5% NaNO2 solution. Afterwards,
5 drops of the sample and 3-5 drops of
10% Na2CO3 was added to the diazo
reagent, then, the appearance of a red
coloration was noted.
Test for Amides
Lastly, the test for amides was done by
adding 1 mL of 20% NaOH to 10 drops
of the sample. Then it was placed the tube
in a boiling water bath. Moistened red
litmus paper was placed over the mouth of
the tube to test the evolution of gas during
heating. Results were noted.
Table 2. Color Reactions
Color Test
Biuret Test
Ninhydrin
Test
Xanthoprot
eic Test
Millons
Test
HopkinsCole Test
Sakaguchi
Test
Nitroprussi
de Test
Fohls Test
Test
for
Amide
Pauly Test

Biuret test

Color Reaction of Intact

Protein
Purple solution
Blue-Violet Solution

Inference

Yellow ppt orange ppt

(excess NaOH)
Flesh to red solution

Positive

Purple ring at Interface

Positive

Red or orange solution

Negative

Yellow ppt

Positive

Brown ppt
Red --> Blue
Paper
Red solution

Litmus

Positive
Positive

Negative

Positive
Positive
Positive

This is used to look the presence

of peptide bonds. The Biuret test is a
positive test for proteins but not for amino
acid. The evidence for the test consists of
formation of a violet-pink complex when
cupric ion, in basic solution is added to
any polymer such as protein which
contains multiple amide bonds. A bluecolored solution indicates a negative test
or those fewer than two peptide bonds are
present.
Ninhydrin test
This is done to know whether there is an
alpha amino acid present. It reacts with
ammonia, a primary amine, or a secondary
amine. Free amino acids can react with
ninhydrin reagent and it will yield a deep
purple solution upon heating with
ninhydrin. The ninhydrin test is positive
for amino acid and some proteins. Most of
amino acids have free amino groups, and
gluten is positive for this test.
Xanthoproteic test
This is used to test for an aromatic
sidechain. The test depends upon a
reaction with a specific type of amino acid
chain. Aromatic rings have the ability to
undergo nitration reaction, which is the
addition of NO2 group to the ring that is
why it is a positive test for side chains in
tyrosine and tryptophan. With the addition
of nitric acid and heat, the reaction will
result to a yellowish-colored solution. The
intensity of the yellow color deepens when
the reaction occurs in basic solution (color
will change to orange). Gluten is positive
of aromatic side chain.
Millons test
This is a test for phenolic group containing
side chain or a test specific for tyrosine,
the only amino acid containing phenol
group. In this 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 red
precipitate or red solution. And since

gluten does not contain tyrosine, the test

resulted negative in the color reactions.
The Hopkins-Cole
This test is specific for trypthopan, the
only amino acid containing an indole
group. The clear violet ring produced is
due to the formation of a compound from
the glyoxylic acid in the reagent and the
tryptophan in the protein. A similar color is
produced when sulphuric acid is added to a
protein solution in the presence of a trace
of formaldehyde.
Gluten showed a positive result for the
presence of tryptophan.
Sakaguchi test
This test is for identifying the presence of
the guanido group of arginine. Since basic
hydrolysis destroys arginine and produces
ornithine and urea, basic hydrolysate must
be negative for this test while all the other samples
are positive. In basic conditions, alpha
naphthol and sodiumhypobromite/chlorite
react with the amino acid containing the
guanido group to form red-orange
complexes. Gluten sample showed
negative result thus, it does not contain an
arginine amino acid.
Nitroprusside test
This is for indicating the presence of
cysteine or free thiol groups. In this test,
cysteine is partially destroyed is evident in
the results of the experiment because it
produces a yellow precipitate. Its principle is
complexation. Gluten yields a positive
result for this test.
Fohls test
This is a test for sulfur containing amino acids.
It also indicates the presence of methionine
and cysteine because those two amino acids
have sulfur in their structures. Its principle is
fusion followed by ionic interaction. A positive
result for this test is the formation of dark
brown or black precipitate from lead sulfide
(PbS). The dark coloration of the samples caused
by the Fohls test indicates that there is
sulfur present

Pauly Test
This test answers for the presence of
tyrosine and histidine residues. Imidazole
group reacts with diazotized aulphanilic
acid
to
form
highly
colored
azocompouds. The diazonium salt formed
couples with either tyrosine or histidine in
alkaline medium to give a red colored
chromogen (azo dye). In this test, gluten
exhibited a positive result.
Test for amides
This is a test for the presence of asparagine

and glutamine. The red litmus paper turned

blue indicates a basic component of the
gluten, thus gluten is positive for the
presence of a basic amino acid.
REFERENCES
Amrita University. (2015). Qualitative
Analysis of Amino Acid. Retrieved from
http://amrita.vlab.co.in/?
sub=3&brch=63&sim=1094&cnt=1
Retrieved
from
http://www.academia.edu/4836795/Qualita
tive_analysis_of_Amino_acids