Isolation and Characterization of Proteins from Milk and Flour

Cruz, A., Dela Vega, E., Dizon, A., Esguerra, M.J., Espiritu, M.A., Garcia, E.M.

Abstract
In the process of isolation and characterization of proteins, there were several types of
proteins that were dessiminated to different groups. The group was assigned with the isolation of
intact gluten using solubilization, wherein proteins are separated through their difference in
solubility. Insoluble proteins are easily isolated, removing other substances that are soluble by
washing. Then proceeds to the the testing of the several samples by performing the qualitative
color reactions. After the qualitative color reactions, paper chromatography was performed for the
separation and identification of amino acid standards based on the polarities of tryptophan,
arginine, proline, cysteine, serine, aspartate, histidine, glycine, and alanine. After performing the
paper chromatography, protein concentration was determined through Bradford Protein Assay.
Albumin standard curve was constructed and the unknown concentration of protein was
determined using linear regression analysis. The graph studied showed the direct relationship of
Bovine Serum Albumin concentration to its absorbance.

I. INTRODUCTION
Proteins are made up of amino acids
and can be separated depending on their
charge, shape, size and physicochemical
properties. Proteins undergo isolation
process to isolate a single protein from its
complex mixture. This process happens to
characterize the acid-base property,
solubility, structure, functions, and
interactions of the protein interest. There are
some common methods used for protein
isolation such as heat denaturation,
solubilization, chromatography, and
isoelectric precipitation.
Gluten is a mixture of glutenin and
gliadin. It is one of the protein component of
flour which is responsible for the strength and
elasticity of the dough[1]. It has visco-elastic
characteristic which obtained by isolation of
starch from wheat dough.
Casein is a phospopotein that
interacts with calcium. It isolated through
isoelectric precipitation method where the pH
of the solution is adjusted to its isoelectric
pH(4.6) which decreases its solubility.
There are qualitative analysis for
proteins such as biuret test (for peptide
linkages), ninhydrin test (detects acidic amino
acid group), xanthoproteic test (detects
aromatic amino acids), millon’s test (detects
ring containing amino acids, Hopkins-sole
test (detects indole containing amino acids),
sakaguchi test (detects guanidines), fohl’s
test (detects sulfur containing amino acids),
nitroprusside test (detects cysteine), test for
amides (detects amides and nitriles), and
pauly’s test (detects histidine and tyrosine).
For quantitative analysis bradford
protein assay and biuret protein assay
performed. Bradford protein assay is used in
determining the total protein concentration of
a sample. Biuret protein assay uses alkaline
copper sulfate which forms a purple complex
when it is bound to the peptide bonds.
II. METHODOLOGY
A. Isolation of Casein from Skimmed Milk
Twenty grams of powdered skimmed
milk was mixed with 50.0 mL of distilled water
into a 100-mL beaker. The mixture was
heated up not exceeding than 40°C then, 10%
acetic acid was added dropwise until curd
was formed. The white curd-like precipitate
was filtered using a funnel and filter paper.
The filtrate was divided into two portions, one
portion was stored for quantitative analysis of
albumin and the portion was placed in beaker
and heated for 75°C for 5 minutes. The filtrate
was discarded and the precipitate was
decanted. The casein residue was dried and
weighed. The percent yield of the dried casein
residue was then calculated and it was stored
for enzymatic hydrolysis.
B. Isolation of Albumin from Skimmed Milk
The mixture was divided into two
equal portions. The first portion contained the
filtrate and was transferred to a beaker from
the isolation of Caseine and the other portion
was set aside for the quantitative analysis of
protein. The filtrate in the beaker was heated
up to 75°C and remained in the hot plate for
about 5 minutes until the temperature was
obtained. The liquid was decant from the
precipitated albumin.
C. Isolation of Gluten from Wheat Flour
One cup of flour was placed in a
beaker and enough water was added to form
a dough. The dough was wrapped with
cheesecloth and was placed under running
water until all the starch is removed. While
washing, the dough was tested with 0.01M
iodine solution. The blue color indicated that
there is still starch and the washing still
continued while yellow color indicated that
there is no more starch and the it is the time
to stop washing and it was stored.
D. Acid Hydrolysis of Intact Proteins
For this type of hydrolysis, 5 ml of the
6M HCl was added to 0.5 g of the isolated
myoglobin in a hard glass test tube. The test
tube was properly labeled according to the
format given. Then cotton was placed to
stopper the tube before it was subjected to
autoclaving at 15 psi for 5 hours. Alternatively,
the protein sample could be placed in a
sealed container containing 6M HCl. The
whole container is then placed in a microwave
oven for about 5-30 minutes with
temperatures up to 200oC. Then, the
appearance of the mixture was noted after
autoclaving. Then, distilled water with a
volume of 10 mL was added and the mixture
was transferred into a 250-mL beaker. The
mixture was neutralized with 1M NaOH and
that neutralized mixture was used for
characterization tests and chromatography.
E. Enzymatic Hydrolysis of Intact Protein
To perform the enzymatic hydrolysis,
a 1g/100 mL distilled water protein mixture
was prepared. A volume of 10 mL of the
protein mixture and 10 mL of the saturated
protease was mixed. Alternatively, a 0.050 g
of protease may be added directly to 50 mL
protein mixture. Then, a 10mL 0.1 M
phosphate buffer with a pH of 7.5 was added.
The tube was incubated in a water bath
maintained at 35-40 oC for about an hour.
Again, alternatively, digestion may be carried
out overnight at room temperature. Then, the
mixture was allowed to cool and was then
used for qualitative procedures for color
reactions and chromatography.
F. Qualitative Color Reactions
Nine groups of test tubes were
prepared for the test. Each group of test tube
contains intact proteins, acid and basic
hydrolysates, added with 1 mL of distilled
water.
The first test performed was the Biuret
test wherein 20 drops of 2.5 M NaOH was
added and mixed to the samples. Then, 2-3
drops of 0.1 M CuSO4 was added.
The second test was the Ninhydrin
test wherein 6-10 drops of 0.1% ninhydrin
solution was placed into the samples then
heated in a boiling water bath.
The third test was the Xanthoproteic
test wherein 10 drops of concentrated HNO3
was slowly added into the samples. After
adding HNO3, 10 drops of NaOH was slowly
added.
The fourth test was the Millon’s test
wherein 5 drops of Millon’s reagent was
added to the samples.
The fifth test was the Hopkins-Cole
test wherein 20 drops of Hopkins-Cole
reagent was mixed to the samples. Then each
test tubes were inclined and 20 drops of
concentrated H2SO4 was slowly added.
The sixth test was the Sakaguchi test
wherein 10 drops of 10% NaOH and 10 drops
of 0.02% napthol solution was added to the
samples. After 3 minutes, 3 drops of 2%
NaOBr was mixed to the samples.
The seventh test was the
Nitroprusside test wherein 0.5 mL of 3 M
NaOH was added to the samples. Then, 0.25
mL of 2% nitroprusside solution was added.
The eighth test was the Fohl’s test
wherein 5 drops of 30% NaOH and 2 drops of
5% (CH3COO)2Pb was added to the samples
then the rest were placed in a boiling water
bath.
The last test performed was the test
for amides wherein 1 mL of 20% NaOH was
added to 10 drops of the sample. The
samples were then placed in a boiling water
bath and red litmus paper was placed and
moistened over the mouth of the tube.
G. Alkaline Hydrolysis of Intact Protein
In alkaline hydrolysis, 10 mL of 4M
NaOH was added to 0.5 g isolated myoglobin
in a hard glass test tube. The test tube was
properly labeled according to the format
given. Like in acid hydrolysis, cotton was
placed to stopper the tube before it was
subjected to autoclaving at 15 psi for 5 hours.
Also, the appearance of the mixture was
considered and noted. Then again, distilled
water with a volume of 10 mL was added in
the mixture then was transferred into a 250-
mL beaker. Nevertheless, the mixture was
neutralized with 1M HCl and that neutralized
mixture was used for characterization tests
and chromatography.
H. Thin Layer Chromatography
In doing this test, 0.5cm margin was
measured from the bottom of the plane
horizontally, the original line was the drawn in
all TLC plates. Equidistant points were then
marked to show the plot of each solution. In
the capillary tubes, the standards were
applied five times while the samples were
applied ten times and made sure that they
were dried in between applications. For the
paper chromatography of the amino acids,
mixture of butanol, acetic acid, and water,
with the ratio of 4:1:5 respectively, was used
as the solvent system. Using a capillary tube,
tryptophan, arginine, proline, cystein serine,
aspartate, histidine, glycine, alanine, acid
hydrolysate and basic hydrolysate was
spotted on the paper chromatogram. When
the chromatography procedure was done, 1%
Ninhydrin solution was sprayed to the
chromatogram to react with the protein and be
identified. The paper was placed over the hot
plate to dry.
I. Quantitative Analysis
Preparation of Albumin Standard (Stock
Solution)
In a 100mg/ml of Bovine serum
albumin, 10mg/ml of BSA stock solution was
prepared by transferring 0.2ml of BSA in a
microtest tube, diluted with 1.8ml of distilled
water.
Bradford and Biuret Total Protein Assay’
In the Biuret Test, 0.8ml was
transferred in to the first microtest tube and
diluted with 1.2ml of distilled water to make
the contentration 4mg/ml. 1ml was drawn to
the first microtestube to be transferred to the
second microtest tube and diluted to 1ml of
water to achieve the concentration of 2mg/ml.
The remaining microtest tubes have the same
procedure that achieved the equivalent
concentrations.
In the Bradford Test, the 10mg/ml
BSA stock solution, 0.2ml was transferred in
to the first micro test tube and diluted with 1.8
ml of distilled water to make the concentration
1mg/ml. From the first microtest tube, 1ml
was then transferred to the second microtest
tube and diluted to 1ml of water to achieve the
concentration of 0.5mg/ml. The remaining
microtest tubes have the same procedure that
achieved the equivalent concentrations.
Preparation of Isolated Albumin (Stock
Solution)
The Biuret test and Bradford was used
in the preparation of the isolated albumin. In
the Biuret Test preparation, the concentration
was 0.01g/ml of the isolated albumin solution,
then 10mg of isolated albumin was weighed
in the analytical balance and was diluted with
1ml of water. On the other hand, in the
Bradford Test, 0.001g/ml was prepared by
transferring 0.1ml of 10mg/ml stock solution
and diluting it with 0.9ml distilled water. After
preparing all of the samples, 0.1ml of each
isolated albumin stock solution and BSA were
transferred to the corresponding well and was
added 01.ml of the Bradford and Biuret
Reagent.
III. RESULTS AND DISCUSSIONS
In the conducted experiment different
types of proteins were isolated from their
sources such as non-fat milk powder and
wheat flour. Intact proteins of gluten and
casein, gluten acid hydrolysates and Casein
enzymatic hydrolysates were all tested to
characterize and determine the functional
groups that they contain. [See table 2]
The tests that were performed had different
concepts behind them. The reactions of the
intact proteins and hydrolysates to the
reagents of each test, depends on their
characteristics. They can either be positive or
negative in a particular test. [See table 1 and
table 2]
A. Qualitative Color Reactions
Fig. 1 Color Reactions Results of Intact
Gluten
Biuret test is a test for proteins and for
detecting peptide linkage (CO-NH group). Its
principle is used to determine the presence of
proteins in biological fluids[2]. The intact
protein should be positive in this test since its
peptide linkage is not yet broken. As seen in
the results, intact proteins of both casein and
gluten produced a violet solution which is a
positive indication of the Biuret test which
would be a correct result since their peptide
linkages was not yet broken since it wasn’t
hydrolyzed yet. But both gluten acid
hydrolysate and casein enzymatic
hydrolysate still had a positive result which
means that their petide linkage was still intact.
This result was not supposed to occur but it
can be because the hydrolysis of gluten and
enzymatic hydrolysis of casein was not
conducted properly which would result for
their peptode linkage not to break.
Ninhydrin test is a for detecting free
alpha amino groups. Its principle is oxidative
deamination and decarboxylation[3]. The
positive result of this test is a blue-violet
coloration of the solution. Intact gluten and
casein, gluten acid hydrolysate, and casein
enzymatic hydrolysate had negative results in
this test. But intact casein and the hydrolates
was supposed to have a positive result in this
test and this error in the result may be caused
by possible contaminations in the samples.
Xanthoproteic test is a test for the
presence of aromatic nucleus such as
phenylalanine, tryptophan and tyrosine in a
protein solution. The expected positive result
of this test is a yellow coloration of the
solution. Although phenylalanine is an
aromatic nucleus, it will not have a positive
result because it gives a negative or weakly
positive reaction[4].Its principle is the nitration
of the phenyl group. Intact casein was the
only one who had a positive for this test. In the
result for this test, there had been an error in
the color of the result of gluten acid
hydrolysate. The explanation for the
variations of result in this test is that some
amino acids may contain any aromatic groups
and other do not.
Millon’s test is a test for the presence
of an hydroxybenzene radical and the only
amino acid with an hydroxybenzene ring is
tyrosine. Its principle is the complexation
reaction between phenolic group and mercury
that is found in the Millon’s reagent. The Nitroprusside test is used for detecting
positive result of this test in the presence of the presence of free thiol groups of cysteine[9].
mercury in a solution is a yellow precipitate[5]. In this test, cysteine is partially destroyed and
In this test, intact gluten and casein, gluten it produced a red solution. Its principle is
acid hydrolysate, and casein enzymatic complexation. In this test, intact gluten and
hydrolysate had negative results. Which casein, gluten acid hydrolysate, and casein
would mean that all of the tested proteins had enzymatic hydrolysate had negative results.
no presence of tyrosine. But then again, there The results indicates that cysteine is absent.
may be errors in conducting the experiment The test for amides which indicates
which affected the result. the presence of primary, secondary and
Hopkins-Cole test is a test for the tertiary amides and nitriles[10]. A positive result
presence of tryptophan. The positive result of for this test is the change in color of litmus
this test is the violet to blue coloration of the paper from red to blue. Its principle is basic
solution when a mixture of protein and an hydrolysis. As seen in the result intact
aldehyde is layered over a concentrated proteins such as gluten, casein, gluten acidic
sulphuric acid[6]. As seen in the results, all of hydrolysates and casein enzymatic
the proteins had a negative result which hydrolysates had positive results for this test.
would mean that there was no presence of The last test conducted was Pauly’s
tryptophan. The error of result in this test may test. It histidine and tyrosine which would yield
be cause by the wrong execution of the a positive result of a red coloration of the
experiment. solution[11]. Intact casein and gluten acid
Sakaguchi test is a test for the hydrolysate had a positive result in this test.
presence of arginine which reacts with the While Intact gluten and casein enzymatic
guanidinium compound[7]. Alkaline or basic hydrolysate had negative results.
hydrolysis destroys arginine and produces
ornithine and urea, thus, basic hydrolysate Table 1. Results of Qualitative Color
must be negative for this test while all the Reaction for Intact Gluten and Intact
other samples are positive. Its principle is the Casein
reaction of Guanido group with α napthol and Test Intact Gluten Intact Casein
an oxidizing reagent. A positive indication for Biuret Purple Purple
this test is a red or orange solution. In this test, solution (+) solution (+)
intact gluten and casein, gluten acid Ninhydrin Turbid Colorless
hydrolysate, and casein enzymatic solution (-) solution (-)
hydrolysate had negative results. The results Xanthoprot Turbid Yellow
eic solution (-) solution (+)
contradicts to what was the expected results
Millon’s Turbid Colorless
and these errors may have been affected by
solution (-) solution (-)
contamination in the samples or mistakes Hopkins- Colourless Colorless
may have been done during the Cole solution (-) solution (-)
experimentation. Sakaguchi Pink solution Colorless
Fohl’s test is a test for sulfur (-) solution (-)
containing proteins. Which indicates the Fohl’s Brown Yellow
presence of methionine and cysteine since solution (-) solution (-)
these two amino acids have sulfur in their Nitroprussi Yellow Orange
structures. Its principle is fusion followed by de solution (-) solution (-)
ionic interaction[8]. The positive result for this Test for Red to blue Red to Red
test is the formation of black precipitate from Amides litmus paper litmus paper(-
lead sulfide (PbS). All of the sample in this (+) )
test produced negative results. But gluten Pauly’s Turbid Red solution
solution (-) (+)
acid hydrolysate had black sediments which
was caused by further reaction of Na2
Table 2. Results of Qualitative Color
Reaction for Gluten Acid Hydrolysate and
Casein Enzymatic Hydrolysate

Test Gluten Acid Casein

Hydrolysate Enzymatic
Hydrolysate
Biuret Violet solution Purple Figure 3. Paper Chromatogram of the
(+) solution (+) Standard Amino Acids, Gluten Acid
Ninhydrin Pink solution Colorless Hydrolysate and Alkaline Myoglobin
(-) solution (-)
Xanthoprot Orange Colorless
eic solution (-) solution (-)
Millon’s Brown Colorless
solution (-) solution (-)
Hopkins- Formation of Colorless
Cole 3 layers in the solution (-)
solution:
Brown,
Figure 4. Paper Chromatogram of Alkaline
Yellow and
Orange (-) Gluten and Acid Myoglobin
Sakaguchi Colorless Colorless
solution (-) solution (-) Based on the results seen on Figure 2
Fohl’s Black Colorless and Figure 3, most of the standard amino
Sediments (-) solution (-) acids only had a short distance travelled from
Nitroprussi Yellow Yellow the origin and this shows that the standard
de solution (-) solution (-) amino acids had a high affinity with the
Test for Red to blue Red to blue stationary phase which is polar which
Amides litmus paper litmus paper suggests that alanine, arginine, aspartic,
(+) (+) cysteine, glycine and histidine are polar. The
Pauly’s Red solution Colorless distance travelled by a spot in a TLC plate can
(+) solution (-)
be calculated with the formula:
B. Chromatography Rf Value = Distance of Compound from the Origin
The Thin Layer Chromatography Distance of Solvent from the Origin
(TLC) is a technique shows the difference on
affinity of the standard amino acids, acidic Table 3. Computed Rf Values of the
hydrolates and alkaline hydrolates of the Standard Amino Acids
samples such as gluten and casein to the
mobile and stationary phase. The results of Standard Rf Value
the paper chromatography are based on the Amino Acid
polarity of the amino acids[12]. Alanine 0.11
Arginine 0.08
Aspartic Acid 0.05
Cysteine 0.17
Glycine 0.08
Histidine 0.06
Proline 0.15
Figure 2. Paper Chromatogram of the
Standard Amino Acids Serine 0.1
 Tryptophan 0.51 Spectrophotometry, we were able to plot each
Tyrosine 0.44 sample and see the relationship of the
concentration of the protein samples and its
From the given standards, tryptophan mean absorbance.
has the biggest Rf value which suggests that Fig. 5 Protein Sample’s Bradford Assay
it has a high affinity to the moving phase Graph Result
which is a 4:1:5 ratio of 1-butanol, acetic acid,
and water which is a non-polar. This suggests
that among the amino acid standards, BRADFORD ASSAY
tryptophan is the most non-polar. On the other 0.800
hand, that standard amino acid Casein had a y = 0.0008x + 0.2843
0.700
faint yellow coloration and this was probably R² = 0.9857
Protein
because it was contaminated by proline 0.600

during the spotting of amino acid standards.
The results of the TLC test of casein
enzymatic hydrolysate, gluten acid
hydrolysate, and alkaline gluten showed
results that are erroneous and this is because
the spots had tailing and casein was not
visible enough for us to see the circumference
of the spot. [See Figure 3 and4]
The tailing could have been resolved
by modifying the polarity of the mobile phase
with a hydrogen bonding solvent component.
The faintness of the color could have been
resolved by spotting the TLC plate with the
right amount of color in the spot. Due to these
errors we were not able to find the Rf values
of casein enzymatic hydrolysate, gluten acid
hydrolysate, and alkaline gluten.
C. Quantitative Analysis (Bradford Protein
Assay)
The Bradford assay is used in
determining the total protein concentration of
a sample. This method is based on the
binding of Coomassie dye to the proteins[13].
The acidic environment of the reagent results
in a spectral shift from the reddish brown of
the dye, which has a maximum absorbance of
465-595 nm, to the blue form of the dye. In the
experiment, after the addition of the dye to our
protein sample solutions in the 96 well
microplate, we did the UV Spectrophotometry
method to get the absorbance of each
sample. From the gathered data in the UV
Mean Absorbance
0.500
0.400
0.300
0.200
0.100
0.000
0 100 200 300 400 500 600
Protein Concentration
After plotting the results we were able
to get the formula y= 0.0008x + 0.2843 and
from this equation we computed the
concentration of our protein sample. The
concentration of our protein was 454.625 with
a mean absorbance of 0.648 which was from
the sample protein assigned for our group and
it was used as the “y” of the equation. The
result shows that as the concentration
increases the absorbance also increases
which shows a linear relationship between the
mean absorbance and protein concentration.
Since our protein sample solution had a high
concentration and it also had a high
absorbance it means that there was a great
amount of protein contained that was
detected by the coomasie dye or the Bradford
reagent.
D. Quantitative Analysis (Biuret Protein
Assay
 Biuret protein assay uses alkaline
copper sulfate which forms a purple complex
when it is bound to the peptide bonds[14]. The
more purple complex is formed in a solution
the more concentrated the protein in the
solution is. We placed our protein solution in
a 96 well microplate with the addition of the
biuret reagent and then we used the UV
Spectrophotometry method to get the
absorbance of each sample. From the data
gathered in the UV Spectrophotometry, we
were able to plot each sample and see the
relationship of the concentration of the protein
samples and its mean absorbance.
Fig. 6 Protein Sample’s Biuret Assay
Graph Result
BIURET ASSAY
0.250
y = 4x10-5x + 0.0521
0.200 R² = 0.9944
Mean Absorbance
0.150
0.100
Protein
0.050
0.000
0 1000 2000 3000
Protein Concentration
After plotting the results we were able
to get the formula y= 4x10-5x+ 0.0521 and
from this equation we computed the
concentration of our protein sample. The
concentration of our protein was 597.5 with a
mean absorbance and also the “y” of the
equation of 0.076 which was from the sample
protein assigned for our group. The result
shows that as the concentration increases the
absorbance also increases which means that
there is a liner relationship between the mean
absorbance and protein concentration. Also,
it can be inferred from the results that there
was great amount of protein contained in the
sample that was detected by the biuret
reagent.
IV. CONCLUSION
Based on the experiment done, the
intact gluten sample had negative results in all
the tests but with the test of amides it showed
a positive result. The results stated that the
intact gluten sample had a positive relation to
the basic hydrolysis. It can be concluded that
the intact gluten has a primary, secondary
and tertiary amide and nitrile present in the
sample.
V. REFERENCES
[1] Wieser H. Chemistry of gluten proteins.
Food Microbiol. 2007;24:115–119. doi:
10.1016/j.fm.2006.07.00
[2] Karki, G. (2018, December 18). Biuret test:
Principle, requirements, Procedure and
Result. Retrieved from
https://www.onlinebiologynotes.com/biuret-
test-principle-requirements-procedure-and-
result/.
[3] Karki, G. (2018, December 18). Ninhydrin
Test: Principle, Requirements, Procedure and
4000 5000
Result. Retrieved from
https://www.onlinebiologynotes.com/ninhydri
n-test-principle-requirements-procedure-and-
result/.
[4] Karki, G. (2018, December 18).
Xanthoproteic test: Objective, Principle,
Reagents, Procedure and Result. Retrieved
from
https://www.onlinebiologynotes.com/xanthop
roteic-test-objective-principle-reagents-
procedure-and-result/.
[5] Karki, G. (2018, December 18). Millon's
test: Objective, Principle, Reagents,
Procedure and Result. Retrieved from
https://www.onlinebiologynotes.com/millons- [13] He, F. (n.d.). Bradford Protein Assay.
test-objective-principle-reagents-procedure- Retrieved from https://bio-
and-result/. protocol.org/bio101/e45.

[6] Karki, G. (2018, December 18). [14] Biuret Protein Assay. (n.d.). Retrieved
Adamkiewicz reaction (Hopkin's-cole test): fromhttps://www.ruf.rice.edu/~bioslabs/meth
Objective, Principle, Reagents, Procedure ods/protein/biuret.html.
and Result. Retrieved from
https://www.onlinebiologynotes.com/adamkie
wicz-reaction-hopkins-cole-test-objective-
principle-reagents-procedure-and-result/.

[7] Karki, G. (2018, December 18). Sakaguchi

test: Objective, Principle, Reagents,
Procedure and Result. Retrieved from
https://www.onlinebiologynotes.com/sakaguc
hi-test-objective-principle-reagents-
procedure-and-result/.

[8] Qualitative Analysis of Amino Acid. (n.d.).

Retrieved from
https://vlab.amrita.edu/?sub=3&brch=63&sim
=1094&cnt=1.

[9] Nitroprusside Reaction. (n.d.). Retrieved

from
https://www.sciencedirect.com/topics/bioche
mistry-genetics-and-molecular-
biology/nitroprusside-reaction.

[10] Detection of Functional Groups. (n.d.).

Retrieved from
https://vlab.amrita.edu/?sub=2&brch=191&si
m=345&cnt=1.

[11] Karki, G. (2018, December 18). Pauly's

test: Objective, Principle, Reagents,
Procedure and Result. Retrieved from
https://www.onlinebiologynotes.com/paulys-
test-objective-principle-reagents-procedure-
and-result/.

[12] THIN LAYER CHROMATOGRAPHY.

(n.d.). Retrieved from
https://www.chemguide.co.uk/analysis/chrom
atography/thinlayer.html.