QUALITATIVE COLOR REACTIONS

Jyle Fraium L. Dino, Franc Ison P. Dy,

Demi Louise L. Go​, Vheverli Whendel P. Go, Marc Lennard G. Ip
Group 4 2A Medical Technology Biochemistry Laboratory

ABSTRACT
The experiment was conducted for the characterization of the protein myoglobin by analyzing the reactions of its side
chains, a-amino, and a-carboxyl groups. Several tests were performed for the investigation which yielded various
colors for each specific test. The Biuret test was used to detect the presence of peptide bonds. This produced a light
blue color for both the acid and enzyme hydrolyzed protein. The Ninhydrin test which is a test for the a-amino acids
showed both negative results for the acidic and enzymatic hydrolysed protein. The Xanthoproteic test for the detection
of side chains of aromatic amino acids and the Millon’s test for the tyrosine and tryptophan residues both produced
negative results as well for both proteins. The Hopkins-Cole test, which was also for the tyrosine and tryptophan
residues did not produce any violet ring. Sakaguchi test was used to determine the arginine amino acid residue. A
yellow and colorless solution was obtained from the acidic and enzymatic hydrolyzed proteins respectively. For the
Nitroprusside test for the thiol group in cysteine, a yellow-orange color was produced for both hydrolyzed proteins.
Fohl’s test for the sulfur amino acids for cysteine and methionine showed positive results for intact protein for
producer a brown precipitate while a negative results for the hydrolysates for giving out a colorless solution. The Test
for Amides displayed the presence of ammonia, detecting the R-groups of asparagine and glutamine. Furthermore, the
Pauly’s test determines the presence of tyrosine and histidine. It results to the formation of highly colored
azo-compounds. After testing, both produced an orange solution.
INTRODUCTION EXPERIMENTAL
Amino acids have a variety of chemically A. Test Compound/s or (Sample/s) used
reactive groups. The reactions for side chains, Intact protein solution, 6M NaOH, 3M NaOH,
a-amino, and a-carbonyl groups can be used to 2.5M NaOH, 20% NaOH, 10% NaOH, Conc.
characterize both free amino acids and proteins. NaOH, 5% N aN O2 , 10% N a2 CO3 , 2%
Amino acids are amphoteric, behaving as amines Nitroprusside solution, 0.02% Naphthol solution,
in some reactions and as carboxylic acids in 1% Sulfanilic acid, 0.1% Ninhydrin solution,
others. Amino acids are critical to life.They have Conc. HN O3 Conc. H 2 SO4 , Hopkins-Cole reagent,
particularly important functions like being the Millon’s reagent, 2% NaOBr, 0.1M CuSO4 , 5%
building blocks of proteins and being the P b(CH 3 COO2 ) 2
intermediates in metabolism. Proteins, also
known as peptides, are organic compounds made B. Procedure
of amino acids arranged in a linear chain.Like 1. Biuret Test
other biological macromolecules, proteins are In this test, 20 drops of 2.5 NaOH were added
essential parts of organisms and participate in into a test tube with the intact protein and two
catalyzing biochemical reactions, structural and separate test tubes with 0.5mL of each acidic
mechanical functions, cell signaling, and other hydrolyzed sample and enzymatic sample. Two
processes within cells. to three drops of 0.1 CuSO4 solution were added
to each test tube. Gently shake the test tubes.
Myoglobin is a protein isolated from beef The colors of the three resulting solutions were
samples. A myoglobin polypeptide is comprised recorded.
of 8 separate right-handed α-helices,designated
A through H, that are connected by short non 2. Ninhydrin Test
helical regions. Amino acid R-groups packed into In this test, 6 - 10 drops of 0.1% ninhydrin
the interior of the molecule are predominantly solution were placed into a test tube with the
hydrophobic in character while those exposed on intact protein and two separate test tubes with
the surface of the molecule are generally 0.5mL of each acidic hydrolyzed sample and
hydrophilic, thus making the molecule relatively enzymatic sample. The three test tubes were
water-soluble. heated in a boiling water bath. The colors were
noted as well if the samples turned blue violet.
3. Xanthoproteic Test sample and enzymatic sample. The test tubes
In this test, 10 drops of the conc. HN O3 were were then placed into a boiling water bath. The
slowly added into a test tube with the intact colors of the resulting solutions were noted as
protein and two separate test tubes with 0.5mL well as the appearance of dark sediments.
of each acidic hydrolyzed sample and enzymatic
sample. Mix well. The colors of the resulting 9. Test for Amides
samples were noted. Ten drops of the conc. In this test, 1mL of the 20% NaOH was added
NaOH were slowly added. Mix well. The colors of into the ​test tube with 10 drops of the intact
the resulting solution were then recorded. protein and two separate test tubes with 10
drops of each 0.5mL of enzymatic sample and
4. Millon’s Test acidic sample. The test tubes were then placed in
In this test, 5 drops of the Millon’s reagent a boiling water bath. The presence of the
were were added into a test tube with the intact evolution of gas during heating was tested on
protein and two separate test tubes with 0.5mL both using litmus paper mounted on the mouth
of each acidic hydrolyzed sample and enzymatic of both test tubes. The change in colors of the
sample. The colors of the resulting solution were three litmus paper were recorded for the intact
noted. protein, acidic hydrolyzed sample and enzymatic
hydrolyzed sample, respectively.
5. Hopkins-Cole Test
In this test, 20 drops of the Hopkins - Cole
reagent were slowly added were added into a test
tube with the intact protein and two separate test
tubes with 0.5mL of each acidic hydrolyzed
sample and enzymatic sample. Mix well. The test
tubes were inclined and 20 drops of the conc.
H 2 SO4 were slowly added along the side. The test
tubes were not mixed. The colors at the interface
were recorded.
Figure 1. ​Litmus paper for the Test for Amides
6. Sakaguchi Test
10. Pauly’s Test
In this test, 10 drops of the 10% NaOH and 10
In this test, the diazo reagent was prepared by
drops of the 0.02% Naphthol solution were added
mixing 3 - 5 drops of 1% sulfanilic acid with 3
into a test tube with the intact protein and two
drops of the 5% N aN O2 solution. Five drops of
separate test tubes with 0.5mL of each acidic
each intact protein, acidic and enzymatic samples
hydrolyzed sample and enzymatic sample. The
and 3 - 5 drops of 10% N a2 CO3 were added to
test tubes were mixed then were left alone for 3
minutes. Three drops of of 2% NaOBr. The the diazo reagent. The appearance of a red
colors of the resulting solutions were recorded. coloration was recorded.

7. Nitroprusside Test
In this test, 0.5mL of the 3M NaOH was added
into a test tube with the intact protein and two
separate test tubes with 0.5mL of each acidic
hydrolyzed sample and enzymatic sample. A
quarter of milliliter of 2% nitroprusside solution
was added to the test tubes. The colors of the
resulting solution were noted.

8. Fohl’s Test
In this test, 5 drops of 30% NaOH and 2 drops
of 5% 5% P b(CH 3 COO2 ) 2 were added into a test Figure 2.​ Qualitative Color Reactions of Acidic
tube with the intact protein and two separate test Hydrolyzed Protein
tubes with 0.5mL of each acidic hydrolyzed
 Test for Redàblue Redàblue Blueàblue
Amide litmus Redàred
paper

Pauly Orange orange Yellow-oran

color ge

1. Biuret Test
​In the Biuret test, peptide bonds are detected.
The presence of proteins turns the solution into a
violet color. The reagent that is composed of
sodium hydroxide and copper sulfate reacts with
gluten and yielded a positive result with a light
blue-violet color.
Figure 3.​ Qualitative Color Reactions of
Enzymatic Hydrolyzed Protein

RESULTS AND DISCUSSION

​The following results shown in table 1 were
obtained from the tests performed for the
characterization of the protein myoglobin,
observing reactions qualitatively.
Figure 4. ​Biuret’s Test
Table 1. ​Qualitative color reactions
2. Ninhydrin Test
Acidic Enzymatic ​This test used ninhydrin to react with amines or
Color Intact Hydrolyse Hydrolysed alpha amino acids. A colored end product is a
Reaction Protein d Protein Protein positive result, with alpha amino acids giving off
a purple reaction while secondary amines give a
Biuret Purple Light blue Light blue yellow-orange reaction [9]. The acidic and
solution
enzymatic hydrolyzed proteins both yielded a
negative result, not undergoing any color
Ninhydrin Yellow-oran yellowish colorless
ge ppt reaction.

Xanthopr HNO- white + colorless + colorless

oteic NaOH- + colorless + colorless
colorless

Millon’s colorless white colorless

Figure 5. ​Ninhydrin Test
Hopkins- No violet No violet No violet
Cole ring ring ring 3. Xanthoproteic Test
​In this test, tyrosine-containing proteins reacts
Sakaguch colorless yellowish colorless
with the added concentrated nitric acid to the
i
substrate being tested. A yellow reaction is
Nitroprus Yellow Yellow-oran Yellow-oran deemed as a positive result for present proteins
side solution ge ge that have amino acids with aromatic rings,
especially tyrosine. A negative result, however,
Fohl’s Brown color colorless colorless was yielded in the experiment.
 Figure 6. ​Xanthoproteic Test

4. Millon’s Test
​This test detects the presence of soluble
proteins, most notably tyrosine and tryptophan.
The color reaction that will be produced is given
by the benzene derivatives wherein hydrogen is
Figure 8. ​Sakaguchi Test
replaced by a hydroxyl group, and a
reddish-brown color depicts a positive reaction.
7. Nitroprusside Test
Mercury (I) and mercury (II), the tyrosine
This test is identified by the reaction of
complexes nitrated by nitric acid forms the red
cysteine to the 3M NaOH and 2% nitroprusside
color [10]. Both acidic and enzymatic hydrolyzed
solution. It also test the presence of amino acids
proteins tested negative for the Millon’s Test.
with a sulfhydryl group (-SH). A positive result
would yield into a red colored solution [1][6][8].
A yellow-orange solutions were obtained from the
intact protein and acidic and enzymatic
hydrolyzed proteins. Myoglobin tested negative
for cysteine.

Figure 7. ​Millon’s Test

5. Hopkins-Cole Test
​The Hopkins-Cole test determines the
presence of tryptophan specifically, which is the Figure 9.​ Nitroprusside Test
only indole group-containing amino acid [10]. A
violet cyclic product is yielded when the the 8. Fohl’s Test
indole ring reacts with a strong acid. Sulfuric acid This test is identified by the reaction of
was used. The acidic and enzymatic hydrolyzed cysteine, cystine, and methionine to the 30%
proteins both did not produce any violet ring, NaOH and 5% Pb(CH​3​COO)​2​. It detects the
resulting to a negative reaction. presence of sulfur containing amino acids. A
positive result would yield into the presence if
6. Sakaguchi Test dark sediments [1][3][6][8]. Intact myoglobin
In this test, the reaction is identified by yielded a positive result because of the presence
guanidinium compound and is particular for of brown precipitate. The acidic and enzymatic
arginine. The 0.02% naphthol and an oxidizing hydrolyzed myoglobin yielded a negative result
agent, sodium hypobromite, reacts with arginine due to the presence of a colorless solution.
to give a positive result of a red colored product
[1][5][6][7][8]. A flesh, yellow, and colorless
solution was obtained from the intact protein,
acidic and enzymatic hydrolyzed proteins
respectively. Myoglobin tested negative for
arginine.

Figure 10.​ Fohl’s Test

9. Test for Amides

In this test, the presence of ammonia,
detecting the R-groups of asparagine and
glutamine, and it can be indicated be indicated
through the changes of the colors of a litmus
paper [1]. The intact myoglobin and the acidic
hydrolyzed myoglobin tested positive for the
presence of amides as it turned red litmus paper
into blue. The enzymatic hydrolyzed myoglobin
tested negative as it failed to produce a color in
the litmus paper.
10. Pauly’s Test
Amines, phenols and imidazole pairs with
diazotized sulfanilic acid to be able to yield highly
colored azo compounds. Diazonium is produced
in a cold environment that’s why the reaction
must be done in a cold condition. Tyrosine or
histidine coupled with diazonium salt in alkaline
condition to form red coloured azo dye [4][8].
The intact myoglobin and the acidic hydrolyzed
myoglobin yielded both orange solutions while
the enzymatic hydrolyzed myoglobin produced a
yellow-orange solution. It can be deduced that
myoglobin tested negative for ​tyrosine and
histidine.
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5th/Ch27/ch27-3-3.html
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Retrieved March 22, 2019, from
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-proteins/13065

Figure 11. ​Pauly’s Test

REFERENCES
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