Qualitative Analysis of Amino Acids and Proteins

Joshua D. Abelgas
James Ryan B. Baring
Mary Grace L. Denatil
Camille C. Mahinay
Chem315.2, BS CHEM 3H1
Instructor: Nesteve John B. Agosto, RCh, MSc

University of Science and Technology of Southern Philippines

September 28, 2021

Page 1 of
Abstract

Page 2 of 19
 Introduction

1.1. Rationale of the Experiment

Proteins are the basic component of living cells. Amino acids are the building blocks of
proteins. They contain the basic elements in living organisms, including carbon, hydrogen,
oxygen, and nitrogen. There are twenty different amino acids. An amino acid structure comprises
the central carbon atom, which bonds with the amine group, the carboxyl group, a hydrogen
atom, and the “R” group, as shown in Figure 1.1. The “R” group in amino acids is what
distinguishes it from other amino acids. The side-chain structure determines the class of the
amino acid: nonpolar, neutral, acidic, or basic (Milio & Loffredo, 1995).

Figure 1.1 Structure of α-amino acids

Proteins are also made up of polypeptides. Polypeptides are formed by amino acids that
have started bonding together. The binding is called a peptide bond (Libretexts, 2021). Amino
acids also connect and form a chain called a polypeptide chain. The peptide bonds between the
carboxyl and amino groups of adjacent amino acid residues hold the amino acids in a polymer
together. Like other biological macromolecules like polysaccharides and nucleic acids, proteins
are fundamental elements of organisms and play a role in almost every function within cells and
serve a variety of purposes (Ayyagari, 2007). Transport proteins (such as hemoglobin), storage
proteins, structural proteins, and proteins involved in muscular contraction are a few examples.
Different proteins are made up of different amino acids.
Specific functional groups in amino acids and proteins can react to produce
characteristically colored products. The color intensity of the product formed by a particular
group varies among proteins in proportion to the number of reacting functional or free groups
present and their accessibility to the reagent (Milio & Loffredo, 1995).
In this experiment, various color-producing reagents were used to qualitatively detect the
presence of certain amino acids in proteins, such as casein and albumin. Casein, the chief protein
in milk and the essential ingredient of cheese. In pure form, it is an amorphous white solid,
tasteless and odourless, while its commercial type is yellowish with a pleasing odour (The
Editors of Encyclopaedia Britannica, 2021). Albumins, on the other hand, are generally soluble
in water and in dilute salt solutions. They can be denatured and coagulated by heat. After casein,
albumins are the second most abundant proteins in milk (Pamarthy et al., 2016). The principles
of these qualitative tests are defined below.
Millon’s Test
Millon’s test is specific to phenol containing structures. Figure 1.2 shows the structure of
tyrosine, which is the only common phenolic amino acid. Millon’s reagent is concentrated
HNO3, in which mercury is dissolved. As a result of the reaction a red precipitate or a red
solution is considered as positive test.

Figure 1.2 Structure of Tyrosine

Xanthoproteic Test
Some amino acids contain aromatic groups that are derivatives of benzene. These
aromatic groups can undergo reactions that are characteristics of benzene and benzene
derivatives. One such reaction is the nitration of a benzene ring with nitric acid. The amino acids
that have activated benzene ring can readily undergo nitration. This nitration reaction, in the
presence of activated benzene ring, forms yellow product. Figure 1.3 below shows the
mechanism for xanthoproteic reaction.

Figure 1.3 Xanthoproteic test reaction mechanism

Biuret Test
The biuret test for protein positively identifies the presence for proteins in solution with a
deep-violet color. Biuret, H2NCONHCONH2, reacts with copper (II) ions in basic solution to
form a deep violet complex. The peptide linkages in proteins resemble those in biuret and also
form
deep violet complexes with basic copper (II) ion in solution. The general or biuret complex
formed between the protein linkages and the copper (II) ion of the biuret test is shown in Figure

1.4.

Figure 1.4 Structure of protein-copper (II) complex

Sulfur Test
When sulfur-containing proteins (such as cysteine and cystine) are heated with lead
acetate in an alkaline medium, a black deposit of lead sulfide, PbS, forms. The structures of
cystine and cysteine are shown in Figures 1.4 and 1.5, respectively.

Figure 1.5 Structure of cystine Figure 1.6 Structure of cysteine

Molisch Test
Molisch's test is a sensitive chemical test for the presence of carbohydrates, based on the
dehydration of the carbohydrate by sulfuric acid or hydrochloric acid to produce an aldehyde,
which condenses with two molecules of a phenol, resulting in a violet ring (Foulger, 1931).
Hopkin’s Cole Test
This test is specific test for detecting tryptophan as shown in Figure 1.7. The indole
moiety of tryptophan reacts with glyoxilic acid in the presence of concentrated sulphuric acid to
give a purple colored product.

Figure 1.7 Structure of Tryptophan

Heller’s Ring Test
 Heller's test is a chemical test that shows that strong acids cause the denaturation of
precipitated proteins. Concentrated nitric acid is added to a protein solution from the side of the
test tube to form two layers. A white ring appears between the two layers if the test is positive
(Dandekar, 2004). Heller's test is commonly used to test for the presence of proteins in urine.

Precipitation Reaction
Proteins are separated or precipitated using different methods such as heating, salting out
effect, use of acids, alcohol, organic solvents, use of positive ions of heavy metals such as Pb 2+,
Zn2+, Cu2+, Cd2+, Hg2+, and Fe3+, and use of alkaloidal reagents producing insoluble proteinate.

1.2 Objectives of the Experiment

The objective of this lab experiment is to qualitatively detect the presence of certain
amino acids in proteins, such as casein and albumin, using various color-producing reagents. The
experiment also addressed the following specific objectives:
1) to identify various chemical components of proteins;
2) to conduct chemical reactions and tests of amino acids; and
3) develop an understanding of the significance of each qualitative test.
Methods and Materials
 Results

The findings of different qualitative tests on proteins casein and albumin are summarized
in the tables below. Various color-producing reagents were used in the experiment to detect the
presence of specific amino acids of the aforementioned proteins. The findings of qualitative tests
for protein precipitation reactions, such as coagulation (denaturation), alcohol effects
(dehydration), neutral salt, and precipitation with alkaloidal reagents, were also tabulated.

3.1 Color Reactions of Proteins

As the name suggests, amino acids are organic compounds that contain amino and
carboxyl groups. The “R” group in amino acids denotes several chemical groups (aliphatic,
aromatic, or heterocycylic), which influence the amino acid's properties. Color tests have long
been used for the qualitative detection of amino acids. Proteins respond to some color reactions
due to the presence of one or more radicals or groups of the complex protein molecule. All
proteins do not contain the same amino acids, and hence various color tests produce responses
that vary in intensity and color depending on the nature of the groups found in the amino acid
under examination. In this experiment, proteins such as casein and albumin were used to
qualitatively detect the presence of certain amino acids in each chemical test.
Table 3.1. Summary of Qualitative Analysis of Proteins
Chemical Test Observable Results
Std. Albumin Isolated Std. Casein Isolated
Albumin Albumin
Xanthoproteic Test It produces a It forms a ring
yellow-colored of pale-yellow
precipitate. color.
Biuret Test A violet color Transparent Transparent A violet color
complex is solution solution complex is
visible. visible.
Molisch’s Test It produces a It produces a It produces a It produces a
white cloudy white cloudy white cloudy white cloudy
layer. layer. layer. layer.
Heller’s Ring Test It forms a white It forms a white
ring with ring.
yellow-colored
layer.
 Table 3.1 summarizes the observable data for protein color responses. Xanthoproteic test
specifically used to detect amino acids containing an aromatic nucleus (tyrosine, tryptophan and
phenylalanine) in a protein solution which gives yellow color nitro derivatives on heating with
concentrated nitric acid, HNO3. The aromatic benzene ring undergoes nitration to give yellow
colored product. Hence, this only confirms the presence of an aromatic amino acid for both
standard albumin and casein, as shown in Appendix B, Figure 1. The Biuret test, on the other
hand, forms a violet complex with substances containing two or more peptide bonds under
alkaline conditions. The absorbance produced is proportional to the number of peptide bonds
reacting and, therefore, to the number of protein molecules present in the reaction system. As
shown in Appendix B, Figure 2, only standard and isolated albumin received a positive result in
this colorimetric technique. Furthermore, Molisch's test is a chemical test used to check for the
presence of carbohydrates in a given analyte. The carbohydrate (if present) undergoes
dehydration upon introducing concentrated hydrochloric or sulphuric acid, resulting in the
formation of an aldehyde. This aldehyde undergoes condensation along with two phenol-type
molecules (such as
𝖺-naphthol, resorcinol, and thymol), resulting in the formation of a purple or reddish-purple
colored complex (BYJU’S, 2021). However, results show a yield of a white-colored layer for all
protein samples, indicating that carbohydrates were not detected in this qualitative test (see
Appendix B, Figure 3). Additionally, as shown in Appendix B, Figure 4, Heller's ring test is a
chemical test that shows that strong acid causes denaturation of precipitated proteins, resulting in
the formation of a white coagulated ring between the two layers of protein solution and the
mineral acid, yielding a positive result for both standard albumin and casein.

3.2 Precipitation of Proteins

The precipitation of a protein occurs in a stepwise process. The addition of a precipitating
agent and steady mixing destabilizes the protein solution. Mixing causes the precipitant and the
target product to collide. Precipitation occurs because the change in pH or hydrophobicity alters
interactions between the protein and the aqueous environment or through binding of salts or
metals to protein functional groups such that intramolecular interactions are disrupted and the
proteins denature, aggregate, and fall out of solution (Nair & Mba, 2016).
 Table 3.2. Qualitative Data for Coagulation Test
Sample Observable Results
Std. Albumin A white coagulum is obtained.
Std. Casein It produces a slightly cloudy solution.
Gelatin After heating, the yellow color of the solution has intensified.

The experimental findings of the coagulation test are shown in Table 3.2. The heat
coagulation test is a less sensitive method that uses heat to denature protein samples. The
formation of a dense coagulum at the upper part of the solution, with the lower part acting as a
control, indicates a positive result for the heat coagulation test. Only standard albumin validated
this result, as shown in Appendix B, Figure 5. Even though both standard casein and gelatin
undergo physical changes, they were unable to get a positive coagulation test result.

Table 3.3. Qualitative Data for Effects of Alcohol

Sample Observable Results
Std. Albumin A cloudy solution formed.

Std. Casein It shows a transparent solution.

Table 3.3 summarizes the qualitative data for the effects of alcohol. When an organic
solvent, such as alcohol, is added to a protein sample, it lowers the dielectric constant of the
solvent and displaces some of the water molecules linked with the protein (dehydration),
lowering the water concentration. The protein sample affected by dehydration in the experiment
is saturated albumin (see Appendix B, Figure 6), whereas standard casein shows no alterations.

Table 3.4. Qualitative Data for Neutral Salt

Test Tube Observable Results
Std. Albumin Std. Casein
1 No white precipitate is obtained Transparent solution
2 A white precipitate is obtained Remains transparent
 The qualitative data for neutral salt is summarized in Table 3.4. The addition of a neutral
salt, such as ammonium sulfate, compresses the solvation layer and promotes protein-protein
interaction, resulting in protein aggregation and the formation of precipitates from the solution
(salting-out method). The addition of ammonium sulfate to the test tube two (2) of standard
albumin produces a white precipitate that results in a positive result for neutral salts, indicating
that albumin has entirely precipitated from its solution by full saturation (see Appendix B, Figure
7). Other test tubes, such as standard casein, went through the same procedure but came out
negative for neutral salt precipitation.

Table 3.5. Qualitative Data for Precipitation with Alkaloidal Reagents

Test Tube Observable Results
Std. Albumin Std. Casein
1 The initial pH is five (5), and When the pH is three (3),
after adding HCl, the pH drops precipitate occurs at the bottom
to two (2). A clear yellow of the yellow-colored solution.
solution is formed.
2 The initial pH is six (6), and The initial pH is five (5), and
after adding HCl, the pH drops after adding HCl, the pH drops
to three (3). The result was a to two (2). A slightly cloudy
cloudy yellow solution. yellow solution is formed.

The qualitative results for the precipitation with alkaloidal reagents are summarized in
Table 3.5. When organic acids are added to a protein solution, the acidic side of the isoelectric
pH causes proteins to precipitate, resulting in the creation of salt. For the first chemical reaction,
the pH of the samples was determined, and all of the test tubes had the same apparent
appearance, which was a homogeneous yellow colored solution. After adding hydrochloric acid
(HCl), the pH was measured again, and changes in each test tube were noticed. According to the
data, only standard casein in test tube one (1) received a positive result for alkaloidal reagent
precipitation (see Appendix B, Figure 8), indicating that casein has been fully precipitated from
its solution by alkaloidal reagents. The solution also became more acidic after the addition of
HCl. Furthermore, the existence of both negative and positive charges, as well as the amphoteric
nature of proteins, is shown by the precipitation of proteins by alkaloidal reagents.
 Discussion
An amino acid is a molecule that contains an amino group and a carboxyl group in the same
molecule. Amino acids found in proteins are α-amino acids. This means the amino group (NH2
—or NH3+ —) is attached to the alpha carbon — this is the carbon next to the carboxyl group.
There are twenty (20) amino acids that differ from each other only in the identity of the side
chain attached to the alpha-carbon. The amino acid side chains can be classified based on
whether they are nonpolar, polar, acidic, or basic. Albumin and casein, for example, respond
differently in different chemical processes based on their function. The primary goal of this
laboratory experiment is to qualitatively detect the presence of specific amino acids in proteins,
such as casein and albumin, using a variety of color-producing reagents, as well as to conduct
protein precipitation reactions using denaturation, dehydration, neutral salt, and alkaloidal
reagents.

4.1 Color Reactions of Proteins

Various colorimetric techniques are frequently used to detect certain amino acids in a
protein qualitatively. The Xanthoproteic test is based on the fact that aromatic groups in the
amino acids are nitrated by heating with concentrated HNO3 to yield intensely yellow-colored
nitro derivative. On the addition of alkali, however, the residue turns orange due to the formation
of a salt of the tautomeric form of the nitro compound. In the Biuret test, proteins create a
complex with Cu2+ ions in a strongly alkaline solution, where cupric copper reacts with the
peptide bonds in proteins to produce a violet color complex that serves as an indicator for protein
identification in the provided sample. Molisch's test, on the other hand, involves the addition of
Molisch's reagent, which causes carbohydrates to dehydrate to form furfural and its derivatives
when reacted with concentrated sulfuric acid (H2SO4), resulting in a purple (violet-red) colored
complex that can be used to detect the formation of carbohydrates as a by-product in various
reactions and distinguish it from other biomolecules. Furthermore, in Heller's ring test, when
native protein solution is treated with concentrated HNO3, it reacts with aromatic rings that are
benzene derivatives, resulting in the typical nitration reaction, which results in a white precipitate
ring due to protein denaturation.
4.2 Precipitation of Proteins
The reactions that precipitate protein from their solution are known as precipitation
reactions. In the coagulation test, when proteins are exposed to heat in acidic conditions, they
undergo structural denaturation, which leads to aggregation, where heat disrupts hydrogen bonds
in secondary and tertiary protein structures while the primary structure remains unaffected. As a
result of denaturation and coagulation, the size of the protein increases, and its solubility
decreases. The effects of alcohol are precipitated by dehydration, denaturation, and charge
removal, where the addition of an organic solvent reduces the solubility of the protein in
solution, causing the protein to precipitate out of solution. For precipitation by neutral salts, the
protein solution is treated with ammonium sulfate, which absorbs water for its solubility, leaving
less water available to participate in the solvation layer around the protein, exposing hydrophobic
parts on its surface. As a result, proteins are easily precipitated out of the solution. Furthermore,
alkaloidal reagent precipitation requires the addition of organic acids that exist as a negative
anion, where protein dissociates as a cation that combines with anions of organic acids, resulting
in a precipitate.
In the laboratory experiment, the color reaction test and protein precipitation both yielded
the predicted findings. However, there is still a chance that an error occurred somewhere along
the line. For example, the furfural aldehyde formed during the acid dehydration reaction step
may interfere with its condensation with the phenolic molecule due to strong nucleophiles
attacking the aldehyde, giving a false negative result when carbohydrates are present for a variety
of reasons in Molisch’s Test. As a result, amino sugar residues do not dehydrate to produce the
correct product when acid is added. Additionally, benzene ring-containing amino acids like
phenylalanine don’t give a positive test to Xanthoproteic test because the phenyl group in
phenylalanine is very stable, which doesn’t react with nitric acid in the conditions of this test.
However, phenylalanine might give a positive result after an extended period of heating.
Furthermore, when doing a Biuret test, the biuret reagent should be made fresh, and
contamination should be avoided since this could produce a precipitate, which would alter the
results.
Conclusion
 References

Ayyagari, A. A. N. (2007). Lab Manual in Biochemistry, Immunology and Biotechnology.

McGraw-Hill.
BYJU’S. (2021, March 22). General Data Protection Regulation(GDPR) Guidelines BYJU’S.
BYJUS. https://byjus.com/chemistry/molischs-test/
Dandekar, D. (2004). Practicals and Viva in Medical Biochemistry. Reed Business Education.
The Editors of Encyclopaedia Britannica. (2021, March 25). casein | Definition, Properties,
Manufacture, & Uses. Encyclopedia Britannica.
https://www.britannica.com/science/casein
Foulger, J. H. (1931). The Qualitative Molisch Test. THE USE OF THE MOLISCH (α-
NAPHTHOL) REACTIONS IN THE STUDY OF SUGARS IN BIOLOGICAL
FLUIDS, 345–346. https://doi.org/10.1016/S0021-9258(18)76522-8
Libretexts. (2021, March 9). Experiment_729_Qualitative Testing of Amino Acids and Proteins
1_2. Chemistry LibreTexts.
https://chem.libretexts.org/Courses/Los_Medanos_College/Chemistry_6_and_Chemistry
_7_Combined_Laboratory_Manual/Experiment_729_Qualitative_Testing_of_Amino_Ac
ids_and_Proteins_1_2
Milio, F. R., & Loffredo, W. M. (1995). Qualitative Tests for Amino Acids and Proteins.
Chemical Education Resources, Incorporated.
Nair, H., & Mba, C. W. P. (2016). Mass Spectrometry for the Clinical Laboratory (1st ed.).
Academic Press. https://doi.org/10.1016/C2013-0-19099-X
Pamarthy, J., Bhat, V., & Sukumaran, M. K. (2016). A Comparative Study on Casein and
Albumin Contents in Cow and Commercial Milk Samples. IOSR Journal of Dental and
Medical Sciences (IOSR-JDMS), 15(1), 102–106. https://doi.org/10.9790/0853-
1513102106
 Appendix A

Experimental Data

Table 3.1. Summary of Qualitative Analysis of Proteins

Chemical Test Observable Results
Std. Albumin Isolated Std. Casein Isolated
Albumin Albumin
Xanthoproteic Test It produces a It forms a ring
yellow-colored of pale-yellow
precipitate. color.
Biuret Test A violet color Transparent Transparent A violet color
complex is solution solution complex is
visible. visible.
Molisch’s Test It produces a It produces a It produces a It produces a
white cloudy white cloudy white cloudy white cloudy
layer. layer. layer. layer.
Heller’s Ring Test It forms a white It forms a white
ring with ring.
yellow-colored
layer.

Table 3.2. Qualitative Data for Coagulation Test

Sample Observable Results
Std. Albumin A white coagulum is obtained.
Std. Casein It produces a slightly cloudy solution.
Gelatin After heating, the yellow color of the solution has intensified.

Table 3.3. Qualitative Data for Effects of Alcohol

Sample Observable Results
Std. Albumin A cloudy solution formed.

Std. Casein It shows a transparent solution.

Table 3.4. Qualitative Data for Neutral Salt
Test Tube Observable Results
Std. Albumin Std. Casein
1 No white precipitate is obtained Transparent solution
2 A white precipitate is obtained Remains transparent

Table 3.5. Qualitative Data for Precipitation with Alkaloidal Reagents

Test Tube Observable Results
Std. Albumin Std. Casein
1 The initial pH is five (5), and When the pH is three (3),
after adding HCl, the pH drops precipitate occurs at the bottom
to two (2). A clear yellow of the yellow-colored solution.
solution is formed.
2 The initial pH is six (6), and The initial pH is five (5), and
after adding HCl, the pH drops after adding HCl, the pH drops
to three (3). The result was a to two (2). A slightly cloudy
cloudy yellow solution. yellow solution is formed.
 Appendix B

Photos of the Experiment

Figure 1. Xanthoproteic Test Results

Figure 2. Biuret Test Results

Figure 3. Molisch’s Test Results

Figure 4. Heller’s Ring Test Results

 Figure 5. Coagulation Test Result

Figure 6. Result of the Effects of Alcohol

Figure 7. Precipitation by Neutral Salts Result

Figure 8. Precipitation by Alkaloidal Reagents Result