ChemLab204: General and Inorganic Chemistry

Group No. 66

Laboratory Report

Date Submitted: March 3, 2016

Date

Proteins and Amino

Acids I: Color
Reactions
Activity No. 11

Marice Abigail J. Marquez

ID No. 150486; BSN-I
ChemLab204A; 2nd Semester, SY 2015-16

RATIONALE
Proteins are the third energy-supplying nutrients and present in every living cell.
Proteins function as structural materials and as enzymes that regulate the countless
chemical reactions taking place in every living organism, including the reactions
involved in the decomposition and synthesis of proteins. All proteins are polymeric
substances that yield amino acids on hydrolysis. Those that yield only amino acids when
hydrolyzed are classified as simple proteins; those that yield amino acids and one or
more additional products are classified as conjugated proteins. Proteins can be also
characterized by their chemical equations. Most proteins are soluble in water, in alcohol,
in dilute base or in various concentrations of salt solutions.
In this activity proteins and amino acids are going to be classified by their color
reactions. And to determine what type of amino acids are present in the every solutions
and how does peptide bonds occur when the reagents was added.

INTERPRETATION:
A. Biuret Reaction
Name of test
A. Biuret

Test Solutions

Reagents Added

Result

1% Albumin

10% NaOH
1% CuSO4

Violet

Reaction
1% Casein

Violet

1% Glycine

Light Blue

1% Peptone

Violet

Urea (s)

Light Blue

________________________________________________________________________________________________________
Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga
University

Biuret test is a chemical test used for detecting the presence of peptide bonds of proteins.
The positive indication for this test is a violet color that is produced when dilute copper
(II) sulfate is added to an alkaline solution of a peptide or protein. At least two peptide
bonds must be present, since amino acids and dipeptides do not give a positive biuret
test. Albumin, casein, and peptone gives a positive result for biuret test it means that
they contain two or more peptide linkages, while glycine and urea (s) do not because
glycine and urea is an amino acid.

INTERPRETATION:
B. Millons Test (Theoretical Result)
Name of test

B. Millons Test

Test Solutions

Reagents Added

1% Albumin

Millons Reagent
[HgNO3 and
Hg(NO3)2 added
with conc. HNO3]

1% Casein
1% Gelatin

Result

Reddish Brown Color

1% Phenol

Millons test is to detect the phenolic group in the amino acid tyrosine. Millons test is
given by any compound containing a phenolic hydroxy group. Consequently, any protein
containing tyrosine will give a positive test of a reddish brown color. The millons
reagent is a solution of mercuric and mercurous ions in nitric and nitrous acids. The
positive indication for this test is a reddish brown color due to the mercury salt of the
nitrated tyrosine.

INTERPRETATION:
Name of test

C. Xanthoproteic
Reaction

Test Solutions

Reagents Added

Result

1% Albumin

Conc. HNO3
Conc. NH4OH

Yellow

1% Casein

Yellow

1% Gelatin

Yellow

1% Phenol

Orange

C. Xanthoproteic Reaction
________________________________________________________________________________________________________
Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga
University

Xanthoproteic test is used to determine the presence of tyrosine, tryptophan, and

phenylalanine to the protein. Aromatic rings in the amino acids such as those of
phenylalanine and tyrosine are readily nitrated. This is the basis of the xanthoproteic
test. The positive indication for this test is a yellow color which turns orange upon the
addition of a base. Albumin, casein, and gelatin only produced a yellow precipitate even
upon the addition of a base unlike phenol it turns orange when it was added with the
base. Proteins with amino acids carrying aromatic groups, especially in the presence of
tyrosine will give a positive result.
Chemical Equations:

OH

OH

HNO3

NH2

HO

NH2

HO
NH2

Tyrosine
O

Yellow Product
O
OH

NH2

NH

H2N

HNO3

OH
NH2

NH

Tryptophan

Yellow Product

O
OH
NH2

+
HNO3

OH
H2N

NH2

Phenylalanine
Yellow Product

________________________________________________________________________________________________________
Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga
University

INTERPRETATION:
D. Test for Sulfur
Test
Solutions

Name of test
D. Test for

1% Albumin

Sulfur

Gelatin (s)

Reagents
Added

Result
Black Precipitate

10% NaOH
5%
Pb(CH3COO)2

White Precipitate

Peptone (s)

Orange Color

The test for sulfur is to determine the sulfur containing amino acids. Cysteine and
cystines in the free state and in proteins give a positive result to the sulfur test. When
sulfur containing amino acids are boiled with an alkali, the sulfyhydryl or disulfide
groups are converted to an inorganic sulfide, NaS. This reacts with lead acetate to form a
black precipitate of PbS. Albumin is the only solution that produce a black precipitate
because the albumin has a sulfur containing amino acids. The gelatin and peptone didnt
produce a black precipitate it remains to its own color.

INTERPRETATION:
E. Hopkins-Cole Reaction
Name of test

E. Hopkins-Cole

Test
Solutions

Reagents
Added

1% Albumin
Conc. H2SO4

Reaction

Result
Violet Ring Formed Between the Two
Layers

1% Casein

Violet Ring Formed Between the Two

Layers

1% Gelatin

No Change

Hopkins-Cole Test determines the presence of the amino acid tryptophan. The
indole ring of tryptophan condenses with glyoxalic acid in the presence of sulfuric acid
________________________________________________________________________________________________________
Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga
University

to form a violet colored complex or a violet ring formed between the two layers. This is
the basis of the Hopkins-Cole Test. In this test the albumin and casein forms the violet
ring between the two layers, this confirms that albumin and casein contained glyoxylic
acid and a protein. And there are some products that do not show the reaction, such as
gelatin.

INTERPRETATION:
F. Ninhydrin Reaction
Name of test

Test Solutions
0.2% Albumin

F.

Reagents
Added

Result
No Change

0.1%
Ninhydrin
Solution

Ninhydrin

Ammonia
Water

Reaction

0.2% Urea

No Change

0.2% Glycine

Ruhemanns Purple

No Change

Ninhydrin test is used to detect the presence of the amino group in alpha amino acids.
When amino acids are heated with ninhydrin (triketohydrindenehydrate), ammonia,
carbon dioxide and aldehyde are produced. The NH3 liberated combines with one mole
of the reduced ninhydrin and one mole of the oxidized ninhydrin to form Ruhemanns
purple. The triketohydrindene hydrate, generally known as ninhydrin is an extremely
sensitive reagent for amino acids. All amino acids, except proline and hydroxyproline,
give a deep blue solution with ninhydrin. Proline and hydroxyproline produce a yellow
solution. For ninhydrin test there are two positive visible results: deep blue or
ruhemanns purple. The color varies slightly from acid to acid, because of the unreacted
acids complex with the pigment. In this test only glycine produced ruhemanns purple
because it is the only solution has the presence of amino group in alpha amino acids.

ANSWERS TO QUESTIONS
1. Why do all proteins give a positive result for Biuret test?
Answer: Because they have more than two peptide linkages and hence, they react
with CuSO4.
2. Why does nitric acid stain the skin with a yellow color?
________________________________________________________________________________________________________
Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga
University

Answer: Nitric acid stain the skin with a yellow color due to a reaction with the
protein keratin. These yellow stains turn orange when neutralized.
3. Will methionine give a positive result for the sulfur test? Explain.
Answer: Yes, because it contains sulfur and it is one of only two amino acids that
contains a sulfur group.
4. What grouping in amino acids or proteins is responsible for the ninhydrin
reaction?
Answer: The amino group, NH2.
5. What color will be produced by proline with ninhydrin? Why?
Answer: Ruhemanns purple. The color varies slightly from acid to acid because
unreacted acids complex with the pigment.

CONCLUSION
Proteins function as structural materials and as enzymes that regulate the countless
chemical reactions taking place in every living organism. And proteins are highly
specific in their functions. The amino acid units in a given protein molecule are arranged
in a definite sequence. Proteins ultimately yield amino acids, when hydrolyzed. And they
can be hydrolyzed by boiling in a solution containing a strong acid such as hydrochloric
acid or in a solution containing a strong base such as sodium hydroxide. Amino acids
will produce positive result depending on their peptide bonds. When the two amino
acids are joined together by a peptide bond or peptide linkage. The product is a
dipeptide. The loss of a proteins natural conformation without peptide bond hydrolysis is
due to the denaturation process. And some test produces positive result depending on
what type of amino acids they are and how they will react with the following reagents.

REFERENCES
1. Hein, M., Best, L., & Pattison, S. (1984). College Chemistry. Monterey, California: Brooks/
Cole Publishing Company.
2. Brown, W., Rogers, E. General Organic, and Biochemistry. Monterey, California: Brooks/
Cole Publishing Company.
3. Boikess, R., Breslauer, K., Edelson, E. Elements of Chemistry. General, Organic, and
Biological.
4. Allen, T., Keefer, R. (1817). Chemistry Experiment and Theory. New York: Harper and
Ron, Publishers.
5. Mousse93. (n.d.). Lab Exam 1. San Francisco: Quizlet Inc. Retrieved February 28, 2016,
from https://quizlet.com/privacy
________________________________________________________________________________________________________
Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga
University

6. The Wall of Biochemistry. (2012, March). Picnonotus. Retrieved February 28, 2016, from
https://fulltimes.wordpress.com/
7. Chemistry- Environment & Ecology- Engineering- Economics- Psychology. (2007). Author.
Retrieved February 28, 2016, from http://www.funqa.com/chemistry/2504-Chemistry4.html
8. Senese, F. (1997). General Chemistry. Amino acid test. Retrieved February 28, 2016 from
http://antoine.frostburg.edu/chem/senese/101/organic/faq/amino-acid-test.shtml

________________________________________________________________________________________________________
Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga
University