Department of Pure and Applied Chemistry

College of Arts and Sciences

Visayas State University

Date Performed: June 20, 2017 Score: ________

Date Submitted: June 29, 2017 Prepared by: BS Chem-2

Experiment No. 5
Amino Acids
Objectives:
 Identify the R group in amino acids.
 Determine the pH of various amino acids in water.
 Use chromatography to separate amino acids in a mixture.
 Use Rf values to identify amino acids.

Results:
A. pH of Amino Acids
Table 1. pHs of different amino acids
Amino
pH Structure Explanation
Acids

Neutral, because the side

chain is nonpolar, which is a
Alanine 6.51
methyl group. Its neutral form exists
in neutral condition.

The side chain is an amino

group, which results to an extra
Lysine 6.27
positive charge in the side chain
itself.

The side chain is a carboxylic

Glutamic group, which is acidic and results
3.48
acid to an extra negative charge in the
side chain itself.
 The side chain is polar,
Cysteine 3.93
uncharged and exists as neutral.

The side chain is carboxylic

Aspartic
3.02 group, which is acidic and results
Acid
to an extra negative charge.

The side chain is uncharged

Tyrosine 6.37 polar one, which is a large
phenolic group.

The side chain is nonpolar

Valine 5.01 one, which is a secondary propyl
group and exists as neutral.

B. Paper Chromatography
Distance from the origin to final solvent line 10.50 cm
Table 2. The Rfs of different Amino Acids
Amino Acids Color Distance Traveled (cm) Rf
Alanine Violet 4.00 3.90x10-1
Glutamic acid Violet 2.30 2.20x10-1
Valine Violet 6.70 6.50x10-1
Lysine Violet 2.20 2.14x10-1
Aspartic acid Violet 1.60 1.55x10-1

Table 2.2 Amino acids present in Unknown & Hydrolysate

Amino
Compound Distance Traveled (cm) Rf
Acid(s)
Glutamic
In Unknown 3.00 2.90x10-1
acid
Aspartic acid
In Dipeptide
& 4.00 3.90x10-1
Hydrolysate
Phenylalanine
 Image 1. Paper Chromatograph of Amino Acids, Unknown amino Acid, and Dipeptide
hydrolysate.

Discussion
Using the pH meter, the pHs of alanine, lysine, glutamic acid, cysteine, aspartic acid, tyrosine,
and valine were measured and the pHs were 6.51, 6.27, 3.48, 3.93, 3.02, 6.37, and 5.01 respectively.
Glutamic acid and aspartic acid had low pHs because of the presence of two carboxyl groups in their
molecule which contribute to the acidity of the two amino acids, thus they both have low pHs. Tyrosine
and cysteine contain uncharged polar side chains. The phenol group of tyrosine participates in
hydrogen bond formation with water present in the solution, giving a pH close to 7. Cysteine gave a
low pH because its side chain accept an H+ ion (proton) and are positive charged. Alanine and valine
amino acids contain nonpolar side chains which do not gain or lose protons or even participate in
hydrogen or ionic bonds. Both amino acids had the experimental pHs because at their given pHs, their
side chains can exist as nonpolar side chains and promote hydrophobic interactions. In the case of
lysine, a basic pH should have been observed since two amino groups are present and the side chain
bears positive charge when it is in an aqueous solution. A human error might have been committed
during the time when the pH of cysteine was measured, like the pH meter was washed properly after
dipping it in an acid solution.
After conducting the chromatography of alanine, glutamic acid, valine, lysine, tryptophan,
unknown amino acid and dipeptide hydrolysate, the distances traveled by the compounds in the
paper chromatograph were measured and their Rf values were calculated and recorded. The Rf
values were calculated since it is helpful to identify substances or components bigger substances. The
amino acid samples had the following distances traveled and Rf values: 4.00 cm, 2.30 cm, 6.70 cm,
2.20 cm, 1.60 cm, 3.90x10-1, 2.20x10-1, 6.50x10-1, 2.14x10-1, and 1.55x10-1. By looking the tabulated values
of the distances traveled and Rf, one amino acid differ from the other. This was because when the
paper was placed in the mobile phase (butanol/acetic acid, & water), the solution (sometimes called
the eluting solvent) begun to rise up on the paper. As the mobile phase rises on the paper it
encountered the spots of amino acids. The fate of each amino acid in the mixture depends on the
affinity of each substance for the mobile and stationary phases. If an amino acid has a higher affinity
for the mobile phase than the stationary phase, it will tend to travel with the solvent and be relatively
unimpeded by the filter paper. In contrast, if the amino acid has a higher affinity for the paper than
the solvent, it will tend to stick to the paper and travel more slowly than the solvent front. And since
amino acids have different affinities in the paper and the solvent, so, such results were obtained in this
set up. Looking on the result produced by the unknown amino acid, the distance that it traveled was
about 3.00 cm and since its Rf value was 2.9x10-1, which is the same with the Rf value of glutamic acid,
it can be said that the unknown amino acid was also glutamic acid. In the dipeptide hydrolysate, the
distance it traveled was about 4.00 cm and the Rf value was 3.90x10-1. Since the dipeptide hydrolysate
sample was prepared from hydrolyzing aspartame, therefore, it can be said that the amino acids
present were phenylalanine and aspartic acid, which actually made up the aspartame compound.

Conclusion
The R group present in an amino acid differs from one specific amino acid to the other. It is the
group that distinguishes the characteristics and sometimes the behavior of an amino acid in a given
condition(s).
The pH of an amino acid in water is greatly affected by its side chain. There are side chains that
are responsible for the acidic pHs of the amino acids, some contribute to the basic pHs of the other
amino acids, and other side chains aid the amino acids to produce neutral pH.
Chromatography is helpful to separate the amino acids from each other because of the
different distances they travel in the paper together with a solvent. It is also helpful to identify unknown
amino acids by just measuring the distance traveled by the amino acid and look for an amino acid(s)
that traveled the same distance(s).
The Rf value can be calculated by dividing the distance traveled by the amino acid, in
centimeters, by the distance traveled by the solvent, in centimeters.
The Rf values are useful to identify amino acids because each amino acid have different R f
value from the other and since no two or more amino acids will have the same or equal Rf values.

Reference:
Ferrier, Denise R. Lippincott’s Illustrated Reviews Biochemistry. Sixth Edition.
Stoker, H. Stephen. General, Organic, & Biological Chemistry. Seventh Edition.
https://www.macalester.edu/~kuwata/Classes/2001-02/Chem%2011/Revised%20Amino%
20Acids%20(9%201%2001).pdf
http://www.encyclopedia.com/science/dictionaries-thesauruses-pictures-and-press-
releases/rf-value
Appendix

Preparation of Dipeptide Prepared Dipeptide Paper Chromatograph

Hydrolysate Hydrolysate of Amino Acids