Department of Pure and Applied Chemistry

College of Arts and Sciences

Visayas State University
Visca, Baybay City, Leyte

Name: Ana Luisa C. Laurente Date Performed: June 13, 2019

Lab Schedule: 7:00 – 10:00 (M-F) Date Submitted: July 15, 2019
Group No: 6 Score:

Experiment No. 5
Amino Acids

A. Abstract
In the experiment, Qualitative Color Reactions was performed in order to analyze the
chemical groups responsible for the color reaction of the protein Gluten. Different tests such
as Biuret and Ninhydrin yielded different colored solutions as results. The different results
were due to the difference in the side chains present in Gluten. Paper Chromatography was
also conducted to be able to separate and determine the amino acid, Glutamic Acid. A 2 cm
margin had been drawn across the longer bottom edge of the chromatograph paper. Seven
equidistant points were plotted along the line where the given amino acids and protein
hydrolysate samples had been applied. 

B. Introduction

Proteins are the most abundant class of organic compounds in the healthy, lean
human body, constituting more than half of its cellular dry weight. Proteins are polymers of
amino acids and have molecular weights ranging from approximately 10,000 to more than
one million. Biochemical functions of proteins include catalysis, transport, contraction,
protection, structure, and metabolic regulation.

Amino acids are the monomeric units, or building blocks, of proteins joined by a
specific type of covalent linkage. The properties of proteins depend on the characteristic
sequence of component amino acids, each of which has distinctive side chains.

Amino acid polymerization requires elimination of a water molecule as the carboxyl

group of one amino acid reacts with the amino group of another amino acid to form a
covalent amide bond. The repetition of this process with many amino acids yields a polymer,
known as a polypeptide. The amide bonds linking amino acids to each other are known as
peptide bonds. Each amino acid unit within the polypeptide is referred to as a residue. The
sequence of amino acids in a protein is dictated by the sequence of nucleotides in a
segment of the DNA in the chromosomes, and the uniqueness of each living organism is due
to its endowment of specific proteins.

C. Methodology

Materials

 Organic model kits  Small beaker

 Amino acids  Large beaker
 Aspartame  Plastic wrap
  Chromatography paper  Thin layer chromatography
 Solvent plates
 Oven
 2% ninhydrin spray

Methods

a. Structure of amino acids and dipeptides

b. pH of amino acids
 put 5-10 drops of amino acid on pH paper
c. Paper chromatography
 Place 1g aspartame in 125ml Erlenmeyer flask
 Add 30 ml 6M HCl and place the flask in a boiling water bath and heat
for 30 minutes
 Pour 10ml butanol/HOAc/water solvent in a large beaker then cover
with plastic wrap
 Obtain chromatography paper (13x20cm)
 Draw pencil line of 2cm from long edge of the paper. Mark 7 points
that are evenly spaced.
 Using the capillary tubes, touch lightly and put into paper
 Join the edges but avoid overlapping the paper.
 Put into the tank and seal for an hour
 Spray ninhydrin and dry
 Compare the color and Rf values produced by the unknown amino
acid and the hydrolysate.

D. Results

STRUCTURE OF AMINO ACIDS

Draw the structure of the amino acid in your model.

 Draw the structure of the dipeptide in your combined model.
O OH
O C CH3
H2
H3C C C CH
H H CH
C C N C CH3
H H2
H 3C H2N

(S)-2-((S)-2-amino-4-methylpentanamido)-4-methylpentanoic acid

Name: Leucine-Leucine Abbreviation: Leu-Leu

pH OF AMINO ACIDS

AMINO ACID pH STRUCTURE EXPLANATION

It’s a polar amino acid; the pH is

Alanine 7.5
neutral

It should be a basic amino acid;

Lysine 6.8
faulty pH paper

Glutamic acid 4.0 It’s an acidic amino acid

It’s a nonpolar amino acid; faulty

Valine 7.6
pH paper
Aspartic acid 3.4 It’s an acidic amino acid

It’s a polar amino acid; it’s pH is

Tryptophan 7
neutral

It’s a polar amino acid; it’s pH is

Glycine 7
neutral

PAPER CHROMATOGRAPHY

Calculations: Rf Values

Distance from origin to final solvent line: 10.8

Amino Acid Color Distance travelled Rf

Alanine Blue violet 2.25 0.20
Glutamic acid Blue violet 2.6 0.24
Valine Blue violet 2.5 0.23
Lysine Blue violet 1.5 0.14

Tyrosine Blue violet 1.75 0.16

Uknown Blue violet 2.5 0.23

 Dipeptide Light violet 2.75 0.26

Amino Acid(s) in Valine

unknown

Amino acid(s) in Aspartic Acid

dipeptide Phenylalanine
hydrolysate

DISCUSSION

Amino acids are compounds that contain the amino group and the carboxylic acid
group. Since an amino acid contains the acidic carboxyl group and the basic amino group,
the proton from the carboxyl group can be transferred to the amino group. When this
happens the amino acid is said to be a zwitterion or in zwitterionic form which occurs at the
pH of 7. The acidity and alkalinity of the amino acid is now primarily dictated by its side chain
or the R group.

Glutamic acid, Valine, and Aspartic acid were observed to be acidic. This was
because of the present of the carboxylic group on its R group. Lysine and Serine were basic
since an animo group is attach to its R group making it basic. Alanine and Tyrosine amino
acids are neutral. This is true because they don’t have any proton donor nor proton acceptor
attached to their R group.

Chromatography is an analytical tool for distinguishing different biomolecule based

on their chemical properties. Paper chromatography was used to characterize the different
amino acids.

In this assay, the stationary phase was an immovable porous solid and an absorbent
paper which was a sheet of filter paper. The filter paper was composed mostly of cellulose
and was very hydrophilic or polar. The components to be distinguished were different amino
acids and were spotted near the bottom of the paper. This location was referred as the
origin. The paper was suspended in the mobile phase, an eluent or a solvent which was
hydrophobic or nonpolar (butanol/acetic acid/ water). The solvent was drawn up the paper by
capillary action. As the solvent moves over the location of the amino acid, it then got
“washed” along the porous solid by the flow of the solvent and begun to move up the paper.

The different amino acids had different polarity due to the difference of their R group.
Hydrophobic or nonpolar ones moved faster because they were more attracted to the
hydrophobic solvent than the hydrophilic paper. On the other hand, hydrophilic or polar
amino acids move slower because they were attracted more to the paper than the
hydrophobic solvent, and therefore do not travel at the same speed through the stationary
phase. This was the result of their difference in polarity. If this was the opposite that the
stationary phase was nonpolar and the eluent was polar it would be vice versa. Remember
that “Like attracts Like”.

The container was covered to prevent evaporation of eluent. The chromatogram was
removed from the eluent before the eluent reaches the top of the paper. The height of how
high the eluent moved up the filter paper was measured 10 cm.

After the solvent evaporated from the paper ninhydrin was sprayed and then reacted
with the amino acids to produce a purple color, allowing us to see where the amino acids
were located. The further the spot from the starting line, the higher the affinity of the amino
acid for the mobile phase and the faster its migration. The finished paper, with its spots, was
a chromatogram.
 Measurements were made from the line on which the original samples were applied
to the tip of the migrated. The different amino acids were identified by its rate of movement.
The rate of movement of an amino during paper chromatography was reported as its relative
mobility (Rf). Rf was simply the distance the amino acid moved through the filter paper
divided by the distance the solvent moved through the paper. This is shown in the formula
below:

distance traveled by amino acid

Rf =
distance traveled by solvent
To best understand why different amino acids have unique Rf values, it is important
to understand the structural features of these molecules. These nonpolar hydrocarbon side
chains are hydrophobic or “water-hating.” Hence, they tend to lower the water solubility of
the corresponding amino acids. Some amino acids have polar but neutral R groups that tend
to promote water solubility. Both acidic and basic R groups tend to promote water solubility,
though the solubility will be pH dependent.

Peptide linkages hold amino acids together. Peptide linkages occur through
condensation of two separate amino acid groups. The formation of each peptide bond
requires the loss of one molecule of H 2O. Likewise, peptide bonds can be broken through
hydrolysis to form individual amino acids.

peptide bond

O O O O

H2N CH C OH H2N CH C OH H2N CH C NH CH C OH H2O

R R' R R'

E. Conclusion

The 20 common amino acids have different R groups which characterize them from each
other. An amino acid can be identified by its R f or its relative mobility. This was determined
using paper chromatography. Chromatography is a convenient and useful method for the
separation of mixtures and for the identification of substances. The method has been
especially valuable for the separation of closely related compounds. Paper chromatography
was especially useful in characterizing amino acids. Like in ours, we identified Glutamic acid
as the amino acid used as the unknown for the paper chromatography. The different amino
acids move at differing rates on the paper because of differences in their R groups thus
giving them their identification.

F. References

Amino Acid and Protein. Experiment 8. S. L. Saeger and M. R. Slabaugh CHEM 209 Lab,
Spring 2005.pdf
Paper Chromatography of Amino Acids.pdf
Chromatographic Separation of Amino Acids.Experiment 11. pdf