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Experiment No. 5
AMINO ACIDS
Abstract
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.
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.
Calculate Rf value for amino acids.
Use Rf values to identify amino acids.
Methodology
Materials
An aromatic and
Tyrosine Neutral hydrophobic amino
acid.
B. PAPER CHROMATOGRAPHY
Fig. 3
Calculations: Rf Values
Table 2.
DISTANCE
AMINO ACID COLOR Rf
TRAVELLED (cm)
Alanine None --- ---
Glutamic Acid Violet 10.4 0.875
Valine Violet 9.9 0.859
Lysine Violet 9.8 0.888
Tryptophan Violet 9.6 0.802
Unknown Orange 10.5 0.876
Aspartic Acid Violet 9.8 0.827
Amino acids in
Violet 10.4 0.875
unknown
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 R f values, it is important
peptide bond
O O O O
R R' R R'
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.
Appendices
References