EXPERIMENT

7 Chromatography
IDENTIFICATION OF AMINO ACIDS BY - A technique that depends on the rate of
PAPER CHROMATOGRAPHY migration of the different amino acids

Amino acids - Also used in the separation and
- Building blocks of proteins quantitative analysis of peptides,
nucleotides, lipids and carbohydrates
- Molecular formula has 2 functional grps
Carboxylic group – acidic properties Principle involved
Amino group – basic properties - based on the division between a
*because of the presence of these two stationary phase, usually a filter paper
functional groups, amino acids are capable that strongly attracts water and a
of internal acid-base reaction resulting to mobile phase, usually organic solvents
formation of zwitterion
Paper Chromatography
Zwitterion - can separate different amino acids
- Compounds that contain both a positive based on their varying solubility in 2
and a negative charge different solvents

250 identified amino acids - in this method, a sample of an amino
acid (or mixture of amino acids) is
only 20 are commonly found in plants and applied as a small spot near one edge of
animals a piece of chromatography paper

10 of these 20 amino acids are known - the edge of the paper is then placed in a
ESSENTIALS to humans because they shallow layer of solvent mixture in a
cannot be synthesized by the body thus chromatography tank
must be obtained thru diet.
Filter paper
R-GROUP - a good material for stationary phase
- Amino acids differ in the R Group because many substances that are
attached on their formula soluble in water or other mixed solvents
are more or less tightly absorbed onto
- These different SIDE CHAINS can be the fibers of the paper
utilized to identify the different amino
acids in the laboratory by
chromatography







As the solvent is drawn up into the paper by Amino acid with a HYDROPHOBIC R-GROUP
CAPILLARY ACTION, 3 possible actions will - Will be more soluble in the mobile non-
take place polar solvent than in water
- It will continually move up to the paper
1. Those substances that are NOT
ABSORBED on the paper will move Different amino acids will move different
at the same rate as the solvent distances up the paper depending upon
their relative solubility in the two solvents,
2. Those substances that ARE BOUND allowing for separation of amino acid
VERY TIGHTLY to the paper will not mixtures
move very far.
Ninhydrin solution
3. Those substances that INTERACT - Used and sprayed on the paper to
WEAKLY with the paper will move, determine clearly the position of amino
but slowly than the solvent. acids. Followed by measuring the
distance of the migration
Solvent mixture
- Contains several components, one of Movement of Amino Acids
which is usually water and another of - Can be defined by a quantity known as
which is a more non-polar solvent Rf value
-measures the movement of an
As the solvent mixture moves up the paper amino acid compared to the
by capillary action, WATER IN THE MIXTURE movement of the solvent
binds to the hydrophilic paper (cellulose)
and creates a liquid stationary phase of At the start of the chromatography, the
many small water droplets amino acid is spotted at what is called the
ORIGIN
The NON-POLAR SOLVENT continues to
move up the paper forming a liquid mobile The chromatography is then performed,
phase and the PROCEDURE IS STOPPED before the
solvent runs all the way up the paper
§ Since amino acids have different R-
groups, they also have different degrees Since Rf value for an amino acid is
of solubility in water vs. the non-polar CONSTANT for a given chromatography
solvent setting, an unknown amino acid can be
identified by comparing its Rf value to those
Amino acid with a POLAR R-GROUP known amino acids
- Will be more soluble in water than in
the non-polar solvent In identifying amino acids, SEVERAL
- It will dissolve more in the stationary FACTORS MAY AFFECT THE LOCATION OF
water phase and will move up the paper THE SUBSTANCE
only slightly 1. Temperature
2. pH and solvent concentration
3. time of chromatography
PROCEDURE 2. Apply several times and make sure to
A. Preparation of Chromatography dry spots first before applying more
Chamber solution

1. Mix n-butanol, glacial acetic acid and 3. Connect the edges of the filter paper to
water in a ratio of a 6:2:2 to form a form a cylinder using a stapler and be
solvent. careful not to overlap the edges

2. Pipette 8mL of the solvent and place it
in a dry 250mL beaker. 4. Using forceps, place the
chromatographic paper into the beaker
containing the solvent
3. Cover the beaker immediately.


5. Cover immediately with watch glass and
4. Let stand for several minutes to become let it stand for several minutes
vapor saturated


6. Remove the filter paper once the
B. Preparation of Paper Chromatogram solvent front is already 1cm away from
1. Obtain a piece of filter paper using the upper edge
forceps that measures:
15cm horizontally
9cm vertically 7. Open up and mark the positions of the
solvents front before they dry up
2. Draw a light pencil line 1cm up to the completely
bottom
8. Spray the paper with 0.2% Ninhydrin
3. Mark 5 equally spaced spots solution and let it dry.

9. If oven is available, place it in and set
C. Experiment Proper the oven to 100 degrees Celsius
1. Using a fine capillary tube, quickly touch
the marked spots with the ff. amino 10. Take note of the color after 30 minutes
acids:
1st spot – 0.5% Aspartic acid
!"
2nd spot - 0.5% Phenylalanine $%&'()*+ '-(.+//+$ 01 (2%)3 (*%$
=
3rd spot - 0.5% Proline $%&'()*+ '-(.+//+$ 01 '4+ &3/.+)' "-3)'
4th spot - 0.5% Lysine
5th spot - 0.5% Unknown solution