Experiment 9: Separation of Amino Acid Mixture by Paper Chromatography
Uploaded by
filzah haziqahExperiment 9: Separation of Amino Acid Mixture by Paper Chromatography
Uploaded by
filzah haziqahEXPERIMENT 9 : SEPARATION OF AMINO ACID MIXTURE BY
PAPER CHROMATOGRAPHY.
NAME : AMIRAH ATHIRAH BT JAMALUDDIN
EXPERIMENT : EXPERIMENT 9 : SEPARATION OF AMINO ACID MIXTURE BY PAPER
CHROMATOGRAPHY.
COURSE CODE : AS 202
GROUP : M4
LAB INSTURCTOR : MADAM HAIRUL AMANI
DATE OF EXPERIMENT : 3RD MAY 2018
ABSTRACT
In this experiment, we need to spot amino acid and the standard mixture. Other than that, to
separate the unknown mixture into the individual component and to identify the components
in the unknown mixture. We are using the chromatography paper in this experiment. Alanine,
leucine, methionine and mixture of amino acids are spots on the chromatography paper and
the chromatography paper is left in the beaker containing solvent mixture. After a while, the
distance of the spots travel are measured and the Rf is determine. We can conclude that the
mixture of the amino acid is containing all the standard solutions which is leucine,
methionine and alanine.
OBJECTIVES
1. To spot amino acid and the standard mixture.
2. To separate the unknown mixture into the individual component.
3. To identify the components in the unknown mixture.
INTRODUCTION
Chromatography is a simple, quick, and inexpensive procedure that gives chemist a quick
answer as to how many components in a mixture. It is also used to support the identity of a
compound in a mixture when the Rf of the mixture is compared with the Rf of the known
compound.
A small amount of the mixture that need to analysed is spotted at the bottom of the line of the
chromatography. It is then placed in the shallow pool of the solvent in the close chamber.
This is the mobile phase where the solvent will slowly rise up the chromatography.
As the solvent moves pass the spot, and equilibrium is established for each component of the
mixture between the molecules of that components which are adsorbed on the solid and the
molecules which are in the solution. In principle, the components will differ in solubility and
in the strength of their adsorption to the adsorbent and some components will be carried
farther up the plate than others. When the chromatography is taken out from the chamber and
is dry, the spot is invisible hence we need to use stain to determine the position of the spots.
In this experiment, we want to determine the components of the mixture of the amino acids
and compared it with three different standard solutions.
PROCEDURE
On the chromatography paper, a line was drawn 3.5 cm from the end of the strip. The filter
paper Whatman no 1 was used. From the edge, four points of line were marked and leaving 3
cm space between them. The amino acid mixture was dropped using capillary tube to obtain
the diameter of 5 mm. The another drop was spotted when the first drop had dried. The
chromatography paper was placed into the tank which contained the solvent mixture
( butanol: glacial ethanol : water, 12 : 3 :5 ) . The solvent was allowed to move upward
through the chromatography paper until it reached about 5 cm from the top end. Tha paper
was taken out and dried in the fume cupboard using the dryer. The chromatography paper
was sprayed with ninhydrin. Then it was dried again. The various spots on the paper was
examined. The spots for amino acid mixture were compared with the standard amino acids
for identification. The distamce from the original point to where the spots stop were
measured. The Rf for each amino acids present were calculated.
JOTTER
DATASHEET
OBSERVATION
Silica column.
Alumina column.
DISCUSSION
In this experiment, we spot the mixture amino acid on the chromatography paper. We then
compare the mixture of the amino acid with the standard solutions. R f we compare the
mixture with the standard amino acids by using the Rf formula which is ;
distance travelled by the solute
Rf = distance travelled by the solvent
The distance travelled for the spot 1 from below of the paper chromatography is 1.8 cm, spot
2 is 4.6 cm and spot 3 is 3.2 cm. the distance travelled by the alanine is 1.7 cm, leucine is 4.7
cm and methionine is 3.5 cm. hence, the Rf for the spot 1 of the mixture from below is 0.22
which is near with the Rf of the standard 1 amino acid, alanine. The Rf for the spot 2 is 0.39.
The spot is belongs to the standard 3 amino acid which is methionine. The R f for the spot 3 on
the paper chromatography is 0.55 which is belong to the standard 2 amino acid, leucine.
CONCLUSION
From this experiment, we have identified the components of the mixture of the amino acid.
Mixture of amino acids contain alanine, leucine and methionine. The spot 1 from the below
of the mixture belongs to alanine, spot 2 belongs to methionine and spot 3 belongs to leucine.
REFERENCES
1. https://www.coursehero.com/file/p3es2se/
2. http://www.desiquintans.com/stoichiometry
3. https://docslide.us/documents/experiment-9-55844f420f72e.html