KENYATTA UNIVERSITY MAIN CAMPUS SCHOOL OF HEALTH SCIENCES PHARMACY AND COMPLEMENTARY/ALTERNATIVE MEDICINE DEPARTMENT

HMB 101 INTRODUCTION TO MEDICAL BIOCHEMISTRY

PRESENTED BY: AGUNDA RUTH ALOO P110/0592/2011

PARTNERS: TERESA NYONGESA VINCENT SAMUEL PAUL

TITLE OF EXPERIMENT: SEPARATION OF AMINO ACIDS BY PAPER CHROMATOGRAPHY

LECTURERS NAME:

PRESENTED ON:

INTRODUCTION Chromatography is used to determine the purity of a compound, to evaluate how far a reaction has proceeded and Analyzing the composition of a mixture. Separation of moderately volatile or non-volatile substances is based on differential absorption on an inert stationery phase immersed in organic solvent or solvent mixture (mobile phase). The components are distributed between the stationery phase and the solvent depending upon the polarities /nature of the compound and solvents.

EXPERIMENTAL OBJECTIVES To relate the observed retardation factor values to the structural differences between the amino acids present.

THEORETICAL PRINCIPLES BEHIND SEPARATION OF COMPONENTS BY USE OF PAPER CHROMATOGRAPHY Paper chromatography is a liquid liquid partitioning technique. A spot is placed near the bottom of a piece of high grade filter paper (whatman no. 1 is used).The solvent ascends the capillary paper by capillary action and moves the components of the spotted mixture upwards at differing rates. Although paper consists of pure cellulose, the cellulose does not function as a stationery phase. Rather the cellulose absorbs water from the atmosphere example; an atmosphere saturated with water vapour.Cellulose can absorb upto 22% of water. It is this water absorbed on the cellulose that functions as a stationary phase. To ensure the cellulose is kept saturated with water, many development solvents used in paper chromatography contain water as a component .As the solvent ascends the paper; the compounds are partitioned between the stationery water phase and the moving solvent. Since water phase is stationery , the components in a mixture that are most highly water-soluble, or those that have the greatest hydrogen bonding capacity, are the ones that are held back and move slowly. Paper chromatography applies mostly to highly compounds or those that are polyfunctional. The separated amino acids on the chromatogram are located by treating the sheet with ninhydrin which reacts with amino acids forming a purple to red spots where the amino acid is located. The reaction depends upon the presence of a free amino group, the nitrogen of the amine reacting and forming a link between two monomers to form the coloured dimer. The reaction below shows the formation of the coloured complex

PROCEDURE Reagents and materials Tank (sweet jar with lid) Filter paper 25 by 25 cm.whatman No. 1 Wire loop or micro pipette Dip tray Clips 0.01M DL-Aspartic Acid 0.01M DL-Leucine 0.01M DL-Lysine Amino Acid mixture Solvent: Ethanol: Ammonia (spgr.0.880)80:10:10 by volume Ninhydrin (dissolve 200mg in 100 ml Acetone, keeps indefinitely in a refrigerator).

The solvent solution was already prepared and poured into the jar by the lab technicians .The lid of the jar was replaced and the tank was allowed to become saturated with solvent vapor. A baseline 2 cm from the edges of the paper was drawn by one of the students wearing a glove. The baseline and the solvent front were indicated. The test solutions (8-10mmin diameter) were applied onto the paper (on the baseline) using a different micropipette for each test solutions and the position of each test solution was noted on the filter paper. The spots were left to dry, afterwards, the filter was rolled to form a cylinder and held by paper clips at the two edges of the filter paper. The cylinder was then placed into the tank with the spotted end down in the tank taking note that the filter paper was not supposed to touch the glass walls nor were the spots supposed to touch the fluid in the bottom. The tank was then replaced with the lid and chromatogram left to run for 2 hours. After 2 hours the chromatogram was removed and the solvent front was marked with a pencil. The paper was opened and dried in an oven. After that the dried chromatogram was dipped into the tray containing Ninhydrin and then it was returned to the oven until purple spots were observed. The position of the amino acid spot were outlined using pencil and the distance between the center of each spot and starting pencil line measured.

RESULTS

Compound

Distance from origin in cm

Distance travelled by solvent front

Retardation factor value

Aspartic Acid Leucine Lysine Amino Acid Mixture

CALCULATIONS The distance travelled by each component is expressed by each as a rate of retardation factor (Rf).Rf values are calculated by dividing the distance between the origin and the distance travelled by the solvent front.

EXPLANATION Polar compounds are strongly attracted to and held by a polar adsorbent .Non polar compounds are held weakly. When non-polar solvent is passed through the adsorbent, non-polar compounds are released easily but polar compounds are retained. When moderately polar solvent is passed through the adsorbent, the polar and non-polar compounds move faster because theres still an attraction between the polar compounds and the polar adsorbent.