Identification of amino acids by chromatography lab
Uploaded by ihatesuchin on Jul 05, 2004
Chromatography is a common technique used by biochemists in separating and identifying different amino acids and helps to reveal the function of cell organelles. Chromatography is particularly approved for its accuracy in distinguishing between each compound, which it does by separating the chemicals according to their Relative Molecular Mass (RMM). The term was introduced in 1906 by Mikhail Tswett and is derived from the Greek words 'chroma´, meaning colour and 'graphein´, meaning to draw. The most popular type of chromatography employs either absorbent paper, or a dried, thin layer of powder on a glass or plastic base.
There are 20 naturally occurring amino acids. The generalised structure of an amino acid is NH2CHRCOOH.
This consists of an amine group (NH2), carboxylic acid group (COOH) and a distinctive R group bonded to the ?-carbon atom. The R group (or 'side chain´) varies in size, shape, charge, hydrogen bonding, capacity and chemical reactivity. The simplest structure is glycine, which has only an extra hydrogen atom in the side chain. Proteins consist of long chains of amino acids which are held together by chemical linkages called peptide bonds
In this experiment, albumen will be used as the chosen protein. Albumen is globular and has a simple structure. Trypsin will be added (the enzyme functioning in the hydrolysis reaction) to the test tube and it will be given a 24 hour incubation at the ideal temperature of 30oC. The trypsin should then have broken up all the albumen into the separate amino acids.
To separate and identify a mixture of amino acids by paper chromatography.
Cut a piece of chromatography paper to about 25cms in length and place on a clean surface. To avoid contamination, hold the paper at the top and wear plastic gloves throughout the whole experiment. Draw a line in pencil three centimetres from the bottom of the paper and then four marks lightly along the line at four centimetre intervals.
Using a micropipette, take the prepared albumen from the test tube and lightly dot a small amount on each pencil mark
The paper should then by wafted swiftly over a blue flame to speed evaporation. This process should then be repeated 40 times, applying the same amount of albumen to the same area, to build up a concentration. This is necessary for the chromatogram results to be clear and distinct. Always remain standing and be careful with the evaporation process, as it is very easy for the...