CHEMISTRY HOME
BLACKBOARD LOGIN

LAB MANUAL HOME

SYLLABUS

Amino acids are the "building blocks" of the body. In addition to building cells and repairing tissues, amino acids combine together to form protein antibodies to combat invading bacteria and viruses; they are part of the enzyme and hormonal system; they build nucleoproteins with RNA and DNA; they form proteins, one of which can carry oxygen throughout the body and others of which participate in muscle activity. When a protein is broken down by digestion this results in the separation of 20 common amino acids. Eight of the known amino acids are considered essential. This means they cannot be manufactured by the body and must be included in a person’s diet and consumed to ensure good health. The rest are considered non-essential and can, therefore, be manufactured by the body with proper nutrition.

Table of amino acids and their Rf values with solvent used in this experiment:

Reference: Aboderin A.A. , Int. J. Biochem. 2:537-544 (1971).

Separation and identification of amino acids can be a lengthy and costly undertaking. The process we will use in today’s experiment is the most inexpensive and least time-consuming of those methods called paper chromatography.

Paper chromatography involves the use of a sheet of porous paper (the stationary phase) and a solvent (the mobile phase) in order to separate substances based on both their polarity and size. A paper chromatogram is “loaded” by placing small spots of the substance to be separated on a defined line and then placing the chromatogram into a tank containing the solvent. As with any porous substance, the paper chromatogram when placed in contact with the solvent will begin to absorb the solvent. The line with the spots of amino acids is initially above the solvent, but as the solvent rises, it carries with it the amino acids. Since the solvent is non-polar, any non-polar substances on the paper will be carried along with the solvent front. Polar amino acids are attracted to the water in the paper and tend to not move as fast as the non-polar amino acids that move with the solvent front. Once separated the amino acids can be identified by comparison to known Rf values from scientific literature (see table on previous page).

An Rf value is simply a ratio of the distance the amino acid traveled from the starting line divided by the distance the solvent front moved from the starting line. Rfvalues are calculated as follows:

where both distances are measured from a predefined starting point. An Rf should have a value between 0 and 1. In the table on the previous page, we multiplied the Rf values by ten.

Another problem in this lab is where to find the amino acids to separate. Amino acids are normally found linked together in proteins and thus require significant hydrolysis (chemical breakdown) to separate them. But they can also be found already separated in various excretions of the human body. The easiest of these excretions to obtain is simple human sweat. The amino acids found in the sweat that creates fingerprints are what police officers use to develop fingerprints on paper and other porous surfaces. We will use your sweat today as a reservoir of amino acids to separate using paper chromatography.