A simple multiday laboratory exercise suitable for use in a high school or community college chemistry course or a biotechnology advanced placement biology course is described. In this experiment students gain experience in the use of column chromatography as a tool for the separation and characterization of biomolecules, thus expanding their exposure to chromatographic procedures beyond those more commonly employed in the high school environment (e.g., paper or thin-layer chromatography). Students chromatographically separate a standard mixture of three highly colored compounds of varying molar mass (vitamin B12, bovine hemoglobin, and blue dextran) on a short Sephacryl S-300 size exclusion column, determining their relative elution volumes. Students later apply a sample of the enzyme wheat germ acid phosphatase to the same column, collect eluant fractions and determine the elution position of the enzyme by use of a rapid and simple enzyme assay carried out in a 96 well plate. The relative molar mass of the enzyme is estimated by comparison of its elution volume to the elution profile of the molecules in the standard mixture.
Supplement
Student handouts; Instructor notes including detailed information on reagent and equipment preparations, CAS and Sigma Chemical Company catalog numbers, an approximate cost analysis for reagents and equipment, and suggestions for variations on the experiment
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