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 B[subscript 12], 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. (Contains 2 figures.)