A Simple Laboratory Experiment on PVP Column Chromatography The separation of compounds employing column chromatography can he a useful experimental topic in an elementary chemistry or biochemistry laboratory course. The exercise is most dramatic when colored solutes are resolved. However, rather than using dyes, the following experiment deals with the separation of three colored biochemicals, two of which are vitamins, the other a. protein. The column support is Bio-Clar G, a.resin composed of poly-N-vinyl pyrrolidone (PVP). I t has recently been found to have applicability in separations of nucleotide derivatives,' aromatic amino acids,%and in desalting nueleic acids.' A section of glass tubing (Pyrex) about 40 cm X 0.8 em i.d. (glass thickness 1 mm) is heated over a Fisher burner and then drawn out. The glass is cut in the center of the capillary portion and again 10 cm from this to form two columns. A plug of glass wool or cotton is inserted and firmly seated in the constricted portion. Approximately 0.15 g of Bio-Clar G (Bio-Rad Laboratories) is slurried in a. minimal volume of deionized water and poured into the column. After the resin settles (few minutes) the water remaining above the 3-cm bed is removed. The sample to he resolved is added immediately and allowed to drain into the bed. This solution consists of several crystals each of vitamin B,. (red) and riboflavin (yellow) plus a. 10-20 fold excessof Type 11, horse heart cytochrome c (orange), all dissolved in three drops of water. These reagents can be obtained from Nutritional Biochemicals Corp. Deionized water is added dropwise to the column. Vitamin B,.begins to appear in the effluent a t 5 min and is completely removed by 7 mi"; riboflavin follows this immediately and its elution is completed by 14 min. At this point, the water remaining above the bed is removed and replaced by a. 1% sodium sulfate solution (w/v). After 3 min cytochrome c begins to emerge from the column and is completely recovered by 8 mi". The total effluent volume amounts to about 5 ml. In PVP column chromatography, solutes with net charge interact with the polymer presumably by weak electrostatic bonding which involves the pyrralidone nitrogen and carbonyl group. Thus, the basic protein cytochrome c (m. w. = 15,600) binds to the support in water but is easily removed by salt solutions; thelatter normalize polar interactions. On the other hand, vitamin BI1 (m. w. = 1357) and riboflavin (m. w. = 376) each with no net charge under the experimental conditions elute rapidly, perhaps by means of gel filtration.
I , J., A N D LERJ.,, DOUGHERTY, T. M., AND SCHEPARTZ, A. I., J . Chromatog., 37,463, (1968). L ~ M MC. L~RNER T. M., A N D SCHEPARTZ, A. I., J. Chromalog., 43, 397 (1969). J., J . Chromatog., 43, 395 (1969). DOUGHRRTY, T. M., AND SCHEPARTZ, A. I.,J. Chromatog., 42,415 (1969). DOUQHERTY, a DOUQHERTY, T. M., AND SCHEPARTZ, A. I., J. Chrmnalog.,40,299 (1969).
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