S.
F.
Darling
Lawrence College Appleton, Wisconsin
I
Demonstrating Enzymatic Hydrolysis of Glucosides by Paper Chromatography
The simple yet widely useful t,echnique of paper chromatography is being introduced more and more into the introductory organic chemistry work.' Our students have used this technique to study sugar mixtures, to identify unknowns containing mono- and disaccharides and to identify amino acids in simple mixtures and protein hydrolysates. Instead of using the ascending techniques described in Fieser2 we have employed the descending technique in large cylindrical or square glass jars equipped with glass trays and stainless steel holders. One such square tank containing four trays gives space for students to develop 16 chromatograms each 4 in. X 22 in. This can be done overnight in a slow moving solvent mixture such as 10-3-3 butanol-pyridinewater or in a few hours with the fast moving solvent 9-2-2 ethyl acetate-acetic acid-water mixture. Both of these mixtures are well adapted for resolving sugars. For amino acid separation we have had excellent results with the 63-10-27 butanol-acetic acid-water mixture. (All the ratios quoted are by volume.) A 4-in.-wide strip of paper enables a student to spot his unknown together with two knowns or two known mixtures an inch apart a t the start and still have the outer spots an inch from the edge. The last precaution avoids confusing effects associated with spots on the edge. Inexpensive but very satisfactory spotting tubes can be made from capillary melting point tubes drawn down to a much smaller diameter. An interesting application of the use of paper chromatography is in the study of enzyme action. Simple B-glucosides are hydrolyzed cleanly and rapidly in the presence of p-glucosidase and this action is easily followed chromatographically using only micro 'See, for example, THE JOURNAL, 37, 49, 156, 293 (1960). FIESEE,LOUIS F., "Experiments in Organic Chemistry," 3rd ed., D. C. Heath & Co., 1955.
509 / Journol of Chemical Education
amounts. I n each of two 13 X 75-mm test tubes place about 5 mg of the glucoside. s a l i ~ i n . ~ Dissolve each sample in about 1 ml of water. To the solution in one of the tubes add one or two milligrams of 0-gluco~idase.~ After one hour spot a few microliters of the contents of each tube and a spot of glucose solution on the chromatographic paper. Develop this chromatogram either overnight in the 10-3-3 BuOH-pyridine-H20 or two hours in the 9-2-2 EtOAc-HOAc-HzO. At the end of this time the chromatograms are removed from the tanks, the solvent front is marked and they are dried in the hood. The spots are brought out with modified silver spray.5 Under the salicin in water alone should appear one spot for salicin at REabout 0.62 in the 10-3-3 solvent mixture. Under the salicin with glucosidase should appear the prominent glucose spot at Rr 0.17 and salicyl alcohol at R,0.87, while complete hydrolysis is shown by the fact that no spot appears a t the R, of salicin. Under glucose there appears the prominent spot a t Rr 0.17. If one desires, an extenstion of the experiment can be conducted as follows: Make duplicate chromatograms as described above. Spray one chromatogram with the modified silver spray and the other with diazotized p-nitraniline followed by sodium carbonate solution. This spray causes the salicyl alcohol spot under the hydrolysate to appear as a bright rose colored spot due to coupling action of the phenol with the diazonium spray. No color appears either under the glucose or the salicin in this case because of the inability of these substances to react with the diazonium spray. a Arbutin can be substituted for salicin in which case both the arieinal -elucoside and the aelucone rive colored mots with the diazonium spray. 4 Obtainable from the Nutritional Biochemicel Corp., Clrveland, Ohio. PEARL, I. A,, AND DARLING, S. F., 3. Or$ Chem., 24, 735 (1959).
