Reverse-Phase Thin-Layer Chromatography The technique of chromatography, particularly thin-layer chromatography (TLC), is presented early in many lshoratory cour~es.',~ The majority of experiments use, as a stationary phase, silica gel containing varying amounts of water. Separations occur by partition (liquid-liquid) chromatography in which the water is held on the stationary phase, while a second mobile liquid is vassed through. In these chromatograms less ~ o l acom~ounds r mierate farther on the plates. Many of the experimenks illu&ate the f a d that all compounds migrate faither as the polarity i f the mobile phase-is increased. Explanations of these different migration phenomena involve discussions of the relative solubilities between the two liquid oharea. We find that the learning from thi~typcufexperimenlcan beenhanced by reversing the polaritsof thestarionaryand n change in the mobilr pltaaer Trralvnrnt of G l i a ye1 with lDro paraffin oil'' dissolvrd in rrher4 readily acromplikh~~ polarity of the stationary phase. Then using a polar mobile phase, migrations of the polar compounds are greatest, while migrations of the nonpolar compounds are least. The principles involved are analogous to those involved with reversephase high performance liquid chromatography, but the experiments use the very simple equipment of TLC. Thus the concepts of reverse-phase chromatography can he introduced earlier in the curriculum. As with the results from running TLC's using standard methods, these results can he explained in terms of different solubilities. For an experimental procedure we have used plant pigments extracted according to the methods outlined in various laboratory manuals from spinach leaf' or carrot2. For the stationary phase, aluminum sheets coated with silica gel 60 F254 with layer thickness 0.2 mm (E M Reagents) were purchased from American Scientific and Chemical. For student use these 20- X 20-em sheets were cut into 6.6- X 2-cm pieces using a straight edge and a sharp knife or a paper cutter. The mobile phase that worked best for separating the pigments of spinach was a mixture of 6 mL of hexane, 3 mL of ethyl acetate, and 1 mL of d i e t h ~ l e r n i n e ~ When ~ . the 6.6- X 2-em TLC plates were dipped into a solution of 10%paraffin oil in ether, dried briefly in air, and the mobile phase was 21 acetone-methanol saturated with paraffin oil, the carotene stayed close to the origin (Rr 0.06), while the more polar pigments (Rr0.68 for chlorophylls) migrated some distance up the plate. Using this reverse-phase technique the separation of carotene from other pigments in the carrot was good, hut the separation of plant pigments from spinach was poor. This added experiment requires only a few minutes of student time and enhances the understandine of the influence of solubilitv in chromatomaohic seoarations.
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'Moore, J. A,; Dalrymple, D. L.; Rodig, 0. R. "Experimental Methods in Organic Chemistry", 3rd ed.; Saunders: Philadelphia, 1982;p p 83-89. Mohrig, J. R.; Neckers, D. C. "Laboratory Experiments in Organic Chemistry", 2nd ed.; Van Nostrand: New York, 1973;p 30. Pavia, D. L.; Lampman, G. M.; Kriz, G. S. "Introduction to Organic Laboratory Techniques"; Saunders: Philadelphia, 1982; pp 282-88. Fieser, L. F.; Williamson, K. L. "Organic Experiments", 4th ed.; Heath: Lexington, MA, 1979;p 300. The paraffin oil can be purchased from E M Science distributed by VWR Scientific or from Fisher Scientific. We used the heavy grade. (a) Stahl, E. "Thin Layer Chromatography"; Stahl, E., Ed.; translated by Ashworth, M. R. F.; Springer-Verlag: New York, 1969; pp 48-52. (h) Bolinger, H.; Konig, A. Ref. 4% pp 266-272.
J. n. Cooley A. L. Wong University ol Idaho MOSCOW, ID 83843
Volume 63
Number 4
April 1986
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353
