RESEARCH
TL Chromatography Shows Reaction Time Thin layer method picks up fleeting intermediates, allows isolating both the intermediates and reaction products Thin layer (TL) chromatography can be used to follow the time course of reactions. Dr. James M. Bobbitt of the University of Connecticut, Storrs, Conn., and co-workers now use the technique to learn how long it takes a reaction to go to completion, quickly pick up fleeting intermediates, and see when maximum product is formed. TL chromatography is an adsorptive microchromatographic technique used widely in Europe but, so far, relatively little in the U.S. Dr. Bobbitt began working with the method when he was at the Organic Chemical Institute of the University of Zurich, Switzerland. Since his return, he has been active in developing TL chromatography as a research tool. Dr. Bobbitt says that the method may be used to determine the purity of materials or to identify the number of components in a system. He has used it to learn quickly whether a multicomponent system can be separated into its individual parts. The tech nique can also be used to analyze frac tions collected from chromatographic columns. And, he notes, it may also be used as a device to isolate and
collect, quantitatively, small amounts of substances found in complex mix tures. TL chromatography is both simple and fast. And it can be used to detect as little as 0.02 microgram of a steroid component or to isolate as much as 10 mg. of diglycerides obtained from di gested milk fats. In Europe, the technique has been used mostly to separate natural prod ucts and to detect impurities. Dr. Bobbitt sees TL chromatography being used as a universal bench tool by both inorganic and organic chemists. The system has the speed of vapor phase chromatography with the reliability of paper chromatography, he adds. Reaction Time. The reaction be tween catalposide and Amberlite IRA400-OH is an example of how TL chromatography can be used to deter mine the time that it takes a reaction to go to completion and to pinpoint intermediates. Catalposide, the ma jor glucoside of the Catalpa genus, and the resin are heated (in water) at 80° C. The reaction is tracked by chromatographing the mixture period ically on a plate prepared from Merck
silica gel "G"; development is with 20% methanol in ether. At first (zero time) only the starting glucoside—an ester of p-hydroxybenzoic acid—appears. After about an hour, the reaction mixture is mostly the ester's alcohol component, a reac tion intermediate. But at the same time, another product begins to show up. This second compound, Dr. Bob bitt says, eventually accounts for the entire reaction product. By choosing reaction times, the Connecticut group has isolated both the intermediate and the final product. Their structures, though, aren't known. Separations. The Connecticut sci entists have been working extensively with TL chromatography as a separa tion tool, too. Speed of separation is a big advantage of the method. Using alumina plates, Dr. A. E. Schwarting, Dr. W. J. Kelleher, and Dr. Anna Rother of the pharmacy school have surveyed the alkaloids oc curring in microbes and higher plants, A crude alkaloid mixture from a Claviceps fungus (ergot) growing in a sub merged culture was shown to contain lysergic acid amide, ergonovine, elymo-
Technique Tracks Reaction's Progress Intermediate and reaction product can be isolated by choosing the right reaction time Hours
0
%
1
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3
5
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I. I .1 I. .
λ •
Vz
Β Starting material (catalposide, an ester, which is reacted with Amberlite IRA-400-OH)
Intermediate (alcohol part of the ester)
Reaction product 42
C&EN
JUNE
12,
1961
9ΨΨ
TL Technique Features Fast Separations with Small Amount of Sample In most cases, thin layer chromatography is adsorption chromatography, as opposed to distribution or partition chromatography like paper chromatography. The main advantages of the TL technique are the speed with which it separates components and its wide quantitative range with small samples. For general use, a glass plate is coated with a thin (250 microns) layer of an adsorbent substance— such as silica gel or alumina—to which a binder (plaster of paris) is added. The adsorbent must have a small particle size—between 1 and 5 microns. Larger particle sizes give results that are hard to duplicate. Clean plates are placed in a special holder and the adsorbent films applied. The plates are then dried. Glass plates of almost any size may be used, but the standard sizes are 5 cm. by 20 cm. and 20 cm. by 20 cm. The test substances, prepared as 0.1, 1.0, or 5 % solutions, are dotted along a line about 2 cm. from the bottom edge of the plate. These dots are from 10 to 20 mm. apart and can have a volume of from 0.1
to 500 microliters. For qualitative work, as much as 100 mg. of material may be dispersed. The spotted plate is then put into a sealed glass jar. The jar's atmosphere is saturated with the proper eluant which, at the same time, covers the bottom to a depth of about 5 cm. The eluant then goes up the coating, taking 20 to 40 minutes to move about 10 cm. for nonpolar substances. Polar substances can be allowed to move about 18 cm. because they diffuse so slowly. To choose the proper eluant, test spots of the material to be chromatographed are placed in a row along the coated plate. Glass capillary tubes, each containing one of many solvents, are prepared; one capillary tube is then placed in the center of each spot. The eluant leaving the capillary forms a circular spot. From the amount of separation observed, the right eluant can be picked. The spots are developed by charring with concentrated sulfuric acid, which serves as a general reagent for all organic materials. Normal developing reagents such as ammonia or
clavine, penniclavine, and some minor alkaloids. And an alkaloid extract of Vinca rosea, when chromatographed twodimensionally, was found to contain at least 70 compounds. The TL technique has also been used effectively at Connecticut to separate glucosides with a methanol-ether carrier. And steroids have been separated with a benzene-ether-pentane carrier. Another possible application for the TL method, Dr. Bobbitt notes, is the initiating and following of reactions that are carried on in situ on the adsorbent. At the agricultural college, Dr. R. G. Jensen and Dr. G. W. Gander have used TL chromatography to separate 1,2-diglycerides, 1,3-diglycerides, and other lipids from partially digested triglycerides. The TL system has also been used by other workers to separate inorganic ions, Dr. Bobbitt notes. In this case, a coordinating compound such as pentanedione must be used in the carrier.
PLATE. Dr. James M. Bobbitt (left) and Dr. Roy J. Gritter examine a plate showing the simultaneous analysis of 23 fractions obtained from a column chromatogram of the products of a free radical oxidation of coprostanol
ninhydrin can be used or the spots may be located by a fluoresceine wash and ultraviolet light. The quantity of material that the system can handle is directly proportional to the thickness of the adsorbent layer; sensitivity is inversely proportional to the thickness. Tests run at 5°, 20°, and 28° C. show no differences (as a function of temperature) in the ability of the TL system to isolate components. Because the TL technique is adsorptive, there is very little spreading of spots on the plates. Thus up to 20 compounds (spots) can be separated on a single large (20 cm. by 20 cm.) plate. And the plates may be used two-dimensionally, including electrophoretically. The adsorbent layer can be prepared as a basic or an acidic layer by mixing the silica gel with a base or an acid or some specially buffered solution, rather than with plain water. Alkaloids, for instance, do not separate well on silica gel which acts as an acidic adsorbent; but they do separate when pH is raised. Alumina may be used as the adsorbent, too.
JUNE
12, 1961 C & E N 43
