RESEARCH
SE-30 Aids Separation of Complex Compounds Gas chromatographic technique uses silicone polymer to detect and separate closely similar steroids and alkaloids Gas chromatography can now separate mixtures of steroids or alkaloids better and more quickly than has been pos sible up to now. Scientists at the National Heart Institute, Bethesda, Md., have cut drastically the long re tention times and high temperatures that were once needed for these molecules. Key factor in the microanalytical technique: small amounts of a new liquid phase, General Electric's methyl silicone polymer SE-30. Dr. E. C. Horning and his associates at N H I - D r . C. C. Sweeley and Dr. W. J. A. VandenHeuvel-hit on SE-30 after trying other polymer phases. In their early attempts with the silicone gum, they used about 7% on Chromosorb W, carried out separations of steroids at 260 e G. But the high tem perature had a bad effect on the hy droxy compounds and acetyl esters. The chromatogram showed broad peaks and multiple components that suggested decomposition. Low SE-30. But with 2 to 3 % SE-30 on the same support, the NHI chemists find a major change in the results. For one thing, they can carry out the separations at a lower tem perature—around 220° C. Also, all of
the compounds are eluted as single components with no decomposition. Another important result: SE-30 sepa rates many steroids that differ only slightly in structure [JACS, 82, 3481 (I960)]. One mixture chromatographed by Dr. Horning and co-workers contained nine steroids (see cut). Among them: androstane, coprostane, cholestane, cholesterol, and stigmasterol. All gave distinct peaks, with retention times varying from about two minutes for androstane to about 54 minutes for stigmasterol. Even more surprising, Dr. VandenHeuvel says, is the excellent separation of steroids that differ only in that the A and Β rings are joined cis in one and trans in the other. One such pair is coprostane (cis) and cholestane (trans). Another example: pregnane-3,20-dione and allopregnane-3, 20-dione. Hydroxy compounds are eluted, without trailing, before the corres ponding ketones, Dr. Horning points out. But ketones do show some trail ing. There is also a sharp difference in retention times for steroids with dif ferent carbon contents, he says.
Standard Procedure. The method used by Dr. Horning and his associ ates doesn't differ markedly from or dinary GC procedures. They use a commercial gas chromatograph equipped with an argon ionization de tector. The U-shaped column is 6 feet by 4 mm. (i.d.). Preparing the pack ing is very simple, they say. These workers dissolve the SE-30—a soft, nearly transparent gum—in an organic solvent at the desired concentration. They slurry prepared Chromosorb W in this solution, then filter and dry it. The coated particles have the same appearance as the uncoated ones, and they flow just as freely. So far, the NHI group has worked only on the micro level—about 5 to 10 micrograms of material. There are many technical problems to solve be fore they can make a big jump to 100 mg. or more, Dr. Horning says. But even so, the microanalytical procedure promises to be very valuable to scien tists in such fields as human metabo lism, biochemical mechanisms, and structure determination. It may also be useful in research on the differences in properties caused by subtle changes in structure, he says.
SE-30 Detects Small Differences In Steroids androstane
coprostane stigmastane
allopregnane-3F20-dione
choIestane-3-one pregnane-3,20-dione
cholestane stigmasterol
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10
.20 M 1NUTES
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GROUP CONFERS. Dr. E. C. Horning (seated) discusses some of their results with members of his group. They are (from left) Kay Maddock, Dr. H. M. Fales, Dr. W. C. Wildman, and Kay Anthony
Another important avenue of re search for the NHI group: adapting the method to capillary chromatog raphy. Dr. Eero Haahti, visiting sci entist from the University of Turku, Finland, is now working along these lines. Still Faster. As soon as the NHI chemists had worked out the system, they began to improve it. By decreas ing the amount of SE-30 on the Chromosorb, they are cutting the retention times to a fraction of those first re ported. Stigmasterol, for example, was obtained in about 54 minutes with 2 to 3 % SE-30 on the column. With 0.47c silicone, it can now be eluted in about six minutes. Also with the faster technique, ster-
COPROSTANE
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Ctlsl J _ J Jl
CHOLESTANE
AN ALKALOID RUN. Dr. Wildman and his assistant, P. F. Highet, check the progress of an alkaloid separation by gas chromatography with SE-30
oids which were obtained in the early part of a run now are eluted much too fast, Dr. VandenHeuvel says. For these compounds, it may be practical to lower the column temperature, he points out. Cutting down on the silicone phase does decrease the column's efficiency, but this has not affected steroid sepa ration to any great extent, Dr. Vanden Heuvel adds. For most of them, he says, it is possible to get good separa tion with only a few thousand theo retical plates. The success of all these runs led Dr. Homing's group to try compounds which, they felt, would really test the method. Their choice: the bile acids. Up to now, procedures for separating these steroids have been long and laborious. Dr. VandenHeuvel turned to a mix ture of the methyl esters of four bile acids: cholic, deoxycholic, hyodeoxycholic, and lithocholic. They differ only in the number and position of their hydroxyl groups. In the first run of this mixture, he says, the column contained 2 to 3 % SE-30. It took about two hours to elute the last of the four componentsmethyl cholate. With 0.8%, this time was cut to about 36 minutes. And with 0.4% silicone phase, this came down even further, to about 13 min utes. Even in this short time, Dr. VandenHeuvel says, the esters came out as single components. Interest Spreads. It wasn't long before Dr. Homing's procedure inter
ested another group in the Laboratory of Natural Products at the National Heart Institute—the alkaloid chemists. The separation of alkaloids has long depended on crystallization, precipita tion, counter cur rent extraction, and either adsorption or liquid phase par tition chromatography. Their high basicity has caused problems in put ting gas chromatography to woric sep arating them. But here again, SE-30 did the trick. Dr. W. C. Wildman, Dr. H. M. Fales, and their associates, Dr. H. A. Lloyd and P. F. Highet, have chromatographed some 45 alkaloids of several classes (JACS, July 20, 1960). Us ing 2 to 3 % SE-30, elution times have varied from 1.5 minutes (lupinine) to about 90 minutes (gnoscopine). Al most all the alkaloids tested have mo lecular weights above 250; all give sharp, single component peaks. A mixture of Papaveraceae alka loids, for example, gives distinct peaks for codeine, morphine, thebaine, laudanosine, and gnoscopine, Dr. Fales says. In addition, Dr. Wildman has chromatographed just about every analgesic compound on the market (and some not in general use) singly and in various mixtures. So far, he finds only a few cases of serious over lap of the peaks. Dr. Wildman has not yet tried the faster technique, using less SE-30. But he has lowered the retention times for some of the alkaloids by raising temperatures and flow rates. He has cut morphine's elution time, for exJULY
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ample, from 11 minutes to six minutes. Structure Clues. One interesting finding by the alkaloid group shows how the technique may help structure studies. It has been known for some time that plotting log retention time vs. molecular weight for a series of homologs gives a straight line. Dr. Wildman and co-workers observe this is likewise true for a series of alkaloids having the same nucleus with varying alkoxyl substitution. But they also find that if, besides the alkoxyl substitution, the alkaloid nucleus is substituted in unhindered positions with the same group—carbonyl, for example—the points for these compounds also fall on a «straight line. And this line is above and parallel to that for the original series. Other substituents will result in other parallel lines, Dr. Wildman points out. If the substituents are in sterically hindered positions, however, the points for these derivatives will not fall on the line with their unhindered analogs, but describe a new line indicating lower retention times. Dr. Wildman and Dr. Fales feel that the technique may give valuable data in structural studies, when combined with other chemical and physical data. Besides its obvious uses in studies of structure, biochemical mechanisms, and the like, Dr. Wildman points to other possible uses for this procedure in the alkaloid field: • Analysis of body fluids may show very quickly the presence of drug metabolites. • The method may pinpoint the particular source of an opium mixture. Using two samples furnished by the UN, Dr. Wildman has shown that one contains laudanosine, while the other shows no sign of this narcotic. He suspects that the "fingerprints" of each opium mixture may be characteristic for the plant variety and geographical region that it comes from.
Etched Films Extend X-Ray Microscopy Combining x-ray and electron techniques gives enlargements up to 40,000 diameters A new technique in x-ray microscopy magnifies up to about 40,000 diameters, may open up exciting possibilities in biology, metallurgy, and other fields. Dr. Saara Asunmaa, working under Dr. Howard H. Pattee, Jr., in Stanford University's biophysics laboratory, achieves this magnification by combining the methods of x-ray microscopy with those of electron microscopy (C&EN, July 11, page 3 5 ) . Until now, the limit of the x-ray microscope has been 2000 diameters. The electron microscope has a limit of 100,000 diameters or more. But many specimens are opaque to electrons; only the outline or surface can be examined this way. Dr. Asunmaa makes a contact xray micrograph on a sensitive film in which the absorbed radiation changes the electron density. This film is then used as a specimen in the electron microscope. The film that does the trick is made of cellulose nitrate activated with silver chloride. Dr. Asunmaa mixes lithium chloride and silver nitrate in an acidic medium with a 2Vc solution of cellulose nitrate in ethanol and ether. From this she casts the thin films—about 1000 A. thick—on glass slides, then ages them in a desiccator. Sensitive Film. To make the micrograph she places the specimen in contact with the film and exposes it to
x-rays from a microfocus tube for 10 minutes to one hour. The silver chloride particles act as radiation traps and are reduced to metallic silver. The reduced silver gives an image that is directly visible under a light microscope, but this optical contrast does not correspond to differences in electron scattering. To get an image that is observable in the electron microscope, the structure of the film must be altered. The radiation absorbed by the silver chloride not only reduces the silver salt but also degrades the cellulose nitrate. Aqueous methanolic sodium cyanide dissolves the soluble low molecular weight fraction of the polymer, while the silver and unreduced silver salts are removed as cyanide complexes. Relief Image. What is left is a relief image with electron optical contrast corresponding to x-ray absorption by the original specimen. This relief image can now be used as the specimen in an electron microscope. The enlargement here is limited only by the resolution obtained in the original x-ray micrograph. Using diatoms as test specimens Dr. Asunmaa has demonstrated resolution down to at least 600 A. and has produced electron micrographs with enlargements up to 40,000 diameters. The technique is too new to have had any applications yet. But many
• The NHI technique may be useful in screening plant extracts for new alkaloids. Several pharmaceutical companies already have plans to use it this way, Dr. Wildman says. Despite the large number of compounds already run and the volume of data built up, Dr. Horning and coworkers feel they have only scratched the surface of the applicability of SE30. The silicone shows such valuable properties, they say, that its use will reach beyond the steroid and alkaloid fields. 42
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DEEPER LOOK. Ordinary electron micrograph (15,000X) at left shows the holes in a diatom shell. The grid structure in some of them has bars mainly of two widths, 600 A. and 300 A. At center is an x-ray micrograph film image enlarged 5000X in the electron microscope. Here, the varying thickness of the shell is visible, but poorer resolution causes loss of the fine grids over the holes. When the film image is enlarged 40,000X (right), the grid network is again visible, although not as sharp as in the micrograph at left. But the resolution is adequate for quantitative work
