High Performance Thin Layer Chromatography Samuel J. Costanzo Norwich Eaton Pharmaceuticals. Inc., Norwich. NY 13815
One of the first ~ o i n tmade s in most analvtical chemistw texts is the distinction between the qualitaiive and quanti"tative aspects of chemical analysis. However, the problems of detecting, identifying, and determining the amounts of sample constituents are not always the initial issue of concern. Often some form of prelimina~yseparation must take place. Iucreasingly, the principles of chromatography are being introduced in many undergraduate science courses. This paper will attempt to clarify where in the scheme of modern chromatography high performance thin layer chromatography fits, and why in some situations it is a viable alternative to gas and liouid chromatoeranhv hieh " ~erformance ~" Until the middle 2 this century, the separation of a sample into its various com~onentswas accomolished in ~ainfullv slow ways, such as paper or column chromatography. This is definitelv not the situation todav. Beeinnine with the advent of pract&al gas chromatograph;in t& 19507s,high efficiency in sample separation became commonolace. After the oioneering workofJustus Kirchnerand ~ b o nStahl, thin laier chromntomaphv .. . . ('I'LC) became important for the se~aration of samples not amenable toanalysk hy gas chrumat&raphy. The rapid of T1.C was slowed during the 19"s with - growth . the corresponding rise in popularity of hig6 pressure liquid chromatography (HPLC). Although both HPLC and TLC are capable of separating the same type of compounds, HPLC is considerably more efficient. Further, capacity factors in HPLC are more reproduciblethan Rrvalues in TLC. However, recent improvements in TLC have removed many of its limitations, and in some situations, it actually gives better results than HPLC. To emphasize the fact that TLC may he a legitimate alternative to HPLC, the relatively new term high performance thin layer chromatography (HPTLC) has been introduced. Just what is high performance thin layer chromatography (1-4)? How is it different from classical TLC?To what extent does HPTLC keep the advantages of classical TLC, such as minimal sample clean UD,flexihilitv in solvent selection and sample detection, high ;Lple through put, and low analysis costs? Many of the recent improvements in TLC may he placed under the heading of high ~erformancethin laver chromatography, as a meins of k&hasizing improved efficiencyand prerision. Zlatkis and Kaiser ( 1 ) define HPTLC as a comhination of improvements in several aspects of TLC. These improvements include optimized plate coating materials, a new method of developing the mohile phase, theways in which samples are applied to the plate, and a novel procedure for conditionine the TLC date. A more concise definition of high performance is given 6y Jupille ( 2 ) .I t was pointed out thoat for a given chromatoera~hicsvstem resolution of samnle components are depeGdeut upon selectivity and efficiency. Since selectivity is determined primarily by interactions hetween the solute, stationary phase, and mobile phase, the efficiency of a system is the best means of establishing performance. High efficiency is the same as high performance. This paper will consider the various ways in which high performance has been achieved in thin layer chromatography, including the new TLC plates, sample application, d a t e developme&, and instrumental te~h&~ue;j3). &
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The HPTLC Plate These new plates may he considered the heart of any HPTLC sg9tem. Halpaap and Ripphahn (5)of E. Merck have documented some of the extensive work put into the development of these plates. The activity of Silica Gel@is largely determined by its pore system (size and distribution) and the degree of loading of silanol groups with water. In addition to these ~ r i m a woarameters of the sorhent. secondarv narameters of partieie size and particle size distribution are also important to the performanceof the plate. To obtain optimum separations with TLC, these secondary factors must he controlled. Also, unlike high pressure liquid chromatography, migration time is affected solely by capillary forces, and not hv external pressure. Extensive studies were made of various layers cover-ing a range of particle size, layer thickness, migration distance, and sample size. In order for valid comparisons to he made, all otherehromatographic conditions, such as type of chamber, temperature, coating procedure, and presentation of the layer, were held constant. From this work several important points were made. 1) Each particle size range of sorbent can be associated with an op-
timum solvent migration distance. 2) Unlike conventional TLC, which used sample volumes of a few microliters, HPTLC requires sample volumes tobe in the nanoliter
range. 3) Because very thin layers will cause considerable sample diffusion, layer thickness of 2 N fim are optimal for HPTLC.
There are indications that the chromatographic differences between HIY'1.C plates and conventional TLC plates may not be as great as expected. A comparison by Brinkmann et (6) of minimum plate height values between conventional and HPTLC plates showed rather small differences. These authors speculate that these results are due to the fact that the particle size of HPTLC and TLC Silica Gels do not differ suhstantially. They point out, for example, that the Merck technical bulletin onlv mentions that the oarticle size ranee for HPTLC plates is heiow that of convent~onalprecoated-TLC plates. As with conventional TLC, most high performance TLC is done on Silica Gel plates. However, some high performance work has been done on reverse phase plates (7-9). Brinkman and DeVeries (7) compared several commercially available plates and evaluated their performance with several different classes of compounds. I t was observed that precoated RP-8 lavers are much softer than conventional and HPTLC silica &l plates. Run times for precoated LiChrosorh RP-2, RP-8, and RP-18 increase very rapidly with increasing water content of the mohile phase. KC18 plates exhibit run times 3-4 times shorter for comparable mobile phases. When the water composition of the mobile phase is M0%, the LiChrosorh RP, and KC18 plates separated several classes of compounds equally weU. At higher levels of water the LiChrosorh RP coated plates could not be used since they were not wetted by the mohile phase. However, by adding 3%NaCl or CaClzto the water, the KC18 plates could be used with more than 30%water in the mohile phase. Precoated cellulose HPTLC plates are commercially availahle. Brinkmann (8) has shown that these plates have smaller plate heights than conventional cellulose plates, and
a.
Volume 61 Number 11 November 1984
1015
considerably shorter development times. These same authors oolvamide sheets for have also comoared re& and HPTLC . " the separation of a series of hydroxy compounds and found considerably longer development times with the high performance systems.
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Sample Application Requirements for a Good Sample Spotting System As stated in the introduction, high performance means high efficiency. If a highly efficient TLC system is to he achieved, the initial spot size must be as small as possible. Kaiser (10) has described this mathematically by a factor which he calls the dosage quality, QD. The expression
of sample also may be avoided by applying a larger volume of samde and then concentrating the s w t after it is on the plate. his is the principle common-to aliof the following spotting techniques: preadsorbent or concentrating zones, chemical focusing, and programmed multiple development. The technique of using a narrow Kieselguhr concentrating layer was first described by Abhott and Thomson (13), and is based on the difference in adsorbing power of the two sorbents. Halpaap and Krebs (14) were the first to study thoroughly a commercial Silica Gel preabsorbent or sample concentrating zone both for conventional and HPTLC plates. This zone extends across the width of the plate for about 25 mm in the direction of develo~mentand is about 150um thick. The material consists of synthetic porous silica of medium pore volume with an extremely large . pore - diameter ( ~ 5 0 0 0 nm) and avery small internal surface area (1 mVg). Samples may he applied directly to the concentrating zone using conventional techniques, such as 2 to 20 p1 glass capillaries. The plate is then developed in a regular TLC chamber. The sample is w r i e d quickly through the concentrating zone and collects at the layer interface as a narrow line, the length of the diameter of the initial spot. Halpaap and Krebs (14) used the separation number of Kaiser (15) to compare the performance of precoated Silica Gel 60 with and without a concentrating zone, and precoated Silica Gel 60 HPTLC plates with and without a concentrating zone. The separation number gives the maximum number of completely separated substances in the range from Rr = 0 to Rt = 1, where separation is considered complete when the interval between-the concentration maxima is equal to or greater than the sum of the two peak widths a t half-height. Using seven lipophilic dyestuffs, an N-chamber without presaturation, toluene as the mohile phase and a development distance of 10.0 cm. these authors comoared the seoaration numbers for various amounts of sample applied using Silica Gel 60 precoated HPTLC plates and Silica Gel 60 precoated HPTLC plates with a concentrating zone. At all levels (0.01 to 50 pg) the HPTLC plates with the concentrating zone gave larger separation numbers than the regular HPTLC plates. At the 50 pg level the separation number for the plates with the concentrating zones was almost 6 times larger than that of the HPTLC plates without a concentrating zone. The chemical focusing, or preliminary run technique, has been described hy Blome (16). The sample is applied in a normal manner, using 1-10 p1 glass capillaries or other spotting devices, and then a strongsolvent carries the sample up the plate a shon distance, concentrating it into a line. If all the components in the sample do not move with the solvent front, the procedure is repeated using another solvent. The plate is thoroughly dried and then developed with a suitatde mobile phase. A concentrating effect similar to that obtained with the preliminary runtechnique is found with Progammed Multiple Development (17). Here the sample is applied to the TLC plate i n a conventional manner, and focused into a sharp line hy the solvent as it moves up and down the TLC plate. Up to this point, emphasis has been given to spotting small volumes of sample (nl),or applying large volumes b l ) followed by a concentration step. Either approach can lead to highly efficient TLC. Another important technique for sample application is automated streaking. Although conventional amounts of sample (pl) are applied which works against high efficiency, automatic streaking has two important advantages. The sample application error is minimized because reproducible amounts of sample are streaked. Secondly, when quantitative densitometry is done, careful matching of the light beam with the sample spot is not required. This latter aspect is even more important when using a densitometer that automatically changes tracks. ~
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~
is a measure of the degree of spot broadening during the chromatographic process, where bl is the final spot diameter and bo is the initial spot diameter. When bo is as small as possible, QD approaches 1, which yields a conservation of seoaration Dower for the svstem. An o~timizeddoswe . technique can be achieved by:. 1) Using as small a dosage volume as possible. (Although a dosage volume of 10 nl is recommended, 100or 200 nl is more realistic of what can easily and reproducihly he achieved.) 2) Use highly concentrated solutions. Samole . S~otiino . Highly efficient sample application can be accomplished in several wavs: self-loading contact spotting, .capillaries, . plates with pieadsorbent or concentrating zones, and spot predevelopment (11). Small volumes of samples may be applied to the HPTLC plate surface using platinum-iridium capillaries, which are commercially available in 100 and 200 nl sizes. These capillaries are inert to most chemicals and show very little memory effects on the plate. Also, the tips can be flamed if necessary. The sample is applied by lightly touching the capillary to the olnte = - - ~surjace. ~ - ~~~~. - ~ atechniaue similar to that used with conventional TLC plates. It should he noted that the direct method of a~plvimz .. . samples to the high performance plate with capillaries is not without disadvantaaen. Although they do an excellent iob of reproducibly applying small aGounGof sample, these capillaries are considerably more expensive than the larger glass capillaries. Because platinum-iridium capillaries are not disposahle, sample application is slowed because of the cleaning required between sample application. Even with cleaning, some sample carryover is possible. Since only small volumes of sample may be spotted directly onto the HPTLC plate, sensitivity may be sacrificed to gain efficiency. Small volumes of sample may also be placed on the TLC plate using microsyringes. The advantage of this approach is that it is possible to vary the sample volume. Additionally, because syringes work by a displacement, rather than a capillary principle, samples may be delivered without touching the plate. As with the non-disposable fixed-volume microcapillary pipets, cleaning and sample carryover problems must he addressed. Hiehlv viscous concentrated biological samples are not easil;applied with either capillary pGets or microsyringes. The technique of contact spotting is a unique way of getting around this difficulty. Briefly, up to 100pl of sample is placed on a nonwettina-. ~olvmer . film forming a symmetrical droplet. The droplet is then concentrated h i evaporation, leaving a much smaller volume, which is transferred to an absorbent layer by contact with the film. Up to 15 samples may be spotted simultaneously using an automated contact spotting device (12). The disadvantages of direct application of a small amount 1016
Journal of Chemical Education
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Plate DeveloDment Plates may he developed in a linear or circular manner. In addition, the techniques of continuous, multiple, and overpressurized development are hecoming more important. A hrief description of each of these follows. The oldest and most familiar form of plate development is accomplished in chambers with relatively large vapor volumes. The lower edge of the plate is suhmergrd in ( ~ & e l ; ~ i wlvent n~ about I cm deep. Capillary action then pulls the mobile phase up the plate. C&e must be taken whenusing these chambers because of the large volume of vapor phase in contact with the plate surface. The more polar components of the vapor will headsorbed preferentially. This can significantly influence the chromatoeraohic oualities of the d a t e . The effect of the vapor phase on t i e plate coating can be minimized by the so-called "sandwich" chamber. Here a second glass plate is placed approximately one millimeter from the surface of the laver, effectivelv eliminatina contact hetween the solvent vaporand coating material. using this approach the chromatoera~heris hetter able to exercise reoroducihle control over ~hekystem. Because the mohile phase is carried through the layer by capillary action, linrar development in sandwich chambers m take place in the classical ascending manner, with the edge of the plate directly in wntact with the developing solvent; or with the plate surface face down, parallel with the bench, and rhesolvent brought to the plate hy sometype of transfer arrangement. This latter arrangement is commonly used with H1"I'I.C ~ l a t e sand has the added advantaee that develo~. ment can take place simultaneously from 60th ends of t i e plate. This effectively doubles the number of analyses per plate. Plates may also be developed in a circular or radial fashion. Blome (16) pointed out some of the advantages of circular TLC. The solvent flow in linear TLC decreases with the square of the distance the solvent front has traveled. In circular development the solvent is pulled out into an increasina place area, and therefore the fiow rate remains relativel; constant. Compounds with low Rrvaluesare hetter separated by radid development. In other words, theseparation number for a group of compounds is higher u,ith radial TLC than with linear TLC. Camag (18)markets a radial development chamber especially designed for high-performance work called a "UChamber." Here the flow of mobile phase is electronically controlled with a steppine motor to insure constant solvent delivery. I t is possihie wch the U-Chamber to obtain very precise RPvalues because of the hiehlv reoroducihle dosaee position, t h e isolation of the m o h " p h a s e from the s&rounding atmosphere, and control of the eas ohase in contact LO miniwith the-plate s&face. Thus, rhe ~ - C h n m b eisahle ; mize most of the difficulties associated with conventional'l'LC systems which cause non-reproducible results. Photodyne Corporation (19) offers a high performance radial TLC system with some of the features of the more expensive Camag system. However, when using mohile phases with high vapor pressures, the solvent flow can he interrupted. Also it is not possible t o control the gas phase of the system. If conditioning of the plate is needed, this can he done prior to development. A similar system offered by Analtech (20) uses wicks to transfer the solvent. This feature ensures continuous solvent flow. Radial chromatoma~hvmav also he run in an anticircular mode. Here, the dir&conof ~ & v e nflow t is from the cireumference to the center of a circle. During development, the area of the plate that the solvent moves into hecomes gradually smaller. This means that when compared to circular TLC, where the solvent spreads out into a larger area, anticircular TLC has a much faster solvent flow rate. Another advantage of anticircular TLC is the lower minimum detectable level of compounds with high Rr values. ~
~
Although the multiple development technique is not new, it has been used mast effectively with HPTLC plates. Multiple development is accomplished with one or more developing solvents. After each development the plate is removed from the chamber, dried, and then developed again. Every subsequent development sweeps the trailing edge of the spot closer to the leading edge. This effectively forces each spot into narrow bands, thus increasing the efficiency of the system. The ability to choose different mohile phases, coupled with improved efficiency gives the chromatographer a very powerful t w l for separating complex mixtures. With conventional plates, m u l t i ~ l edevelooment can he verv time consumine. ~owe;er, because of the shorter deveiopment distances needed with the hiah performance dates, this is no loneer a prohlem. For example,~eeet al. (21(were able to separateand quantitate five antiarrhythmia drugs in serum using two different mohile phases. The first development took only 7 min, and the second 13 min. Multiple development of a plate mav be done automaticallv by the &chniqui of programmed ~ u l t i p l e~ e v e l o p m e n i (PMD). In practice PMD is accomplished by a series of short developments, each development slightly longer than the previous one. During the advance of the solvent up the plate, solute concentration takes place as the molecules in the lower portion of each spot are carried uo the plate. As the solvent is removed from-the plate (by heat o i g a s flow), a second concentration step takes place as the molecules of the upper portion of each spot are the first to become "dry and fixed" while the lower molecules are still in contact with the mohile phase. A commercially available PMD apparatus is manufactured by Regis Chemical Company (22). Continuous development (23) is another form of TLC ideally suited for high efficiency plates. Emphasis is placed on resolving power rather than on speed of analysis. A portion of the plate is extended out of the development chamber, allowing evaporation a t the solvent front and thus causing continuous movement of the mohile phase. Solvent strength for continuous development systems is weaker than comparahle conventional TLC systems. But with weaker solvents the selectivity often improves. The price paid for hetter selectivity is the slow rate a t which the sample travels up the plate. T o a considerable extent this problem can he overcome by allowing the mohile phase to travel only short distances. This takes full advantage of the fast flow rate near the origin. Modern high efficiency plates are ideally suited for this situation. A good example of the resolving power and flexibility of continuous development chromatography is the simultaneous analysis of 13 mycotoxins of widely different polarity (21). Complete separation and analysis took only 1 hr. After 5 min the first continuous development separated three of these compounds, leaving all others a t the origin. One was quantitated hy absorbance, while the other two were determined by fluorescence. After two additional continuous developments with the original solvent, the fourth component separated and was subsequently determined in the fluorescence mode. A fourth continuous development with the same solvent system separated three more mycotoxins, each determined by ahsorhance a t different wavelengths. Finally, three continuous developments with a more polar solvent svstem eave baseline resolution of the remaining six aflatoxins, all d2ermined by fluorescence. The limit of detection was in the low nanoaram range for the absorbance mode, while analyses by f l ~ o r ~ e n c e yielded limits in the low picogram range. Analysis of twelve individual spots using the data pair technique (24) gave relative standard deviations between 0.7 and 2.2%. Even if these same analyses could be accomplished hy gas or high pressure liquid chromatography, the lack of flexibility of these techniques makes it an open question as to whether all 13 compounds could he assayed in 1 hr. Overpressured thin layer chromatography (OPTLC) is a Volume 61 Number 11 November 1984
1017
relatively new technique that holds a lot of promise (2.5). OPTLC addresses one of the major shortcomings of all TLC systems, i.e., the development solvent is pulled through the place matrix by capillary forces, thus giving the chromatographer almost no contn~lover the flow rate. Further hecause this rate decreases with the squareof the distance thesolvent travels, the most efficient HPTLC separations have short development distances. Overpressured TLC combines the techniques of thin layer chromatography and high pressure liquid chromatography. Using a pressurized ultramicro chamber (PUM chamber), the sorbent layer is completely covered by a plastic membrane held closely to the surface by a layer of pressurized gas. Similar to HPLC systems, mobile nhase can then be introduced under nressure. As exoected because the solvent flow rate is faster than with nonpiessurized TLC systems, development time and spot diffusion are both much less. This is especially true of species with high Rr values. where develonment times are 5-20 times faster. The linear relationship of development distance and time makes 0PTI.C ideal for the transfer of conditions from TLC to HPLC. However, in order to take full advantage of the better resolution offered hv OPTLC, the use of hiah performance . . plates is essential. Ouantitatlve Evaluation of TLC Plates Direct quantitative evaluation of TLC plates is accomplished by the interaction of UV or visible radiation with sample within the plate matrix. The nature of the plate coating causes the radiation to be reflected, much the same as light passing through an opaque o b j e c t . ~ h i type s of reflected radiation is called diffuse since it is scattered in all directions. (Specular reflectance occurs in a single plane, like light being reflected by a mirror.) In conventional L'V-visible absorption or fluorescent spectroscopy the level of scatter is insignificant and the pathlength that the radition travels remains constant. However, when a light absorbing species is impregnated within a diffusely scattering material, such as Silica Gel. the radiation has a wide ranpe of nathlensths. Thus. the ~ e e r - ~ a m b eequation rt does not ideqiately apply to this situation. A more accurate descrintion is eiven bv Kubelka's equation for diffusely reflected iight w&h has both light ahsorotion and scatterine coefficients and is the basis for in situ &antitation by spekrodensitometry. This important aspect of HPTLC has been described elsewhere (26,27). A major shortcoming of densitometry is the very short linear range. The same amount of material distributed differently within the layer will cause different densitometric readings. HPTLC does much to overcome irregular sample distribution problems by employing superior spotting techniques, better control of plate development, and high efficiency plates. A New Look at TLC Several interesting..papers have recentlv appeared (28-30) . which considef the factors important for the-optimization of TLC separation. From a theoretical treatment of plate height, a c o m p d s o n was made of the average plate height versus development length for several different particle sizes of coating materials. Plates with oarticles less than 10 um are significantly influenced by the diffusion of the solute; and as a conseauence, their efficiencv is decreased with increasing development distnnce. In contrast plates with coarser panicl& are not as easilv influenced bv the diffusion of the solute and resolution is -improved with increase in development length. Experimental verification of these theories has been found in several TLC systems (6.30). Easy separatibns, incliding dyestuffs, pyrene-chrysene, chloroanalines, and tocopherols were best separated on small particle HI'1'I.C plates. This behavior is explained hy the fact that with dmaller particles, the relociry of the mobile phase is decreased to such an extent that molecular diffusion of the solute controls spot broadening. With eady separations (low diffusion coefficients) it is advantageous to separate com1018
Journal of Chemical Education
nounds usine small narticles for short distances. More difficult separations 6 a r g &ffusiou coefficients) are better performed on conventional TLC plates with large particles. Examples include bis(chlorophenyl)triazeue, aflatoxins, 2,4- and 2,5dimethylphenyls, and citropten-bergapten. These results raise the question of the need to distinguish formally between "conventional" and "high nerformance" TLC. In summary, the direitibn of TLC is toward more highly efficient chromato~raphicsvstems; a much greater use of instrumentation; andttaiing f k l advaLtatge O ~ T L Cflexibility. ,~ Efficiency is being attained by the use of better plates, improved spotting techniques, and more highly controlled developing chambers. Instrumentation is occurring with all aspects of TLC: spotting and streaking devices-new linear, circular, anticircular, and overpressured developing chambers; and automatic track-changing spectrodensitometers. Perhaps most impressive are the many ways in which chromatoeranhers have been makine use of TLC's flexibilitv. While muih effort has been given recently to the optimization of HPLC mobile phases (31,32), the actual choice of solvents is severely limited by their physical and chemical properties. For example, the UV absorbing ability of benzene makes it a poor LC solvent, while the vapor pressure of ethyl ether makes Bases. such as i t difficult to work with under high - oressure. . sodium hydroxide, are commonly used in thin layer chromatography hut rarely used in high pressure liquid chromatography. Post-column derivatization techniques are still not routinely done with HPLC, whereas TLC makes use of hundreds of post-development reactions, many of which are specific and give additional information about the species being chromatographed. Finally, continuous and multiple development techniques offer the chromatographer unique control over fairlv comnlicated senarations. As was indicated above, the flexibiiity of these approaches is limited only by the creativity of the chromatographer. Llterature Cited (1) Zlatkis, A. and Kaiaer, R. E. (Editors). HPTLCHigh Performam Thin Layer Chmmavgraphy, elrr~er.Amiu,dam.olfnrd.Ncv York,and lnrriwvof Chm. msrmaphy. Baa Dwkcim. 1977. InlrldurLion 121 JuD.IIP.T.H . ( . R ~ ' ~ ' r i ~ Riwd w e i n ,\naln~:alrhrm~a~n.6.325~1977. . ' (3) ~ e k o mD.. C. and Davis. C. ~ . . A n o lc~&L, . 53. 2 5 2 (1981i. ~ (4) Berfsh, W.. Hars. S., Kaiser, R.E.. andZlatkia,A. (Edi~ora),inshumantalHfTLC. Huthig. Heidelberg. Band, and New York, 1980. (5) Hslpaap,H. andRipphahn,J. in (Editom ZlatLia,AandKeiaer.RE.),-H'gb Performanee Thin Layer Chmmatography.Ekvi~,,Amsterdam, w a r d , New Vork, and Institute of Chromatography.Bad Durkeim, 1977, p. 95. (8) Brinkmen, Th. V. A,, DeVries, G.. and Cupem. R., J. Chmm., 198,421 (1980). (7) Kaiser. R. E. end Rider, R.J. Chromotogr.. 142,411 (1977). (8) Brinkman,Th. V. A., DeVries, 0.. and Cuperus, R., J Chmmotogr.,192,311 (1980). (9) Brinkman,U.A.T. and DcVrier, 0.. J. HighRooolut. Chhhmfogr. Chmm~ogr.CCC., z
",C
"*9
,so*.,,
(10) Kaiser,R.E.,i" (Editors:Zlatkis,A.andKaiaer,R. E.),HPTLCHighPerformanee Thin Laye Chmmatography,Elae"ier. Amatordam. Oxford. Nnr York and Institute of Chromatography,Bad Durkheim, I977.p. 85. (11) Berueh, W., Ham, S., Kaiser. R.E., and Zlatkia, A. (Edifor4,lashumentalHPTCC. Huthia. Heidolbere. Bssel. and New York. 1 9 S 0 , ~81. . (12) Clarke ~nalyticsl~$tems,SantaClara. CA. (13) Ahhatt, D. C. and Thomaor, J., Chsm. Ind., 310 (1965)
(29) (30) (31) (32)
W i s Chemical Company. Morton Gmve, Illinois. Perry, J. A., J. Chromntogr, 165,117 (1979). Berthke, H., Sanli, W., Fmi, R. W., J. Chromotogr. Sci., 12,392 (1974). Tyihak,E., Mincsovics,E.,Kalasz,H.,Nagy, J.. J Chmmotagr.,211,95 (1981). Pollak, V., Adwrncer in Chmmotogrophy,17.1(1979). Coddens, M.E., Butler. H. T.,Scheufte, S. A., Poo1e.C.F. Liquid Chmmtogr HPLC Magazine, 1,282 (1983). Guioehon. G.. Siouffi, A,, Engelhardt,H. and Halam. J.. J. Chromtam S c i , 16.152 (1978). Guiochon, G., and Sioufli, A,. J. Chromotogr. Sci.. 16,170 (1978). Sioufli, A., Brissolle, F., and Guioehon, G., J. Chmmatogr.,209,129 (1981). Glaich, J. L.,Kirkland, J. J., and Snyder, L. R., J Chromomr., 238,269 (1982). Kirkland, J. J.,and Glajeh, J. L., J'Chromologr, 255.27 (1983).
