930
Anal. Chem. 1982, 5 4 , 930-934
Derivatization of Kel-F Particles with n -Butyllithium for Liquid Chromatography Jeffrey A. Huth and Neil D. Danlelson" Department of Chemistty, Miami Universiiy, Oxford, Ohio 45056
n -Butylllthlum will react easily with Kel-F (poly(chlorotrlfluoroethylene) or PCTFE) to form n -butyl-Kel-F and LICI. Kel-F 6300, a copolymer of PCTFE and 3 % vinylidene fluoride, was 85% derlvatlzed causing the surface area to lncrease to 14 m2/g. Although n-butyl-Kel-F 6300 showed promise as a column packing material for llquld chromatograpy (LC), column lifetime due to Insufficient rigidity of the polymer was short. Kel-F 6061 (PCTFE) upon reaction with n-butyllithlum was only derlvatlzed to an extent of 25 % with a surface area Increase from 1.6 to 3.6 m*/g. However, a variety of substituted aromatic compounds could be separated on a 25 cm X 4.6 mm 1.d. column packed with n-butyl-Kel-F 6061 and the column lifetime was excellent. Both n-butylKel-F polymers were markedly better LC column packlngs than underlvatlzed Kel-F for small molecules.
The majority of the packing materials presently used for high-performance liquid chromatography (HPLC) consist of porous silica particles to which an organic modifier is bound via a siloxane bond. Although a variety of groups have been bound to the silica support, aliphatic substituenb such as the octadecyl derivative have been used most often to synthesize reversed-phase packings. These materials are highly efficient, reproducible, and versatile with respect to many separation problems. However, the presence of residual silanols, particularly in the shorter chain packings, can alter the retention of solutes and cause tailing of peaks (1). Another limitation of silica-based HPLC packings is the mobile phase pH constrajnta between 2 and 7 due to hydrolysis of the siloxane bond a t low pH and dissolution of the silica at high pH. In addition, Wehrli et al. (2) have shown that ion-pairing reagents such as quaternary amines can shorten the lifetime of reversed-phase columns. Therefore, a support that is rigid, stable to wide variations of ionic strength and pH, and completely hydrophobic in nature would be ideal for reversed-phase packings. The fluorocarbon polymer Kel-F has found some application particularly in biochemistry as a column packing material for liquid chromatography. Pearson et al. (3) coated Kel-F with Adogen 464, a trialkylmethylammonium chloride, to facilitate the separation of transfer RNA molecules. Since then, many others have used similar columns for the separation of transfer RNA (4, 5 ) , nucleosides, nucleotides, oligonucleotides (6-8), and DNA fragments (9-12). Despite success in the separations attempted, column lifetimes were limited by the "bleeding" of the coating. To avoid this problem, Usher (13)used uncoated Kel-F as a support for the separation of DNA fragments and ribooligonucleotides. However, the use of Kel-F for separating mildly hydrophobic small molecules was not recommended. In order to prepare a carbon HPLC packing, Zwier and Burke (14) used a reduction procedure developed by Jansta et al. (15,16)for Teflon to reduce Kel-F with lithium amalgam. Functionalization of the carbon via the surface hydroxyls was then carried out using a Grignard reaction after prior chlorination of the surface. Considerable success was achieved 0003-2700/82/0354-0930$0 1.25/0
in demonstrating the improved adsorptive properties of the carbon support over that of silica. In an attempt to increase the surface area and the versatility of Kel-F as a column packing material, we have discovered ( I 7) that organolithium and organomagnesium reagents will derivatize the polymer with a variety of functional groups. The intent of this paper is to describe the chromatographic properties of one of these derivatives, n-butyl-Kel-F. The reaction of n-butyllithium with two types of Kel-F was studied, and the chromatographic capabilities of the derivatized Kel-F polymers were compared. In addition, the application of one of these packings for the separation of substituted aromatic compounds was demonstrated.
EXPERIMENTAL SECTION Materials. Kel-F 6300 (100-200 mesh), a copolymer of poly(chlorotrifluoroethy1ene) containing 3% vinylidine fluoride, was obtained as a gift from the 3M Co. (Minneapolis, MN). Initially, the polymer was ground with a standard ball mill and sieved into two fractions, less than and greater than 44 pm. For chromatographic use, the smaller material was ground further with a Pilamec high-energyvibration ball mill until the particles were less than 37 pm. Prior to the reaction, particles of about 20-37 pm were obtained from the bulk of the material by separation on an elutriation column (18) using a methanol solvent. Kel-F 6061 (80-100 mesh), 100% poly(chlorotrifluoroethylene), was obtained from A. M. Plastics (Rockaway, NJ). This material was jet ground (Alnort, Inc., Camden, NJ) so that better than 93% of the particles were less than 325 mesh. Sieving indicated most of the material was between 20 and 37 pm. Gold-label tetrahydrofuran (THF) and n-butyllithium (1.6 M in hexane) were obtained from Aldrich Chemical Co. (Milwaukee, WI). Methanol, HPLC grade, was purchased from Burdick and Jackson (Muskegon, MI). Tetrabutylammonium hydrogen sulfate (TBAHS) was obtained from Sigma (St. Louis, MO). Solutes used for the separations were obtained from various suppliers and were reagent grade or better. Procedure. A 7-g quantity of Kel-F (6300 or 6061) in 125 mL of THF was purged, using N2or He, in a three-neck 500-mL round-bottom flask equipped with a thermometer, condenser,and a rubber septum over a period of 30 min at 35 "C. Depending on the stoichiometry of the reaction, either 120 mL (3:l stoichiometry) or 40 mL (1:l stoichiometry) of n-butyllithium was injected via a syringe through the septum. Immediately the reaction temperature rose to 60 "C, at which it was held for 30 min. The reaction was terminated by the careful1 addition of acetone or methanol to neutralize any unreacted n-butyllithium. The n-butyl-Kel-F particles were then filtered and washed with 100-200 mL portions of hexane, acetone, methanol, water, and finally methanol. The particles appeared unchanged except for a color change from white to gold-brown. In scale-up studies of this reaction, a ratio of at least 15 mL of THF per gram of Kel-F should be employed. Use of solvent in amounts less than this would lead to Kel-F aggregates which could not be easily broken down to the original particle size. Elemental (Atlantic Microlab, Atlanta, GA) and surface area analyses (Particle Data Laboratories, LTD., Elmhurst, IL)were carried out in addition to polymer characterization by IR spectrometry. Chromatography. Columns 20 cm X 4.6 mm i.d. were packed at approximately 4000 psi using a Micromeritics Model 705 column packer (Micromeritics,Norcross, GA) and a LDC Model 396-57 Simplex high-pressurepump (MiltonRoy Co., Riviera Beach, FL). All separations were carried out using an Altex Model llOA 0 I982 American Chemical Soclety
ANALYTICAL CHEMISTRY, VOL. 54, NO. 6,MAY 1982
high-pressure pump equiplped with an Altex Model 153 UV detector, constant wavelength (254 nm) (Altex, Berkeley, CA). The injector useld was a Rheodyne Model 7010 equipped with a 7011 loop filler port (Rheodyne Inc., Berkeley, CA). Peaks were recorded by an Omniscribe Model B-5000 recorder (Houston Instrument, Austin, TX).
RESULTS AND DISCUSSION Reactivity of Kel-F Polymers. Infrared spectrometry proved to be very useful for the qualitative characterization of the reacted Kel-F. In the region of 3000-2800 cm-l, the Kel-F IR spectrum had no absorption bands due to the absence of C--Hbonds in the structure. However, the spectrum of Kel-F derivatized witlh n-butyllithium showed bands at 2956, 2926, 2868, and 2866 cm-l due to the presence of the n-butyl groups on the polymer. The reduction of the C-C1 band a t 9b0 cm-' was also indicative of the extent of the derivatization, particularly if essentially all of the chlorine had reacted. The increase in carbon and hydrogen and the loss in chlorine wlere also clearly indicated by elemental analysis. Unreacted Kel-F 6300 was 21.3% C, 29.4% C1, 0% H, and 49.3% F (by difference) in composition. After duplicate reactions of 44 pm Kel-F 6300 particles and elemental analysis showed 27.2 f 0.1% C, 23.0 f 0.5% C1, 1.3 f 0.2% H, and 48.0 f 1% F (by difference) to be present. In both cases, the percent range for each element indicated the derivatization reaction was reproducible. As expected, the smaller particles were derivatized to a greater extent than the larger ones because of the higher surface area. In addition, no significmt change in percent F betwieen Kel-F and derivatized Kel-F can be concluded. Previously, it ha8 been reported tlhat organic fluorides are rarely reactive toward lithium metal or organolithium reagents (19). The chemical reaction occurring between Kel-F and n-butyllithium was believed to be the following: F
F
I-
F
On the basis of theoretical calculations of the elemental composition of Kel-F with varying degrees of chlorine substitution, the extent of derivatization of the n-butyl-Kel-F could be estimated. If a Kel-F chain of four monomeric units is assumed, then a replacement of one chlorine on one monomer by a n-butyl group would constitute 25% derivatization. By use of the molecular weight of the 25% derivatized polymer, the weight percentage of each element can be determined. Weight percentages of each element corresponding to 50%, 75%, and 100% derivatization were also calculated. Figure 1 shows the linear approximations of the various elemental weight percentages as a function of percentage derivatization. Ideally, thle experimental points a t a given percentage of derivatization should fall on a horizontal line. This figure also illustrates the differences in derivatizations obtained by varying the stoichiometry. On the basis of carbon percentage, a 3:l stoichiometry of n-butyllithium to Kd-F 6300 monomer yielded approximately 85% derivatization, whereas a 1:l stoichiometry produced approximately 40% derivatization. In addition, approximately 25 % derivatization was found for a 3:l stoichiiometry of n-butyllithium to Kel-F 6061 monomer. Quantitative differenceti in alkyl substitution depending on the type of Kel-F and reaction conditions are expressed in Table I as millimoles of C4/gram of packing. The similar increase in carbon and hydrogen corresponded to the fact that
931
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Flgure 1. Theoretical linear approximation plots of the weight percent of hydrogen (---), chlorine (-), and carbon (.e.) of n-butyCKeCF 6300 and 6061 as a function of percent derivatization. For hydrogen, weight percent Is multiplled by 10. The experimental weight percentages frorn elemental analysis are for 3:l n-butyCKe1-F 6300 (0),1:l n-butyCKeCF 6300 (X), and 3:l n-butyl-Kel-F 6061 (A).
Table I. Addition of n-Butyl Groups to Kel-F Based on Elemental Analyses of C, H, and C1 reaction Kel-F stoichi(20-37 Mm) ometry carbon
hydrogen
chlorine
1:1 2.6 6300 2.8 3.4 3:1 5.7 6.5 6300 8.3 3:l 1.7 6061 1.9 1.9 a mmol of C4/g of packing based on carbon analysis (% C Kel-F product - % C Kel-F reactant)/(100 x 4 x 12). b mmol of C4/g of packing based on hydrogen analysis (% H Kel-F product - % H Kel-F reactant)/(100 x 9 x 1). C mmol of Cl/g of packing reacted ( % C1 Kel-F reactant -. % C1 Kel-F product)/(100 x 1 x 35.5). The change in molecular weight of Kel-F reactant and product was considered negligible.
primarily n-butyl groups were derivatizing the polymer. The decrease in chlorine ahso correlates with the reaction previously proposed. For the 6061 polymer, very good correlation was obtained between the loss of chlorine and the gain of carboin and hydrogen via n-butyl groups. However, for the 6300 polymer, the loss in chlorine was greater than the corresponding gain in butyl groups. Since the infrared spectrurn did not indicate a presence of either OH or C=O moieties, possibly reduction occurred at some reaction sites instead olf substitution by butyl groups (17). Because the crystallinit,y is less for the 6300 polymer due to fact it is a copolymer, better interaction of the solvent with this polymer was expected (20). In addition to derivatizing Kel-F, the reaction also roughened the KeEF surface. A similar result (21) has been achieved for Teflon when treated with sodium in the presence of THF and naphthalene. Electron microscopy of Kel-F 6300 itself showed a somewhat smooth, nonporous surface, whereas the 3:l derivatized particles of 6300 Kel-F were pitted and porous in appearance. The decrease in density of Kel-F 6061 upon reaction from 1.9 to 1.5 also indicated an increase in porosity. Surface area measurements for unreacted Kel-F 6061, 3:l
032
ANALYTICAL CHEMISTRY, VOL. 54,NO. 6, MAY 1982
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Flgure 2. Separation of a mixture containing (1) p-nitroaniline (1.40 mg/mL), (2) toluene (12.5 mg/mL), and (3) phenanthrene (0.36 mg/mL) on 20 cm X 4.6 mm i.d. columns packed with n-butyl-Kel-F 6300 obtained from stoichiometric reactions of 0.3:l (A), 1:l (B), and 3:l (C) n-butyliithium-Kel-F ratios: mobile phase 60% methanol-20% water; flow rate 1.0 mL/min; injection volume 20 pL; au = absorbance units.
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Time (min 1 Flgure 3. (A) Same as Figure 2 on underlvatized KeCF 6300. (E)Same as (A) but with mobile phase of 40% methanol-60% water.
n-butyl-Kel-F6061, and 3:l n-butyl-Kel-F6300 were 1.6 m2/g, 3.6 m2/g, and 14.4 m2/g, respectively. Because of the high degree of n-butyl substitution (Table I) and the low surface area values, not all the n-butyl groups are likely to be present on the surface. For example, since almost complete loss of chlorine was possible upon reaction of Kel-F 6300 with excess n-butyllithium (Figure l ) , apparently the reaction solvent THF can swell the polymer allowing n-butyl substitution to occur inside the Kel-F particles. Chromatographic Properties of II -Butyl Kel-F. The chromatographicutility of derivatized Kel-F 6300 was studied first. Figure 2 shows the separation of p-nitroaniline, toluene, and phenanthrene on columns packed with n-butyl-Kel-F6300 of different reaction stoichiometries. As can be seen, the 3:l derivatized packing generated the best separation. However, this packing, as well as the 1:l packing, were not stable. After 2 to 3 days of use, the pressure increased from 2000 to 5000 psi and resulted in the plugging of the column. In an attempt to maintain rigidity, a lower stoichiometry of 0.31 was used. Although this packing could withstand several weeks of use, the separation capabilities of this material were marginal (Figure 2A). To illustrate that the reaction does indeed influence the separating properties of Kel-F, we chromatographed the same sample mixture on Kel-F itself. With a mobile phase of 80% methanol-20% water, only one peak (Figure 3A) was obtained for the same three solutes. After the water content was increased to 60%, a separation of the three solutes was obtained (Figure 3B). However, the separation of the p-nitroaniline and toluene was incomplete, and
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,
,
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Separation of solutes in Flgure 2 on the 3:l n-butyl-Kei-F 6061 column: mobile phase 75% methanol-26% 0.01 M TBAHS; flow rate 0.7 mL/min. I is an impurlty of nitrobenzene. Flgure 4.
the phenanthrene peak was extremely broad. The chromatographic properties of n-butyl-Kel-F6061 were found to be more favorable than those of the 6300 polymer. Figure 4 shows the separation of p-nitroaniline, toluene, and phenanthrene. A small impurity of nitrobenzene was also present and separated. As can be seen, the separation was much better than that obtained with the 6300 polymer. More importantly, the lifetime of this column was much improved. Use of this column at flow rates of 0.7-1.0 mL/min produced pressures of 800-1300 psi but no increase in pressure or plugging occurred after several weeks of use. The improved rigidity of the n-butyl-Kel-F 6061 was most likely due to the nature of the polymer. The higher the degree of crystallinity for a polymer, the greater hardness the polymer will exhibit (20). This is evident in the hardness ( R values) reported for the two Kel-F polymers. The crystalline form of Kel-F 6300 has an R value of 92, whereas the R value for crystalline Kel-F 6061 is about 110 (22).
ANALYTICAL CHEMISTRY, VOL. 54, NO. 6, MAY 1982
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Flgure 5. Separation of a mixture containing 10 mg/mL of (1) benzene, (2) cumenst, and (3) tetramethylbenzene on 3:1 n-butyl-Kel-F 6061 column: mobile phase 85 % methanol- 15 % 0.01 M TBAHS; flow rate 1.O mL/min. Two leading peaks (I) are solvent impurities. Injection volume = 20 pL.
Time (min )
Flgure 7. (A) Same as Figure 4 on underivatized KeCF 6061. (B)Same as (A) but with mobile phase of 40% methanol-60% water, 0.025 M TBAHS.
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20 T i m e (min ) Flgure 6. Separation of a mixture Containing ( I ) aniline (0.21 mg/mL), (2) nitrobenzene (0.25 mgJmL), (3) toluene (10.0 mg/mL), and (4) o-xylene (10.0 mg/mL) on 3 1 n-butyl-Kel-F 6061 column. Conditions are the sarne as Flgure 4.
Figurer) 5 and 6 show the separations of various other substituted aromatic hydrocarbons on the n-butyl-Kel-F 6061 column. Figure 5 illustrates the separation of benzene, cumene, and tretramethylberuene. In addition, two impurities were eluted early but were separated from the compounds of interest. In Figure 6,aniline, nitrobenzene, toluene, and o-xylene were separated in less than 20 min. Again to demonstrate the effect of the reaction of n-butyllithium on Kel-F, underivatized Kel-F 6061 was packed in a column. With a mobile phase of 80% methanol-20% 0.01 M TBAHS, only one peak was obtained for p-nitroaniline, toluene, and phenanthrene (Figure 7A). Increasing the water content to 60%) but maintaining a constant TBAHS concentration, these three solutes could be separated (Figure 7H),but unsatisfactorily. The peaks exhibited irregular shapes and tailing. Tetrabutylammoniurn hydrogen sulFate (TBAHS) was
added to the mobile phase for several of the previous separations on the n-butyl-KeEF 6061 column. Due to the extreme hydrophobicity of Kel-F, water itself will not wet the surface, although methanol will. However, the addition of a surfactant appeared to increase further the interaction of the mobile phase with the packing. In addition, in the absence of TBAHS, the pressure would increase slightly after several hourr of use, however, not nearly to the same extent as thlat for the derivatized 6300 packing. At a flow of 0.7 mL/min, the pressure increased from 800 psi to about 1200 psi in 6 h with a methanol-water mobile phase but returned to 800 psi with the addition of TBAHS. Despite the excellent lifetime of the n-butyl-Kel-F 6061 colurnns, the use of THF and CHC13 in the mobile phase should be avoided. TJse of these solvents resulted in a rapidly increasing pressure and subsequent plugging of the column. The high pressure could be alleviated by passing methanol or acetonitrile through the column. Burke (14) observed1 a similar result for THF with his carbon packings derived from Kel-F. This appears to be the result of a solvation effect of these solvents with n-butyl-Kel-F. In addition, it has been reported (23) that a few halogenated solvents can swell or dissolve Kel-F at elevated temperatures.
CONCLUSIONS The results indicated that n-butyl-Kel-F was definitely a more effective HPLC packing than underivatized Kel-F. It is likely the retention mechanism for the separation of molecules on Kel-F itself was primarily adsorption due to iits nonporous nature and lack of a bonded alkyl phase. The retention mechanism for n-butyl-Kel-F could be either adsorptive or partitioning in nature. An exclusion mechanism is unlikely since solutes similar in size could be separated. Because the butyl chains are quite short and not all were on the surface, it is unlikely a dense enough liquid layer was present for partitioning. The increase in the separation capability of n-butyl-Kel-F 6300 with extent of derivatization was probably the result of an increase in surface area on whilch adsorption of the solute may occur. Although the 3:l n-butyl-Kel-F 6300 was higher in surface area than the 3:l n-butyl-Kel-F 6061,compression of the former packing during chromatographic use probably caused poor interaction of the
934
Anal. Chem. 1982, 5 4 , 934-938
solute with the stationary phase resulting in broader peaks as compared to the latter packing. Although the chromatographic performance of n-butylKel-F is presently inferior to present silica packings, some similarities such as surface area do exist between n-butyl-Kel-F and pellicular packings (24). Two major limitations in the column performance of n-butyl-Kel-F were undoubtedly the nonuniform particle distribution and the packing technique. Lately, use of a high-pressure pump has indicated derivatized 6061 Kel-F can be packed at 10000 psi. However, recent examination of derivatizedKel-F particles by light microscopy showed more fines than expected. Careful fractionation both before and after the derivatization reaction will apparently be required to achieve a narrower particle size distribution. It is expected that with the preparation of better packing materials n-butyl-Kel-F would be ideal for applications which now utilize pellicular packings such as purity verification, routine and/or rapid analyses, and initial screening of unknowns. In addition, chemically modifed Kel-F is relatively inexpensive and, therefore, would be useful in guard columns or for screening samples suspected of containing components which could irreversibly damage microparticle silica columns.
ACKNOWLEDGMENT The authors thank D. D. Anderson of the 3M Co. for the sample of 6300 Kel-F and W. Kuhn, Department of Material Science, University of Cincinnati, Cincinnati, OH, for the use of the Pilamec mill. LITERATURE CITED (1) Klrkland, J. J.; Snyder, L. R. "Introduction to Modern Llquld Chromatography", 2nd ed.; Wlley-Intersclence: New York, 1979; Chapters 5, 7. (2) Wehrll, A.; Hildenbrand, J. C.; Keller, H. P.; Stamfll. R.; Frel, R. W. J . Chromatogr. 1978, 149, 199-210. (3) Pearson, R. L.; Weiss, J. F.; Kelmers. A. D. Blochim. Biophys Acta 1971, 228, 770-774.
.
(4) Kelmers, A. D.; Heatherly, D. E. Anal. Biochem. 1971, 44, 486-495. (5) Roe, B.; Marcu, K.; Dudock, B. Biochim. Biophys. Acta 1973, 319, 25-36. (6) Slnghal, R. P. Biochim. Blophys Acta 1973, 3 79, 11-24. (7) Egan, B. 2. Biochim. Blophys Acta 1973, 299, 245-252. (8) Shum, B. W.; Crothers, D. M. Nucleic Acids Res. 1978, 5 , 2297-231 1. (9) Hardies, S. C.; Wells, R. D. R o c . Nafl. Acad. Sci. U.S.A. 1978, 73, 3117-3121. (10) Landy, A.; Foeller, C.; Reszelbach, R.; Dudock, B. Nucleic Acids Res. 1978, 3, 2575-2592. (11) Eshaghpour, H.; Crothers, D. M. Nucleic Acids Res. 1978, 5 , 13-21. (12) Blna, M.; Radonovich, M. F.; Roe, B. A. Anal. Blochem. 1981, 714, 105-1 11. (13) Usher, D. A. Nucleic Aclds Res. 1979, 6 , 2289-2306. (14) Zwier, T. A.; Burke, M. F. Anal. Chem. 1981, 53, 812-8113, (15) Plzak, 2.; Dousek, F. P.; Jansta, J. J . Chromatogr. 1978, 747, 137- 142. (16) Smolkova, E.; Zima, J.; Dousek, F. P.; Jansta, J.; Plzak, 2 . J. Chromatogr. 1980, 191, 61-69. (17) Danielson, N. D.; Taylor, R. T.; Huth, J. A,; Slerglej, R. W.; Galloway, J. G.; Paperman, J. B., Miami Universify, Oxford, OH,Sept 1981, unpublished work. (18) Scott, C. D. Anal. Biochem. 1988, 24, 292-298. (19) Wakeflekl, B. J. "The Chemistry of Organollthium Compounds"; Pergamon Press: Oxford, 1974; p 22. (20) Billmeyer, F. W., Jr. "Textbook of Polymer Science", 2nd ed.; WlleyInterscience: New York, 1971; Chapter 7. (21) Cagle, C. V. "Handbook of Adhesive Binding"; McGraw-Hill: New York, 1973; Chapter 19. (22) Deehan, D., 3M Co. Nov 1981, personal communication. (23) Saunders, K. J. "Organic Polymer Chemistry"; Chapman and Hall: London, 1973; Chapter 7. (24) Klrkland, J. J. J. Chromatogr. Sci. 1971, 9 , 206-214.
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RECEIVED for review November 30,1981. Accepted February 4,1982. This work was supported in part by grants from the Faculty Research Committee of Miami University, the Research Corporation, and the donors of the Petroleum Research Fund, administered by the American Chemical Society. J. A. Huth gratefully acknowledges support provided by a Dissertation Fellowship from Miami University. This work was partially presented at the Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, March 1981.
Steroid Structure and Solvent Composition in Thin-Layer Chromatography Slobodan M. Petrovlb," Ljlljana A. Kolarov, and Eva S. Traljld Institute of Mlcroblologlcal Processes and Applled Chemlstry, Faculty of Technology, University of Novi Sad, V. VlahoviEa 2, 2 1000 Novi Sad, Yugoslavia
Julljana A. Petrovlb Instltute of Chemistry, Unlverslty of Novl Sad, V. VlahoviEa 2, 21000 Novi Sad, Yugoslavia
To study the reiatlons between the chemical structure of solutes and their retention behavlor In thin-layer chromatography, we determlned the retention behavior of 15 mono-, di-, tri-, and tetrasubstltuted sterold derlvatives as a function of the composition of eight binary solvent systems. The slopes and Intercepts of the linear relations between the retention constant (RM) and the logarithm of the volume fraction of the polar solvent have been calculated and dlscussed In relation to the solute and solvent characteristics. The RM and seiectivlty parameter (ARM) of steroids largely depend on the retentlon behavior of the hydroxyl group.
The effects of solvent composition on retention behavior have great theoretical and practical significance since the 0003-2700/82/0354-0934$01.25/0
mobile phases employed in both partition and adsorption chromatography are usually mixed solvents. They have therefore been of interest to chromatographers since the beginnings of chromatographic techniques. Several treatments of this subject have been applied to liquid-solid chromatography (1-7). Some of them demonstrated a linear relationship between the retention and the binary solvent properties (1-6). The treatments of Soczewinski and co-workers (2-4) are based on the concept of a competition between solute and solvent molecules in the liquid phase for a place on the adsorbent surface. For adsorption from solution as a result of the competition between the solute and an electron donor solvent (S) for the active sites on the adsorbent surface, they derived the equation.
RM
= c - n log X s
0 1982 Amerlcan Chemical Soclety
(1)
