Anal. Chem. 1995,67, 3284-3292
Influence of Stationary Phase Chemistry on Shape Recognition in Liquid Chromatography Lane C. Sander* and Stephen A. Wise Chemical Science and Technology Laboratory, Analytical chemistry Division, National Institute of Standards and Technology, Gaithersburg, Maryland 20899-0007
Molecular shape recognition is examined for a series of CIS columns prepared using a variety of synthetic a p proaches. Mono-, di-, and trifunctional silanes are used to prepare stationary phases through monomeric and polymeric surface modificationprocedures, including an approach employing self-assembled monolayer technology. Shape discrimhationproperties of the columns were investigated with various nonplanar, planar, and linear polycyclic aromatic hydrocarbon solute probes. Chromatographic retentionbehavior is examined in the context of recently proposed statistical mechanical “interphase” retention models.
ences are most strongly influenced by the type of surface modification chemistry employed. It has been recognized that certain cl8 columns provide enhanced separations of structural i~omers.15,’~ An early example is provided in Environmental Protection Agency @PA) method 610, for the determination of priority pollutant polycyclic aromatic hydrocarbons (PAHs) from aqueous effluents.17 This method specifies the use of a specific cl8 column. Although not recognized at the time, the unique properties of this column were the result of the polymeric surface modification procedure used in the synthesis of the bonded phase, since c18 columns prepared using monomeric surface modification chemistry do not exhibit the required selectivity characteristics and are unable to resolve all of the components in the priority pollutant PAH mixture. In Retention behavior in liquid chromatography is influenced by general, better separations of PAH isomers can usually be a wide variety of physical and chemical properties of both the achieved by use of polymeric CIS columns than with monomeric chromatographic system and the solute. The development of c 1 8 columns. retention models reflects an effort to describe the interaction The unique chromatographic properties of polymeric processes between the solute and the stationary and mobile columns have received surprisingly little attention in the literature, phases which are responsible for Differences in although procedures for the synthesis of polymeric stationary separations that are commonly observed among supposedly phases were reported over ten years a g ~ ~and, the ~ ~columns J ~ similar columns have been attributed to differences in silanol have been available commercially even longer. However, the a~tivity,~ carbon loading? substrate composition, stationary phase advantages offered by polymeric c18 columns toward isomer morphology, and bonding chemistry.6 separations are widely recognized by analysts involved in enviFor isomers and other solute classes with similar physical and ronmental measurement of PAHS,~O,~~ as well as the separation of chemical properties, molecular shape can sometimes provide a carotenoid isomer^.^*-^^ Polymeric c18 columns have been largely basis for separation. Parameters affecting shape selectivity have been studied in some detail by our research group and other~?-~~~-ll ignored outside of these specialties, perhaps because differences in retention behavior are less dramatic for other classes of and reviews of shape selectivity have been presented.12J3 A few compounds, compared with monomeric c18 columns. Compounds trends can be summarized. Shape selectivity is enhanced by increased phase loading, longer chain length bonded phase not constrained to rigid conformations (i.e., with free rotation ligands, reduced column temperature, increased organic modifier about single bonds) often exhibit similar retention behavior on composition in the mobile phase, and the use of polymeric phases. monomeric and polymeric c18 columns. For example, methylene The use of mobile phase additives such as cholesterol can also unit selectivity for alkylbenzene homologs is comparable on influence shape r e ~ o g n i t i o n .However, ~~ shape selectivity differmonomeric and polymeric column typesz5 (1) Martire, D. E.; Boehm, R. E. J. Phys Chem. 1983,87,1045-1062. (2) Yan, C.; Martire, D. E. J. Pkys. Chem. 1992,96, 3489-3504. (3) Yan, C.; Martire, D. E. Anal. Chem. 1992,64, 1246-1253. (4) Dill, K. A. J. Phys. Chem. 1987,91, 1980-1988. (5) Walters, M. J. J. Assoc. Off: Anal. Chem. 1987,70, 465-469. (6) Sander, L. C.; Wise, S. A. Anal. Chem. 1984,56, 504-510. (7) Sander, L. C.; Wise, S. A. LC-GC 1990,8, 378-390. (8) Sander, L. C.; Wise, S. A. Anal. Chem. 1989,61, 1749-1754. (9) Wise, S. A.; Bonnett. W. J.; Guenther, F. R.; May, W. E. J. Ckromatogr. Sci. 1981,19, 457-465. (10) Cole, S. R;Dorsey, J. G. J. Chromatogr. 1993,635, 177-186. (11) Sentell, K. B.; Dorsey, J. G. J. Ckromatogr. 1989,461, 193-207. (12) Sander, L. C.; Wise, S. A. J. Ckromatogr. 1993,656,335-351. (13) Dorsey, J. G.; Cooper, W. T. Anal. Chem. 1994,66, 857A-867A. (14) Cole, S. R.: Dorsey, J. G. 44th Pittsburgh Conference on Analytical Ckemistly and Applied Spectroscopy, Atlanta, GA, 1994; Abstr. 1002.
3284 Analytical Chemistry, Vol. 67, No. 78, September 75, 7995
(15) Ogan, K. L.; Katz. E. D. J. Chromatogr. 1980,188, 115-127. (16)Amos, R.J. Chromatogr. 1981,204,469-478. (17) EPA Test Method, Polynuclear Aromatic Hydrocarbons-Method 610, U S . Environmental Protection Agency, Environmental Monitoring and Support Laboratory: Cincinnati, OH, 1982. (18) Majors, R. E.; Hopper, M. J. J. Chromatogr. Sci. 1974,12, 767-778. (19) Verzele, M.; Mussche, P. J. Chromatogr. 1983,254,117-122. (20) Dong, M. W.; DiCesare, J. L. J. Chromatogr. Sci. 1982,20, 517-522. 439-442. (21) Fetzer, J. C.; Biggs, W. R.; Jinno, K. Chromatographia 1986,21, (22) Matus, 2.; Ohmacht, R. Chromatographia 1990,30, 318-322. (23) Epler, K. S.; Sander, L. C.; Ziegler, R. G.; Wise, S. A.; Craft, N. E. J. Chromatogr. 1992,595, 89-101. (24) Lesellier, E.; Tchapla, A.; Krstulovic, A. M. J. Chromatogr. 1993.645,2939. (25) Sander, L. C.; Wise, S. A. 14th Intemational Symposium on Column Liquid Chromatography Boston, MA, 1990; Abstr. P109. This article not subject to U.S. Copyright. Published 1995 Am. Chem. SOC.
Table 1. Reaction Conditions and Propertles of stationary Phases
no. M-1 M-2 M-3 M-4
designation monomeric49 monomeric monomeric monomeric (end capped)6
P-1 solution polymerized6 P-2 solution polymerized
silane silica functionality (g) silane monochloro 3.50 10.22 g monochloro 3.56 10.35 g trichloro 6.94 10 mL HMDS 3.55 10mL trichloro dichloro
coverage solvent, vol reaction condi% @mol/ a”/ plates (mL) time (h) tions carbon m2) BOP (N) xylene, 100 heptane, 100 heptane, 110 xylene, 100
3.5 10 mL xylene, 100 3.15 10 mL xylene, 100
12.45 11.88 7.77 9.20
3.51 3.32 2.03
1.82 1.78 1.43 1.82
4 2
reflux reflux
17.07 15.10
5.26 4.48
0.73 12258 0.5 mL of HzO added 1.48 9940 0.5 mL of HzO added
ambient 19.84 19.90 16 ambient 19.54 17 ambient 17.31 17.37 16 ambient 17.67 7 days ambient 21.90 7 days ambient 38.09
6.45 6.48 6.32 5.34 5.37 5.49 7.44 19.76
0.31 6009 humidified silica
3.61 10 mL heptane, 100 22
S 2 surface p ~ l y m e r i z e dtrichloro ~~~~~ S 3 surface polymerized dichloro
3.1 10 mL heptane, 100 3.55 10 mL heptane, 100
S 4 surface polymerized S 5 surface polymerized S 6 surface polymerized
3.1 3.0 3.0
dichloro trichloro trichloro
10.3 g heptane, 100 10 mL heptane, 100 10mL heptane, 100
comments
reflux ambient ambient reflux
S1 surface p ~ l y m e r i z e d trichloro ~~,~~
Recently Wirth and Fatunmbi reported a procedure for the preparation of mixed alkyl ligand stationary phases with enhanced hydrolytic ~ t a b i l i t y .The ~ ~ ~synthetic ~~ approach is based on the self-assembled monolayer research of Maoz and SagivzSand Wasserman et al.B These research groups described the reaction of humidified silica with octadecyltrichlorosilane (termed “horizonal polymerization”) to yield surfaces with properties comparable to Langmuir-Blodgett films. The surfaces were characterized by ellipsometry and X-ray reflection, and they were found to have a thickness of 22.6-27.6 A30 and a surface density of 21 & 3 Wirth and Fatunmbi viewed these surfaces as “much too dense for chromatographic applications”,and prepared instead mixed-ligand stationary phases using propyl- and octadecyltrichlorosilane with the intention of increasing the space between individual CIS chain^.^^.^^ NMR studies confirmed the spacing, and chromatographic performance (i.e., efficiency and selectivity) was comparable to monomeric CIScolumns. Although Wirth and Fatunmbi predicted that horizonal polymerization of octadecyltrichlorosilane would result in surfaces that could not be used in liquid chromatography, we have utilized this approach for the preparation of homogeneous self-assembled monolayer phases for comparison to alternative bonding schemes. We have also utilized difunctional silane reagents with both polymeric and selfassembled monolayer modification approaches. Since crosslinking is not possible with difunctional silanes, the type and extent of surface modification can be expected to differ from polymeric phases prepared with trifunctional silanes. This work describes the preparation of CISstationary phases by a variety of procedures utilizing mono-, di-, and trichlorosilane reagents. A procedure utilizing homogeneous self-assembled monolayer technology will be compared with more conventional monomeric and polymeric syntheses. Selectivity characteristics for each phase will be examined by use of shape-relevant PAH probes, and retention behavior is discussed in light of recently proposed retention models. (26) Wirth, M. J.; Fatunmbi, H. 0. Anal. Chem. 1992,64, 2783-2788. (27) Wirth, M. J.; Fatunmbi, H. 0. Anal. Chem. 1993,65, 822-826. (28)Maoz, R.; Sagiv, J. J Colloid Interface Sci. 1984,100, 465. (29) Wasseman, S. R;Whitesides, G. M.; Tidswell,I. M.; Ocko, B. M.: Pershan, P. S.; Axe. J. D.J. Am. Chem. SOC.1989,5852. (30) Wasserman, S R.; Tao, Y. T.; Whitesides, G. M. Longmuir 1989,5, 1074.
9473 8812 6694 6024
2 mL of 2,Mutidine added humidified silica anhydrous M-3 end capped
23 22 24 2
0.34 humidified silica, replicate of S1 1.05 10360 humidified silica 1.04 humidified silica, replicate of S 3 0.35 3423 72 pL of HzO added 630 pL of HzO added
EXPERIMENTAL SECTION
Materials and instrumentation. Reagents were obtained from the following sources: dimethyloctadecylchlorosilane,methylodadecyldichlorosilane,and odadecyltrichlorosilanefrom United Chemical Technologies, Inc. (formerly Huls America, Bristol, PA); 2,&lutidine,biphenyl, l,&diphenylhexatriene, triphenylene, tetraphenylmethane, and triptycene from Aldrich Chemical Co. Inc. (Milwaukee, WI); pyrene and 1,3,5triphenylbenzene from Fluka Chemical Co. (Ronkonkoma, NY); o-terphenyl from Analabs, Inc. (New Haven, CT); and pterphenyl from Eastman Organic Chemicals (Rochester, NY). PAH isomers having a molecular weight of 278 are from sources previously identified.31 Standard Reference Material (SRM) 869, “Column Selectivity Test Mixture for Liquid Chromatography”, was obtained from the Standard Reference Materials Program (NIST, Gaithersburg, MD) . HPLC grade solvents were used in the chromatographic separations. All reagents were used as received without further purification. Silica used in the preparation of stationary phases was from a single lot of YMC SIL-2OO-S3 spherical silica (YMC, Inc., Wilmington, NC). This silica has a nominal particle size of 3 pm, a pore diameter of 200 A, and a surface area of 200 m2/g. Pore diameter and surface area values determined by the manufacturer for this lot of silica are 174 A and 200 m2/g, respectively. Surface coverage values were calculated from carbon determinations, which were carried out by Galbraith Laboratories, Inc. (Knoxville, TN). Commercial CIScolumns were obtained from the following sources: Zorbax CISfrom MacMod Analytical Inc. (Chadds Ford, PA), pBondapak CISfrom Waters (Milford,MA), Cosmosil5Cl&P from Nacalai Tesque (Kyoto, Japan), Phenomenex ODS (20) from Phenomenex, Inc. (Torrance, CA), Beckman ODS from Beckman Instruments (San Ramon, CA), YMC S5 6Ow ODS from yMC, Inc., and Adsorbosphere HS C18 from Alltech Associates, Inc. (Deerfield, IL). Separations were carried out using a liquid chromatograph consisting of a reciprocating piston pump, autosampler, and variable-wavelength ultraviolet absorbance detector. Bonded Phase Synthesis. A variety of approaches were utilized to prepare CIScolumns with different characteristics. A summary of these stationary phases and their physical characteristics is presented in Table 1. Monomeric and polymeric (31) Wise, S. A; Sander, L. C. HRC CC,J. High Resolut. Chromatogr. Chromatogr. Commun. 1985,8, 248-255. Analytical Chemistry, Vol. 67, No. 78, September 75, 7995
3285
Solution Polymerized
Surface Polymerized Figure 1. Diagram distinguishing lwo approaches to the synthesis of polymeric stationary phases. Silane polymerization occurs in Solution or at the silica sultace. depending on the order of addition of
reagents. syntheses were carried out using procedures similar to those previously described.’ For all syntheses, silica was dried at 150 “C for 4 h under reduced pressure prior to use. At the completion of each synthetic procedure, the modified silica was filtered and washed with several portions each (-50 mL) of nonpolar and polar solvents. A typical washing sequence was as follows: heptane or xylene, acetone, methanol, water, methanol, and ‘pentane. Columns were slurry packed at 62 MPa (9ooo psi) using pentane. Monomeric synthesis M-1 was carried out by adding silica to a solution of xylene containing the silane. 2,gLutidie was added, and the mixture refluxed for 23 h. Monomeric synthesis M-2 was prepared in a similar fashion, except that prior to reaction, the silica was equilibrated with humid air so that a layer of water was adsorbed (termed “humidified silica?. To accomplish this, the silica was placed in a sintered glass frit funnel and aspirated for -3 h. Using more sophisticated apparatus, Widh and Fatunmbiz’ demonstrated that a steady state for the surface adsorbed water is achieved withii 2 h of exposure (see discussion below). No reaction catalystwas used, and the reaction was carried out under ambient conditions. Monomeric synthesis M-3 was carried out using octadecyltrichlorosilane under anhydrous condition. Sufficient silica was used so that at the completion of the reaction, half of the material could be end capped for comparison (synthesis M-4). The end capping reaction was carried out with hexamethyl. disilazane. The procedures developed for polymeric phase syntheses summarized in Table 1 have been classified as ”solution polymerized” and “surface polymerized” to distinguish how water is introduced to initiate polymerization (see FIgure 1). For solution polymerization,water is added to a slurry of the silica containing the di- or trichlorosilane. Polymerization occurs in solution, with subsequent deposition onto the silica Surface polymerization describes a synthetic procedure in which water is added to dry silica either through exposure to humid air or by direct addition, with the ‘%et” silica introduced into a solution cantainiing the silane. Both procedures have appeared in the literatnre,6193*and are used by commercial column manufacturers. Recently, Wirth and Fatunmbi described a procedure for adsorbing a monolayer 3286 Aflalyiical Chemistry, Vol. 67, No. 18, September 15, 1995
of water onto dry silica, followed by reaction with a mixture of short and long chain length trichloro~ilanes.2~2~ We have used a variation of this procedure to coat silica with water (syntheses Sl-S4), as well as a different procedure for direct addition of measured quantities of water to silica (syntheses $5 and SS). Synthesis Fl was carried out by “solution polymerization”? The order of addition of reagents and application of reflux influencesthe reaction, and the procedure is as follows. Dry silica is dispersed in xylene and octadecyltrichlorosilane is added. Water is added and the sluny mixed. Polymerization is allowed to proceed at room temperatnre for 5 min. Finally, the slurry is heated to reflux for 4 h. At the completion of the reaction, the slurry is filtered while hot so that unbonded silane polymer remains dissolved and can be removed by filtration and washing. Synthesis P-2 was carried out in an identical manner, except methyloctadecyldichlorosilane was used instead of octadecyltrichlorosilane. Syntheses S1and S2 (replicate) were performed by “surface polymerization”. Silica hydrated with an adsorbed monolayer of water was dispersed in heptane, to which octadecyltrichlorosilane was subsequently added. The slurry was allowed to react at ambient temperature, with occasional resuspension. Syntheses $3 and S4 (replicate) were carried out under identical conditions, except methyloctadecyldichlorosilane was used instead of the trifunctional silane. Syntheses S5 and S6 are surface polymerization reactions in which an excess quantity of water (compared to the amount required for monolayer coverage) was equilibrated with the silica. A weighed quantity of dry silica was equilibrated with humid air to achieve monolayer water adsorption. The silica was again weighed and the mass of adsorbed water determined. This mass was used as the basis for the addition of water in larger amounts. For synthesis S5, silica was equilibrated with a mass of water equivalent to 2 times a monolayer coverage, and for synthesis %,lo times this mass of water was used. Water was added to the silica with mixing and sealed in a container for 24 h prior to reaction. RESULTS AND DISCUSSION We have reported two approaches for the synthesis of polymeric C18 bonded phases, distinguished by the order of addition of water to the reaction mi~lure.’3~,”In the firstmethod (solution polymerization),water is added to a solution containing silica and a trichlorosilane,and the slurry refluxed? In the second method (surface polymerization), water is added directly to the silica and equilibrated prior to reaction. This ”wef silica is added to a solution containing the trichlorosilane and the reaction refluxed.’.” Columns prepared by this process exhibited poor peak shape and low efficiency, whereas columns prepared by solution polymerization exhibited performance comparable to columns with monomeric bonding. We have observed that surface coverage values in excess of -6.5 flmol/m2 are difficult to achieve by solution polymerization, even with the addition of relatively large quantities of water during synthesis? By contrast, surface coverage values can exceed theoretical limits for monclayers with surface polymerization reactions (see reaction SS, Table 1) and thus suggests the formation of multiple layers. (32) Sander. L C.; Wise. S. A Crif. Rev. A d . C h m . 1987. 18. 299-415. (33) Sander. L C.: Wise. S. A/. Ckmmfogr. 1984.316. 163-181. (34)Sander. L C.: Wise. S. A Adunnru in Chmmtogrophy: Giddings. J. C.. Gmshka. E..CazqJ.. Brown. P.R.Edr:Marcel Dekker NewYork, 1986: Vol. 25. pp 139-218.
Surface coverage values for each of the stationary phases are listed in Table 1 and range from 2.0 to 7.4 pmol/m2. An upper limit to bonded phase density is given by the space requirement for alkyl chains in normal paraffin crystals. This value has been estimated by Nyburg and Liith to be 20.6 Az (8.08 pmol/m2), which is also approximately the spacing of silanols at the silica surface.35 The bonding density for phases S1 (6.45 pmol/m2) and S 2 (6.32 pmol/m2) are somewhat lower than the predicted values; however, the excellent reproducibility of these syntheses suggest that surface hydration was uniform and complete. This discrepancy may be the result of the curvature of the silica within the pores. A similar proposal was made by Berendsen et al. in a description of the effect of surface curvature on bonding density, as a function of alkyl phase length.36 Berendsen and co-workers observed that the bonding density for monochlorosilanes decreased with increasing ligand length and attributed this trend to the increased space requirement for extended chains bonded to a curved surface. This effect should be most pronounced for narrow-pore substrates and less important with increasing pore size. The surface coverage for synthesis S6 (carried out with added water in excess of monolayer coverage) was greater than is possible for monolayer coverage. To achieve this level of loading, portions of the stationary phase must consist of bulkpolymerized silane (i.e., silane polymer extending away from the silica surface). In previous work, the influence of bonding chemistry on retention behavior was demonstrated in some detail for two classes of stationary phases: monomeric and polymeric cl8 phases. These differences are most apparent for solutes with rigid molecular shapes, such as PAHs. For other classes of molecules (e.g., alkyl hydrocarbons), these differences are less signiiicant. Meaningful evaluation of shape selectivity depends on the selection of appropriate solute probes. Standard Reference Material (SRM) 869 “Column Selectivity Test Mixture for Liquid Chromatography” has been developed for this p ~ r p o s e . This ~ ~ , mixture ~~ consists of three PAHs: benzo[a]pyrene (BaP planar shape), phenanthro [3,4cIphenanthrene (PhPh; nonplanar shape), and tetrabenzonaphthalene (TBN; nonplanar shape). The elution order of these compounds has been shown to correlate with stationary phase shape recognition performance and permits classification of phases into monomeric and polymeric categories.7,37,38 The selectivity coefficient aTBN/BaP is defined as the ratio k ’ T B N / ~’ B ~ Pand has been used as a numerical descriptor of shape selectivity. Values of aTBI\’/BaP typically range from 0.3 to 2.2. For polymeric CIS columns, which exhibit a high degree of shape recognition, aTBI\’/Bap < 1, and for monomeric C@columns (low shape recognition), aTBN/hp > 1.7. Selectivity coefficients (a“/Bop) are listed in Table 1and will be described below. To supplement the study of retention behavior, additional probes were selected (see Figure 2). These compounds were selected on the basis of differences in retention behavior that are expected to occur for planar and nonplanar, linear and nonlinear, rigid and nonrigid, and square and narrow molecules. (35) Nyburg, S. C.; Luth, H.Acta Crystallogr. 1972,B28,2992-2995. (36) Berendsen, G. E.; Pikaart, K. A; de Galan, L. J. Liq. Chromatogr. 1980,3, 1437-1464. (37) Sander, L. C.; Wise, S. A. Certificate of Analysis, SRM 869, Standard Reference Materials Program, NIST, Gaithersburg, MD, 1990. (38) Sander, L. C.; Wise, S. A. HRC CC,J. High Resolut. Chromatogr. Chromatogr. Commun. 1988.11, 383-387.
Blphenyl
p-Terphenyl
1,8-DlphenyIhexatrlene
Pyrene
Trlphenylene
o-Terphenyl
Trlptycene
Tetraphenylmethane
Figure 2. Structures of solute probes utilized in the evaluation of column shape selectivity.
It is instructive to compare the separation of the compounds in Figure 2 for several commercial CIS columns (Figure 3). Separations were carried out under the same mobile phase conditions; however, the figure is presented so that retention is normalized to the last component, 1,3,5triphenylbenzene (time scales remain correct). Even though absolute retention varies by as much as a factor of 4 among the various columns, selectivity for the probes remains remarkably constant. Separation of o-terphenyl and pyrene is achieved with only three of the columns, so differences do exist, but overall selectivityis best characterized as similar rather than dissimilar. Separations of the nine-component mixture are presented in Figure 4 for several of the columns listed in Table 1. The chromatograms are ordered by increasing aTBN/Bap values (top to bottom). The values range from 0.31 to 1.82, indicating changes in elution order of TBN and BaP with phase density. The separation for column M-1, monomeric CIS,is very similar to the commercial columns shown in Figure 3. It is not surprising that these columns have all been characterized as monomeric in nature in previous studies (selectivity coefficient aTBN/BaP values range from 1.8to 2.1).7,37338 S i c a n t changes in selectivity are apparent among the columns represented in Figure 4. The elution behavior of the last four components is of particular interest. A trend can be observed if the retention of the bulky solutes [tetraphenylmethane (TPM) and 1,3,5triphenylbenzene (l’PB)l is compared with the retention of the extended solutes [P-terphenyl and l,& Analytical Chemistry, Vol. 67, No. 18, September 15, 1995
3287
h.,dn
1
Cosmosll C1I 0
5
0
10
IO
I5
20
30
Phmnonunsx ODs (20)
Beckman ODS
0
0
20
IO
10
30
30
IO
IO
so
60
10
Figure 3. Separation of the nine-component shape selectivity mixture on commercial CIScolumns. Mobile phase composition, 60: 40 acetonitrile/water.
diphenylhexatriene @ P H q I. Retention of TPM and TPB decreases relative to pterphenyl and DPHT, with decreasing values of aTBN/BaP (increasing shape selectivity). Similarly, the absolute retention of fi-terphenyl and DPHT is observed to increase with decreasing aTBN/BaP. This change in retention is continuous, and at various intermediate points (e.g., columns P-2, S3 and P-l), the elution order of these four compounds changes. Pyrene and o-terphenyl are separated on all columns except the monomeric column, and the elution of biphenyl and triptycene is unchanged on all of the columns. The retention behavior observed for the columns in Figure 4 can be discussed in terms of recently advanced models of retention. Martire and co-workers described a model of solute retention based on statistical thermodynamic^.'-^ This model is intended to describe the retention of rigid “blocklike”molecules such as PAHs on ordered stationary phases. Retention depends on repulsive and attractive contributions of the stationary and mobile phase species with the solute molecules and can be expressed in terms of state variables and molecular parameters. Solute parameters include the effective contact area, the van der Waals volume, and the m i n i u m cross-sectional area. This theory predicts that in liquid chromatography shape selectivity should increase with increases in stationary phase order (Le., chain straightening and lengthening). The following order for shape descrimination among solutes is predicted: rods > plates > flexible chains. An empirical “slot model” of retention was advanced by Wise and Sander based on the retention behavior of planar and nonplanar PAHs.~~ In this model, the stationary phase is represented as consisting of a number of slots into which solute 3288 Analytical Chemistry, Vol. 67, No. 78, September 75, 7995
molecules penetrate during retention. For slots of finite size, planar solutes penetrate more slots and will be retained in preference to nonplanar solutes, which are excluded from the slots. By similar reasoning, long narrow molecules with large length to breadth ratios (LIB) will be retained in preference to square molecules. Both the rigorous treatment of Martire et and the empirical model of Wise and Sandel3I are descriptive of the trends observed in Figure 4. For the solutes utilized in the test mixture, DPHT has the largest LIB ratio (2.6), and unlike biphenyl and fi-terphenyl, DPHT is planar. DPHT elutes last for the most densely loaded columns (Sl,P-l), but elutes earlier on the less densely loaded monomeric column @I-1), as might be expected on the basis of its molecular weight. TPM elutes relatively early on the densely loaded columns, which is in accord with its overall “globular”shape. Apparently greater interaction of TPM with the stationary phase results from wider interchain spacing, as with columns M-1 and P-2, and TPM has increased retention. It is interesting to note that the bulky and rigid solute triptycene elutes early with all of the columns, perhaps indicating poor interaction with even relatively widely spaced alkyl chains of the monomeric column M-1. The models of Martire et al. and of Wise and Sander provide logical explanations of the observed trends: bulky solutes elute before planar species, and long, narrow solutes elute after square-shaped species. Several columns not included in Figure 4 deserve comment. Separation of the nine-component test mixture for the end-capped column M-4 was nearly identical to the monomeric column M-1. We have observed that end capping has little effect on shape recognition for nonpolar solutes.12 Two of the syntheses were repeated to give an indication of the reproducibility of the procedures. Also, the carbon values for S1 and S3 represent duplicate measurements of the same sample, submitted for analysis over a 2-month interval. As indicated in Table 1, columns S1 and S2 and columns S3 and S4 have nearly identical percent carbon loadings and aTBN/BnP values. This is a good indication that the silica “hydration” step is not a critical parameter (or at least is not difficult to reproduce) in the surface polymerization reactions. Column S5, prepared by surface polymerization with silica equilibrated with a 2-fold excess of water (compared to monolayer coverage), exhibited very poor chromatographic performance. Peak tailing was severe for each of the nine solutes, and absolute retention for all of the compounds was signiticantly reduced. A column could not be prepared from the bonded silica from synthesis S 6 due to complete blockage (i.e., loss of flow) during packing. In general, peak shape appears to be degraded for the most heavily loaded stationary phases (Sl, S5) and is most severe for strongly retained solutes, particularly with large L / B values. The origin of this effect needs further study; however, the trend would appear to indicate reduced mass transfer and/or overloading of extended solutes within densely loaded stationary phases. NMR and neutron scattering studies have indicated that the mobility of segments of immobilized alkyl chains increases with the distance from the silica s u r f a ~ eand ~ ~with , ~ decreasing ~ bonded phase Diffusion within high-density bonded phases may be reduced compared with more conventional stationary (39) Sindorf, D. W.; Maciel, G. E. /. A m . Chem. SOC.1983,105, 1848-1851. (40)Beaufils. J. P.: Hennion, M. C.: Rosset, R.Anal. Chem. 1985,57,25931596. (41) Gangoda. M. E.: Gilpin, R. K. J. Mugn. Reson. 1983,53,140-143.
s-1 Surface polymerized trichlorosiiane
Y
I1
U
19.8% c 6.45 pmol/mz qsHISP = 0.31 ,
0
10
20
30
"
"
I
"
'
~
40
1
"
"
,
"
"
,
"
50
P-1 Solution polymerized trichlorosilane 17.1% C 5.25 pmoilm' hew+ = 0.73
Blphmyl
Mphnylnu
I 1.l,bTrlphnyl-
s-3 Surface polymerized dichlorosilane 17.3% C 5.34 pmoVm2 %EHlS,P = *05
1""1""1""1""l""1""1""1 0
10
20
30
20
30
Blphmyl
Rlphucylom
P-2 Solution polymerized dichlorosilane 15.1 % C 4.48 pmol/m* %swee = 1-48
1""1""1""1""1""1""I""I 0
10 Mplunylmo
M-1 Monomeric
1,5,bTdphnylbmrm
12.5% C 3.51 pmoUm' arews* = 1.a2 l " " l " " i " ' ~ l " i ' l " ' ~ l ' ' ~ ' i ' 0
10
20
30
Figure 4. Separation of the nine-component shape selectivity mixture on selected columns listed in Table 1. Mobile phase composition, 60:40 acetonitrile/water.
phases, resulting in peak tailing for highly retained solutes. The efficiency of monomeric (M-1,N = 9473) and polymeric e-1,N = 12258) CIS columns are comparable (N is the number of
theoretical plates, determined from the peak width at half-height for triphenylene). These columns are similar in performance to conventional monomeric and polymeric CIS columns from comAnalytical Chemistry, Vol. 67,No. 18, September 15, 1995
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& 1.24
s-1
Surface polymerized trlchlororilme 19.8% c 6.46 pmoUm' , a = 0.31 1 " " l ' " ' l " " l ' " ' l " " l " " l " ' ' 1 ' ' " l " ' ' l " " i
0
10
20
30
40
50
P-1 Solutlon polymerized trlchloroallmr 17.1% C 5.25 PmoUm' = 0.73
I 0
" ' r " " " ~ " " " ~ " " 1 " ' " ~ ' ~ ~ 1 " ' ~ ~ ~ ~ ' ~ 10 20 30 40
s-3 Surface polymerlzed dlchlororllrne 17.3% C 5.34 pmoVm' a,I1.05 1 " " l ' " ' l ' " ' 1 ' " ' I '
0
10
20
P-2 Solution polymerlzed dlchlororilme
M-1 Monomerlc
15.10 % C 4.46 pmoVm' am,,,,, = 1.46
12.5% C 3.51 pmoUm' = 1.82 1 " " 1 ~ " ' 1 " " 1 " ~ ' 1 ' ' " 1 " ' 1 ' 1 0 10 20
30
Figure 5. Separation of MW 278 PAH isomers on selected columns. Mobile phase composition, 85:15 acetonitrile/water.
mercial sources. This is contrary to numerous arguments that have been advanced against polymeric CIS columns, i.e., that polymeric CIS columns are inefficient due to poor mass transfer characteristics of the polymeric stationary phase. These arguments are widely professed without the support ofdata. Since the thickness of typical polymeric CIS stationary phases is comparable to monomeric CISstationary phases, mass transfer should not be appreciably different between the two stationary phase types based on differences in stationary phase thickness. The differences in chain mobility determined through NMR experiments for these two phase types43do not appear to be sufiicient to significantly affect column efficiency. A similar NMR experiment for homogeneous octadecyl self-assembled monolayers would provide additional insight. An additional comparison of differences in column selectivity is shown in Figures 5 and 6 for the separation of PAH isomers having a molecular weight of 278. PAH isomer mixtures are commonly utilized to illustrate differences in shape discrimination since better separations are usually possible with shape-selective column^.^^^^^ The separations in Figure 5 were all carried out (42) Albert, K.; Evers, B.; Bayer, E. J. Magn. Reson. 1 9 8 5 , 62, 428-436. (43) Fatunmbi, H. 0.;Bruch, M. D.; Wirth. M. J. Anal. Chem. 1 9 9 3 , 6 5 , 2 0 4 8 2054. (44) Wise, S. A; Sander, L. C.; Lapouyade, R.; Garrigues, P. J. Chromafogy. 1990, 514, 111-122. (45) Wise, S. A.; Sander, L. C.; Chang, H.; Markides, K. E.; Lee. M. L. Chromatogmphia 1 9 8 8 , 2 5 , 473-480.
3290 Analytical Chemistry, Vol. 67, No. 18, September 15, 1995
under the same conditions, while for Figure 6, mobile phase strength was varied to increase retention on columns with a reduced carbon loading. Significant differences in absolute retention are apparent among the columns, and little separation of the isomer mixture is possible for the monomeric CIScolumn (M-1). Better separations are possible for columns with increasing surface coverage (and decreasing selectivity coefficient amN/kp). The use of weaker mobile phase compositions (Figure 6) did not appreciably affect column shape selectivity toward the isomer mixture. It is interesting to compare columns P-l (solution polymerized, trichlorosilane, 17.1% carbon) and S 3 (surface polymerized, dichlorosilane, 17.3%carbon). The separations in Figures 5 and 6 for these columns are quite different, yet the carbon loadings and resulting surface coverage values are very similar. Even if the stationary phase loading difference of 0.2% carbon is significant, the trend is opposite that expected, namely, increasing shape recognition and decreasing amN/kp with increasing phase loading. The fact that columns S3 and P-l have nearly identical carbon loading but exhibit significantly different chromatographic retention behavior suggests different alkyl chain organization exists between the columns. Two stationary phases with the same carbon loading might exhibit different selectivityif the alkyl chains are spaced differently. A uniform ligand distribution would result in larger interchain spacing than for isolated clusters of ligands.46 This idea was first proposed by Lochmuller and Wilder, who
4
1.24
1.32
1.33
/ aRP7
s.1 Surfow polytnerized trichiororllrne 19.0% C 6.45 pmoilm' a,= 0.31 05:15 ACN:H20 1 " ' ' 1 " ' ' 1 " " 1 ' " ' 1 " " 1 " ' ' 1 ' ' ' ' 1 ' " ' I ' " ' I ' ' " I
0
P-I Solution polymerized trlchlororiiane 17.1% c 5.25 pmoVm' a,,, = 0.73 75:25 ACN:H20
10
20
30
40
50
1 " ' ' I ' ' ' ' I " " I ' " ' 1 " ' ' 1 " ' ' 1 " " 1 ' " ' 1 " ' ' 1 ' ' " ~
0
10
20
30
40
50
30
40
50
30
40
s-3
Sutfrw polymerized dichlororilene 17.3% C 5.34 pnollm' a, = 1.05 7030 ACN:H20 1 " " 1 " " 1 " " 1 ' " " ' ' " I ' " ' I " " I " " 1 " " 1 ' ' ' ' 1
0
10
20
P.2 Solution polymerized dichlorosllme 15.1 % C 4.48 pmollm' a,- = 1.40 6595 ACN:HZO
1 ' ~ " 1 " " 1 ' ' " 1 ' " ' I " " I ' " ' I ' ~ " I " " i
0
10
20
M-I Monomeric 12.5% C 3.51 pmol/m' a,= 1.82 6040 ACN:H20 1 " " 1 " " 1 " " 1 ' " ' 1 ' " ' 1 " " 1 ' ' ' ' 1 ' " ' I ' " ' I " ' ' I
0
10
20
30
40
50
Figure 6. Separation of MW 278 PAH isomers on selected columns. Mobile phase composition has been adjusted to increase retention for columns with lower stationary phase loadings.
described the isolated clusters as liquid dr0plets.4~ This model is not unreasonable for solution polymerization, for which silane polymers form in solution and are deposited on (and ultimately bonded to) the silica surface (see Figure 1). The distribution of ligands for surface polymerization reactions may be more uniform due to the initial monolayer of water on the silica surface. A comparison of the absolute retention for various solutes on the d8erent columns provides further insight into solute retention mechanisms. The retention model of Dill4 predicts that solute retention will increase with stationary phase ligand density to a point, and then at higher ligand densities, retention will decrease as the "energetic cost" of creating a cavity for the solute in the stationary phase becomes large. This retention behavior was observed experimentally by Sentell and Dorsey48 for a series of monomeric CISphases with stationaryphase loadings of 1.6-4.1 pmol/mz. Surface coverages of monomeric and polymeric columns in Table 1 span a wider range, namely, 2.03-7.44 pmoll (46) Lochmiiller, C. H.; Colbom, A S.: Hunnicutt, M. L.; Harris, J. M. Anal. Chem. 1 9 8 3 , 5 5 , 1344-1348. (47) Lochmtiller. C . H.: Wilder, D. R J. Chromatogr. Sei. 1979, 17,574-579. (48) Sentell, K. B.; Dorsey, J. G. Anal. Chem. 1989, 61,930-934.
m2. Plots of k ' vs surface coverage are shown in Figure 7 for solutes listed in Figure 2. This plot is not directly comparable to the research of Sentell and Dorsey since k ' is plotted rather than the partition coefficient K (determination of K requires knowledge of the phase ratio, which is controversial for polymeric stationary phases). In spite of this difference, the fact that a maximum in k ' is observed supports the retention model of Dill and is in agreementwith the findings of Sentell and Dorsey.48 It is perhaps more interesting to examine trends in retention suggested by these data. If the curves in Figure 7 are examined carefully, it is apparent that maximum retention occurs at different surface coverage values for different solutes. Several solutes exhibit maximum retention at or near 4 pmol/m2, whereas other solutes exhibit a maximum at much higher phase loadings. Bulky solutes such as TF'B, TPM and o-terphenyl are examples of solutes for which maximum retention is observed at lower surface coverages. Extended solutes such as DPHT and bterphenyl exhibit maximum retention at much higher phase loadings approaching 7 pmol/ (49) Sentell, K B.: Barnes, K. W.; Dorsey, J. G. J. Chromatogr. 1988, 455,95104.
Analytical Chemistry, Vol. 67,No. 18, September 15, 1995
3291
chain spacing for partitioning within the stationary phase compared with planar or linear solutes.4 The retention of bulky molecules is reduced for stationary phases with high surface coverages due to the higher energetic cost of cavity creation compared with planar or linear molecules. As the space between bonded alkyl chains decreases, solute/stationary phase interactions are favored for extended and linear solutes.
60 1,6-DiphenylhexatrIene m pTerpheny1 0 1,3,5-Triphenylbenzene A
50
-
40
-
*
Tetraphenylmethane Trlphenylene
k
r-
I
1
1
I
I
I
I
1
2
3
4
5
6
7
8
ACKNOWLEDGMENT The authors thank Robert Cooley (YMC Inc., Wilmington, NC) for gifts of YMC SIL200 silica. Certain commercial equipment, instruments, or materials are identified in this report to specify adequately the experimental procedure. Such identification does not imply recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the materials or equipment identilied are necessarily the best available for the purpose.
Surface Coverage (pnoVm?) Figure 7. Plot of retention as a function of surface coverage for various Cj8 columns (Table 1) and selectivity probes (Figure 2).
Received for review April 6, 1995. Accepted June 21, 1995.8
AC950345D
m2. This retention behavior might be expected on the basis of interchain distances. Bulky solutes should require larger inter-
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Analytical Chemistry, Vol. 67, No. 18, September 15, 1995
F S
Abstract published in Advance ACS Abstracts, August 1, 1995.
