Polar Polymeric Stationary Phases for Normal-Phase HPLC Based on

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Anal. Chem. 2003, 75, 1011-1021

Polar Polymeric Stationary Phases for Normal-Phase HPLC Based on Monodisperse Macroporous Poly(2,3-dihydroxypropyl methacrylate-co-ethylene dimethacrylate) Beads Mingcheng Xu,† Dominic S. Peterson,‡ Thomas Rohr,‡ Frantisek Svec,†,‡ and Jean M. J Fre´chet*,†,‡

Department of Chemistry, University of California, Berkeley, California 94720-1460, and E. O. Lawrence Berkeley National Laboratory, Materials Sciences Division, Berkeley, California 94720

The effect of variables such as shape template size, porogen composition and percentage, content of crosslinking monomer, and polymerization temperature on the properties of uniformly sized 3-µm porous poly(glycidyl methacrylate-co-ethylene dimethacrylate) beads prepared by the staged templated suspension polymerization technique has been studied. The porous properties of the beads including surface morphology, pore size distribution, and specific surface area have been optimized to obtain highly efficient stationary phases for normal-phase HPLC. A column packed with diol stationary phase obtained by hydrolysis of poly(glycidyl methacrylate-coethylene dimethacrylate) beads affords an efficiency of 67 000 plates/m for toluene using THF as the mobile phase. The retention properties and selectivity of the diol beads are easily modulated by changes in the composition of the mobile phase. The performance of these beads is demonstrated with the separations of a variety of polar compounds including positional isomers, aniline derivatives, and basic tricyclic antidepressant drugs. The first chromatographic separations carried out a century ago1 involved a stationary phase that was more polar than the mobile phase. This separation mode, today called normal-phase liquid chromatography (NPLC), is well suited for the separation of organic compounds such as positional isomers or enantiomers that differ only slightly in their structures for which the more common reversed-phase LC mode is not suitable.2-4 Many of these compounds featuring minute structural differences have strong biological activity and are of interest to the pharmaceutical, agrochemical, and food industries. Some of these compounds also dissolve well in nonpolar organic solvents while their solubility in aqueous media and polar solvents is rather poor. This and the * Corresponding author. E-mail: [email protected]. † University of California, Berkeley. ‡ E. O. Lawrence Berkeley National Laboratory. (1) Tsvett, M. S. Ber. Deut. Bot. Gessel. 1906, 24, 322-327. (2) Neue, U. HPLC Columns: Theory, Technology, and Practice; Wiley: New York, 1997. (3) Valko, K. Handbook of Analytical Separations, Vol. 1, Separation Methods in Drug Suntheses and Purification; Elsevier: Amsterdam, 2000. (4) Subramanian, G. Chiral Separation Techniques. A Practical Approach; WileyVCH: New York, 2001. 10.1021/ac026216w CCC: $25.00 Published on Web 01/18/2003

© 2003 American Chemical Society

need for sensitive analytical methods allowing their separation has triggered a revival in the interest in the “classical” NPLC mode.3,5 The remarkable selectivity of NPLC results from a host of highly specific interactions of functionalities of the surface of the separation medium with the analytes. The general mechanism of NPLC based on adsorption phenomena was postulated by Snyder in the 1970s.6,7 Since its inception, NPLC and its relatives hydrophilic interaction chromatography8shave been used for the separations of a broad spectrum of compounds including both small molecules and polymers. 5,9-16 Although alumina and titania have also been used as column packings for NPLC,17 current commercial NPLC separation media are usually based on silica.18-20 Silica-based stationary phases provide a large surface area and high column efficiency while affording excellent mechanical resistance.21,22 Macroporous rigid polar organic beads are commonly used for aqueous size exclusion chromatography (SEC) as well as for hydrophobic interaction and affinity chromatography of biopolymers.23 However, these stationary phases are rarely used under (5) Ballschmiter, K.; Wossner, M. Fresenius J. Anal. Chem. 1998, 361, 743755. (6) Snyder, L. R. Anal. Chem. 1974, 46, 1384-1393. (7) Snyder, L. R.; Kirkland, J. J. Introduction to Modern Liquid Chromatography; Wiley: NewYork, 1979. (8) Alpert, A. J. J. Chromatogr., A 1990, 499, 177-196. (9) Churms, S. C. J. Chromatogr., A 1996, 720, 75-91. (10) Jandera, P. J. Chromatogr., A 1998, 797, 11-22. (11) Yoshida, T.; Okada, T. J. Chromatogr., A 1999, 840, 1-9. (12) Jandera, P.; Holcapek, M.; Kolarova, L. J. Chromatogr., A 2000, 869, 6584. (13) Pilc, J. A.; Sermon, P. A. J. Chromatogr., A 1987, 398, 375-380. (14) Jandera, P.; Urbanek, J.; Prokes, B.; Churacek, J. J. Chromatogr., A 1990, 504, 297-318. (15) Glockner, G. Gradient HPLC of Copolymers and Chromatographic CrossFractionation; Springer: Berlin, 1991. (16) Desbene, P. L.; Desmazieres, B. J. Chromatogr., A 1994, 661, 207-213. (17) Winkler, J.; Marme, S. J. Chromatogr., A 2000, 888, 51-62. (18) Dorsey, J. G.; Foley, J. P.; Cooper, W. T.; Barford, R. A.; Barth, H. G. Anal. Chem. 1994, 66, 500R-546R. (19) Hanai, T. Advances in Chromatography; Dekker: New York, 2000; Vol. 40, p 316. (20) Lacourse, W. R. Anal. Chem. 2002, 74, 2813-2831. (21) Unger, K. K.; Janzen, R. J. Chromatogr. 1986, 373, 227-264. (22) Unger, K. K. Packings and Stationary Phases in Chromatographic Techniques; M. Dekker: New York, 1990. (23) Svec, F. Organic Polymer Support Materials. In HPLC of Biological Molecules; Gooding, K. M., Regnier, F. E., Eds.; Marcel Dekker: New York, 2002; pp 17-48.

Analytical Chemistry, Vol. 75, No. 4, February 15, 2003 1011

Chart 1

NPLC conditions.24-28 In our previous studies, we have demonstrated the potential of macroporous monodisperse poly(2,3dihydroxypropyl methacrylate-co-ethylene dimethacrylate) beads for the NPLC separations of low molecular weight compounds29 as well as both nonpolar and polar polymers.30,31 We also used several new porous polar polymer supports for the preparation of highly selective stationary phases for chiral HPLC.32-35 However, our previous communications did not explore the materials properties, their control, and their effect on performance in the NPLC mode. In this report, we explore the effects of variables of the polymerization process such the size of polystyrene shape templates, the types of porogens, the polymerization temperature, and the percentage of cross-linking monomer used in the preparation of 3-µm monodisperse polar beads on their morphology, porous properties, and chromatographic performance. We also report our findings concerning the conditions that cause the shape template to be expelled from the porous beads during polymerization. This now enables the preparation of beads with no structural defects (holes) on their surface. Finally, we show the effect of the packing procedure on the performance of columns packed with these new polymer-based stationary phases and demonstrate both the vastly improved column efficiency and selectivity of these beads in several NPLC separations. EXPERIMENTAL SECTION Chemicals. The glycidyl methacrylate (GMA) and ethylene dimethacrylate (EDMA) monomers (Aldrich) were distilled under vacuum. Azobisisobutyronitrile (AIBN), poly(vinyl alcohol) (PVA; 87-89% hydrolyzed, MW 85 000-146 000), and other chemicals were obtained from Aldrich and used directly. Hydrochlorides of basic drugs doxepin, cloropramine, and nortriptyline (Chart 1) were purchased from Spectrum Chemical Manufacturing Corp. (Gardena, CA). All solvents used in chromatographic experiments (24) Rolls, W.; Svec, F.; Fre´chet, J. M. J. Polymer 1990, 31, 165-174. (25) Yang, Y. B.; Regnier, F. E. J. Chromatogr. 1991, 544, 233-247. (26) Yang, Y. B.; Harrison, K.; Kindsvater, J. J. Chromatogr., A 1996, 723, 1-10. (27) Hargitai, T.; Reinholdsson, P.; Tornell, B.; Isaksson, R. J. Chromatogr. 1993, 630, 79-94. (28) Smigol, V.; Svec, F.; Fre´chet, J. M. J. J. Liq. Chromatogr. 1994, 17, 259267. (29) Petro, M.; Svec, F.; Fre´chet, J. M. J.; Haque, S. A.; Wang, H. C. J. Polym. Sci., Polym. Chem. 1997, 35, 1173-1180. (30) Petro, M.; Svec, F.; Fre´chet, J. M. J. Anal. Chem. 1997, 69, 3131-3139. (31) Svec, F.; Petro, M.; Fre´chet, J. M. J. Collect. Czech. Chem. Commun. 2001, 66, 1047-1061. (32) Liu, Y.; Svec, F.; Fre´chet, J. M. J.; Juneau, K. N. Anal. Chem. 1997, 69, 61-65. (33) Lewandowski, K.; Murer, P.; Svec, F.; Fre´chet, J. M. J. Anal. Chem. 1998, 70, 1629-1638. (34) Lewandowski, K.; Murer, P.; Svec, F.; Fre´chet, J. M. J. J. Comb. Chem. 1999, 1, 105-112. (35) Murer, P.; Lewandowski, K.; Svec, F.; Fre´chet, J. M. J. Anal. Chem. 1999, 71, 1278-1284.

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Analytical Chemistry, Vol. 75, No. 4, February 15, 2003

Scheme 1

were HPLC grade. Aqueous dispersions of 1.5- and 0.6-µm polystyrene latex particles (14 and 10% solid, respectively) were purchased from Bangs Laboratories (Fishers, IN). Preparation of Beads. A modified staged templated suspension polymerization process described in detail elsewhere36,37 was used for the preparation of the uniformly sized beads. In one example of the implementation, the 0.6-µm monodisperse polystyrene templates (0.15 mL) were preswollen by adsorption of an emulsion of dibutyl phthalate (0.105 mL) in 7.5 mL of 0.25 wt % aqueous sodium dodecyl sulfate (SDS) solution. After the swelling was completed and the droplets of the emulsified dibutyl phthalate disappeared (typically 12 h), a mixture containing GMA (1.0366 g), EDMA (2.4188 g), cyclohexanol (1.0123 g), toluene (4.1708), and AIBN (40 mg) emulsified by sonication in 20 mL of 0.25% aqueous SDS solution was added to the dispersion of the preswollen templates in a 100-mL Erlenmeyer flask. The mixture was stirred at room temperature until the monomer emulsion was completely transferred into the templates. After adjusting the concentration of the system to 1% by addition of 10 mL of 4% aqueous PVA solution, the dispersion was purged with nitrogen for 20 min and the flask was sealed. Polymerization was carried out in the flask placed in an orbiting shaker bath set to 250 rpm and the desired temperature for 24 h. The beads were then repeatedly decanted in water and methanol until the supernatant liquid was clear and, unless indicated otherwise, washed three times with tetrahydrofuran (THF) to remove the linear polystyrene of the original template. Drying in a vacuum at 50 °C afforded 3.2 g of monodisperse beads for a 90% yield. Hydrolysis of Epoxide Functionalities. The epoxide groups of poly(glycidyl methacrylate-co-ethylene dimethacrylate) beads (2 g) were hydrolyzed by shaking in 20 mL of 0.5 mol/L aqueous sulfuric acid at 60 °C for 3 h to afford diol functionalities (Scheme 1). Under these conditions, the polymethacrylate ester groups are completely stable and are not hydrolyzed.38 The hydrolyzed beads were washed several times with distilled water until its pH was close to neutral, dispersed in 10 mL of 0.1 mol/L aqueous sodium hydroxide, and then washed again with distilled water to neutral reaction. The beads were then dried in a vacuum at 50 °C overnight. Complete hydrolysis was confirmed by IR spectroscopy with the appearance of a strong hydroxyl band at 3487 cm-1 and the disappearance of bands typical of epoxy group at 907 and 845 cm-1.39 Characterization of the Porous Structure. The pore size distribution of dry beads was determined using an Autopore III 9400 mercury intrusion porosimeter. The surface area was calculated from the BET isotherms of nitrogen adsorption and desorption using ASAP 2010. Both instruments were from Mi(36) Smigol, V.; Svec, F. J. Appl. Polym. Sci. 1992, 45, 1439-1448. (37) Smigol, V.; Svec, F. J. Appl. Polym. Sci. 1993, 48, 2033-2039. (38) Kalalova, E.; Radova, Z.; Svec, F.; Kalal, J. Eur. Polym. J. 1977, 13, 287291. (39) Hrudkova, H.; Svec, F.; Kalal, J. Br. Polym. J. 1977, 1977, 238-242.

cromeritics (Norcross, GA). Single measurements were used for the determination of porous properties. Alternatively, the pore size distribution of the beads swollen in THF was determined by means of inverse size exclusion chromatography (ISEC) using well-defined polystyrene standards. Since retention of all standards with molecular weights Mw exceeding 1 007 000 were equal, the retention volume of this standard was arbitrarily set as the void volume V0, while the pore volume Vi was calculated from the difference of retention volumes for toluene VTOL and V0. The distribution coefficients KD for each standard were calculated from their retention volumes VR using eq 1. The pore diameters D were also estimated from the

KD ) (VR - V0)/(VTOL - V0)

(1)

molecular weight of the polystyrene standards according to the empirical function shown in eq 2.40 Scanning electron microscopy

D ) 0.62M0.59 w

(2)

images were obtained using a JEOL JSM6300 electron microscope. Chromatography. Poly(2,3-dihydroxypropyl methacrylate-coethylene dimethacrylate) beads (1.3 g) were dispersed under sonication in 10 mL of a 60:40 cyclohexanol/acetone mixture and then slurry packed into a 150 × 4.6 mm i.d. stainless steel column using acetone as the packing solvent at a constant pressure of 20 MPa for 30 min. The packed columns were conditioned using a 50:50 hexane/THF mixture for 1 h followed by hexane for 2 h at a flow rate of 1.0 mL/min. The efficiency of these columns was evaluated from the peak of toluene using THF as the mobile phase and calculated by the software from the peak width at the halfheight. A Waters HPLC system consisting of two 515 pumps, a 717 plus autosampler, and a 486 UV detector, all controlled by Millenium 3.2 software, was used for the chromatographic experiments. RESULTS AND DISCUSSION Effect of Template Size on Porous Properties. To obtain monodisperse final beads, uniformly sized latex particles must be used as shape templates. These are pre-swollen with dibutyl phthalate and then swollen with the other components of the polymerization mixture. Linear polymers such as those used as shape templates are known to be porogens that enable the production of macroporous polymers with large pores and the porous properties of poly(styrene-co-divinylbenzene) beads can be tuned by varying the size and molecular weight of the polystyrene templates.41-44 It is also assumed that the size and nature of the template particle have a significant effect on the structural integrity of the surface of the beads they afford. In particular, numerous preparations of beads by a seeded polym(40) Halasz, I.; Martin, K. Angew. Chem., Int. Ed. Engl. 1971, 17, 901. (41) Wang, Q.; Svec, F.; Fre´chet, J. M. J. Polym. Bull. 1992, 28, 569-576. (42) Hosoya, K.; Fre´chet, J. M. J. J. Polym. Sci., Polym. Chem. 1993, 31, 21292141. (43) Wang, Q.; Svec, F.; Fre´chet, J. M. J. Anal. Chem. 1994, 66, 4308-4315. (44) Liang, Y. C.; Svec, F.; Fre´chet, J. M. J. J. Polym. Sci., Polym. Chem. 1997, 35, 2631-2643.

erization led to a final product in which a surface defectsa holes was clearly visible on the surface of the beads. In this study, we compare the effect of polystyrene templates having two different sizess1.5 and 0.6 µmson the properties of porous poly(glycidyl methacrylate-co-ethylene dimethacrylate) beads. Given the notoriously poor compatibility of most polymeric materials, we assumed that the polystyrene templates would have a larger effect on the porous as well as surface properties of methacrylate-based beads. Indeed, the use of template particles with a diameter of 1.5 µm affords porous beads 1 with a larger pore size than observed for beads 3 prepared using 0.6-µm templates. This is documented in Table 1 by the almost complete disappearance of pores with a size smaller than 7 nm in beads 1. Because the size of the final beads was kept at 3 µm, the larger polystyrene templates have a volume that represents 15% of total volume of the final bead. Since our target was to prepare highly efficient stationary phases for the HPLC separations of small molecules, the creation of beads with excessively large pores and therefore a small surface area was not desirable. In contrast, the 0.6-µm templates represent only 0.8% of the overall final volume and also keep the percentage of the soluble polymer porogen low. Beads prepared using the smaller size templates have a considerably larger volume of smaller pores that may be detected by nitrogen adsorption/desorption, and therefore, they also have a larger surface area. This effect of the percentage of polystyrene in the polymerization mixture was further confirmed by experiments in which the 1.5-µm templates were used to prepare 7-µm beads 2. In this case, the templates only represent 1.0% of the bead volume. As a result, the porous properties of beads 2 and 3 are very similar. The noticeable effect of the content of polymeric porogen dictated by the size of the templates is best demonstrated by parallel measurements of efficiency using columns packed with 3-µm beads 1 and 3. While the column packed with beads prepared using the 1.5-µm templates afforded only 15 300 theoretical plates/m for toluene using tetrahydrofuran as the mobile phase, this number almost doubled to 25 900 for the column packed with beads prepared from the smaller 0.6-µm templates. In theory, the use of templates even smaller than those we have used in this study would be attractive since the porogenic effect of the template polymer would be further reduced. Although monodisperse templates with sizes much less than 0.6 µm are readily accessible using controlled emulsion polymerization, a significant technical challenge would have to be overcome in order to use them in the staged templated polymerization process. The theory of swelling of polymer particles derived by Ugelstad45 suggests that in order to achieve fast and complete transfer of these components into the template domain the size of droplets of the emulsified components in the swelling steps should be smaller than that of the templates. To obtain monodisperse beads, the swelling process must be fast and complete. Unfortunately, the sonication procedure we currently use to prepare our mixture of monomers and liquid porogens is not suitable for the preparation of very fine emulsified droplets. Therefore, no further decrease in the template size was attempted in this study and all (45) Ugelstad, J.; Mork, P. C.; Nordhuus, I.; Mfutakamba, H.; Soleimany, E. Macromol. Chem. Phy., Suppl. 1985, 10/11, 215-234.

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Table 1. Porous Properties of 3-µm Poly(glycidyl methacrylate-co-ethylene dimethacrylate) Beads Prepared from 0.6-µm Polystyrene Templates Preswollen with Dibutyl Phthalate Using Polymerization at a Temperature of 70 °C for 24 h

beads

GMA

1g 2g,h 3 4 5 6 7 8 9 10 11i 12i 13i 14i

24 24 24 24 33 38 21.5 21.5 21.5 21.5 10.8 11.3 12.4 16.4

polymerization mixture, wt % EDMA TOa 16 16 16 16 22 25 14.5 14.5 14.5 14.5 25.2 26.1 28.9 38.4

0 0 0 60 45 37 57.4 51.5 48.3 13.4 51.5 50.4 47.2 36.4

CHa

Dp-L,b nm

Vp-L,c mL/g

Vp-S,d mL/g

Sg,e m2/g

efficiency,f plates/m

60 60 60 0 0 0 6.6 12.5 15.7 50.6 12.5 12.2 11.5 8.8

20.6 27.1 27.8 80.1 49.6 28.4 100.9 73.0 51.2 49.4 40.6 39.7 36.9 34.3

0.37 0.39 0.48 0.50 0.48 0.35 0.66 0.63 0.66 0.40 0.78 0.75 0.69 0.45

0.004 0.056 0.067 0.016 0.072 0.079 0.009 0.015 0.058 0.056 0.162 0.162 0.168 0.191

59.3 86.4 75.2 51.2 74.5 78.9 24.4 39.1 55.7 63.2 140.1 140.3 147.6 151.9

15 300 11 900 25 900 39 800 44 800 45 200 43 500 52 000 49 000 31 200 67 000 66 900 67 000 63 600

a TO, toluene; CH, cyclohexanol. b Specific surface area determined by nitrogen absorption/desorption (BET). c Volume of pores larger than 7 nm determined by mercury intrusion porosimetry. d Volume of pores smaller than 7 nm determined by nitrogen adsorption/desorption. e Median pore diameter determined by mercury intrusion porosimetry. f Efficiency of column packed with diol beads obtained by hydrolysis of epoxide groups. g Polystyrene templates with a diameter of 1.5 µm were used for these experiments. h Particle size 7 µm. i Beads were prepared by polymerization at a temperature of 70 °C for 8 h and continued at 95 °C for 16 h.

of the porous beads described in the following sections were prepared using 0.6-µm templates. Effect of the Co-Porogens on Porous Properties and Surface “Hole” Formation. Since the content of polystyrene porogen is fixed through the sizes of both the templates and the final beads, the porous properties can be modulated by changing the composition of the porogenic solvents and their percentage in the polymerization mixture. Cyclohexanol and toluenessolvents with large differences in their solvency for the poly(glycidyl methacrylate-co-ethylene dimethacrylate)swere chosen as coporogens. Toluene is a thermodynamically poor solvent for the polymer, which produces large pores, while cyclohexanol is a better solvent, as seen from the comparison of beads 3 and 4 (Table 1). While the median pore diameter of beads 3 prepared in the presence of cyclohexanol is ∼30 nm, toluene affords beads with a pore diameter of over 80 nm. Simultaneously, the specific surface area decreases from 75.2 to 51.2 m2/g as a result of a 4-fold decrease in the volume of pores smaller than 7 nm. This effect can be compensated by using a smaller percentage of toluene in the polymerization mixture as demonstrated with beads 5 and 6. However, this approach yields beads with a smaller overall pore volume. Therefore, the best alternative to control the porous properties is the use of binary mixtures of both porogenic solvents. For example, the specific surface area increases with the cyclohexanol-to-toluene ratio as demonstrated with beads 7-10. This increase in specific surface area is a consequence of the lowering of median pore size and the simultaneous increase in the volume of pores smaller than 7 nm as a result of an earlier onset of phase separation during polymerization. Figure 1 shows the significant initial effect of cyclohexanol, even when used in small amounts in the porogenic mixture with toluene. However, as the cyclohexanol-to-toluene ratio exceeds 1:3, further changes have little effect on both the surface area and the pore size. Figure 2 shows SEM micrographs of beads 1-4. Holes with a size of ∼1 µm are clearly observed for both the 3- and 7-µm beads prepared using 1.5-µm polystyrene templates. A decrease 1014 Analytical Chemistry, Vol. 75, No. 4, February 15, 2003

Figure 1. Effect of cyclohexanol/toluene ratio on specific surface area and median pore diameter of poly(glycidyl methacrylate-coethylene dimethacrylate) beads. Conditions: 0.6-µm polystyrene shape templates preswollen with dibutyl phthalate; polymerization mixture glycidyl methacrylate 21.5%, ethylene dimethacrylate 14.5%, total porogens 64%, initiator azobisisobutyronitrile (1% with respect to monomers); reaction time 24 h; temperature 70 °C.

in the template size to 0.6 µm leads to the formation of much smaller holes that have a size close to that of this template. It is clear that formation of the hole is directly related to the presence of the template and its exclusion from the final bead. The reason for the expulsion of the seed from the domain of the porous polymer during polymerization is thought to be related to its incompatibility with the newly formed cross-linked polymer. Segregation of incompatible components leads to their mutual exclusion and, ultimately, leads to the formation of a hole visible on the outer surface of the bead. These holes have been observed in many different monodisperse porous beads prepared via swelling of preformed template particles.36,42-44,46,47 It is worth noting that beads 1-3 with holes were prepared using cyclohexanol as the porogen. In contrast to cyclohexanol, (46) Ugelstad, J.; Mork, P. C.; Kaggerud, K. H.; Ellingsen, T.; Berge, A. Adv. Colloid Interface Sci. 1980, 13, 101-140.

Figure 2. Scanning electron micrographs of poly(2,3-dihydroxypropyl methacrylate-co-ethylene dimethacrylate) beads 1-4 prepared under different conditions. Conditions: polystyrene shape templates preswollen with dibutyl phthalate; polymerization mixture glycidyl methacrylate 24%, ethylene dimethacrylate 16%, total porogens 60%, initiator azobisisobutyronitrile (1% with respect to monomers); reaction time 24 h; temperature 70 °C. (A) bead size 3 µm, porogen cyclohexanol, templates 1.5 µm; (B) bead size 7 µm, porogen cyclohexanol, templates 1.5 µm; (C) bead size 3 µm, porogen cyclohexanol, templates 0.6 µm; (D) bead size 3 µm, porogen toluene, templates 0.6 µm.

which is a poor solvent for polystyrene, toluene is a good solvent for polystyrene and its use avoids the formation of holes as seen with beads 4. Figure 3 shows a micrograph of porous poly(glycidyl methacrylate-co-ethylene dimethacrylate) beads prepared in the presence of cyclohexanol and washed only with methanol after polymerization. Clearly, each porous bead has a smaller particle attached to its surface. To our knowledge, these micrographs are the first showing the conjugates of newly formed beads paired with their rejected templates. The methanol used in the workup of the beads after polymerization does not dissolve polystyrene, and therefore, the template particle remains attached to the bead surface. Since all other workup protocols reported in the literature included washing with a solvent for polystyrene such as THF, benzene, or toluene,36,42-44,46,47 the rejected template polymer was dissolved during this treatment and its removal from the surface left a hole behind. The bond between the porous particle and the rejected polystyrene is weak and breaks upon sonication in methanol. This enables both the separation of the porous beads from the small secondary particles and the analysis of the latter. IR spectroscopy shows no CdO stretch or other carbonyl band near 1720 cm-1, indicating that the small particles consist of pure polystyrene with no methacrylate incorporation. Figure 4 shows that the shape of the recovered template polymer is different from that of the original latex particles used in the swelling procedure. This suggests that the template is indeed dissolved in the polymerization mixture during the initial swelling procedure and then reinstated as a secondary spherical entity as the polymerization proceeds, before being fully rejected. If the rejection of the (47) Wang, Q.; Hosoya, K.; Svec, F.; Fre´chet, J. M. J. Anal. Chem. 1992, 64, 1232-1238.

Figure 3. Scanning electron micrographs of poly(glycidyl methacrylate-co-ethylene dimethacrylate) beads with rejected template particles. Conditions: 1.5-µm polystyrene shape templates preswollen with dibutyl phthalate; polymerization mixture glycidyl methacrylate 24%, ethylene dimethacrylate 16%, cyclohexanol 60%, initiator azobisisobutyronitrile (1% with respect to monomers); reaction time 24 h; temperature 70 °C; workup with methanol only.

polystyrene molecules proceeded continuously during polymerization without restoring the template particle first, beads with core-shell morphology would be more likely to form. The weight-average molecular weight determined by SEC of the excluded polystyrene template particles collected after methanol wash was found to be 365 000, a value that is slightly higher than that measured for the original polystyrene latex templates (Mw ) 290 000). The difference is even more notable for Mn as an increase from 79 000 to 134 000 is measured. The higher molecular weights of the recovered polystyrene result from the fact that low molecular weight components of the original template polymer have a higher tendency to dissolve in the porogen and therefore to be removed by the methanol washing. Since the polystyrene template is rejected from the final methacrylate beads, the IR spectrum of the porous beads exhibits no peaks that would suggest the presence of aromatic rings originating from polystyrene. In sharp contrast with the formation of surface holes prepared using cyclohexanol as porogen, no holes are observed for beads Analytical Chemistry, Vol. 75, No. 4, February 15, 2003

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Figure 5. Effect of porogenic mixture consisting of toluene and cyclohexanol in various proportions on the appearance of 3-µm poly(2,3-dihydroxypropyl methacrylate-co-ethylene dimethacrylate) beads prepared by hydrolysis of poly(glycidyl methacrylate-co-ethylene dimethacrylate) particles. Conditions: 0.6-µm polystyrene shape templates preswollen with dibutyl phthalate; glycidyl methacrylate 21.5%, ethylene dimethacrylate 14.5%, total porogens 64%, initiator azobisisobutyronitrile (1% with respect to monomers); reaction time 24 h; temperature 70 °C. Toluene/cyclohexanol ratio 90:10 (A), 80:20 (B), 75:25 (C), and 20:80 (D).

Figure 4. Scanning electron micrographs of 1.5-µm polystyrene shape templates (A) and polystyrene particles rejected from poly(glycidyl methacrylate-co-ethylene dimethacrylate) beads (B).

prepared using toluene as porogen. IR analysis of beads prepared with toluene, worked up in methanol, and finally sonicated shows that polystyrene is still present within the methacrylate beads as evidenced by a peak at 1600 cm-1 corresponding to the carboncarbon stretch of aromatic rings. This indicates that as a result of their affinity for the toluene porogen the polystyrene molecules of the template are not rejected from the domain of the porous polymer and remain in the beads 4. Thus, no holes are observed on the bead surface. However, this peak characteristic of polystyrene disappears completely after treatment of the beads with THF, a process that dissolves the polystyrene and effects its complete removal from the porous beads. We have noted earlier that the template had significant effects on the ultimate porous structure as explored by size exclusion chromatography.42 In contrast, the effect of surface holes on the efficiency of the HPLC column has not yet been studied since beads without holes were not available for a comparison. Therefore, columns were packed with beads 3 and 4, and their efficiencies were measured for toluene used as an unretained analyte. Indeed, a 15% increase in efficiency from 28 000 to 32 000 plates/m for columns packed with beads 3 and 4, respectively, confirms the negative effect of holes. While this finding is significant, even more improvements in efficiency can be achieved by fine-tuning the overall preparation process. 1016 Analytical Chemistry, Vol. 75, No. 4, February 15, 2003

Figure 5 shows the significant difference in bead surface morphology observed for beads prepared with cyclohexanol and with toluene. It is clear that the bead surface is much smoother for beads prepared with cyclohexanol, while it is rather rough for those polymerized using a higher percentage of toluene as a co-porogen. This likely results from the poor solvency of toluene for poly(glycidyl methacrylate-co-ethylene dimethacrylate). Therefore, we explored the use of mixtures of the two porogens to avoid formation of surface holes and simultaneously obtain beads with a smoother surface. Figure 5 shows micrographs of beads obtained by polymerization of mixtures containing the same overall percentage of porogenic solvent (64%) but with different proportions of cyclohexanol and toluene (beads 7-10, Table 1). Clearly, the surface of beads 8 and 9 is smoother than that of beads 7 prepared with a lower proportion of cyclohexanol. In addition, the smoother beads always exhibit enhanced chromatographic properties. For example, an efficiency of 52 000 plates/m was observed for a column packed with beads 8 versus 43 500 plates/m for beads 7. Although additional improvements in surface morphology might be achieved by further increases in the cyclohexanol content, the incompatibility of this co-porogen with the template polymer leads again to the formation of beads with holes as observed in the SEM of beads 10. The monomer-to-porogen ratio is another variable that directly affects the porous properties of the beads. Since the overall pore volume in macroporous beads is close to the volume of added porogen, a higher percentage of porogen in the polymerization mixture affords beads with higher porosities as confirmed by the data of Table 1. Despite significant changes in the volume of large

pores, this variable only has a limited effect on the column efficiency that varies between 63 000 and 67 000 plates/m. Since the chemical composition of the polymer in beads 11-14 remains fixed and the specific surface area does not change too much either, this indicates that the large pores do not have a significant effect on the chromatographic properties. Effect of the Polymerization Temperature. Typically, the porous properties of macroporous beads are controlled by the type of porogen and the percentages of both porogen and cross-linker in the polymerization mixture.48-50 These variables are closely related to the solubility of the polymer molecules in the polymerization system. In contrast, much less is known about the effect of the kinetics of the polymerization that, in turn, depend on both the concentration and type of free-radical initiator and the polymerization temperature.51-56 Our systematic work concerning the effect of temperature on the porous properties of beads prepared by “classical” suspension polymerization has demonstrated that temperature is probably the most convenient variable to regulate the pore size distribution of macroporous media since it allows for significant adjustment without requiring a change in the composition of the reaction mixture.57 None of the previous studies mentioned above concerned monodisperse beads or provided a correlation between polymerization temperature and chromatographic performance of the resulting stationary phases. Therefore, we carried out a series of polymerizations with different times of reaction at temperature levels of 70 and 95 °C. The results are summarized in Table 2. In agreement with our previous findings, the median pore diameter (60.6 nm) found for beads polymerized for 24 h at a temperature of 70 °C is larger than that measured for beads prepared at 95 °C (44.2 nm). Interestingly, beads with an intermediate pore diameter of 52.7 nm are obtained after a polymerization process involving 16 h at 70 °C followed by 8 h at 95 °C. Similar observations of pore sizes located between the two extremes were made for beads polymerized consecutively for various periods of time at the two temperatures. Kinetic studies published earlier58,59 have demonstrated that all monomers present in the polymerization mixture are incorporated in the network in less than 8 h during the course of a typical suspension polymerization of glycidyl methacrylate and ethylene dimethacrylate at 70 °C in the presence of porogens. This has led to the initial perception that the porous polymer network (48) Seidl, J.; Malinsky, J.; Dusek, K.; Heitz, W. Adv. Polym. Sci. 1967, 5, 113213. (49) Guyot, A.; Bartholin, M. Prog. Polym. Sci. 1982, 8, 277-332. (50) Okay, O. Prog. Polym. Sci. 2000, 25, 711-779. (51) Horak, D.; Svec, F.; Bleha, M.; Kalal, J. Angew. Macromol. Chem. 1981, 95, 09-115. (52) Horak, D.; Svec, F.; Ilavsky, M.; Bleha, M.; Kalal, J. Angew. Macromol. Chem. 1981, 95, 116-127. (53) Takeda, K.; Akiyama, M.; Yamamizu, T. Angew. Makromol. Chem. 1988, 157, 123-136. (54) Yamamizu, T.; Akiyama, M.; Takeda, K. Kobunshi Ronbunshu 1989, 45, 29-35. (55) Reinholdsson, P.; Hargitai, T.; Isaksson, R.; To ¨rnell, B. Angew. Makromol. Chem. 1991, 192, 113-132. (56) Horak, D.; Svec, F.; Tennikova, T. B.; Nahunek, M. Angew. Macromol. Chem. 1992, 195, 139-150. (57) Svec, F.; Fre´chet, J. M. J. Macromolecules 1995, 28, 7580-7582. (58) Svec, F.; Hradil, J.; Coupek, J.; Kalal, J. Angew. Macromol. Chem. 1975, 48, 135-143. (59) Horak, D.; Svec, F.; Ribeiro, C. M. A.; Kalal, J. Angew. Macromol. Chem. 1980, 87, 127-136.

Table 2. Porous Properties of 3-µm Poly(glycidyl methacrylate-co-ethylene dimethacrylate) Beads Prepared by Polymerization Started at a Temperature of 70 °C and Continued at 95 °C

beads 15 16 17 18 19

polymerization time, h 70 °C 95 °C 24 20 16 8 0

0 4 8 16 24

Dp-L,a nm

Vp-L,b mL/g

Vp-S,c mL/g

Sg,d m2/g

efficiency,e plates/m

60.6 57.1 52.7 53.1 44.2

0.77 0.77 0.65 0.62 0.65

0.062 0.066 0.058 0.033 0.021

78.5 76.7 51.1 45.9 47.7

36 800 41 800 46 500 58 800 62 800

a Median pore diameter determined by mercury intrusion porosimetry. b Volume of pores larger than 7 nm determined by mercury intrusion porosimetry. c Volume of pores smaller than 7 nm determined by nitrogen adsorption/desorption. d Specific surface area determined by nitrogen absorption/desorption (BET). e Efficiency of column packed with diol beads obtained by hydrolysis of epoxide groups.

prepared from a mixture containing a high percentage of crosslinker is rather rigid and that no significant changes in its structure are likely to occur after complete conversion of monomers into polymer. However, a more focused study has revealed that structural rearrangements may still take place within the beads even after the disappearance of all monomers from the polymerizing droplets.59 The steep and continuous decrease in the volume of pores smaller than 7 nm accompanied by the concomitant decrease in surface area observed in the present study indicates that these structural rearrangements probably also involve the unreacted double bonds originating from the cross-linker. For example, the volume of small pores decreases from 0.062 mL/g for beads prepared by reaction at 70 °C for 24 h to only 0.033 mL/g after 8 h of polymerization at 70 °C followed by 16 h at 95 °C. At the same time, the specific surface area decreases from 78.5 to 45.9 m2/g. Such changes are not unexpected since the process of delayed incorporation of dangling double bonds in the surrounding polymeric matrix is a chemical reaction that must be affected by temperature. This observation also explains the changes in the porous structure of our beads prepared using different temperature regimes (Table 2). The temperature regime also affects the surface appearance of the beads. The surface of the beads polymerized for 24 h at 70 °C is rougher that that of beads prepared at 95 °C. However, some changes are also observed for beads prepared using a polymerization process that starts at 70 °C and continues at 95 °C. Given that the incorporation of all monomers in the polymer is completed within 8 h, it is somewhat surprising that even the surface morphology can change after heating to a higher temperature. Chromatographic evaluation of these beads also underlines the considerable changes that take place when polymerization of the beads is completed at a temperature of 95 °C. For example, the column efficiency is 36 800 plates/m for beads prepared by polymerization at 70 °C for 24 h while a value of 41 800 plates/m is achieved for beads by polymerization for 20 h at 70 °C followed by 4 h at 95 °C. Extending the polymerization time at 95 °C to 16 h while keeping the overall polymerization time at 24 h affords stationary phase with an even higher efficiency of 58 800 plates/ m. Analytical Chemistry, Vol. 75, No. 4, February 15, 2003

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Table 3. Effect of Extent of Cross-Linking on Porous Properties of 3-µm Poly(glycidyl methacrylate-co-ethylene dimethacrylate) Beads Prepared by Polymerization at a Temperature of 70 °C for 8 h Followed by 16 h at 95 °C beads

GMA

18 20 21 11 22

21.5 18.0 14.5 10.8 7.2

polymerization mixture, wt % EDMA TOa 14.5 18.0 21.5 25.2 28.8

51.5 51.5 51.5 51.5 51.5

CHa

Dp-L,b nm

Vp-L,c mL/g

Vp-S,d mL/g

Sg,e m2/g

efficiency,f plates/m

12.5 12.5 12.5 12.5 12.5

53.1 53.3 48.9 40.6 27.3

0.62 0.69 0.68 0.78 0.92

0.033 0.054 0.101 0.162 0.237

45.9 58.0 89.9 140.1 215.0

58 800 63,000 66 800 67 000 63 600

a TO, toluene; CH, cyclohexanol. b Specific surface area determined by nitrogen absorption/desorption (BET). c Volume of pores larger than 7 nm determined by mercury intrusion porosimetry. d Volume of pores smaller than 7 nm determined by nitrogen adsorption/desorption. e Median pore diameter determined by mercury intrusion porosimetry. f Efficiency of column packed with diol beads obtained by hydrolysis of epoxide groups.

Figure 6. Inverse size exclusion calibration curves for 3-µm poly(2,3-dihydroxypropyl methacrylate-co-ethylene dimethacrylate) beads prepared by hydrolysis of poly(glycidyl methacrylate-co-ethylene dimethacrylate) particles containing 40 (9), 50 (4), 60 (2), and 70% (0) ethylene dimethacrylate. Chromatographic conditions: polystyrene standards, THF, flow rate 1 mL/min.

Effect of Cross-Linking. In contrast to the effects of temperature and porogenic solvent that affect the porous properties of the resulting material without affecting its composition, variations in the monovinyl/divinyl monomer ratio induce not only the formation of different porous structures but obviously lead to beads with different compositions. A higher content of cross-linker leads to the formation of beads with both smaller microglobules and pores, therefore translating into larger surface areas. However, this also leads to a decrease in the number of desired functionalities originating from the monovinyl monomer. Experiments with polymerization mixtures containing glycidyl methacrylate and ethylene dimethacrylate in ratios between 8:2 and 4:6 (Table 3) clearly document the shift in the pore size distributions toward smaller pore sizes as the percentage of cross-linker is increased. Simultaneously, the volume of pores smaller than 7 nm increases from 0.033 to 0.237 mL/g and the specific surface area also increases from 45.9 to 215.0 m2/g. Changes in porous structure are also reflected in the inverse size exclusion chromatography calibration curves shown in Figure 6. Beads prepared with 40% ethylene dimethacrylate exhibit poor selectivity in both the low and high molecular weight ranges. Increasing the cross-linking to 50% significantly improves the 1018 Analytical Chemistry, Vol. 75, No. 4, February 15, 2003

Figure 7. Effect of cross-linking density on van Deemter plots for the columns packed with 3-µm poly(2,3-dihydroxypropyl methacrylateco-ethylene dimethacrylate) beads prepared by hydrolysis of poly(glycidyl methacrylate-co-ethylene dimethacrylate) particles. Conditions: 0.6-µm polystyrene shape templates preswollen with dibutyl phthalate; polymerization mixture glycidyl methacrylate + ethylene dimethacrylate 36%, toluene 51.5%, cyclohexanol 12.5%, initiator azobisisobutyronitrile (1% with respect to monomers); reaction time 24 h; temperature 70 °C. Ethylene dimethacrylate in polymerization mixture: 14.5 ([), 18 (9), 21.5 (2), 25.2 (0), and 28.8% (]).

selectivity in the high molecular weight range with a lesser effect on low molecular weight range. The calibration curves show little change as the percentage of cross-linker is increased further. Increasing the percentage of cross-linking monomer not only has a significant effect on the porous structure, but it also impacts column efficiency with variations between 58 800 and 67 000 plates/m being observed. The best efficiency is achieved with 70% cross-linked beads 11. More importantly, the shape of the van Deemter plots shown in Figure 7 is also affected by cross-linking. While the least cross-linked stationary phases rapidly lose their efficiency at higher flow velocities, the plot for 80% cross-linked beads is rather flat. Since the rising part of the curve is mostly affected by the mass-transfer kinetics within the pool of the stagnant mobile phase located in the pores (C term), our measurements indicate that mass transfer is enhanced despite the overall decrease in the pore size. This can be explained by the swelling of both the loose chain ends and the less cross-linked polymer layer located at the pore surface of the less cross-linked beads. Although this “swellable polymer layer” cannot be detected in measurements that are carried out in the dry state (e.g., mercury intrusion porosimetry or nitrogen adsorption/desorption)

Figure 8. Effect of composition of the mobile phase and cross-linking density of 3-µm poly(2,3-dihydroxypropyl methacrylate-co-ethylene dimethacrylate) beads prepared by hydrolysis of poly(glycidyl methacrylate-co-ethylene dimethacrylate) particles on retention factors of toluene (]), nitrobenzene (0), phenol (4). and aniline (O). Conditions: 0.6-µm polystyrene shape templates preswollen with dibutyl phthalate; polymerization mixture glycidyl methacrylate + ethylene dimethacrylate 36%, toluene 51.5%, cyclohexanol 12.5%, initiator azobisisobutyronitrile (1% with respect to monomers); reaction time 24 h; temperature 70 °C. Ethylene dimethacrylate in polymerization mixture: 14.5 (A), 18 (B), 25.2 (C), and 28.8% (D). Chromatography: column 150 × 4.6 mm; mobile phase hexane/THF; flow rate 1 mL/min; UV detection at 254 nm.

this swelling changes the effective pore size.60 However, evidence for swelling can be obtained from inverse size exclusion chromatography measurements carried out with THF as the mobile phase as shown in Figure 6. Indeed, the upper size exclusion limit for the 40% cross-linked beads is ∼320 000 while for 50% cross-linking it is ∼1 000 000 despite the equal median pore size of 53 nm measured for both types of beads in the dry state. Efficiency itself is not a sufficient characteristic for NPLC stationary phases. This must also include an evaluation of the chromatographic performance in terms of column selectivity and retention. While the former depends mainly on the type of surface chemistry, the latter is a function of both pore surface area and surface coverage with polar functionalities. Figure 8 shows the retention factors of stationary phases with diol functionalities prepared by hydrolysis of poly(glycidyl methacrylate-co-ethylene dimethacrylate) beads (Chart 1) containing various percentages of the cross-linker for four model analytess toluene, nitrobenzene, phenol, and aniline. As expected, almost no retention of toluene is seen for any of these phases regardless of mobile phase. Similarly, aniline is highly retained but its retention does not change with cross-linking. The selectivity for the separation of phenol and aniline on stationary phases 18 and 22 cross-linked with 40 and 80% ethylene dimethacrylate, respectively, is not sufficient to achieve good resolution. Except for these two stationary phases, the retention changes only little with increasing cross-linking despite the fact that surface area increases and surface coverage with diol functionalities decreases. The lack of significant change may be explained by the very high surface coverage of all of our beads with diol functionalities. The overall content of hydroxyl groups in these beads prepared from a matrix including 60% glycidyl methacrylate is 1.89 mmol/mL.29 Even if the percentage of this monomer is reduced by half in beads 22, the beads still contain 0.95 mmol/mL hydroxyl groups, which is

much more than the surface coverage of 0.16 mmol/mL measured earlier for the typical bare silica stationary phase Nucleosil.29 Despite the “dilution” of the diol functionalities, their number remains sufficient to afford good retention. Column Packing Procedure. Earlier evidence has shown that the performance of HPLC columns packed with a ODS stationary phase depends largely on packing density and external porosity, which is determined by the packing pressure.61 Since our porous polymer beads are mechanically very stable, we packed columns with diol beads 8 using a high pressure of 20 MPa and studied the effect of various pushing solvents selected from a group consisting of acetone, toluene, methanol, and tetrahydrofuran. The highest column efficiency of 35 000 plates/m was achieved with acetone. However, since acetone has a rather low viscosity, we also used it in combination with cyclohexanol to form a liquid with both increased density, which prevents the packing material from rapid sedimentation by gravity, and increased viscosity for higher friction. Therefore, mixtures of acetone with 40-80% cyclohexanol were also tested. The van Deemter plots (not shown) indicate a significant effect of the eddy diffusion and flow variations (A term) on the band spreading. The highest column efficiency of 52 000 plates/m was obtained for a column packed using a 40:60 acetone/cyclohexanol mixture. Back pressure in the column is also closely related to the quality of the packing. Figure 9 shows back pressure versus flow velocity plots for a 150 × 4.6 mm i.d. column packed with beads 18 and 11 (40 and 70% cross-linked, respectively) and for different mobile phases typical of NPLC. Since no solvation of the diol beads occurs in hexane, column permeability is highest when this solvent is used. In contrast, THF swells the beads, leading to a decrease in the size of interparticular voids and a concomitant increase in back pressure. Values obtained for the 1:1 THF/ hexane mixture are between those for the pure solvents.

(60) Svec, F. Angew. Macromol. Chem. 1986, 144, 39-49.

(61) Guan-Sajonz, H.; Guiochon, G. J. Chromatogr., A 1996, 743, 247-259.

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Figure 9. Effect of the mobile phase on back pressure at different linear flow velocities for columns packed with 3-µm poly(2,3-dihydroxypropyl methacrylate-co-ethylene dimethacrylate) beads 18 (closed points) and 11 (open points) with a cross-linking density of 40 and 70%, respectively; column equilibrated in the mobile phase for 2 h at a flow rate of 0.5 mL/min. Mobile phase: THF (diamonds), 1:1 THF/hexanes (squares), and hexanes (triangles). Figure 11. Separation of tricyclic antidepressants in column packed with 3-µm 70% cross-linked diol stationary phase obtained from poly(glycidyl methacrylate-co-ethylene dimethacrylate) beads 11. Chromatographic conditions: column 150 × 4.6 mm; mobile phase 24.8: 74.8:0.4 mixture of THF, hexane, and triethylamine; injection 10 µL of a solution containing 20 mg of each analyte in 1 mL of the mobile phase, flow rate 1 mL/min; flow rate 1 mL/min; UV detection at 254 nm. Peaks: doxepine (1), clomipramine (2), and nortriptyline (3); see Chart 1 for the structures.

Figure 10. Separation of toluene (peak 1), nitrobenzene (2), phenol (3), and aniline (4) using column packed with 3-µm 70% cross-linked diol stationary phase obtained from poly(glycidyl methacrylate-coethylene dimethacrylate) beads 11. Chromatographic conditions: column 150 × 4.6 mm; mobile phase 80:20 hexane/THF; injection 10 µL of a solution containing 20 mg of each analyte in 1 mL of the mobile phase, flow rate 1 mL/min; UV detection at 254 nm.

The straight lines observed in Figure 9 confirm the high mechanical stability of our beads since any nonlinearity of the plots would indicate compression of the stationary phase. Even a pressure of 25.5 MPa does not lead to the collapse of the bed. As expected, columns packed with the 40% cross-linked beads exhibit higher back pressure than those packed with 80% cross-linked beads since the former are more prone to swelling. Chromatographic Separations. The performance of a column packed with poly(2,3-dihydroxypropyl methacrylate-co-ethylene dimethacrylate) beads 11 is demonstrated on the separation of a 1020 Analytical Chemistry, Vol. 75, No. 4, February 15, 2003

Figure 12. Effect of composition of the mobile phase on retention of 4-hydroxybenzyl alcohol (curve 1), 2-hydroxybenzyl alcohol (2), 3-hydroxybenzyl alcohol (3), 4-nitrophenol (4), and 2-nitrophenol (5) in column packed with 3-µm 70% cross-linked diol stationary phase obtained from poly(glycidyl methacrylate-co-ethylene dimethacrylate) beads 11. Chromatographic conditions: column 150 × 4.6 mm; mobile phase hexane/THF; injection 10 µL of a solution containing 20 mg of each analyte in 1 mL of hexane/THF (1:1); flow rate 1 mL/ min; UV detection at 254 nm.

model mixture consisting of toluene, nitrobenzene, phenol, and aniline (Figure 10). All analytes are baseline separated with very high resolution. The column has an efficiency of 52 000 and 34 0000 plates/m for toluene (almost unretained) and aniline (most retained), respectively. Although a small tailing of peaks can be

documents that the retention of a column packed with poly(2,3dihydroxypropyl methacrylate-co-ethylene dimethacrylate) beads can easily be modulated through the composition of the mobile phase. The elution power provided by a typical binary solvent may not be sufficient for the separation of multicomponent mixtures. A mobile-phase gradient helps to achieve the separation of such mixtures in a reasonable period of time. Figure 13 shows the separation of six aniline derivatives using a gradient elution mixture of THF and hexane in which the percentage of THF increases linearly from 15 to 40% in 40 min. Less polar molecules such as 3,5-bis(trifluoromethyl)aniline and 4-triphenylmethylaniline, which have weaker interactions with the stationary phase, elute prior to very polar analytes such as 2,4-dimethoxyaniline and 3,4,5-trimethoxyaniline, confirming that a retention mechanism typical of normal-phase chromatography rules the separation.

Figure 13. Separation of aniline derivatives using column packed with 3-µm 70% diol stationary phase obtained from poly(glycidyl methacrylate-co-ethylene dimethacrylate) beads 11. Chromatographic conditions: column 150 × 4.6 mm; mobile phase, linear gradient of 15-40% THF in hexane in 40 min; injection 10 µL of a solution containing 35 mg of each analyte in 1 mL of hexane/THF (6:4); flow rate 1 mL/min; UV detection at 254 nm. Peaks (in order): 3,5-bis(trifluoromethyl)aniline (1), 4-triphenylmethylaniline (2), aniline (3), 3-benzyloxyaniline (4), 2,4-dimethoxyaniline (5), 3,4,5-trimethoxyaniline (6).

seen in the chromatogram, the peak asymmetry does not exceed 15%. Peak symmetry can be improved significantly by addition of a small percentage of triethylamine to the mobile phase as demonstrated in Figure 11 for the separation of the tricyclic antidepressant drugs clomipramine, doxepine, and nortriptyline (Chart 1). Baseline separation is achieved using a 24.8:74.8:0.4 mixture of THF, hexane, and triethylamine as the mobile phase. The percentage “window” for triethylamine is rather narrow since very broad and severely tailing peaks are obtained in the mobile phase with no addition of triethylamine while clomipramine and doxepine coelute at a triethylamine concentration higher than 1%. The immense selectivity typical of normal-phase HPLC that allows the separation of compounds with only very small structural differences is well known. Figure 12 shows the retention factors for positional isomers of hydroxyl-substituted benzyl alcohols and nitrophenols under isocratic conditions as examples of the selectivity of the diol beads for small molecules. Figure 12 also

CONCLUSIONS The optimization of porous and surface properties as well as cross-linking density of 3-µm methacrylate-based beads affords polymeric stationary phases useful in normal-phase HPLC. The highly cross-linked beads are characterized by their monodispersity and rigidity that enable their use in a broad range of flow rates since the column packing does not compress under the pressure required to achieve the desired flow. The diol functionalities obtained by hydrolysis of epoxide groups of poly(glycidyl methacrylate-co-ethylene dimethacrylate) beads provide for high adsorption activity in normal-phase HPLC. In contrast to typical bare silica NP stationary phases, the surface coverage of our polymeric beads with uniform diol functionalities is high and homogeneous and endows this type of polymeric stationary phase with excellent chromatographic properties. Columns packed with this polymeric medium afford efficiencies exceeding 67 000 plates/ m. Due to the high retentivity and efficiency of the separation medium and columns, even very weak interactions can be utilized for the separation. ACKNOWLEDGMENT Support of this work by the Office of Nonproliferation Research and Engineering of the U.S. Department of Energy under Contract DE-AC03-76SF00098 and the National Institute of General Medical Sciences, National Institutes of Health (GM-44885) is gratefully acknowledged. Received for review October 8, 2002. Accepted December 19, 2002. AC026216W

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