Anal. Chem. 1996, 68, 2857-2868
Retention Characteristics of Polybutadiene-Coated Zirconia and Comparison to Conventional Bonded Phases Jianwei Li and Peter W. Carr*
Department of Chemistry, Kolthoff and Smith Halls, 207 Pleasant Street, SE, University of Minnesota, Minneapolis, Minnesota 55455
This paper presents a detailed study of retention on a reversed-phase material made by coating polybutadiene (PBD) on porous zirconia. PBD-coated zirconia particles with six different carbon loads (0.25-5.6% carbon by weight) were prepared by evaporatively depositing and cross-linking PBD on microparticulate porous zirconia. Retention data of a homologous series of alkylbenzenes were obtained on the six PBD phases as a function of mobile phase composition in methanol-water and acetonitrile-water mixtures from 20 to 50% (v/v). The results obtained for the phase were compared to those for conventional octadecylsilane (ODS) bonded phases, and the effect of the amount of PBD on retention was studied in detail. We find that, per amount of bonded phase, the PBD phase is less retentive than is the ODS phase, but it has comparable hydrophobic selectivity. Furthermore, the PBD phase has about the same sensitivity toward changes in mobile phase composition as does the ODS phase, and its solute shape selectivity is similar to that of a monomeric ODS phase. Finally, we conclude that retention arises primarily from a partition-like process. Reversed-phase liquid chromatography is the most popular operating mode of liquid chromatography practiced today.1 This popularity is attributed mainly to the development of chemically stable, silica-based microparticulate bonded phases that provide rapid mass transfer and a high degree of reproducibility.1 However, a significant limitation of silica-based bonded phases is their lack of pH stability. Silica supports dissolve in basic solutions,2 and the majority of all commercial supports are also unstable in acid due to hydrolysis of siloxane bonds.3 These situations are particularly troublesome in biopreparative-scale work, in which contaminated columns must be cleaned, sterilized, and depyrogenated with acids and bases.4 There are several ways to improve or to replace silica-based supports. These methods include (1) the acid and base pretreatment of silica,5 (2) the use of sacrificial presaturation columns,6 (1) Melander, W. R.; Horva´th, C. In High-Performance Liquid Chromatographys Advances and Perspectives; Horva´th, C., Ed.; Academic Press: New York, 1980; Vol. 2, pp 113-319. (2) Krummen, K.; Frei, R. W. J. Chromatogr. 1977, 132, 27-36. (3) Glajch, J. J.; Kirkland, J. J.; Ko ¨hler, J. J. Chromatogr. 1987, 384, 81-90. (4) Weary, M.; Pearson, F. BioPharm. 1988, 1, 22-29. (5) Ko¨hler, J.; Kirkland, J. J. J. Chromatogr. 1976, 125, 115-127. (6) Atwood, J. G.; Schmidt, G. J.; Slavin, W. J. Chromatogr. 1979, 171, 109115. (7) Wickramanayake, P. P.; Chatt, A.; Aue, W. A. Can. J. Chem. 1981, 59, 10451050. S0003-2700(95)01178-4 CCC: $12.00
© 1996 American Chemical Society
(3) cladding the silica surface with alkaline-stable metal oxides such as zirconia and titania,7-15 (4) the use of mechanically and chemically stable, synthetic polymer phases based on macroporous polystyrenes and styrene-divinylbenzene copolymers (PS-DVs)16-19 and other polymeric materials,19-24 (5) the use of carbon phases in the form of activated charcoal or graphite as a reversed-phase materials,25-29 including the graphitized silica,30-36 and (6) the immobilization of nonpolar polymers such as polybutadiene,37-39 polystyrene,40-44 and others45-49 on silica and alumina as a method (8) Aigner-Held, R.; Aue, W. A.; Pickett, E. E. J. Chromatogr. 1980, 189, 139144. (9) Tomb, W. H.; Weetall, H. H. U.S. Patent 3 783 101, 1974. (10) Stout, R. W.; DeStefano, J. J. J. Chromatogr. 1985, 326, 63-78 (11) Stout, R. W. U.S. Patent 4 600 646, 1986. (12) Marsh, D. R.; Tsao, G. T. Biotechnol. Bioeng. 1976, 18, 349-362. (13) Messing, R. A.; Weetall, H. H. U.S. Patent 3 519 538, 1970. (14) Stout, R. W.; Sivakoff, S. I.; Ricker, R. D.; Palmer, H. C.; Jackson, M. A.; Odiorne, T. J. J. Chromatogr. 1986, 352, 381-397. (15) Barkatt, A.; Macedo, P. B. U.S. Patent 4 648 975, 1987. (16) Pietrzyk, D. J.; Chu, C. H. Anal. Chem. 1977, 49, 757-764. (17) Popl, M.; Dolansky´, V.; Fa¨hnrich, J. J. Chromatogr. 1978, 148, 195-201. (18) Smith, R. M. J. Chromatogr. 1984, 291, 372-376. (19) Dawkins, J. W.; Lopd, L. L.; Warner, E. P. J. Chromatogr. 1986, 352, 157167. (20) Hanai, T.; Arai, Y.; Hirukawa, M.; Noguchi, K.; Yanagihara, Y. J. Chromatogr. 1985, 349, 323-329. (21) Hirayama, C.; Ihara, H.; Yoshinga, T.; Hirayama, A.; Motozato, Y. J. Liq. Chromatogr. 1986, 9, 945-954. (22) Jandera, P.; Chura´cˇek, J.; C ˇ a´slavsky´, J.; Voja´cˇkova´, M. Chromatographia 1980, 13, 734-740. (23) Kruempelman, M.; Danielson, N. D. Anal. Chem. 1985, 57, 340-346. (24) Benson, J. R.; Woo, D. J. J. Chromatogr. Sci. 1984, 22, 386-. (25) Lin, C. K. In Advances in Chromatography; Giddings, J. C., Ed.; Marcel Dekker: New York, 19xx; Vol. 32, pp 1-20. (26) Knox, J. H.; Kaur, B. J. Chromatogr. 1986, 352, 3-25. (27) Knox, J. H.; Kaur, B. In High Performance Liquid Chromatography; Brown P. R., Hartmick, R. A., Eds.; John Wiley & Sons: New York, 1989; Chapter 4, pp 1-36. (28) Knox, J. H.; Unger, K. K.; Mueller, H. J. Liq. Chromatogr. 1983, 6, 1-36. (29) Gibert, M. T.; Knox, J. H.; Kaur, B. Chromatographia 1982, 16, 138-146. (30) Colin, H.; Eon, C.; Guiochon, G. J. Chromatogr. 1976, 119, 41-54; 1976, 122, 223-242. (31) Ciccioli, P.; Tappa, R.; di Corcia, A.; Liberti, A. J. Chromatogr. 1981, 206, 35-42. (32) Colin, H.; Guiochon, G. J. Chromatogr. 1977, 137, 19-33. (33) Plzak, Z.; Douszk, F. P.; Jansta, J. J. Chromatogr. 1978, 147, 137-142. (34) Smolkova, E.; Zima, J.; Sousek, F. P.; Jansta, J.; Plzak, Z. J. Chromatogr. 1980, 191, 61-69. (35) Unger, K.; Roumeliotis, P.; Mueller, H.; Gotz, H. J. Chromatogr. 1980, 202, 3-14. (36) Knox, J. H.; Kaur, B.; Millward, G. R. J. Chromatogr. 1986, 352, 3-25. (37) Hanson, M.; Unger, K. K.; Schomburg, G. J. Chromatogr. 1990, 517, 269284. (38) Bien-Vogelsang, U.; Deege, A.; Figge, H.; Ko ¨hler, J.; Schomburg, G. Chromatographia 1984, 19, 170-179. (39) Figge, H.; Deege, A.; Ko¨hler, J.; Schomburg, G. J. Chromatogr. 1986, 351, 393-408.
Analytical Chemistry, Vol. 68, No. 17, September 1, 1996 2857
to make a wide range of packing materials for reversed-phase HPLC. Although the above-mentioned techniques have resulted in improvements in column stability, almost all of them have drawbacks. For example, polymeric phases are generally lower in efficiency than are chemically bonded alkylsilica phases and tend to shrink and swell upon changes in solvent;24 carbon materials are limited by the low mechanical strength, unless graphitized, and by the common occurrence of irreversible adsorption.25 Nonetheless, silica and alumina modified with nonpolar polymers have shown not only enhanced stability at high pH but also practically no silanophilic interactions with strongly basic compounds.50 Although polymer-coated silicas and aluminas are more stable in alkaline solution than are uncoated materials, zirconia is a better choice as a support material. That is, zirconia has the desired physical and mechanical stability of other metal oxides, but it has much better chemical and thermal stability.51 The unique properties of zirconia have motivated us51 and others52 to develop zirconia-based supports. We have been interested in a variety of chromatographic modes based on modified zirconia.53 Rigney et al. coated polybutadiene on zirconia to prepare a reversed-phase support.54,55 They found that the polymer-coated zirconia was stable in alkaline solution; there was no evidence for degradation of the support, even when it was exposed for several hours to a mobile phase of 1 M sodium hydroxide at 100 °C. More recently, polybutadiene-coated zirconia has been effectively used to separate peptides and proteins in very acidic conditions, and the columns can be completely recovered by washing with concentrated acids and bases.56 The purpose of the present study is to gain a better understanding of retention on polybutadiene-coated zirconia, including absolute and relative retention, sensitivity of retention toward changes in the mobile phase, solute shape selectivity, hydrophobicity, and the nature of the retention process. In addition, conventional silica-based ODS and zirconia-based PBD phases are compared. (40) Kurganov, A. A.; Kuzmenko, O.; Davankov, V. A.; Eray, B.; Unger, K. K.; Tru ¨ dinger, U. J. Chromatogr. 1990, 506, 391-400. (41) Harino, H.; Kimura, K.; Tanaka, M.; Shono, T. J. Chromatogr. 1990, 522, 107-116. (42) Kurganov, A. A.; Tevlin, A.; Davankov, V. A. J. Chromatogr. 1983, 261, 223-233. (43) Davankov, V. A.; Kurganov, A. A.; Unger, K. K. J. Chromatogr. 1990, 500, 519-530. (44) Abuelafiya, R.; Pesek, J. J. J. Liq. Chromatogr. 1989, 12, 1571-1578. (45) Saburov, V. V.; Muidinov, M. C.; Guryanov, S. A.; Kataev, A. D.; Turkin, S. I.; Zubov, V. B. Zh. Fiz. Khim. 1991, 65, 2692-2698. (46) Sander, L. C.; Wise, S. A. J. Chromatogr. 1984, 316, 163-181. (47) Schomburg, G.; Deege, A.; Ko¨hler, J.; Bien-Vogelsang, J. J. Chromatogr. 1983, 282, 27-39. (48) Schomburg, G.; Ko ¨hler, J.; Figge, H.; Deege, A.; Bien-Vogelsang, U. Chromatographia 1984, 18, 265-274. (49) Stewart, H. N. M.; Perry, S. G. J. Chromatogr. 1968, 37, 97-98. (50) Petro, M.; Berek, D. Chromatographia 1993, 37, 549-561. (51) Nawrocki, J.; Rigney, M. R.; McCormick, A.; Carr, P. W. J. Chromatogr. 1993, 657, 229-282. (52) Hanggi, D. A.; Marks, N. R. LC-GC 1993, 11, 128-138. (53) Nawrocki, J.; Dunlap, C. J.; Carr, P. W.; Blackwell, J. A. Biotechnol. Prog. 1994, 10, 561-573. (54) Rigney, M. P.; Weber, T. P.; Carr, P. W. J. Chromatogr. 1989, 484, 273291. (55) Rigney, M. P.; Funkenbusch, E. F.; Carr, P. W. J. Chromatogr. 1990, 499, 291-304. (56) Sun, L. Ph.D. Thesis, University of Minnesota, Minneapolis, MN, 1994.
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THEORETICAL SECTION It is well known in reversed-phase liquid chromatography that, over a reasonable range in mobile phase composition, the capacity factor of a homologous series of solutes can be approximately described by57
ln k′ ) a + bnCH2 + cφ + dnCH2φ
(1)
Here, nCH2 is the number of methylene groups, φ is the volume fraction of organic modifiers in the mobile phase, and a, b, c, and d are empirical fitting coefficients. From this equation, we can derive several related equations under specific situations. First, when the composition of mobile phase is fixed, eq 1 becomes
ln k′ ) (a + cφ) + (b + dφ)nCH2 ) A + BnCH2
(2)
A is the absolute retention, and the slope B is the hydrophobic selectivity.58,59 Equation 2 allows us to compare the absolute and relative retention of two stationary phases at a given mobile phase composition. In addition, the slope B is proportional to the free energy of transfer (∆G°) per methylene group from the mobile phase to the stationary phase (∆G° ) -BRT; R is the gas constant, and T is the temperature).60 Second, if the solute is fixed, eq 1 yields
ln k′ ) (a + bnCH2) + (c + dnCH2)φ ) C + Dφ
(3)
C is the intercept, and D is the slope related to the sensitivity of retention to the mobile phase composition. Equation 3 can be rewritten in a more conventional manner:61-65
log k′ ) log k′w - Sφ
(4)
Here, k′w is the extrapolated capacity factor of a solute when the mobile phase is pure water, and S is -D/2.303. Since k′w refers to retention in pure water (φ ) 0), it should be independent of the nature of the modifier. In general, this is not so and is a sign of nonlinearity of eq 4.66 We will use eq 4 to compare the absolute mobile phase sensitivities. Third, the differentiation of eq 2 with respect to nCH2 at a fixed mobile phase concentration for a homologous series generates (57) Cozk, M.; Engelhardt, H. Chromatographia 1989, 27, 5-14. (58) Karger, B.; Gant, J. R.; Hartkopf, A.; Weiner, P. H. J. Chromatogr. 1976, 128, 65-78. (59) Vigh, G.; Varga-Puchony, Z. J. Chromatogr. 1980, 196, 1-9. (60) Melander, W. R.; Horva´th, Cs. Chromatographia 1982, 15, 86-90. (61) Jandera, P.; Chura´cˇek, J. J. Chromatogr. 1974, 91, 207-221. (62) Tijssen, R.; Billeiet, H. A. H.; Schoenmakers, P. J. J. Chromatogr. 1976, 128, 65-78. (63) Schoenmakers, P. J.; Billeiet, H. A. H.; Tijssen, R.; de Galan, L. J. Chromatogr. 1978, 149, 519-537. (64) Jandera, P.; Chura´cˇek, J. In Advances in Chromatography; Giddings, J. C., Ed.; Marcel Dekker: New York, 1981; Vol. 19, pp 125-260. (65) Jandera, P.; Chura´cˇek, J.; Svoboda, L. J. Chromatogr. 1979, 174, 35-50. (66) Schoenmakers, P. J.; Billiet, A. H.; de Galan, L. J. Chromatogr. 1983, 282, 107-121.
ln
[
]
k′(nCH2 + 1) k′(nCH2)
) ln RCH2 ) b + dCH2φ
(5)
Here, RCH2 is the k′ of a methylene group (methylene group selectivity), and dCH2 is the sensitivity of a methylene group to changes in mobile phase composition on a given stationary phase. Next we consider how the absolute retention is related to the retention process. For the moment, we will simply assume that the retention of a homologous series of alkylbenzenes on PBD-coated zirconia can be treated as a partition process. That is, we treat both the mobile and stationary phases as bulk liquids. The capacity factor (k′), as given by a partition model, is1
k′ ≡
ns Vs Cs ) ) ΦpKp nm Vm Cm
(6)
n, V, and C denote the number of moles of a solute, the volume of a phase, and the concentration of a solute at equilibrium, respectively; subscripts s and m denote the stationary and mobile phases, respectively; Kp is the partition coefficient; and Φp is the phase ratio. Furthermore, the partition coefficient should be independent of polymer load: Kp ) k′/Φp ) constant. Kp will be used later to examine the retention process on the PBD phase and to compare the absolute retention on the PBD and ODS phases. EXPERIMENTAL SECTION Preparation of PBD-Coated Zirconia and Columns. PBDcoated zirconia was prepared by depositing PBD on bare zirconia according to the protocol of Sun.67 The zirconia particles (PICP7) were synthesized by the polymerization-induced colloid aggregation method.68 The postsynthetic treatments included curing at 170 °C for 24 h, followed by pyrolysis at 375 °C for 2 h in air. The particles were then sintered at 750 °C for 6 h and at 900 °C for 3 h. Finally, the sintered particles were washed in 0.5 M nitric acid and then 0.5 M sodium hydroxide. The bare zirconia particles are about 2.5 µm in diameter (SEM) and have a pore size of 200 Å, a surface area of 34 m2/g, and a pore volume of 0.17 mL/g (N2 BET). Table 1 shows the characteristics of these materials, including carbon (polymer) loads obtained by elemental analysis. The zirconia particles were used to pack columns of different dimensions (100 × 4.6, 50 × 4.6, and 50 mm × 2.1 mm i.d.) by an upward stirred slurry method.67 The packing conditions were exactly the same as Sun’s.67 All column blanks, distributors, and frits were obtained from Alltech (Alltech Associates Inc., Deerfield, IL). Physical Characterization of PBD-Coated Zirconia Particles. BET nitrogen experiments were carried out using a Micrometrics ASAP 2000 (Micromeritics Instrument Corp., Norcross, GA) to determine the surface characteristics. Prior to BET analysis, about 1 g of each material was heated at 150 °C under vacuum for 8 h to remove any adsorbed gases. The results were computed by both the BET and BJH methods.69,70 (67) Sun, L.; McCormick, A.; Carr, P. W. J. Chromatogr. 1994, 658, 464-473. (68) Sun, L.; Annen, M. J.; Porras, F. L.; Carr, P. W.; McCormick, A. J. Colloid Interface Sci. 1994, 163, 464-473. (69) Sing, S. J.; Gregg, K. S. W. Adsorption Surface Area and Porosity, Academic Press, NY, 1982.
Methanol-water mixtures were used in particle wetting experiments. A small amount of PBD-zirconia was added to the wetting solvent in a vial and was then vigorously shaken. If the particles remained suspended for a considerable time, we concluded that the particles were not fully wetted. For the phosphate adsorption experiments, about 0.1 g of each type of particle was weighed in a vial, and then 10 mL of 1.25 mM/L of phenyl phosphate (C6H5PO42-) in 50:50 (v/v) methanolwater was added to each vial. The vials were sonicated under vacuum for 10 min and then shaken vigorously by hand every 30 min for the next 3 h. Thereafter, the slurry was allowed to settle and equilibrate for more than a week. Finally, the supernatant was withdrawn and analyzed for phosphorus by ICP. Chromatographic Apparatus. All chromatographic experiments were carried out on a Hewlett Packard 1090 liquid chromatograph with an autosampler, a temperature controller, a filter photometric UV detector, and a 3393 integrator-based data station (Hewlett Packard S.A., Wilmington, DE). All chromatographic operations and data collection were performed using the data station. Reagents. All reagents used were obtained from commercial sources and were reagent grade or better, unless noted below. Polybutadiene of molecular weight 5000 (20% vinyl, 80% cis- and trans-1,4-) and dicumyl peroxide were from Aldrich (Aldrich Chemical Co., Milwaukee, WI). Hexane (EM Sciences, Gibbstown, NJ), toluene (Fisher Chemical Co., Fair Lawn, NJ), and 2-propanol (Mallinckrodt Chemical Co., Paris, KY) were all HPLC grade. Organic solvents used in liquid chromatography were ChromAR HPLC grade methanol and acetonitrile. Deionized (DI) water was filtered by a filtration apparatus with a 0.45 µm filter (Gelman Sciences Inc., Ann Arbor, MI) and then boiled to remove carbon dioxide before use. All solvents were filtered a second time on a 0.45 µm filtration disk. Methanol was used to dissolve all the test solutes. Solutes used in this study were mainly a homologous series of alkylbenzenes (Aldrich). Deuterium oxide (D2O) and deuterated methanol and acetonitrile (CIL Cambridge Isotope Laboratories, Woburn, MA) were used to determine the column void volume. Three PAHs (SRM 869) were used to test the solute shape selectivity of the PBD phase: benzo[a]pyrene (BaP), 1,2:3,4:5,6: 7,8-tetrabenzonaphthalene (TBN), also known as dibenzo[g,p]chrysene, and phenanthro[3,4-c]phenanthrene (PhPh). They were donated by NIST (National Institute of Standard and Technology, Office of Reference Materials, Gaithersburg, MD). Chromatographic Conditions. We usually used 100 mm × 4.6 mm i.d. columns to obtain the retention data; however, short columns were sometimes used for PBD phases with high polymer loads and low organic mobile phases. Four sets of retention data were collected in this study. The first set of data was collected with a homologous series of small alkylbenzenes for all six PBD phases in Table 1. The mobile phase compositions were 50, 40, 30, and 20% (v/v) of organic modifiers (methanol and ACN). When retention times were too short for these solutes on 100 mm × 4.6 mm i.d. columns, and the intercept and slope of ln k′ vs nCH2 could not be reliably determined, higher homologues were used. All experiments were duplicated. The second set of retention data was obtained for a homologous series of 16 alkylbenzenes on columns of 5.6, 2.7, and 1.5% (w/w) carbon, and (70) Barret, E. P.; Joyner, L. G.; Halenda, P. P. J. Am. Chem. Soc. 1951, 73, 373-380.
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Table 1. Characteristics of PBD-Coated Zirconia with Different Carbon Loadsa %PBD addedb
%carbon loadc
VPBD (mL/g)d
VPBD/VBETd,e (%)
hydrophobicityf
Ag (m2/g)
Vporeg (mL/g)
Dporeg (Å)
dfd,h (Å)
0.0i 0.5 1.0 2.0 2.0j 4.0 6.0 8.0
0.0 0.25 0.75 1.53 2.00 2.68 3.91 5.59
0.0 0.0033 0.0098 0.016 0.026 0.035 0.051 0.074
0.0 1.9 5.8 9.4 15.3 20.6 30.0 43.2
na na 5 10 na 20 30 40
34.3 31.9 28.3 28.1 26.0 23.1 20.6 14.7
0.170 0.178 0.151 0.147 0.137 0.125 0.126 0.094
198 223 214 209 210 217 245 256
0.0 1.0 2.9 4.7 7.6 10.2 14.9 21.4
a Experimental conditions: 120 mL of HPLC grade hexane as solvent; 12.3 g of zirconia (PICA7); the weight of cross-linking initiator (DCP) was 2.5% of the weight of PBD added. b Relative amount of PBD (MW 5000) added to zirconia. c Relative amount of carbon on zirconia obtained by elemental analysis. d Volume of PBD on zirconia calculated from the amount of immobilized PBD, assuming no change in PBD density (0.89 g/mL) as a result of immobilization and cross-linking. e Fraction of interior volume occupied by PBD calculated from zirconia pore volume (0.17 mL/g based on BET adsorption of N2) and the amount of immobilized PBD. f Hydrophobicity of PBD-coated zirconia particles as percent of methanol needed to wet particles. More than 50% methanol is needed to wet ODS-silica particles. na, not available. g BET adsorption results. A is the specific surface area, Vpore is the specific pore volume, and Dpore is the pore diameter, computed as 4Vpore/A.69,70 h df is film thickness calculated from zirconia’s surface area and the amount of immobilized PBD, assuming formation of a continuous, homogeneous flat film. i Uncoated zirconia. j A coating not chromatographically studied.
the mobile phases were 75, 65, and 60% (v/v) ACN, respectively. The third set of data was collected on three carbon loads (5.6, 2.7, and 1.5% (w/w)) at a mobile phase composition of 85% ACN to evaluate the solute shape selectivity of PBD phase. Finally, the fourth set of retention data of benzene and toluene from 0 to 100% (v/v) of two organic modifiers was obtained on a PBD phase of 5.6% carbon. k′ values at high volumes of organic modifiers were estimated by extrapolation based on the higher homologues. For most experiments, the injection volume was typically 1 µL, the concentrations of test solutes were usually 3 mg/mL, and the flow rate was 1 mL/min. Chromatograms were detected by at 254 nm. Operation temperature was controlled at 25 ( 0.3 °C. Column void volumes were obtained by measuring the retention volumes of distinguishable isotopes of all of the components of the eluent, as proposed by Knox.71 All peaks were detected by a HP 1047A RI detector (Hewlette Packard), and they were then used to compute the void volume. The extracolumn volume was determined by the injection of D2O in the methanol-water mobile phase (50% methanol) without a column. RESULTS AND DISCUSSION Physical Characteristics of PBD-Coated Zirconia. Polymer Loading on the Zirconia Particles. Both the retention and efficiency of a polymeric stationary phase on a porous particle support can be significantly affected by the amount of polymer impregnated into the pores.50 A very small amount of polymer will be unable to fully cover the particle surface. However, an excessive amount of polymer will not produce uniform coating and may even block pores. Column efficiency would probably be poor in both situations. To at least partly characterize the PBD coatings, we carried out elemental analysis to determine the gross amount of loaded polymer. The results are shown in Table 1. Figure 1 is a plot of the amount of the polymer loaded and the amount of polymer offered to the zirconia. It is obvious from Figure 1 that the amount of polymer deposited is nearly linearly proportional to the amount of PBD offered to zirconia over the range (0-8% w/w) examined (see Table 1). The slope of this plot is 0.7, indicating that about 70% of the polymer offered to the zirconia is permanently incorporated. The linear relationship in Figure 1 also suggests that the pore (71) Knox, J. H.; Kaliszan, R. J. Chromatogr. 1985, 349, 211-234.
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Figure 1. Plot of the carbon load vs the amount of PBD offered to zirconia. Both axes are denoted by weight percent relative to zirconia. The solid line is the linear regression fit with a slope of 0.7.
space is not completely filled even at the highest amount of polymer. This conclusion is also supported by the calculated fraction of the interior volume occupied by the polymer (see Table 1). At 5.6% (w/w) carbon, less than 43% of the pore space is occupied by polymer. It should be mentioned at this point that we have also carried out 13C solid state NMR experiments on PBD-coated zirconia to determine the degree of cross-linking for the six PBD phases. We found that, on average, about 50% of the carbon-carbon double bonds of the polymer disappear upon cross-linking, i.e., the degree of cross-linking is, on average, about 50%. In addition, at the lower weight fractions of polymer, the degree of crosslinking was greater. BET Surface Analysis of PBD-Coated Zirconia Particles. To examine how the surface area, pore volume, and pore diameter of the particles vary with the amount of polymer deposited, we conducted BET measurements (see Table 1). Figure 2 illustrates how the amount of carbon loaded affects the surface characteristics of the particles. As the amount of polymer is increased, both the surface area and pore volume decrease approximately linearly. Consequently, the first conclusion we draw from Figure 2 is that the pores on the PBD-coated zirconia particles are not entirely filled by the amount of polymer used here. This agrees with the data on elemental analysis (see Figure 1). We also include in Figure 2 a theoretical line (dot-dash line) for the pore
Figure 2. Plots of the surface area and pore volume as a function of the carbon load. Symbols: 9 and the right ordinate, the surface area; 0 and the left ordinate, the pore volume. The dotted and solid lines are the least-squares fits to the surface area and pore volume data, respectively. The dash-dot line denotes the pore volume theoretically predicted by the amount of polymer loaded (see Table 1).
volume computed on the basis of the weight of carbon loaded onto the zirconia. The theoretical line almost completely overlaps the linear regression line for the measured pore volume. This implies that all the pores, big and small, are fully accessible to the nitrogen in the BET measurements, and that the polymer has not merely blocked pore openings without actually occupying the pore space. This interpretation is supported by plots of pore volume and surface area against the pore diameter at the six different carbon loads. Parts A and B of Figure 3 show the differential pore volume and incremental surface area against pore diameter, respectively. Although we have carried out BET measurements on eight zirconia samples (see Table 1), only the results on three samples are included in the figure for clarity. Figure 3 clearly show that all pores, large and small, contribute to the total pore volume and surface area for all carbon loads. However, the small pores (e.g., 8) bonded phase is primarily attributed to partition effects.80 Before we discuss the retention mechanism on PBD phases, let us summarize the related findings so far. As the amount of polymer on zirconia is increased, the hydrophobic selectivity (B) remains virtually constant; however, the absolute retention (k′) on the PBD phases increases monotonically with the amount of carbon loaded. It should be mentioned here that, compared with bonded phase (relative retention varies with chain length), the
relative retention of the PBD phase clearly demonstrates that there are only minor changes in the retention process for PBD phases as the amount of coating is varied (see Figure 7). Partition versus Adsorption Models on the PBD Phase. If we consider that the retention process on a PBD phase is partitionlike, then as the amount of polymer loaded is increased, the phase ratio (Φp) must increase, and this will result in an increase in k′, as suggested by eq 6. However, for an adsorption process, the phase ratio is defined as the ratio of surface area of the stationary phase to volume of the mobile phase. As suggested by the BET data (Table 1) and the data related to the mobile phase volume (Figure 4), the surface area of PBD-coated zirconia decreases significantly (up to 57% relative to bare zirconia) as the amount of carbon increases; however, the volume (Vm) of the mobile phase only decreases, on average, about 0.22 mL (about 16% relative to the bare zirconia column). Thus, the phase ratio for an adsorption process should decrease as the amount of carbon is increased. This implies that the observed increase in k′ with carbon load for an adsorption model must be due to a significant increase in the distribution equilibrium constant. In other words, the adsorption energy of nonpolar solutes on the PBD phase must increase significantly as the carbon load increases to account for the increase in k′. This is physically unreasonable. As the amount of carbon goes up, there is less interfacial effect from zirconia on solute adsorption. Moreover, since the solutes used in this study are alkylbenzenes, they should suffer minimal interactions with Lewis acid sites on zirconia surface.51 In fact, we have measured the retention of alkylbenzenes on PBD phases with the same composition of organic modifiers as used in this study, but with fluoride added to block Lewis acid sites. We found that there are only negligible differences in the retention of alkylbenzenes with and without Lewis bases in the mobile phases. Thus, we conclude that residual interactions with zirconia’s surface are negligible. From the above discussion, we qualitatively conclude that the partition effect is the dominant retention process on the PBD phase. Next we will present more quantitative evidence to support the partition model. The free energy of transfer of a methylene group from a mobile phase to a stationary phase is -BRT,60 and it should be comparable to that for a similar ideal partition system. Here, we chose an aqueous phase (consisting of a mixture of methanol and water) in contact with a bulk liquid hexadecane organic phase as our model system,86 and the relevant data are shown in Table 2. The data in Table 2 clearly indicate that the free energy of transfer of a methylene group to the PBD phase is very close to that found for a bulk hexadecane liquid phase, and, most importantly, the PBD phase is even closer to the bulk phase than is a polymeric ODS phase. Because the free energies of transfer of a methylene group to the three phases (PBD, ODS, and hexadecane) are comparable (Table 2), the retention mechanism on the ODS phase is a partition process,79 and hexadecane is a pure liquid phase, we can draw the conclusion that the retention on the PBD phase is a partition process. Partition Coefficient of Benzene between Methanol-Water Mobile Phase and the PBD Stationary Phase. If a partition process is the retention mechanism on the PBD phase, then the free energy of transfer of a solute from a mobile phase to the PBD phase should also be comparable to that of an ideal partition-like system. Here, again, we use the hexadecane partition system as the reference
(85) Dill, K. A. J. Phys. Chem. 1987, 91, 1980-1988.
(86) Li, J. Ph.D. Thesis, University of Minnesota, Minneapolis, MN, 1992.
Table 5. Evaluation of Solute Shape Selectivity of PBD-Coated Zirconiaa polymer loadb
BaPc
1.53 2.68 5.59
0.32 0.77 1.97
capacity factor (k′) PhPhd TBNe 0.36 0.84 2.15
0.99 2.43 6.56
selectivity, k′TBN/k′BaP 3.13 3.18 3.33
a Based on the test results by SRM 869 sample.83 The mobile phase consisted of 85% ACN and 15% water by volume. b Carbon load on zirconia by elemental analysis, see Table 1. c Benzo[a]pyrene solute. d Phenanthro[3,4-c]phenanthrene solute. e 1,2:3,4:5,6:7,8-Tetrabenzonaphthalene solute.
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Figure 11. Plot of the ratio of the partition coefficients vs the volume fraction of methanol. The ratio of partition coefficients is defined as KRPLC(mobilefstationary)/Khexadecane(mobilefhexadecane), in which KRPLC values computed as (k′/Φp) are listed in Table 2. Khexadecane values are computed from the work of Li.86 The data for the ODS phases are computed on the basis of the results of Cozk and Engelhardt.57 Symbols: 9, PBD phase; b, polymeric ODS phase; and 2 monomeric ODS phase.
system, and Khexadecane(mobilefhexadecane) values are computed on the basis of the work of Li.86 Based on eq 6 for a partition process, the partition coefficient (Kp ≡ k′/Φp) should be independent of the amount of carbon. The data in the second part of Table 2 (partition coefficients computed for all polymer loads and mobile phase compositions in methanol-water mobile phase) confirm our view that retention on PBD-zirconia is primarily due to partitioning. The partition coefficients of benzene are, indeed, independent of the polymer load and are affected only by the mobile phase compositions. We therefore conclude that the increase in k′ of nonpolar solutes with carbon load results mainly from an increase in the phase ratio. Next, we compare the partition coefficients of benzene obtained on a PBD phase to those with hexadecane liquid phase; the ratios of the partition coefficients [KRPLC(mobilefstationary)/Khexadecane(mobilefhexadecane)] are shown in Figure 11. For the purpose of comparison, we have also included data for ODS phases in the figure. We can see from Figure 11 that the ratio for the PBD phase is constant at about 0.5, indicating that the partition coefficient of benzene between the mobile phase and the PBD phase is surprisingly close to that between the mobile phase and bulk hexadecane. The lack of exact agreement may be due to many factors, not the least of which is residual unsaturation in (87) Martire, D. E.; Boehm, R. E. J. Phys. Chem. 1983, 87, 1045-1062. (88) Bo¨hmer, M. R.; Koopal, L. K.; Tijssen, R. J. Phys. Chem. 1991, 95, 62856297.
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the PBD phase. It is important to emphasize that the ratios for the ODS phases are between 2 and 5, and vary with the mobile phase composition. Even though the ratios are large, the dominant retention mechanism on the ODS phase is still a partition process.1,79,87 As an aside, it is somewhat surprising that the absolute partition coefficient of benzene into the ODS phase exceeds that into the bulk hexadecane phase, since the bonded phase is more ordered and should suffer a greater decrease in entropy upon solute insertion.87,88 Accordingly, we can conclude that the retention process on the PBD phase is a partition process, considering the facts that (1) the hexadecane system is a pure partition system and (2) the retention mechanism on the ODS phase is a partition process, too. In summary, both qualitative and quantitative evidence strongly support the conclusion that the partition process is dominant retention mechanism on the PBD phase. CONCLUSIONS In this study, the retention characteristics of PBD-coated zirconia have been determined, and some of its features have been compared to those of conventional ODS phases. The PBD phase acts as a true reversed chromatographic stationary phase. It has a hydrophobic selectivity very comparable to that of an ODS phase but is overall less retentive toward nonpolar solutes than are ODS phases. The sensitivities of both PBD and ODS phases toward a change in the mobile phase composition are similar; however, the PBD phase tends to be less sensitive to solute shape than is an ODS phase. The similarity between conventional ODS phases and PBD-coated phases suggests that it should be relatively straightforward to adopt analytical methods based on conventional phases to these new, highly stable phases. Finally, the retention process for nonpolar solutes on PBD phases is best described as a partition process. ACKNOWLEDGMENT The authors gratefully acknowledge the support by Grant GM 45988-05 from the National Institutes of Health. We also thank Dr. Eric Munson and Mr. Matt Doscotch for running 13C solid state NMR on our PBD-coated zirconia samples. Finally, we thank Professor Alon McCormick from the Department of Chemical Engineering and Material Sciences, University of Minnesota, for his help in understanding our BET data. Received for review December 5, 1995. Accepted May 23, 1996.X AC951178K X
Abstract published in Advance ACS Abstracts, July 1, 1996.
