Characterization and Evaluation of C18 HPLC Stationary Phases

Waters Corporation, 34 Maple Street, Milford, Massachusetts 01757-3696. The characterization and evaluation of three novel 5-µm. HPLC column packings...
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Anal. Chem. 2003, 75, 6781-6788

Characterization and Evaluation of C18 HPLC Stationary Phases Based on Ethyl-Bridged Hybrid Organic/Inorganic Particles Kevin D. Wyndham,* John E. O’Gara, Thomas H. Walter, Kenneth H. Glose, Nicole L. Lawrence, Bonnie A. Alden, Gary S. Izzo, Christopher J. Hudalla, and Pamela C. Iraneta

Waters Corporation, 34 Maple Street, Milford, Massachusetts 01757-3696

The characterization and evaluation of three novel 5-µm HPLC column packings, prepared using ethyl-bridged hybrid organic/inorganic materials, is described. These highly spherical hybrid particles, which vary in specific surface area (140, 187, and 270 m2/g) and average pore diameter (185, 148, and 108 Å), were characterized by elemental analysis, SEM, and nitrogen sorption analysis and were chemically modified in a two-step process using octadecyltrichlorosilane and trimethylchlorosilane. The resultant bonded materials had an octadecyl surface concentration of 3.17-3.35 µmol/m2, which is comparable to the coverage obtained for an identically bonded silica particle (3.44 µmol/m2) that had a surface area of 344 m2/g. These hybrid materials were shown to have sufficient mechanical strength under conditions normally employed for traditional reversed-phase HPLC applications, using a high-pressure column flow test. The chromatographic properties of the C18 bonded hybrid phases were compared to a C18 bonded silica using a variety of neutral and basic analytes under the same mobile-phase conditions. The hybrid phases exhibited similar selectivity to the silica-based column, yet had improved peak tailing factors for the basic analytes. Column retentivity increased with increasing particle surface area. Elevated pH aging studies of these hybrid materials showed dramatic improvement in chemical stability for both bonded and unbonded hybrid materials compared to the C18 bonded silica phase, as determined by monitoring the loss in column efficiency through 140-h exposure to a pH 10 triethylamine mobile phase at 50 °C. In recent years, a large number of improvements have been realized for reversed-phase HPLC packing materials that address chromatographic problems attributed to either the silica support or the bonded phase.1 For example, the widespread use of highpurity silica has greatly reduced the peak tailing due to metallic impurities (i.e., Fe, Na, Al). Updated bonded-phase chemistries based on trifunctional (i.e., CnH2n+1SiCl3, where n ) 8, 18)2 or sterically hindered monofunctional silanes (i.e., CnH2n+1SiR2Cl, * To whom correspondence should be addressed: (e-mail) kwyndham@ waters.com. (1) (a) Majors, R. E. LC-GC 1997, 15, 1008-1015. (b) Ko ¨hler, J.; Kirkland, J. J. J. Chromatogr. 1987, 385, 125-150. 10.1021/ac034767w CCC: $25.00 Published on Web 11/15/2003

© 2003 American Chemical Society

where n ) 8, 18 and R ) isopropyl or isobutyl)3 have both been shown to greatly increase the chemical stability of silica-based reversed-phase columns when exposed to low-pH mobile phases (pH 1-3). Embedded polar group bonded phases (e.g., amides, carbamates, ureas) also have been used to reduce peak tailing factors of basic analytes and to modify the selectivity of separations on silica-based reversed-phase columns.4 As a result of these improvements, silica-based packing materials today offer high chromatographic efficiency and excellent mechanical stability. However, contemporary silica-based reversed-phase HPLC packing materials still have two major limitations: peak tailing of basic analytes due to interactions with residual silanol groups and poor chemical stability in mobile phases outside of the pH 2-8 range.3,5 When mobile phases are used with pH 8, particle erosion can occur due to the dissolution of the base silica particle, which can results in a loss of column efficiency, an increase in column back pressure, and bed collapse of the silica packing material.6 The improvement of particle stability of bonded silica particles for alkaline pH chromatographic applications has proven to be a challenging problem. Increasing the density of a C18 bonded phase on silica provides little deterrent toward particle dissolution for silica particles exposed to pH >8 mobile phases for extended periods of time. While the recent use of polymerized bonded phases on silica has shown some improvement in particle and bonded phase stability at low pH, the use of such materials may result in columns that have lower efficiency and display poor mass transfer.7 Many groups have started to explore base particles other (2) Sagliano, J. N.; Floyd, T. R.; Hartwick, R. A.; Dibussolo, J. M.; Miller, N. T. J. Chromatogr., A 1988, 443, 155-172. (3) Kirkland, J. J.; Glajch, J. L.; Farlee, R. D. Anal. Chem. 1989, 61, 2-11. (4) (a) O’Gara, J. E.; Walsh, D. P.; Phoebe, C. H., Jr.; Alden, B. A.; Bouvier, E. S. P.; Iraneta, P. C.; Capparella, M.; Walter, T. H. LC-GC 2001, 19, 632642. (b) O’Gara, J. E.; Alden, B. A.; Walter, T. H.; Petersen, J. S.; Niederla¨nder, C. L.; Neue, U. D. Anal. Chem. 1995, 67, 3809-3813. (c) Czajkowska, T.; Jaroniec, M.; Buszewski, B. J. Chromatogr., A 1996, 728, 213-224. (d) Czajkowska, T.; Hrabovskya, I.; Buszewski, B.; Gilpin, R. K.; Jaroniec, M. J. Chromatogr., A 1995, 691, 217-224. (e) Czajkowska, T.; Jaroniec, M. J. Liq. Chromatogr. 1996, 19, 2829-2841. (f) Czajkowska, T.; Jaroniec, M. J. Chromatogr., A 1997, 147, 147-158. (5) (a) Nawrocki, J. J. Chromatogr., A 1997, 779, 29-71. (b) McCalley, D. V. J. Chromatogr. 1997, 769, 169-178. (c) McCalley, D. V. LC-GC 1999, 17, 440-455. (6) Kirkland, J. J.; Henderson, J. W.; De Stefano, J. J.; van Straten, M. A.; Claessens, H. A. J. Chromatogr., A 1997, 762, 97-112.

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than silica in order to access the upper half of the pH scale for reversed-phase HPLC separations. Chromatographic columns based on organic polymers, graphitic carbon, alumina, titania, and zirconia have been demonstrated to have little or no performance degradation when employed with high-pH mobile phases (pH 8-14) at elevated temperatures (60-120 °C).8,9 Unfortunately, the use of these nonsiliceous materials often presents other complications to reversed-phase chromatography. Organic polymers generally exhibit significantly reduced efficiency and decreased mechanical stability. Graphitic carbon shows strong adsorption for many polar compounds and inferior peak shapes for many analytes. Alternative metal oxides also have been shown to exhibit strong adsorption of acids or bases due to acidic or basic surface sites. These alternative materials all suffer from lack of predictable chromatographic performance using typical C18 silica methods (i.e., mobile phase, pH, solvent additives), which makes method development difficult. As a means of developing reversed-phase columns that are chemically stable toward higher pH mobile phases, we have recently explored the use of a unique class of organic/inorganic hybrid materials (denoted hybrids).10 This class of hybrid materials is normally prepared from a sol-gel synthesis of monosubstituted organofunctional silanes (e.g., YSi(OR)3, where Y ) alkyl or aryl and R ) methyl or ethyl) or internal organofunctional bridging disilanes (e.g., (RO)3Si-X-Si(OR)3, where X ) alkyl or aryl and R ) methyl or ethyl) in either a homocondensation or mixed condensation with tetraethoxysilane (TEOS).11 Hybrid materials have been employed for the synthesis of monoliths and thin films12 and nonporous and mesoporous particles,13 as well as used as coatings for capillary GC columns and open tubular LC columns.14 In our initial development of spherical porous hybrid particles for use as reversed-phase packing materials, we explored the co(7) (a) Petro, M.; Berek, D. Chromatographia 1993, 37, 549-561. (b) Ohtsu, Y.; Shiojima, Y.; Okumura, T.; Koyama, J.-I.; Nakamura, K.; Nakata, O.; Kimata, K.; Tanaka, N. J. Chromatogr. 1989, 481, 147-157. (c) Trammell, B.; Ma, L.; Luo, H.; Jin, D.; Hillmyer, M.; Carr, P. Anal. Chem. 2002, 74, 4634-4639. (8) (a) Tanaka, N.; Araki, M. Adv. Chromatogr. 1989, 30, 81-122. (b) Knox, J. H.; Kaur, R. P. Adv. Chromatogr. 1997, 37, 73-119. (9) (a) Haky, J. E.; Vemulapalli, S.; Wieserman, L. F. J. Chromatogr. 1990, 505, 307-318. (b) Tru ¨ dinger, U.; Mu ¨ ller, G.; Unger, K. K. J. Chromatogr. 1990, 535, 111-125. (c) Nawrocki, J.; Rigney, M. P.; McCormick, A.; Carr, P. J. Chromatogr., A 1993, 657, 229-282. (10) (a) Cheng, Y.-F.; Walter, T. H.; Lu, Z.; Iraneta, P.; Alden, B. A.; Gendreau, C.; Neue, U. D.; Grassi, J. M.; Carmody, J. L.; O’Gara, J. E.; Fisk, R. LC-GC 2000, 11, 1162-1172. (b) Neue, U. D.; Walter, T. H.; Alden, B. A.; Jiang, Z.; Fisk, R. P.; Cook, J. T.; Glose, K. H.; Carmody, J. L.; Grassi, J. M.; Cheng, Y.-F.; Lu, Z.; Crowley, R. Am. Lab. 1999, 31, 36-39. (c) Wyndham, K. D.; O’Gara, J. E.; Walter, T. H.; Glose, K. H.; Lawrence, N. L.; Hudalla, C.; Xu, Y.; Izzo, G. PMSE Prepr. 2002, 87, 274-275. (11) Brinker, C. J.; Scherer, G. W. Sol-Gel Science; Academic Press: London, 1990. (12) (a) Oviatt, H. W.; Shea, K. J.; Small, J. H. Chem. Mater. 1993, 5, 943-950. (b) Loy, D. A.; Carpenter, J. P.; Alam, T. M.; Shaltout, R.; Dorhout, P. K.; Greaves, J.; Small, J. H.; Shea, K. J. J. Am. Chem. Soc. 1999, 121, 54135425. (c) Boury, B.; Corriu, R. J. P. Adv. Mater. 2000, 12, 989-992. (13) (a) Cintro´n, J. M.; Colo´n, L. A. Analyst 2002, 127, 701-704. (b) Buchel, G.; Gru ¨ n, M.; Unger, K. K.; Matsumoto, A.; Tsutsumi, K. Supramol. Sci. 1998, 5, 253-259. (c) Unger, K. K.; Becker, N.; Roumeliotis, P. J. Chromatogr. 1976, 125, 115-127. (d) Feng, Q.; Xu, J.; Dong, H.; Li, S.; Wei, Y. J. Mater. Chem. 2000, 10, 2490-2494. (e) Guan, S.; Inagaki, S.; Ohsuna, T.; Terasaki, O. J. Am. Chem. Soc. 2000, 122, 5660-5661. (14) (a) Wang, D.; Chong, S. L.; Malik, A. Anal. Chem. 1997, 69, 4566-4576. (b) Rodrı´guez, S. A.; Colo´n, L. A. Chem. Mater. 1999, 11, 754-762. (c) Guo, Y.; Colo´n, L. A. J. Microcolumn Sep. 1995, 7, 485-491.

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condensation of methyltriethoxysilane (1 equiv) with TEOS (2 equiv).10 By utilizing synthetic conditions similar to those used in the production of many high-purity porous silica packings, we were able to synthesize spherical porous methyl hybrid particles, having an empirical formula SiO2(CH3SiO1.5)0.5. These methyl hybrid materials displayed pore characteristics similar to those of contemporary silica-based packing materials and could be surface C18 and C8 modified using common chlorosilane-bonding protocols. Chromatographic evaluation of bonded methyl hybrid columns indicated chromatographic performance (e.g., efficiency, selectivity, retentivity) equivalent to many silica-based reversedphase materials, while employing the same methods (e.g., mobile phase composition, temperature, run times). Advantages of these methyl hybrid materials over silica-based packing materials include decreased tailing factors for basic analytes and dramatic improvements in high-pH chemical stability of these packings in comparison to silica-based packings. In this article, we will characterize and evaluate the use of a new ethyl-bridged hybrid particle as a reversed-phase packing material. This hybrid particle, having an empirical formula SiO2(O1.5SiCH2CH2SiO1.5)0.25, is synthesized by the co-condensation of 1,2-bis(triethoxysilyl)ethane (BTEE, 1 equiv) with TEOS (4 equiv). Because this material is based on a disilane precursor (EtO)3SiCH2CH2Si(OEt)3, a higher molar ratio of TEOS was employed in this reaction in order to maintain a comparable 1:2 ratio of RSiO1.5 to SiO2 groups with respect to the methyl hybrid material. In this report, analytical and chromatographic characterization is detailed for this novel hybrid material. To demonstrate the unique properties that arise from ethyl-bridged hybrid particles, comparisons will be made to identically bonded porous silica. EXPERIMENTAL SECTION Apparatus. Elemental analyses were performed using an Exeter Analytical Inc. combustion analyzer model CE-440. The specific surface areas (SSA), specific pore volumes (SPV), and average pore diameters (APD) were measured using the multipoint nitrogen sorption method (Micromeritics ASAP 2405) as follows; the SSA was calculated using the BET method, the SPV was determined at a single point for P/Po >0.98, and the APDdes was calculated from the desorption leg of the nitrogen isotherm and APDads was calculated from the adsorption leg of the nitrogen isotherm using the BJH method. Unless otherwise noted, pore diameters in this report are based on APDdes. Helium pycnometry measurements were obtained as an average of three measurements by Micromeritics (Norcross, GA) using an AccuPyc 1330 instrument. Particle size analyses were carried out using a Beckman Coulter Multisizer 3 analyzer (30-µm aperture, 70 000 counts). Multinuclear (13C, 29Si) CPMAS NMR spectra were obtained using a Bruker Instruments Avance-300 spectrometer (7-mm double-broadband probe). The spinning speed was typically 5.0-6.5 kHz, recycle delay was 5 s, and the cross-polarization contact time was 6 ms. Reported 13C and 29Si CPMAS NMR spectral shifts were recorded relative to tetramethylsilane using the external standards adamantane (13C CPMAS NMR, δ 38.55) and hexamethylcyclotrisiloxane (29Si CPMAS NMR, δ -9.62). Relative areas of different silicon peaks were estimated by spectral

deconvolution using DMFit software.15 Scanning electron microscopic (SEM) images were obtained using a JEOL JSM-5600 instrument at 7 kV using Electroimage software (v. 1.6). Chromatographic data were recorded using a Waters Alliance 2690 Separations Module (with built-in vacuum degasser and column heater) and a Waters 2487 dual-wavelength absorbance detector. Unless otherwise specified, column temperatures were set at 23.4 °C ((0.1 °C) using a Neslab RT-111 water bath. Instruments were controlled by Waters Millennium Chromatography Software, and data were corrected for system volume and dispersion effects. Reagents. All reagents were used without further purification, and all procedures were carried out in a chemically safe fume hood. Octadecyltrichlorosilane and trimethylchlorosilane were purchased from Aldrich. All solvents employed in the synthesis and derivatization of ethyl-bridged hybrid particles, and for chromatographic evaluation were HPLC grade, and were used as supplied from J. T. Baker. Test solutes (purity g98%) and other reagents used for mobile-phase preparation were purchased from Aldrich and were used without any further purification. Procedures. The syntheses of ethyl-bridged hybrid particles,16 having an empirical formula (SiO2)(O1.5SiCH2CH2SiO1.5)0.25 and porous silica particles,17 have been previously reported. In short, ethyl-bridged hybrid particles are prepared in a two-step process. During the first step of this process, a polyethoxysiloxane oil phase is prepared from the acid-catalyzed hydrolytic condensation of a 1:4 molar ratio of BTEE and TEOS using a deficient amount of water. Highly spherical porous hybrid particles are then prepared by further condensation under alkaline conditions in an oil-in-water (o/w) emulsion. After sizing and postsynthetic surface-ripening steps, surface derivatization of these hybrid materials was performed using standard surface-bonding protocols.4a,16 In a first reaction step, the surface silanol groups are reacted with octadecyltrichlorosilane (under nitrogen). In a second reaction step, unreacted surface silanol groups were end-capped with trimethylchlorosilane. Columns containing C18 bonded materials were packed into stainless steel columns using conventional slurrypacking methods.18 Mechanical stability studies of C18 bonded particles (0.25 g) were performed in 3.9 × 10 mm steel columns that were initially slurry packed with methanol (50 mL) at low pressures (3.5 MPa) to ensure that no fracturing occurred. These columns were then subjected to increasing pressures (3.5-62 MPa) using a constantpressure pump (model 10-500, SC Hydraulics) and methanol as the solvent. At elevated pressures, columns containing beds of weak materials compact or crush resulting in a restriction of the flow. Flow rates were manually recorded at each pressure. As a means to even out slight differences in particle size and packing parameters, and assess the influence of column pressure on the mechanical stability of the different materials, the flow rates and pressures were normalized to the data obtained for these columns at 7 MPa back pressure (16-20 mL/min). Independent comparisons were made to columns packed with mechanically strong, low-porosity silica, 4-µm Nova-Pak C18 particles (Waters Corp.) (15) Massiot, D.; Fayon, F.; Capron, M.; King, I.; Le Calve´, S.; Alonso, B.; Durand, J.-O.; Bujoli, B.; Gan, Z.; Hoatson, G. Magn. Reson. Chem. 2002, 40, 70-76. (16) Jiang, Z.; Fisk, R. P.; O’Gara, J. E.; Walter, T. H.; Wyndham, K. D. U.S. Patent Application No. 09/924,399, 2001. (17) Unger, K. K. Porous Silica; Elsevier: Amsterdam, 1979. (18) Neue, U. D. HPLC Columns; Wiley-VCH: New York, 1997.

and a highly porous polymeric material, 7-µm Ultrastyragel 106-Å particles (Waters Corp.). Chromatographic parameters including plate number, N, retention factor, k, and tailing factors, Tf (determined using the USP method)19 were determined for each column using a standard test protocol.20 In this isocratic procedure, packed 4.6 × 150 mm stainless steel columns were evaluated with a standard test mixture using a 60:40 methanol/20 mM KH2PO4/K2HPO4 buffer solution (pH 7.00), at 23.4 ( 0.1 °C. To attain higher linear velocities, van Deemter analyses were performed on shorter 2.1 × 50 mm columns using decanophenone (3 mg/mL) in isocratic 70% acetonitrile solutions at 30 ( 1 °C. Alkaline (pH 10) column degradation studies were performed using materials packed in 4.6 × 150 mm steel columns. In this procedure, columns were challenged with a 50 mM triethylamine (TEA) solution in water for 60 min (2 mL/min, 50 °C) and then purged with water (2 mL/ min, 50 °C) and methanol (2 mL/min, 50 °C) for 10 min each. The efficiency of the column was then tested using a standard test mixture (65:35 methanol/20 mM KH2PO4/K2HPO4 pH 7 buffer solutions, 50 °C). For C18 bonded materials, the efficiency loss for acenaphthene was reported, while for unbonded ethylbridged hybrid particles, the efficiency loss for the void marker, uracil, was reported. RESULTS AND DISCUSSION Characterization of the Materials. The materials studied were all based on ∼5-µm porous silica (SiO2) or ethyl-bridged hybrid (SiO2)(O1.5SiCH2CH2SiO1.5)0.25 particles. The hybrid particles used in these studies had uniform spherical morphology, as determined by SEM. Nitrogen sorption studies of unbonded ethyl-bridged hybrid particles showed type IV H1-hysteresis loops that are characteristic of mesoporous amorphous materials with nonordered cylindrical pores.21 The SSA (BET method, 140-344 m2/g), SPV (0.71-0.85 cm3/g), and APDdes (100-185 Å) for individual materials are summarized in Table 1. Skeletal density measurements for ethyl-bridged hybrids (2.00 g/mL) and the silica sample (2.13 g/mL) were determined by helium pycnometry measurements. The unbonded silica sample (S) used in this study has similar particle properties and was prepared in a manner similar to Waters Symmetry silica. For the three unbonded ethylbridged hybrid materials (H1, H2, H3), we observed 6.20-6.50% C, which is in reasonable agreement with the calculated carbon content (6.45% C) based on the empirical formula for a hybrid material containing 20 mol % ethyl-bridging groups. The three unbonded ethyl-bridged hybrid particles listed in Table 1 differ in surface properties, which were obtained from modifications in the synthetic procedures.16 For example, the surface area for H1 (270 m2/g) is higher than that of H2 (187 m2/g) or H3 (140 m2/g) and is closest to that of the silica-based particle S (344 m2/g). The desorption average pore diameter of H1 (108 Å) is also lower than that of H2 (148 Å) and H3 (185 Å), which is in good agreement with normal surface area trends (19) The United States Pharmacopeia; 22nd ed.; Mack Printing Co.: Easton, PA, 1990; pp 1558-1568. (20) (a) Neue, U. D.; Serowik, E.; Iraneta, P.; Alden, B. A.; Walter, T. H. J. Chromatogr., A 1999, 849, 87-100. (b) Neue, U. D.; Phillips, D. J.; Walter, T. H.; Capparella, M.; Alden, B. A.; Fisk, R. LC-GC 1994, 12, 468-480. (21) Gregg, S. J.; Sing, K. S. W. Adsorption, Surface Area and Porosity; Academic Press: New York, 1982.

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Table 1. Characterization Data for Unbonded and C18 Bonded 5-µm Particles bonded material unbonded material a

S H1 H2 H3 a

step 1

dp (µm)

% Cb

SSA (m2/g)

SPV (cm/g)

APDdes (Å)

APDads (Å)

4.9 4.9 4.6 5.5

0.00 6.30 6.50 6.20

344 270 187 140

0.85 0.81 0.79 0.71

100 108 148 185

116 127 180 247

S-C18 H1-C18 H2-C18 H3-C18

step 2

% C1

∆% Cb

coverage (µmol/m2)

% C2

∆% Cc

18.72 19.80 16.37 14.30

18.72 13.50 9.87 8.10

3.44 3.21 3.17 3.35

19.20 21.40 17.32 15.00

0.48 1.60 0.95 0.70

50 vol %. b ∆% C ) % C1 - % Cb. c ∆% C ) % C2 - % C1.

(SA ) 4 PV/PD), considering that the pore volume (0.71-0.81 cm3/g) did not vary significantly for these three hybrid materials. After C18H37SiCl3 bonding reactions, marked increases were found for the carbon content of the silica (step 1, ∆% C ) 18.72) and ethyl-bridged hybrid materials (step 1, ∆% C ) 8.10-13.50). A linear dependence of the carbon content as a function of particle surface area was observed for the 5-µm ethyl-bridged hybrid particles. The highest surface area hybrid, H1-C18 had a higher carbon increase (step 1, ∆% C, 13.50) than H2-C18 (step 1, ∆% C, 9.87) and H3-C18 (step 1, ∆% C, 8.10). A similar increase in carbon content was observed for higher surface area trimethylsilylend-capped hybrids (step 2, ∆% C ) 1.60-0.70). The determination of the octadecylsilyl (C18) surface coverage for the silica and ethylbridged hybrids materials after bonding was based on a modified version of the Berendsen and de Galan equation.22

C18 surface coverage (µmol/m2) ) ∆% C × 106 (70.98 - % Cb)(304.57 g/mol) × SSA

The equation modification takes into account the initial carbon content of the unbonded particles (% Cb) by introducing a change in carbon content during the step 1 bonding (∆% C ) % C1 - % Cb).23 Other variables for this equation include the effective molecular weight (304.57) and percent carbon (70.98%) of surfacebound trifunctional C18 silane (considering that all chlorosilyl groups have reacted or hydrolyzed to provide an average T2 surface attachment), as well as the SSA for the unbonded particle. Because differences in SSA are included in the coverage calculation, similar step 1 surface coverages (3.21-3.35 µmol/m2, Table 1) were determined for all hybrid particles. The C18 surface coverage for ethyl-bridged hybrids is close to the coverage obtained for S-C18 (3.44 µmol/m2), signifying a similar environment of accessible surface silanol groups. In contrast, the C18 surface coverages reported for C18 bonded methyl hybrid particles (2.5 µmol/m2) are significantly lower than the ethyl-bridged hybrid material, which indicates fewer accessible surface silanols for the methyl hybrid particle due to the presence of surface methylsilyl groups.10 To better understand the chemical environment of these amorphous ethyl-bridged hybrid materials, 29Si CPMAS NMR spectroscopy was employed. Representative 29Si CPMAS NMR (22) Berendsen, G. E.; de Galan, L. J. Liq. Chromatogr. 1978, 1, 561-586. (23) Sandoval, J. E. J. Chromatogr., A 1999, 852, 375-381.

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Figure 1. 29Si CPMAS NMR spectra for 5-µm unbonded (a) ethylbridged hybrid (H2) and (b) silica (S). (c) Table containing relative peak areas for resonances attributed to ethyl-bridged silicon atoms (Tn) and inorganic silicon atoms (Qn).

spectra for unbonded ethyl-bridged hybrid (H2) and silica (S) particles are shown in Figure 1. In the 29Si NMR spectra of unbonded ethyl-bridged hybrid particles, several distinct silicon environments are observed between -46 and -120 ppm. Using the common Tn and Qn notation, the resonances commonly ascribed to partially condensed ethyl-bridged hybrid units T1 (δ -46) and T2 (δ -56) are downfield of the completely condensed T3 resonance (δ -65).12 Incompletely condensed Q2 (δ -92) and Q3 (-101) and fully condensed Q4 (δ -110) inorganic silicon environments are observed significantly upfield of the resonances attributed to ethyl-bridged species. By comparing the relative intensities of the different silicon resonances (determined by spectral deconvolution), we can estimate the relative concentrations of the different environments around silicon. For example, by comparing T2 and T3 resonances for H2, it was determined that greater than 80% of the ethyl-bridged silicon atoms are within completely condensed T3 environments. Due to this high degree of condensation for bridging ethylsilyl groups, the majority of the ethyl-bridged hybrid silicon atoms do not bear hydroxyl groups

Figure 2. Mechanical stability testing of porous particles at increasing column pressures of methanol flow: theoretical ideal (open triangles); 4-µm Nova-Pak C18 column (gray squares); H1-C18 (gray diamonds); H2-C18 (open circles); H3-C18 (closed circles); S-C18 (closed triangles); and 7-µm Ultrastyragel 106-Å column (open squares). Conditions: 3.9 × 10 mm columns packed at 3.5 MPa. Flow rates for each column were determined at different pressures (3.5-62 MPa) and were normalized for flow rate data acquired at 7 MPa.

and, hence, are not reactive to surface modification with chlorosilanes. The 29Si CPMAS NMR spectra of C18 bonded hybrid particles displayed an increase in Tn resonances due to surface octadecylsilyl groups, and a decrease in Q2 and Q3 resonances. In the solid-state 13C NMR spectra obtained for the unbonded ethyl-bridged hybrid, a single predominant resonance is observed that is characteristic of the ethyl-bridging group (δ 4.5). After bonding and end-capping reactions were performed, new resonances emerge that are characteristic of C18 surface groups. Particle Strength Evaluations. Mechanical instability of HPLC packing materials at elevated pressures can result in particle fracturing or compression deformation, which in turn results in higher observed column back pressures, column clogging, or particle percolation through the outlet of the column.18,24 The mechanical stability of any new packing material is therefore an important variable for evaluation. Considering that the material properties for most hybrid organic/inorganic materials range between the properties normally observed for inorganic (i.e., SiO2)18 and polymeric materials (i.e., poly(styrene-co-divinylbenzene)),25 one may expect that the mechanical stability of ethylbridged hybrid particles would be greater than polymeric materials and less than pure silica-based particles. To assess the mechanical stability of our hybrid particles, we measured the flow through a short (10 mm) column as a function of pressure, as illustrated in Figure 2. In this series of experiments, the normalized flow trends for columns packed with H1-C18, H2-C18, H3-C18, and S-C18 were determined at increasing column pressures up to 62 MPasa pressure that exceeds the (24) Groh, R. Diplomarbeit, Universita¨t des Saarlandes, 1975. (25) Dawkins, J. V.; Lloyd, L. L.; Warner, F. P. J. Chromatogr. 1986, 352, 157167.

pressure limit of conventional HPLC, providing a better approximation to pressures that a column may be exposed to during the column packing process.18 The normalized flow trend observed were compared to the mechanically strong, low-porosity, sol-based silica, 4-µm Nova-Pak C18. In this experiment, high mechanical strength was associated with a higher and more linear flow profile. For example, the theoretical ideal for mechanical strength in this test would display a linear trend as shown in Figure 2. The highest normalized flow was found for the test performed using Nova-Pak C18. The three C18 bonded hybrid columns (H1-C18, H2-C18, H3-C18) displayed very similar flow curves that deviated from the curve obtained for the Nova-Pak C18 column at pressures greater than 14 MPa. This deviation in normalized flow rates, when compared to Nova-Pak C18 columns, becomes more pronounced at higher column pressures. For example, at 35 MPa, H2-C18 columns had flow rates that were 11% lower, and at 62 MPa, the H2-C18 columns flow rates were 22% lower when compared to Nova-Pak C18 columns. The similar flow curves for the columns packed with the different hybrid particles indicate that the changes in the pore structure do not influence the mechanical stability of these materials. Postexperimental inspection of the hybrid columns showed no evidence of voiding on the tops of these columns, but a small amount of particle fracturing was observed on the outlet frit (via SEM). Attempts to repeat these pressure experiments on the same column resulted in a further reduced flow rate. We can therefore conclude from these tests that degradation of the C18 bonded ethyl-bridged hybrid columns was irreversible and was due to particle fracturing caused by the high-pressure treatment. The normalized flow curves obtained for the silica-based column (S-C18) and an Ultrastyragel 106-Å polymeric column showed greater deviations from Nova-Pak C18 columns. At 35 MPa, the silica column flow rate was 17% lower, while at 62 MPa, the same column had 35% reduced flow rate when compared to the Nova-Pak C18 columns. A similar flow trend to S-C18 was obtained when 5-µm Symmetry C18 columns were tested (not shown), a result that was expected considering that S has material properties similar to Symmetry silica. Normalized flow curves attained for the polymeric Ultrastyragel 106-Å packings were relatively flat throughout the pressure range. While the decreased mechanical stability of columns packed with S-C18 may be due to the higher particle porosity (65%) with respect to the hybrid materials (5961%),18 the low-flow trend observed for the polymeric column is indicative of particle crushing. Considering that silica-based materials having physical properties similar to those of S-C18 have been routinely shown to have no ill effects when used in HPLC applications (column pressures 2.3) for these bases due to ion exchange interactions with residual unbonded silanols. In contrast, the hybrid columns display significantly reduced tailing factors for propranolol (0.94-1.07) and amitriptyline (1.52-1.72). This decrease in peak tailing for basic analytes on the ethyl-bridged hybrid columns is ascribed to a lower acidity of surface silanols groups of hybrid particles (pKa >8), with respect to silica (pKa 3.5-6.8). Similar results have been reported for the methyl hybrid packing.26 Van Deemter analyses were performed to compare differences in the efficiency and mass-transfer characteristics of amorphous silica and hybrid packing materials. A representative curve for S-C18 and H2-C18 is shown in Figure 4 as a plot of reduced plate height (h) versus reduced linear velocity (v). Both columns (26) (a) Mendez, A.; Bosch, E.; Roses, M.; Neue, U. D. J. Chromatogr., A 2003, 986, 33-44. (b) Neue, U. D.; Phoebe, C. H.; Tran, K.; Cheng, Y.-F.; Lu, Z. J. Chromatogr., A 2001, 925, 49-67.

Figure 4. Representative h-v curve comparisons of H2-C18 (open diamonds) and S-C18 (closed diamonds). Inset shows van Deemter coefficients determined by curve fitting (half-height method). Conditions: 2.1 × 50 mm column, decanophenone (3 mg/mL) in 70% acetonitrile at 30 ( 1 °C. k S-C18 ) 16.82; k H2-C18 ) 11.60.

show typical behavior, which may be fit using the van Deemter equation:27

h ) a + (b/v) + cv

The cv term accounts for all phenomena that result in an increase in h with increasing linear velocity. This includes the kinetics of analyte transfer in to and out of the pore network and the bonded phase layer. The c-term for H2-C18 (0.049) is very similar to S-C18 (0.046), indicating comparable mass-transfer characteristics of these two columns. Van Deemter analysis of H1-C18 and H3C18 also shows low c-terms (0.044 and 0.047, respectively) that are similar to many highly efficient commercially available silicabased reversed-phase columns. The b-terms for H2-C18 (8.92) and S-C18 (9.19) are in good agreement with the expected values for well-retained analytes, such as decanophenone (k ) 11.6016.82),27 and were confirmed using arrested flow methods.28 High-pH Column Aging Studies. The generally accepted failure mechanism for bonded silica-based materials at elevated pH is base-catalyzed particle dissolution. To compare the stability of packing materials described here, we used an accelerated column aging study employing a pH 10 mobile phase. This procedure involved exposing materials packed in 4.6 × 150 mm columns to a 50 mM TEA (pH 10, 2 mL/min) mobile phase at elevated temperature (50 °C). The condition of the column was then determined by periodically stopping the flow of TEA and flushing the column with water and methanol to remove any soluble hydrolysis products. After equilibration with a phosphate buffer/methanol test mobile phase, a mixture of nonpolar and basic analytes was injected onto the column, and the chromatogram was recorded. Under these test conditions, the major change observed is a loss of efficiency and severe distortion (fronting) of the peaks. Similar observations have been reported by other (27) (a) Knox, J. H. J. Chromatogr., A 1999, 831, 3-15. (b) Knox, J. H.; Scott, H. P. J. Chromatogr. 1983, 282, 297-313. (c) Knox, J. H. J. Chromatogr. Sci. 1977, 15, 352-364. (28) Berthod, A.; Chartier, F.; Rocca, J.-L. J Chromatogr. 1989, 469, 53-65.

Figure 5. Loss of efficiency (5σ method) for acenaphthene on H2C18 (closed squares) and S-C18 (closed circles) columns exposed to 50 mM triethylamine solution at 50 °C. For unbonded H2 (open squares), the efficiency loss of the void marker, uracil, was reported. Test conditions are described in the Experimental Section.

researchers carrying out high-pH stability studies under different conditions.6 In most cases, column efficiency stays fairly constant for a period of time and then abruptly drops off. Visual inspection of the columns after this drop in efficiency shows a 1-10-mm void at the head of the column, which results from partial dissolution of the particles and resettling of the packed bed. Representative results from this accelerated aging test are shown in Figure 5 as a plot of relative efficiency (% N remaining), measured for acenaphthene, versus the total time that columns S-C18 and H2-C18 were exposed to TEA. For S-C18, acenaphthene efficiency was maintained at greater than 90% for the first 10-h exposure to the TEA solution, at which time a drastic loss in plate count was observed. This catastrophic loss in column efficiency is due to dissolution of the silica particle. At 17 h, this silica-based column displayed less than 50% of the initial column efficiency. In sharp contrast, H2-C18 maintained greater than 90% of the original column efficiency over 100-h exposure to the pH 10 mobile phase. After this time, a gradual decline in efficiency for acenaphthene could be observed. When this test was terminated at 140 h, H2-C18 still maintained 81% of its original column efficiency. Considering that S-C18 and H2-C18 were C18 bonded using the same procedure, we attributed this great increase in high-pH column stability of H2-C18 to an increase in the chemical stability of the hybrid matrix toward base-catalyzed dissolution. Increases in column stability of 36-40 h using this test have also been reported using methyl hybrid C18 columns.10 Other researchers have noted the excellent chemical stability of ethyl-bridged hybrid films toward corrosion.29 To confirm this theory, we tested the elevated pH stability of columns packed with unbonded H2 under similar test conditions. As shown in Figure 5, the efficiency stability of H2 (based on the void marker, uracil) closely followed the trend observed for H2-C18. After 100 h of exposure to triethylamine at 50 °C, H2 columns still displayed 94% of their original efficiency. Because similar performance was obtained for unbonded and bonded hybrid particles, we can conclude that the (29) (a) van Ooij, W. J.; Child, T. F. Chemtech 1998, 28, 26-35. (b) Subramanian, V.; van Ooij, W. J. Corrosion 1998, 54, 204-215.

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presence of a bonded phase group does not significantly change the efficiency stability of these columns when aged under these conditions. The chemical makeup of the ethyl-bridged hybrid particle provides the substantial improvement in column stability that we have observed. CONCLUSIONS Ethyl-bridged hybrid particles are exceptional packing materials for use in both standard and elevated pH reversed-phase applications. These hybrid materials are spherical and mechanically strong and can be surface bonded using protocols common to silica materials. Preliminary chromatographic evaluation of columns packed with these materials shows separations that are comparable to silica-based columns using the same methods and displays equivalent selectivities and mass-transfer capabilities. Major improvements in peak tailing factors for basic analytes are observed on these hybrid columns because of the reduced acidity of unreacted surface silanol groups. Variations in surface area of these hybrid particles have a linear influence on carbon content after bonding and on chromatographic retentivity. However, these modifications did not impact the mechanical stability, chromatographic efficiencies, relative retentions, peak tailing factors, or mass-transfer characteristics of columns packed with these materials. Significant enhancements in column stability at elevated pH are achieved with these hybrid packing materials. This improvement is due to the increased hydrolytic stability of the ethylbridged group within the particle matrix, which may facilitate the use of these columns in unique chromatographic applications (e.g., elevated pH separations using NaOH as the mobile-phase modifier).

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We have only begun to explore the use of ethyl-bridged hybrid materials for use as improved reversed-phase HPLC packing materials, but the high mechanical and chemical stability of these particles offers many interesting possibilities in areas of very highpressure LC, supercritical fluid chromatography, and manipulation of selectivity using the entire pH range. Our work exploring the column stability at low pH (e.g., 1% TFA, 80 °C), the use of sodium hydroxide-based mobile phases, and the use of different bonded phases will be reported separately. ACKNOWLEDGMENT We acknowledge Yuehong Xu, Thomas Brady, Daniel O′Shea, Patricia David, and Joomi Ahn for acquiring combustion, TGA, and nitrogen sorption data. We thank Michael Savaria, Steve Shiner, and Cheryl Boissel for column packing support and for securing chromatographic data. A special thanks is given to Raymond Fisk for his pioneering work into the field of hybrid particle technology. SUPPORTING INFORMATION AVAILABLE Plots of pore property influence on C18 surface coverage and retentivity, 29Si and 13C CPMAS NMR spectroscopic analysis, SEM evaluation, nitrogen sorption isotherms for ethyl-bridged hybrid particles, and a complete table of van Deemter data. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review July 8, 2003. Accepted October 9, 2003. AC034767W