Anal. Chem. 1998, 70, 4344-4352
Bidentate Silane Stationary Phases for Reversed-Phase High-Performance Liquid Chromatography J. J. Kirkland*
Little Falls Analytical Division, Hewlett-Packard Company, Newport Site, 538 First State Boulevard, Newport, Delaware 19804 J. B. Adams, Jr.
Adams Research, 759 Morris Road, Hockessin, Delaware, 19707 M. A. van Straten and H. A. Claessens
Department of Chemistry, Eindhoven University of Technology, 5600 MB, Eindhoven, The Netherlands
A family of new silica-based bidentate silane stationary phases shows distinct advantages as reversed-phase chromatography column packings. Column efficiency, reproducibility, and selectivity characteristics are equivalent to those of conventional monofunctional silane stationary phases, but column stability is measurably improved. A silica-based bidentate-C18/C18 column packing exhibits excellent stability with both low- and intermediate-pH mobile phases, but is especially notable for high-pH separations. Highly basic compounds such as basic drugs can be separated routinely at high pH as free bases. Our studies have defined how the structure of this bidentate silane should be designed to position C18 ligands for optimum solute interaction and column efficiency. The characteristics of these new phases have been determined to describe areas of most useful chromatographic applications. Monofunctional silanes are widely used for bonded stationary phases in high-performance liquid chromatography (HPLC) columns because of the ability to reproducibly prepare efficient columns with silica supports.1 Monofunctional stationary phases often are preferred by users because of their superior kinetic properties and the ability to be closely reproduced in surface reactionssone molecule of silane reacts with one silanol group. Polymeric2 and horizontally polymerized silanes3 have been proposed as superior stationary phases in terms of stability in both low- and high-pH mobile phases, presumably because of multiple attachment to the silica support. While these materials have not shown higher stability than the monofunctional sterically protected silanes in low-pH environments,1,4,5 they have demonstrated better (1) Snyder, L. R.; Kirkland, J. J.; Glajch, J. L. Practical HPLC Method Development, 2nd ed; Wiley-Interscience: New York, 1997; Chapter 5. (2) Hetem, M. J.; De Haan, J. W.; Claessens, H. A.; Cremers, C. A.; Deege, A.; Schomberg, G. J. Chromatogr. 1991, 548, 53. (3) Wirth, M. J.; Fatunmbi, H. O. Anal. Chem. 1993, 65, 822.
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stability than many monofunctional stationary phases at high pH.2 An exception may be densely bonded, double end-capped C8 and C18 stationary phases,6-8 although appropriate comparative data are not yet available. There is considerable interest in conducting reversed-phase HPLC separations at high pH (>9), especially for basic compounds. Here, the pH can be adjusted so that the compounds are free bases, unable to interact deleteriously by ion exchange with totally ionized unreacted silanol groups remaining on the silica support surface. Operation at a pH well above the pKa value of basic compounds also should produce more repeatable separations, since retention changes due to the formation of ionized forms is not possible.6,8 Therefore, in terms of retention reproducibility, separations at high pH should be equivalent to those carried out in the often-recommended low-pH mobile phases where both ionizable solutes and silanol groups are fully protonated.6 To improve the stability of monofunctional silica-based column packings in high-pH mobile phases further, we have developed and studied new bidentate silane stationary phases. These bidentate phases retain the benefits of typical monofunctional silane phasesshigh column efficiency and excellent reaction repeatabilityswhile demonstrating good stability in high-, intermediate-, and low-pH mobile phases. This paper describes the preparation and characterization of some useful bidentate phases. We also report the reversed-phase HPLC characteristics of these materials and describe some important aspects of structure in determining the kinetic nature of C18-modified stationary phases. The stability of these new column packing materials at high pH was defined by two approaches that also were used in previous (4) Kirkland, J. J.; Glajch, J. L.; Farlee, R. D. Anal. Chem. 1989, 61, 61. (5) Boyes, B. E.; Kirkland, J. J. Pept. Res. 1993, 6, 249. (6) Kirkland, J. J.; DeStefano, J. J. Git Special, Chromatography International 96, GIT, Darmstadt, June 1996; p 62. (7) Kirkland, J. J.; Henderson, J. W.; DeStefano, J. J.; van Straten, M. A.; Claessens, H. A. J. Chromatogr. 1997, 762, 97. (8) Kirkland, J. J.; van Straten, M. A.; Claessens, H. A. J. Chromatogr., A 1998, 797, 111. S0003-2700(97)01380-2 CCC: $15.00
© 1998 American Chemical Society Published on Web 09/19/1998
studies.7-10 First, the rate of silica support solubility with continuously flowing, aggressive mobile phase containing an aggressive pH 11 phosphate buffer was measured using the well-known silicomolybdate colorimetric method. Second, in comparable column-aging studies, the change in chromatographic properties of the column packings was determined. EXPERIMENTAL SECTION Silica Support Dissolution Studies. As in previous studies,7-10 mobile phase was continuously purged through the columns with a model 100A pump (Beckman, Fullerton, CA). Fractions of the eluent were collected with a Waters P/N 37040 fraction collector (Waters, Milford, MA). Absorbance measurements were with a Pye Unicam LC3 detector (ATI Unicam, Cambridge, U.K.). All chemicals, solvents, and silicate standard solutions were of analytical grade from Merck (Darmstadt, Germany). Buffers and reagent solutions were prepared from deionized water from a Milli-Q purification system (Millipore, Bedford, MA). The purge solutions for the dissolution studies were composed of acetonitrile-0.02 M potassium phosphate buffer, pH 11.0 (50:50 v/v). To duplicate actual chromatographic practice, columns were purged continuously at 1.5 mL/min at 25 °C with eluents and not recycled. All columns were flushed for 10 min with acetonitrilewater (50:50, v/v) prior to the dissolution experiments. After starting a specific dissolution study, we sampled the effluent after ∼1 L had passed through the column, using a fraction collector. Column effluent samples for silicate analysis were collected for a 6-min period (9 mL total). Silica concentrations dissolved from the columns were measured colorimetrically at 410 nm for collected fractions, using the silicomolybdate complex method.11 For these silica measurements, standard silicate mixtures were prepared in the buffermodifier purge solutions used for the dissolution studies. Absorbance values were measured using blank solutions as reference. The potential interference of phosphate on the color reaction was eliminated by removing the phosphate prior to silica measurements.9 Results from the colorimetric measurements for the concentration of dissolved silica in the eluents were plotted as a function of eluent volume. First, the volume of eluent (V) between two consecutive fractions was calculated using the relationship (Vi+1 - Vi). With this value and the measured concentrations of silica in the two consecutive fractions, the average silica concentration for two consecutive fractions (Ci) was determined. Plots of the amount of silica dissolved (m) vs eluent volume (V) then were obtained by integrating the silica concentration vs eluent volume (∆m/∆V vs V) using the expression m ) ∑Ci∆iV, where ∆iV represents the effluent volume difference corresponding to two consecutive fractions. Column Characterizations and Equipment. Chromatographic data for the plate height vs mobile-phase velocity plots were obtained with a Hewlett-Packard model 1090 II instrument (Wilmington, DE). Data for the van’t Hoff plots were obtained (9) Claessens, H. A.; van Straten, M. A.; Kirkland, J. J. J. Chromatogr. 1996, 728, 259. (10) Kirkland, J. J.; van Straten, M. A.; Claessens, H. A. J. Chromatogr. 1995, 691, 3. (11) Iler, R. K. The Chemistry of Silica; John Wiley: New York, 1979; p 97.
using a Beckman model 100A pump (Beckman Instruments, Fullerton, CA), a Rheodyne model 7125 injector with a 5-µL sample loop (Cocati, CA), a Spark Mistral model column oven (Spark, Emmen NL), a Linear UVIS 200 UV detector (Linear Instruments, Reno, NV), and a Nelson 3000 data system (Perkin-Elmer, Cupertino, CA). A 5-µL sample of 10-4-10-5 M uracil (Fluka Chemie, AG, Buchs, CH), toluene (Aldrich, Milwaukee, WI), and trimipramine (Sigma, St. Louis, MO) was used for these tests. Molecular models for bidentate structures were prepared with HyperChem software from Hypercube, Inc. (Waterloo, Canada). Chromatographic Reagents. Analytical-grade methanol and buffer components were from J. T. Baker (Phillipsburg, NJ). EM Science supplied HPLC-grade methanol and acetonitrile. Tricyclic antidepressants and β-blocker test solutes from Sigma were used as received. Phosphate buffer was prepared by mixing appropriate K2HPO4 and KOH solutions to obtain the desired pH 11. The 0.05 M 1-methylpiperidine buffer initially was made by titrating a solution of the free base (Aldrich) to pH 11 with hydrochloric acid.6,8 The 0.05 M pH 11.5 pyrrolidine buffer was prepared similarly from the free amine (99%, Aldrich). Purging studies with 1-methylpiperidine-buffered mobile phases prepared from the commercial amine showed that columns were slowly fouled with unknown impurities in this free base. Therefore, this amine was purified by forming the oxalic acid salt, recrystallizing, freeing the base with sodium hydroxide solution, and distillation. Buffer made with this purified amine then was used for long periods without difficulty. Column-aging (purging) studies were performed with a Shimadzu model LC-600 pump (Tokyo, Japan). Chromatographic testing was with a Hewlett-Packard model 1050 instrument and a DuPont Instruments column thermostat (Wilmington, DE). Chromatographic data were processed with ChromPerfect version 6.02 software (Justice Innovation, Palo Alto, CA). Plate height calculations were determined by the half-peak height method (eq 2.8a of ref 12). Peak asymmetry values were determined by the 10% peak height (Figure 5.19 of ref 1). Samples were injected with a Rheodyne model 7125 sampling valve. Columns. All 15 × 0.46 cm columns were prepared at the Hewlett-Packard Newport site. The physical and surface properties of the low-acidity, highly purified type B Zorbax Rx-Sil silica support used for these columns have previously been reported.13 Surface area for this silica support typically is 180 m2/g, with pores of 8 nm. The bidentate silane reagents used in this study also were synthesized at the Newport site.14 Reactions with the silica support were conducted by proprietary methods to generate densely bonded surfaces that could not be further reacted because of steric limitations. All packings were exhaustively double-endcapped with dimethyl- and trimethylsilane groups,7 and columns were formed by conventional slurry-packing methods.15 A series of bidentate C18-containing stationary phases was prepared to investigate the effect of different alkyl functional groups, as listed in Table 1 (chromatographic implications from (12) Snyder, L. R.; Kirkland, J. J.; Glajch, J. L. Practical HPLC Method Development, 2nd ed; Wiley-Interscience: New York, 1997; Chapter 2. (13) Kirkland, J. J.; Dilks, C. H., Jr.; DeStefano, J. J. J. Chromatogr. 1993, 635, 19. (14) Adams, J. B.; Kirkland, J. J., patent allowed. (15) Snyder, L. R.; Kirkland, J. J. Introduction to Modern Liquid Chromatography; John Wiley: New York, 1979; Chapter 5.
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Table 1. Summary of Results for Bidentate Column Packingsa amitriptylinec phase
%
Cb
N
k
As
Zorbax XDB-C8 7.15 9000 3.77 1.53 Zorbax XDB-C18 9.38 9100 6.44 1.47 C1/C18 bidentate 9.08 8800 6.02 1.84 C4/C18 bidentate 9.32 9900 5.98 1.39 C8/C18 bidentate 10.50 10000 5.55 1.27 C18/C18 bidentate 12.09 6000 5.22 2.07 C18/C18P bidentatee 11.94 9400 5.31 1.57
toluened N
k
As
13400 12900 12200 12500 12900 11400 12100
0.93 1.46 1.29 1.36 1.38 1.22 1.48
1.05 0.97 1.18 1.23 1.20 1.45 1.21
a Ethylene bridge between silicon atoms for bidentate packings unless noted; dimethyl-C8 and -C18 (Zorbax XDB) packings shown for comparison; all packings double end-capped. b Standard deviation, 0.01%, absolute. c 60% acetonitrile-40% 0.01 M sodium phosphate buffer, pH 7.0, 1.5 mL/min, 40 °C. d 80% methanol-20% water, 1.0 mL/ min, ambient temperature. e P ) propylene bridge.
Figure 2. Stability comparison for C18 bonded-phase columns at pH 0.9 and 90 °C: columns, 15 × 0.46 cm; purge mobile phase (continuous at 1.5 mL/min), 1:1 (v/v) methanol-water with 1.0% trifluoroacetic acid; temperature, 90 °C; test mobile phase, 60:40 methanol-water; flow rate, 1.0 mL/min; temperature, ambient; solute, toluene. Figure 1. General structure of bidentate stationary phases: R ) methyl, n-butyl, n-octyl or n-octadecyl; Q ) -CH2CH2- or -CH2CH2CH2-.
these data are discussed later). When reacted with the silica support, the bidentate silanes produced a surface that assumed the structure of Figure 1, where R is an alkyl group and Q is a -CH2CH2- or -CH2CH2CH2- bridging group. Complete reaction of both reactive groups of the bidentate silane was observed under the reaction conditions used, based on elemental analysis. This absence of reactive functionalities on the reacting bidentate silicon atoms is taken as evidence that both silicon atoms for the bidentate are attached to the silica surface. Additional evidence (described below) is that the bonded bidentates show unusual stability in aggressive low- and high-pH environments. Column-Aging Studies. Columns were continuously purged at 1.5 mL/min (not recycled) with a 50% acetonitrile-50% 0.017 M potassium phosphate pH 11 buffer mixture at ambient temperature (23 °C). These columns were periodically tested with toluene using a 80% methanol-20% deionized water mobile phase at 1.0 mL/min (ambient temperature). Tests also were made with a 5-µL mixture of tricyclic antidepressant drugs (doxepin, trimipramine, amitriptyline, and nortriptyline at 0.025, 0.25, 0.025, and 0.25 mg/mL, respectively) or β-blocker basic drugs (pindolol, metoprolol, oxyprenolol, and propranolol, pKa ) 9.5-9.7, at 0.008, 0.165, 0.413, 0.413, and 0.083 g/µL, respectively, in 1:1 methanolwater) at 40 °C. Before chromatographic testing, each column was first flushed with at least 20 column volumes of methanolwater (50:50) before equilibrating with ∼20 column volumes of the new mobile phase. Temperature Studies. Data for van’t Hoff plots were obtained using a mobile phase of 60% acetonitrile-40% 0.02 M sodium phosphate buffer, pH 7.0, at a flow rate of 1.0 mL/min. Uracil was used as a t0 marker for k value measurements of the test solutes, which were determined as the arithmetic mean of triplicate runs. Measurements were performed by stepwise increasing the column temperature. 4346 Analytical Chemistry, Vol. 70, No. 20, October 15, 1998
RESULTS AND DISCUSSION Characteristics at Low pH. A potential advantage for bidentate silane stationary phases on silica supports is superior stability with low-pH mobile phases, as a result of the covalent anchoring by two siloxane bonds to the silica support. The 2-fold attachment assists in minimizing the normal attrition of conventional monofunctional stationary phases that results from the hydrolysis of attaching siloxane bonds and loss of the stationary phase. This feature was demonstrated in an earlier report characterizing bidentate phases prepared from commercially available silanes.4 The stability of the bonded bidentate structure at low pH is further illustrated with the data in Figure 2. Here, three different types of C18 stationary phases on the same type of silica support were continuously purged (“aged”) under highly aggressive conditions with a methanol-1% trifluoroacetic acid (pH ∼0.9) mobile phase at 90 °C. We found that the bidentate-C18 was almost as stable as the sterically protected diisobutyl-C18 silane, which has been documented as a highly stable silane stationary phase.4,5 Figure 2 also shows that the bidentate-C18 packing clearly possesses greater stability than a conventional monofunctional dimethyl-C18 packing prepared on the same type silica. If the bidentate silane was only attached to the silica support by a single bond, one would expect that the packing stability would more nearly be that of a conventional monofunctional-C18 material. Therefore, the excellent stability of the bidentate-C18 packing is taken as additional evidence of attachment of the bidentate structure in two places on the silica support, as illustrated in Figure 1. We also speculate that the formation of a thermodynamically favorable ring structure (Figure 1) may also contribute to the drive for reaction of both ends of the bidentate structure. The attractive low-pH stability of the bidentate configuration also is coupled with the excellent mass transfer and high column efficiency that is characteristic of conventional monofunctional silane stationary phases, as illustrated by the low-pH (∼2) chromatogram in Figure 3 for the bidentate-C8/C18 column
Figure 3. Separation of drugs with bidentate C8/C18 column: column, 15 × 0.46 cm; mobile phase, 45% acetonitrile-55% 0.1% trifluoroacetic acid; flow rate, 1.0 mL/min; temperature 23 °C; UV detector, 254 nm; pressure, 75 bar; sample, 10-µL solution of components; N, plate number; Tf5, tailing factor at 5% of peak height.12
packing (R ) n-octyl, Q ) ethyl in Figure 1). Similar results at low pH (not given here) also were found for other bidentate structures. Results at Intermediate pH. Chromatographic results for the bidentate column packings prepared in this study are summarized in Table 1, together with comparable data from conventional monofunctional dimethyl-C8 and dimethyl-C18 columns also double end-capped and prepared with the same type of silica support. Two chromatographic tests were used: the highly basic drug, amitriptyline, with acetonitrile-pH 7.0 phosphate buffer, and neutral toluene with a simple methanol-water mobile phase. It should be noted that no attempt was made to optimize the packing method used to prepare the bidentate columns, while the dimethylC8 and -C18 columns were commercial units whose packing methods presumably had been optimized. Despite the lack of column-packing optimization for the bidentate materials, data obtained on four separate experimental lots of bidentate -C18P packing resulted in columns showing peak asymmetry values with standard deviations of 4.5 and 5.3% for toluene and amitriptyline, respectively. Plate number variations were similar for these columns, with standard deviations of 4.4 and 4.8%, respectively, for toluene and amitriptyline. As might be expected, asymmetry and plate number deviations were smaller for columns made from the same lot of bidentate-C18P packing. Note that the method used in calculating the plate numbers in Table 1 results in somewhat higher than actual values for some peaks. Peaks with asymmetry values of
