Overloading Study of Bases Using Polymeric RP-HPLC Columns as

Overloading Study of Bases Using Polymeric RP-HPLC Columns as an Aid to ... explanation of why band shape changes with sample size and mobile-phase pH...
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Anal. Chem. 2002, 74, 4672-4681

Overloading Study of Bases Using Polymeric RP-HPLC Columns as an Aid to Rationalization of Overloading on Silica-ODS Phases Stephan M. C. Buckenmaier and David V. McCalley*

Centre for Research in Biomedicine, University of the West of England, Frenchay, Bristol BS16 1QY, U.K. Melvin R. Euerby

AstraZeneca R&D Charnwood/Lund, Pharmaceutical and Analytical R&D, Bakewell Road, Loughborough, Leicestershire LE11 5RH, U.K.

The separation of ionized bases by reversed-phase liquid chromatography with alkyl silica columns often leads to severely tailed bands that are highly detrimental. Band shape and its dependence on sample mass are notably different when mobile-phase pH is changed, and this behavior has not been previously explained. Ionized silanols present in the stationary phase have been credited with a role in determining peak shape. In the present study, separations on two different polymer columns were compared with those previously obtained on alkyl silica phases. Because silanols are absent from polymer columns, this comparison enabled us to assess the role of silanols in separations on alkyl silica phases and to offer an explanation of why band shape changes with sample size and mobile-phase pH for both polymer and silicabased phases. Reversed-phase (RP) separations using silica phases bonded with octadecylsilyl (ODS) ligands have for many years dominated the field of HPLC, due to the many advantages of this technique. However, a problem exists in analyzing basic compounds, of which many pharmaceuticals constitute an important group, due to detrimental interactions with the stationary phase that can lead to poor peak shapes and low separation efficiency. These detrimental interactions have been claimed to result from the presence of residual silanol groups on the surface of the silica support.1 Giddings2 and others3 proposed that tailing can be produced by the presence of a few strong sites of high adsorption energy in the presence of a large number of sites of low adsorption energy. For the case of RP-LC with alkyl silica columns, strong interactions might involve protonated bases and ionized silanols versus weaker hydrophobic interactions between solute and alkyl ligands. Given the presence of these strong and weak retention * To whom correspondence should be addressed. Fax: (UK code) 117 3442904. E mail: [email protected]. (1) Leach, D. C.; Stadalius, M. A.; Berus, J. S.; Snyder, L. R. LC-GC Int. 1988, 1, 22-30. (2) Giddings, J. C. Dynamics of Chromatography; Marcel Dekker: New York, 1965. (3) Fornstedt, T.; Zhong, G.; Guiochon, G. J. Chromatogr., A 1996, 741, 1-12.

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sites, band tailing could result from either overload of the strong sites or much slower sorption-desorption of solute molecules from the strong sites compared with the weak sites. In contrast with the extensive literature on overloading in preparative chromatography,4 little work has been reported for ionized solutes in analytical situations. Snyder and co-workers5,6 showed that the column saturation capacity ws (equal to the maximum sample mass in milligrams that the column can hold) for the basic peptide angiotensin II was ∼60 times lower than that for the nonionogenic compound benzyl alcohol, leading to much wider tailing bands for angiotensin II as sample size was increased. The authors suggested that this could be explained by an overloading of a small number of ionized silanols at pH 2-3 (which serve as strong retention sites for angiotensin II). However, an alternative explanation proposed by the same authors5,7 is that initially adsorbed charged molecules discourage further sorption of sample molecules of the same charge, i.e., a mutual repulsion effect between sorbed ions. Furthermore, McCalley showed for retention of strong bases at pH 3 on new-generation, pure alkyl silica columns (type B) that silanols contributed little to retention, yet the column saturation capacity was still quite small, and band tailing accompanied by reduced retention of bases was obtained with increasing sample load.8-10 If bases are not retained on silanol sites on type B silicas at low pH, the question arises as to how overload of silanols can be the reason for reduced retention of high loads of bases at low pH. Much higher loading capacities could be obtained by operating columns with a pH 7 mobile phase, where at least 10-20 µg could be injected without substantial deterioration in efficiency. Column overload is of special interest to the pharmaceutical industry, e.g., in the determination of impurities. Here, it is necessary to inject large sample sizes to enable detection of small (4) Fornstedt, T.; Guiochon, G. Anal. Chem. 2001, 73, 608A-617A. (5) Eble, J. E.; Grob, R. L.; Antle, P. E.; Snyder, L. R. J. Chromatogr., A 1987, 384, 45-79. (6) Snyder, L. R.; Cox, G. B.; Antle, P. E. Chromatographia 1987, 24, 82-96. (7) Cox, G. B.; Snyder, L. R. J. Chromatogr. 1989, 483, 95-110. (8) McCalley, D. V. J. Chromatogr., A 2000, 902, 311-321. (9) McCalley, D. V. J. Chromatogr., A 1998, 793, 31-46. (10) McCalley, D. V.; Brereton, R. G. J. Chromatogr., A 1998, 828, 407-420. 10.1021/ac0202381 CCC: $22.00

© 2002 American Chemical Society Published on Web 08/14/2002

impurity peaks, which, however, may be obscured by broad or tailing signals from the major constituents. Porous polymer columns (made entirely from organic polymers) can be compared with alkyl silica columns as a means of clarifying the mechanism of band tailing and overloading for basic solutes. The chemical composition of these columns (not to be confused with silica phases having polymeric ODS layers) is completely different from that of silica; they have no silanol groups at all. We have not studied totally polymeric columns before and are unaware of any other reports describing the overloading behavior of basic compounds on such phases. We initially selected Hamilton PRP-1, a porous poly(styrene-divinylbenzene) (PSDVB) copolymer, for study. We also studied Asahipak ODP-50, a poly(vinyl alcohol) (PVA) phase whose free alcohol groups are esterified with stearic acid, to provide a functionality similar to ODS. However, some free alcohol groups might conceivably remain underivatized on such a phase. If silanol overload is the major cause of low capacity of protonated bases at acid pH on silica, polymeric phases should give completely different loading behavior. Since no detailed information was available from the manufacturers, ionic sites might exist on either polymeric phase. Therefore, we decided first to investigate whether polymers offer a pure hydrophobic retention surface, as is often supposed. Indeed, our previous work in column characterization indicated that Astec C18, a polymer phase based on PVA (as with Asahipak), has some ion-exchange capacity at pH 7.6.11 A secondary aim was to investigate retention and overloading of polymeric phases, which have considerable uses in their own right, as both HPLC and solidphase extraction materials. Despite the advantages of polymeric phases however, shrinking and swelling effects giving lower efficiency, and also lower pressure stability, have somewhat limited their impact.12 EXPERIMENTAL SECTION Equipment and Reagents. The HPLC system was a model 1100 (Agilent, Waldbronn, Germany) comprising autosampler, high-pressure binary pump, heated column thermostat, and variable-wavelength UV detector (1-µL flow cell, 5-mm path length). Injections of 5 µL were made, and the column was maintained at 30 °C. Void volume was measured by injection of uracil. pH adjustment of the mobile phase was made prior to addition of the organic solvent by making solutions of KH2PO4 or K2HPO4 and adjusting with concentrated phosphoric acid or KOH. For pH 2-3 and pH 7 buffers, adjustment was made in such a way as to keep [K+] known and constant, on the assumption that any ionic interaction between columns and bases was likely to be cation-exchange interactions with negatively charged sites on the columns. The Hamilton PRP-1 column, length 15 cm × 0.41 cm i.d., particle size 5 µm, surface area of dry packing 415 m2 g-1, was obtained from Fisher (Loughborough, U.K.), and the Asahipak ODP-50 column length 12.5 cm × 0.4 cm i.d., particle size 5 µm, surface area of dry packing 100 m2 g-1, was obtained from Esslab (Hadleigh, U.K.). The pH stability range claimed by the manufacturers was 1-13 for PRP-1 and pH 2-13 for Asahipak. Columns were generally used conservatively over the range pH 2-12 for the former and pH 3-12 for the latter. (11) Euerby, M. R.; Petersson, P. LC.-GC Eur. 2000, 13, 665-677. (12) Neue, U. D. HPLC Columns; Wiley-VCH: New York, 1997.

Acetonitrile (far-UV HPLC grade), THF, and phosphate salts (HPLC grade) were obtained from Fisher Scientific (Loughborough, U.K.). All test solutes were obtained from Sigma-Aldrich (Poole, U.K.) and were of the highest available grade. Pyridine is toxic (particularly to liver and kidneys) and requires handling in a fume hood using protective equipment; this chemical requires specialist disposal (high-temperature incineration). Beer-Lambert Law Deviations. Small reduced-path length detector cells give reduced extracolumn band spreading but require higher concentrations of solute to yield a given absorbance. Higher concentrations can give deviations from the BeerLambert law due to phenomena such as self-absorption. Such overload can affect peak shape measurements but can usually be avoided by appropriate choice of detection wavelength. Measurement of Column Efficiency and Column Saturation Capacity. We used the decline in efficiency with sample mass as the primary means of assessing column overload, with changes in retention as a secondary measure. It is important to establish the apparent effect of overload when various measures of column efficiency are used. We studied overload plots using the efficiency at a given load relative to the maximum efficiency (e.g., at small sample mass), N/No, against load when efficiency was calculated by a number of different methods. These included the half-height method (at 50% of peak height), the Dorsey-Foley procedure (10%),13 the 5-σ method (4.4%), the tailing method (5%), the tangent method (0%), and the statistical moments method. In addition, the method as used by Snyder for assessment of peaks with rightangled triangle shapes was also used to calculate the efficiency of peaks approaching this shape.6 All measurements were made using the Agilent Chemstation. A variety of solutes was investigated at different pH to reflect the different peak profiles obtained under overload conditions (see below). Although considerable variations occurred in absolute measurements of efficiency, differences were small when the relative efficiency (N/No) was utilized. However, results obtained using the statistical moments procedure were less reproducible than those from other methods. Thus, for graphical display of loss in efficiency with load, we chose the half-height method, due to its high reproducibility and general use in chromatography for making efficiency measurements. For determining ws for heavily overloaded peaks that gave right-angled triangle shapes, we used the specific method advocated by Snyder and co-workers.6,14 ws can be calculated from the formula

Wbase )

16t02(1 + k0)2 6t02k02wx + N0 ws

(1)

where Wbase is the peak width at base, wx is the sample mass of an overloaded peak, t0 is the column dead time, and k0 is the retention factor for a small sample. The end of an overloaded band was taken as the end of a small band, as recommended.6 RESULTS AND DISCUSSION Retention Mechanism on Polymeric Columns. We initially investigated the ionic character of the polymeric columns by (13) Foley, J. P.; Dorsey, J. G. Anal. Chem. 1983, 55, 730-737. (14) Snyder, L. R.; Kirkland, J. J.; Glajch, J. L. Practical HPLC Method Development; Wiley: New York, 1997.

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Figure 1. k versus the reciprocal of buffer cation concentration in (a) acetonitrile-phosphate (pH 2; 30:70, v/v) for nortriptyline (2), (10: 90, v/v) quinine (×), and benzylamine (0) with the PRP-1 phase; (b) acetonitrile-phosphate (pH 3; 35:65, v/v) for nortriptyline (2) with Asahipak.

determining the retention factor k of bases as a function of the buffer cation strength. Plots of k against the inverse of buffer cation concentration have been shown to give a straight line with positive slope on silica-ODS phases,15 which follows from a contribution of ion exchange to retention. The slope of such plots and its extrapolation to infinite buffer cation concentration allows assessment of the contributions of ion-exchange and hydrophobic interactions to overall retention. In previous work, using a pure silica (type-B) RP-LC column,8 we showed that such plots had virtually zero slope at pH 3, indicating little contribution of ion exchange to retention. Such results at acid pH are not typical for older RP-LC phases, which may contain higher concentrations of strongly acidic silanol groups.11,15 In contrast, at pH 7, strong bases (which remain positively charged) showed decreased retention as buffer cation concentration was increased, giving plots with a positive slope. This result indicates increasing ionization of silanol sites as the pH is raised, which is expected from an approximate pKa ) 7 for silanols.16 For separation on polymeric columns, on the other hand, there are no silanols to ionize. Figure 1 shows plots for nortriptyline, quinine, and benzylamine on PRP-1 at pH 2 and for nortriptyline on Ashipak at pH 3. There is little if any effect of buffer cation concentration on retention for either polymeric column, since the various plots are fairly flat; thus, ion exchange is unimportant, similar to modern alkyl silica columns at low pH.8 In contrast, Figure 2 shows retention decreases (15) Cox, G. B.; Stout, R. W. J. Chromatogr. 1987, 384, 315-336. (16) Nawrocki, J. J. Chromatogr., A 1997, 779, 29-71.

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Figure 2. k versus the reciprocal of buffer cation concentration in (a) acetonitrile-phosphate (pH 7; 40:60 v/v) for diphenhydramine (]) and quinine (×) with PRP-1; (b) for nortriptyline (2), quinine (×), and diphenhydramine (]) with Asahipak; and (c) acetonitrile-phosphate (pH 12; 50:50, v/v) for diphenhydramine (]), quinine (×), and benzylamine (0) with PRP-1.

noticeably with increasing buffer cation concentration for both polymeric columns at pH 7. Thus, it appears that cation-exchange sites exist on polymeric phases at higher pH, as is also the case with silica phases. The moderate curvature in the plots at higher buffer concentration may be due to “salting out” effects. Most commercial PVA is prepared by hydrolysis of polyvinyl acetate. For both PVA and PS-DVB, free radical generators such as dibenzoyl peroxide may be used in the production process. Residues of these or other reagents used could introduce charged species, such as carboxylate groups, into the polymer. At high pH (pH 12), however, the solutes themselves are uncharged, since this is well above the pKa of the most basic solute (nortriptyline, pKa ) ∼10.0) and ionic retention is unlikely. Figure 2c confirms

Table 1. Maximum Efficiencies (No in Plates/Column), Sample Sizes (in µg) Necessary To Produce Reduction in No by 10%, Best Peak Symmetry (As(min)), Maximum Retention (ko), Ratio of Minimum Retention to Maximum Retention (k/ko(min)), and Column Capacity ws (in mg Obtained under the Conditions of Figure 4)

nor diph quin benz proc Figure 3. Superposition of peak profiles for increasing sample mass for nortriptyline (wx ) 0.1-20 µg) on Asahipak, mobile phase acetonitrile-100 mM phosphate (pH 3; 25:75, v/v). For benzylamine on PRP-1 (wx ) 0.1-20 µg), mobile-phase acetonitrile-30 mM phosphate (pH 7) (15:85, v/v), and for quinine (wx ) 2.6-20 µg) on PRP-1 mobile phase acetonitrile-30 mM phosphate (pH 12; 43:57, v/v).

this hypothesis, showing a zero slope of the plots of k versus buffer cation strength. It thus appears that the retention of protonated bases on polymeric columns is mainly a hydrophobic process at low pH (solutes charged but column sites uncharged) and high pH (solutes uncharged but column sites charged), while cation exchange contributes additionally to retention at pH 7 (strongly basic solutes charged, column sites charged). Overview of the Effect of Sample Load of Bases on Peak Profile at Low, Intermediate, and High pH. Figure 3 shows representative results of superposition of peak profiles when increasing amounts (up to ∼20 µg) of solute are injected at low, high, and neutral pH. For purposes of simplicity, only five to eight plots at different sample load are shown for each solute, although about twice this number of data points were actually obtained. At low pH (2-3), peaks became increasingly right-angled triangle in shape with increasing sample load together with increasing peak width and tailing (“overload tailing”). Retention times move to shorter and shorter values as sample load is increased, although the end of the peak occurs at a common point. This behavior is similar to that shown by Snyder and co-workers,5-7 who studied mostly neutral compounds. At pH 7, a somewhat different pattern was obtained. Peaks showed a more complex profile, with pronounced exponential tailing. This tailing, which clearly differs from that observed at pH 3, appears to be indicative of kinetic effects, e.g., as a result of ion-exchange interactions additional to hydrophobic interactions. Decreasing retention with sample load is again observed at pH 7, as at pH 2. At pH 12, no significant change in peak shape or retention with load up to 20 µg was indicated. This result can be attributed to the uncharged nature of the base at this high pH, as a result of which column overload does not occur for this range of sample masses. The small peak at higher retention for quinine is an impurity (hydroquinine). A detailed consideration of overload at each pH and a comparison with the behavior of neutral and anionic solutes on the same columns follows.

nor (pH 3) diph (pH 3)

No-10% (µg)

N0 (p/c)

As(min)

5850 (0.10) 5160 (0.09) 2670 (0.08) 6530 (0.08) 2880 (0.11)

Hamilton PRP-1 1.6 7.9 0.3 (0.10) (0.10) 1.7 4.6 0.6 (0.09) (0.09) 1.8 2.6 1.0 (0.08) (0.08) 1.8 0.4 0.6 (0.08) (0.08) 2.1 0.2 7.0 (0.05) (0.05)

0.90 (9.9) 0.84 (22.7) 0.90 (20.0) 0.85 (20.1) 0.93 (21.2)

5250 (0.10) 5740 (0.08)

Asahipak ODP-50 1.4 9.4 0.8 (0.05) (0.05) 1.3 3.3 0.5 (0.08) (0.08)

0.84 (20.4) 0.84 (20.9)

ko

k/ko(min)

ws (mg) 2.8 2.5 3.6 1.2 2.5

3.0 2.8

Effect of Sample Load of Bases at Low pH (pH 2-3). Figure 4 shows plots of N/No and As/As(min) (asymmetry factor divided by minimum asymmetry factor) against sample load for five bases analyzed on PRP-1 in mobile phases buffered at pH 2 in combination with acetonitrile. A representative plot of k/ko (retention factor divided by retention factor for small sample load) against sample mass is also shown for benzylamine. We chose a relatively high concentration of phosphate buffer (0.06 M) and a pH around the first pKa of phosphate (pKa ) 2.1), to achieve good buffering capacity. This precaution was taken in order to eliminate any possible effects of buffer overload (rather than column overload) on the results. Table 1 shows overloading behavior for bases on PRP-1 at acid pH, with some additional results for Asahipak included. Note a higher buffer concentration (0.1 M instead of 0.06 M) was used for Asahipak at pH 3 to compensate for worse buffer capacity of phosphate, further away from its pKa. The value in parentheses below the value of the actual parameter measured is the respective load (µg) at which it was taken. Nortriptyline, diphenhydramine, benzylamine, and quinine gave similar “trumpet-shaped” plot profiles (as in Figure 4) with rapidly deteriorating column efficiency and increase in peak asymmetry with sample load. For benzylamine, the apparent improvement in As at sample loads above 10 µg may be due to peak distortion (simultaneous tailing and fronting). When a high load (20 µg) was used, a reduction in k of g10% compared to its small mass value (ko) occurred for these analytes (see Table 1). Procainamide followed a similar pattern but showed much less tendency to overload. Thus it was not possible to produce 50% loss in efficiency of No for accurate ws calculation6 or reduction of k by 10% of ko at the highest sample loads investigated (∼20 µg, Table 1). This observation can be attributed in part to its low k. Since k is a measure of the amount of sample in the stationary phase divided by the amount of sample in the mobile phase at a given time, overload is more likely for high k compounds (see eq 1). To measure ws accurately for procainamide, much higher sample loads6 were necessary (up to 125 µg used to deduce the ws value reported in Table 1). Analytical Chemistry, Vol. 74, No. 18, September 15, 2002

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Figure 4. N/No (2), As/As(min) (]), and k/k0 versus sample size wx (µg) for five basic solutes on PRP-1 using acetonitrile-60 mM phosphate (pH 2) (30:70, v/v) for nortriptyline and diphenhydramine and (10:90 v/v) for procainamide, quinine, and benzylamine.

The overloading profiles for diphenhydramine and nortriptyline on Asahipak (not shown) were similar to those on PRP-1; thus, there seems no reason to suspect fundamental differences in the performance of these two phases. ws values for the polymeric columns are similar, and of the same order as reported previously for the same compounds on silica ODS, although they cover a rather narrower range. The general similarity in overloading behavior for polymeric phases and pure alkyl silica (type B) columns is striking, suggesting the possibility of a common overloading mechanism; that is likely to be mutual ionic repulsion of protonated bases held on the hydrophobic surface of the stationary phase. 4676 Analytical Chemistry, Vol. 74, No. 18, September 15, 2002

Comparison of Column Loadability for Neutrals. Figure 5 shows overloading behavior for the neutrals toluene, phenol, naphthalene, and benzyl alcohol on PRP-1. Pyridine is essentially neutral at pH 7 (loading profile, Figure 6), and its performance can also be compared. The presence of organic solvent has been clearly shown to reduce still further the expected degree of protonation of pyridine (aqueous pKa ) 5.2) in the mobile phase.17 Table 2 shows the performance parameters for these compounds obtained under the conditions of Figure 5, and Table 3 and Figure 6 for pyridine at pH 7. Pyridine (pH 7), phenol (pH 2), and benzyl (17) McCalley, D. V. J. Chromatogr. 1994, 664, 139-147.

Figure 5. Superposition of peak profiles obtained for increasing sample mass and corresponding plots of N/No (2) and As/As(min) (]) versus sample size wx (µg) for neutrals on PRP-1 using acetonitrile60 mM phosphate buffer pH 2 (30:70, v/v) for phenol and (60:40, v/v) for toluene, (30:70, v/v) for benzyl alcohol, and (73:27, v/v) for naphthalene.

alcohol are of particular interest since they were analyzed in a mobile phase of organic content (30% acetonitrile) similar to that used for bases at pH 2. This factor negates gross differences in behavior of the phase caused by polymer swelling/shrinking effects in the organic solvent. Due to their higher retention, toluene and naphthalene results were obtained in mobile phases containing substantially more acetonitrile (60 and 73%, respectively). The peak profiles did not change markedly within the range of sample size (up to ∼20 µg) in which the bases at pH 2 had given right-angled triangle shapes. Nevertheless, gradual deterioration leading to a right-angled triangle was still obtained, for example with benzyl alcohol on PRP-1 but using considerably higher loads (240-475 µg) (Figure 5). Similarly, comparing Tables 1 and 2 shows that ws for benzyl alcohol was at least 80-250 times larger than that of bases when chromatographed at acid pH. This difference is of the same order as that reported by Snyder5 between benzyl alcohol and a basic peptide at acid pH on silicaODS columns. Pyridine at pH 7 (Table 3) produced a similarly large ws value (225 mg). Although a higher range loading experiment was not performed for phenol so that ws could be measured accurately, clearly its ws is similarly much higher than for protonated bases (Figure 5). ws was not as high for naphthalene on PRP-1 as expected (see Table 2). It is possible that this result is influenced by unfavorable

diffusion into PS-DVB itself, due to similar values of δ, the solubility parameter.19-21 In contrast, higher ws was found using Asahipak (PVA matrix). The low efficiencies found for naphthalene and toluene may be due to swelling of the polymers in high concentrations of organic solvent, making overloading results less comparable with those for protonated bases. It has been suggested that peak shapes on PS-DVB phases may be improved by substituting THF for a small proportion of the mobile-phase modifier content (e.g., 5%).19,22 Some beneficial solvent-induced swelling of the polymer matrix may occur by sorbed THF, giving less hampered diffusion of sample molecules. Alternatively, some type of selective binding or blocking of the smallest micropores by THF occurs, which may beneficially render these regions inaccessible to sample molecules;19,23 possibly these factors influence overload behavior. Thus, while all previous overloading experiments had been performed using acetonitrile as organic modifier, we measured ws for nortriptyline at pH 2 using PRP-1 with 5% THF substituted for 5% of the acetonitrile content. All other conditions were identical to those used previously. Overloading patterns with and without THF, however, were very similar and ws around 3 mg for nortriptyline was again obtained. It was concluded that phase loadability was much higher for neutrals than protonated bases and that this conclusion was probably unaffected by the presence of micropores in the polymer. In further experiments, we continued to use acetonitrile exclusively as organic modifier, without addition of THF. Effect of Sample Load of Bases at pH 7. Figure 6 and Table 3 show the effect of sample load on the performance of PRP-1 at pH 7. We adjusted the organic modifier concentration to give approximately the same k value for each individual compound as had been obtained at pH 2. Overloading depends on k, and in this way, a rough comparison of performance can be made without the necessity of calculating ws. Pyridine has already been discussed above. Quinine (pKa ) 8.5), diphenhydramine (pKa ) 9.0), benzylamine (pKa ) 9.3), and procainamide (pKa ) 9.2) are considerably stronger bases that remain significantly protonated at pH 7, despite organic solvent effects. These reduce the effective pKa of bases and increase the effective pH of the phosphate buffer.17,18 Clearly, loading behavior is quite different from that shown at pH 2 on the same column. The plots of N/No and As/As(min) for three compounds (quinine, pyridine, procainamide) show only very small decreases in column efficiency with loads up to 20 µg, whereas diphenhydramine and benzylamine actually show increases in efficiency. Note in Table 3 that No sometimes occurred at highest sample loads in contrast to lowest sample loads as found at low pH. Nevertheless, considerable reduction in k was shown with increasing sample size, particularly for benzylamine and quinine (k/ko ) 0.53 and 0.54, respectively using a 20-µg sample load). These decreases in retention were generally more pronounced than those at low pH. The base loading behavior at pH (18) Canals, I.; Oumada, F. Z.; Rose´s, M.; Bosch, E. J. Chromatogr., A 2001, 911, 191-202. (19) Gawdzik, B.; Osypiuk, J. Chromatographia 2001, 54, 595-599. (20) Ells, B.; Wang, Y.; Cantwell, F. F. J. Chromatogr., A 1999, 835, 3-18. (21) Li, J.; Cantwell, F. F. J. Chromatogr., A 1996, 726, 37-44. (22) Bowers, L. D.; Pedigo, S. J. Chromatogr. 1986, 371, 243-251. (23) Tanaka, N.; Ebata, T.; Hashizume, K.; Hosoya, K.; Araki, M. J. Chromatogr. 1989, 475, 195-208.

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Figure 6. Loading plots for PRP-1 as in Figure 4. Mobile phase acetonitrile-30 mM phosphate (pH 7) (50:50 v/v) for quinine, (15:85, v/v) for benzylamine, (30:70, v/v) for pyridine, (40:60, v/v) for diphenhydramine, and (20:80, v/v) for procainamide.

7 was very similar to that found for the same compounds under similar conditions using pure silica-ODS (type B) phases.8 Our studies of retention as a function of buffer cation strength (Figure 2) indicated that the retention mechanism of stronger bases on polymeric phases at pH 7 is a combination of ionexchange and hydrophobic retention. The slow kinetics of the (strong) ion-exchange sites relative to the fast kinetics of (weak) hydrophobic sites give rise to exponential tailing and low efficiency with small sample load. The apparent improvement in efficiency with load shown particularly for benzylamine and diphen4678 Analytical Chemistry, Vol. 74, No. 18, September 15, 2002

hydramine (albeit initially from poor values of 200-500 plates) may be rationalized by theoretical explanations given by Giddings2 for a surface containing a small number of strong adsorption sites in the presence of a larger number of weaker sites. As the sample load is increased, the small number of strong sites would essentially become saturated, and the additional adsorbate forced onto the weak sites, serving to increase the fraction of the solute on the latter. If the weak sites are themselves not overloaded (see below), their influence may swamp that of the strong sites. In this case, it is possible that peak shape can improve. Indeed, the

Table 2. Overloading of Neutrals (for Conditions, See Figure 5)

toluene (pH 2) phenol (pH 2) napht benzylalc

napht

No-10% (µg)

N0 (p/c)

As(min)

4850 (0.16) 6860 (0.10) 1340 (0.08) 6880 (0.11)

Hamilton PRP-1 1.7 8.0 5.0 (0.32) (0.05) 1.3 3.8 7.4 (0.10) (0.08) 2.5 7.7 5.1 (0.08) (0.08) 1.3 2.4 15 (0.50) (0.11)

0.92 (20.0) 0.99 (20.4) 0.82 (101) 0.96 (475)

high

diph

high

quin

9.2

benz

300

proc

2790 (0.08)

Asahipak ODP-50 1.8 2.2 51 (0.08) (0.08)

0.92 (101)

high

nor

ko

k/ko(min)

ws (mg)

Table 3. Overloading of Bases at pH 7 (for Conditions, See Figure 6 and Figure 8)

diph quin benz proc pyr 2NSA

Table 4. Overloading of Bases at pH 12 (for Conditions, See Figure 7)

N0 (p/c)

As(min)

240 (20.2) 65 (0.32) 520 (15.5) 940 (1.1) 7020 (0.10) 4090 (1.1)

4.5 (0.32) 5.8 (0.32) 5.0 (0.10) 3.8 (0.08) 1.5 (0.10) 1.9 (0.32)

ko

No-10% (µg)

Hamilton PRP-1 6.2 (0.32) 2.7 5.0 (0.32) 0.7 (0.10) 0.5 7.0 (0.05) 1.1 5.3 (0.10) 1.1 5.1 (0.10)

k/ko(min) 0.75 (20.2) 0.54 (20.0) 0.53 (20.1) 0.74 (21.0) 0.93 (639) 0.86 (9.9)

ws (mg) high high high high 225 2.4

peak shapes we obtained resemble those predicted by Giddings. Swamping of strong sites is also very likely to explain the marked decreases in retention that occur as sample load is increased. The question of why driving the solute onto the hydrophobic sites does not apparently cause strong overload of these sites must finally be answered. First, the degree of ionization of even the stronger bases is reduced as the pH is raised. Organic solvent effects (see above) reduce ionization of strong bases further than might be expected from consideration of a purely aqueous system.17,18 Thus, a substantial fraction of the base exists in the unprotonated form. The retention factor of a partially protonated base is a composite of the retention factors of the protonated (low k) and unprotonated (considerably higher k) forms, weighted according to the fraction of molecules in each state. Since k (protonated) is much smaller,24 then the fraction of protonated base molecules in the stationary phase is smallsmost ionized molecules are in the mobile phase. Thus, the phase is more difficult to overload, although its composite (larger) k value might indicate that overloading should take place more easily. Other factors may be involved in increased sample capacity at pH 7, for instance, the existence of further secondary ionic retention sites available for population in addition to the usual hydrophobic sites or neutralization of solute positive charge by the opposite charge on the phase. (24) Wilson, N. S.; Nelson, M. D.; Dolan, J. W.; Snyder, L. R.; Wolcott, R. G.; Carr, P. W. J. Chromatogr., A 2002, 961, 171-193.

No (p/c) 780 (20.8) 1300 (5.2) 1240 (20.1) 2560 (16.7) 3020 (19.9)

As(min) Hamilton PRP-1 3.0 (0.80) 2.7 (0.08) 6.1 (0.80) 2.4 (16.7)

ko 6.4 (6.9) 2.8 (0.08) 1.1 (0.32) 0.6 (0.08)

Asahipak ODP-50 1.9 5.9 (9.9) (0.32)

k/ko(min) 1 1 0.62 (20.1) 0.98

0.97 (19.9)

Effect of Sample Load of Bases at High pH (pH 12). The high-pH stability of polymeric columns allows investigation of loadability at much higher pH than with silica-based phases. Figure 7 and Table 4 show the effect of sample load for bases at pH 12. For diphenhydramine, procainamide, quinine, and nortriptyline, sample load had relatively little effect on plate count and asymmetry factor, rather less than for pH 7, and much less than at pH 2. In addition, virtually no change occurred in k with sample loads up to 20 µg. This behavior was expected in that these compounds are all likely to be uncharged at pH 12, as is pyridine at pH 7. The behavior of benzylamine was anomalous, with peak shape improving and k reducing considerably with increasing sample mass, both in a fashion similar to that at pH 7. The reason for this behavior is obscure. A comparison of Table 4 with Tables 1 and 3 shows that maximum efficiencies for the compounds are higher at pH 12 than at pH 7 but inferior to those obtained at low pH. It is likely that this result is merely due to the higher concentrations of organic solvent necessary for elution at pH 12, resulting in swelling of the polymer and loss of efficiency (see arguments above). A disadvantage of work at pH 12 was the excessive equilibration times required to obtain stable values of column performance parameters for some compounds (∼20 h in the case of nortriptyline). Effect of Sample Load and Retention Mechanism for an Anionic Compound. If charge repulsion is the main contributory factor to overload (rather than being merely due to overload of cation-exchange sites present on polymeric columns), then it would be expected that anionic solutes might experience similar overloading effects to cationic solutes such as protonated bases. We selected 2-naphthalenesulfonic acid (2-NSA) as a compound that is negatively charged at pH 7 and the PRP-1 phase. Prior to the loading studies, we investigated the effect of increasing phosphate anion concentration on retention of 2-NSA at pH 7 to determine whether any anion-exchange sites existed on the phase. However, we failed to show decreased retention of 2-NSA with anion concentration (results not shown) indicating the absence of such sites. Figure 8 and Table 3 indicate clear evidence of reduction in retention and rapid deterioration in peak shape as sample load is increased up to 20 µg of injected solute. The general loading Analytical Chemistry, Vol. 74, No. 18, September 15, 2002

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Figure 7. Loading plots as in Figure 4. Mobile phase acetonitrile-30 mM phosphate (pH 12) (43:57,v/v) for quinine, (55:45, v/v) for benzylamine, (60:40, v/v) for diphenhydramine, (40:60, v/v) for procainamide on PRP-1, and (50:50, v/v) for nortriptyline on Asahipak.

behavior is very similar, and ws (2.4 mg) is comparable to the values obtained for protonated bases at low pH. This result adds weight to the proposal that mutual repulsion of charged ions on the hydrophobic surface may be largely responsible for the overloading effects observed on polymeric columns. CONCLUSIONS Although overload of ionized silanols cannot be entirely discounted by the present study, overload caused by ionic repulsion probably accounts for the majority of the increased band 4680

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tailing (“overload tailing”) and reduced efficiency that occurs on both pure alkyl silica (type B) and polymeric columns with increasing sample mass at low pH. Cation-exchange sites (slow kinetics) exist on both polymer and alkyl silica columns at pH 7. These give rise to significant exponential tailing (“kinetic tailing”), not present at low pH. As sample mass is increased at pH 7, saturation of cation-exchange sites occurs, resulting in a greater proportion of the sample being retained on hydrophobic sites (fast kinetics). This factor rationalizes the apparent improvement in efficiency and also the large concomitant decrease in k with sample

concentration required to elute the simultaneously occurring neutral species; neutralization of solute charge by the opposite charge on the stationary phase may be another factor. At high pH, solutes are unprotonated, leading generally to fewer detrimental effects. Columns that maintain good efficiency at high pH are necessary to exploit these advantages. It is possible that silanol overload may play a greater part on older, impure alkyl silica phases (type A). Experiments with such phases, as well as on pH-stable type B silica phases, are envisaged to investigate further some aspects of the results of the present study.

ACKNOWLEDGMENT The authors thank Alan McKeown (AstraZeneca R&D Charnwood) and Nicole Kirsch (UWE) for many helpful discussions. Figure 8. Loading plots for 2-NSA on PRP-1 (as in Figure 4), mobile phase acetonitrile-30 mM phosphate buffer (pH 7; 23:77, v/v).

load. Mutual ionic repulsion of protonated bases at pH 7 is much less likely due to their low k in mobile phases of sufficient organic

Received for review April 11, 2002. Accepted June 29, 2002. AC0202381

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