overloading study of bases using polymeric rp-hplc columns as an aid to rationalization of...

Overloading Study of Bases Using PolymericRP-HPLC Columns as an Aid to Rationalization ofOverloading 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 liquidchromatography with alkyl silica columns often leads toseverely tailed bands that are highly detrimental. Bandshape and its dependence on sample mass are notablydifferent when mobile-phase pH is changed, and thisbehavior has not been previously explained. Ionizedsilanols present in the stationary phase have been creditedwith a role in determining peak shape. In the presentstudy, separations on two different polymer columns werecompared with those previously obtained on alkyl silicaphases. Because silanols are absent from polymer col-umns, this comparison enabled us to assess the role ofsilanols in separations on alkyl silica phases and to offeran explanation of why band shape changes with samplesize and mobile-phase pH for both polymer and silica-based phases.

Reversed-phase (RP) separations using silica phases bondedwith octadecylsilyl (ODS) ligands have for many years dominatedthe field of HPLC, due to the many advantages of this technique.However, a problem exists in analyzing basic compounds, of whichmany pharmaceuticals constitute an important group, due todetrimental interactions with the stationary phase that can leadto poor peak shapes and low separation efficiency. These detri-mental interactions have been claimed to result from the presenceof residual silanol groups on the surface of the silica support.1

Giddings2 and others3 proposed that tailing can be producedby the presence of a few strong sites of high adsorption energyin the presence of a large number of sites of low adsorptionenergy. For the case of RP-LC with alkyl silica columns, stronginteractions might involve protonated bases and ionized silanolsversus weaker hydrophobic interactions between solute and alkylligands. Given the presence of these strong and weak retention

sites, band tailing could result from either overload of the strongsites or much slower sorption-desorption of solute moleculesfrom the strong sites compared with the weak sites. In contrastwith the extensive literature on overloading in preparative chro-matography,4 little work has been reported for ionized solutes inanalytical situations. Snyder and co-workers5,6 showed that thecolumn saturation capacity ws (equal to the maximum samplemass in milligrams that the column can hold) for the basic peptideangiotensin II was ∼60 times lower than that for the nonionogeniccompound benzyl alcohol, leading to much wider tailing bandsfor angiotensin II as sample size was increased. The authorssuggested that this could be explained by an overloading of a smallnumber of ionized silanols at pH 2-3 (which serve as strongretention sites for angiotensin II). However, an alternative explana-tion proposed by the same authors5,7 is that initially adsorbedcharged molecules discourage further sorption of sample mol-ecules of the same charge, i.e., a mutual repulsion effect betweensorbed ions. Furthermore, McCalley showed for retention ofstrong bases at pH 3 on new-generation, pure alkyl silica columns(type B) that silanols contributed little to retention, yet the columnsaturation capacity was still quite small, and band tailing ac-companied by reduced retention of bases was obtained withincreasing sample load.8-10 If bases are not retained on silanolsites on type B silicas at low pH, the question arises as to howoverload of silanols can be the reason for reduced retention ofhigh loads of bases at low pH. Much higher loading capacitiescould be obtained by operating columns with a pH 7 mobile phase,where at least 10-20 µg could be injected without substantialdeterioration in efficiency.

Column overload is of special interest to the pharmaceuticalindustry, e.g., in the determination of impurities. Here, it isnecessary to inject large sample sizes to enable detection of small

* To whom correspondence should be addressed. Fax: (UK code) 1173442904. 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.

(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.

Anal. Chem. 2002, 74, 4672-4681

4672 Analytical Chemistry, Vol. 74, No. 18, September 15, 2002 10.1021/ac0202381 CCC: $22.00 © 2002 American Chemical SocietyPublished on Web 08/14/2002

impurity peaks, which, however, may be obscured by broad ortailing signals from the major constituents.

Porous polymer columns (made entirely from organic poly-mers) can be compared with alkyl silica columns as a means ofclarifying the mechanism of band tailing and overloading for basicsolutes. The chemical composition of these columns (not to beconfused with silica phases having polymeric ODS layers) iscompletely different from that of silica; they have no silanol groupsat all. We have not studied totally polymeric columns before andare unaware of any other reports describing the overloadingbehavior of basic compounds on such phases. We initially selectedHamilton 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 areesterified with stearic acid, to provide a functionality similar toODS. However, some free alcohol groups might conceivablyremain underivatized on such a phase. If silanol overload is themajor cause of low capacity of protonated bases at acid pH onsilica, polymeric phases should give completely different loadingbehavior. Since no detailed information was available from themanufacturers, ionic sites might exist on either polymeric phase.Therefore, we decided first to investigate whether polymers offera pure hydrophobic retention surface, as is often supposed. Indeed,our previous work in column characterization indicated that AstecC18, a polymer phase based on PVA (as with Asahipak), has someion-exchange capacity at pH 7.6.11 A secondary aim was toinvestigate retention and overloading of polymeric phases, whichhave considerable uses in their own right, as both HPLC and solid-phase extraction materials. Despite the advantages of polymericphases however, shrinking and swelling effects giving lowerefficiency, and also lower pressure stability, have somewhat limitedtheir impact.12

EXPERIMENTAL SECTIONEquipment and Reagents. The HPLC system was a model

1100 (Agilent, Waldbronn, Germany) comprising autosampler,high-pressure binary pump, heated column thermostat, andvariable-wavelength UV detector (1-µL flow cell, 5-mm pathlength). Injections of 5 µL were made, and the column wasmaintained at 30 °C. Void volume was measured by injection ofuracil. pH adjustment of the mobile phase was made prior toaddition of the organic solvent by making solutions of KH2PO4 orK2HPO4 and adjusting with concentrated phosphoric acid or KOH.For pH 2-3 and pH 7 buffers, adjustment was made in such away as to keep [K+] known and constant, on the assumption thatany ionic interaction between columns and bases was likely to becation-exchange interactions with negatively charged sites on thecolumns.

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, wasobtained from Fisher (Loughborough, U.K.), and the AsahipakODP-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 manu-facturers was 1-13 for PRP-1 and pH 2-13 for Asahipak. Columnswere generally used conservatively over the range pH 2-12 forthe former and pH 3-12 for the latter.

Acetonitrile (far-UV HPLC grade), THF, and phosphate salts(HPLC grade) were obtained from Fisher Scientific (Loughbor-ough, U.K.). All test solutes were obtained from Sigma-Aldrich(Poole, U.K.) and were of the highest available grade. Pyridine istoxic (particularly to liver and kidneys) and requires handling ina fume hood using protective equipment; this chemical requiresspecialist disposal (high-temperature incineration).

Beer-Lambert Law Deviations. Small reduced-path lengthdetector cells give reduced extracolumn band spreading butrequire higher concentrations of solute to yield a given absor-bance. Higher concentrations can give deviations from the Beer-Lambert law due to phenomena such as self-absorption. Suchoverload can affect peak shape measurements but can usually beavoided by appropriate choice of detection wavelength.

Measurement of Column Efficiency and Column Satura-tion Capacity. We used the decline in efficiency with sample massas the primary means of assessing column overload, with changesin retention as a secondary measure. It is important to establishthe apparent effect of overload when various measures of columnefficiency are used. We studied overload plots using the efficiencyat a given load relative to the maximum efficiency (e.g., at smallsample mass), N/No, against load when efficiency was calculatedby a number of different methods. These included the half-heightmethod (at 50% of peak height), the Dorsey-Foley procedure(10%),13 the 5-σ method (4.4%), the tailing method (5%), the tangentmethod (0%), and the statistical moments method. In addition,the method as used by Snyder for assessment of peaks with right-angled triangle shapes was also used to calculate the efficiencyof peaks approaching this shape.6 All measurements were madeusing the Agilent Chemstation. A variety of solutes was investi-gated at different pH to reflect the different peak profiles obtainedunder overload conditions (see below). Although considerablevariations occurred in absolute measurements of efficiency,differences were small when the relative efficiency (N/No) wasutilized. However, results obtained using the statistical momentsprocedure were less reproducible than those from other methods.Thus, for graphical display of loss in efficiency with load, we chosethe half-height method, due to its high reproducibility and generaluse in chromatography for making efficiency measurements.

For determining ws for heavily overloaded peaks that gaveright-angled triangle shapes, we used the specific method advo-cated by Snyder and co-workers.6,14

ws can be calculated from the formula

where Wbase is the peak width at base, wx is the sample mass ofan overloaded peak, t0 is the column dead time, and k0 is theretention factor for a small sample. The end of an overloaded bandwas taken as the end of a small band, as recommended.6

RESULTS AND DISCUSSIONRetention Mechanism on Polymeric Columns. We initially

investigated the ionic character of the polymeric columns by

(11) Euerby, M. R.; Petersson, P. LC.-GC Eur. 2000, 13, 665-677.(12) Neue, U. D. HPLC Columns; Wiley-VCH: New York, 1997.

(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 Develop-

ment; Wiley: New York, 1997.

Wbase )16t0

2(1 + k0)2

N0+

6t02k0

2wx

ws(1)

Analytical Chemistry, Vol. 74, No. 18, September 15, 2002 4673

determining the retention factor k of bases as a function of thebuffer cation strength. Plots of k against the inverse of buffer cationconcentration have been shown to give a straight line with positiveslope on silica-ODS phases,15 which follows from a contributionof ion exchange to retention. The slope of such plots and itsextrapolation to infinite buffer cation concentration allows assess-ment of the contributions of ion-exchange and hydrophobicinteractions to overall retention. In previous work, using a puresilica (type-B) RP-LC column,8 we showed that such plots hadvirtually zero slope at pH 3, indicating little contribution of ionexchange to retention. Such results at acid pH are not typical forolder RP-LC phases, which may contain higher concentrations ofstrongly acidic silanol groups.11,15 In contrast, at pH 7, strong bases(which remain positively charged) showed decreased retentionas buffer cation concentration was increased, giving plots with apositive slope. This result indicates increasing ionization of silanolsites as the pH is raised, which is expected from an approximatepKa ) 7 for silanols.16 For separation on polymeric columns, onthe other hand, there are no silanols to ionize. Figure 1 showsplots for nortriptyline, quinine, and benzylamine on PRP-1 at pH2 and for nortriptyline on Ashipak at pH 3. There is little if anyeffect of buffer cation concentration on retention for eitherpolymeric column, since the various plots are fairly flat; thus, ionexchange is unimportant, similar to modern alkyl silica columnsat low pH.8 In contrast, Figure 2 shows retention decreases

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

(15) Cox, G. B.; Stout, R. W. J. Chromatogr. 1987, 384, 315-336.(16) Nawrocki, J. J. Chromatogr., A 1997, 779, 29-71.

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) withAsahipak.

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 (×), anddiphenhydramine (]) with Asahipak; and (c) acetonitrile-phosphate(pH 12; 50:50, v/v) for diphenhydramine (]), quinine (×), andbenzylamine (0) with PRP-1.

4674 Analytical Chemistry, Vol. 74, No. 18, September 15, 2002

this hypothesis, showing a zero slope of the plots of k versus buffercation strength.

It thus appears that the retention of protonated bases onpolymeric 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 cationexchange contributes additionally to retention at pH 7 (stronglybasic solutes charged, column sites charged).

Overview of the Effect of Sample Load of Bases on PeakProfile at Low, Intermediate, and High pH. Figure 3 showsrepresentative results of superposition of peak profiles whenincreasing amounts (up to ∼20 µg) of solute are injected at low,high, and neutral pH. For purposes of simplicity, only five to eightplots at different sample load are shown for each solute, althoughabout twice this number of data points were actually obtained. Atlow pH (2-3), peaks became increasingly right-angled trianglein shape with increasing sample load together with increasingpeak width and tailing (“overload tailing”). Retention times moveto shorter and shorter values as sample load is increased, althoughthe end of the peak occurs at a common point. This behavior issimilar to that shown by Snyder and co-workers,5-7 who studiedmostly neutral compounds. At pH 7, a somewhat different patternwas obtained. Peaks showed a more complex profile, withpronounced exponential tailing. This tailing, which clearly differsfrom that observed at pH 3, appears to be indicative of kineticeffects, e.g., as a result of ion-exchange interactions additional tohydrophobic interactions. Decreasing retention with sample loadis again observed at pH 7, as at pH 2. At pH 12, no significantchange in peak shape or retention with load up to 20 µg wasindicated. This result can be attributed to the uncharged natureof the base at this high pH, as a result of which column overloaddoes not occur for this range of sample masses. The small peakat higher retention for quinine is an impurity (hydroquinine).

A detailed consideration of overload at each pH and acomparison with the behavior of neutral and anionic solutes onthe same columns follows.

Effect of Sample Load of Bases at Low pH (pH 2-3).Figure 4 shows plots of N/No and As/As(min) (asymmetry factordivided by minimum asymmetry factor) against sample load forfive bases analyzed on PRP-1 in mobile phases buffered at pH 2in 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 arelatively high concentration of phosphate buffer (0.06 M) and apH around the first pKa of phosphate (pKa ) 2.1), to achieve goodbuffering capacity. This precaution was taken in order to eliminateany possible effects of buffer overload (rather than columnoverload) on the results.

Table 1 shows overloading behavior for bases on PRP-1 at acidpH, with some additional results for Asahipak included. Note ahigher buffer concentration (0.1 M instead of 0.06 M) was usedfor Asahipak at pH 3 to compensate for worse buffer capacity ofphosphate, further away from its pKa. The value in parenthesesbelow the value of the actual parameter measured is the respectiveload (µg) at which it was taken. Nortriptyline, diphenhydramine,benzylamine, and quinine gave similar “trumpet-shaped” plotprofiles (as in Figure 4) with rapidly deteriorating columnefficiency and increase in peak asymmetry with sample load. Forbenzylamine, the apparent improvement in As at sample loadsabove 10 µg may be due to peak distortion (simultaneous tailingand fronting). When a high load (20 µg) was used, a reduction ink of g10% compared to its small mass value (ko) occurred for theseanalytes (see Table 1). Procainamide followed a similar patternbut showed much less tendency to overload. Thus it was notpossible to produce 50% loss in efficiency of No for accurate ws

calculation6 or reduction of k by 10% of ko at the highest sampleloads investigated (∼20 µg, Table 1). This observation can beattributed in part to its low k. Since k is a measure of the amountof sample in the stationary phase divided by the amount of samplein the mobile phase at a given time, overload is more likely forhigh k compounds (see eq 1). To measure ws accurately forprocainamide, much higher sample loads6 were necessary (up to125 µg used to deduce the ws value reported in Table 1).

Figure 3. Superposition of peak profiles for increasing sample massfor nortriptyline (wx ) 0.1-20 µg) on Asahipak, mobile phaseacetonitrile-100 mM phosphate (pH 3; 25:75, v/v). For benzylamineon PRP-1 (wx ) 0.1-20 µg), mobile-phase acetonitrile-30 mMphosphate (pH 7) (15:85, v/v), and for quinine (wx ) 2.6-20 µg) onPRP-1 mobile phase acetonitrile-30 mM phosphate (pH 12; 43:57,v/v).

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

N0(p/c) As(min) ko

No-10%(µg) k/ko(min)

ws(mg)

Hamilton PRP-1nor 5850 1.6 7.9 0.3 0.90 2.8

(0.10) (0.10) (0.10) (9.9)diph 5160 1.7 4.6 0.6 0.84 2.5

(0.09) (0.09) (0.09) (22.7)quin 2670 1.8 2.6 1.0 0.90 3.6

(0.08) (0.08) (0.08) (20.0)benz 6530 1.8 0.4 0.6 0.85 1.2

(0.08) (0.08) (0.08) (20.1)proc 2880 2.1 0.2 7.0 0.93 2.5

(0.11) (0.05) (0.05) (21.2)

Asahipak ODP-50nor (pH 3) 5250 1.4 9.4 0.8 0.84 3.0

(0.10) (0.05) (0.05) (20.4)diph (pH 3) 5740 1.3 3.3 0.5 0.84 2.8

(0.08) (0.08) (0.08) (20.9)


The overloading profiles for diphenhydramine and nortriptylineon Asahipak (not shown) were similar to those on PRP-1; thus,there seems no reason to suspect fundamental differences in theperformance of these two phases. ws values for the polymericcolumns are similar, and of the same order as reported previouslyfor the same compounds on silica ODS, although they cover arather narrower range. The general similarity in overloadingbehavior for polymeric phases and pure alkyl silica (type B)columns is striking, suggesting the possibility of a commonoverloading mechanism; that is likely to be mutual ionic repulsionof protonated bases held on the hydrophobic surface of thestationary phase.

Comparison of Column Loadability for Neutrals. Figure 5shows overloading behavior for the neutrals toluene, phenol,naphthalene, and benzyl alcohol on PRP-1. Pyridine is essentiallyneutral at pH 7 (loading profile, Figure 6), and its performancecan also be compared. The presence of organic solvent has beenclearly shown to reduce still further the expected degree ofprotonation of pyridine (aqueous pKa ) 5.2) in the mobile phase.17

Table 2 shows the performance parameters for these compoundsobtained under the conditions of Figure 5, and Table 3 and Figure6 for pyridine at pH 7. Pyridine (pH 7), phenol (pH 2), and benzyl

(17) McCalley, D. V. J. Chromatogr. 1994, 664, 139-147.

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.


alcohol are of particular interest since they were analyzed in amobile phase of organic content (30% acetonitrile) similar to thatused for bases at pH 2. This factor negates gross differences inbehavior of the phase caused by polymer swelling/shrinkingeffects in the organic solvent. Due to their higher retention,toluene and naphthalene results were obtained in mobile phasescontaining substantially more acetonitrile (60 and 73%, respec-tively).

The peak profiles did not change markedly within the rangeof sample size (up to ∼20 µg) in which the bases at pH 2 hadgiven right-angled triangle shapes. Nevertheless, gradual dete-rioration leading to a right-angled triangle was still obtained, forexample with benzyl alcohol on PRP-1 but using considerablyhigher loads (240-475 µg) (Figure 5). Similarly, comparing Tables1 and 2 shows that ws for benzyl alcohol was at least 80-250 timeslarger than that of bases when chromatographed at acid pH. Thisdifference is of the same order as that reported by Snyder5

between benzyl alcohol and a basic peptide at acid pH on silica-ODS columns. Pyridine at pH 7 (Table 3) produced a similarlylarge ws value (225 mg). Although a higher range loadingexperiment was not performed for phenol so that ws could bemeasured accurately, clearly its ws is similarly much higher thanfor protonated bases (Figure 5).

ws was not as high for naphthalene on PRP-1 as expected (seeTable 2). It is possible that this result is influenced by unfavorable

diffusion into PS-DVB itself, due to similar values of δ, thesolubility parameter.19-21 In contrast, higher ws was found usingAsahipak (PVA matrix). The low efficiencies found for naphthaleneand toluene may be due to swelling of the polymers in highconcentrations of organic solvent, making overloading results lesscomparable with those for protonated bases.

It has been suggested that peak shapes on PS-DVB phasesmay be improved by substituting THF for a small proportion ofthe mobile-phase modifier content (e.g., 5%).19,22 Some beneficialsolvent-induced swelling of the polymer matrix may occur bysorbed THF, giving less hampered diffusion of sample molecules.Alternatively, some type of selective binding or blocking of thesmallest micropores by THF occurs, which may beneficially renderthese regions inaccessible to sample molecules;19,23 possibly thesefactors influence overload behavior. Thus, while all previousoverloading experiments had been performed using acetonitrileas organic modifier, we measured ws for nortriptyline at pH 2 usingPRP-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 verysimilar and ws around 3 mg for nortriptyline was again obtained.

It was concluded that phase loadability was much higher forneutrals than protonated bases and that this conclusion wasprobably unaffected by the presence of micropores in the polymer.In further experiments, we continued to use acetonitrile exclu-sively as organic modifier, without addition of THF.

Effect of Sample Load of Bases at pH 7. Figure 6 and Table3 show the effect of sample load on the performance of PRP-1 atpH 7. We adjusted the organic modifier concentration to giveapproximately the same k value for each individual compound ashad been obtained at pH 2. Overloading depends on k, and inthis way, a rough comparison of performance can be made withoutthe necessity of calculating ws. Pyridine has already beendiscussed above. Quinine (pKa ) 8.5), diphenhydramine (pKa )9.0), benzylamine (pKa ) 9.3), and procainamide (pKa ) 9.2) areconsiderably stronger bases that remain significantly protonatedat pH 7, despite organic solvent effects. These reduce the effectivepKa of bases and increase the effective pH of the phosphatebuffer.17,18

Clearly, loading behavior is quite different from that shown atpH 2 on the same column. The plots of N/No and As/As(min) forthree compounds (quinine, pyridine, procainamide) show onlyvery small decreases in column efficiency with loads up to 20 µg,whereas diphenhydramine and benzylamine actually show in-creases in efficiency. Note in Table 3 that No sometimes occurredat highest sample loads in contrast to lowest sample loads as foundat low pH. Nevertheless, considerable reduction in k was shownwith increasing sample size, particularly for benzylamine andquinine (k/ko ) 0.53 and 0.54, respectively using a 20-µg sampleload). These decreases in retention were generally more pro-nounced than those at low pH. The base loading behavior at pH

(18) Canals, I.; Oumada, F. Z.; Roses, 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.

Figure 5. Superposition of peak profiles obtained for increasingsample mass and corresponding plots of N/No (2) and As/As(min) (])versus sample size wx (µg) for neutrals on PRP-1 using acetonitrile-60 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) fornaphthalene.


7 was very similar to that found for the same compounds undersimilar 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 strongerbases on polymeric phases at pH 7 is a combination of ion-exchange 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 efficiencywith small sample load. The apparent improvement in efficiencywith load shown particularly for benzylamine and diphen-

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 sitesin the presence of a larger number of weaker sites. As the sampleload is increased, the small number of strong sites wouldessentially become saturated, and the additional adsorbate forcedonto the weak sites, serving to increase the fraction of the soluteon the latter. If the weak sites are themselves not overloaded (seebelow), their influence may swamp that of the strong sites. Inthis case, it is possible that peak shape can improve. Indeed, the

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) forbenzylamine, (30:70, v/v) for pyridine, (40:60, v/v) for diphenhydramine, and (20:80, v/v) for procainamide.


peak shapes we obtained resemble those predicted by Giddings.Swamping of strong sites is also very likely to explain the markeddecreases in retention that occur as sample load is increased.

The question of why driving the solute onto the hydrophobicsites does not apparently cause strong overload of these sites mustfinally be answered. First, the degree of ionization of even thestronger bases is reduced as the pH is raised. Organic solventeffects (see above) reduce ionization of strong bases further thanmight be expected from consideration of a purely aqueoussystem.17,18 Thus, a substantial fraction of the base exists in theunprotonated form. The retention factor of a partially protonatedbase is a composite of the retention factors of the protonated (lowk) and unprotonated (considerably higher k) forms, weightedaccording to the fraction of molecules in each state. Since k(protonated) is much smaller,24 then the fraction of protonatedbase molecules in the stationary phase is smallsmost ionizedmolecules are in the mobile phase. Thus, the phase is moredifficult to overload, although its composite (larger) k value mightindicate that overloading should take place more easily. Otherfactors may be involved in increased sample capacity at pH 7, forinstance, the existence of further secondary ionic retention sitesavailable for population in addition to the usual hydrophobic sitesor neutralization of solute positive charge by the opposite chargeon the phase.

Effect of Sample Load of Bases at High pH (pH 12). Thehigh-pH stability of polymeric columns allows investigation ofloadability at much higher pH than with silica-based phases.Figure 7 and Table 4 show the effect of sample load for bases atpH 12.

For diphenhydramine, procainamide, quinine, and nortriptyline,sample load had relatively little effect on plate count and asym-metry factor, rather less than for pH 7, and much less than at pH2. In addition, virtually no change occurred in k with sample loadsup to 20 µg. This behavior was expected in that these compoundsare all likely to be uncharged at pH 12, as is pyridine at pH 7.The behavior of benzylamine was anomalous, with peak shapeimproving and k reducing considerably with increasing samplemass, both in a fashion similar to that at pH 7. The reason forthis behavior is obscure.

A comparison of Table 4 with Tables 1 and 3 shows thatmaximum efficiencies for the compounds are higher at pH 12 thanat pH 7 but inferior to those obtained at low pH. It is likely thatthis result is merely due to the higher concentrations of organicsolvent necessary for elution at pH 12, resulting in swelling ofthe polymer and loss of efficiency (see arguments above).

A disadvantage of work at pH 12 was the excessive equilibra-tion times required to obtain stable values of column performanceparameters for some compounds (∼20 h in the case of nortrip-tyline).

Effect of Sample Load and Retention Mechanism for anAnionic Compound. If charge repulsion is the main contributoryfactor to overload (rather than being merely due to overload ofcation-exchange sites present on polymeric columns), then itwould be expected that anionic solutes might experience similaroverloading effects to cationic solutes such as protonated bases.We selected 2-naphthalenesulfonic acid (2-NSA) as a compoundthat is negatively charged at pH 7 and the PRP-1 phase. Prior tothe loading studies, we investigated the effect of increasingphosphate anion concentration on retention of 2-NSA at pH 7 todetermine whether any anion-exchange sites existed on the phase.However, we failed to show decreased retention of 2-NSA withanion concentration (results not shown) indicating the absenceof such sites.

Figure 8 and Table 3 indicate clear evidence of reduction inretention and rapid deterioration in peak shape as sample load isincreased up to 20 µg of injected solute. The general loading

(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.

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

N0(p/c) As(min) ko


ws(mg)

Hamilton PRP-1toluene (pH 2) 4850 1.7 8.0 5.0 0.92 high

(0.16) (0.32) (0.05) (20.0)phenol (pH 2) 6860 1.3 3.8 7.4 0.99 high

(0.10) (0.10) (0.08) (20.4)napht 1340 2.5 7.7 5.1 0.82 9.2

(0.08) (0.08) (0.08) (101)benzylalc 6880 1.3 2.4 15 0.96 300

(0.11) (0.50) (0.11) (475)

Asahipak ODP-50napht 2790 1.8 2.2 51 0.92 high

(0.08) (0.08) (0.08) (101)

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

N0(p/c) As(min) ko


ws(mg)

Hamilton PRP-1diph 240 4.5 6.2 0.75 high

(20.2) (0.32) (0.32) (20.2)quin 65 5.8 2.7 5.0 0.54 high

(0.32) (0.32) (0.32) (20.0)benz 520 5.0 0.7 0.53 high

(15.5) (0.10) (0.10) (20.1)proc 940 3.8 0.5 7.0 0.74 high

(1.1) (0.08) (0.05) (21.0)pyr 7020 1.5 1.1 5.3 0.93 225

(0.10) (0.10) (0.10) (639)2NSA 4090 1.9 1.1 5.1 0.86 2.4

(1.1) (0.32) (0.10) (9.9)

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

No(p/c) As(min) ko k/ko(min)

Hamilton PRP-1diph 780 3.0 6.4 1

(20.8) (0.80) (6.9)quin 1300 2.7 2.8 1

(5.2) (0.08) (0.08)benz 1240 6.1 1.1 0.62

(20.1) (0.80) (0.32) (20.1)proc 2560 2.4 0.6 0.98

(16.7) (16.7) (0.08)

Asahipak ODP-50nor 3020 1.9 5.9 0.97

(19.9) (9.9) (0.32) (19.9)


behavior is very similar, and ws (2.4 mg) is comparable to thevalues obtained for protonated bases at low pH. This result addsweight to the proposal that mutual repulsion of charged ions onthe hydrophobic surface may be largely responsible for theoverloading effects observed on polymeric columns.

CONCLUSIONSAlthough overload of ionized silanols cannot be entirely

discounted by the present study, overload caused by ionicrepulsion probably accounts for the majority of the increased band

tailing (“overload tailing”) and reduced efficiency that occurs onboth pure alkyl silica (type B) and polymeric columns withincreasing sample mass at low pH. Cation-exchange sites (slowkinetics) 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 greaterproportion of the sample being retained on hydrophobic sites (fastkinetics). This factor rationalizes the apparent improvement inefficiency and also the large concomitant decrease in k with sample

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.


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

concentration required to elute the simultaneously occurringneutral species; neutralization of solute charge by the oppositecharge on the stationary phase may be another factor. At highpH, solutes are unprotonated, leading generally to fewer detri-mental effects. Columns that maintain good efficiency at high pHare necessary to exploit these advantages.

It is possible that silanol overload may play a greater part onolder, impure alkyl silica phases (type A). Experiments with suchphases, as well as on pH-stable type B silica phases, are envisagedto investigate further some aspects of the results of the presentstudy.

ACKNOWLEDGMENTThe authors thank Alan McKeown (AstraZeneca R&D Charn-

wood) and Nicole Kirsch (UWE) for many helpful discussions.

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

AC0202381

Figure 8. Loading plots for 2-NSA on PRP-1 (as in Figure 4), mobilephase acetonitrile-30 mM phosphate buffer (pH 7; 23:77, v/v).


overloading study of bases using polymeric rp-hplc columns as an aid to rationalization of...

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