Journal of Chromatography A, 957 (2002) 149164 www.elsevier.

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Characterization and comparison of the chromatographic performance of conventional, polar-embedded, and polar-endcapped reversed-phase liquid chromatography stationary phases
J. Layne*
Phenomenex Inc., 2320 W. 205 th St., Torrance, CA 90501, USA Received 28 August 2001; received in revised form 15 January 2002; accepted 20 February 2002

Abstract We have evaluated and compared the performance of several conventional C 18 phases with those possessing either a polar-endcapping group or a polar-embedded group within the primary alkyl ligand and found distinct differences in the chromatographic behavior among the three groups, as well as a high degree of variability within each group. The trend is for the polar-endcapped phases to display similar hydrophobic retention characteristics as the conventional C 18 columns, but to express higher hydrogen bonding capacities and silanol activity. The polar-embedded phases displayed the opposite behavior, with a greatly reduced hydrophobic nature compared to the conventional and polar-endcapped C 18 phases, and also a very much reduced silanol activity. Most interestingly, it appears that ionic or dipole interactions play a signicant role in the overall retention behavior of the polar-embedded phases towards basic and acidic analytes. 2002 Elsevier Science B.V. All rights reserved. Keywords: Stationary phases, LC; Polar-endcapped phases; Polar-embedded phases

1. Introduction Liquid chromatography has become an indispensable tool for both routine analysis and research in the pharmaceutical, biomedical, and biotechnology industries. On an analytical level, reversed-phase chromatography (RP-HPLC) is the most widespread technique, probably due to the broad applicability of that mode of separation to a wide range of compounds and sample matrices. One distinct advantage of RP-HPLC over other HPLC techniques such as
* Present address: 50 West Church St., West Chazy, NY 12992, USA. E-mail address: laynej@labs.wyeth.com (J. Layne).

ion-exchange (IE-HPLC) or normal-phase chromatography (NPC) is the vast number of stationary phases available, many of which may offer a unique selectivity that can facilitate the separation and analysis of particular chemical mixtures. Ironically, the wide range of possible stationary phases may also lead to potential problems, as there are so many different phases available it is often difcult for the analyst to determine which would be best suited for a given situation. Although many HPLC column manufacturers supply chromatographic information about their phases, this information is not always useful as it may be biased towards their own products, and comparisons between different manufacturers HPLC column data are not always possible

0021-9673 / 02 / $ see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0021-9673( 02 )00193-0

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because of differences in testing conditions. Thus, the analyst is often left to screen several different stationary phases to nd the appropriate one for a separation. In order to simplify this column selection process, there have been several attempts to create test mixtures and protocols to evaluate and characterize column chemistries [19]. These evaluations typically rely on the use of several test probes that have been selected to identify specic column characteristics such as hydrophobicity or silanol activity. Although the test procedures seek to evaluate similar stationary phase characteristics, there are distinct differences in the methodologies that they use to determine those characteristics. For instance, Engelhardt [5] proposed using the selectivity factor (a ) between ethylbenzene and toluene in a mobile phase of 55:45 (v / v) methanol / water as an indicator of stationary phase hydrophobicity, while Kimata et al. [1] suggested that this same hydrophobicity value be determined from the selectivity (a ) between amylbenzene and butylbenzene in a mobile phase of 80:20 (v / v) methanol / water. Likewise, while Neue et al. suggest that the selectivity value (a ) between a base such as amitriptyline, chlorpheniramine, or propranolol and a neutral probe such as acenaphthene be used to gauge silanol activity [6], the Tanaka test [1] uses the selectivity value between benzylamine and phenol to evaluate this same characteristic. Despite the differences in methodologies used for the phase characterizations, it was found that there was a relatively high correlation between the results of four separate HPLC column characterization procedures for the evaluation of hydrophobic interactions [7]. However, there was little correlation between the results of the same test methods for the characterization of the silanol activity of different phases [7]. Most of these test procedures have focused on evaluating several fundamental chromatographic characteristics arising from either hydrophobic or silanol interactions. The hydrophobic interactions between a given stationary phase and analyte and will be determined largely by the surface area, carbon load, and bonding density of a given stationary phase. Hydrophilic interactions, including hydrogen bonding, dipole interactions, and ion-exchange interactions will be determined by the number (sur-

face area) and accessibility of residual silanol groups on the surface of the silica. Based upon an evaluation of these previous works, we have dened several fundamental chromatographic parameters which include: (1) hydrophobicity, (2) hydrophobic or methylene selectivity, (3) hydrogen bonding capacity, (4) silanol activity at low pH and (5) silanol activity at neutral pH (5total ion-exchange capacity) which are best described in the work of Kimata et al. (the Tanaka test mix) [1]. Because of the relatively widespread acceptance (or at least recognition) of the Tanaka test mix, we have chosen to use a modied version of these tests for several of our evaluations in this current work. Within the eld of reversed-phase chromatography (RP-HPLC), silica-based octadecyl (ODS) bonded phases remain among the most widely used of all of the available stationary phases. However, despite the widespread popularity and acceptance of ODS phases, there seems to remain a need, or at least an opportunity, for alternative column chemistries. Two types of newer stationary phases that are gaining popularity are the polar-embedded and the polar-endcapped stationary phases. These phases are modications of the classical C 18 chemistry with the additional of a polar functional group, such as an amide or carbamate group, within the alkyl chain itself, or a polar functional group used as an endcapping agent (Fig. 1). Perhaps due to the relatively recent introduction of these types of phases, there

Fig. 1. Representative structures for a conventional ODS phase (A), a polar-embedded (amide) phase (B), and a polar-endcapped C 18 phase (C).

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have been few studies that have attempted to critically evaluate or characterize their performance. Some of the earliest references to these types of phases appear to be from the work of Jaroniec, Buszewski, and co-workers [1015]. In general, these polarembedded phases are reputed to offer certain advantages such as stability under highly aqueous conditions, improved peak shape for basic compounds, and unique selectivity compared to conventional C 18 phases [16]. McCalley [17] evaluated three polarembedded phases, including one amide-linked C 16 phase and two carbamate-linked phases (C 8 and C 18 ), and reported that the polar-embedded phases displayed lower hydrophobicity, alternative selectivity, and some improved performance for basic compounds compared to conventional C 18 phases. Similarly, OGara et al. [18] described the synthesis and chromatographic behavior of a carbamate-linked phase and found that the phases were stable in highly aqueous conditions, offered a unique selectivity compared to conventional alkyl-bonded phases, and offered improved peak shape for some basic drugs. Given the relative lack of critical information about these polar-embedded and polar-endcapped stationary phases, the goals of this study were to attempt to gain an understanding of how these phases differ from conventional alkyl-bonded phases. We attempted to characterize these phases using a series of assays designed to elucidate fundamental chromatographic behavior and have included several applications that may further illustrate the differences between these phases.

2.2. Columns and reagents

The columns evaluated in this study are detailed in Table 1. To make basic statistical comparisons we evaluated seven columns from each category of stationary phase: conventional C 18 phases, polarembedded phases, and polar-endcapped phases. All of the columns used in the study were based upon the newer, high-purity, so-called base-deactivated silica. As is apparent from the column descriptions, not all of the manufacturers were willing to divulge the nature of their column chemistries. Column efciencies (plates m 2 1 ) were determined using naphthalene (0.05 mg) as the probe under isocratic running conditions (65% acetonitrile in 20 mM potassium phosphate, pH 7, 1 ml min 2 1 ow-rate). The acetonitrile and methanol used in these analyses were HPLC grade (EM Science, Gibbstown, NJ, USA), and the de-ionized water was prepared using an E-Pure water purication system (Barnstead / Thermolyne, Dubuque, IA, USA). All reagents were of the highest possible purity and were purchased from SigmaAldrich (St Louis, MO).

2.3. Chromatographic tests, probes, and running conditions

In order to characterize the fundamental chromatographic qualities of these stationary phases, we ran a series of test methods based loosely upon the work of Kimata et al. [1] including hydrophobicity, methylene selectivity, hydrogen bonding capacity, silanol activity at low pH, and silanol activity at neutral pH. The primary differences between the tests run in this study and the conditions of the tests used by Kimata et al. [1] lay in our use of buffered mobile phases for all of the tests (20 mM potassium phosphate) and some slight changes to organic modier compositions (detailed in text below). To illustrate how these fundamental chromatographic characteristics might be reected in more realistic test conditions, we also ran several comparisons using acidic and basic compounds under a variety of running conditions. The test mixtures and running conditions, as well as their distinctions from the test mixtures used by Kimata et al. [1], included the following.

2. Experimental

2.1. Equipment
The HPLC system used was an HP 1100 LC system (Agilent Technologies, Palo Alto, CA, USA) consisting of an HP 1100 in-line degasser, an HP 1100 autosampler, an HP 1100 column thermostat set to 30 8C, an HP 1100 quaternary pump, and an HP 1100 variable wavelength detector. HP ChemStation (version 6.03) was used for data acquisition and analysis. Statistical analyses (paired t -tests; a 5 0.05 for all comparisons) were carried out using SigmaPlot 5.0 (SPSS Inc., Chicago, IL, USA).

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Table 1 Columns used and general nature of bonding chemistry Column name Luna 5 mm C 18 (2) Inertsil 5 mm ODS(3) Zorbax 5 mm XDB C 18 Symmetry 5 mm C 18 Hypurity Elite 5 mm C 18 Zorbax 5 mm SB C 18 Prodigy 5 mm ODS(3) Zorbax 5 mm Bonus-RP Polaris 5 mm C 18 A Discovery 5 mm RP-Amide SymmetryShield 5 mm RP18 Supelco 5 mm ABZ1 Experimental 10 mm urea Polaris 5 mm Amide C 18 Keystone 5 mm Aquasil Aqua 5 mm C 18 YMC 5 mm Hydrosphere YMC 5 mm ODS-Aq Prontosil 5 mm C 18 Aq Metasil 5 mm Aq Synergi 4 mm Hydro-RP Surface area (m 2 g 2 1 ) 400 450 180 335 200 180 400 Carbon load (%) 17.5 15 10 19 13 10 19 Nature of bonding Conventional ODS Conventional ODS Conventional ODS Conventional ODS Conventional ODS Conventional ODS Conventional ODS Amide linked C 14 Unknown linked C 18 Amide linked C 16 Carbamate linked C 18 Amide linked RP Urea linked C 18 Amide linkage C 18 Polar-endcapped ODS Polar-endcapped ODS Polar-endcapped ODS Polar-endcapped ODS Polar-endcapped ODS Polar-endcapped ODS Polar-endcapped ODS Manufacturer Phenomenex GL Sciences Agilent Waters Hypersil Agilent Phenomenex Agilent Metachem Supelco Waters Supelco Phenomenex Metachem Keystone Phenomenex YMC YMC Bischoff Metachem Phenomenex N (plates m 2 1 ) 116 889 96 669 95 997 91 515 87 816 84 568 108 384 110 156 105 481 115 577 109 530 95 551 54 458 87 744 109 457 87 666 109 430 90 049 92 800 88 846 113 187

335 170 400

15 12 32 12 15 16

320 300

475

19

2.3.1. Hydrophobicity ( kBB ) This is a measure of the retentivity of a phase based upon hydrophobic interactions with a neutral analyte and was determined from the k -value of butylbenzene (0.05 mg) in a mobile phase of 65:35 acetonitrile / 20 mM KH 2 PO 4 K 2 HPO 4 , pH 7. Kimata et al. [1] used the k -value of amylbenzene (kAB ) run in 80% methanol to determine a similar measure of phase hydrophobicity.

2.3.2. Methylene selectivity (aCH 2 ) We dene this value as the ability of a phase to distinguish between two compounds based upon a single methylene (CH 2 ) unit substitution, and was determined by rst injecting a series of alkylbenzene homologues (benzeneamylbenzene; ca. 0.05 mg each) in a mobile phase of 65:35 acetonitrile / 20 mM KH 2 PO 4 K 2 HPO 4 , pH 7. The slope of the regression through the data points of a plot of log k versus no. methylene units (Fig. 2) was used as the methylene selectivity value. Kimata et al. [1] used the alpha (a ) value of amylbenzene / butylbenzene in 80% methanol to determine this value.

Fig. 2. Example of method used to obtain methylene selectivity values (aCH2 ) for Symmetry C 18 ( o ), YMC ODS-Aq ( j ), and Zorbax Bonus-RP ( d ). Linear regression data for these three columns: y 50.181x 10.059, r 2 50.99 (Symmetry C 18 ); y 5 0.171x 10.037, r 2 50.99 (YMC ODS-Aq); y 50.147x 20.152, r 2 5 0.99 (Zorbax Bonus-RP).

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2.3.3. Hydrogen bonding capacity (aC / P ) This is a measurement of the amount of accessible hydrogen bonding sites (from silanol groups) on the silica surface after bonding and is determined from the selectivity (a ) between caffeine (0.05 mg) and phenol (0.07 mg) in a mobile phase of 15:85 acetonitrile / 20 mM KH 2 PO 4 K 2 HPO 4 , pH 7. Kimata et al. [1] used these same test probes in a mobile phase of 30% methanol to determine the aC / P value in their investigation.

mg) and phenol (0.07 mg) in 30:70 acetonitrile / 20 mM KH 2 PO 4 H 3 PO 4 , pH 2.5.

2.3.5. Silanol activity at pH 7 (aB / P pH 7 ) The total ion-exchange capacity under conditions in which a large percentage of residual silanols are deprotonated and ionized was determined using the same selectivity value, but in a mobile phase of 30:70 acetonitrile / 20 mM KH 2 PO 4 K 2 HPO 4 , pH 7. We also ran several illustrative applications including (structures in Fig. 3) the following. 2.3.6. Polar acids These contained p -hydroxybenzoic acid (0.2 mg), sorbic acid (0.2 mg), benzoic acid (5 mg), salicylic acid (5 mg), and p -toluic acid (0.15 mg) in a mobile phase of 75:25 20 mM KH 2 PO 4 H 3 PO 4 pH 2.5 /

2.3.4. Silanol activity at pH 2.5 (aB / P pH 2.5 ) The extent of silanophilic interactions with basic analytes under conditions in which the majority of the residual silanol groups is protonated was determined from the selectivity of benzylamine (0.05

Fig. 3. Structures of the analytes used in the illustrative applications. (1) Chlorpheniramine, (2) Diphenhydramine, (3) Triprolidine, (4) p -Hydroxybenzoic acid, (5) Sorbic acid, (6) Benzoic acid, (7) Salicylic acid, (8) p -Toluic acid, (9) Desipramine, (10) Nortriptyline, (11) Amitriptyline, (12) Clomipramine.

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acetonitrile. We would expect that differences in the hydrogen bonding capacities of the phases and / or any secondary interactions arising from ionic interactions might be reected in the retention and selectivity for these compounds.

2.3.7. Basic drugs at low pH These were run to simulate the typical running conditions that most analysts would use for the analysis of basic pharmaceutical compounds. We evaluated the capacity factors (k ) and peak asymmetry values for triprolidine (0.5 mg), chlorpheniramine (0.5 mg), and diphenhydramine (0.5 mg) using a mobile phase of 75:25 20 mM KH 2 PO 4 H 3 PO 4 pH 2.5 / acetonitrile. Under low pH conditions, we would expect that the majority of residual silanol groups to be protonated and, therefore, interactions with these basic probes should be a reection primarily of differences in hydrogen bonding capacities, or may reect differences in the acidity of the base silicas. 2.3.8. Basic drugs at neutral pH Although most analysts typically analyze basic compounds using a low-pH buffered mobile phase in order to minimize the peak tailing associated with ionic interaction with deprotonated silanol groups, there are instances where it may be necessary or desirable to operate at higher mobile phase pH where silanols may be ionized. Thus, we also evaluated capacity factor (k ) and peak asymmetry values using a series of tricylic antidepressants (desipramine, nortriptyline, amitriptyline, and clomipramine, each 0.5 mg) in a mobile phase of 35:35:30 acetonitrile / methanol / 20 mM KH 2 PO 4 K 2 HPO 4 , pH 7. Presumably, differences in the silanol activity (primarily ion-exchange interactions) of the stationary phases will be reected in the peak shapes and / or retention of these basic compounds.

3. Results and discussion

3.1. Overview of fundamental chromatographic performance

The overall retention behavior of a phase will be due to both hydrophobic interactions that occur

between the bonded phase and analyte molecules as well as polar and ionic interactions that may result between residual silanol groups and the basic functional groups of the analyte molecules. In the case of the polar-embedded and polar-endcapped C 18 phases, one would also suspect that polar and / or ionic interactions might occur between the functional groups that are used as the endcapping or embedded group and sample molecule polar moieties. By evaluating both the hydrophobic character of these stationary phases (hydrophobicity and methylene selectivity) and the hydrophilic nature of the phases (hydrogen bonding, silanol activity), we have attempted to elucidate the mechanisms by which these polar-embedded and polar-endcapped phases differ from conventional C 18 phases (Table 2). From these data, it is clear that there is a large degree of variability not only between the three different classes of RP phases, but also within each class. For example, the conventional RP phases had hydrophobicity values (k BB ) ranging from 2.96 to 9.82, and the polar-endcapped phases displayed hydrogen bonding capacities (aC / P ) ranging from 0.240 to 0.725. Other authors have also noted the high degree of variability between columns that would seem to be at least nominally similar (e.g. all C 18 phases) [1,3]. It is for this reason that we have chosen a relatively large sample size (n 57) for each class of column chemistry in the hope that the mean values would more closely reect, in general, the behavior of a related group of HPLC phases. The overall hydrophobic nature of a stationary phase is largely a function of the total surface area of the base silica gel, the bonding density of the stationary phase ligands, and the nature of the stationary phase itself [1]. These physical and chemical differences should be reected chromatographically in differences in overall capacity factors (k BB ) and selectivity for neutral solutes (aCH2 ). Amongst the three groups of RP phases, there was no signicant difference in the hydrophobicity values (k BB ) between then conventional C 18 phases (mean k BB 5 7.86662.368 SD) and the polar-endcapped phases (mean k BB 57.43461.800 SD). However, the polarembedded phases displayed signicantly lower hydrophobicity (mean k BB 54.42461.568 SD) than either of the other two phases (Fig. 4). Similarly, the polar-embedded phases displayed signicantly lower

J. Layne / J. Chromatogr. A 957 (2002) 149164 Table 2 Results of tests for fundamental chromatographic qualities of the phases Column Luna 5 mm C 18 (2) Inertsil ODS(3) Zorbax XDB C 18 Symmetry C 18 Hypurity Elite C 18 Zorbax SB C 18 Prodigy ODS(3) Mean SD Keystone Aquasil Aqua C 18 YMC Hydrosphere YMC ODS-Aq Prontosil C 18 AQ Metasil AQ Synergi Hydro-RP Mean SD Zorbax Bonus-RP Polaris C 18 -A Discovery RP-Amide SymmetryShield RP18 Supelco ABZ1 Experimental urea Polaris Amide C 18 Mean SD k BB 9.110 8.630 8.570 9.230 2.960 6.740 9.820 7.866 2.368 4.410 8.670 6.250 7.830 7.040 7.770 10.070 7.434 1.800 3.860 4.380 3.340 6.150 1.880 6.250 5.110 4.424 1.568

155

aCH2
0.176 0.171 0.187 0.181 0.155 0.179 0.184 0.176 0.011 0.147 0.176 0.167 0.171 0.173 0.158 0.174 0.167 0.011 0.147 0.165 0.150 0.152 0.143 0.157 0.139 0.150 0.009

aC / P
0.210 0.261 0.213 0.224 0.310 0.283 0.210 0.244 0.040 0.725 0.251 0.270 0.262 0.303 0.217 0.240 0.324 0.179 0.201 0.200 0.166 0.164 0.182 0.133 0.123 0.167 0.030

aB / P

pH 2.5

aB / P

pH 7

0.059 0.045 0.088 0.049 0.061 0.079 0.051 0.062 0.016 0.131 0.094 0.055 0.094 0.079 0.073 0.063 0.084 0.025 0.000 0.102 0.050 0.013 0.000 0.000 0.000 0.024 0.039

0.131 0.147 0.134 0.147 0.378 0.412 0.130 0.211 0.126 1.659 0.149 0.140 0.169 0.254 0.162 0.203 0.391 0.561 0.167 0.121 0.097 0.098 0.230 0.800 0.080 0.228 0.258

methylene selectivity (aCH2 ) values compared to the other two phases (mean aCH2 50.17660.011 SD, 0.16760.011 SD, and 0.15060.009 for the conventional, polar-endcapped, and polar-embedded phases, respectively) (Fig. 5). These data are consistent with the studies of OGara and co-workers [18] who found that carbamate-embedded C 8 phases displayed reduced k -values for neutral, polar, and basic probes compared to conventionally bonded and endcapped C 8 phases. McCalley [3] obtained similar results using a series of basic analytes and carbamate-embedded, amide-embedded, and conventional alkylbonded phases. From the two results of this study, as well as the results of previous works [3,18], it appears that the presence of the polar functional group located within

the alkyl ligand of the polar-embedded phases causes a signicant reduction in the hydrophobic nature of that phase, while the presence of a polar group as an endcapping agent does not seem to inuence the overall hydrophobic nature of the polar-endcapped stationary phases. However, the fact that many of the polar-embedded phases also use shorter ligands (C 14 C 18 ) may also be contributing to this diminished hydrophobicity. If one considers hydrophobic interactions to comprise the primary interactive mechanism in RP-HPLC, it is likely then that the polar-endcapped phases are going to display fairly similar retention behavior to conventional C 18 phases, while the polar-embedded phases should be expected to behave quite differently due to their reduced hydrophobicity and methylene selectivity.

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Fig. 4. Hydrophobicity values (k BB ) for the test columns.

Fig. 5. Bar chart for methylene selectivity (aCH2 ) values of the test columns.

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There was no signicant difference in the hydrogen bonding capacity (aC / P ) between the conventional C 18 phases (mean aC / P 50.22460.040 SD) and the polar-endcapped phases (mean aC / P 50.32460.179 SD), although the mean values would seem to indicate a trend towards a higher hydrogen bonding capacity on the polar-endcapped phases (P 50.32) (Fig. 6). In contrast, the polar-embedded phases did display a signicant lower hydrogen bonding capacity than the other two phases (mean aC / P 5 0.16760.030 SD). These results are similar to those obtained by Euerby and Petersson under similar testing conditions [19]. Assuming that the test probes and running conditions used do indeed succeed in evaluating hydrogen bonding capacities, these results would seem to indicate that the polar-embedded phases either: (a) have fewer residual, accessible silanol groups than the other two classes of stationary phases, or (b) that the polar-embedded groups somehow reduce the accessibility of the surface silanol groups to our test probes.

3.1.1. Silanol activity at pH 2.5 (aB / P pH 2.5 ) The polar-embedded phases displayed a signicantly lower silanol activity at low pH than the polar-endcapped phases (mean aB / P pH 2.5 5 0.02460.039 SD for the polar-embedded phases and 0.08460.025 SD for the polar-endcapped phases). However, there was no signicant difference between the conventional C 18 phases and the other two classes of stationary phases (mean aB / P pH 2.5 5 0.06260.016 SD) (Fig. 7). The low silanol activity is consistent with several published studies [3,16,18,19] and has been cited as contributing to an improvement in the peak shape of basic drugs using these types of phases. It is most interesting to note that the polar-embedded phases Zorbax Bonus-RP, Supelco ABZ1, Experimental Urea, and Polaris Amide C 18 displayed no silanol activity as measured by this assay, and SymmetryShield RP18 displayed exceedingly low silanol activity as compare to the other phases. The low / zero values result from the fact that

Fig. 6. Hydrogen bonding capacities (aC / P ) of the conventional, polar-embedded, and polar-endcapped phases.

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Fig. 7. Silanol activity at pH 2.5 (aB / P pH 2.5 ). The polar-embedded phases Zorbax Bonus-RP, Supelco ABZ1, Experimental urea, and Polaris Amide C 18 displayed zero values for silanol activity at low pH as benzylamine was eluted in the void with thiourea (t 0 marker).

benzylamine, the probe for ion-exchange interactions, eluted in the void volume of the column with the t 0 marker (thiourea) (Fig. 8). A possible explanation for the low silanol activity of the polar-embedded phases may be that the polar-embedded group acquires a positive charge and, consequently, there may be a degree of ionic repulsion that occurs between that group and the basic benzylamine probe. Under low pH conditions, the carbonyl oxygen of an amide group can become protonated, and the resulting structure is stabilized through resonance of the lone electron pair electrons of the nitrogen atom [15,18]. This shielding of the silanols through ionic repulsion of basic analytes may also be a possible explanation for the improvement in peak shapes for basic compounds which other authors have noted when using polar-embedded phases [3,16]. An alternative explanation is that the low silanol activity noted on some of the polar embedded phases may be due to the presence of residual amino groups bonded to the base silica. Amide-embedded phases can be synthesized in a two-step reaction in which the rst step is the bonding of aminopropyl groups to

the base the silica [10]. In the second step, the resulting aminopropyl phase is reacted with an acylchloride to yield the amide-linked alkyl phase. However, this second reaction may not be 100% complete, resulting in the presence of residual amino groups which, below pH 9, will be protonated [10,15] and therefore may impart an ion-exchange characteristic to the phase. For instance, Czajkowska et al. [15] found that the retention of 5ethylpyridinecarboxylic acid (EPDC) decreased as a function of increasing pH on conventional alkyl phases, but the retention of the dicarboxylic acid actually increased on two amide-linked phases as the pH increased and the ionization of the acid increased. However, the retention of the EPDC methyl ester was pH-independent, indicating that the increased retention of the acid formed was the result of an ion-exchange mechanism. These data would support the premise that residual amino groups can contribute to ion-exchange interactions with test probes, resulting in either increased retention of acidic probes or decreased retention of basic analytes through ionic repulsion. Because the ion-exchange interactions resulting from residual amino groups can

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Fig. 8. Chromatograms illustrating the dipole or ionic repulsion effect on the conventional C 18 phase (A) and a polar-embedded phase (B).

have a detrimental effect on chromatographic separations, some manufacturers now offer polar-embedded phases which are synthesized via a one-step bonding reaction [18] which, presumably, reduces or eliminates the presence of unwanted residual amino groups in the bonded phase.

any signicant statistical effect (mean aB / P 0.39160.561 SD).

pH 7

3.2. Illustrative applications 3.2.1. Polar, acidic compounds Comparisons of the mean capacity factors (k ) for a group of low molecular mass acids (Fig. 10) indicated very few differences amongst the three classes of stationary phaseonly sorbic acid displayed a phase-dependent retention behavior (order of retention was: polar-embedded.polar-endcapped. conventional C 18 ). The lack of signicant statistical difference amongst the three classes of stationary phase is an interesting result in itself as it may shed some light on the possible retention mechanism of the polar-embedded phases. Using a conventional C 18 phase, the overall retention of these acidic compounds can be expected to result from a combination of hydrophobic interactions between the carbon skeleton of the analyte and the alkyl ligand of

3.1.2. Silanol activity at pH 7.0 (aB / P pH 7 ) Interestingly, under neutral pH conditions, this shielding of the silanols does not appear to be as strong and there is no signicant difference in silanol activity between the conventional and polar-embedded RP phases (mean aB / P pH 7 50.21160.13 SD and 0.22860.258 SD for the conventional and polarembedded phases (Fig. 9)). Thus, it appears that the shielding effect noted for the polar-embedded phases is pH-dependent, as was noted by Czajkowska et al. [15]. While the relatively high mean silanol activity at pH 7 values for the polar-endcapped phases did seem to indicate a trend for increased silanol activity, the high degree of variability negated

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Fig. 9. Silanol activity of the test columns at pH 7 (aB / P

pH 7

).

the bonded phase, as well as hydrogen bonding between the carboxyl group and the residual silanols. Thus, we would predict that phases that displayed a high hydrophobic index (k BB ) and high hydrogen

Fig. 10. Mean capacity factors (k ) for a series of polar acidic compounds on the conventional C 18 , polar-endcapped, and polarembedded phases.

bonding capacity (aC / P ) should display the greatest retention for these probes. However, the polar-embedded phases, which as a group displayed signicantly lower hydrophobic retentivities and lower hydrogen bonding capacities than both the conventional and polar-endcapped phases (from Table 2), had essentially equivalent retention for these acidic probes (representative chromatograms in Fig. 11). One possible explanation for these results is the presence of a third interactive mechanism present only on the polar-embedded phases. Under the low pH conditions (pH 2.5) in which this application is run, we have hypothesized that the nitrogen-containing carbamate or amide embedded groups may be protonated, leading to a degree of ionic interaction between these groups and the acidic analytes. In addition, the presence of residual amino groups, as alluded to earlier, can also be contributing to the retention of these acidic compounds. This explanation is consistent the work of Czajkowska et al. [15] who also noted an enhanced retention for acidic compounds on amide-linked stationary phases. However, the fact that the carbamate-linked polar-embed-

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Fig. 11. Representative chromatograms for the separation of a group of polar acids using a conventional C 18 (A, Luna 5 mm C 18 (2)), a polar-endcapped phase (B, YMC Hydrosphere), and polar-embedded phase (C, SymmetryShield RP-18). Mobile phase: 25:75 acetonitrile / 20 mM potassium phosphate pH 2.5, ow-rate51.5 ml / min; (1) p -hydroxybenzoic acid, (2) sorbic acid, (3) benzoic acid, (4) salicylic acid (5) p -toluic acid.

ded phase (SymmetryShield RP18) displayed this enhanced retention of basic compounds despite a low hydrophobicity and low hydrogen bonding capacity would seem to contradict the contribution of residual amino groups to the retention of these compounds. While the synthesis of the amide-linked phases may lead to the presence of residual amino groups on the surface, the carbamate-linked phase is reputedly bonded in a one-step process that is unlikely to have any residual reactive groups on the surface. In addition, even cleavage or hydrolysis of this carba-

mate group would most likely leave a carboxyl group [16,18]. Therefore, it is possible that the enhanced retention of these acidic probes would most likely be due to the positive character of the carbamate group itself [18], which could interact with acidic compounds via an ion-exchange or dipoledipole interaction.

3.2.2. Basic compounds under low pH conditions Comparison of the mean capacity factors for a group of basic drugs shows very interesting trends

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(Fig. 12). For all three compounds, the polar-endcapped phases displayed signicantly higher capacity factors than either the conventional or polar-embedded phases. For the rst two analytes, triprolidine and chlorpheniramine, there was no difference in retention times between the conventional and polarembedded phases, but the last analyte, diphenhydramine, was more strongly retained on the conventional phases. Presumably, the enhanced retention on the polar-endcapped phases is due to the relatively high silanol activity and hydrogen bonding capacity of these phases (from Figs. 8 and 9). If we consider a representative set of columns (Fig. 13), the polarendcapped phase (Keystone Aquasil) has approximately two-thirds the hydrophobic retentivity of the conventional C 18 phase (Luna C 18 (2)), yet these basic compounds are signicantly more retained on that polar-endcapped phase. In contrast, a comparison of the same polar-endcapped phase and the polar-embedded phase (Polaris Amide C 18 ) shows exactly opposite results. The polar-embedded phase has a slightly higher hydrophobicity than the polarendcapped phase, yet the three basic analytes are much less retainedin fact, the rst two analytes actually elute in the void volume. In addition, maleic acid, the conjugate salt of chlorpheniramine maleate, elutes in the void on the conventional and polarendcapped phases, but is strongly adsorbed on the

polar-embedded phase and elutes as a broad, tailing peak. We may attribute the weak retention for bases and the adsorption of the acid, to the positive character of the nitrogen-containing embedded group of Polaris Amide C 18 , which is consistent with our previous data from silanol activity at low pH. Comparison of the mean peak asymmetry values for the three phase classes (Fig. 14) indicates that there is no signicant difference between the phases under low pH conditions. At low pH, it appears that, despite the higher silanol activity of the polar-endcapped phases and the increased inertness due to shielding of the polar-embedded phases, there is no signicant improvement in peak shape from conventional C 18 phases. It may be possible that, despite the contributions of the polar-endcapping or polar-embedded groups to the net interactions that dictate peak shape, the residual silanols play the most signicant and overriding role in predicting peak shapes for basic compounds. Thus, as long as conditions are such that the silanols remain protonated, basic compounds will be eluted from all the columns with similar peak shapes.

Fig. 12. Mean capacity factors (k ) for a group of basic compounds under low pH conditions (pH 2.5) using the conventional C 18 , polar-endcapped, and polar-embedded phases.

3.2.3. Basic compounds at neutral pH At neutral pH conditions, the residual silanol groups of base silica will be ionized and should interact relatively strongly with basic compounds. Under these conditions there was no difference in the mean capacity factors for four tricyclic antidepressants between the polar-endcapped and conventional C 18 phases. However, the polar-embedded phases displayed signicantly lower retention than both of those groups of phases (Fig. 15). While we may attribute some of this behavior to the ionic or dipole repulsion from the shielding effect, we must also consider the fact that, as a group, the polar-embedded phases also displayed lower hydrophobic retentivity. The mean peak asymmetry values (Fig. 16) mirror those for the retention factors, with no signicant difference between the conventional and polar-endcapped C 18 phases and signicantly improved peak shape on the polar-embedded phases (e.g. mean Asy for amitriptyline was 1.2860.38 for the polar-embedded phases, 1.5660.36 for the conventional C 18 phases, and 2.2160.46 for the polar-endcapped phases). Again, we may attribute this improvement

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Fig. 13. Representative chromatograms for the separation of basic compounds at low pH using a polar-endcapped phase (B, Aquasil C 18 ), a conventional C 18 phase (A, Luna C 18 (2)), and a polar-embedded column (C, Polaris C 18 -Amide). Mobile phase: 25:75 acetonitrile / 20 mM potassium phosphate pH 2.5, ow-rate51.5 ml / min; (1) maleic acid, (2) triprolidine, (3) chlorpheniramine, (4) diphenhydramine.

in peak shape to an increased phase inertness resulting from ionic or dipole repulsion of basic compounds by the polar-embedded group. Several other authors [1619] have also noted an improvement in the peak shape of basic drugs when using polar-embedded phases, and these results would seem consistent with the previous data.

4. Conclusions We attempted to characterize and compare the

behavior of conventional C 18 , polar-embedded phases, and polar-endcapped phases using a series of diagnostic probes and several generic applications. Relative to conventional C 18 phases, polar-endcapped phases were typied by equivalent hydrophobic interaction capacity and methylene selectivity, enhanced hydrogen bonding and silanol activity, some increased selectivity for acidic compounds, presumably through the enhanced hydrogen bonding, increased retention for basic compounds at low pH without a compromise in peak shape and, at neutral pH, increased retention and peak tailing of basic

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Fig. 14. Mean peak asymmetry values for the basic compounds at low pH on the conventional, polar-endcapped, and polar-embedded phases. Fig. 16. Mean peak asymmetry values for the basic drugs at neutral pH using the three types of stationary phase.

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Fig. 15. Mean capacity factors for a group of tricyclic antidepressants under neutral pH conditions on the three classes of stationary phase.

compounds. The polar-embedded phases were typied by signicantly reduced hydrophobicity and methylene selectivity, reduced hydrogen bonding capacity and silanol activity, and a unique selectivity due to ion-exchange or dipole interaction with nitrogen-containing embedded groups. Due to the presence of a positive character to the nitrogencontaining polar-embedded phases, acidic compounds may display enhanced retention relative to neutral probes, and basic compounds may actually experience ionic or dipole repulsion, leading to greatly reduced retention factors, especially at low pH conditions.