Characterization c18

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957 (2002) 149164Journal of Chromatography A,www.elsevier.com/locate/chroma

Characterization and comparison of the chromatographicperformance 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, US A

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 phases with those possessing either a18polar-endcapping group or a polar-embedded group within the primary alkyl ligand and found distinct differences in thechromatographic behavior among the three groups, as well as a high degree of variability within each group. The trend is forthe polar-endcapped phases to display similar hydrophobic retention characteristics as the conventional C columns, but to18express higher hydrogen bonding capacities and silanol activity. The polar-embedded phases displayed the oppositebehavior, with a greatly reduced hydrophobic nature compared to the conventional and polar-endcapped C phases, and also18a 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 ScienceB.V. All rights reserved.

Keywords : Stationary phases, LC; Polar-endcapped phases; Polar-embedded phases

1. Introduction ion-exchange (IE-HPLC) or normal-phase chroma-tography (NPC) is the vast number of stationary

Liquid chromatography has become an indispens- phases available, many of which may offer a uniqueable tool for both routine analysis and research in the selectivity that can facilitate the separation andpharmaceutical, biomedical, and biotechnology in- analysis of particular chemical mixtures. Ironically,

dustries. On an analytical level, reversed-phase chro- the wide range of possible stationary phases maymatography (RP-HPLC) is the most widespread also lead to potential problems, as there are so manytechnique, probably due to the broad applicability of different phases available it is often difcult for thethat mode of separation to a wide range of com- analyst to determine which would be best suited for apounds and sample matrices. One distinct advantage given situation. Although many HPLC columnof RP-HPLC over other HPLC techniques such as manufacturers supply chromatographic information

about their phases, this information is not alwaysuseful as it may be biased towards their own* Present address: 50 West Church St., West Chazy, NY 12992,products, and comparisons between different manu-USA.

E -mail address : [email protected] (J. Layne). facturers HPLC column data are not always possible

0021-9673/02/$ see front matter 2002 Elsevier Science B.V. All rights reserved.P I I : S 0 0 2 1 - 9 67 3 ( 0 2 ) 0 0 1 9 3 - 0


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957 (2002) 149164150 J . Layne / J . Chromatogr . A

because of differences in testing conditions. Thus, face area) and accessibility of residual silanol groupsthe analyst is often left to screen several different on the surface of the silica. Based upon an evaluationstationary phases to nd the appropriate one for a of these previous works, we have dened severalseparation. fundamental chromatographic parameters which

In order to simplify this column selection include: (1) hydrophobicity, (2) hydrophobic orprocess, there have been several attempts to create methylene selectivity, (3) hydrogen bonding capaci-test mixtures and protocols to evaluate and character- ty, (4) silanol activity at low pH and (5) silanolize column chemistries [19]. These evaluations activity at neutral pH ( 5 total ion-exchange capacity)typically rely on the use of several test probes that which are best described in the work of Kimata et al.have been selected to identify specic column (the Tanaka test mix) [1]. Because of the relativelycharacteristics such as hydrophobicity or silanol widespread acceptance (or at least recognition) of theactivity. Although the test procedures seek to evalu- Tanaka test mix, we have chosen to use a modiedate similar stationary phase characteristics, there are version of these tests for several of our evaluations indistinct differences in the methodologies that they this current work.use to determine those characteristics. For instance, Within the eld of reversed-phase chromatography

Engelhardt [5] proposed using the selectivity factor (RP-HPLC), silica-based octadecyl (ODS) bonded(a ) between ethylbenzene and toluene in a mobile phases remain among the most widely used of all of phase of 55:45 (v/ v) methanol/ water as an indicator the available stationary phases. However, despite theof stationary phase hydrophobicity, while Kimata et widespread popularity and acceptance of ODSal. [1] suggested that this same hydrophobicity value phases, there seems to remain a need, or at least anbe determined from the selectivity ( a ) between opportunity, for alternative column chemistries.amylbenzene and butylbenzene in a mobile phase of Two types of newer stationary phases that are80:20 (v /v) methanol/ water. Likewise, while Neue gaining popularity are the polar-embedded and theet al. suggest that the selectivity value ( a ) between a polar-endcapped stationary phases. These phases arebase such as amitriptyline, chlorpheniramine, or modications of the classical C chemistry with the18propranolol and a neutral probe such as acenaph- additional of a polar functional group, such as anthene be used to gauge silanol activity [6], the amide or carbamate group, within the alkyl chainTanaka test [1] uses the selectivity value between itself, or a polar functional group used as an endcap-benzylamine and phenol to evaluate this same ping agent (Fig. 1). Perhaps due to the relativelycharacteristic. Despite the differences in methodolo- recent introduction of these types of phases, theregies used for the phase characterizations, it wasfound that there was a relatively high correlationbetween the results of four separate HPLC columncharacterization procedures for the evaluation of hydrophobic interactions [7]. However, there waslittle correlation between the results of the same testmethods for the characterization of the silanol activi-ty of different phases [7].

Most of these test procedures have focused onevaluating several fundamental chromatographiccharacteristics arising from either hydrophobic orsilanol interactions. The hydrophobic interactionsbetween a given stationary phase and analyte andwill be determined largely by the surface area,carbon load, and bonding density of a given station-ary phase. Hydrophilic interactions, including hydro- Fig. 1. Representative structures for a conventional ODS phasegen bonding, dipole interactions, and ion-exchange (A), a polar-embedded (amide) phase (B), and a polar-endcappedinteractions will be determined by the number (sur- C phase (C).18


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Table 1Columns used and general nature of bonding chemistry

Column name Surface area Carbon load Nature of bonding Manufacturer N 2 2 1 2 1(m g ) (%) (plates m )

Luna 5 mm C (2) 400 17.5 Conventional ODS Phenomenex 116 88918Inertsil 5 mm ODS(3) 450 15 Conventional ODS GL Sciences 96 669Zorbax 5 mm XDB C 180 10 Conventional ODS Agilent 95 99718Symmetry 5 mm C 335 19 Conventional ODS Waters 91 51518Hypurity Elite 5 mm C 200 13 Conventional ODS Hypersil 87 81618Zorbax 5 mm SB C 180 10 Conventional ODS Agilent 84 56818Prodigy 5 mm ODS(3) 400 19 Conventional ODS Phenomenex 108 384Zorbax 5 mm Bonus-RP Amide linked C Agilent 110 15614Polaris 5 mm C A Unknown linked C Metachem 105 48118 18Discovery 5 mm RP-Amide Amide linked C Supelco 115 57716SymmetryShield 5 mm RP18 335 15 Carbamate linked C Waters 109 53018Supelco 5 mm ABZ 1 170 12 Amide linked RP Supelco 95 551Experimental 10 mm urea 400 32 Urea linked C Phenomenex 54 45818Polaris 5 mm Amide C Amide linkage C Metachem 87 74418 18Keystone 5 mm Aquasil 12 Polar-endcapped ODS Keystone 109 457Aqua 5 mm C 320 15 Polar-endcapped ODS Phenomenex 87 66618YMC 5 mm Hydrosphere Polar-endcapped ODS YMC 109 430YMC 5 mm ODS-Aq 300 16 Polar-endcapped ODS YMC 90 049Prontosil 5 mm C Aq Polar-endcapped ODS Bischoff 92 80018Metasil 5 mm Aq Polar-endcapped ODS Metachem 88 846Synergi 4 mm Hydro-RP 475 19 Polar-endcapped ODS Phenomenex 113 187

2.3.1. Hydrophobicity ( k ) BBThis is a measure of the retentivity of a phase

based upon hydrophobic interactions with a neutralanalyte and was determined from the k -value of butylbenzene (0.05 mg) in a mobile phase of 65:35acetonitrile/ 20 m M KH PO K HPO , pH 7.2 4 2 4Kimata et al. [1] used the k -value of amylbenzene(k ) run in 80% methanol to determine a similarABmeasure of phase hydrophobicity.

2.3.2. Methylene selectivity ( a ) CH 2 We dene this value as the ability of a phase to

distinguish between two compounds based upon a

single methylene (CH ) unit substitution, and was2determined by rst injecting a series of alkylbenzenehomologues (benzeneamylbenzene; ca. 0.05 mgeach) in a mobile phase of 65:35 acetonitrile/20 m M KH PO K HPO , pH 7. The slope of the regres-2 4 2 4

Fig. 2. Example of method used to obtain methylene selectivitysion through the data points of a plot of log k versusvalues ( a ) for Symmetry C ( ), YMC ODS-Aq ( j ), andCH2 18no. methylene units (Fig. 2) was used as the methyl-Zorbax Bonus-RP ( d ). Linear regression data for these three

ene selectivity value. Kimata et al. [1] used the alpha 2columns: y5 0.181 x1 0.059, r 5 0.99 (Symmetry C ); y5182 2(a ) value of amylbenzene /butylbenzene in 80% 0.171 x1 0.037, r 5 0.99 (YMC ODS-Aq); y 5 0.147 x2 0.152, r 5

methanol to determine this value. 0.99 (Zorbax Bonus-RP).


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957 (2002) 149164 153 J . Layne / J . Chromatogr . A

2.3.3. Hydrogen bonding capacity ( a ) mg) and phenol (0.07 mg) in 30:70 acetonitrile/20C / PThis is a measurement of the amount of accessible m M KH PO H PO , pH 2.5.2 4 3 4

hydrogen bonding sites (from silanol groups) on thesilica surface after bonding and is determined from 2.3.5. Silanol activity at pH 7 ( a ) B / P pH 7

the selectivity ( a ) between caffeine (0.05 mg) and The total ion-exchange capacity under conditionsphenol (0.07 mg) in a mobile phase of 15:85 in which a large percentage of residual silanols areacetonitrile/ 20 m M KH PO K HPO , pH 7. deprotonated and ionized was determined using the2 4 2 4Kimata et al. [1] used these same test probes in a same selectivity value, but in a mobile phase of mobile phase of 30% methanol to determine the a 30:70 acetonitrile/20 m M KH PO K HPO , pH 7.C / P 2 4 2 4value in their investigation. We also ran several illustrative applications in-

cluding (structures in Fig. 3) the following.

2.3.4. Silanol activity at pH 2.5 ( a ) 2.3.6. Polar acids B / P pH 2.5 The extent of silanophilic interactions with basic These contained p -hydroxybenzoic acid (0.2 mg),

analytes under conditions in which the majority of sorbic acid (0.2 mg), benzoic acid (5 mg), salicylic

the residual silanol groups is protonated was de- acid (5 m

g), and p

-toluic acid (0.15 m

g) in a mobiletermined from the selectivity of benzylamine (0.05 phase of 75:25 20 m M KH PO H PO pH 2.5/ 2 4 3 4

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 between the bonded phase and analyte molecules ashydrogen bonding capacities of the phases and/or well as polar and ionic interactions that may resultany secondary interactions arising from ionic interac- between residual silanol groups and the basic func-tions might be reected in the retention and selectivi- tional groups of the analyte molecules. In the case of

ty for these compounds. the polar-embedded and polar-endcapped C phases,18one would also suspect that polar and /or ionic

2.3.7. Basic drugs at low pH interactions might occur between the functionalThese were run to simulate the typical running groups that are used as the endcapping or embedded

conditions that most analysts would use for the group and sample molecule polar moieties. Byanalysis of basic pharmaceutical compounds. We evaluating both the hydrophobic character of theseevaluated the capacity factors ( k ) and peak stationary phases (hydrophobicity and methyleneasymmetry values for triprolidine (0.5 mg), chlor- selectivity) and the hydrophilic nature of the phasespheniramine (0.5 mg), and diphenhydramine (0.5 (hydrogen bonding, silanol activity), we havemg) using a mobile phase of 75:25 20 m M attempted to elucidate the mechanisms by whichKH PO H PO pH 2.5/ acetonitrile. Under low pH these polar-embedded and polar-endcapped phases2 4 3 4

conditions, we would expect that the majority of differ from conventional C phases (Table 2).18residual silanol groups to be protonated and, there- From these data, it is clear that there is a largefore, interactions with these basic probes should be a degree of variability not only between the threereection primarily of differences in hydrogen bond- different classes of RP phases, but also within eaching capacities, or may reect differences in the class. For example, the conventional RP phases hadacidity of the base silicas. hydrophobicity values ( k ) ranging from 2.96 toBB

9.82, and the polar-endcapped phases displayed2.3.8. Basic drugs at neutral pH hydrogen bonding capacities ( a ) ranging fromC / P

Although most analysts typically analyze basic 0.240 to 0.725. Other authors have also noted thecompounds using a low-pH buffered mobile phase in high degree of variability between columns thatorder to minimize the peak tailing associated with would seem to be at least nominally similar (e.g. allionic interaction with deprotonated silanol groups, C phases) [1,3]. It is for this reason that we have18there are instances where it may be necessary or chosen a relatively large sample size ( n5 7) for eachdesirable to operate at higher mobile phase pH where class of column chemistry in the hope that the meansilanols may be ionized. Thus, we also evaluated values would more closely reect, in general, thecapacity factor ( k ) and peak asymmetry values using behavior of a related group of HPLC phases.a series of tricylic antidepressants (desipramine, The overall hydrophobic nature of a stationarynortriptyline, amitriptyline, and clomipramine, each phase is largely a function of the total surface area of 0.5 mg) in a mobile phase of 35:35:30 acetonitrile/ the base silica gel, the bonding density of themethanol/ 20 m M KH PO K HPO , pH 7. Pre- stationary phase ligands, and the nature of the2 4 2 4sumably, differences in the silanol activity (primarily stationary phase itself [1]. These physical and chemi-ion-exchange interactions) of the stationary phases cal differences should be reected chromatograph-will be reected in the peak shapes and /or retention ically in differences in overall capacity factors ( k )BB

of these basic compounds. and selectivity for neutral solutes ( a ). AmongstCH 2the three groups of RP phases, there was no signi-cant difference in the hydrophobicity values ( k )BB

3. Results and discussion between then conventional C phases (mean k 518 BB7.8666 2.368 SD) and the polar-endcapped phases

3.1. Overview of fundamental chromatographic (mean k 5 7.4346 1.800 SD). However, the polar-BB performance embedded phases displayed signicantly lower

hydrophobicity (mean k 5 4.424 6 1.568 SD) thanBBThe overall retention behavior of a phase will be either of the other two phases (Fig. 4). Similarly, the

due to both hydrophobic interactions that occur polar-embedded phases displayed signicantly lower


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Table 2Results of tests for fundamental chromatographic qualities of the phases

Column k a a a a BB CH2 C / P B / P pH 2.5 B / P pH 7

Luna 5 mm C (2) 9.110 0.176 0.210 0.059 0.13118Inertsil ODS(3) 8.630 0.171 0.261 0.045 0.147Zorbax XDB C 8.570 0.187 0.213 0.088 0.13418Symmetry C 9.230 0.181 0.224 0.049 0.14718Hypurity Elite C 2.960 0.155 0.310 0.061 0.37818Zorbax SB C 6.740 0.179 0.283 0.079 0.41218Prodigy ODS(3) 9.820 0.184 0.210 0.051 0.130

Mean 7.866 0.176 0.244 0.062 0.211SD 2.368 0.011 0.040 0.016 0.126

Keystone Aquasil 4.410 0.147 0.725 0.131 1.659Aqua C 8.670 0.176 0.251 0.094 0.14918YMC Hydrosphere 6.250 0.167 0.270 0.055 0.140YMC ODS-Aq 7.830 0.171 0.262 0.094 0.169Prontosil C AQ 7.040 0.173 0.303 0.079 0.25418Metasil AQ 7.770 0.158 0.217 0.073 0.162Synergi Hydro-RP 10.070 0.174 0.240 0.063 0.203

Mean 7.434 0.167 0.324 0.084 0.391SD 1.800 0.011 0.179 0.025 0.561

Zorbax Bonus-RP 3.860 0.147 0.201 0.000 0.167Polaris C -A 4.380 0.165 0.200 0.102 0.12118Discovery RP-Amide 3.340 0.150 0.166 0.050 0.097SymmetryShield RP18 6.150 0.152 0.164 0.013 0.098Supelco ABZ 1 1.880 0.143 0.182 0.000 0.230Experimental urea 6.250 0.157 0.133 0.000 0.800

Polaris Amide C 5.110 0.139 0.123 0.000 0.08018

Mean 4.424 0.150 0.167 0.024 0.228SD 1.568 0.009 0.030 0.039 0.258

methylene selectivity ( a ) values compared to the the alkyl ligand of the polar-embedded phases causesCH2other two phases (mean a 5 0.176 6 0.011 SD, a signicant reduction in the hydrophobic nature of CH 20.167 6 0.011 SD, and 0.150 6 0.009 for the conven- that phase, while the presence of a polar group as antional, polar-endcapped, and polar-embedded phases, endcapping agent does not seem to inuence therespectively) (Fig. 5). These data are consistent with overall hydrophobic nature of the polar-endcappedthe studies of OGara and co-workers [18] who stationary phases. However, the fact that many of the

found that carbamate-embedded C phases displayed polar-embedded phases also use shorter ligands8reduced k -values for neutral, polar, and basic probes (C C ) may also be contributing to this dimin-14 18compared to conventionally bonded and endcapped ished hydrophobicity. If one considers hydrophobicC phases. McCalley [3] obtained similar results interactions to comprise the primary interactive8using a series of basic analytes and carbamate-em- mechanism in RP-HPLC, it is likely then that thebedded, amide-embedded, and conventional alkyl- polar-endcapped phases are going to display fairlybonded phases. similar retention behavior to conventional C 18

From the two results of this study, as well as the phases, while the polar-embedded phases should beresults of previous works [3,18], it appears that the expected to behave quite differently due to theirpresence of the polar functional group located within reduced hydrophobicity and methylene selectivity.


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

Fig. 5. Bar chart for methylene selectivity ( a ) values of the test columns.CH2


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There was no signicant difference in the hydro- 3.1.1. Silanol activity at pH 2.5 ( a ) B / P pH 2.5 gen bonding capacity ( a ) between the convention- The polar-embedded phases displayed a signi-C / Pal C phases (mean a 5 0.2246 0.040 SD) and the cantly lower silanol activity at low pH than the18 C / Ppolar-endcapped phases (mean a 5 0.3246 0.179 polar-endcapped phases (mean a 5C / P B / P pH 2.5SD), although the mean values would seem to 0.024 6 0.039 SD for the polar-embedded phases andindicate a trend towards a higher hydrogen bonding 0.084 6 0.025 SD for the polar-endcapped phases).capacity on the polar-endcapped phases ( P 5 0.32) However, there was no signicant difference be-(Fig. 6). In contrast, the polar-embedded phases did tween the conventional C phases and the other two18display a signicant lower hydrogen bonding capaci- classes of stationary phases (mean a 5B / P p H 2 . 5ty than the other two phases (mean a 5 0.062 6 0.016 SD) (Fig. 7). The low silanol activityC / P0.167 6 0.030 SD). These results are similar to those is consistent with several published studiesobtained by Euerby and Petersson under similar [3,16,18,19] and has been cited as contributing to antesting conditions [19]. Assuming that the test probes improvement in the peak shape of basic drugs usingand running conditions used do indeed succeed in these types of phases.evaluating hydrogen bonding capacities, these results It is most interesting to note that the polar-embed-

would seem to indicate that the polar-embedded ded phases Zorbax Bonus-RP, Supelco ABZ1

, Ex-phases either: (a) have fewer residual, accessible perimental Urea, and Polaris Amide C displayed18silanol groups than the other two classes of station- no silanol activity as measured by this assay, andary phases, or (b) that the polar-embedded groups SymmetryShield RP18 displayed exceedingly lowsomehow reduce the accessibility of the surface silanol activity as compare to the other phases. Thesilanol groups to our test probes. low / zero values result from the fact that

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


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Fig. 7. Silanol activity at pH 2.5 ( a ). The polar-embedded phases Zorbax Bonus-RP, Supelco ABZ 1 , Experimental urea, andB / P p H 2 .5Polaris Amide C displayed zero values for silanol activity at low pH as benzylamine was eluted in the void with thiourea ( t marker).18 0

benzylamine, the probe for ion-exchange interac- the base the silica [10]. In the second step, thetions, eluted in the void volume of the column with resulting aminopropyl phase is reacted with anthe t marker (thiourea) (Fig. 8). A possible explana- acylchloride to yield the amide-linked alkyl phase.0tion for the low silanol activity of the polar-embed- However, this second reaction may not be 100%ded phases may be that the polar-embedded group complete, resulting in the presence of residual aminoacquires a positive charge and, consequently, there groups which, below pH 9, will be protonatedmay be a degree of ionic repulsion that occurs [10,15] and therefore may impart an ion-exchangebetween that group and the basic benzylamine probe. characteristic to the phase. For instance, CzajkowskaUnder low pH conditions, the carbonyl oxygen of an et al. [15] found that the retention of 5-amide group can become protonated, and the re- ethylpyridinecarboxylic acid (EPDC) decreased as asulting structure is stabilized through resonance of function of increasing pH on conventional alkylthe lone electron pair electrons of the nitrogen atom phases, but the retention of the dicarboxylic acid[15,18]. This shielding of the silanols through actually increased on two amide-linked phases as theionic repulsion of basic analytes may also be a pH increased and the ionization of the acid in-

possible explanation for the improvement in peak creased. However, the retention of the EPDC methylshapes for basic compounds which other authors ester was pH-independent, indicating that the in-have noted when using polar-embedded phases creased retention of the acid formed was the result of [3,16]. an ion-exchange mechanism. These data would

An alternative explanation is that the low silanol support the premise that residual amino groups canactivity noted on some of the polar embedded phases contribute to ion-exchange interactions with testmay be due to the presence of residual amino groups probes, resulting in either increased retention of bonded to the base silica. Amide-embedded phases acidic probes or decreased retention of basic analytescan be synthesized in a two-step reaction in which through ionic repulsion. Because the ion-exchangethe rst step is the bonding of aminopropyl groups to 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 phase (A) and a polar-embedded phase (B).18

have a detrimental effect on chromatographic sepa- any signicant statistical effect (mean a 5B / P p H 7rations, some manufacturers now offer polar-embed- 0.391 6 0.561 SD).ded phases which are synthesized via a one-stepbonding reaction [18] which, presumably, reduces or 3.2. Illustrative applicationseliminates the presence of unwanted residual aminogroups in the bonded phase. 3.2.1. Polar , acidic compounds

Comparisons of the mean capacity factors ( k ) for a3.1.2. Silanol activity at pH 7.0 ( a ) group of low molecular mass acids (Fig. 10) indi- B / P pH 7

Interestingly, under neutral pH conditions, this cated very few differences amongst the three classesshielding of the silanols does not appear to be as of stationary phaseonly sorbic acid displayed astrong and there is no signicant difference in silanol phase-dependent retention behavior (order of reten-

activity between the conventional and polar-embed- tion was: polar-embedded . polar-endcapped .ded RP phases (mean a 5 0.211 6 0.13 SD and conventional C ). The lack of signicant statisticalB / P pH 7 180.228 6 0.258 SD for the conventional and polar- difference amongst the three classes of stationaryembedded phases (Fig. 9)). Thus, it appears that the phase is an interesting result in itself as it may shedshielding effect noted for the polar-embedded some light on the possible retention mechanism of phases is pH-dependent, as was noted by Czajkow- the polar-embedded phases. Using a conventionalska et al. [15]. While the relatively high mean silanol C phase, the overall retention of these acidic18activity at pH 7 values for the polar-endcapped compounds can be expected to result from a combi-phases did seem to indicate a trend for increased nation of hydrophobic interactions between the car-silanol activity, the high degree of variability negated bon skeleton of the analyte and the alkyl ligand of


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Fig. 9. Silanol activity of the test columns at pH 7 ( a ).B / P p H 7

the bonded phase, as well as hydrogen bonding bonding capacity ( a ) should display the greatestC / Pbetween the carboxyl group and the residual silanols. retention for these probes. However, the polar-em-Thus, we would predict that phases that displayed a bedded phases, which as a group displayed sig-high hydrophobic index ( k ) and high hydrogen nicantly lower hydrophobic retentivities and lowerBB

hydrogen bonding capacities than both the conven-tional and polar-endcapped phases (from Table 2),had essentially equivalent retention for these acidicprobes (representative chromatograms in Fig. 11).

One possible explanation for these results is thepresence of a third interactive mechanism presentonly on the polar-embedded phases. Under the lowpH conditions (pH 2.5) in which this application is

run, we have hypothesized that the nitrogen-con-taining carbamate or amide embedded groups may beprotonated, leading to a degree of ionic interactionbetween these groups and the acidic analytes. Inaddition, the presence of residual amino groups, asalluded to earlier, can also be contributing to theretention of these acidic compounds. This explana-tion is consistent the work of Czajkowska et al. [15]who also noted an enhanced retention for acidicFig. 10. Mean capacity factors ( k ) for a series of polar acidiccompounds on amide-linked stationary phases. How-compounds on the conventional C , polar-endcapped, and polar-18

embedded phases. ever, the fact that the carbamate-linked polar-embed-


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(Fig. 12). For all three compounds, the polar-end- polar-embedded phase and elutes as a broad, tailingcapped phases displayed signicantly higher capacity peak. We may attribute the weak retention for basesfactors than either the conventional or polar-embed- and the adsorption of the acid, to the positiveded phases. For the rst two analytes, triprolidine character of the nitrogen-containing embedded group

and chlorpheniramine, there was no difference in of Polaris Amide C , which is consistent with our18retention times between the conventional and polar- previous data from silanol activity at low pH.embedded phases, but the last analyte, diphenhydra- Comparison of the mean peak asymmetry valuesmine, was more strongly retained on the convention- for the three phase classes (Fig. 14) indicates thatal phases. Presumably, the enhanced retention on the there is no signicant difference between the phasespolar-endcapped phases is due to the relatively high under low pH conditions. At low pH, it appears that,silanol activity and hydrogen bonding capacity of despite the higher silanol activity of the polar-end-these phases (from Figs. 8 and 9). If we consider a capped phases and the increased inertness due torepresentative set of columns (Fig. 13), the polar- shielding of the polar-embedded phases, there isendcapped phase (Keystone Aquasil) has approxi- no signicant improvement in peak shape frommately two-thirds the hydrophobic retentivity of the conventional C phases. It may be possible that,18

conventional C phase (Luna C (2)), yet these despite the contributions of the polar-endcapping or18 18basic compounds are signicantly more retained on polar-embedded groups to the net interactions thatthat polar-endcapped phase. In contrast, a compari- dictate peak shape, the residual silanols play theson of the same polar-endcapped phase and the most signicant and overriding role in predictingpolar-embedded phase (Polaris Amide C ) shows peak shapes for basic compounds. Thus, as long as18exactly opposite results. The polar-embedded phase conditions are such that the silanols remain proton-has a slightly higher hydrophobicity than the polar- ated, basic compounds will be eluted from all theendcapped phase, yet the three basic analytes are columns with similar peak shapes.much less retainedin fact, the rst two analytesactually elute in the void volume. In addition, maleic 3.2.3. Basic compounds at neutral pH acid, the conjugate salt of chlorpheniramine maleate, At neutral pH conditions, the residual silanolelutes in the void on the conventional and polar- groups of base silica will be ionized and shouldendcapped phases, but is strongly adsorbed on the interact relatively strongly with basic compounds.

Under these conditions there was no difference in themean capacity factors for four tricyclic antidepres-sants between the polar-endcapped and conventionalC phases. However, the polar-embedded phases18displayed signicantly lower retention than both of those groups of phases (Fig. 15). While we mayattribute some of this behavior to the ionic or dipolerepulsion from the shielding effect, we must alsoconsider the fact that, as a group, the polar-embed-ded phases also displayed lower hydrophobic re-

tentivity.The mean peak asymmetry values (Fig. 16) mirrorthose for the retention factors, with no signicantdifference between the conventional and polar-end-capped C phases and signicantly improved peak 18shape on the polar-embedded phases (e.g. mean Asyfor amitriptyline was 1.28 6 0.38 for the polar-em-bedded phases, 1.56 6 0.36 for the conventional C 18Fig. 12. Mean capacity factors ( k ) for a group of basic compoundsphases, and 2.21 6 0.46 for the polar-endcappedunder low pH conditions ( pH 2.5) using the conventional C , 18phases). Again, we may attribute this improvementpolar-endcapped, and polar-embedded phases.


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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 ), a 18conventional C phase (A, Luna C (2)), and a polar-embedded column (C, Polaris C -Amide). Mobile phase: 25:75 acetonitrile/ 20 m M 18 18 18potassium phosphate pH 2.5, ow-rate 5 1.5 ml/min; (1) maleic acid, (2) triprolidine, (3) chlorpheniramine, (4) diphenhydramine.

in peak shape to an increased phase inertness re- behavior of conventional C , polar-embedded18sulting from ionic or dipole repulsion of basic phases, and polar-endcapped phases using a series of

compounds by the polar-embedded group. Several diagnostic probes and several generic applications.other authors [1619] have also noted an improve- Relative to conventional C phases, polar-end-18ment in the peak shape of basic drugs when using capped phases were typied by equivalent hydro-polar-embedded phases, and these results would phobic interaction capacity and methylene selectivi-seem consistent with the previous data. ty, enhanced hydrogen bonding and silanol activity,

some increased selectivity for acidic compounds,presumably through the enhanced hydrogen bonding,

4. Conclusions increased retention for basic compounds at low pHwithout a compromise in peak shape and, at neutral

We attempted to characterize and compare the pH, increased retention and peak tailing of basic


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Fig. 14. Mean peak asymmetry values for the basic compounds atlow pH on the conventional, polar-endcapped, and polar-embed-ded phases.

Fig. 16. Mean peak asymmetry values for the basic drugs atneutral pH using the three types of stationary phase.

References

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101.[7] S. Rogers, J.G. Dorsey, J. Chromatogr. A 892 (2000) 57.[8] H. Claessens, M. van Straten, C. Cramers, M. Jezierska, B.

Buszewski, J. Chromatogr. A 826 (1998) 135.Fig. 15. Mean capacity factors for a group of tricyclic antidepres- [9] R. Vervoort, A. Debets, H. Claessens, C. Cramers, G. Desants under neutral pH conditions on the three classes of stationary Jong, J. Chromatogr. A 897 (2000) 1.phase. [10] B. Buszewski, J. Schmid, K. Albert, E. Bayer, J. Chroma-

togr. 552 (1991) 415.[11] B. Buszewski, M. Jaroniec, R. Gilpin, J. Chromatogr. A 673

(1994) 11.compounds. The polar-embedded phases were[12] P. Kasturi, B. Buszewski, M. Jaroniec, R. Gilpin, J. Chroma-

typied by signicantly reduced hydrophobicity and togr. A 673 (1994) 261.

methylene selectivity, reduced hydrogen bonding [13] B. Buszewski, M. Jaroniec, R. Gilpin, J. Chromatogr. A 668(1994) 293.capacity and silanol activity, and a unique selectivity[14] T. Czajkowska, I. Hrabovsky, B. Buszewski, R. Gilpin, M.due to ion-exchange or dipole interaction with

Jaroniec, J. Chromatogr. A 691 (1995) 217.nitrogen-containing embedded groups. Due to the [15] T. Czajkowska, M. Jaroniec, J. Chromatogr. A 762 (1997)presence of a positive character to the nitrogen- 147.

[16] J. OGara, D. Walsh, C. Phoebe, B. Alden, E. Bouvier, P.containing polar-embedded phases, acidic com-Iraneta, M. Capparella, T. Walter, LC/GC 19 (2001) 632.pounds may display enhanced retention relative to

[17] D. McCalley, J. Chromatogr. A 844 (1999) 23.neutral probes, and basic compounds may actually[18] J. OGara, B. Alden, T.Walters, J. Petersen, C. Niederlander,

experience ionic or dipole repulsion, leading to U. Neue, Anal. Chem. 67 (1995) 3809.greatly reduced retention factors, especially at low [19] M. Euerby, P. Petersson, LC/ GC Europe September (2000)

665.pH conditions.

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