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Journal of Chromatography A, 1216 (2009) 6285–6294

Contents lists available at ScienceDirect

Journal of Chromatography A

journa l homepage: www.e lsev ier .com/ locate /chroma

escription of retention characteristics of calixarene-bonded stationaryhases in dependence of the methanol content in the mobile phase

hristian Schneider, Thomas Jira ∗

nstitute of Pharmacy, Pharmaceutical/Medicinal Chemistry, Ernst-Moritz-Arndt-University Greifswald, Friedrich-Ludwig-Jahn-Str. 17, D-17487 Greifswald, Germany

r t i c l e i n f o

rticle history:eceived 16 April 2009eceived in revised form 29 June 2009ccepted 1 July 2009

a b s t r a c t

Calixarene-bonded stationary phases in HPLC are known to support additional interactions compared toconventional alkyl-bonded phases (�–� interactions, complex-building interactions). Thus it cannot bepresumed that the same mechanisms of retention apply and that retention can be predicted in similarways. Here 31 solutes of highly various molecular structures have been analysed at different mobile

vailable online 9 July 2009

eywords:PLCalixarenesechanism of retention

phase compositions (0–98% (v/v) methanol) in order to characterise the chromatographic behaviour ofthe novel stationary phases and to test the applicability of established models predicting retention factors.The influence of a change of the methanol content is discussed for non-polar, polar and ionic solutes anddifferences of their behaviour on the differing column types are shown. Additionally estimates aboutunderlying retention mechanisms are given.

artitiondsorption

. Introduction

The description of chromatographic retention with mathe-atical models is of high interest since decades. Especially the

orrelation of the retention factor k with the volume fraction ofrganic modifier in the mobile phase was studied intensively andumerous mathematical models have been developed. Some ofhem are empirically derived and some are based on partition ordsorption models. They can be used to enhance optimization pro-edures and their regression analyses can give estimates aboutnderlying retention mechanisms. The knowledge about the lat-er can be a valuable tool then new chromatographic problems

ust be solved. Therefore 31 solutes have been analysed on a setf novel calixarene-bonded stationary phases over a wide range ofethanol concentrations (0–98%). The chromatographic behaviour

f the stationary phases is characterised via regression analyses ofn k vs. ϕ and via comparisons between predicted and extrapolatedata. Thereby a complete overview can be given about the retentionharacteristics of non-polar, polar and ionic solutes over the wholeange of methanol concentration on the novel calixarene-bondedhases. The results are compared to data of standard alkyl-bonded

hases and differences in retention mechanisms are shown. Special

nterest is given to the extreme ranges of methanol content becausehe biggest changes and differences have to be expected there.

∗ Corresponding author. Tel.: +49 3834/864850; fax: +49 3834/864843.E-mail address: jira@uni-greifswald.de (T. Jira).

021-9673/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.chroma.2009.07.004

© 2009 Elsevier B.V. All rights reserved.

2. Theory

A well known empirical, linear equation was introduced by Sny-der and co-workers [1,2] in 1979:

ln k = ln kW − S · ϕ (1)

It has been widely used to describe changes of k when thevolume fraction of organic modifier ϕ is changed [3–12]. This rela-tionship is true for most solutes in a range of 0.2–0.8 ϕ. But itwas also found that deviations from linearity can occur at loweror higher percentages of organic modifier [5,13–18] and for ionicsolutes even at medium modifier concentrations [19–21].

Schoenmakers et al. [13,22,23] developed a model taking thesenon-linearities into consideration:

ln k = Aϕ2 + Bϕ + C + E√

ϕ (2)

It is based on the solubility parameter concept [24]. The termsAϕ2 and E

√ϕ account for non-linear behaviour at high and low

modifier concentrations, respectively.Other equations were, for example, developed by Johnson et

al. [25] and by Bosch et al. [26] both based on the Dimroth andReichardt polarity parameter [27]. However, Johnson et al. did notcorrelate ln k with ϕ, but with ET(30), a measure of the polarityof the mobile phase. They concluded that the stationary phase is

altered by the mobile phase and hence solutes will find differentconditions in/at the stationary phase when different modifiers areused.

A set of equations was tested by Nikitas et al. [28–30]. Theirapplicability to describe chromatographic retention was analysed

6 atogr

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l

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286 C. Schneider, T. Jira / J. Chrom

ia regression analyses of different sets of solutes and columns.hey used already known systems as well as new models, developedssuming either partition or adsorption models. The most satisfac-ory results were obtained with an equation based on an adsorption

odel.

n k = a − ln(1 + b ϕ) − ϕc

1 + b ϕ(3)

However, as the authors point out this is no evidence thatdsorption is the dominant retention mechanism because modelsased on partition also showed good results.

.1. Partition and adsorption

There is a discussion since years which model (partition ordsorption) describes HPLC-retention more adequately, how is thetationary phase structured and what are the mechanisms of reten-ion?

According to the well known solvophobic model, adapted forPLC by Horváth et al. [31,32], the stationary phase consists of

solated solvated hydrocarbon chains [33]. The single chains aressumed to be in an extended or a collapsed state, depending onhe water content of the mobile phase. They act as lipophilic adsor-ents for the solutes. Retention and selectivity are mainly driveny the mobile phase, caused by the hydrophobic effect of water. In

ight of the solvophobic model there is no differentiation betweendsorption and partition.

However, partition is assumed by the models of Lochmüller andilder [34] and Martire and Boehm [35]. Following Lochmüller andilder complete partition is only possible for small analytes and

tationary phases with ligands longer than 12 carbon atoms. In theiew of Martire and Boehm the carbon chains can adopt extended orollapsed states as expected in the solvophobic model. Yet the col-apsed state shall not prevent partition, it is only thought to hindert. Thus the stationary phase is assumed to enable steric selectivity.

Dill’s model [36] is even more differentiated. It also takes con-ormational changes of the alkyl ligands into consideration andxpects an anisotropic carbon density and an anisotropic distribu-ion of adsorbed mobile phase depending on the distance to theilica surface. This is known as the interphase model of the station-ry phase. Dill concludes that partition is the dominating retentionechanism in HPLC, but the occurrence of adsorption processes is

ossible.Partition mechanisms in HPLC have been intensively examined

y Carr et al. [37–40]. They compared the chromatographic reten-ion process with bulk-partitioning in Hexadecan. Although it wasoncluded that changes of the retention induced by changes of theobile phase occur because of effects in the mobile phase, the con-

ribution of the stationary phase to the overall retention processhall be bigger than the contribution of the mobile phase. Fur-hermore partition is expected as dominating mechanism up to a

ethanol concentration of 70%. At higher concentrations the mech-nism of retention gets more “adsorption like”, probably becausef increased sorption of modifier. The sorption or intercalation ofrganic modifier and of water molecules is furthermore believedo influence retention and selectivity by introducing more order tohe stationary phase [41].

Additionally dipole–dipole and hydrogen-bonding interactions,specially with adsorbed mobile phase, have to be considered forolar solutes. Thus the interface region is extraordinarily important,s it was also noted by Jaroniec [42] and Tijssen et al. [23].

In contrast to the results of Carr et al. [37–40], Nikitas et al.28–30] found partition like mechanisms for mobile phases with

ore than 70% methanol. For ϕ < 0.6 data rather indicate adsorp-ion. Possibly the mechanism successively changes to partition asong as ϕ is less than 0.7.

. A 1216 (2009) 6285–6294

2.2. Changes of the retention factor with the methanol content

As mentioned, Snyder and co-workers found a linear correlationbetween ln k and ϕ at least at medium modifier concentrations. Formost solutes this nearly linear range reaches from about 10/20% to80/90% methanol. However, it depends on the solutes and columnsused if and in what range a linear model will give good correlations[15,16]. Thus non-linear behaviour must be expected for mobilephases with less than 10% or more than 90% methanol.

At low methanol concentrations a concave plot is often observedfor polar solutes [5,13–18]. Accordingly measured retention fac-tors are higher than the extrapolated factors. The differences canbe large. Jandera and Kubát [43] calculated a difference of 80%between the measured log KW and the extrapolated value (extrapo-lation done with data between 20% and 80% methanol). In contrast,non-polar solutes show convex plots [13,15,16]. Schoenmakers etal. [13] found a difference of −4.09 between measured and extrap-olated log KW.

Different reasons are proposed for that in literature. Changesof the alkyl chains like incomplete solvation [14,44], conforma-tional changes [30,43] or even the collapse of the stationary phase[15] are given as explanations. However, Hammers and co-workers[14] point out that increased retention factors of polar analytescould be caused by interactions with adsorbed methanol via Lewis-acid–base interactions. Hsieh and Dorsey [15] furthermore suspecthindered diffusion into pores and increased hydrogen-bondinginteractions to hydroxyl groups. The latter shall be increasedbecause of limited solvation of the stationary phase. FollowingSchoenmakers et al. [13] the reason for non-linear behaviour is thelimited sorption of methanol at low concentrations. Because thesorption of methanol decreases the polarity of the stationary phase(less water is adsorbed or silanol groups may be less shielded), thelimited sorption results in a more polar phase showing increasedand decreased retention times for polar and non-polar solutes,respectively.

The most distinct deviations from linearity were found for ionicanalytes. Here parabolic plots of ln k vs. ϕ are found [19–21]. Thepositions of the minima depend on the pH value of the mobilephase and the lipophilicity of the analyte [19]. The more lipophilicthe solute, the higher is the concentration of methanol giving aminimum, thus the longer is the nearly linear part of the parabola.Certainly interactions with dissociated silanols are involved here[44–46]. The ionisation of the solute and the silanol group as wellas its shielding and hydrate shell may be of influence.

3. Experimental

3.1. Reagents and chemicals

35 different solutes have been used in the study. The soluteshave been selected to support a wide range of interactions for alkyl-bonded and calixarene-bonded silicas.

Benzene, toluene, phenol and pentanol were purchased fromRiedel-de-Haën (Seelze, Germany). Ethylbenzene and anthracenewere obtained from Berlin-Chemie (Berlin, Germany). Propylben-zene, ephedrine and N,N-dimethylacetamide were from Fluka(Neu-Ulm, Germany). O-, m- and p-cresol, triphenylene andphenanthrene were obtained from Acros Organics (NJ, USA). Naph-thalene, sodium hydroxide, phosphoric acid, ethanol, propanol,methyl- and ethylbenzoate were obtained from Merck (Darmstadt,Germany). Butanol was from AppliChem (Darmstadt, Germany).

O-terphenyl, biphenyl and benzoic acid were from Sigma–Aldrich(Steinheim, Germany). Propranolol was purchased from SigmaChemical (St. Louis, USA). Diclofenac was from 3 M Medica Pharma(Borken, Germany). Naproxen and salicylic acid were obtainedfrom Fagron (Barsbüttel, Germany). Amitriptyline hydrochloride

C. Schneider, T. Jira / J. Chromatogr. A 1216 (2009) 6285–6294 6287

Table 1Deviations of measured from extrapolated logarithmic retention factor.

�ln k′ , 00% methanola C18 AI AII AIII BI BII BIII Sci Res LiC Krm

Benzene −1.039 0.345 0.303 0.436 n.c. n.c. −0.022 n.c. −0.092 −0.495 −0.741Procaine n.c. 2.471 2.369 2.219 n.c. n.c. n.c. 3.011 n.c. 3.116 n.c.Ephedrine 0.672 1.053 1.462 1.293 n.c. n.c. n.c. 1.688 1.606 n.c. n.c.Dimethylacetamide 1.762 1.225 1.422 1.308 1.537 1.729 1.522 1.501 1.331 1.652 n.c.Phenol 0.076 0.650 0.635 0.680 0.427 0.478 0.301 0.554 0.261 −0.200 n.c.o-Cresol 0.135 0.909 0.780 0.896 n.c. n.c. 0.477 0.782 0.358 0.064 n.c.Benzoic acid 0.730 1.193 n.c. 0.969 n.c. n.c. 0.688 n.c. 0.486 0.113 n.c.Salicylic acid 0.742 1.326 1.076 1.109 n.c. n.c. 1.024 1.100 0.435 −0.161 n.c.

�ln k′ , 98% methanolla

Benzene −0.085 −0.096 0.290 0.409 −0.301 0.030 −0.067 0.103 0.171 −0.298 −0.271Toluene 0.049 0.106 0.435 0.560 −0.124 0.227 0.040 0.271 0.323 −0.104 −0.094Ethylbenzene 0.127 0.222 0.576 0.710 0.031 0.272 0.212 0.272 0.416 −0.013 −0.017Propylbenzene 0.345 0.320 0.721 0.814 0.088 0.365 0.301 0.201 0.531 0.122 0.117Naphthalene 0.412 0.477 0.726 0.879 0.274 0.556 0.376 0.478 0.666 0.238 0.114Anthracene 0.780 0.569 0.921 0.948 0.584 0.519 0.497 0.568 1.270 0.453 0.447Phenanthrene 0.744 0.570 0.898 0.948 0.328 0.601 0.502 0.560 1.227 0.434 0.418Biphenyl 0.539 0.360 0.737 0.959 0.284 0.518 0.522 0.308 0.799 0.203 0.187o-Terphenyl 0.739 0.452 0.785 0.931 −0.113 0.439 0.415 0.668 0.757 0.306 0.205Triphenylene 1.001 0.488 0.577 0.838 0.027 0.479 0.539 0.622 0.741 0.548 0.687Propranolol 4.845 0.883 2.258 1.654 n.c. n.c. n.c. 1.475 2.112 n.c. 3.656Promazine 4.685 1.015 3.208 2.835 1.606 2.222 n.c. 0.955 1.952 n.c. 3.998Chlorpromazine 4.196 1.222 2.956 2.588 2.538 1.889 4.132 1.169 2.330 n.c. 3.865Promethazine 4.662 2.466 2.854 2.758 3.682 2.574 n.c. 1.888 1.983 n.c. 3.950Amitriptyline 4.324 1.099 1.811 1.074 3.313 2.892 3.249 1.612 1.055 n.c. 3.927Nortriptyline 4.336 2.243 3.286 2.298 2.535 0.715 n.c. 1.268 5.157 n.c. 3.814Dimethylacetamide −0.559 0.376 0.481 0.443 0.159 0.353 0.188 0.305 0.461 1.933 n.c.Phenol −0.631 −0.202 0.363 0.484 −1.448 −0.006 −0.284 −0.040 0.235 −3.196 −1.795o-Cresol −0.301 −0.058 0.478 0.602 −0.850 0.132 −0.144 0.093 0.302 −0.947 −0.747Benzoic acid −0.432 0.216 0.610 0.782 −0.526 0.417 0.311 0.392 0.569 −1.157 −0.959Naproxen 0.282 1.052 1.136 1.267 0.470 0.894 0.733 0.968 1.364 −0.136 −0.652D 270

n ined.–70%

whhLs

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3

(cttrtbkw(u

1

iclofenac 0.907 1.129 0.988 1.164 0.

.c., not computable; too less data points for correlation or ln k′ could not be determa Extrapolation was done with retention data from the medium modifier part (30

as obtained from Salutas Pharma (Barleben, Germany). Promet-azine hydrochloride, promazine hydrochloride, chlorpromazineydrochloride and nortriptyline hydrochloride were obtained fromundbeck (Kopenhagen, Denmark). Prednisolone and hydrocorti-one were from Schering (Berlin, Germany).

All solutes were of the highest available analytical grade.HPLC gradient grade Methanol was purchased from Merck

Darmstadt, Germany) or from Acros Organics (NJ, USA). Water wasbtained by bi-distillation.

.2. Equipment

The data have been collected on two HP 1090 series II chro-atographs (Hewlett Packard, Waldbronn, Germany) equippedith diode array detectors.

.3. Columns

The study included seven different calixarene-bonded phasesCaltrex AI—calix[4]aren; Caltrex AII—calix[6]aren; Caltrex AIII—alix[8]aren; Caltrex BI—p-tert-butyl-calix[4]aren, Caltrex BII—p-ert-butyl-calix[6]aren, Caltrex BIII—p-tert-butyl-calix[8]aren, Cal-rex Science—calix[4]aren and p-tert-butyl-calix[4]aren in a 1:1atio), a resorcinarene-bonded phase (Caltrex resorcinaren) andhree alkyl-bonded phases (two Caltrex Kromasil C18 of differentatches; a LiChrospher 100 RP-18). The Caltrex-columns were allindly supplied by Syntrex GbR (Greifswald, Germany). The ligands

ere immobilized via hydrophobic spacers on endcapped silica

Kromasil Si 1 0 0, 5 �m, specific surface area/BET: 300 m2/g, man-facturer: EKA Chemicals (Bohus, Sweden).

All columns had particle diameters of 5 �m and dimensions of25 mm × 4 mm.

0.679 0.843 0.774 1.344 −0.055 −0.015

methanol).

3.4. Chromatography

Experiments were done with mobile phases consisting of mix-tures of methanol/water pH 3 at 0, 2, 5, 10, 30, 40, 50, 60, 70, 90 and98% (v/v) methanol. The pH value was adjusted with phosphoricacid or sodium hydroxide prior to mixing. Mixing was performedon-line after degassing the solvents ultrasonically. The temperaturewas thermostated to 40 ◦C in all experiments and elution was car-ried out at a flow-rate of 1 ml/min isocratically. Column hold-uptimes were determined using a linearization procedure for homol-ogous series [47] (n-alcohols). Additionally the hold-up time of thechromatograph was determined by injecting pure methanol with-out a column installed and has been subtracted from all retentiondata.

4. Results and discussion

The following discussion is based on the primary interactionswhich the solutes support in HPLC. At first the linear correlationswith Eq. (1) will be discussed. For the analysis the plot is dividedinto three parts, the middle part between 30% and 70% methanol inthe mobile phase, the high and low methanol part. The deviationsfrom linearity are calculated in relation to the linear correlation ofthe medium part. Possible conclusions about underlying retentionmechanisms are given. Afterwards correlations are discussed usingEqs. (2) and (3).

It must be noted that drawn conclusion should be consid-

ered carefully. The regression analysis of retention factors vs. themethanol concentration of the mobile phase cannot give proof forthe occurrence of one retention mechanism or another. Neverthe-less deviations from linear correlations can give hints to changesof interactions. Thus the given conclusions should be considered

6288 C. Schneider, T. Jira / J. Chromatogr

Fig. 1. Deviations of the measured retention factor at 98% to the extrapolatedretention factor (30–70% methanol) in dependence of the carbon number oftetC

aw

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4

4

cEdat

we−ttspa(

iiictdqaw

iotf

oaTbm

he solutes. Solutes in order of increasing carbon number: benzene, toluene,thylbenzene, propylbenzene, napthalene, biphenyl, anthracene/phenanthrene, o-erphenyl/triphenylene. Stationary phases: (�) Kromasil C18; (�) Caltrex BI; (�)altrex AIII; (�) Caltrex BIII.

s possible retention effects and might be taken as base for furtherork.

More detailed information about retention models and theironnection to retention mechanism can be drawn from compre-ensive review articles [48,49].

.1. Linear correlations

.1.1. Hydrophobic solutesAs expected, the hydrophobic solutes show very good linear

orrelations at medium methanol concentrations (all r2 > 0.999).ven if the whole modifier range is observed, there are only feweviations (most r2 > 0.999). However, it has only been possible tonalyse one non-polar solute, benzene, at low modifier concentra-ions because of very long retention times.

Benzene indeed shows distinct deviations from linearity at highater content. The difference between measured ln k and the value

xtrapolated from data within the range of 30–70% methanol is1.039 on the Kromasil C18. This means the extrapolated reten-

ion factor is threefold too high. Similar behaviour can be found onhe other examined alkyl-bonded phases. Although deviations areomewhat less, they are still considerable. On calixarene-bondedhases there is no such behaviour. Here deviations are minornd mostly measured values deviate to longer retention timesTable 1).

Accordingly it is probable that reduced sorption of methanols not the determining factor for the non-linear plots because annverse behaviour of calixarene- and alkyl-bonded phases concern-ng methanol adsorption seems unlikely. Thus a connection withonformational changes of the alkyl ligands is reasonable. In con-rast calixarenes have a high degree of internal order and thereforeo not undergo comparable conformational changes. As a conse-uence ln k vs. ϕ keeps mostly linear. However, these conclusionsre drawn from data of only one analyte and therefore additionalork is needed to proof these results.

Deviations can also be found at high methanol content. Heret must be emphasized that measured data at a methanol contentf 98% of course include a larger error. Therefore single observa-ions cannot be judged. Nevertheless conclusions can be drawnrom general trends found for different phases and solutes.

The observed deviations from linearity depend on the solute and

n the stationary phase. Concerning the benzene derivatives devi-tions rise with increasing carbon number on all phases (Fig. 1).his can be connected with conformational changes on alkyl-onded phases again (stretching of the chains [33,35]). Largerolecules would get more benefit from elongated chains than small

. A 1216 (2009) 6285–6294

molecules. But again this is unlikely for calixarene-bonded phases.In fact the high methanol content will not determine conforma-tional changes of the calixarenes, but an increased adsorption ofmethanol could support the interaction with the stationary phase.That is the penetration into or between the cavities may be facili-tated.

Further increase of the size of the solutes leads to furtherincreased deviations. However, for calixarene-bonded phases,especially for the smaller calix[4]- and calix[6]-arenes, there is amaximum difference for biphenyl, anthracene or phenanthrene.Larger analytes show smaller �ln k in most cases (slight increaseson Caltrex BIII and Science). Contrary differences keep increas-ing up to triphenylene on alkyl-bonded phases, while o-terphenylgives smaller �ln k as it was found for calixarene-bonded phases.Probably the beneficial effects of high methanol concentrations arelimited by steric effects.

On alkyl-phases the flexible and non-planar o-terphenyl is morestrongly excluded from the stationary phase than the planar triph-enylene, as described by the slot model [50]. Thus it gets less benefitfrom the high methanol concentration.

On calixarene-bonded phases the limiting effect is probablyrelated to the size of the cavities: It is known that small solutes likebenzene and toluene are complexed by tert-butylcalix[4]arenes.However, phenanthrene and anthracene will only be complexedpartly in a cavity with a diameter of about 6 Å. The larger o-terphenyl and triphenylene will be even more sterically hinderedforming complexes with calixarenes. Naturally complex buildingwill be easier for bigger solutes with bigger calixarenes. Thus stericeffects could hinder large molecules like triphenylene from a ben-eficial interaction.

Support for this steric influence comes from the deviations onsmall calix[4]arenes which are less than the deviations on calix[6]-and calix[8]arenas. Additionally differences are higher for non-substituted than for substituted calixarenes at similar ring sizebecause the tert-butyl-groups hinder the penetration into the sta-tionary phase.

Unfortunately it is difficult to elucidate differences betweenhydrophobic and steric effects by analysing plots of ln k vs. ϕ. Togain more insight into the role of steric effects on the retention ofcalixarene-bonded stationary phases further work is in progress.

4.1.2. Ionic solutesIn contrast to hydrophobic solutes distinct deviations from

linearity were found for small, protonated analytes (procaine,ephedrine) even at medium modifier concentrations. A parabolicplot can be estimated, as it was also observed by El Tayar et al.[19] (Fig. 2a). Plots of large, protonated solutes like promethazineor amitriptyline are less curved and widely linear in that modifierrange, as can be seen from the high coefficients of determination(Table 2). But large differences are again found if the methanolcontent is increased (Fig. 2b). Obviously the hydrophobicity of thesingle solute, and with it the part the polar or ionic interaction hason the overall interaction, has influence on the curvature of the ln kplot. The bigger the ionic part of the solute is in relation to the wholemolecule, the more the plot is curved.

Besides the solute dependency a dependency on the station-ary phases was found. Deviations from linearity are highest on theLiChrospher and on the Caltrex resorcinarene column for the samesolute. Both are strongly ionic phases. On Caltrex A phases differ-ences are less, and they are least on the Kromasil C18 and CaltrexB phases. Since the latter phases are also least ionic this shows

that deviations from linearity tend to increase with increasing ioniccharacter of the stationary phase.

Recapitulatory, non-linear plots of ln k vs. ϕ for ionic solutescan also be shown on calixarene-bonded phases, as it is describedfor alkyl-bonded phases [19–21]. Furthermore the curvature gets

C. Schneider, T. Jira / J. Chromatogr. A 1216 (2009) 6285–6294 6289

Fig. 2. Plots of the logarithmic retention factors of ionic solutes. (a and b) (�) Procaine and (�) promethazine on Caltrex AIII. (c) (�) Procaine Kromasil C18; (�) procaineCaltrex Science; (�) promazine Caltrex AI; (�) promethazine Caltrex AI. (d) (�) promethazine Caltrex AIII; (�) promethazine Caltrex BI. Data points of procaine on theKromasil between 0.4 and 0.7 ϕ do not represent measured values! Actual ln k cannot be calculated because procaine eluted before column void time. Values were set to −4.0t

TC

r

BTNAoTPEPADPBNDP

rBTNAoTPEPADPBNDP

n

o illustrate the parabolic nature of the plot.

able 2oefficients of determination of linear correlations of ln k vs. ϕ.

2 30–70% methanol C18 AI AII AIII BI

enzene 0.999 1.000 1.000 1.000 1.000oluene 0.999 0.999 1.000 1.000 1.000aphtalene 1.000 0.996 0.999 0.997 0.999nthracene 1.000 0.998 0.998 1.000 0.997-Terphenyl 1.000 1.000 1.000 1.000 1.000riphenylene 1.000 1.000 1.000 1.000 1.000rocaine n.c. 0.938 0.932 0.900 n.c.phedrine n.c. 0.997 0.973 0.984 n.c.romethazine 0.994 0.984 0.988 0.987 0.996mitriptyline 0.994 0.987 0.986 0.986 0.999imethylacetamide 0.994 0.993 0.987 0.974 0.997henol 1.000 0.998 0.999 0.998 1.000enzoic acid 0.988 0.999 0.998 0.996 0.999aproxen 0.995 0.998 0.997 0.995 0.996iclofenac 1.000 1.000 0.998 0.998 1.000rednisolone 0.972 0.987 0.993 0.988 0.993

2 00–98% methanolenzene 0.985 0.998 0.997 0.991 0.998oluene 1.000 0.999 0.997 0.995 1.000aphtalene 0.998 0.997 0.994 0.989 0.999nthracene 0.996 0.998 0.994 0.993 0.998-Terphenyl 0.998 0.999 0.997 0.996 1.000riphenylene 0.995 0.999 0.998 0.996 1.000rocaine n.c. 0.901 0.834 0.849 n.c.phedrine 0.548 0.928 0.888 0.909 n.c.romethazine 0.640 0.905 0.722 0.798 0.841mitriptyline 0.745 0.969 0.939 0.948 0.914imethylacetamide 0.973 0.927 0.891 0.864 0.960henol 0.996 0.995 0.989 0.979 0.983enzoic acid 0.996 0.984 0.987 0.971 0.995aproxen 0.998 0.989 0.987 0.980 0.995iclofenac 0.995 0.992 0.993 0.988 0.997rednisolone 0.985 0.976 0.975 0.957 0.994

.c. not computable; too less data points for correlation or ln k could not be determined.

BII BIII Sci Res LiC Krm

0.999 1.000 0.999 1.000 0.998 0.9990.999 1.000 0.998 1.000 0.999 0.9990.997 0.999 0.997 0.999 0.999 0.9990.996 1.000 1.000 0.994 0.999 0.9981.000 1.000 1.000 1.000 1.000 1.0001.000 1.000 1.000 1.000 1.000 1.0000.884 n.c. 0.883 n.c. 0.013 n.c.n.c. n.c. 0.967 0.856 0.691 0.9200.997 0.972 0.987 0.988 0.655 0.9950.997 0.997 0.990 0.992 1.000 0.9970.984 0.986 0.985 0.988 0.992 0.9510.999 1.000 0.999 0.996 0.991 0.9860.997 0.998 0.996 0.996 0.999 0.9950.996 0.995 0.993 0.992 0.997 0.9970.998 0.998 0.999 0.991 0.998 0.9990.991 0.989 0.987 0.987 0.997 0.998

1.000 0.999 1.000 1.000 0.991 0.9890.999 0.999 0.998 0.998 0.999 0.9990.997 0.997 0.997 0.994 0.999 0.9990.998 0.998 0.998 0.987 0.999 0.9981.000 0.999 0.999 0.997 1.000 1.0000.999 0.999 0.998 0.996 0.999 0.9980.944 n.c. 0.854 n.c. 0.881 n.c.n.c. n.c. 0.874 0.649 n.c. n.c.0.931 n.c. 0.933 0.077 0.655 0.6090.927 0.939 0.963 0.879 0.982 0.7060.908 0.909 0.914 0.876 0.810 0.9730.996 0.996 0.995 0.994 0.896 0.9570.993 0.991 0.989 0.987 0.987 0.9860.993 0.992 0.990 0.978 0.995 0.9950.997 0.995 0.996 0.984 0.998 0.9980.982 0.984 0.976 0.964 0.999 0.999

6 atogr

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290 C. Schneider, T. Jira / J. Chrom

tronger the more ionic solute and stationary phase are and thushe bigger the part of ionic interaction on the overall interaction is.

Concerning the whole methanol range more differencesetween the stationary phases were observed. While all ionicolutes show parabolic plots on alkyl-bonded phases, some plateausere observed on calixarene-bonded phases when the methanol

oncentration is increased to 98%. Partly retention factors evenecrease (Fig. 2c). All in all strongest curvatures, and thus highestifferences to the extrapolated values, were found on the Kromasil18 phases (Table 1). (No data can be given for the LiChrospherP-18 since retention data could not be obtained due to its extraor-inary ionic character.) Retention times are two- or threefold highert 98%, which conforms to several void volumes, compared withata at 70% or 90% methanol.

This may be partly due to changes of the ionisation of theases. With a decreasing degree of ionisation the hydrophobic-

ty of the solutes increases and hence their retention times rise.he different hydrophobicities of the stationary phases explain theifferent deviations. Therefore same analytes should experience aigher increase of retention on a more hydrophobic phase. Indeedhe highest increases were found on the most hydrophobic alkyl-onded phases, but the Caltrex A phases show higher deviationshan the more hydrophobic Caltrex B phases (Table 1). Further-

ore, if deviations were exclusively driven by the mobile phase,on-linearities would begin at the same methanol concentrationsn all stationary phases. Indeed, retention data show deviations onaltrex A phases already at ϕ = 0.9, while data are often linear oretention even decreases (elution on or before void time) on tert-utyl-calixarene phases at this methanol concentration (Fig. 2d).his indicates an influence of the stationary phases.

An effect caused by the bonded ligands seems improbableecause differences are found on both alkyl- and calixarene-bondedhases. Hence interactions on the silica surface are likely respon-ible and accordingly silanophilic interactions may be involved, asescribed by other authors [44–46]. They might be extraordinarilytrengthened, if the low amount of water in the mobile phase is con-idered. This already low amount will be even more reduced, if thetructure of methanol–water mixtures is taken into consideration51]. Free methanol, free water as well as methanol–water asso-iates exist in the mobile phase. Thus the effective concentration ofree water is even less than ϕ suggests. Therefore only few water isvailable for hydrogen-bonding with the silica support and espe-ially with the silanol groups. It is furthermore known from studiesith different organic modifiers, that the tailing of basic solutes is

tronger, the less the ability of the modifier to form hydrogen bondss [52]. Thus water will shield silanol groups effectively and its veryow amount in the mobile phase could lead to disproportionately

ncreased ionic interactions.

Furthermore the polarity of the stationary phase could havehanged in relation to the mobile phase, caused by a layer ofightly bound water on the silanol surface. This could also lead ton increased affinity of polar and ionic solutes, while hydropho-

Fig. 3. Plots of the logarithmic retention factors of the hydrogen donor p

. A 1216 (2009) 6285–6294

bic interactions with the bonded ligands would not change. Ofcourse both effects must occur on all stationary phases. However,differences can result from different bonding densities and dif-ferent steric hindrances. Such influence of the bonding densitywas reported by Gritti and Guichon [53], who found a decreasedinfluence of silanols on the retention of basic compounds withincreasing bonding density. Certainly the same must apply foradsorbed water.

At high water content an analysis is more difficult because ofvery long (LiChrospher RP-18 and Caltrex resorcinarene) or veryshort (Caltrex B phases) retention times. Nevertheless deviations tolonger retention factors are obvious and differences between alkyl-and calixarene-bonded phases (less deviations on alkyl-bondedphases) can be estimated (Table 1). The generally increased reten-tion times again imply an increased affinity to ionised silanols. Aneffect driven by the mobile phase is unlikely, since neither the ion-isation of the solutes will decrease nor should the hydrophobiceffect of the mobile phase be disproportionately strengthened. Con-cerning the stationary phase the decreased adsorption of methanolproposed by Schoenmakers et al. [13] could increase the polarity ofthe phase.

4.1.3. Hydrogen-bond donorsThe acidic, polar hydrogen-donating solutes (benzoic acid,

naproxen . . .) as well as the weakly acidic, polar hydrogen-donatingsolutes (phenol, prednisolon . . .) show a highly correlating, lin-ear plot of ln k (Table 2). The correlations are slightly weaker forthe acids on the Caltrex resorcinarene and for the polar steroidson all stationary phases. This once more shows that the curvatureincreases with the polarity of the solute and the stationary phase.

As expected, linearity does also not apply for ϕ < 0.1 or ϕ > 0.9.However, �ln k values of polar benzene derivatives on the KromasilC18 are very small at high water concentrations (Table 1). In con-trast positive differences were found on calixarene-bonded phases(Fig. 3). The same differences between the stationary phases canbe seen for small, acidic solutes but at generally higher values.These differences probably arise from the decreased adsorptionof methanol and the concurrently increased adsorption of water.Though this is an effect related to the silica gel it should also befound on alkyl-phases.

Here a compensating effect takes places. Obviously even smallpolar solutes are not only exclusively retained via polar interac-tions, but also via hydrophobic interactions. Furthermore it wasfound that hydrophobic solutes are disproportionately less retainedon alkyl-bonded phases at high water content. Consequently thenearly linear plots of phenol and the cresols on Kromasil C18 arelikely to result from one increased and one decreased interaction

rather than from an unchanged mechanism of interaction.

The behaviour of hydrogen-donating solutes is quite different athigh methanol concentrations. Positive as well as negative devi-ations to the extrapolated retention factors can be found. Thedeviations rise with increasing size of the solutes on all station-

henol. (a) On Kromasil C18. (b) (�) On Caltrex AI; (�) on Caltrex BI.

C. Schneider, T. Jira / J. Chromatogr

FNB

ahaafpaatdtbtpBamsiF[s

Fl

ig. 4. Plots of the logarithmic retention factors of the hydrogen acceptor solute,N-dimethylacteamide (�) on LiChrospher Rp-18; (�) on Caltrex AI; (�) on CaltrexI; (�) on Kromasil C18.

ry phases. However, this is certainly related to the increasedydrophobic interaction at high methanol content. Consequently, ifnything the polar interaction between hydrogen-donating solutesnd the stationary phases is decreased. Some possible reasonsor that can be excluded: (i) Negative deviations are found on allhases, thus an influence of the ligands is not likely (ii) dissoci-ted silanols are no possible partners for an interaction and (iii)n interaction with protonated silanols should be rather increasedhan decreased. Hence the adsorption of mobile phase seems to beecisive. However, as stated above (see ionic solutes) it is unlikelyhat the polarity of the silica surface disproportionately decreasesecause the hydrophilic surface tightly binds water. Thus adsorp-ion in the interface region could be of special importance forolar solutes, as previously supposed [23,42,54,55]. Klatte andeck [56] showed that methanol molecules near to the station-ry phase are highly ordered and following Sun et al. [57] theirethyl-groups are directed towards the hydrophobic ligands. This

tate of increased organization begins about 20 Å above the sil-ca support, i.e. about 3 Å above the bonded alkyl chains [56,58].urthermore also water molecules are adsorbed in this region59] and the distribution of both species varies with the compo-ition of the mobile phase. Although these results were found for

ig. 5. Comparison between (a and b) linear regression and regression according to Eq. (2ine. Data points are from (a) benzene Kromasil C18; (b) procaine Caltrex AII; (c and d) (�

. A 1216 (2009) 6285–6294 6291

alkyl-bonded phases, the similar processes can be estimated forcalixarene-bonded phases. Hence interactions in the interphaseregion can occur with adsorbed methanol and adsorbed water.Exact knowledge about the composition of an eluent-surface-phaseis not available, but influences of the bonded ligands are proba-ble. Consequently, the large fraction of 98% methanol could lead toa disproportionately high decrease of adsorbed water and shoulddecrease the affinity of polar solutes (hydrogen donors and accep-tors). Moreover, the loss of adsorbed water upon an increase ofthe methanol concentration should be connected to the differenthydrophobicities of the bonded layers, with the alkyl phases beinghighly sensitive. Accordingly they show the most distinct deviations(Table 2).

4.1.4. Hydrogen-bond acceptorsLinear correlations of the hydrogen-bond acceptor N,N-

dimethylacetamide at medium methanol concentrations areweaker than for hydrophobic solutes (Table 2), indicating the slightcurvature of the ln k vs. ϕ plots (Fig. 4). Only minor differences canbe found between the stationary phases.

This is also true for the high water region. Similar positive devi-ations indicate an influence of the silica gel. This had to be expectedsince the polar interaction probably takes place on protonatedsilanols or adsorbed water. The overall disproportionate increaseis likely also connected to a decreased adsorption of methanol bysimultaneously increased adsorption of water. On the one hand thedecreased amount of methanol could increase hydrogen-bondinginteractions to silanolic groups and on the other hand the addition-ally adsorbed water can itself function as hydrogen donator.

At high methanol concentrations N,N-dimethylacetamid’sbehaviour is different from phase to phase. Retention increases onthe LiChrospher RP-18, is widely unchanged on Caltrex phases andeven decreases on the Kromasil C18 then ϕ is changed from 0.9

to 0.98 (Fig. 4). The first result is certainly related to the strongpolar and ionic character of the LiChrospher phase. Interestinglythe deviation on the Caltrex resorcinaren phase is distinctly less,although both stationary phases mostly show similar character-istics. Obviously the hydroxyl groups on the resorcinarene-calix

) and (c and d) between regression according to Eq. (2) solid line and Eq. (3) dotted) phenol Caltrex BI; (�) promethazine Caltrex AII.

6292 C. Schneider, T. Jira / J. Chromatogr. A 1216 (2009) 6285–6294

Table 3Coefficients of determination of correlations according to Eqs. (2) and (3).

r2 00–98% methanol with Eq. (2) C18 AI AII AIII BI BII BIII Sci Res LiC Krm

Benzene 0.999 1.000 0.999 0.999 1.000 1.000 0.999 1.000 1.000 0.999 0.999Toluene 1.000 1.000 1.000 1.000 1.000 1.000 0.999 1.000 1.000 1.000 0.999Phenol 0.999 0.999 1.000 0.999 0.994 0.999 0.998 0.999 0.999 0.956 0.982o-Cresol 1.000 0.999 1.000 1.000 0.998 1.000 0.999 1.000 1.000 0.998 0.997Naphtalene 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0.998Anthracene 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000o-Terphenyl 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000Triphenylene 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000Dimethylacetamide 0.995 0.999 1.000 0.999 0.999 0.999 0.998 0.999 0.999 0.969 0.994Ephedrine 0.877 0.994 0.998 0.996 1.000 0.999 0.999 0.997 0.998 n.c. 0.841Promethazine 0.946 1.000 0.993 0.998 0.991 0.977 1.000 0.994 0.979 0.877 0.979Amitriptyline 0.961 0.994 0.998 0.985 0.996 1.000 1.000 0.999 0.979 1.000 0.983Benzoic acid 0.998 1.000 0.999 0.999 0.997 0.999 0.999 1.000 1.000 0.995 0.992Salicylic acid 0.990 0.999 0.998 0.999 0.993 0.998 0.997 0.999 0.999 0.979 0.986Naproxen 0.999 1.000 1.000 1.000 0.999 1.000 0.999 1.000 1.000 0.998 0.997Diclofenac 0.999 1.000 1.000 1.000 0.999 1.000 1.000 1.000 1.000 1.000 1.000Prednisolone 0.995 1.000 1.000 1.000 0.998 1.000 0.998 1.000 1.000 1.000 1.000

r2 00–98% methanol with Eq. (3)Benzene 0.994 0.999 0.999 0.997 1.000 1.000 0.999 1.000 1.000 0.999 0.999Toluene 1.000 1.000 1.000 0.999 1.000 1.000 0.999 1.000 1.000 1.000 0.999Phenol 0.998 0.997 0.998 0.997 0.990 0.997 0.996 0.997 1.000 0.850 0.983o-Cresol 0.999 0.998 0.999 0.998 0.996 0.999 0.997 0.998 1.000 0.992 0.994Naphtalene 1.000 1.000 1.000 1.000 1.000 1.000 0.999 1.000 1.000 1.000 1.000Anthracene 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000o-Terphenyl 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000Triphenylene 1.000 1.000 0.999 1.000 1.000 1.000 1.000 1.000 0.999 1.000 1.000Dimethylacetamide 0.990 0.996 0.998 0.995 0.993 0.998 0.997 0.999 0.998 0.957 0.994Ephedrine 0.771 0.992 0.994 0.995 0.999 0.991 0.998 0.990 0.891 n.c. 0.501Promethazine 0.802 0.993 0.865 0.943 0.955 0.962 0.997 0.993 0.098 0.637 0.798Amitriptyline 0.876 0.988 0.996 0.975 0.981 0.993 0.998 0.999 0.913 0.965 0.862Benzoic acid 0.997 0.999 0.999 0.998 0.995 0.999 0.998 0.999 1.000 0.992 0.993Salicylic acid 0.987 0.998 0.996 0.997 0.990 0.997 0.995 0.998 0.999 0.981 0.987NDP

dlba

rh

4

aErfh(sst

pwamitodnd

aproxen 0.998 1.000 1.000 1.000iclofenac 0.999 1.000 1.000 1.000rednisolone 0.985 1.000 1.000 0.999

o not lead to strongly increased polar interactions, which under-ines the assumption of protonated silanols or/and adsorbed watereing responsible for the hydrogen-bonding acidity of the station-ry phases.

Furthermore hydrogen-bonding interactions in the interfaceegion could lead to the observed differences, as discussed forydrogen-bond donors.

.2. Additional correlations

Application of Eq. (2) of course yields improved correlations forll solutes and all stationary phases. The additional terms Aϕ2 and√

ϕ describe curvatures at high and low methanol concentrations,espectively. Accordingly the improvements are relatively minoror non-polar solutes. However, the deviations from linearity atigh and low methanol concentrations are adequately describedFig. 5a). Naturally, improvements are larger for polar and ionicolutes (Fig. 5b). Especially for calixarene-bonded phases regres-ions can be done very exactly with Schoenmaker’s equation forhe majority of the analyses (Table 3).

However, exact regressions of the non-linear parts will only beossible, if all data points are available. If regression analyses is doneith data points between 0.3 and 0.7 ϕ only, predictions for high

nd low methanol regions will be insufficient. Obviously the infor-ation about the development of the plot in these regions is not

ncluded in the data of medium methanol concentrations and hence

he prediction is random. Partly convex plots are estimated insteadf concave plots and differences to measured values are higher thanifferences obtained with linear correlation. Accordingly, Eq. (2) isot suitable for the prediction of retention from medium modifierata alone. Information about the high and low methanol regions

0.997 0.999 0.998 0.999 1.000 0.995 0.9960.997 0.999 0.999 1.000 1.000 0.998 0.9980.994 0.999 0.997 1.000 1.000 0.999 0.999

must be introduced to the regression either by including additionaldata points or by estimating start-values for the parameters. Thiscan be done, if the behaviour of similar solutes is known.

Application of Eq. (3) again gives better results than linearregressions because curvatures can be included (Table 3). The goodapplicability found by Nikitas et al. [29] can be confirmed for non-polar and slightly polar solutes. However, better regressions thanthose obtained with Eq. (2) are hard to find. Additionally con-vex plots at low methanol concentrations cannot be describedand concave plots are less exactly described (Fig. 5). Further-more the regression of the retention data of protonated solutesis deficient because parabolic plots cannot be calculated with Eq.(3). All in all regression curves calculated with Eq. (3) less accu-rately represent the measured data points than the curves obtainedwith Schoenmaker’s equation. Thus it is less suited for a descrip-tion of non-polar, polar and ionic solutes on calixarene-bondedcolumns.

5. Conclusion

Different locations of interaction and different effects on reten-tion can be supposed for the different interactions (Table 4).

The wide linearity of the plots of ln k vs. ϕ of non-polar solutesimplies an unchanged mechanism of retention for a broad rangeof methanol concentrations in the mobile phase. However, thereis a change when ϕ approaches 0.9–0.98. The concavely shaped

plot is probably not related to adsorption effects because the devi-ations from linearity increase with the size of the solutes andtend to decrease again if a particular size, which depends onthe kind of stationary phase, is reached. This is not feasible foradsorption processes. Accordingly partition processes seem to rise

C. Schneider, T. Jira / J. Chromatogr. A 1216 (2009) 6285–6294 6293

Table 4Effects of the single interactions on retention at very low and high methanol concentrations and their possible origins.

Low concentrations of methanol High concentrations of methanol

Hydrophobic interaction • Retention decreases on Kromasil C18a • Retention increases, probably due to facilitated partitioncaused by additionally adsorbed methanol

• Retention keeps nearly unchanged on calixaren-bondedphases

• Increase depends on size/hydrophobicity of the analytesand on steric characteristics of the stationary phases

• Origin of the decreases on Kromasil C18 supposedly areconformational changes of the alkyl chains

Ionic interaction • Retention increases on all stationary phases because ofincreased interaction at dissociated silanols

• Also increase of retention through:

• Less increase on C18 because of shielding effects of“collapsed” alkyl chains and possibly because of acompensating effect through reduced hydrophobicinteractions

• Higher hydrophobicity of less protonated bases and

• Increased interaction at silanols, probably due to lesshydrate shells• Differences between stationary phases result fromdifferent steric hindrances during diffusion to the silicasurface

Polar interaction (solute is proton-acceptor) • Retention increases on all stationary phases • Different behaviour of the phases• Because of increased polarity of the stationary phases as aconsequence of less adsorbed methanol

• Reasons probably are increased interaction withprotonated silanols or with adsorbed water• Decreases can be related to effects in the interface region:i.e. differences of the composition of adsorbed mobile phase

Polar interaction (solute is proton-donator) • Retention keeps unchanged on C18 or increases • Retention increases in dependence of the hydrophobicityof the solutes, but decreases were also observed

• Retention increases on other stationary phases • i.e. the polar interaction effects a decrease of retention(increase caused by hydrophobic interaction)

• Increased polarity of the stationary phase is again causative • Like above, the reason probably is the composition of theadsorbed mobile phase at the interface

• Increased polarity is partly compensated on C18 (see

creasee

dc

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stic

oilsaicTia

tmmc

[

ionic solutes)

a All statements concerning retention are given in relation to a linear plot, e.g. “inxtrapolation.

isproportionately at high methanol concentrations, at least onalixarene-bonded stationary phases.

The decreases of retention at low methanol concentrations,pecific for alkyl-bonded phases, also point out a change in theartition process. This is likely related to conformational changesf the alkyl-chains, as proposed by Martire and Boehm [35]. Aelation to adsorption processes is unlikely because the strongerolding of the chains would not distinctly change the accessible sur-ace. Calixarene-bonded phases are of advantage here. They do notndergo conformational changes and hence the retention mecha-ism for non-polar solutes keeps widely constant, facilitating therediction of retention factors.

Recapitulatory, the non-linear behaviour of non-polar soluteseems to be influenced by changes of partition effects. This confirmshe results of Nikitas et al. [30], who found a successive shift toncreased partition with increasing ϕ for alkyl-bonded silicas. Thisan be confirmed for calixarene-bonded phases.

Besides the hydrophobic interaction, ionic interactions are obvi-usly crucial for protonated solutes. Accordingly their degree of

onisation and the activity of dissociated silanols are decisive. Theatter depends on the adsorption of mobile phase, the size ofurrounding hydrate-shells and the steric hindrance of bonded lig-nds. Furthermore hydrophobic interactions still take place also foronic solutes. Hence their retention characteristics also depend onhanges of hydrophobic effects and changes can be compensated.hat is why the degree of ionisation is of special importance. All

n all, ln k plots of ionic analytes are more curved the more ionicnalyte and stationary phase are.

The retention data of polar compounds imply a dependence onhe polarity of the stationary phase and hence on the adsorption of

obile phase, as proposed by Schoenmakers et al. [13] and Ham-ers et al. [14]. A decreasing adsorption of methanol at very low

oncentrations in the mobile phase leads to increased polarity and

[

[[[

of retention” means: retention increases more strongly than is predicted by linear

to higher retention times of polar solutes. But again the hydrophobicinteraction can have compensating influence.

Additionally the interface region, i.e. the mobile phase adsorbedon top of the bonded ligands, influences retention, as can bededuced from data of the high methanol region. This has previouslybeen supposed by Jaroniec [42] and Tijssen et al. [23]. A layer ofbonded mobile phase and its composition will obviously influencethe retention. Changes of this composition can lead to non-linearplots of ln k at high methanol content. Especially polar soluteswill be influenced because they interact via hydrogen-bonding towater and methanol. Thus the formation of an eluent-surface-phaseshould be considered concerning the mechanism of retention ofpolar solutes.

Further work is in progress to gain even more insight into theretention characteristics and mechanisms of calixarene-bondedstationary phases.

References

[1] L.R. Snyder, J.W. Dolan, J.R. Gant, J. Chromatogr. 165 (1979) 3.[2] J.W. Dolan, J.R. Gant, L.R. Snyder, J. Chromatogr. 165 (1979) 31.[3] B.L. Karger, J.R. Gant, A. Martkopf, P.H. Weiner, J. Chromatogr. 128 (1976) 65.[4] M. Harnisch, H.J. Mockel, G. Schulze, J. Chromatogr. 282 (1983) 315.[5] T. Braumann, B. Jastorff, J. Chromatogr. 350 (1985) 105.[6] M.T.W. Hearn, M.I. Aguilar, J. Chromatogr. 392 (1987) 33.[7] A. Opperhuizen, T.L. Sinnige, J.M.D. van der Steen, O. Hutzinger, J. Chromatogr.

388 (1987) 51.[8] K. Valkó, J. Liq. Chromatogr. Relat. Technol. 10 (1987) 1663.[9] K. Valkó, T. Cserhati, I. Fellegvari, J. Sagi, A. Szemzo, J. Chromatogr. 506 (1990)

35.10] A. Robbat, T.-Y. Liu, J. Chromatogr. 513 (1990) 117.

[11] G. Cimpan, M. Hadaruga, V. Miclaus, J. Chromatogr. A 869 (2000) 49.12] X. Liu, H. Tanaka, A. Yamauchi, B. Testa, H. Chuman, J. Chromatogr. A 1091 (2005)

51.13] P.J. Schoenmakers, H.A.H. Billiet, L.d. Galan, J. Chromatogr. 282 (1983) 107.14] W.E. Hammers, G.J. Meurs, C.L. De Ligny, J. Chromatogr. 246 (1982) 169.15] M.-M. Hsieh, J.G. Dorsey, J. Chromatogr. 631 (1993) 63.

6 atogr

[[[[[[[

[[

[[[

[[[[[[[[[[[

[[

[[[

[[[

[[[

[[[54] M. Jaroniec, D.E. Martire, J. Chromatogr. 351 (1986) 1.

294 C. Schneider, T. Jira / J. Chrom

16] P.A. Tate, J.G. Dorsey, J. Chromatogr. A 1042 (2004) 37.17] P. Jandera, J. Churacek, L. Svoboda, J. Chromatogr. 174 (1979) 35.18] M.J.M. Wells, C. Randall Clark, J. Chromatogr. 235 (1982) 31.19] N. El Tayar, H. van de Waterbeemd, B. Testa, J. Chromatogr. 320 (1985) 293.20] K.E. Bij, C. Horvath, W.R. Melander, A. Nahum, J. Chromatogr. 203 (1981) 65.21] S. Eksborg, H. Ehrsson, U. Lonroth, J. Chromatogr. 185 (1979) 583.22] P.J. Schoenmakers, H.A.H. Billiet, R. Tussen, L. De Galan, J. Chromatogr. 149

(1978) 519.23] R. Tijssen, H.A.H. Billiet, P.J. Schoenmakers, J. Chromatogr. 122 (1976) 185.24] J.H. Hildebrand, R.L. Scott, The Solubility of Non-electrolytes, 3rd ed., Dover

Publications, New York, 1964.25] B.P. Johnson, M.G. Khaledi, J.G. Dorsey, Anal. Chem. 58 (1986) 2354.26] E. Bosch, P. Bou, M. Roses, Anal. Chim. Acta 299 (1994) 219.27] K. Dimroth, C. Reichardt, T. Siepmann, F. Bohlmann, Justus Liebigs Ann. Chem.

661 (1963) 1.28] P. Nikitas, A. Pappa-Louisi, P. Agrafiotou, J. Chromatogr. A 946 (2002) 9.29] P. Nikitas, A. Pappa-Louisi, P. Agrafiotou, J. Chromatogr. A 946 (2002) 33.30] P. Nikitas, A. Pappa-Louisi, P. Agrafiotou, J. Chromatogr. A 1034 (2004) 41.31] C. Horvath, W. Melander, I. Molnar, J. Chromatogr. A 125 (1976) 129.32] C. Horváth, W. Melander, J. Chromatogr. Sci. 15 (1977) 393.

33] A. Vailaya, C. Horvath, J. Chromatogr. A 829 (1998) 1.34] C.H. Lochmüller, D.R. Wilder, J. Chromatogr. Sci. 17 (1979) 574.35] D.E. Martire, R.E. Boehm, J. Phys. Chem. 87 (1983) 1045.36] K.A. Dill, J. Phys. Chem. 91 (1987) 1980.37] P.W. Carr, J. Li, A.J. Dallas, D.I. Eikens, L.C. Tan, J. Chromatogr. A 656 (1993) 113.38] L.C. Tan, P.W. Carr, J. Chromatogr. A 775 (1997) 1.

[[[[[

. A 1216 (2009) 6285–6294

39] P.W. Carr, L.C. Tan, J.H. Park, J. Chromatogr. A 724 (1996) 1.40] J.H. Park, Y.K. Lee, Y.C. Weon, L.C. Tan, J. Li, L. Li, J.F. Evans, P.W. Carr, J. Chromatogr.

A 767 (1997) 1.[41] L. Limsavarn, J.G. Dorsey, J. Chromatogr. A 1102 (2006) 143.42] M. Jaroniec, J. Chromatogr. A 656 (1993) 37.43] P. Jandera, J. Kubát, J. Chromatogr. 500 (1990) 281.44] F. Tsopelas, A. Tsantili-Kakoulidou, M. Ochsenkuhn-Petropoulou, Talanta 73

(2007) 127.45] R. Kaliszan, Quant. Struct. Act. Relat. 9 (1990) 83.46] A. Nahum, C. Horvath, J. Chromatogr. 203 (1981) 53.47] P.J.M.v. Tulder, J.P. Franke, R.A.d. Zeeuw, J. High Res. Chromatogr. Chromatogr.

Commun. 10 (1987) 191.48] P. Nikitas, A. Pappa-Louisi, J. Chromatogr. A 1216 (2009) 1737.49] M. Rosés, X. Subirats, E. Bosch, J. Chromatogr. A 1216 (2009) 1756.50] S.A. Wise, L.C. Sander, J. High Resolut. Chromatogr. Chromatogr. Commun. 8

(1985) 248.[51] Y.C. Guillaume, C. Guinchard, Anal. Chem. 70 (1998) 608.52] H.A. Claessens, E.A. Vermeer, C.A. Cramers, LC-GC Int. 6 (1993) 692.53] F. Gritti, G. Guiochon, J. Chromatogr. A 1132 (2006) 51.

55] M. Jaroniec, D.E. Martire, J. Chromatogr. 387 (1987) 55.56] S.J. Klatte, T.L. Beck, J. Phys. Chem. 100 (1996) 5931.57] L. Sun, J.I. Siepmann, M.R. Schure, J. Phys. Chem. B 110 (2006) 10519.58] R. Meyer, C. Schneider, Th. Jira, Pharmazie 63 (2008) 619.59] K. Ban, Y. Saito, K. Jinno, Anal. Sci. 20 (2004) 1403.