selectivity of calixarene-bonded silica phases in hplc: description of special characteristics with...

Research Article

Selectivity of calixarene-bonded silicaphases in HPLC: Description of specialcharacteristics with a multiple term linearequation at different methanolconcentrations

Retention and selectivity characteristics of different calixarene-, resorcinarene- and alkyl-

bonded stationary phases are examined by analyzing a set of test solutes covering the

main interactions (hydrophobic, steric, ionic, polar) that apply in HPLC. Therefore Dolan

and Snyder’s multiple term linear equation has been adapted to fit the properties of

calixarene-bonded columns. The obtained parameters are used to describe retention and

selectivity of the novel Caltrexs phases and to elucidate underlying mechanisms of

retention. Here, differences of stationary phase characteristics at different methanol

concentrations in the mobile phases are examined. Both selectivity and retention were

found to depend on the methanol content. Differences of these dependencies were found

for different stationary phases and interactions. The differences between common alkyl-

bonded and novel calixarene-bonded phases increase with increasing methanol content.

Keywords: Calixarenes / HPLC / Methanol concentrations / Retention mechanism /Stationary phase characteristicsDOI 10.1002/jssc.201000497

1 Introduction

Calixarenes are cavity-shaped cyclic molecules consisting of

phenol units linked via methylene bridges. They will act as a

reversed phase in HPLC, if they are bonded to silica gel. As

special, receptor-like molecules they differ from common alkyl-

bonded phases by supporting additional interactions [1–11].

Their ability to form inclusion complexes [1, 2, 12–18]

and provide p–p interactions [2–4, 13] or p-electron transfer

[1, 3, 4, 12, 13, 19] makes them a valuable tool for HPLC-

analyses. Additionally, the variable possibilities of modifying

the calixarenes, e.g. a variable ring size, different substi-

tuents, different conformations and pH-depending p-elec-

tron densities, further enable an enhanced interaction

spectrum and can additionally improve the specificity of the

host–guest interaction. This yields a broader spectrum of

possible interactions compared to alkyl phases.

To select suitable columns for new separation problems,

it is beneficial to describe the retention characteristics and

the selectivity of each column with suitable column tests

and parameters. Therefore, the characteristics and proper-

ties of the novel commercially available calixarene- and

resorcinarene-bonded stationary phases (Caltrexs) have

been intensively examined in the recent years [20–23].

Unfortunately, there is no universally accepted chromato-

graphic test to choose an appropriate packing material for a

particular separation problem until now [24].

Moreover, established column-tests for alkyl-bonded

phases are inappropriate to cover all aspects of calixarene-

bonded phases [22]. To overcome this, mathematical models

can be used to numerically describe retention and selectivity

characteristics. Numerous work have been done in that field

and mathematical models relating the retention factor to

properties of the column, the solute and the mobile phase

were developed [25–32]. The retention has been related to

physicochemical parameters or, in more empiric ways, to

parameters derived from retention data.

In a recent article, we reported the use of a modified

version of the multiple-term Dolan–Snyder [33] equation

and compared the properties of calixarene-, resorcinarene-

and alkyl-bonded phases at different pH values. Underlying

retention mechanisms have been estimated.

Here, we report the comparison of these stationary phases

at different concentrations of organic modifier methanol with

respect to retention and selectivity mechanisms.

2 Theory

As described previously [33] a HPLC-specific system,

introduced by Dolan–Snyder and co-workers [31, 34–36],]

Christian SchneiderThomas Jira

Institute of Pharmacy,Pharmaceutical/MedicinalChemistry, Ernst-Moritz-Arndt-University Greifswald,Greifswald, Germany

Received July 6, 2010Revised July 6, 2010Accepted July 22, 2010

Correspondence: Professor Thomas Jira, Ernst-Moritz-Arndt-University Greifswald, Institute of Pharmacy, Pharmaceutical/Medicinal Chemistry, F.-L.-Jahn-Str. 17, D-17487 Greifswald,GermanyE-mail: [email protected]: 149-3834/864843

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com

J. Sep. Sci. 2010, 33, 2943–2955 2943

was adapted for the use with calixarene-bonded columns.

An additional term has been added to the original equation

and this resulted in

log a ¼ log k0 � log k0ref

¼ Z0H 1 s0rSr 1 s0f Sf 1 b0A 1 a0B 1 k0C ð1Þ

Here, Z0H reflects the hydrophobic interactions, s0rSr is

related to rigid steric interactions and s0rSr to flexible steric

interactions. k0C represents ionic interactions and hydrogen

bonding is displayed by a0B for interactions between a donor

solute and the stationary phase as well as by b0A for

interactions between acceptor solutes and the stationary

phase. Capital letters represent contributions of the

stationary phases/the chromatographic system, whereas

Greek letters represent for contributions of the solutes.

Additionally, the retention of a reference solute log k0ref was

originally included. As a result, it is not necessary to include

the phase volume or other parameters reflecting differences

between the examined columns. Yet, the reference

substance should not exhibit specific interactions for

comparisons of different chromatographic systems. It

should be exclusively hydrophobic. Otherwise specificities

of the columns could be biased.

However, some more changes had to be made to adopt

the equation to the requirements of calixarene phases: (i) to

exclude p–p interactions and forming of inclusion

complexes of the reference solute as far as possible, trans-decaline was chosen instead of ethylbenzene. (ii) The point

of origin has been included in every log a versus log acorrelation and the limit of the standard error has been

widened to 0.02 as the critical criterion to account for the

greater variability of log a values on the more differing

columns. (iii) The steric parameter has been split into a

rigid and a flexible steric parameter since this better reflects

the properties of the bonded calixarenes.

For the upcoming interpretation of the parameters, it

must be noted that this is a relative and logarithmic

system.

Thus, the zero point for the calculated parameters is

only determined by the reference solutes, it does not imply

an absence of interaction. Consequently, negative values

don’t stand for negative influences on retention, but just a

lower influence compared to the retention of the reference.

Furthermore, column parameters cannot be arbitrarily

compared without further considerations. Their influence

on retention or selectivity results not until the product with

the respective solute parameter is calculated. Since solute

parameters will be different from solute to solute and from

interaction to interaction, an equal column parameter at

another interaction can have differing influence. This is also

true for the same interaction at different methanol

concentrations. Same solute parameters cannot be

presumed here either and must be considered in the

calculation. For different comparisons it follows:

(i) Same parameters at the same methanol concentration

(e.g. which stationary phase is more hydrophobic at

50% methanol?) can be compared without further

calculation. On each stationary phase, the column

parameters would have to be multiplied with the same

solute parameters.

(ii) Same parameters at different concentrations of metha-

nol need further considerations. Solute parameters

must be taken into consideration. They have been

individually calculated at every methanol concentra-

tion. It can be differentiated between the influence on

retention and on selectivity, related to the interpreta-

tion of Wilson et al. [31]. The influence of a column

parameter on retention (Hret, Srety) is the product of

the column parameter and its respective average solute

parameter. In case of hydrophobic interaction, addi-

tionally the logarithmic retention factor of trans-deca-

line must be added because it is per definition purely

hydrophobic:

Hret ¼ Z0avg �H1 ln k0tdc ð2Þ

For calculation of the influence on selectivity (Hsel,

Csely) the solute parameters on every column are sorted

according to their retention times on each individual

column. Now selectivity of two peaks is the difference of the

logarithmic retention factors a ¼ ln k1 ¼ � ln k2. With Eq.

(1) this results in:

a ¼ ðln kref 1 Z02H 1 s0r2Sr 1 � � �Þ� ðln kref 1 Z01H 1 s0r1Sr 1 � � �Þ ð3Þ

Consequently, the contribution of a single interaction to

selectivity is calculated from the difference of the solute

parameters multiplied with the column parameter:

a ¼ ðZ02 � Z01ÞH1ðs0r2 � s0r2ÞSr1 � � � ð4Þ

For the contribution of a column toward an average solute,

the average value of the differences of all examined n solutes

i in the test-system can be used. Yet, the absolute values

must be used since a negative difference can likewise yield

selectivity as a positive difference does. Hence, positive and

negative differences of different pairs of peaks must not

compensate each other:

Hsel ¼Xn

i¼1

ðjZ0i11 � Z0ijÞ �H ð5Þ

(iii) Different parameters at the same methanol concentra-

tions should be handled as described under (ii). Again

equal solute parameters cannot be presumed.

3 Materials and methods

3.1 Chemicals and analytes

Thirty-five different solutes have been used in the study. The

solutes were selected to support a wide range of interactions.

Benzene, toluene, phenol and pentanol were purchased

from Riedel-de-Haen (Seelze, Germany). Ethylbenzene and

anthracene were obtained from Berlin-Chemie (Berlin,

J. Sep. Sci. 2010, 33, 2943–29552944 C. Schneider and T. Jira


Germany). Propylbenzene, ephedrine and N,N-dimethyl-

acetamide were from Fluka (Neu-Ulm, Germany). o-, m- and

p-cresol, triphenylene and phenanthrene were obtained

from Acros Organics (NJ, USA). Naphthalene, 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

Sigma Chemical (St. Louis, MO, USA). Diclofenac was from

3M Medica Pharma (Borken, Germany). Naproxen and

salicylic acid were obtained from Fagron (Barsbuttel,

Germany). Amitriptyline hydrochloride was obtained from

Salutas Pharma (Barleben, Germany). Promethazine

hydrochloride, promazine hydrochloride, chlorpromazine

hydrochloride and nortriptyline hydrochloride were

obtained from Lundbeck (Kopenhagen, Denmark). Predni-

solone and hydrocortisone were from Schering (Berlin,

Germany). All solutes were of the highest available analy-

tical grade.

HPLC gradient grade methanol was purchased from

Merck or from Acros Organics. Water was obtained by bi-

distillation.

3.2 Instruments

The data have been collected on two HP 1090 series II

chromatographs (Hewlett Packard, Waldbronn, Germany)

equipped with diode array detectors.

3.3 Columns

The study included seven different calixarene-bonded

phases (Caltrexs BI – p-tert-butyl-calix[4]arene, Caltrexs

BII – p-tert-butyl-calix[6]arene, Caltrexs BIII – p-tert-butyl-

calix [8]arene; Caltrexs AI – calix[4]arene; Caltrexs AII –

calix[6]arene; Caltrexs AIII – calix[8]arene, Caltrexs Science –

calix[4]arene and p-tert-butyl-calix[4]arene in a 1:1 ratio),

a resorcinarene-bonded phase (Caltrexs Resorcinarene

(RES)) and three alkyl-bonded phases (two Caltrexs

Kromasils C18 (C18) (KRM); a LiChrosphers 100 RP-18

(LIC)). The Caltrexscolumns were all kindly supplied by

Syntrex GbR (Greifswald, Germany). The ligands were

immobilized via hydrophobic spacers on endcapped silica

(Kromasils Si 100, 5 mm, specific surface area/BET:

300 m2/g, manufacturer: EKA Chemicals (Bohus, Sweden).

All columns had particle diameters of 5 mm and

dimensions of 125� 4 mm.

3.4 Chromatography

In all experiments, the mobile phase consisted of mixtures

of methanol/water, pH 3. The pH-value was adjusted with

phosphoric acid prior to mixing. Mixing was performed on-

line after degassing the solvents ultrasonically. The

temperature was thermostated to 401C in all experiments

and elution was carried out at a flow-rate of 1 mL/min

isocratically. Column hold-up times were determined using

a linearization procedure for homologous series [37]

(n-alcohols). Additionally, the hold-up time of the chroma-

tograph was determined by injecting pure methanol without

a column installed. This time has been subtracted from all

retention data.

Analyses were done at six different combinations of

mobile phases methanol/water, pH 3 v/v: 40:60; 50:50;

60:40; 70:30; 90:10 and 98:02. At lower methanol concen-

trations, retention times exceeded reasonable values.

4 Results and discussion

In comparison to previous analysis [33] of the properties

of calixarene-bonded phases with Eq. (1), here we only

used mobile phases at pH 3.0. Hence, more acidic solutes

can be used, and were found, as references for proton-

donators.

Correlations of Dlog a values according to Wilson et al.[31] led to benzoic acid, naproxen and diclofenac as a group

of similar interacting solutes which obviously defines the

interaction of proton-donators with the stationary phases.

Concerning the other interactions no different, determining

solutes were observed. The hydrophobic interaction is

represented by the ‘‘ideal’’ analytes trans-decaline, benzene,

toluene and ethylbenzene, the rigid steric interaction by

naphthalene, anthracene and triphenylene, the flexible

steric interaction by biphenyl and o-terphenyl, the ionic

interaction by propranolol, promethazine and amitriptyline

and finally solely N,N-dimethylacetamide was found as

representing proton acceptor.

Additionally the calculation of intermediate Zavg values

could be done here over all used columns. This was carried

out only over the mainly hydrophobic alkyl-columns at pH 7

because of highly diverse interactions. Hence, the newly

calculated parameters presented here should not be directly

compared to the previously reported parameters.

4.1 Comparison regarding individual parameters

Distinct differences between the stationary phases were

found concerning the hydrophobic properties. As expected

alkyl phases are strongly hydrophobic. Above methanol

concentrations of 50–60% v/v, they are more hydrophobic

than any other phase. Hydrophobic interactions are

dominating here.

Only at 40 and 50%, Caltrexs BI and BII are more

hydrophobic (Table 1).

On calixarene phases, additional interactions are more

probable and hence alkyl phases receive higher H. Between

substituted and non-substituted calixarenes, the tert-butyl

phases are more hydrophobic. The substitution yields

J. Sep. Sci. 2010, 33, 2943–2955 Liquid Chromatography 2945


more ‘‘alkyl-like’’ stationary phases. Thus columns can

be clearly classified concerning H at least at higher

modifier concentrations. Hydrophobic character decreases

from alkyl over tert-butyl to unsubstituted calixarene phases.

However, at lower methanol concentrations differences

between the phases diminish and Caltrexs B phases

become slightly more hydrophobic than alkyl phases.

Generally, characteristic differences between the stationary

phases decrease with the methanol concentration of the

mobile phase (Fig. 1).

This results from widely stable H on alkyl phases, while

parameters increase on calixarene- and the resorcinarene

phase. However, only the ratios of H at different methanol

concentrations can be directly compared (see above), but not

the absolute values. Therefore, the influence on retention

and selectivity has been calculated (Table 2).

Now it is obvious that the hydrophobic influence on

retention decreases with increasing methanol concentration

on all stationary phases, although constant H on alkyl

column had suggested a constant influence of the hydro-

phobic interaction. Unfortunately, the terms ‘‘hydrophobic

interaction’’ and ‘‘hydrophobicity’’ are not always clearly

distinguished in HPLC.

On a thermodynamic basis, ‘‘hydrophobicity’’ can be

seen as the transfer of a solute from the liquid phase or a

solution in a non-polar medium into water. This transport

comprises the breaking of bindings between solute and non-

polar solvent, closing of the cavity in the solvent, creation of

a cavity in water, formation of new bonds between solute

and water and the ordering of water molecules around the

solute [38]. With the stationary phase as non-polar solvent

and the mobile phase as polar phase (water), the hydro-

Table 1. Column parameters of the stationary phases at different methanol concentrations in the mobile phase

C18 AI AII AIII BI BII BIII Sci Res LiC Krm

H 40a) 1.00 0.88 0.90 0.83 1.03 1.05 0.91 0.97 0.89 0.98 1.00

50 1.01 0.88 0.86 0.79 0.99 1.01 0.87 0.97 0.85 0.96 1.01

60 0.97 0.80 0.79 0.68 0.92 0.89 0.82 0.87 0.75 0.96 1.02

70 0.99 0.72 0.75 0.60 0.93 0.90 0.76 0.83 0.70 1.00 1.04

90 1.02 0.50 0.52 0.35 0.78 0.63 0.47 0.55 0.45 0.89 0.90

98 1.04 0.49 0.38 0.23 0.79 0.55 0.36 0.48 0.34 0.97 1.04

Sr 40 �0.28 0.18 0.32 0.35 �0.20 �0.06 �0.10 �0.08 0.33 �0.24 �0.35

50 �0.40 0.07 0.37 0.36 �0.13 �0.03 �0.05 �0.15 0.42 �0.25 �0.44

60 �0.49 0.04 0.39 0.39 �0.13 0.04 �0.07 �0.08 0.43 �0.40 �0.54

70 �0.50 0.14 0.35 0.36 �0.13 0.00 �0.11 �0.07 0.42 �0.44 �0.58

90 �0.80 0.12 0.28 0.30 �0.25 �0.09 �0.06 �0.06 0.39 �0.47 �0.60

98 �0.91 0.02 0.27 0.34 �0.48 �0.17 �0.02 �0.06 0.47 �0.76 �0.96

Sf 40 �0.38 0.15 0.18 0.34 0.09 0.24 0.23 0.10 �0.09 �0.51 �0.46

50 �0.51 0.12 0.34 0.29 0.20 �0.28 0.18 0.09 �0.10 �0.47 �0.54

60 �0.63 0.10 0.36 0.35 0.14 0.26 0.15 0.10 �0.01 �0.53 �0.65

70 �0.63 0.15 0.30 0.30 0.18 0.25 0.13 0.08 0.02 �0.56 �0.65

90 �0.79 0.12 0.26 0.24 0.05 0.11 0.10 0.09 �0.01 �0.54 �0.61

98 �0.79 0.13 0.24 0.24 �0.09 0.04 0.07 0.01 0.02 �0.71 ��0.80

C 40 �0.41 0.94 0.92 1.15 �0.87 �0.68 �1.03 0.09 2.58 1.54 �0.59

50 �0.38 0.92 0.94 1.26 �0.82 �0.83 �1.22 0.24 3.14 1.87 �0.58

60 �0.35 0.99 1.07 1.31 �0.91 �0.95 �1.91 0.41 3.59 2.97 �0.31

70 �0.59 0.80 0.97 1.21 �1.94 �1.83 �4.13 0.23 3.61 3.53 �0.71

90 �1.58 0.42 0.59 0.97 �4.58 �3.21 �5.49 �0.28 3.84 3.39 �0.90

98 0.05 �0.27 0.78 0.98 �1.86 �2.04 �1.50 �0.52 4.09 4.18 0.31

B 40 �0.64 0.35 0.43 0.35 0.04 0.19 0.15 0.10 �0.04 �0.48 �0.62

50 �0.49 0.31 0.45 0.37 0.06 0.14 0.19 0.12 �0.24 �0.58 �0.53

60 �0.55 0.27 0.41 0.34 0.08 0.12 0.18 0.13 �0.20 �0.55 �0.55

70 �0.63 0.19 0.42 0.29 0.10 0.13 0.17 0.13 �0.14 �0.54 �0.55

90 �0.69 0.19 0.39 0.21 0.12 0.14 0.13 0.11 �0.11 �0.71 �0.71

98 �0.68 0.33 0.30 0.22 �0.11 0.10 0.07 0.12 �0.07 �1.14 �0.99

A 40 �0.80 0.29 0.12 0.38 �0.25 �0.09 0.16 0.06 1.10 �0.33 �0.78

50 �0.83 0.48 0.13 0.35 �0.26 �0.18 0.05 0.22 1.11 �0.65 �0.92

60 �1.04 0.44 0.17 0.39 �0.40 �0.39 0.50 0.18 1.04 �0.51 �1.14

70 �1.02 0.24 0.18 0.36 �0.37 �0.08 0.22 0.14 0.98 �0.42 �1.03

90 �1.26 0.29 0.26 0.29 �0.59 �0.10 0.03 0.11 1.02 �0.40 �1.49

98 �2.59 0.45 �0.07 �0.01 �0.83 �0.33 �0.14 �0.05 0.95 0.60 �3.64

a) Methanol concentration in the mobile phase in percent (v/v).



phobic interaction comprises influences of the mobile and

the stationary phase in HPLC.

The influence of the mobile phase can be described by

the ‘‘hydrophobic effect’’ of water. It means the low solu-

bility of apolar solutes in water caused by a large loss of

entropy during solvation (rigid ordering of water molecules

around the solute) at room-temperature described as

‘‘iceberg-effect’’ or ‘‘iceberg-model’’ by Frank and Evans

[39]. Release of the water molecules results in a gain of

entropy and therefore the mobile phase promotes retention.

On the other hand, the transfer of a methylene group

from organic solvent to water is not only characterized by a

loss of entropy but also by a positive change of enthalpy at

room temperature [40]. Since, in contrast, the transfer from

gas phase to water is realized under a negative change of

enthalpy, the positive change of enthalpy at the transfer

from organic solvent to water must be connected to the

breaking of bonds between solute and non-polar solvent.

Hence, a considerable part of the unfavorable free energy

change results from that. Thus, their formation at contra-

riwise transfers (water to organic solvent) yields a loss of

enthalpy and consequently the organic solvent, i.e. the

stationary phase, is also involved in the retention process. It

promotes retention.

In conclusion for the ‘‘hydrophobic interaction’’ nega-

tively, repulsive, entropy-based effects of the mobile phase

as well as attracting, enthalpy-based effects of the stationary

phase should be considered.

Now a large part of the hydrophobic influence on

retention Hret is probably related to the mobile phase.

Especially in medium modifier regions, conformational

changes of the conformations of bonded ligands or differ-

ences of adsorption of mobile phase components will not

change the interaction with the stationary phase that much.

In contrast, the repelling hydrophobic effect is large at a

composition of 40/60% methanol/water (v/v), for example.

It decreases with increase of methanol content since the

hydrophobic effect is mainly connected with water and its

small molecular volume. Hence, hydrophobic influence on

retention becomes lower at high methanol concentrations

(Table 2). This may also explain why differences of H at

higher methanol concentrations are more pronounced:

With higher amounts of water the hydrophobic effect is

large. Hence, the mobile phase’s influence is large and

differences between stationary phases are less important. If,

in contrast, the hydrophobic effect is low, interaction with

the stationary phase (van der-Waals bonds with lipophilic

ligands) will become more important. Hence, differences

between stationary phases become more distinct.

The influence on selectivity Hsel does not show a

generally decreasing tendency (Table 2). Obviously, the

decreasing influence on retention is not necessarily

connected with a decreasing influence on selectivity. This

supports the assumption concerning the hydrophobic effect.

It will have no big impact on selectivity. That is why

mainly a large influence on retention is supposed for the

hydrophobic interaction, but only a smaller influence for

selectivity [41, 42]. With a decreasing hydrophobic effect at

increasing methanol concentrations, its influence on

retention diminishes, but hydrophobic selectivity is main-

tained. Probably, it results primarily from interactions with

the stationary phase. They are still ongoing at higher

methanol concentrations.

In conclusion, retention is mainly affected by the

hydrophobic effect and selectivity mainly from interactions

with the stationary phases.

The steric column parameters considerably reflect the

strong steric character of stationary phases with bonded

supramolecules (Table 1). All calixarene and the resorci-

narene phase show higher Sr and Sf than alkyl phases at the

individual modifier concentrations. This reflects the possi-

bilities for complexations, which are sterically influenced by

properties of host and guest molecules. Differences between

different calixarene phases result from substitution [33].

However, the resorcinarene’s Sr value is even higher

than for Caltrexs A phases, while its flexible parameter is

less than for the phases with bonded macromolecules. It

shows extraordinarily high affinity to large, rigid solutes.

Differences between unsubstituted calixarenes and resoci-

narenes are the hydroxyl groups at the upper rim and the

different method of bonding to the silica gel for resorci-

narenes. Here, longer spacers are used (C11) and they are

attached to the bridging methyl group instead of the

benzene rings. Both types of steric interactions could benefit

from the latter because of enhanced possibilities to move the

benzene rings. Yet, Sf is actually not increased and therefore

non-planarity seems disadvantageous. Hence, the increased

chain length of the spacers could be causative. It may

facilitate interactions with large hydrophobic solutes, similar

to alkyl phases, if solutes penetrate deep enough into the

bonded layer. For that, steric characteristics (planarity/non-

planarity) surely are important.

However, the characteristic differences of the ratios of

the parameters at different methanol concentrations, which

were found for H, do not appear here. They are similar at

medium and high volume fraction of methanol. Also, the

Figure 1. Hydrophobic column parameters H at differentmethanol concentrations. & at 40%, � at 90% methanol in themobile phase (v/v).



steric influences on retention change only less over the

different methanol concentrations (Fig. 2A and B).

Alkyl phases and phases with unsubstituted calixarenes

namely seem to show opposite behavior, but the changes are

far less than they are for the hydrophobic interaction.

Therefore, steric influence on overall retention is relatively

small at medium modifier concentrations, but it gains in

importance (because hydrophobic importance decreases)

with the elution strength of the mobile phase (Table 2).

Characteristics of Caltrexs B phases lie between those of

alkyl and Caltrexs A phases.

Influences on selectivity are more variable, at least on

alkyl phases (Fig. 2C and D). Where the influence on

retention was constant or even slightly increasing, the

influence on selectivity distinctly decreases. Thus, the

slightly increasing interactions on alkyl phases mostly are

not selective. On Caltrexs A phases, again opposite behavior

was found. Here, Srelr and Srel

f increase with increasing

methanol concentration, influences on retention slightly

decreased. Thus, steric interactions yield higher selectivity,

although its influence on retention decreases for unsub-

stituted calixarenes. This effect is more pronounced for

larger calixarenes, which is reasonable since larger calixar-

enes provide more possibilities for interactions with larger,

sterically active solutes. Again, characteristics of Caltrexs B

phases lie between those of alkyl- and Caltrexs A phases.

These dependencies show that higher support of

retention does not need to induce higher selectivity as well

and the other way round that higher selectivity may be

supported although retention is less influenced.

As already reported, the stationary phases can be clearly

differentiated by their ionic properties [23, 33]. Therefore,

Table 2. Hydrophobic and steric influences on retention and selectivity


Retention

Hret 40a) 3.66 2.87 3.36 2.98 3.80 4.00 3.11 3.54 3.15 3.55 3.71

50 2.77 1.96 2.40 2.08 2.75 2.99 2.13 2.50 2.26 2.64 2.82

60 1.90 1.10 1.50 1.25 1.76 1.98 1.20 1.58 1.41 1.70 1.86

70 1.04 0.36 0.72 0.57 0.85 1.10 0.47 0.77 0.70 0.90 1.08

90 �0.29 �0.95 �0.47 �0.43 �0.74 �0.32 �0.74 �0.55 �0.42 �0.44 �0.23

98 �0.86 �1.59 �0.91 �0.78 �1.51 �0.97 �1.46 �1.17 �0.87 �1.15 �0.97

S retr 40 0.02 �0.01 �0.02 �0.03 0.01 0.00 0.01 0.01 �0.02 0.02 0.03

50 �0.03 0.00 0.03 0.03 �0.01 0.00 0.00 �0.01 0.03 �0.02 �0.03

60 �0.16 0.01 0.12 0.13 �0.04 0.01 �0.02 �0.02 0.14 �0.13 �0.17

70 �0.14 0.04 0.10 0.10 �0.04 0.00 �0.03 �0.02 0.12 �0.13 �0.17

90 �0.16 0.02 0.06 0.06 �0.05 �0.02 �0.01 �0.01 0.08 �0.09 �0.12

98 0.19 0.00 �0.05 �0.07 0.10 0.03 0.00 0.01 �0.10 0.15 0.19

S retf 40 �0.04 0.02 0.02 0.03 0.01 0.02 0.02 0.01 �0.01 �0.05 �0.05

50 �0.06 0.01 0.04 0.03 0.02 0.03 0.02 0.01 �0.01 �0.06 �0.07

60 0.10 �0.02 �0.06 �0.06 �0.02 �0.04 �0.02 �0.02 0.00 0.09 0.11

70 0.09 �0.02 �0.04 �0.04 �0.02 �0.04 �0.02 �0.01 0.00 0.08 0.09

90 0.02 0.00 �0.01 �0.01 0.00 0.00 0.00 0.00 0.00 0.02 0.02

98 �0.23 0.04 0.07 0.07 �0.03 0.01 0.02 0.00 0.01 �0.21 �0.23

Selectivity

Hsel 40 0.39 0.51 0.58 0.53 0.43 0.43 0.58 0.28 0.78 0.72 0.43

50 0.39 0.51 0.56 0.54 0.38 0.38 0.41 0.32 0.78 0.75 0.40

60 0.43 0.45 0.50 0.55 0.47 0.34 0.44 0.24 0.86 1.00 0.47

70 0.45 0.34 0.45 0.32 0.55 0.50 0.37 0.20 0.54 0.76 0.50

90 0.73 0.23 0.27 0.24 0.56 0.34 0.36 0.23 0.31 0.61 0.65

98 1.03 0.42 0.38 0.22 0.78 0.48 0.33 0.28 0.25 0.81 1.00

S selr 40 �0.21 0.13 0.24 0.26 �0.14 �0.04 �0.06 �0.05 0.27 �0.17 �0.27

50 �0.19 0.03 0.18 0.18 �0.06 �0.01 �0.03 �0.07 0.22 �0.11 �0.22

60 �0.27 0.02 0.23 0.26 �0.08 0.02 �0.05 �0.05 0.18 �0.19 �0.31

70 �0.35 0.11 0.26 0.25 �0.08 0.00 �0.07 �0.05 0.21 �0.28 �0.38

90 �0.79 0.12 0.29 0.30 �0.25 �0.08 �0.06 �0.06 0.38 �0.43 �0.66

98 �1.83 0.04 0.57 0.70 �0.97 �0.26 �0.03 �0.12 0.79 �1.32 �1.97

S self 40 �0.20 0.07 0.09 0.21 0.05 0.12 0.11 0.05 �0.05 �0.25 �0.25

50 �0.28 0.08 0.20 0.16 0.11 0.14 0.09 0.04 �0.06 �0.26 �0.32

60 �0.36 0.07 0.30 0.29 0.08 0.15 0.09 0.07 0.00 �0.33 �0.39

70 �0.55 0.15 0.31 0.26 0.13 0.21 0.10 0.07 0.01 �0.50 �0.57

90 �1.02 0.18 0.36 0.37 0.06 0.13 0.12 0.11 �0.01 �0.72 �0.87

98 �1.30 0.23 0.35 0.35 �0.15 0.04 0.11 0.01 0.04 �1.06 �1.38




Caltrexs B phases constantly show least C, followed by

Kromasils C18 phases, the Caltrexs Science, the Caltrexs

A phases and finally the extraordinarily ionic Caltrexs

Resorcinarene and the LiChrospher RP-18 (Table 1). The

influence of these properties on retention decreases up to

methanol concentrations of 90% v/v on most phases.

Increases were only found on the strongly ionic columns

(Fig. 3).

At very high elution strength, the behavior is not

completely uniform. Mainly ionic influences rise, especially

on both ionic phases consistent with former considerations

[23], but on some phases decreasing changes continue

(Table 3).

However, the occurrence of strengthened ionic inter-

actions is obvious. All in all, the ionic interaction also has only

minor influence on the whole retention at medium modifier

concentrations compared to Hret. This part continuously

increases with the modifier concentrations since Hret

decreases much faster. Obviously, this especially applies for

RES and LIC with their increasing ionic influences.

Unlike the above-mentioned interactions, the influence

on selectivity here changes according to the influence on

retention (Fig. 3). There seem to be no differences between

ionic retention and selectivity. Thus, if an ionic interaction

applies, it mostly is selective.

Polar interactions divide into interactions with hydro-

gen-bond donators and hydrogen-bond acceptors. The

interaction with acidic, proton-donating solutes that corre-

sponds to the basic column parameter B again is widely

stable over the methanol concentrations (Table 1). The

characteristic differences of the properties reported earlier at

60% methanol concentration [33] are also valid over the

Figure 3. Influence of the ioniccharacteristics on A) retentionand B) selectivity. & Kroma-sils C18, � Caltrexs Resorci-narene, m Caltrexs AI, ~Caltrexs BII.

Figure 2. Influenceof the steric charac-teristics on (A, B)retention and (C, D)selectivity. (A, C)Rigid steric interac-tion, (B, D) flexiblesteric interaction; &

Kromasils C18, �LiChrosphers RP-18,m Caltrexs AII, ~Caltrexs BIII.



whole range from 40 to 90%, probably up to 98%. Even if

their influence on retention or selectivity is calculated, the

methanol content is widely not decisive (Table 3). Only on

alkyl phases, both Bret and Bsel diminish when the water

content of the mobile phase is drastically lowered (Fig. 4).

This supports the earlier proposed connection of the

polar interaction with adsorbed mobile phase [23, 33]. Polar

solutes may interact with an adsorbed layer of mobile phase

molecules, especially with adsorbed water. The constitution

of such bonded layer should depend on the composition of

the mobile phase and also on the bonded ligands of the

respective stationary phase. Drastic decrease of the water

concentration will result in loss of adsorbed water and hence

in less influence of B. This effect may be especially

pronounced on phases with exclusively hydrophobic ligands

(alkyl chains) yielding decreasing basic characteristics.

Moreover, additionally possible polar interactions in the

calixarene cavities could further stabilize Bret and Bsel on

calixarene columns.

Like for the ionic interaction, the column’s basic influ-

ence on retention and selectivity changes widely propor-

tional. Thus, unlike for hydrophobicity, both probably result

from the same physicochemical effects.

The ratio of column’s acidic parameters A at different

methanol concentrations also does not change strongly.

Only on alkyl phases, distinct changes were observed;

nevertheless, they show lowest parameters (except LIC at

98% showing drastically increased A, which is probably

related to a parabolic change of ln k versus j (volume

fraction of methanol in the mobile phase) on this column

caused by a high amount of unbound silanols). Besides

interaction with protonated silanols, again interactions with

Table 3. Ionic and polar influences on retention and selectivity


Retention

Kret 40a) �0.09 0.20 0.20 0.25 �0.19 �0.15 �0.22 0.02 0.56 0.33 �0.13

50 �0.07 0.17 0.17 0.23 �0.15 �0.15 �0.23 0.05 0.58 0.35 �0.11

60 �0.05 0.13 0.14 0.17 �0.12 �0.12 �0.25 0.05 0.47 0.39 �0.04

70 �0.08 0.11 0.13 0.16 �0.26 �0.25 �0.55 0.03 0.48 0.47 �0.10

90 �0.24 0.06 0.09 0.15 �0.70 �0.49 �0.84 �0.04 0.59 0.52 �0.14

98 0.01 �0.07 0.20 0.25 �0.48 �0.53 �0.39 �0.13 1.05 1.08 0.08

Bret 40 �0.35 0.19 0.24 0.20 0.02 0.11 0.09 0.06 �0.02 �0.27 �0.34

50 �0.17 0.11 0.16 0.13 0.02 0.05 0.07 0.04 �0.08 �0.20 �0.18

60 �0.24 0.11 0.18 0.15 0.03 0.05 0.08 0.06 �0.09 �0.24 �0.24

70 �0.26 0.08 0.17 0.12 0.04 0.05 0.07 0.05 �0.06 �0.22 �0.23

90 �0.28 0.08 0.16 0.08 0.05 0.06 0.05 0.04 �0.05 �0.29 �0.28

98 �0.51 0.25 0.22 0.17 �0.08 0.08 0.06 0.09 �0.06 �0.86 �0.74

Aret 40 0.03 �0.01 0.00 �0.01 0.01 0.00 �0.01 0.00 �0.04 0.01 0.03

50 �0.06 0.03 0.01 0.02 �0.02 �0.01 0.00 0.02 0.08 �0.05 �0.06

60 �0.14 0.06 0.02 0.05 �0.05 �0.05 0.07 0.02 0.14 �0.07 �0.16

70 �0.16 0.04 0.03 0.06 �0.06 �0.01 0.03 0.02 0.15 �0.07 �0.16

90 �0.22 0.05 0.05 0.05 �0.10 �0.02 0.01 0.02 0.18 �0.07 �0.26

98 0.06 �0.01 0.00 0.00 0.02 0.01 0.00 0.00 �0.02 �0.01 0.08

Selectivity

Ksel 40 �0.12 0.42 0.41 0.44 �0.31 �0.24 �0.43 0.03 0.76 0.52 �0.21

50 �0.09 0.33 0.29 0.45 �0.25 �0.26 �0.30 0.09 0.74 0.53 �0.17

60 �0.09 0.25 0.24 0.40 �0.24 �0.18 �0.36 0.11 0.74 0.55 �0.08

70 �0.10 0.20 0.25 0.21 �0.36 �0.35 �0.21 0.06 0.36 0.35 �0.12

90 �0.40 0.10 0.18 0.24 �0.60 �0.41 �0.63 �0.08 0.57 0.40 �0.24

98 0.01 �0.15 0.32 0.45 �0.96 �0.70 �0.39 �0.25 1.03 0.98 0.09

Bsel 40 �0.41 0.23 0.34 0.28 0.02 0.12 0.10 0.08 �0.03 �0.30 �0.47

50 �0.28 0.20 0.28 0.18 0.04 0.09 0.11 0.07 �0.12 �0.25 �0.30

60 �0.29 0.14 0.30 0.23 0.04 0.06 0.13 0.08 �0.11 �0.30 �0.30

70 �0.43 0.14 0.36 0.21 0.06 0.09 0.12 0.10 �0.09 �0.40 �0.42

90 �0.60 0.24 0.37 0.24 0.12 0.14 0.15 0.10 �0.09 �0.62 �0.66

98 ��0.57 0.43 0.41 0.32 �0.13 0.12 0.06 0.14 �0.08 �1.09 �1.10

Asel 40 �0.24 0.08 0.04 0.11 �0.08 �0.03 0.05 0.02 0.37 �0.10 �0.23

50 �0.16 0.09 0.03 0.07 �0.05 �0.04 0.01 0.04 0.23 �0.11 �0.16

60 �0.30 0.12 0.05 0.11 �0.11 �0.10 0.14 0.05 0.27 �0.11 �0.33

70 �0.22 0.07 0.05 0.08 �0.08 �0.02 0.04 0.03 0.21 �0.08 �0.23

90 �0.30 0.10 0.08 0.10 �0.15 �0.02 0.01 0.03 0.26 �0.10 �0.36

98 �0.40 0.09 �0.01 0.00 �0.18 �0.05 �0.02 �0.01 0.15 0.11 �0.61




adsorbed mobile phase can be the origin of this interaction

as well as interactions in the cavities, as reported by Bauer

and Gutsche [43, 44].

The influence on retention Aret does not change

strongly, like Bret (Fig. 5).

Yet, at very high methanol concentrations, values on the

alkyl phases increase. This is opposite to basic influence.

This means that both effects cannot be solely influenced by

adsorbed water. At least one more source of column’s

acidity must be differentially influenced with increasing

methanol concentrations between alkyl and calixarene

columns. Perhaps, steric hindrance effects while diffusion

to the silica gel and the protonated silanols account here.

At least it shows that there is more than one source of

interaction involved. The dependence of the selectivity

clearly shows the different properties of the columns

toward N,N-dimethylacetamide, but the respective

influences are not decisively dependent on methanol

concentration.

4.2 Comparison of the interaction spectra at indivi-

dual methanol concentrations

The comparison of the parts each of the characteristics of

the columns has on retention and selectivity may be further

useful for selecting suitable stationary phases for different

separation problems. It should be possible to choose a

highly retentive and more important a highly selective

column for specific kinds of analytes (hydrophobic, ionic,

etc.).

Between 40 and 60% v/v methanol concentration in the

mobile phase, ratios of influences do not change signifi-

cantly. As already noted, the hydrophobic retention is by far

most determining for retention at these medium modifier

concentrations (Fig. 6).

This is true for all examined stationary phases and

therefore differences between them become less consider-

able. Additional interactions in fact are slightly higher on

calixarene-bonded phases even here, but this does not have

big impact on global retention. Only the ionic interaction

can considerably influence retention, mainly on RES and

LIC and obviously only for protonable solutes. The differ-

ences between Caltrexs A phases (more ionic) and Caltrexs

B phases (barely ionic) are also noticeable. Polar and steric

interactions are hardly of influence on retention.

Differences are more distinct concerning selectivity. On

C18, the hydrophobic interaction, and thus their hydro-

phobic properties, is nearly exclusively determining for

selectivity. Ionic interactions add to them on the strongly

ionic alkyl phase LIC. Besides that, it behaves like other

alkyl phases. Generally, the influences on selectivity on

alkyl-bonded phases are ordered as followed:

HSel444CSelt4ASel � SSelr 4BSel � SSel

f :

On the Caltrexs Resorcinarene, the ionic influence is even

higher. For ‘‘average’’ solutes, it becomes nearly as impor-

tant as the hydrophobic. Thus, it is clearly defining for ionic

solutes, but for non-ionic solutes the hydrophobic inter-

action still will be. Moreover, this in fact describes selectiv-

ity, but not the impact on resolution. Indeed, a strong tailing

comes together with the high retention of protonated bases

Figure 5. Influence of the hydrogen-donating characteristics on(A) retention and (B) selectivity. & Kromasils C18, � Caltrexs

Resorcinarene, m Caltrexs AI, ~ Caltrexs BII, . LiChrosphers

RP-18.

Figure 4. Influence of thehydrogen-accepting character-istics on (A) retention and (B)selectivity. & Kromasils C18,� Caltrexs Resorcinarene, m

Caltrexs AII, ~ Caltrexs BII, .

LiChrosphers RP-18.



on ionic phases. Therefore, a good resolution is not neces-

sarily connected. Moreover, another two unexpected prop-

erties of the RES are visible. First, it generates selectivity

from rigid steric interaction to a remarkable extent. This will

probably result from longer spacers in contrast to calixar-

enes. Second, it is the only one of the examined phases

which remarkably gains selectivity from A. Obviously, the

hydroxyl groups at the upper rim are potent locations to

generate selectivity of non-protonated amines. For an aver-

age solute, the importance of the interactions on Caltrexs

Resorcinarene is

HSel � CSelt4ASel � SSelr 4SSel

f 4BSel:

Increased selectivity of protonated bases is further to achieve

on Caltrexs A phases. In contrast, they do not play a role on

tert-butyl-substituted Caltrexs B phases. Ionic properties are

effectively suppressed there.

All in all, Caltrexs A phases support the broadest

spectrum of interactions. Their steric and hydrogen-bond-

ing acceptor properties are also pronounced:

HSel4CSelt � BSel � SSelf 4SSel

r 4ASel:

Caltrexs B phases can only offer similar values for Sself and

partly for Bsel. Generally they become more ‘‘alkyl-like’’

caused by the substitution with tert-butyl groups, concern-

ing retention and selectivity. Here applies

HSel4CSelf � BSel4SSel

r � ASel4CSelt:

Moreover, it can be seen that the mixed layer of the Science

phase with substituted and non-substituted calixarenes

yields medium values.

The hydrophobic interaction still has dominating

influence on retention with 70% methanol in the mobile

phase (Fig. 7).

However, additional interactions rise in importance,

especially on Caltrexs A phases and the Resorcinarene. In

particular, ionic influence increased distinctly. Yet for

Caltrexs B and the alkyl phases (except the ionic interaction

on LIC), the dominance of Hret is unbroken.

On alkyl phases, this also applies for selectivity (Fig. 7).

It mainly results from hydrophobic interactions. In relation

to medium modifier concentrations, Hsel even increases:

HSeldCSelt4ASel4SSel

r � BSel � SSelf :

Again, additional interactions are far more important on

stationary phases covered with non-substituted calixarenes.

Here, Hsel decreases in relation to other interactions. A

fairly balanced spectrum is supported, even if the hydro-

phobic interaction still is highest. On these phases, the

difference to alkyl phases becomes particularly obvious.

Selectivity can result substantially stronger from steric, ionic

and polar interactions:

HSel � BSel � SSelf � SSel

r � CSelt4ASel:

This also applies for more ‘‘alkyl-like’’ B-phases, but to a less

extent. Only Sself and Bsel, barely Ssel

r or Asel and hardly ever

Csel have a share in selectivity:

HSel4SSelf 4BSel4SSel

r � ASel4CSelt:

Moreover, it attracts attention that rigid steric and polar

acidic properties of the RES did rise in relation to Hsel, but

Csel decreased. Hence, the spectra of interactions change

Figure 7. Comparison of the influences of the different interac-tions on retention and on selectivity at 70% methanol concen-tration. Influences of interactions displayed for every stationaryphase from left to right: hydrophobic interaction, rigid stericinteraction, flexible steric interaction, ionic interaction, interac-tion with proton donators, interaction with proton acceptors.

Figure 6. Comparison of the influences of the different interac-tions on retention and on selectivity at 50% methanol concen-tration. Influences of interactions displayed for every stationaryphase from left to right: hydrophobic interaction, rigid stericinteraction, flexible steric interaction, ionic interaction, interac-tion with proton donators, interaction with proton acceptors.



differentially for different stationary phases as well as for

different interactions:

HSel4CSelt4ASel � SSelr 4SSel

f � BSelt:

Generally, additional interactions become more important

on calixarene-bonded phases with increasing methanol

concentrations, while they become less important on alkyl

phases, except the ionic interaction. The latter does take

increased influence on retention but decreased on selectivity

in relation to hydrophobic influence on all stationary

phases. This once more shows that increased retention not

necessarily leads to increased selectivity.

Conditions particularly changed at methanol concen-

trations of 90% (Fig. 8).

The hydrophobic interaction is not the dominating

factor for retention any more. In contrast, it rather is of least

influence because of a weaker hydrophobic effect of the

mobile phase.

The other interactions are on alkyl phases

(SRetf 4SRet

r 4ARet � CRet � BRet � HRet) still weaker than

on calixarene phases (B-phases: BRet � SRetr � SRet

r �ARet � HRet4CRet; A-phases: BRet4CRet � SRet

r � ARet �SRet

f 44HRet). All of them, especially the interaction with

protonated, acidic solutes, increased in importance on the

latter phases. This trend continues at even higher j.

However, it was found neither on RES (CRet44ARet4SRetr �

SRetf � BRet44HRet) nor on alkyl phases. This confirms that

parameter B probably cannot solely be based on interactions

with adsorbed water. Perhaps, this is the only mechanism

on alkyl phases (this would explain the decreasing influence

at higher methanol concentrations) but on calixarene phases

likely interactions with the cavities are also involved.

On both strongly ionic columns, the respective inter-

action now is distinctly dominant. However, this naturally

only applies for protonable solutes. The disproportionally

high influences are based on the enormous retention times

of ionic solutes even at very high modifier concentrations

[23]. Protonated and neutral solutes therefore can be

successfully separated at high modifier concentrations on

these columns.

Differences between the stationary phases keep

increasing concerning selectivity. Hsel further increases in

relation to other influences on alkyl phases. As described,

the influence on selectivity does not necessarily decrease

with the influence on retention:

HSeldASel4CSelt4BSel4SSel

r 4SSelf :

However, hydrophobic influences on both criteria (selectivity

and retention) decrease on calixarene phases. This obviously

leads to raised importance of other interactions at the same

time. Nevertheless, the influence does not become small. Even

on Caltrexs A phases, where the spectra of interactions are

most diversified, Hsel keeps in the same range as the addi-

tional interactions. But for all that, the biggest part in selec-

tivity the steric and the hydrogen-accepting properties have.

BSel � Sself 4SSel

r � HSel4CSelt4ASel:

The RES again shows high involvement of ionic and polar

acidic properties, as at lower methanol concentrations.

However, their importance even further increased:

CSelt4SSelr � HSel � ASel4SSel

f � BSel:

Phases with tert-butyl groups still support mainly flexible

steric as well as interactions with protonated acids besides

the hydrophobic:

HSel4BSel � SSelf 4SSel

r � ASel4CSelt:

Further increase of j only results in few changes of the

described trends. The influence of the hydrophobic inter-

action further decreases on retention and particularly ionic

influence increases.

Concerning selectivity, the spectra of influences does not

change significantly on Caltrexs B phases. On Caltrexs A

phases and RES, influences of mainly supported interactions

further increase. However, this only holds true for the calix-

arenes of larger ring size, not for AI. On this column, and

similarly on BI, Hsel increases as on alkyl phases. Obviously,

there are also differences between different ring sizes. The

hydrophobic influence decreases the stronger, the larger

the cavities are. In contrast, as steric influences increase, the

larger the cavities, substituted or not, are. This applies not

only at high methanol concentrations.

5 Concluding remarks

With increasing methanol concentration of the mobile

phases, the influence of the hydrophobic interaction

Figure 8. Comparison of the influences of the different inter-actions on retention and on selectivity at 90% methanolconcentration. Influences of interactions displayed for everystationary phase from left to right: hydrophobic interaction, rigidsteric interaction, flexible steric interaction, ionic interaction,interaction with proton donators, interaction with protonacceptors.



on retention drastically decreases. This leads to higher

influences of additional interactions from about 90%

on, probably earlier on calixarene phases, later on alkyl

phases. Moreover, the importance of the ionic interaction

rises on strongly ionic columns in relation to other

interaction. Obviously, that only applies for protonable

solutes.

In contrast, selectivity is strongly influenced by hydro-

phobic properties of the stationary phases at all examined

methanol concentrations. This influence even increases

with j on alkyl columns; on other columns it is little

changed or decreases. This decrease leads to increased

importance of additional interactions.

The most balanced spectrum of interactions is suppor-

ted by Caltrexs A phases, probably because of unhindered

access to the cavity. This facilitates complex building and

generally partition into the bonded layer. Furthermore, the

Caltrexs Resorcinarene offers possibilities to account for

special characteristics (ionic or proton-acceptor properties)

of solutes.

We gratefully thank Syntrex GbR, Greifswald for providingthe Caltrexs-columns and the above-mentioned companies forthe kind supply of solutes.

The authors have declared no conflict of interest.

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