characterization of calixarene-bonded stationary phases

Research Article

Characterization of calixarene-bondedstationary phases

Calixarene-bonded stationary phases received growing interest in HPLC as stationary

phases with special retention characteristics and selectivity. The commercially available

unsubstituted and p-tert-butyl-substituted Caltrexs columns have been intensively studied

and characterized in our workgroup. They can be used as reversed phases, yet they

support additional interactions. Especially, their steric, polar and ionic properties differ

from conventional alkyl-bonded phases. However, also the hydrophobic interaction shows

differences since adsorption and partition interactions on or in a bonded layer of calix-

arenes are not similar to those of alkyl-bonded layers. The relative strength of the

hydrophobic properties of the stationary phases has been found depending on the

methanol concentration of the mobile phase. Generally, the dependencies of their inter-

action strengths on mobile-phase conditions, e.g. the change of the intensity of the

hydrogen-bonding abilities with decreasing methanol content, are not similar from phase

to phase either. This probably gives calixarene-bonded stationary phases enhanced suit-

ability for analyses at extreme compositions of the mobile phase. An overview about the

synthesis, retention and selectivity properties of Caltrexs columns is given here.

Keywords: Calixarene-bonded stationary phases (Caltrexs) / Column com-parison / Column selectivity / HPLC / Retention mechanismsDOI 10.1002/jssc.201000281

1 Introduction

Calixarenes, the cavity-shaped cyclic molecules, are the third

generation of supramolecules following cyclodextrines and

crown ethers and are used in GC, CE and HPLC as

stationary phases. They consist of phenol units linked viamethylene bridges. Calixarenes, as special, receptor-like

molecules, can form inclusion complexes [1–11] like the

other host supramolecules and support additional interac-

tions compared with conventional HPLC phases [2, 4,

12–20].

The resulting specific interactions can influence the

retention factors and improve the selectivity of the solutes.

Additionally, the variable possibilities of modifying the

calixarenes, e.g. a variable ring size, different substituents,

different conformations and pH-depending p-electron

densities, further enable an enhanced interaction spectrum

and can improve the specificity of the host–guest interaction

further with.

All these different possible characteristics make calix-

arenes a valuable tool for chromatographic tasks. In order to

select a suitable material for a specific application, the

description of their chromatographic properties is reason-

able.

Unfortunately, there is no universally accepted chro-

matographic test to choose an appropriate packing material

for a particular separation problem until now [21]. In

reversed-phase chromatography, many descriptors can give

certain information to estimate the chromatographic beha-

viour of the stationary phases, i.e. the type of the bonded

ligand and its bondage to the surface, the surface coverage,

the surface area and the support material are used to explain

the differing properties of the chromatographic materials

[22]. Nevertheless, the use of empirically based test mixtures

is often inevitable because phases behave differently than

expected by their chemical and physical parameters. Test

runs can provide information concerning the hydrophobic

(hydrophobic retention capacity, hydrophobic selectivity and

steric selectivity) and polar properties (silanol group activity,

polar selectivity, ion exchange selectivity and complexation

capacity) [23] of a column.

Moreover, the calculation of parameters describing the

characteristics of the stationary phases for the single inter-

actions via mathematical models can give deeper insight

into retention mechanisms [24–32]. Additionally, these

parameters provide a fast and easy possibility for comparing

different stationary phases.

Christian Schneider1

Ulf Menyes2

Thomas Jira1

1Institute of Pharmacy,Pharmaceutical/MedicinalChemistry, Ernst-Moritz-Arndt-University of Greifswald,Greifswald, Germany

2Syntrex GbR, Greifswald,Germany

Received April 23, 2010Revised June 1, 2010Accepted June 2, 2010

Abbreviation: DMCS, dimethylchlorosilane

Correspondence: Prof. Dr. Thomas Jira, Ernst-Moritz-Arndt-University Greifswald, Institute of Pharmacy, Pharmaceutical/Medicinal Chemistry, Friedrich-Ludwig-Jahn-Street 17, D-17487Greifswald, GermanyE-mail: [email protected]: 149-3834-864843

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

J. Sep. Sci. 2010, 33, 2930–29422930

Interest in calixarene- and resorcinarene-bonded

stationary phases in HPLC for the separation of positional

[1–3, 33–35] and geometric isomers [1, 36–39] and other

solutes of pharmaceutical interest [2, 40, 41] is growing.

Even, optical isomers were discriminated by specifically

modified chiral phases [42–44]. Some selectivities were due

to interactions between analytes and cavities formed by the

supramolecules. Hence, not only hydrophobic but also more

specific interactions are responsible for the higher selectivity

of particular analytes on these phases.

Besides analytical methods, the application field of

calixarenes also covers the medical and ecological sector as

well as preparative chemistry, for example, and is a rapidly

developing area of supramolecular chemistry.

Here, we give an overview about different commercially

available calixarene-bonded stationary phases (Caltrexs

columns). The synthesis of the calixarene-bonded silica gel

is presented as well as concluding results of comprehensive

chromatographic analysis of recent years.

2 Materials and methods

2.1 Syntheses of calixarene-modified silica (Caltrexs

HPLC phases)

2.1.1 Synthetic approach

For synthesis of Caltrexs HPLC separation materials, we

used the heterogeneous hydrosilylation strategy. For this

way, it is necessary to use calixarenes which are modified

with olefin-containing groups. We utilized calixarenes from

Syntrex GbR with linkers at the oxygen group via ether

function. In case of resorcinarenes, the olefin function is the

end group of the side chain of the bridged group between

the resorcine units. The hydroxyl groups are free, which

results in special behaviour of the resulting chromato-

graphic materials.

In case of calixarenes (unmodified or with tert-butyl

groups on the upper (broader) rim), the phenolic hydroxyl

groups are not free. Here, these positions were alkylated

with olefin linkers by Syntrex GbR. The number of phenolic

units, which build the calixarene cavities, differs from 4–6–8

units. A change of the number of phenolic units results in

an enhanced flexibility and size of the calixarene ring.

Calix[4]arene molecules are relatively rigid. Especially with

the spacers on the lower rim, no transformation of the so-

called cone conformation to the other three conformations

is possible. In case of calix[6] and calix[8]arenes, all possible

conformations can be adopted. This can be seen in NMR

investigations from the signals of the both hydrogen atoms

at the bridged carbon between the phenolic rings. In case of

calix[4]arenes, two separate signals are observed after

modification with olefin linkers. This shows the stable cone

conformation of the calix[4]arenes [45, 46]. In case of larger

calixarenes, one broad signal is observed at these chemical

shift, showing increased flexibility [47].

The modification procedure was optimized according to

the behaviour of the calixarenes based on the syntheses

strategy of Pesek et al. [48–50] for the preparation of chro-

matographic materials. This procedure is described in [51],

resulting in a monomolecular coverage of the silica surface.

Other methods for calixarene silica surface modification are

given by Glennon and co-workers in [52, 53], among others.

The usage of different olefin groups containing calix-

arenes for modification of the silica gels leads to a number

of different Caltrexs HPLC phases. For better handling of

the names of different Caltrexs phases, Syntrex GbR

introduced short names for these materials. Caltrexs A

materials are modified with unsubstituted calixarenes. In

accordance to this, tert-butyl-modified calixarenes lead to the

Caltrexs B phases. Additionally, roman numerals I, II and

III stand for calixarenes with 4, 6 or 8 phenolic units,

respectively.

In case of Caltrexs Science materials, a 50:50 w/w

mixture of calix[4]arenes and p-tert-butylcalix[4]arenes was

used for the modification of the silica gel. Thus, both

selectivities of unmodified and modified calix[4]arenes are

combined in one material. This leads to a good starting

material for phase testing of Caltrexs columns, giving

‘‘medium’’ results between Caltrexs AI and Caltrexs BI

phases.

Syntheses of calixarene-modified silicas follow a three-

step procedure after a catalytic heterogeneous hydrosilyla-

tion procedure.

In step 1, the free hydroxyl groups on the silica surface

were modified with dimethylchlorosilane (DMCS) to receive

the silane functions. The silica gel was activated by heating

under reflux in 0.1 mol HCl for 2 h, washed with distilled

water twice and dried in a vacuum drying chamber at 0.5 bar

for 12 h at 1101C.

According to the determined specific surface of the

Kromasils silica of 310 m2/g and 2.5 mM DMCS per gram

were used for silanisation.

In a 2.5 L vessel, 1000 mL of well-dried toluene and

100 g of silica have been mixed. Afterwards, 76.4 mL of

triethylamine were added to the suspension and slowly

55.5 mL of DMCS were added under stirring with a drop-

ping funnel. The suspension was again heated under reflux

for 60 h under slow nitrogen stream. The nitrogen stream

was directed through a washing funnel containing sodium

hydroxide for removing gaseous HCl. After cooling down,

500 mL of distilled water were added and the silica gel was

filtered. In a cleanup step, the silica gel was washed with an

acetone/water mixture twice, subsequently washed with

acetone and finally dried at 1401C in the vacuum-drying

chamber.

After drying, a Brunauer-Emmett-Teller (BET) surface

area measurement and an analysis of carbon content were

executed.

In the second step, 1000 mL of toluene and 100 g of

silanized silica were mixed. In total, 20 g of the selected

calixarene were solved or suspended in 200 mL toluene and

added under stirring. Then a solution of 0.25 g rhodium

J. Sep. Sci. 2010, 33, 2930–2942 Liquid Chromatography 2931


catalyst in 100 mL toluene was slowly added to the

suspension and it was heated under reflux for 24 h (refluxed

funnel closed with a drying tube), allowed to cool down and

filtered. The filter cake was washed with fresh toluene twice

and then several times with methylene chloride until the

filtered solution is clear and colourless. Finally, the calixar-

ene-modified silica gel was washed with acetone several

times and then dried under vacuum in a separation funnel

at 801C.

A BET measurement and analysis of total organic

carbon content followed.

The third step is a so-called endcapping step in order to

reduce the residual silanol activity.

At this step, the dried, calixarene-modified silica gel is

heated in toluene (1000 mL toluene/100 g modified silica

gel). Previously, 25.22 mL triethylamine were added and the

suspension was moderately stirred at room temperature for

30 min. Afterwards, 21.1 mL trimethylchlorosilane were

slowly added. After boiling under reflux for 24 h, cooling to

room temperature, slowly adding 500 mL water and stirring

for 30 min, the crude material was filtered and several

washing steps were applied (once with acetone, twice with

methylene chloride, once with acetone, two times with

acetone/water 50:50 v/v and once again with acetone).

Finally, the material was dried in a vacuum oven at 1401C

for 2 h.

2.1.2 Materials for synthesis

Kromasils silica gel (Si120 5 mm) was purchased from Eka

Nobel (Bohus, Sweden). DMCS and trimethylchlorosilane

were obtained from ABCR (Karlsruhe, Germany), triethyl-

amine from Acros Organics (NJ, USA). All calixarenes

are products from Syntrex GbR and produced after

modified procedures of Gutsche et al. [7, 54, 55]. Chloro-

tri-(triphenyl-phosphine)-rhodium catalyst was purchased

from ICT (Bad Homburg, Germany). Other chemicals such

as solvents, acids and bases are used from commercial

distributors.

2.2 Chromatography

Experimental details for the newly calculated data of Tables

1 and 3 are given here. For other conditions, see references.

2.2.1 Conditions

Experiments were performed with mobile phases consisting

of mixtures of methanol/water pH 3 at 40, 50, 60, 70, 90 and

98% v/v methanol. The pH value was adjusted with

phosphoric acid or sodium hydroxide prior to mixing.

Mixing was performed online after degassing the solvents

ultrasonically. The temperature was thermostated to 401C in

all experiments and elution was carried out isocratically at a

flow rate of 1 mL/min. Column hold-up times were

determined using a linearization procedure for homologous

series [98] (n-alcohols). Additionally, the hold-up time of the

chromatograph was determined by injecting pure methanol

without a column installed and has been subtracted from all

retention data.

2.2.2 Apparatus

Data have been collected on two HP 1090 series II

chromatographs (Hewlett Packard, Waldbronn, Germany)

equipped with diode array detectors.

2.2.3 Columns

The study included seven different calixarene-bonded

phases (Caltrexs AI – calix[4]arene; Caltrexs AII –

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

Science – calix[4]arene and p-tert-butyl-calix[4]arene in a 1:1

ratio), a resorcinarene-bonded phase (Caltrexs Resorcinar-

ene, RES) and an alkyl-bonded phase (Kromasils C18). The

Caltrexs columns were all kindly supplied by Syntrex GbR

(Greifswald, Germany). The ligands were immobilized viadescribed procedure.

All columns had particle diameters of 5 mm and

dimensions of 125� 4 mm.

2.2.4 Chemicals and analytes

Benzene, toluene, phenol and pentanol were purchased

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

anthracene were obtained from Berlin-Chemie (Berlin,

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

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

p-Cresol, p-hydroxybenzoic acid, triphenylene and phenan-

threne were obtained from Acros Organics. Naphthalene,

sodium hydroxide, phosphoric acid, ethanol, propanol,

trans-decalin, methyl-, ethyl- and propylbenzoate were

obtained from Merck (Darmstadt, Germany). Butanol was

from AppliChem (Darmstadt, Germany). o-Terphenyl,

biphenyl, butylbenzoate and benzoic acid were from

Sigma-Aldrich (Steinheim, Germany). Propranolol was

purchased from Sigma Chemical (St. Louis, MO, USA).

Diclofenac was from 3 M Medica Pharma (Borken,

Germany). Naproxen, ketoprofen, ibuprofen 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 Lund-

beck (Copenhagen, Denmark). Prednisolone and hydrocor-

tisone were from Schering (Berlin, Germany), fluvastatin

from Sandoz Pharma (Basel, Switzerland).

All solutes were of the highest available analytical grade.

Methanol (HPLC gradient grade quality) was purchased

from Merck or from Acros Organics. Water was obtained by

bidistillation.

J. Sep. Sci. 2010, 33, 2930–29422932 Ch. Schneider et al.


3 Results and discussion

The retention and selectivity characteristics of the novel,

commercially available calixarene-bonded stationary phases

(Caltrexs) have been extensively studied in our workgroup.

Special interest has been given to the single interactions

(hydrophobic, polar, etc.) and the column’s support.

Therefore, different test systems and analysis methods have

been used.

With varying groups of analytes (phenols, polyaromatic

hydrocarbons (PAHs), alkyl-substituted aromatics, benzoic

acid esters, barbituric acid derivatives and xanthines) and

organic modifiers, SokolieX et al. [40] determined the basic

chromatographic behaviour of the novel Caltrexs columns.

The retention order and selectivities of the analytes as well

as the calculation of methylene/phenyl selectivities and

ln k – ln k correlations were used to compare the different

calixarene phases among each other and to phenyl and

alkyl phases.

Afterwards different, already known and established,

column tests were used to investigate the hydrophobic

interaction capabilities that are more detailed elsewhere [56].

Furthermore, the steric properties were examined by the

analysis of planar/aplanar, nonpolar solutes and thiox-

anthenes/steroids.

More recently, the retention characteristics of seven

calixarene-bonded, one resorcinarene-bonded and three alkyl-

bonded columns were investigated and compared [57].

Therefore, 31 solutes have been analysed over nearly the

whole range of methanol concentrations (0–98%). The chro-

matographic behaviour of the stationary phases was char-

acterised via regression analyses of ln k versus j (volume

fraction of methanol in the mobile phase) and via compar-

isons between predicted and extrapolated data. A complete

overview could be given about the retention characteristics of

nonpolar, polar and ionic solutes on alkyl-bonded and calix-

arene-bonded stationary phases. Special interest has been

given to the extreme ranges of methanol content.

As a reference and base for the evaluation, the well-

known, linear model

ln k ¼ ln kw � S � j ð1Þ

by Snyder et al. was used [58]. This equation has

been widely used to describe the changes of the retention

factor k with the methanol fraction of the mobile phase

[13, 59–67]. It is correct for the majority of analytes in

the range of 0.2–0.8 j, but for lower and higher concen-

trations often nonlinearity occurs [65, 68–73], for ionic

solutes even at medium modifier concentrations [74–76].

In particular, these nonlinearities can be interpreted as

indications to the changes of the occurring mechanisms

of interaction.

In order to gain more detailed information about

underlying retention mechanisms concerning the single

interactions, a multiple-term linear equation, originally

introduced by Dolan and co-workers [26], was adapted to be

used for calixarene stationary phases [77].

The adapted version

log a ¼ log k0 � log k0ref ¼ Z0H1s0S1b0A1a0B1k0C ð2Þ

includes one more term for steric interactions and is based

on different reference solutes as well as some different

conditions. The single terms represent the hydrophobic

Z0H, rigid steric s0rSr, flexible steric s0fSf and ionic inter-

action k0C as well as hydrogen-bonding interactions

between donor solutes and an acceptor group in the

stationary a0B and vice versa b0A. Capital letters (column

parameters) represent contributions of the stationary

phases/the chromatographic system, whereas Greek letters

represent contributions of the solutes (solute parameters).

These parameters characterize the different stationary

phases in terms of underlying interactions and can be used

to determine the part each interaction has on retention and

selectivity.

By that, six calixarene- and five alkyl-bonded phases

were compared at low and neutral pH value [77]. Further

study is in progress to elucidate the role of the methanol

content of the mobile phase towards the single interactions.

First results will be given here.

All in all, an overview about the results, especially in

comparison to the characteristics of common alkyl-bonded

phases, is shown.

3.1 Properties of the single interactions

As reversed phases, calixarene-bonded stationary phases

show linear behaviour of nonpolar solutes at medium

modifier concentrations like conventional alkyl phases

according to Eq. (1). However, they differ in their

hydrophobic strength.

Generally, Caltrexs B phases of the same ring size are

more hydrophobic than nonsubstituted Caltrexs A phases,

due to the bonded tert-butyl groups at the upper rim

(Table 1).

This is at least true for methanol concentrations higher

than 40% and probably also for lesser concentrations. But

Table 1. Hydrophobic column parameters at different methanol

concentrationsa)

H 40% 50% 60% 70% 90% 98%

Kromasils C18 0.997 1.006 0.973 0.988 1.016 1.043

Caltrexs AI 0.880 0.883 0.799 0.722 0.503 0.494

Caltrexs AII 0.896 0.858 0.785 0.752 0.516 0.376

Caltrexs AIII 0.826 0.786 0.679 0.604 0.350 0.234

Caltrexs BI 1.027 0.994 0.915 0.931 0.778 0.792

Caltrexs BII 1.048 1.011 0.893 0.901 0.628 0.549

Caltrexs BIII 0.915 0.867 0.819 0.761 0.467 0.359

Caltrexs Science 0.967 0.967 0.869 0.828 0.551 0.480

Caltrexs Resorcinarene 0.892 0.853 0.753 0.701 0.449 0.337

a) All parameters are newly calculated with a different set of

solutes and some changed conditions compared with [77].

More additional studies are in progress.



the column parameters show a different picture regarding

the comparison to alkyl phases. With increasing methanol

content, calixarene phases and the resorcinarene phase

become increasingly less hydrophobic compared with

the alkyl columns. However, at 40 and 50% methanol

(and probably below), Caltrexs BI and BII columns tend to

be even more hydrophobic than C18 phases. (Here, it

shall be noted that column parameters of different

columns may be easily compared at one condition, but

comparison must be done carefully between different

conditions because both column and solute parameters

have been calculated separately for every condition. But

only their combination gives the impact on retention or

selectivity [77]. More detailed information will be given

in a forthcoming publication.) In general, hydrophobicity

declines from C18- over tert-butyl-calixarene to calixarene

phases for mobile phases with more than 50–60%

methanol. The lesser hydrophobicity of the calixarene

phases is related to their broader spectrum of supported

interactions. Because solutes are retained via more addi-

tional interactions (steric and polar), a smaller part remains

for hydrophobic interaction. Yet these individual differences

between the stationary phases become smaller at higher

water content.

In HPLC, both mobile and stationary phases contribute

to the overall hydrophobic interaction [29]. Because of the

hydrophobic effect of water, the mobile phase mainly has an

exclusionary effect on hydrophobic solutes and presses the

analyte to the stationary phase. The stationary phase itself

can undergo attractive interactions with apolar solutes. It is

discussed up to now which has more influence on retention

and whether retention is governed by adsorption or parti-

tioning [29, 33, 78–85]. However, it is reasonable that the

hydrophobic effect of the mobile phase is stronger at higher

water content since it is mainly caused by water with its

small molecular volume. Thus, the mobile phase plays a

bigger role at higher water content, suppressing the differ-

ences between the stationary phases.

The influence of the pH value is small for calixarene-

and alkyl-bonded columns, as expected because underlying

van-der-Waals forces and the hydrophobic effects are widely

independent of pH value (Table 2).

But there are additional differences at extreme compo-

sitions of the mobile phase.

At very high water content, convex plots of ln k versus jwere found for nonpolar benzene on alkyl-bonded columns.

This means that extrapolated retention factors from the

linear part are too high and measured values showed a

decrease. These effects do not occur on calixarene-bonded

phases (Fig. 1).

Probably, decreases are related to conformational

changes of the alkyl ligands on conventional phases, i.e.incomplete solvation, stronger folding and collapsing of the

alkyl chains [69, 70, 85–87]. Similar conformational changes

are not probable on calixarene phases because of their high

degree of internal order. They can be advantageous here

because the linearity of ln k versus j facilitates the prediction

of the retention and thus the development and optimization

of chromatographic methods.

At high methanol content, solute- and column-depen-

dent increases of the measured retention data occur

compared with linear extrapolation (Fig. 2).

Increases rise with increasing size of the apolar solutes.

However, for calixarene phases, especially smaller calix[4]-

and calix[6]arenes, there is a maximum increase for biphe-

nyl, anthracene or phenanthrene. Larger molecules like

triphenylene do not show higher increases. Probably, the

increases in general are based on increased sorption of

methanol, resulting in stronger partitioning of the solutes.

More hydrophobic solutes can take more advantage from

the facilitated partitioning, resulting in increased retention.

However, partitioning effects are influenced by steric

effects. Thus, there is a maximum possible benefit from

facilitated partitioning for large solutes, especially on

columns with smaller calixarenes since large analytes do not

fit correctly into the stationary phase.

Obviously, steric interactions become more important for

larger solutes. Interpretation of known column tests

concerning steric selectivity of calixarene phases and the

Table 2. pH-dependent variability of the column parameters

Column parameters

pH 7–3a)

DH DSr DSf DC DB DA

Kromasils C18 2.82 40.09 36.95 103.86 �82.20 256.06

Caltrexs AI 10.87 1.99 �38.71 63.54 �13.36 �77.77

Caltrexs AII 16.61 �87.67 �111.22 69.59 61.31 37.90

Caltrexs AIII 19.99 �92.63 �98.81 55.42 93.19 69.27

Caltrexs BI 5.30 6.04 �30.17 109.92 60.70 153.89

Caltrexs BII 0.87 �12.44 �15.14 121.32 �65.61 127.34

Caltrexs BIII 18.74 �3.19 �47.55 124.12 5.22 �82.44

a) Differences of column parameters between pH 7 and 3 are

displayed in percent of the average column parameter [77].

Figure 1. Comparison of ln k of benzene on alkyl- andcalixarene-bonded phases at low methanol concentrations.Stationary phases: & Kromasils C18, � Caltrexs AI.



analysis of the retention of thioxanthenes and steroids have

shown that these test methods are insufficient to evaluate

the potential of calixarene-bonded phases [56]. It can be

described more exactly with the parameters from Eq. (2).

In light of Eq. (2), steric interactions should not be

understood as ‘‘repulsive’’, ‘‘exclusive’’ or otherwise ‘‘nega-

tive’’ interaction. Indeed, steric interactions are mostly

interpreted as ‘‘steric hindrance’’, but they can also be seen

as ‘‘positive’’, ‘‘additive’’ interactions [77]. In short, steric

interactions do not need to be understood as the hypothe-

tical, but not occurred, interaction which an analyte cannot

perform because it does not fit completely into a stationary

phase. It can also be understood as the interaction which an

analyte can perform because it fits to the stationary phase at

all. Thus, it can be described as the additional interaction

which a solute performs in relation to a hypothetical inter-

action without fitting into the stationary phase at all.

However, in both interpretations, the steric interactions are

no own class of interactions. They are additionally occurring

(or not occurring) hydrophobic, polar or ionic interactions

an analyte or stationary phase supports because of their

steric characteristics.

These steric characteristics are particularly high for

stationary phases with bonded macromolecules (Table 3).

All calixarene phases and the resorcinarene phases

show higher rigid and flexible steric parameters at the

individual methanol concentrations, reflecting the increased

possibilities for additional interactions, mainly viacomplexations with the calixarene cavity, influenced by

steric effects.

Moreover, the steric properties of Caltrexs B columns,

particularly the rigid properties, are less than for Caltrexs A

columns. This is the result of hindered inclusions into the

bonded layer caused by the tert-butyl groups at the upper

rim of the cavities. However, this lowering effect is not as

big for the flexible steric interaction. Probably, flexible

molecules, such as biphenyl or o-terphenyl, can interact

more effectively with substituted calixarenes than rigid

planar molecules because the flexible benzene rings can

adopt preferable conformations. This is the reason why the

flexible steric parameters are substantially higher than rigid

parameters on phases with bonded tert-butyl-calixarenes,

but they are lower on phases with unsubstituted calixarenes.

Furthermore and contrary to the expectations, the

resorcinarene column even showed more intensive rigid

steric interactions than the unsubstituted calixarene phases.

Obviously, it exhibits extraordinary affinity to large, rigid

molecules. On the contrary, its flexible steric parameters are

less for all the calixarene phases. This may be either related

to the free hydroxyl groups or related to the different

methods of binding to the silica gel. For resorcinarenes,

longer spacers are used and they are not attached to the

benzene rings, but to the bridging molecules (see above).

This could enhance the possibilities for the resorcinarene to

move its benzene rings and possibly to adopt different

conformations. Both types of steric interactions should

benefit from that, giving an explanation for the high values

of Sr. Yet Sf is actually not increased, suggesting that

nonplanarity is disadvantageous. This suggests a relation-

ship to the increased chain length of the spacers. They may

facilitate the interaction to hydrophobic solutes, if the

solutes can penetrate deep enough into the bonded layer.

Therefore, planarity/nonplanarity surely is of importance.

Comparing the different pH values, parameters are

generally higher at low pH value. But this is probably not

Figure 2. Deviations from linearly extrapo-lated retention factors at high methanolconcentrations (extrapolation done withvalues from 0.3 to 0.7 j). Solutes from leftto right for each stationary phase: benzene,toluene, ethylbenzene, propylbenzene,naphthalene, biphenyl, phenanthrene,anthracene, o-terphenyl and triphenylene.

Table 3. Column parameters at 60% methanol in the mobile

phase, pH 3a)

Sr Sf C B A

Kromasils C18 �0.485 �0.632 �0.349 �0.548 �1.041

Caltrexs AI 0.040 0.096 0.989 0.266 0.437

Caltrexs AII 0.387 0.357 1.070 0.413 0.174

Caltrexs AIII 0.390 0.345 1.315 0.342 0.391

Caltrexs BI �0.132 0.138 �0.906 0.075 �0.395

Caltrexs BII 0.044 0.261 �0.945 0.122 �0.392

Caltrexs BIII �0.072 0.149 �1.905 0.180 0.505

Caltrexs Science �0.078 0.101 0.412 0.134 0.181

Caltrexs Resorcinarene 0.435 �0.007 3.592 �0.205 1.040

a) These parameters are also calculated with a different set of

solutes and some changed conditions compared with [77].



directly caused by weaker steric interactions, but by very

strong ionic interactions at pH 7 because of a higher

number of dissociated silanols. They lead to a relative

decrease of the other interactions in the multiple-term

equation, becoming less important. Nevertheless, the way of

interaction keeps essentially the same, there is no change in

the mechanism of the steric interactions between low and

neutral pH value.

Differences between the stationary phases are far more

distinct regarding the ionic interaction of protonated bases

(Table 3).

The ionic activity of Caltrexs B phases is very low.

Indeed, protonated bases often elute shortly after or with the

void volume of the column. Endcapped Kromasils C18

columns are slightly more ionic. However, there is a distinct

difference to Caltrexs A phases, which show remarkable

retention of ionic solutes. The interaction takes place at

dissociated silanols. Because normal silanols with a pKa

value of 7.270.2 [88] are not dissociated at pH 3.0, the ionic

activity is related to acidic silanols, which can be dissociated

even below pH 2 because of metal contamination of the

silica gels [89–91].

Nevertheless, differences in the amount of dissociated

silanols cannot be the reason for the diverse ionic char-

acteristics. All calixarene phases as well as the resorcinarene

and the Kromasils C18 phase are based on the same silica

gel. It is described by Gritti and Guiochon that silanol

activity and the interaction of cationic compounds depend

on the surface coverage of the stationary phase [92]. Hence,

differences will be related to sterical hindrance effects,

which occur during diffusion to the silica surface and

therefore to the type of bonded ligands.

Likely the bulky calixarenes, particularly the alkyl-

substituted calixarenes, shield the silanol surface more

effectively in comparison to alkyl phases, leading to reduced

ionic activity at dissociated silanols. However, Caltrexs A

phases show increased activity, although less shielding than

through alkyl chains seems unlikely.

Here, interactions could take place in the calixarene

cavity. Endo-complexation of amines with calixarenes are

known [8, 9], as well as interactions with cations [93]. This

interaction should be strengthened, if the electron density in

the cavity is increased or a negative charge is existent or

possible through dissociation of a phenolic hydroxyl. Even a

very small amount of unbound hydroxyls could remarkably

influence retention because of the high stability of ionic

interactions.

Obviously, steric effects will have influence here

because the protonated part of the molecule must enter the

bonded layer. Thus, calixarenes generally lower the ionic

activity of silica-based phases through effective shielding.

Particularly, substituted calixarenes will have distinct

effects. However, additional ionic interactions may take

place in the cavity of calixarenes. But again, this interaction

is sterically hindered on substituted calixarenes because the

protonated groups must enter the bonded layer. This means

both ionic interactions (at dissociated silanols and in the

cavity) are effectively shielded on Caltrexs B columns,

resulting in lowest values of C. On alkyl phases, the

common interaction with protonated silanols takes place,

but the analyte must reach the silica gel. On Caltrexs A

phases, the shielding of the silica surface will not be as

effective as on Caltrexs B columns. Additionally, interaction

of protonated bases can occur in the calixarene cavity, which

is located higher in the bonded layer and better accessible.

The most ionic stationary phase is the Caltrexs Resor-

cinarene. Their extreme affinity for protonated solutes

results from the two free hydroxyls at every resorcine unit.

If the whole range from 0 to 98% v/v methanol

concentration is observed, plots of ln k versus j are less

linear than for hydrophobic analytes. Parabolic plots can be

found, also on calixarene phases, as they have been observed

for alkyl phases [74–76]. The plots are more curved for

smaller, protonated analytes than for larger, more hydro-

phobic analytes (Fig. 3).

Obviously, curvature is related to the hydrophobicity,

and with that to the part the ionic interaction has on overall

retention, of the single solutes. The more ionic the solute,

the bigger the ionic part of the molecule is in relation to the

whole solute, the more the plot is curved.

Figure 3. Plots of logarithmic retention factors of ionic solutes.Solutes: (A) procaine, (B) promethazine; stationary phases: & ,Kromasils C18; �, Caltrexs AII; m, Caltrexs BII; ~, Caltrexs

Science; values of procaine on the Kromasils C18 at 0.4–0.7 jwere set to �5 to illustrate the plot, but could not be actuallycalculated, since procaine eluted before the void volume.



Besides that solute dependency, a dependency from

the stationary phases occurs. Curvatures rise with the

ionic activity of the stationary phases. Moreover, not all

phases show increases of ln k values at high methanol

concentrations and if so they do not necessarily begin at

the same methanol concentrations, indicating that this

is not solely driven by the mobile phase. Probably, silano-

philic interactions are extraordinarily strengthened or

the general polarity of the stationary phase is decreased

because of differences in the adsorption of mobile-phase

molecules as a result of the low amount of water. Surely,

both effects would occur on all stationary phases, but inter-

column variability could result from different bonding

densities, different hydrophobicities of bonded ligands,

different steric effects and, in relation to the hydrophobicity

of the ligands, differences in the adsorption of mobile-phase

molecules.

Besides, differences between neutral and low pH value

obviously occur. Values are clearly larger at neutral pH,

since about 50% of the common silanols are dissociated in

addition to the acidic silanols. The large number of disso-

ciated silanols cannot be shielded effectively.

The retention factors k of polar, hydrogen-donating

solutes change very linearly with increasing methanol

content of the mobile phase in medium ranges of methanol

concentration. Nevertheless, deviations were observed below

10% and above 90% methanol (Fig. 4).

Below 10% methanol measured values are increased on

calixarene columns. On the contrary, deviations to an

extrapolated linear behaviour are low on alkyl phases, or

mostly at least less than on calixarene phases. It can be

proposed that this is related to increased polarity of the

stationary phase because of less adsorption of methanol, as

suggested by Schoenmakers et al. [68].

Of course, this will occur on all stationary phases, yet a

compensating effect occurs on alkyl columns. All tested

solutes, even small, polar ones, are not exclusively retained

by polar interactions, but also by hydrophobic interactions.

Now, the hydrophobic interaction is reduced on alkyl

columns at high water content. In combination with the

increase of the polar interaction, this can give linear plots.

On calixarene phases, no compensation takes place and

their affinity for polar analytes is increased.

Above 90% methanol deviations from linearity also

depend on the stationary phase and additionally on the

analyte. Positive and negative deviations have been found.

On alkyl phases, least retention factors were measured, they

are higher on Caltrexs B columns and highest on Caltrexs

A columns. Concerning different solutes, deviations rise

with increasing size, or more exactly with increasing

hydrophobic interaction (Fig. 5).

On alkyl-bonded phases, a rather steady increase was

found from phenol to diclofenac, i.e. with increasing

retention times. However, on calixarene-bonded phases

ketoprofen and naproxen are often more retained than

ibuprofen and differences between ketoprofen, naproxen,

ibuprofen and diclofenac are less than on alkyl-bonded

phases. This is probably related to specific, sterically influ-

enced interactions, but nevertheless reflects the strength of

the hydrophobic interaction. Thus, it can be presumed that

increasing deviations are related to increased hydrophobic

interactions, additionally influenced by steric effects, at high

methanol concentrations, as it was proposed for purely

hydrophobic solutes. Consequently, it is reasonable that the

polar interactions are decreased at high methanol concen-

trations.

This can be explained with adsorption effects in the

interface region between mobile and stationary phases.

Special importance of the interface region and the form-

ing of an eluent-surface–phase have been supposed

previously [94–97]. In that region, mobile-phase mole-

cules, water and methanol, are adsorbed. Polar solutes will

interact with them via hydrogen-bonding interactions. Very

high methanol content could lead to disproportionately

reduced sorption of water, which itself would reduce

polar interactions. Although the exact composition of such

an inter-phase is not available, it is reasonable that the

bonded ligands influence their composition. More polar

phases should adsorb a higher fraction of water. Corre-

spondingly, more polar Caltrexs A phases show

more positive deviations than Caltrexs B phases, substi-

tuted with apolar alkyl groups. The C18 phases, with their

Figure 4. Plots of logarithmic retention factors of a protondonator. Solutes: o-cresol; stationary phases: (A) Kromasils C18,(B) & , Caltrexs AII; �, Caltrexs BIII.



purely hydrophobic alkyl ligands, consequently adsorb least

water and show least retention factors in relation to linear

extrapolation.

The named differences in the strength of the polar

interaction at high methanol concentrations are also

reflected in the calculated column parameters, not only at

extreme mobile-phase compositions (Table 3).

The weakest interactions are found with alkyl phases,

this means they show least polar basic characteristics.

Caltrexs B phases are considerably more polar, and

Caltrexs A phases even more. The value of the Caltrexs

Science lies between both types (Caltrexs AI and BI), as

expected.

This could be related to the mechanism mentioned

above (interaction with adsorbed mobile phase in the

interface region) and in addition to specific interactions with

calixarenes. Proton donators may additionally interact with

ether oxygens, or free hydroxyls, at the lower rim of the

calixarene cavities, as was suggested by Li et al. [4, 14].

Hence, steric effects would obviously be of influence again.

Both models can explain the physicochemical origin of

column basicitiy. Probably, it is a combination of both

effects:

Taking into consideration the interactions with calixar-

enes solely, this explains the increased basicities of these

columns. However, it does not explain the negative devia-

tions at high methanol concentrations. Moreover, this is

likely not the reason for the relatively high values of B of

Caltrexs B columns because direct interactions with calix-

arene cavities are sterically hindered here.

Thus the importance of the inter-phase region for polar

solutes seems to be evident.

On the other hand, adsorbed mobile phase as the only

influence is also not probable. The Caltrexs Resorcinarene

shows opposite behaviour towards acids and bases.

Although its basicity B is quite low, its acidity is high. The

increased acidity is surely related to the additional hydroxyls.

However, with these groups, the phase is very polar and

should also show increased basicity, if it is only related to

adsorbed water. Indeed, its values of B are all smaller than

the B of the Caltrexs A column, which also consists of four

benzene units, but without the additional hydroxyls, what in

turn should reduce B because of less adsorbed water.

Possibly, the hydroxyl groups on the resorcinarene hinder

the interaction with the cavity.

In conclusion, column basicity of calixarene phases is

likely related to adsorbed mobile phase (water) and to direct

interaction with the ligands, the deviations at high and low

water content probably to adsorption effects of water and

methanol.

Calixarene phases also differ from alkyl phases regard-

ing their acidity (Table 3).

Alkyl phases support distinctly less interactions to

hydrogen-bond acceptors like N,N-dimethylacetamid.

Values of A of Caltrexs B phases are far higher, values of

Caltrexs A phases are even more higher and the values of

Caltrexs Resorcinarene are clearly the highest. As

mentioned above, this results from interactions with the

hydroxyls at the upper rim of the resorcinarene cavity,

which are next to protonated silanols, a partner for inter-

actions with proton acceptors.

Differences in the number of accessible protonated

silanols are no explanation for the mentioned differences,

since silica gels are identical and the better shielding calix-

arenes show higher acidity. Thus, again direct interactions

of calixarenes with amines, which are reported in the

Figure 5. Deviations from linearly extrapo-lated retention factors (extrapolation donewith values from 0.3 to 0.7 j) of protondonators at high methanol concentrations.Solutes from left to right for each stationaryphase: phenol, benzoic acid, o-cresol, keto-profen, naproxen, ibuprofen and diclofenac.

Figure 6. Plots of the logarithmic retention factors of thehydrogen acceptor N,N-dimethylacetamide stationary phases:& on Kromasils C18, � on Caltrexs AII and m on Caltrexs BII.



Tab

le4.

Co

ncl

ud

ing

com

pari

son

of

stati

on

ary

ph

ase

pro

pert

ies

reg

ard

ing

the

sin

gle

inte

ract

ion

sta

kin

gp

lace

inH

PLC

.

Dev

iatio

nsfr

omlin

eari

tyat

low

conc

entr

atio

nsof

met

hano

la)

Dev

iatio

nsfr

omlin

eari

tyat

high

conc

entr

atio

nsof

met

hano

la)

Phy

sico

chem

ical

orig

inof

the

inte

ract

ion

Ord

erof

stre

ngth

ofin

tera

ctio

n

and

effe

ctof

chan

geof

pHva

lue

Hyd

roph

obic

inte

ract

ion

Ret

entio

nde

crea

ses

onK

rom

asils

C18

Ret

entio

nke

eps

near

lyun

chan

ged

on

calix

aren

e-bo

nded

phas

es

Ret

entio

nin

crea

ses,

prob

ably

due

to

faci

litat

edpa

rtiti

onin

gca

used

by

addi

tiona

llyad

sorb

edm

etha

nol

Res

ults

from

the

hydr

opho

bic

effe

ct

ofw

ater

(mai

nly)

and

van-

der-

Waa

ls

inte

ract

ions

with

bond

edlig

ands

Med

ium

mod

ifier

:A

lkylZ

Cal

trex

s

B4

Cal

trex

sAE

Res

orc

Hig

hm

odifi

er:

Alk

yl*

Cal

trex

s

Ori

gins

ofth

ede

crea

ses

onK

rom

asils

C18

supp

osed

are

conf

orm

atio

nal

chan

ges

Incr

ease

depe

nds

onsi

ze/h

ydro

phob

icity

ofth

ean

alyt

esan

don

ster

ic

B4

Cal

trex

sAE

Res

orc

Min

orin

fluen

ceof

pHva

lue

chan

ge

ofth

eal

kyl

chai

nsch

arac

teri

stic

sof

the

stat

iona

ryph

ases

Ste

ric

inte

ract

ion

Influ

ence

sth

eam

ount

ofad

ditio

nal

hydr

opho

bic

inte

ract

ion

No

disc

rete

inte

ract

ion

Pos

sibi

lity

ofa

stat

iona

ryph

ase

or

Rig

idst

eric

inte

ract

ion:

Res

orc4

Cal

trex

sA4

Cal

trex

sB*

Alk

yl

Influ

ence

spo

ssib

lepo

lar

and

ioni

c

inte

ract

ions

inth

eca

lixar

ene

cavi

ties

aso

lute

for

addi

tiona

lot

her

inte

ract

ions

Can

beun

ders

tood

addi

tivel

y:ba

seis

Flex

ible

ster

icin

tera

ctio

n:C

altr

exs

AC

altr

exs

B4

Res

orc*

Alk

yl

ahy

poth

etic

alpo

int

with

out

any

inte

ract

ion

influ

ence

dby

ster

icef

fect

s

Mai

nly

decr

ease

sat

high

erpH

valu

efo

rC

altr

exco

lum

ns

Or

subt

ract

ive:

base

isa

hypo

thet

ical

poin

tin

clud

ing

all

poss

ible

inte

ract

ions

influ

ence

dby

ster

icef

fect

s

Incr

ease

son

Cal

trex

Kro

mas

ilsC

18

Ioni

c inte

ract

ion

Ret

entio

nin

crea

ses

onal

lst

atio

nary

phas

es

beca

use

ofin

crea

sed

inte

ract

ion

at

diss

ocia

ted

sila

nols

Als

oin

crea

seof

rete

ntio

nth

roug

h:

Hig

her

hydr

opho

bici

tyof

less

prot

onat

ed

base

san

d

Elec

tros

tatic

inte

ract

ion

betw

een

prot

onat

edba

ses

and

the

stat

iona

ry

phas

e

Res

orc*

Cal

trex

sA*

Alk

yl4

Cal

trex

sB

Dis

tinct

lyin

crea

sed

athi

ghpH

valu

e

Less

incr

ease

onC

18be

caus

eof

shie

ldin

g

effe

cts

of‘‘c

olla

psed

’’al

kylc

hain

san

dpo

ssib

ly

Incr

ease

din

tera

ctio

nat

sila

nols

,pr

obab

ly

due

tole

sshy

drat

esh

ells

At

diss

ocia

ted

sila

nols

and

prob

ably

inth

eca

lixar

ene

cavi

ties

Diff

eren

ces

betw

een

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iona

ry

phas

esdi

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ish

wid

ely

beca

use

ofa

com

pens

atin

gef

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ugh

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ced

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opho

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ract

ions

Diff

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betw

een

stat

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ltfr

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ndra

nces

duri

ngdi

ffus

ion

toth

esi

lica

surf

ace

Pol

arin

tera

ctio

n

(Sol

ute

is

prot

on-

acce

ptor

)

Ret

entio

nin

crea

ses

onal

lst

atio

nary

phas

es

Bec

ause

ofin

crea

sed

pola

rity

ofth

est

atio

nary

Diff

eren

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havi

ours

ofth

eph

ases

Rea

sons

prob

ably

are

incr

ease

d

Pol

arin

tera

ctio

nof

prot

on

dona

tors

with

Cal

trex

sA4

Cal

trex

sB4

Res

orc4

Alk

yl

phas

esas

aco

nseq

uenc

eof

less

adso

rbed

met

hano

l

inte

ract

ion

with

prot

onat

edsi

lano

ls

orw

ithad

sorb

edw

ater

Ads

orbe

dm

obile

phas

em

olec

ules

(mai

nly

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and/

or

Dec

reas

esca

nbe

rela

ted

toef

fect

sin

the

inte

rfac

ere

gion

:i.e

.diff

eren

ces

ofth

e

com

posi

tion

ofad

sorb

edm

obile

phas

e

The

calix

aren

eca

vitie

spr

obab

lybo

th

effe

cts

sim

ulta

neou

sly

onca

lixar

ene

phas

es

Unc

lear

influ

ence

ofhi

gher

pH,

incr

ease

sas

wel

las

decr

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s

Pol

arin

tera

ctio

n

(sol

ute

is

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on-

dona

tor)

Ret

entio

nke

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ange

don

C18

orin

crea

ses

Ret

entio

nin

crea

ses

inde

pend

ence

ofth

e

hydr

opho

bici

tyof

the

solu

tes,

but

decr

ease

sw

ere

also

obse

rved

Pol

arin

tera

ctio

nbe

twee

npr

oton

-

acce

ptor

san

d

Res

orc*

Cal

trex

sA4

Cal

trex

sB*

Alk

yl

Ret

entio

nin

crea

ses

onot

her

stat

iona

ry

phas

es

i.e.

the

pola

rin

tera

ctio

nef

fect

sa

decr

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tent

ion

(incr

ease

caus

edby

hydr

o

phob

icin

tera

ctio

n)

Mai

nly

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onat

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drox

yls

(sila

nols

,

arom

.

hydr

oxyl

sof

reso

rcin

aren

es)

Incr

ease

dpo

lari

tyof

the

stat

iona

ryph

ase

isag

ain

caus

ativ

e

Incr

ease

dpo

lari

tyis

part

lyco

mpe

nsat

ed

onC

18(s

eeio

nic

solu

tes)

Like

abov

e,th

ere

ason

prob

ably

isth

e

com

posi

tion

ofth

ead

sorb

edm

obile

phas

eat

the

inte

rfac

e

Add

ition

alpo

ssib

lelo

catio

nsar

e

Ads

orbe

dm

obile

phas

e(w

ater

)

The

calix

aren

eca

vitie

s

Als

ono

clea

rin

fluen

ce,

but

mai

nly

incr

ease

sat

high

erpH

,ev

entu

ally

rela

ted

toin

crea

sed

pola

rity

a)

All

state

men

tsco

nce

rnin

gre

ten

tio

nare

giv

en

inre

lati

on

toa

lin

ear

plo

t,e.g

.‘‘

incr

ease

of

rete

nti

on

’’m

ean

s:re

ten

tio

nin

crease

sm

ore

stro

ng

lyth

an

isp

red

icte

db

yli

near

extr

ap

ola

tio

n.



literature [8, 9], and adsorbed mobile phase, in the interface

region and on the silica surface, can be causative.

Regarding different pH values, parameters are mostly

higher at neutral pH value. This has been somewhat

unexpected because a lower number of protonated silanols

should have lowered the number of accessible interaction

partners for proton acceptors. Perhaps, this is associated

with increased adsorbed water, as a consequence of the

increased polarity, which is in turn related to the number of

dissociated silanols.

A relationship to the amount of adsorbed water and

accordingly the amount of adsorbed methanol and the

polarity of the stationary phase was also found at the high-

water region of the ln k versus j plots. All stationary phases

show increased retention times, which is in accordance to

the assumption of increased polarity because of reduced

sorption of methanol (Fig. 6).

At high methanol content, the stationary phases show

similar conditions than for hydrogen donators. Alkyl phases

show the lowest retention factors in relation to the linear

extrapolation, substituted calixarene phases show higher

factors and unsubstituted calixarene phases the highest.

Consistently, adsorption of water and methanol in the

interface region of the mobile phase is likely determining

here, as it is expected for hydrogen-bond donators.

In summary, the polar interaction between the hydro-

gen-bond acceptors and the stationary phase is surely based

on the interaction with protonated silanols, but not alone.

On both alkyl and calixarene phases, adsorbed water and

methanol can also work as hydrogen-bond donator.

Furthermore, calixarenes can directly interact with amines,

which is probably causative for differences between the

stationary phases. Additionally, the polarity of the stationary

phase and thus the adsorption of mobile phase, especially in

the interface region, seem to be the reason for deviations at

very high and low methanol content.

4 Concluding remarks

In the recent years, calixarene-bonded Caltrexs columns

have been intensively studied in our workgroup.

Although they act as reversed phases in HPLC, they

support additional interaction compared with conventional

alkyl-bonded phases. Towards different kinds of solutes,

they show particular behaviour. The physicochemical

origins of the single interactions, their changes in the

extreme ranges of mobile phase composition and their

individual strength of the occurring interactions often differ

from C18 phases (Table 4).

This makes them valuable tools for the separation of

solutes, which are difficult to analyse on conventional

columns. Especially not only for large, bulky, and with that

steric active, solutes but also for polar molecules, they can

exhibit increased selectivity because of their individual

characteristics. Moreover, different substitutions and cavity

dimensions increase the diversity of possible applications.

Further study about the influence of different methanol

concentrations on the single interactions and their impact

on retention and selectivity is in progress.

The authors thank the companies mentioned above for thefriendly supply of analytes.

The authors have declared no conflict of interest.

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characterization of calixarene-bonded stationary phases

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