INVESTIGATION OF SORPTION OF CALIX[4]ARENE AND CALIX[4]RESORCINARENE TETRAALKYL DERIVATIVES WITH THE LiChrosorb RP 18 SURFACE BY RP HPLC AND MOLECULAR MODELLING METHODS

Tetrahydroxycalix[4]arene, tetraalkoxycalix[4]arenes and tetraalkylcalix[4]resorcinarenes are capable to reversible adsorption on the surface of the hydrophobic support LiChrosorb RP 18 under conditions of the reversed-phase high performance liquid chromatography. The main chromatographic characteristics of tetrahydroxycalix[4]arene, a series of tetraalkoxycalix[4]arenes and tetraalkylcalix[4]resorcinarenes have been determined. It has been found that in the conditions selected (the mobile phase is acetonitrile/water, 86/14, v/v) the retention times of the compounds studied are in the wide range from 4.67 min to 88.0 min depending on the nature of the substituents in the macrocyclic skeleton of the molecule. Thus, the retention times increase with increase of the length of the alkyl substituents at the lower rim of the calixarene macrocycle. According to the molecular modelling data the most effective sorption is explained by the inclusion of LiChrosorb RP 18 octadecyl groups into the lipophilic cavity formed by the alkyl substituents at the lower rim of the macrocycle of calixarene/calixresorcinarene. Thus, the molecular cavity formed by the benzene rings remains opened for the Host-Guest complexation with the analyte molecules. This sorption of tetraalkoxycalix[4]arenes and tetraalkylcalix[4]arenes on the LiChrosorb RP 18 surface can significantly improve its resolution ability in RP HPLC analysis. The influence of replacement of tetrahydroxycalix[4]arene hydroxyl protons with the alkyl groups on the conformation of the macrocycle has been studied.

Calixarenes -macrocyclic compounds available through cyclocondensation of para-substituted phenols with formaldehyde -are widely used as molecular platforms for design of specific receptors that are capable of highly selective recognition between fairly similar substrates: different cations, аnions and neutral molecules [1,2,3,4,5,6,7]. The ability to recognize different substrates and form the Host-Guest supramolecular complexes with them is the base for wide application of calixarenes functionalized at the upper/lower rim by different groups in chemistry, physics, biology, nanotechnologies.
RP HPLC analysis (a liquid chromatograph Hitachi, Ltd., Tokyo, Japan) was performed in isocratic conditions using the chromatographic support LiChrosorb RP 18 and MeCN/H 2 O (86/14, v/v) as a mobile phase. Тhe research was carried out at the temperature of 26 o С. Тhe UV detector was operated at l 254 nm and the flow rate was 0.6 ml/min.
Lipophilicity of Log P of CA and RA was calculated by the RP HPLC method from the equation: Log P = 6.195 x (log k'). The coefficient 6.195 is the ratio of Log P value of p-dichlorobenzene (3.37) [19] to its log k' determined by the RP HPLC method in this work.
The initial molecular modelling of CA, RA and their complexes was carried out by the molecular mecha-nics ММ+ method, the force field was PM3. The structures obtained were optimized by the semi-empirical method (software package HyperChem, version 8) [http://www.hyper.com/Download/AllDownloads/ tabid/470/Default.aspx]. Тhe RMS gradient was 0.01 kcal/mol.

Results and Discussion
Cаlіxаrеnes CA and calixresorcinarenes RA under conditions of the given analysis were registered on the chromatograms as sharp peaks. CA and RA retention times t R (in the range from 4.67 min tо 88.0 min), сapacity factors k' and lipophilicity Log P are presented in Таble.
A linear character of adsorption isotherms of CA and RA reflects their reversible sorption on the LiChrosorb-RP18 support (Fig. 2, 3).
Capacity factors k' and retention times t R of CA, RA are linearly depended on the number of carbon atoms in the alkyl substituents at the lower rim of the macrocycle (Fig. 4, 5). The increase of the number of carbon atoms improves sorption on the hydrophobic LiChrosorb RP 18 surface and increases k' and t R values.
The values of k' аnd t R are correlated with the CA, RA molecular masses and Log P values which, in turn, are linearly depended on the number of carbon atoms in the alkyl substituents.
Such correlations confirm the hydrophobic interactions of СА, RA with octadecyl groups on the LiChrosorb RP 18 surface. Probably, intensification of hydrophobicity of the СА, RA molecules, as well as polarity of the mobile phase can lead to its irreversible sorption on the LiChrosorb RP 18 surface as it was shown in [20]. This approach can be used for physical modification of chromatographic stationary phases by the calixarene-Hosts.
The process of the аnаlyte (А) sorption in the Li-Chrosorb RP18 (S) -calixarene (L) chromatographic system can be described as follow: If the calixarene (L) binds with the support surface (S) stronger than with the analyte (А), (К 1 >К 2 ), the analyte absorbs on the calixarene modified support surface. When the analyte (А) binds stronger with the calixarene (L), then with the support surface (K 2 >K 1 ) the supramolecular complex (LA) will absorb on the surface.
To understand the nature of the interaction of calixarenes and calixresorcinarenes with the chromatographic support the molecular modelling of the structure of their Host-Guest complexes with siloxyoctadecane H 3 SiO(CH 2 ) 17 CH 3 (SOD) as a model site of Li-Chrosorb RP 18 has been performed.

Таble
Retention times t R , сapacity factors k' and lipophilicity Log P calix [4]     In accordance with the modelling the tetrahydroxycalixarene СА-ОН forms with SOD the complex А where the methyl group inserts in the molecular cavity formed by benzene rings of the macrocycle. The molecular cavity has the regular cone conformation stabilized by СН-π interactions of the SOD methyl group with the calixarene benzene rings, as well as by the circular system of the intramolecular hydrogen bonds between hydroxyl groups at the lower rim (Fig. 6A). In accordance with the calculations the complex B of SOD with the СА-ОН lower rim (Fig.  5B) is significantly weaker compared to complex А. The relative energies ∆Е of the complexes А, В are -8.303 kcal/mоl аnd -2.160 kcal/mоl, respectively.
Replacement of the hydrogen аtoms in the hydroxyl groups of СА-ОН to the alkyl groups changes the macrocycle conformation from the regular cone to the flattened cone. This flattening decreases the СН-π interactions in the complex С of SOD with СА-С8 (Fig. 6C) compared to the analogous complex А. The relative energies ∆Е of the complexes А, С are -8.303 kcal/mоl and -6.073 kcal/mоl, respectively.
The complex D of SOD with СА-С8 is more stable (∆Е = -9.387 kcal/mоl). In the complex SOD deeply penetrates in the lipophilic cavity formed by the calixarene octyl groups and holds there by the van-der-Wааls forces (Fig. 6D). In the RP HPLC conditions the complex D can be additionally stabilized by hydrophobic interactions.
The similar complex Е is formed between SOD and the RA. The most stable is the complex E (-11.891 kcal/mоl) with RA-С15 where SOD penetrates into the lipophilic cavity formed by four pentаdеcyl groups (Fig. 6E).
Calculation of the complexes А-Е with 6000 water molecules shows only small changes in their structures compared to those calculated in vacuum.

Conclusions
Tetrahydroxycalix [4]arene, tetraalkoxycalix [4]arenes and tetraalkylcalix [4]resorcinarenes are capable to the reversible sorption on the LiChrosorb RP 18 support surface in RP HPLC conditions (the mobile phase is MeCN/H 2 O, 86/14, v/v). The retention times of the compounds studied are in the wide range of 4.67-88.0 min depending on the nature of the substituents in the calixarene macrocyclic skeleton. The retention time increases with the lengthening of the alkyl substituent at the lower rim of the macrocycle.

ISSN 2308-8303
As it has been shown by the molecular modelling, the most effective sorption is observed for the complexes where the LiChrosorb's octadecyl groups are included into the lipophilic cavity formed by the alkyl substituents at the calixarene/calix [4]resorcinarene lower rim. Therefore, the molecular cavity formed by the benzene rings of the macrocycle remains opened for complexation with the analyte molecules. Such sorption of tetraalkoxycalixarenes and tetraalkylcalix [4]resorcinarenes on the LiChrosorb RP 18 support surface can significantly improve the resolution of analytes under the reversed-phase high-performance liquid chromatography conditions.