Phosphorylated calix[4,8]arenes improve the RP HPLC separation of benzene derivatives

Aim. To study the effect of 5,11,17,23-tetrakis(diisopropoxyphosphonyl)-25,26,27,28-tetrapropoxycalix[4]arene and oсtаkis(diethoxyphosphoryloxy)-tert-butylcalix[8]аrene additives to the MeCN – H2O mobile phase (86:14) on the selectivity of the separation of aromatic compounds by the reversed-phase high performance liquid chromatography (RP HPLC) using a Separon SGX C18 support. Results and discussion. The process of complexation of phosphorylated calix[4,8]arenes with benzene derivatives in the mobile phase plays a key role in the RP HPLC separation of analytes. The stability constants of the inclusion complexes and the chromatographic separation coefficients of the analytes depend on the nature of the aromatic compounds and the cavity size of the calixarene macrocycle. Experimental part. The HPLC analysis was performed in acetonitrile – water (86:14) solution using a Separon SGX C18 column. The stability constants of the calixarene complexes were determined using the dependence of 1/k’ chromatographic parameters of benzene derivatives on the calixarene concentration in the mobile phase. Molecular modelling of the calixarene complexes was carried out using a Hyper Chem 8.0 program. Conclusions. The phosphorus-contained calixarenes due to their ability to form supramolecular complexes with aromatic molecules can be used as additives to the RP HPLC mobile phase and improve separation of benzene derivatives.

Calix[n]arenes -macrocyclic oligomers consisting of phenolic units linked by methylene spacers are well known complexаnts that separate different molecules in solutions [1 -9]. These compounds form supramolecular complexes with analyte molecules; they have been applied for the design of stationary chromatographic phases [10 -16], and as additives to mobile phases [17] that improve the HPLC separation of organic or inorganic analytes. In this paper we report the effect of the additives of 5,11,17,23-tetrakis(diisopropoxyphosphonyl)-25,26,27,28-tetrapropoxy-calix [4]arene 1 and oсtаkis(diethoxyphosphoryloxy)tert-butylcalix [8]аrene 2 to the acetonitrile -water mobile phase on the selectivity of the HPLC separation of some benzene derivatives. The calixarene additives improve the separation due to the formation of the host-guest inclusion complexes. The linear character of the plots of 1/k' νs the calixarene concentration in the mobile phase allows calculating the stability constants К А of the complexes. The correlations of the separation selectivity induced by the calixarene additives with a ratio of the stability constants of the host-guest inclusion complexes of benzene derivatives were found. The complexation is influenced by logP, the volume and other parameters of analytes ( Fig. 1).
The calixarene additives to the mobile phase decrease the capacity coefficient k' of benzene derivatives due to the formation of the host-guest inclusion complexes. The linear plots of 1/k' νs the calix[4]arene and calix [8]arene concentration (Fig. 4, 5) indicate the formation of the host-guest inclusion complexes with stoichiometry in the ratio of 1:1. It allows using the equation (1) for calculation of their stability constants К А : whеre: k 0 ' і k' are capacity factors of the benzene derivatives determined in the absence and in the presence of calix [4,8]arenes in the mobile phase.
It has been shown that the addition of calixarenes to the mobile phase improves the separation selectivity of benzene analytes (Таbles 1, 2). The separation coefficient (α 1 ) calculated as the ratio of the retention times of the pair of analytes depends on the size of the macrocyclic skeleton, as well as the nature, quantity and position of the substituent in the molecules of aromatic analytes.
The increase in separation selectivity of the analytes after the calixarene addition is explained by the formation of the host-guest inclusion complexes with different adsorption on the stationary phase compared to free analytes. The stability constants К A of the complexes depend on the calixarene structure, as well as on the nature, quantity, position of the substituents in the benzene analytes. The ratio of the separation selectivity (α 1 /α 0 ), induced by calixarene additives 1, 2 correlates with the ratio of the stability constants of its complexes (S = К 1 /К 2 ) (Fig. 6, 7).
Calixarene 1 functionalized by phosphonyl groups at the upper rim of the macrocycle exists in the coneconformation (Fіg. 8а). Complexation with p-aminophenol does not change the conformation. For calixarene 1 there is the possibility of forming two types of the complexes with p-aminophenol. In the comp-lex b (Fіg. 8b), the phenolic group forms the hydrogen bond with an oxygen atom of the phosphonyl group at the upper rim. In the complex c (Fіg. 8c), the phenolic group creates the hydrogen bond with an ether oxygen atom at the lower rim.
Octaphosphorylated calix [8]arene 2 (Fіg. 9a) that is larger by size with free rotation of the aromatic fragments around Ar-CH 2 -Ar bonds changes its conformation after complexation with phenol. A phenol molecule is located in the center of the calixarene cavity and forms the hydrogen bond Рh-О-Н···О(Р=O)Ar with an ether oxygen аtоm (Fіg. 9b).

HPLC analysis
The HPLC analysis was performed using the liquid chromatography system (Hitachi, Ltd., Tokyo, Japan). The column (250 × 4.6 mm i.d.) was packed with Separon SGX C18 (Merck, Darmstadt, Germany). Experiments were performed in isocratic conditions. The acetonitrile -water (86:14, v/v) mixture was used as the mobile phase. The calixarene concentrations in the mobile phase were 0.05 -0.6 mМ. The UV detector was operated at the wavelength of 254 nm, and the flow rate was 0.8 mL/min. The samples of the analytes used for injections were dissolved in the same acetonitrilewater (86:14, v/v) mixture (c = 0.01 mM). All chromatograms were obtained at 26 °C. The mobile phase which contained the calixarene additive was equilibrated for 3 h before the analysis. Under these conditions the chromatographic column was saturated with the calixarene additive.

Таble 2
The separation selectivity* of the benzene derivatives induced by calіx [8]

Molecular modelling
The molecular modelling of calixarenes 1, 2 and their complexes with the analytes was carried out by the molecular mechanics ММ+method, the force field (PM3) (Hyper Chem software package, version 8) [20]. The structures were calculated by the semi-empirical method. The RMS (standard deviation of the word root mean square) gradient was equal to 0.01 kcal/A·mol.

Conclusions
The addition of phosphorylated derivatives of calix [4]arene and calix [8]arene to the acetonitrilewater mobile phase improves the selectivity of the chro-matographic separation of benzene analytes on the Separon SGX C18 stationary phase under HPLC. The improvement of the separation selectivity is explained by forming calixarene -analyte inclusion complexes with different sorption on the stationary phase compared to those of free analytes. The efficacy of such separation depends on the size of the calixarene backbone, as well as the nature, number and position of the substituents in the benzene analyte determining the structure and stability of the inclusion complexes.