Mechanism of enantioseparation on zeolite-supported chiral stationary phases in the presence of lipophilic buffer

Summary Chiral stationary phases have been prepared on a zeolite A support. In addition to polar hydrophilic chiral selector molecules derived from DNB-L-Leu and DNB-L-Phe amides, dynamic modification of the underivatized free surface hydroxyl (silanol) groups of the zeolite can be used to increase the overall hydrophobic character. On these non-capped chiral stationary phases, in the presence of lipophilic buffer salts in the aqueous organic phase, the enantioseparation of ion-paired D,L-mandelic acid was facilitated and was studied in more detail. The enantioseparation might be based on hydrogen bonding and π-π interaction supported by simultaneous hydrophobic interaction. Presented at: Balaton Symposium on High-Performance Separation Methods, Siófok, Hungary, September 3–5, 1997.     

Using principal component analysis for the study of the retention behaviour of phenol derivatives under reversed-phase conditions

Summary The retention time of 11 ring-substituted phenol derivatives was measured on six different reversed-phase HPLC columns and the log k, theoretical plate number (N) and asymmetry factor (F) values were calculated for each solutes on each column. The similarities and dissimilarities among the columns and solutes were elucidated by principal component analysis followed with nonlinear mapping technique and cluster analysis. Calculations indicated that the retention characteristics of porous graphitized carbon stationary phase considerably deviate from those of octadecyl- and hexyl-coated silica, octadecyl-coated polystyrene-divinylbenzene polymer and polybutadiene-coated alumina. The differences among these columns were markedly smaller. The retention behaviour of aminophenols differed from those of other phenol derivatives proving the importance of molecular polarity in the retention. It was established that the mode of calculation slightly modifies the similarity and dissimilarity among the columns and solutes, therefore, the use of more than one calculation method is proposed.     

Enantioselective chromatography: Selection of chiral recognition models

In this brief overview diverse chiral recognition models and chiral host-guest (selector-selectand, SO-SA) relationships which are used in enantioselective chromatography are discussed. In particular it is focussed on aspects of chiral interactions on (a) small molecular brush type chiral stationary phases (CSPs) and on (b) biopolymer and synthetic polymer type CSPs. The importance and the great variability of intermolecular SO-SA bindings via complementary contact sites, also in connection with molecule conformations, is stressed. Some representative and selected examples of chromatographic enantioseparations are presented.     

Optimized separation of pesticide enantiomers by chiral high-performance liquid chromatography

Summary A computer-assisted method is described for optimization of multi-component, mobile phase selection for separating enantiomers of four pesticides in normal-phase HPLC. The method is based on the triangle, solvent-selection concept using a statistical scanning method. The optimization of the separation over the experimental region is based on a special polynomial estimation from seven experimental runs, and resolution (R_s) is used as the selection criterion. Excellent agreement was obtained between predicted and experimental data.     

Retention characteristics of octadecylsilyl silica,trimethylsilyl silica and phenyldimethylsilyl silica in reversed-phase liquid chromatography

Summary The hydrophobic retention characteristics of stationary ligands for reversed-phase high-performance liquid chromatography have been evaluated from the slope (r-value) of the plots relating the capacity factors (log k) of selected aliphatic and aromatic compounds with the reciprocal of methanol concentration (log (1/[MeOH])) in aqueous mobile phase. Octadecylsilyl (ODS), trimethylsilyl (TMS) and phenyldimethylsilyl (phenyl) groups were selected as the stationary ligands bonded to silica support.On ODS or TMS silicas, unlike on phenyl silica, aliphatic compounds gave slightly larger r-values than aromatic compounds, indicating that the shape of the ligand recognizes the hydrophobic surfaces of aliphatic and aromatic solute molecules. On TMS and phenyl silicas, the degree of solute hydrophobicity contributing to its retention is about 90% and 85% of that on ODS silica, respectively. On the other hand, on TMS and phenyl silicas, the polar functional group on the solute molecule brought about a smaller decrease in retention than on ODS silica.     

Use of a displacement model for solvent sorption to study non-specific selectivity in reversed-phase liquid chromatography

Summary A thermodynamic equation is derived for the non-specific selectivity of alkyl bonded phases as a function of the mobile phase composition using a displacement mechanism to model the sorption of solvents into the bonded phase. The equation is used to calculate the thermodynamic parameters which characterize the incremental behavior of a hydrophobic group in ethyl alkanoate and methyl perfluoroalkanoate ester solutes chromatographed with water-methanol and water-acetonitrile mobile phases on both octyl and octadecyl bonded phases.     

Dependence of the methylene selectivity on the composition of hydro-organic eluents for reversed-phase liquid chromatographic systems with alkyl bonded phases

Summary A theoretical treatment is presented which considers differences between the composition of the mobile phase and solvents that are incorporated into the bonded phase via preferential sorption. Equations are derived and used to analyze retention data for various homologs chromatographed under reversed-phase conditions using alkyl bonded phases and combinations of water-methanol, water-acetonitrile and watertetrahydrofuran as mobile phases. In the case of water-methanol the surface phase and bulk mobile phase compositions are similar. However, significant differences in composition between the two phases are observed when binary combinations of water-acetonitrile and water-tetrahydrofuran are used as the cluents.     

Comparison and evaluation of HIC columns of different hydrophobicity

Summary Retention and selectivity in hydrophobic interaction chromatography (HIC) depend both on the type of stationary phase and on the mobile phase. In the last few years various high performance packing materials and columns have been introduced for HIC resulting in a range of different retentions and selectivity. We have investigated the effect of the stationary phase on the retention of various proteins. The retention of some solutes of different hydrophobicities were measured on three commercial HIC columns (TSK-Phenyl, Synchropack-Propyl, CAA-HIC) under isocratic conditions using water-methanol mixtures as eluent. The log k_w values determined according to the literature were devalues determined according to the literature were dependent on the type and structure of the stationary phase and indicated a much less hydrophobic character for these columns than that obtained for reversed phase columns. Gradient separations were then carried out on a standard protein mixture using ammonium sulfate and sodium citrate to change the gradient time. In order to compare the effect of the stationary phase and the two salts investigated apparent capacity factors (k_g) were determined and plotted against the gradient time obtained for the three columns in the two eluent system. It was shown that the type of stationary phase had a significant effect on the retention of proteins. In addition, the effect of the mobile phase composition, i.e. salt type, was considerably different on the various stationary phases. In order to exploit the potential of HIC to modulate selectivity for the separation of proteins, the combined effect of the stationary phase and the type of salt should be taken into account.Dedicated to Professor Leslie S. Ettre on the occasion of his 70th birthday.     

Combined effect of organic modifier concentration and column temperature on retention in reversed-phase ion-pair liquid chromatography

Summary The retention behavior of phenylamine and naphthylamine sulphonic acid was evaluated in reversed-phase ion-pair liquid chromatography as a function of organic modifier concentration and column temperature. It has been observed that the logarithm of capacity factors decrease linearly with organic modifier concentration, and there is a good linear relationship between the intercept and slope for this relationship. Phenylamine and naphthylamine sulphonic acid retention decreases with increase in column temperature. A linear dependence of lnk _ip on the reciprocal of the absolute temperature, the Van't Hoff plot, was observed over the column temperature range studied, and the standard enhalpic change (H^o) for these sulphonic acid transfers from the mobile phaser to the stationary phase was determined. H^o was dependent on the solute structure and in the range from 2.5 Kcal/mol to 5.5 Kcal/mol, which is close to that observed in RP-HPLC. The enthalpy/entropy compensation effect was evaluated by plotting lnk _ip(T) vs. –H^o, and the apparent differences in retention mechanisms between the analytes were observed, which may arise from the significant differences in their configuration, hydrophobicity and the charges of the solutes as well as the complex retention processes of RP-IPC.     

High-performance ligand-exchange chromatography of amino acids on chiral stationary phases

Three chiral stationary phases, obtained by grafting silica gel with (-)-trans-1,2-cyclohexanediamine, were studied for the resolution of α-amino acids by ligand-exchange chromatography. The packings were prepared by bonding the chiral ligand to silica gel via different hydrocarbon spacers. Separation of the optical isomers was accomplished by eluents containing a constant concentration of copper(II) acetate (0.05mM). The elution sequence of amino acids was found to be dependent on the grafting reaction selected to prepare the chiral packings.     

A refined physico-chemical model of solute retention in reversed-phase high-performance liquid chromatography with ternary mobile phases

Summary In our previous publication we have introduced a new model of solute retention in RP-HPLC systems with ternary mobile phases of the B+AB₁+AB₂ type (B: acetonitrile or tetrahydrofuran; AB₁: methanol; AB₂: water). That model proposed no stoichiometric differentiation between acetonitrile and tetrahydrofuran, alternatively present in the solvent system; moreover, it made some very rough assumptions only as to the intermolecular interactions among the mobile phase constituents.This paper introduces a significant refinement to the already established retention model, which is based on the simple quantitative relationships between acetonitrile and tetrahydrofuran, and the remaining components of the ternary liquid system. The refined model is tested with same experimental data.     

Enantioseparation characteristics of the chiral stationary phases based on natural and regenerated chitins

Natural and regenerated chitins were derivatized with 3,5‐dimethyphenyl isocyanate. The corresponding chiral stationary phases were prepared by coating the resulting chitin derivatives on 3‐aminopropyl silica gel. The swelling capacity of the chitin derivatives, enantioseparation capability, as well as eluents tolerance of the chiral stationary phases were evaluated. The results demonstrated no remarkable difference in enantioseparation capability between natural and regenerated chitins based chiral stationary phases. The similar enantioseparation characteristics of two chiral stationary phases could be understood by comparing the IR spectra of related chitin derivatives. The one of the two chiral stationary phases prepared by coating the chitin derivative with a lower molecular weight generally provided better enantioseparations. All chiral stationary phases can work in 100% chloroform, 100% ethyl acetate, 100% acetone, and the mobile phases containing a certain amount of tetrahydrofuran. The chiral stationary phase prepared from the chitin derivative with the highest swelling capacity exhibited better enantioseparations than others. This chiral stationary phase was damaged by flushing with 100% tetrahydrofuran, however, the enantioseparation capability was recovered again after the column was allowed to stand for 1 month. Furthermore, the recovered chiral stationary phase provided better enantioseparations for some chiral analytes than before.     

Molecular shape recognition capability of liquid-crystal bonded phases in reversed-phase high performance liquid chromatography

Summary The chromatographic retention behaviour of two liquidcrystal bonded phases have been evaluated using polycyclic aromatic hydrocarbons (PAHs) as the probe samples in reversed-phase high performance liquid chromatography (RP-HPLC). The results clearly indicate that these phases have better planarity and shape recognition capabilities than commercially-avaialble polymeric octadecylsilica (ODS) phases whose strong planarity and shape selectivities were found earlier. It can also be concluded from the chromatographic observations that the shape recognition capability of these phases is dependent on both mobile phase composition and column temperature, but that the effect of mobile phase and temperature on the shape selectivity work independently. The retention behaviour can be explained by changes in the phase structure with changes of eluent composition and temperature.     

Characterization of the properties of stationary phases for liquid chromatography in aqueous mobile phases using aromatic sulphonic acids as the test compounds

We investigated the effects of the concentration of naphthalene sulphonic acids (NSAs) as anionic test compounds in the injected sample and of the salt additives to the mobile phase on ion-exclusion. The retention behaviour of NSAs sensitively reflects even minor changes in the ionic and hydrophobic interactions and can be useful for predicting the effects of the stationary phases in reversed-phase chromatography of polar and ionic compounds, both small ones and biopolymers, e.g., oligonucleotides. We studied chromatographic properties of several stationary phases intended for separations in aqueous mobile phases: a C18 column end-capped with polar hydrophilic groups, a densely bonded C8 column doubly end-capped with short alkyl groups, a short alkyl stationary phase designed to keep full pore accessibility in highly-aqueous mobile phases and a Bidentate column with “bridged” C18 groups attached to the silica hydride support. The chemistry and pore structure of various types of column packing materials and of the salt additives to the mobile phase affect the proportion of the pore volume non-accessible to anions due to ion-exclusion and consequently the peak asymmetry and hydrophobic selectivity in reversed-phase chromatography of organic acids. We also addressed the problems connected with the determination of column hold-up volume in aqueous mobile phases. The accessibility of the stationary phase for anionic compounds in contact with the sample zone is affected by ion-exclusion due to repulsive interactions with the negatively charged surface in the pores of the stationary phase. The accessible part of the stationary phase increases and consequently the migration velocity along the column decreases with increasing concentration of the sample in the zone moving along the column. Because of a limited access to the stationary phase, its capacity can be easily overloaded. The combination of the column overload and ion-exclusion effects may result in fronting or tailing peak asymmetry. To explain this behaviour, we proposed a modified Langmuir model, respecting the variation of the column capacity due to the effects of sample concentration on ion-exclusion.     

Evaluation of novel dendrimer chiral stationary phases using HPLC

Summary The reversed phase chromatographic properties of the [G1]-L-glutamic and ethyl ester-AC-silica (1), [G2]-L-glutamic acid ethyl ester-AC-silica (2) and the [G1]-L-glutamic acidt-butyl ester-AC-silica (3) dendrimer stationary phases were evaluated. Initial studies involved the comparison between these phases with a classic reversed phase (i.e. ODS1) by the separation of a standard reversed phase test mixture composed of dimethylphthalate, nitrobenzene, anisole, diphenylamine and fluorene. Separations were achieved with comparable performance to those obtained with the conventional reversed phase (ODS1). However, it was apparent that the chromatographic selectivity exhibited by the dendrimer stationary phases was different from that of the ODS1 phase. On a per mole basis, the dendrimers exhibited similar (and sometimes greater) affinity for these analytes compared with the ODS1 ligand. Subsequent chromatographic experiments were conducted upon the dendrimer chiral stationary phases using chiral analytes under reversed phase and normal phase conditions. Chiral resolution was not observed.     

Statistical evaluation of the validity of a method for characterizing stationary phases for reversed-phase liquid chromatography based on retention of homologous series

Summary The validity of a method for characterizing stationary phases for reversed-phase, liquid chromatography, based on the use of homologous series, has been evaluated. The method is based on a retention model which describes the dependence of the logarithm of the capacity factor on mobile phase composition and the carbon number of specific homologous series. A first-order as well as a second-order version of this model was investigated. The second-order model proved to be a significant improvement on the first-order model, even for smaller mobile-phase ranges. Nevertheless both models showed a significant lack of fit, reflecting the incompleteness of these models. Therefore, it is very questionable whether this method is suitable to describe HPLC-column characteristics like hydrophobicity and hydrophylicity.     

Optimization of enantiomeric separations with chiral bonded phases

Summary The preparation and properties of chiral bonded phases with amino acid groups are described. These phases were used in non-polar eluents and in aqueous systems in the presence of Cu²⁺ ions in the ligand-exchange mode. The optimization of chiral resolution is demonstrated for both cases. With non-polar eluents similarity between bonded groups and solutes is required. The elution could be accelerated by non-protonic moderators. Ligand-exchange separation is influenced by the copper content of the eluent and the stationary phase, the organic moderator concentration, the pH and ionic strength of the buffer and by the temperature. Structural requirements of both the bonded groups and of the solutes for chiral separation are discussed.     

Enantioselective liquid-solid extraction for screening of structurally related chiral stationary phases

Summary An enantioselective liquid-solid batch extraction method is described for the screening of novel chiral stationary phases (CSPs) during optimization studies of chiral selectors derived form a common lead structure. Extraction enantioselectivity (α) values can be calculated from the enantiomeric excess ee-values of the selectand, which are measured in the liquid phase by enantioselective HPLC. Extraction α-values have been correlated with chromatographic α-values. The influence was studied of several experimental parameters of the assay (pH_a, buffer concentration, temperature, selector/selectand and phase ratio) on the enantiomeric excess (ee) values of the selectands and the enantioselectivity of the CSPs, respectively. The derived statistically significant model has then been implemented to predict chromatographic α-values of novel CSPs. For example, an ee of 89.3% for DNB-Leu as selectand could be achieved in batch extraction for a novel synthesized but mechanistically similarly-acting CSP derived form quinine. This corresponds to a calculated extraction α-value of 17.7. Based on this α_extraction a chromatographic α-value of 28.8 was predicted by the linear correlation model; the experimental HPLC α-value of 31.7 was in good agreement and demonstrated the validity of the proposed screening method. The method is particularly helpful in SO optimization studies.     

High-performance liquid chromatographic investigation of the enantioselectivity and mechanism of chiral recognition of new cholic acid-based stationary phases

Summary To elucidate the mechanism of chiral recognition of cholic acid-based stationary phases, four new cholic acid derivatives, with differently substituted carbamate or three acetoxy groups, were bonded to a hydrosilyl-modified silica gel. Their capacity to discriminate between enantiomers was evaluated in normal-phase high-performance liquid chromatography. The results were compared with those from equivalent separations on trihydroxy- and 3α-phenylcarbamate-substituted cholic acid-based bonded phases. The influence of mobile phase composition of the separation of the enantiomers of amino alcohols was shown. Different mechanisms of chiral discrimination are discussed, highlighting the influence of the nature of the carbamate on enantioselectivity.