On the chromatography mechanism of reversed-phase liquid chromatography

Summary An equation is derived which can describe how the retention of solutes is influenced by the composition of the mobile phase in reversed-phase liquid chromatography, the retention of solutes in alkyl bonded stationary phase regarded as the complexation between solute molecule and the active sites on the surface of the stationary phase. When the stationary phase is not fully saturated by the organic modifier, the activity of the active sites, the activity coefficient of the adsorbed solute as well as the activity coefficient of the solute in the mobile phase depend on the composition of the mobile phase. However, when the stationary phase is fully saturated, the composition of the mobile phase mainly influences the activity coefficient of the solute in the mobile phase. In addition, the selectivity of retention is discussed in terms of the derived equation.     

Convergence of retention for small solutes in reversed-phase liquid chromatography

Summary It is shown theoretically that when the concentration of organic solvent in the mobile phase increases, or solute size decreases, log k values of small solutes in reversed-phase liquid chromatography (RPLC) will tend to have a minimum value called the convergence point. A theoretical model for evaluating the convergent coordinates of small solutes is presented by using a stoichiometric displacement model for retention (SMDR). The physical meaning of the coordinates of each kind of convergence are also elucidated. The convergence points have either two-dimensional coordinates with a common ordinate (the logarithm of the phase ratio of the column, log ) or threedimensional corrdinates with two common axes: — log and the logarithm of the molar concentration of the pure displacing agent in mobile phase, log a_D. The other axis relates to the nature of the solutes, such as carbon number of a homolog, van der Waal's surface area, hydrophobic fragment constant etc. for the latter and those and/or concentration axis for the former. The model was tested with published data and found to give a good fit.     

Retention models for isocratic and gradient elution in reversed-phase liquid chromatography

One- and multi-variable retention models proposed for isocratic and/or gradient elution in reversed-phase liquid chromatography are critically reviewed. The thermodynamic, exo-thermodynamic or empirical arguments adopted for their derivation are presented and discussed. Their connection to the retention mechanism is also indicated and the assumptions and approximations involved in their derivation are stressed. Special attention is devoted to the fitting performance of the various models and its impact on the final predicted error between experimental and calculated retention times. The possibility of using exo-thermodynamic retention models for prediction under gradient elution is considered from a practical point of view. Finally, the use of statistical weights in the fitting procedure of a retention model and its effect on the calculated elution times as well as the transferability of retention data among isocratic and gradient elution modes are also examined and discussed.     

An expert system for the selection of criteria for selectivity optimization in high-pressure liquid chromatography

Summary Systematic procedures for the optimization of chromatographic selectivity require objective criteria to characterize the quality of separation in a chromatogram. Numerous criteria have been suggested. Different criteria yield different results and the choice will depend on a large number of factors. It is genuinely difficult to select the most suitable criterion in a particular situation. For these reasons, an expert system has been developed to assist chromatographers in the selection of optimization criteria. A structured representation of the required knowledge and its implementation in an expert-system shell are presented in this paper.     

Indirect detection in liquid chromatography. II. Response models for reversed-phase non-ionic systems

Summary Uncharged solutes devoid of inherent detectable properties can be monitored and quantified in reversed-phase chromatographic systems by including a non-ionic detectable component (probe) in the eluent. Response models have been developed, which are based on the fact that the distribution of the solute and the probe are connected to each other by common interaction effects, such as competition for the binding surface. The direction and magnitude of the indirect response can be predicted quantitatively by means of expressions which clearly show the influence of the retention of the solute relative to the probe as well as the effect of the solid phase coverage by the probe. The relatively low response obtained in non-ionic systems may be due to a low loading of the probe on the solid phase and to a simultaneous distribution of the solute in complexed and uncomplexed forms, the two distribution modes giving opposite response patterns.     

Factors influencing retention and resolution of substituted alkylbenzenes in reversed-phase liquid chromatography

Summary The retention data of isomeric xylenes, ethyltoluenes and diethylbenzenes, and of mesitylene, benzene, toluene and ethylbenzene were obtained on a reversedphase column using methanol-water and ethanol-water mobile phases at four different temperatures. This database was used to relate the dependence of solute retention and resolution on the polarity of the mobile phase, solute dipole moment, and column temperature. The additivity of the free energy of the transfer of solute molecules or solute segments from the stationary phase to the mobile phase, was examined for the isomeric compounds. For this, the logarithm of the net retention volume was substituted for the free energy. Deviations from the additivity of free energies indicate that the separation of isomeric substituted alkylbenzenes is governed by their differential interactions with both the polar mobile phase and nonpolar stationary phase. Among the disubstituted alkylbenzenes,ortho-isomers favor the mobile phase more andpara-isomers tend to prefer the stationary phase more. Themeta-isomers are found to follow the additivity rule closely. These trends are amplified as the polarity of the mobile phase is increased indicating that these isomers are resolved better in water-rich mobile phases. These findings are substantiated by analogous results from gas-liquid chromatographic retention data, estimation of dipole moment effects, and examination of the entropic and enthalpic contributions to the net retention volume.Dedicated to Professor Leslie S. Ettre on the occasion of his 70th birthday.     

Retention behavior of dipeptide isomers in reversed-phase high-performance liquid chromatography

Summary The effects of eluent pH and organic modifier concentration on the capacity factor (k) and selectivity of dipeptide isomers were investigated. It has been observed that the variation in the logarithm of the capacity factor of the dipeptide isomers is linearly dependent on the organic modifier concentration (C_b), however, the selectivity is almost independent of it. Both capacity factor and selectivity were seriously affected by the pH of the eluent. Both the capacity factor and the intercept of the ln k vs. C_b plot increased with increasing van der Waals volume of the non-polar amino acid subunit of the dipeptides.     

Correlation between retention and the C18 silica surface coverage in reversed-phase liquid chromatography

Summary Members of three homologous series and five non-homologous solutes with various functional groups were chromatographed on a series of well-characterized C₁₈ reversed stationary phases with a range of methanol-water mobile phases. Measured capacity factors of solutes were correlated with the concentration of C₁₈ ligands on the stationary phase. A linear relationship with the slope depending on a solute molecular structure and the volume fraction of methanol in the mobile phase was obtained. A method for the evaluation of phase ratio is also proposed. Presented at the 17th International Symposium on Chromatography, September 25–30, 1988, Vienna, Austria.     

Validation of a strategic approach to high-performance liquid chromatographic method selection

Summary A recently reported chromatographic method selection strategy has been validated using fifteen drug formulations selected randomly from the Belgium Compendium, 1992. On the basis of the hydrophobic and the acidic — basic properties of the sample components, reversed-phase was recommended as the first choice mode for all formulations. For two multicomponent formulations the range of analyte polarity dictated the need for gradient elution. The commerical software DryLab G/plus^® was used for selection of optimum gradient conditions. The results obtained by both isocratic and gradient chromatography are discussed, as is the usefulness of a tailing suppressor in both modes.     

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.     

Two- and three-parameter equations for representation of retention data in reversed-phase liquid chromatography

Two-parameter equations that describe the dependence of ln kappa upon psi, where kappa is the retention factor and psi the volume fraction of the organic modifier in the mobile phase, are examined in what concerns the underlying approximations and their performance to fit experimental data obtained from reversed-phase liquid chromatography. Using 293 experimental systems, it was found that the performance of these equations to describe ln kappa versus psi data is rather low, since the percentage of the systems that can be described satisfactorily ranges from 40 to 60% depending on the fitting equation. This percentage may be raised to 75%, if the discreteness effect is properly taken into account. A further improvement to 90% of the systems studied can be achieved only by the use of three-parameter equations, which may arise by refinements of the rough approximations of the two-parameter equations. Although the refinements do not lead always to better equations, we developed a new three-parameter expression of In kappa that works more satisfactorily, since it combines simplicity, linearity of its adjustable parameters and the highest applicability.     

Correlation of the retention data of polyaromatic hydrocarbons obtained on various stationary phases used in normal- and reversed-phase liquid chromatography

Summary The correlation between the retention data of polyaromatic hydrocarbons (PAH) obtained in normal-and reversed-phase liquid chromatography is investigated in order to determine the dominant factors controlling the retention. It is clear that the separation of PAHs on various chemically-bonded packing materials in normal- and/or reversed-phase modes is primarily controlled by the molecular structure and shape. The -electron interaction between the solute and the stationary phase also contributes to the retention, although pure silica shows a somewhat different behavior.     

Effect of the organic modifier on the retention of retinoids in reversed-phase liquid chromatography

Summary The retention of retinoids in reversed-phase liquid chromatography was studied using aqueous mobile phases of different composition (methanol 94–86% and acetonityrile 92–82%) at five temperatures (40–60 °C). With both organic modifiers the effect of the molecular structure increased as the water content and the polarity of the mobile phase increased. The temperature-dependence increased in the same manner with aqueous acetonitrile mobile phases. The - interactions between the retinoids and acetonitrile diminish when the water content of the mobile phase is increased, as happens also to the hydrophobic interactions with both organic modifiers. The net effect of these changes depends on the composition of the mobile phase. There was excellent correlation of retention with all polarity parameters studied(, P, x_e, x_d, x_n, E _T ^N , _T, , _o and _d), when the calculations were made separately with methanol and acetonitrile. The volume fraction of the organic modifier, , was the only parameter describing the retention well in both organic modifiers simultaneously.     

Polysiloxane-encapsulated stationary phase for reversed-phase high-performance liquid chromatography

Summary Silica beads of 6-μm average diameter were silanized with methylvinyldiethoxysilane and then subjected to encapsulation with poly(methylvinylsiloxane). The resulting product is a new stationary phase for reversed-phase high performance liquid chromatography (RP-HPLC) which has superior ability for the separation of polar, non-polar and basic compounds. The chromatographic peaks are symmetric. Its stability has been studied; after continuous use for three months the carbon content and chromatographic behaviour of the phase were unchanged. on to the silica surface to given an uniform organic film. Material prepared in this way has both good chromatographic behaviour and superior selectivity. Because contact of the silica matrix with the mobile phase is avoided, the alkali-resisting ability of the stationary phase is increased. The non-specific adsorption of alkaline solutes on to the silica surface is also avoided because of the complete coverage of surface silanol groups. Reports of stationary phases encapsulated with polystyrene [6], polybutadiene [I] and octadecylsiloxane polymers have recently appeared in the literature [3]. In this paper we report the encapsulation of poly-(methylvinylsiloxane) (analogous to the phase SE-31 often used in GC) on to a silica matrix previously modified with methylvinyldiethoxysilane. The resulting phase has superior performance in reversed-phase HPLC.     

Indirect detection in reversed-phase liquid chromatography: Ion-pair retention models for cations on polystyrene polymers

Summary The distribution equilibria of cationic compounds in reversed-phase chromatographic systems (ion-pair chromatography) have been studied on the basis of their effect on a detectable mobile phase component. The solid phase was a polystyrene-divinylbenzene copolymer and the detectable component, a quaternary ammonium ion, 1-methylpyridine. The solutes were mono- and divalent amines and quaternary ammonium ions. The cations can be retained by ion-pair adsorption and ion exchange. Expressions for the ion-pair retention of the solutes and the mobile phase cation (system peak) have been developed assuming Langmuir distribution of ion pairs to a solid phase with one kind of binding site. The validity of the expressions has been tested by evaluation of ion-pair distribution constants using non-linear curve fitting techniques. Good agreement for the constants of common ion pairs was obtained from different kinds of capacity ratio expressions. Ion exchange retention can appear beside ion-pair retention, and it has been observed in the pH range 1.6–6.1. The effect depends not only on cations in the mobile phase, but also on the nature of the buffering systems.     

Simple models for the effect of aliphatic alcohol additives on the retention in reversed-phase liquid chromatography

Four retention models for the effect of aliphatic alcohol additives on the retention of analytes in reversed-phase liquid chromatography have been developed following either a semi-thermodynamic treatment or an empirical approach. Their performance was tested using the experimental retention times of six non-polar analytes (alkylbenzenes) and ten o-phthalaldehyde derivatives of amino acids under different isocratic chromatographic runs when a small amount of ethanol, 1-propanol, 1-butanol, 1-pentanol, 1-hexanol or 1-heptanol was added to methanol/water mixtures containing a constant amount of methanol. It was shown that for the structurally simple alkylbenzenes all the models can be adopted for retention prediction with good results. In contrast, just one out of four models, that with the fewest approximations, predicts satisfactorily the retention properties of amino acids derivatives. However, the most interesting feature is that this model can predict the effect of an alcohol-additive on the retention properties of solutes, even if this additive was not used in chromatographic runs done for the fitting procedure, provided that it belongs to the same homologous series of alkanols. This feature is also observed in all models described the retention of alkylbenzenes.     

Preparation and retention characteristics of different phenyl phases for reversed-phase liquid chromatography

Summary Three different phenyl phases were prepared. The amount of organic moiety bound on silica support was determined from thermogravimetric curves of the modified silica gel. The specific surface areas of gels were obtained from nitrogen sorption measurement at –196°C. The effect on separation and selectivity of the different bonded-phenyl functional groups was studied. The selectivity of the phenyl-bonded silica gels was examined and compared with octadecyl (ODS) silica in liquid chromatography. Corresponding to the high surface concentration of functional groups, the capacity factors of solutes, normalized to unit surface area of the adsorbent, k/S_BET were found to decrease in the sequence phenylmethyl>diphenyl>triphenyl. Polar solutes are retained in greater extent on the phenyl phases than on the ODS phase.