Selectivity In Reversed-phase Liquid Chromatography Research Articles

Enhancement of selectivity in reversed-phase liquid chromatography

In an effort to gain insight into the relationship between stationary phase solvation and selectivity, the use of short- and medium-chained-length alcohols (methanol, n-propanol, n-butanol, and n-pentanol) as mobile phase modifiers in reversed-phase liquid chromatography (RPLC) was investigated to determine their impact on chromatographic selectivity. A wide range of mobile phase compositions was evaluated because of the large effect exerted by solvent strength on selectivity. Employing a set of six vanillin compounds as retention probes, evidence is presented to support the view that an increase in the hydrophobicity of the organic modifier used in RPLC can increase the selectivity of the C 18 alkyl bonded phase while simultaneously decreasing the retention time of the eluting solutes. Thus, we are presented with an interesting paradox: higher selectivity and shorter retention times, which can be attributed to changes in either solvent selectivity and/or stationary phase solvation by the organic modifier.

The influence of column temperature on selectivity in reversed-phase liquid chromatography for shape-constrained solutes

Relationships between column temperature and retention behavior are reviewed for separations in reversed-phase liquid chromatography on hydrocarbonaceous bonded phases. Changes in selectivity are observed to occur with changes in temperature for a wide variety of stationary phase types. The largest temperature effects are observed for long alkyl chain length stationary phases and liquid crystalline stationary phases. Changes in stationary phase conformational structure are characterized by spectroscopic approaches, and correlations are made with retention behavior. In general, improved separations of isomers are usually possible at reduced column temperature. Applications are presented for classes of compounds that exhibit well-defined (constrained) molecular structure.

Evaluation of trifluoroacetic acid as an ion-pair reagent in the separation of small ionizable molecules by reversed-phase liquid chromatography

This paper describes the results of a study on trifluoroacetic acid (TFA) as an effective ion-pair reagent in the separation of small ionizable compounds by reversed-phase liquid chromatography (RPLC). Retention times were obtained when the amount of TFA in the mobile phase was gradually increased in the separation of a series of basic compounds by gradient elution. For the purpose of comparison, phosphoric acid was also used in the mobile phase. We conclude that TFA can be used as not only a mobile phase pH stabilizer but also sometimes as an effective ion-pair reagent to control both retention and selectivity in the separation of small ionizable solutes. The number of charges (or p K a values) on the solutes is not the only factor responsible for the effect of TFA. The other factor comprises the mutual accessibility of TFA and the positively charged functional groups and subsequent non-coulombic interaction. Both factors equally control the ion-pair effect. The results of the study should be applicable to other fluorinated acids, such as heptafluorobutyric acid, for the control of retention and selectivity in RPLC.

Effect of temperature on selectivity in reversed-phase liquid chromatography, a thermodynamic analysis

This paper describes the results of a theoretical analysis of the effect of temperature on selectivity for several complicated retention processes in reversed-phase liquid chromatography (RPLC). Situations included in this study are: (1) chromatography of ionizable compounds such as carboxylic, amine, amino acid, and peptide compounds; (2) chromatography of solutes that experience two independent retention modes such as amine compounds; and (3) ion-pair chromatography of carboxylic acids. The focus of the study is to evaluate the effect of temperature-dependent enthalpy change of the more retained solute on selectivity. We find that for chromatography of these ionizable compounds an improvement in selectivity at elevated temperature may be expected at high mobile phase pH, and the degree of the improvement for carboxylic compounds is roughly related to the mobile phase pH, with basic mobile phase more beneficial. Furthermore, it is found that buffers can play a very important role in affecting the retention, selectivity and change in selectivity with temperature, particularly for co-eluted compounds. For solutes that undergo two independent retention processes, a similar but more significant change in selectivity can be expected if the secondary interaction is not fully eliminated. For ion-pair chromatography of carboxylic acids, high temperature can either significantly improve or further decrease selectivity, depending on the ion-pair complex formation and its retention enthalpy. If the ion-pair complex formation and its retention enthalpy is dominant, a significant decrease in selectivity is expected with increasing temperature, otherwise an improvement in selectivity may be seen.

Influence of temperature on retention and selectivity in reversed phase liquid chromatography

The influence of temperature on retention and selectivity in reversed-phase liquid chromatography using a porous organic polymer stationary phase is quantitatively characterized using the solvation parameter model. The predominant influence of increasing temperature (20–60 °C) is to decrease the relative difference in the ease of cavity formation between the aqueous mobile phase and solvated sorbent and to decrease the hydrogen-bond acidity of the aqueous mobile phase, with other polar interactions affected to a lesser extent. Temperature variation and composition variation produce similar trends in retention, but within the easily accessible range for both variables, the capacity to change retention is much greater for composition variation than temperature variation. Temperature variation is a useful parameter for fine tuning an isocratic separation with an outcome that is easily predicted using the solvation parameter model.

Quadrupolar Effects on the Retention of Aromatic Hydrocarbons in Reversed-Phase Liquid Chromatography

Because of their large molecular quadrupole moments, aromatic hydrocarbons can give rise to strong electrostatic interactions with polar molecules such as acetonitrile and water. Such interactions may have some significant effects on the retention and separation of the aromatic compounds in reversed-phase liquid chromatography. We report here an experimental investigation of these effects in which we studied the retention behavior of a series of aromatic hydrocarbons in comparison with that of aliphatic hydrocarbons which have no appreciable quadrupole moments. The elution order of benzene, cyclohexane, and n-hexane was determined and shown to follow inversely the increasing magnitude of the molecular quadrupole moments. A linear dependence of retention on molecular polarizability was observed for both the aliphatics and aromatics, the latter being consistently less retained than the former under the same chromatographic conditions. The molecular polarizability and quadrupole moment were regressed as independent variables against the logarithmic retention factors of both the aliphatic and aromatic solutes, and an excellent correlation was found with all five different chromatographic systems studied. These results clearly show that, while the retention of aromatic hydrocarbons is primarily governed by the dispersion interactions with the nonpolar stationary phase, the electrostatic interactions of the aromatic solutes with the polar mobile phase act to reduce the retention and thus contribute favorably to the control of the relative retention and selectivity in reversed-phase liquid chromatography.

The calculation of retention and selectivity in reversed-phase liquid chromatography II. Methanol-water eluents

To calculate retention in reversed-phase, high-performance liquid chromatography a method based on the molecular structure of the analyte and the characteristics of sorbents and mobile phases has been employed. Characteristics of different ODS-columns in water-methanol eluents have been determined.

Method selectivity in reversed-phase liquid chromatography

Method selectivity in reversed-phase liquid chromatography

Method for characterization of selectivity in reversed-phase liquid chromatography : V. Calibration of the retention scale for chromatographic systems with low concentrations of organic solvents in the mobile phase

The method for the characterization and prediction of selectivity and absolute retention in reversedphase liquid chromatography based on two indices (a lipophilic and a polar one) has been previously applied to chromatographic systems with mobile phases containing 50% and more of one or two organic solvent(s). Because of a high retention, the homologues series of n-alkylbenzenes is not suitable for the direct calibration of the retention scale in mobile phases with lower concentrations of organic solvents, and the extrapolation of the calibration parameters based on the data for this series in mobile phases with low water content yields predicted retention data that are subject to significant errors. To circumvent this inconvenience and to expand the application possibilities of the above method to mobile phases with higher concentrations of water, three homologous series were tested as potential candidates for calibration standards: 3- n-alkyl-6-methyluracils, 3-alkoxycarbonyl-2-pyrazolines and alkan-2-ones. All three homologous series tested were found to be suitable substitutes for n-alkylbenzenes as calibration standards in mobile phases containing 25–50% methanol in water. The selection of suitable calibration standards for acetonitrile—water mobile phases is more difficult because of a curvature of the dependence of log k′ on the concentration of acetonitrile at low concentrations of this solvent in aqueous mobile phases. Here, the alkoxycarbonyl pyrazolines and alkan-2-ones proved useful as calibration standards in mobile phases containing 30–50% acetonitrile. The agreement between the predicted and experimental k′ was within 10% or better of the experimental k′.

Calculation of retention and selectivity in reversed-phase liquid chromatography

A method has been developed to calculate retention in reversed-phase high-performance liquid chromatography based on the molecular structure of the analyte and characteristics of the sorbents and mobile phases. A simple approach based on solvophobic theory is used.

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

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.

Entropy dominated subambient temperature separations in reversed-phase liquid chromatography

Subambient temperature selectivity in reversed-phase liquid chromatography (RP-LC) is an entropy dominated process. Since reduced temperature results in a more ordered stationary phase as well as more effective intramolecular interactions, it is useful for resolving solutes which exhibit similar retentions at ambient and higher temperatures, such as isomeric compounds. We have been investigating the effects of temperature on RP-LC selectivity for polycyclic aromatic hydrocarbon (PAH) and polyphenyl isomers over the temperature range of −20 to 70°C using methanol-water mobile phases and monomeric octadecyl stationary phases. We have observed that a high bonding density monomeric reversed-phase column (4.4 μmol m −2) exhibits improved selectivity over a low density column (1.5 μmol m −2). Shape selectivity for both columns is enhanced at temperatures of 25° C or less and this is especially demonstrated by the high density phase. Van 't Hoff plots (ln k′ vs. 1/ T) have been constructed to observe whether solute retention behavior is linear over the temperature range studied. This is observed over the entire temperature range for the low bonding density stationary phase, yet distinct curvature is noted at ca. 25°C for the high density phase. One explanation of this observation is a “phase transition” of the bonded octadecyl chains from a liquid-like state at higher temperatures to a more ordered and extended crystalline-like state at lower temperatures.

Fully automatic high-performance liquid chromatographic optimization

A good optimization routine should correctly find the best chromatographic separation conditions for mixtures of known or unknown constituents. The chromatographer must define the number of the parameters to be optimized and their ranges. However, the more parameters to be optimized and the more they interact, the more difficult and time-consuming the optimization procedure will be. A system capable of performing fully automated optimization of mobile phase selectivity in reversed-phase liquid chromatography was built. The optimization routine searches for the best conditions (trying to maximize a chromatographic response function) and for the points, inside defined experimental borders, where the least available experimental information is available. By conducting the experiment under the predicted optimum conditions and an additional experiment under conditions corresponding to the least density of information, the system was forced not to search for a local maximum, but to approach the global optimum. Peak tracking, an important part of any optimization process in high-performance liquid chromatography, was an integral part of the optimization process in high-performance liquid chromatography, was an integral part of the optimization software and was based on fuzzy theory. This implementation of an on-line identification of the sample components made a fully automated optimization of the mobile phase composition possible. Once a suitable separation had been achieved, it was necessary to validate the procedure, special attention being focused on robustness. The robustness test appraises the outcome of small variations in method conditions on the analytical performance. An important feature of this robustness analysis was the three-dimensional representation of the data as the hypersurface which helps to relate robustness to elution characteristics.

Effects of stationary phase structure on retention and selectivity in reversed-phase liquid chromatography of flavonoid compounds with methanol as organic modifier

Retention of an extended set of flavonoid compounds by octyl, octadecyl, phenyl and cyanopropyl-bonded, reversed-phase columns, with methanol, as the organic mobile phase modifier and acetic acid as the acid modifier is reported. Solvent strengths and useful ranges for both isocratic and gradient elution are determined. Relative retention is found to be independent of methanol volume fraction in the mobile phase for all the columns examined. Correlations between retention on different columns, specific selectivity effects and their dependence on molecular structure are analyzed. Practical applications for separation and identification are discussed.

Simultaneous optimization of variables influencing selectivity and elution strength in micellar liquid chromatography : Effect of organic modifier and micelle concentration

Previously, the simultaneous enhancement of separation with elution strength was reported in micellar liquid chromatography (MLC) using the hybrid eluents of water—organic solvent—micelles The practical implication of this phenomenon is that better separations can be achieved in shorter analysis times by using the hybrid eluents. Since both micelle concentration and volume fraction of organic modifier influence selectivity and solvent strength, only an investigation of the effects of a simultaneous variation of these parameters will disclose the full separation capability of the method, i.e. the commonly used sequential solvent optimization approach of adjusting the solvent strength first and then improving selectivity in reversed-phase liquid chromatography is inefficient for the case of MLC with the hybrid eluents. This is illustrated in this paper with two examples: the optimization of the selectivity in the separation of a mixture of phenols and the optimization of a resolution-based criterion determined for the separation of a number of amino acids and small peptides.The large number of variables involved in the separation process in MLC necessitates a structured approach in the development of practical applications of this technique. A regular change in retention behavior is observed with the variation of the surfactant concentration and the concentration of organic modifier, which enables a successful prediction of retention times. Consequently interpretive optimization strategies such as the iterative regression method are applicable.

Subambient temperature modification of selectivity in reversed-phase liquid chromatography

L'effet de la temperature de la colonne sur la selectivite est etudie pour la separation de melanges d'hydrocarbures aromatiques polycycliques. Trois colonnes commerciales C18 avec des surfaces de monomeres et de polymeres sont comparees

Selectivity in Reversed-Phase Liquid Chromatography

Abstract Utilizing experimentally estimated capacity factors and calculated activity coefficients of the solutes in the mobile phase, as well as octadecylsilica stationary phases of varying coverage, the selectivity of separation in a reversed-phase liquid chromatographic system is studied. The retention times were measured for n-alkanes (C5-C7), n-alcohols (C5-C7) and benzene derivatives in methanol - water mobile phases. A strictly linear relationship between the experimentally determined selectivity factor (α) and the calculated selectivity factor in the mobile phase (αm) is observed. The relative retention in the stationary phase (αs) is independent of the mobile phase strength and the silica surface coverage, i.e., αs is merely a function of the molecular structure of related solutes. Therefore, a change in surface coverage does not lead to a change in α.

Methods for characterization of selectivity in reversed-phase liquid chromatography : IV. Retention behaviour of oligomeric series

The retention behaviour of oligomeric series in reversed-phase systems with respect to the number of repeat structural units and the mobile phase composition can be described by the same set of equations as for homologous series, but the constants of these equations are strongly dependent on the structure of both the repeat structural unit and the structural residue in the molecule. The separation selectivity for the individual oligomers is determined mainly by the size (molar volume) and polarity (interaction index) of the repeat units and of the structural residue, but it also depends on the type and concentration of the organic solvent in the mobile phase. If the repeat structural unit increases both the size and polarity of the oligomer molecules, the selectivity may vary as a function of the structural parameters of the structural residue and of the mobile phase composition, and the retention may even decrease with increasing number of repeat structural units in a given series. This behaviour can be explained, characterized and even predicted (at least qualitatively) from the structural constants V x, I x, on the basis of the interaction indices model. This retention behaviour was investigated and verified for oligostyrenes, oligoethylene glycols and oligoethylene glycol nonylphenyl ethers on various C 18 and C 8 columns in mobile phases containing methanol, 1,4-dioxane or 2-propanol in water. The effectiveness of gradient elution for the separation of oligomeric series depends strongly on their structure. The presence of cetyltrimethylammonium bromide (CTAB) in the mobile phase has only a minor influence on the separation of non-sulphated oligoethylene glycol nonylphenyl ethers, but it leads to an increase in the retention of sulphated ethers, with a separation selectivity similar to that of non-sulphated ethers. The individual oligomers in mixtures containing sulphated and non-sulphated surfactants of this type can be separated in a single run using CTAB-containing mobile phases.

Method for chromaterization of selectivity in reversed-phase liquid chromatography: I. Derivation of the method and verification of the assumptions.

A new method is suggested for characterization of selectivity in reversed-phase liquid chromatography. It makes use of selectivity scale, calibrated by a reference homologous series ( n-alkylbenzenes), and of toluene as the reference standard. The selectivitiy is comprised of lipophilic and polar contributions, which are characterized quantitatively by constants related to the solute structure. For certain types of compounds, these constants can be calculated from the additive increments of structural substituents. In diagrams, where the constants Δq, characterizing the polar contribution to selectivity in one binary solvent system (such as acetonitrile—water), are plotted as a function of Δ in another binary solvent system (such as methanol—water), the compounds with an equal functional group occupy narrow, limited areas and can be distiguished from compounds with other functional groups. This may offer an aid to the identification of functional gropus in simple organic compounds from reversed-phase chromatographic data.

Method for characterization of selectivity in reversed-phase liquid chromatography: II. Possibilities for the predictiion of retention data

The method suggested for characterization of selectivity, based on an adequate calibration homologous series, is used for predictions of relative retentions of various solutes. It allows the prediction of possible changes in the order of elution with changing composition of mobile phase (selectivity cross-overs) from the combination of constants characterizing lipophilic and polar contributions to selectivity. It also allows the characterization and prediction of selectivity in ternary mobile phases containing two different organic solvents and water. Using this method, it is possible to predict the retention on one column from the constants that characterize the lipophilicites and polarities of the functional groups of the solutes measured on an- other column (with bonded alkyl chains of the same length) with a probable relative error between 10 and 20%.