Retention In Reversed-phase Liquid Chromatography Research Articles

Peculiarities of an imidazole derivative retention mechanism in reversed-phase liquid chromatography: β-cyclodextrin concentration and temperature considerations

The retention mechanism of a series of imidazole derivatives in reversed-phase liquid chromatography (RPLC) was investigated over a range of column temperatures and β-cyclodextrin concentrations. The changes in van't Hoff plots as a function of the β-cyclodextrin concentration were examined. Enthalpy, entropy and the Gibbs free energy were determined for the two physicochemical processes: (i) the solute transfer from the mobile to the stationary phases, (ii) the solute complexation by β-cyclodextrin. These thermodynamic data showed that the solute retention mechanism was dependent on the β-cyclodextrin concentration in the mobile phase. Enthalpy–entropy compensation revealed that the main parameter determining retention increased as follow: β-cyclodextrin⇄solute complexation>RP 18 stationary phase⇄solute interaction. This fact confirms that the main parameter determining retention in RPLC is the distribution of the solute molecule in the mobile phase, whereas the interactions with the stationary phase play a minor role.

Study of retention in reversed-phase liquid chromatography using linear solvation energy relationships: II. The mobile phase

The applicability of linear solvation energy relationships (LSERs) to reversed-phase liquid chromatography (RPLC) was studied by examining the retention of a wide variety of aliphatic and aromatic compounds over the range of 20–50% (v/v) acetonitrile, methanol and tetrahydrofuran. The role of cavity formation, dispersion interaction, polarity/polarizability, hydrogen bond acidity, and hydrogen bond basicity in determining the retention behavior as the mobile phase composition was changed has been investigated. The LSER coefficients were then examined in terms of the corresponding properties of the mobile phase (cohesive energy density, surface tension, the Abraham solvophobic parameter, polarity/polarizability, hydrogen bond basicity, and hydrogen bond acidity) and from these the influence of mobile phase and stationary phase on the retention behavior was explored. In order to chemically interpret the RPLC retention results we compared them to alkane–water and octanol–water partition coefficients.

Retention Behavior of p-Alkylphenols in Reversed-Phase Liquid Chromatography Using Ethanol/Water Mixtures.

The retention behavior of p-alkylphenols was studied in reversed-phase liquid chromatography using octadecylsilyl-silica gel and ethanol/water mobile phases. The results were compared with those in methanol/water systems. Linear correlations were observed between the logarithmic retention factor (ln k) and the carbon number of side chains in the solutes, irrespective of the type and volumetric fraction (φ) of the organic modifiers in the mobile phases. The straight lines mea-sured at different φ converged at almost one point. By analyzing the slope of the linear correlations, a functional contribution of a methylene group to k and a reduction of the hydrophobic surface area of the solutes and octadecylsilyl ligands due to the retention were estimated. It was also attempted to analyze the characteristics of the retention in reversed-phase liquid chromatography on the basis of solvophobic theory.

Mixture-design approach to retention prediction using the solvation parameter model and ternary solvent systems in reversed-phase liquid chromatography

A new approach for the prediction of retention in reversed-phase liquid chromatography using ternary mobile phase compositions is presented. The solvation parameter model is used to generate system constants characteristic of the capability of the chromatographic system for defined solvent–solvent and solute–solvent interactions. A mixture-design approach is then used to construct system surfaces for each system constant over all mobile phase compositions. These surfaces are smooth and devoid of irregular features. Models derived from these surfaces allowed us to predict the retention of a varied group of 36 solutes on a porous polymer sorbent, PLRP-S 300, at a methanol–acetonitrile–water composition not included in the data used to construct the system surfaces. The accuracy of the predicted retention factors was similar to that obtained by fitting the solvation parameter model to the same experimental data.

Effect of mobile-phase composition on pressure-induced shifts in solute retention for LC separations using β-cyclodextrin stationary phases

Modest pressure (<350 bars) has a significant impact on reversed-phase liquid chromatographic retention using β-cyclodextrin stationary phase. Demonstrated here for separations of the positional isomers of nitrophenol and naphthol as well as the enantiomers of hexobarbital and mephobarbital, the effect of small pressure perturbations on solute capacity factor ranges from +11 to −14%. These changes in retention arise from pressure-induced shifts in solute equilibria and are used to estimate the change in molar volume upon solute binding with β-cyclodextrin (ΔVcomp). In this article, the effect of the mobile phase on pressure-induced shifts in solute retention is examined. Mobile-phase composition is observed to have a significant influence on the change in solute capacity factor with pressure and therefore on ΔVcomp. As the composition of water in the mobile phase increases, the change in partial molar volume of complexation is observed to become more positive for both positional isomer and chiral separations. Although these studies imply that changes in solvent composition have a general effect on the observed ΔVcomp and therefore on the change in pressure perturbation with solvent composition, it is clear that more studies are needed to establish the exact nature and breadth of this effect. © 1998 John Wiley & Sons, Inc. J Micro Sep 10: 647–652, 1998

Effect of mobile‐phase composition on pressure‐induced shifts in solute retention for LC separations using β‐cyclodextrin stationary phases

Modest pressure (<350 bars) has a significant impact on reversed-phase liquid chromatographic retention using β-cyclodextrin stationary phase. Demonstrated here for separations of the positional isomers of nitrophenol and naphthol as well as the enantiomers of hexobarbital and mephobarbital, the effect of small pressure perturbations on solute capacity factor ranges from +11 to −14%. These changes in retention arise from pressure-induced shifts in solute equilibria and are used to estimate the change in molar volume upon solute binding with β-cyclodextrin (ΔVcomp). In this article, the effect of the mobile phase on pressure-induced shifts in solute retention is examined. Mobile-phase composition is observed to have a significant influence on the change in solute capacity factor with pressure and therefore on ΔVcomp. As the composition of water in the mobile phase increases, the change in partial molar volume of complexation is observed to become more positive for both positional isomer and chiral separations. Although these studies imply that changes in solvent composition have a general effect on the observed ΔVcomp and therefore on the change in pressure perturbation with solvent composition, it is clear that more studies are needed to establish the exact nature and breadth of this effect. © 1998 John Wiley & Sons, Inc. J Micro Sep 10: 647–652, 1998

Revisionist look at solvophobic driving forces in reversed-phase liquid chromatography: II. Partitioning vs. adsorption mechanism in monomeric alkyl bonded phase supports

The partitioning and adsorption mechanisms of retention in reversed-phase liquid chromatography (RPLC) were examined based on the free energy of transfer of a methylene group from organic aqueous mixtures to bulk hexadecane. Retention on monomeric bonded phases with octyl chains or longer are dominated by a partitioning-like mechanism. An adsorption-like mechanism contributes to retention in monomeric bonded phases with short bonded chains or with low surface coverage density. The retention mechanism becomes more adsorption-like as the length of the bonded chains decrease.

Revisionist look at solvophobic driving forces in reversed-phase liquid chromatography IV. Partitioning vs. adsorption mechanism on various types of polymeric bonded phases

The partition and adsorption mechanisms of retention in reversed-phase liquid chromatography have been examined based on a comparison of the free energy of transfer of methylene groups from aqueous-organic mixtures to bulk hexadecane with those to a variety of polymeric bonded phases. The stationary phases studied include: conventional silica-based polymeric phases of various alkyl chain lengths, a so-called “horizontally polymerized” octadecyl phase on silica and a series of polybutadiene-coated zirconia phases. The data indicate that for methylene groups a partition-like mechanism is dominant on all phases. On the polybutadiene-coated zirconia and “horizontally polymerized” octadecyl phases the partition mechanism holds at all mobile phase compositions. In contrast on conventional polymeric silica phases the retention mechanism seems to become more adsorption-like at methanol compositions greater than about 70% (v/v).

Study of Retention Behavior of Pesticides for Reversed Phase Liquid Chromatographic Separation by Quantitative Structure-Retention Relationships

Regression models that are useful for the explanation of the reversed phase liquid chromatographic retention behavior of the pesticides; triazines, phenoxyacetic esters and acetanilides were studied by quantitative structure - retention relationships (QSRRs). Descriptors were theoretically calculated by MM+ and AM1 method. The retention of triazines and phenoxyacetic esters in methanol-water was explained by solvation energy of specific site of solute, total solvation energy and polarizability which were chosen as nonspecific descriptors for acetonitrilewater. The retention of acetanilides was dependent not only on solvent-accessible surface area but on conformation of solute.

Temperature Effects on Retention in Reversed-phase Liquid Chromatography of Nucleosides and Their Bases Using Water as the Mobile Phase

Using elevated temperature, a number of hydrophobic organic compounds were separated rapidly by reversed-phase liquid chromatography (RP–LC) using a conventional octadecylsilane (ODS) stationary phase and pure water as the mobile phase. Thirteen nucleosides and their bases were used as representative hydrophobic analytes and two types of polymeric ODS-packed columns were chosen as representative reversed-phase columns. The experimental results showed that the partitioning behaviour of the hydrophobic analytes between the stationary and mobile phases was extremely dependent on the temperature of the columns. By elevating the temperature of the columns or by using temperature programming methods, the analytes which were bound by the stationary phase could be released to the mobile phase without need for any organic solvent in the mobile phase. That is, the separation could be achieved with pure water as the mobile phase. The observed effects were attributed to two effects. First, the elevated temperature enhanced the mobility of the bonded stationary phase, resulting in reduced hydrophobic interactions between the analytes and the stationary phase. Second, elevating the temperature enhanced the water solubility of the analytes, thereby increasing their transfer to the mobile phase. This technique can be employed in order to reduce or even eliminate the use of organic solvents in some RP–LC separations.

Study of retention in reversed-phase liquid chromatography using linear solvation energy relationships I. The stationary phase

In contrast to almost all previous work in which data sets of retention for reversed-phase liquid chromatography were limited to aromatic solutes, in this work the retention of 87 highly variegated, aliphatic and aromatic solutes have been studied using linear solvation energy relationships. Results show excellent statistical fits for these retention data obtained in 50:50 (v/v) acetonitrile-water, on five bonded phase columns differing in silanol group acidity. The fits are equally good when aliphatic and aromatic solute subsets were examined separately. The most important retention-governing solute parameters are the solute volume and hydrogen bond acceptor basicity. The solute dipolarity/polarizability and hydrogen bond donor acidity are statistically significant but chemically minor factors. The relative importance of the solute parameters in explaining the data does not differ for the various bonded phases studied, but there are some subtle differences between the aliphatic and aromatic solute subsets.

Temperature Dependence of Retention in Reversed-Phase Liquid Chromatography on a Porous Acrylic Support

The solute retention mechanism of reversed-phase liquid chromatography is examined by using newly synthesized hydrophilic porous copolymer beads as a stationary phase. It is shown that when water is used as an eluent, the retention time for solutes such as alcohols, ketones and polyethylene glycols increases with temperature. Adsorption behaviour of polyethylene glycol onto the copolymer beads shows that the adsorption also increases with temperature. These observations are explained on the basis of the hypothesis that hydrophobic interaction plays a vital role in solute retention.

Effect of the eluent pH on the thermospray molecular ion intensity of nucleosides

The protonated molecular ion intensities of 15 nucleosides obtained by thermospray ionisation have been measured using 0.1 M ammonium acetate mobile phase at neutral and acidic pH. To explain the dependence of the molecular ion intensity on the mobile phase pH, the hydrophobicity, the pK a values and reversed-phase high-performance liquid chromatographic retention data (log k′) at neutral and acidic pH values were studied. Significant correlations (above 95% probability level) were found between the change in the protonated molecular ion intensity and the hydrophobicity as well as the p K a values of the compounds. The reversed-phase chromatographic retention parameter (log k′) obtained at pH 3.5, showed significant correlation together with the p K a values to the molecular ion intensity change caused by decreasing the mobile phase pH. None of the investigated nucleosides showed an increased molecular io intensity change at low pH when more than 5% methanol was present in the mobile phase.

Binding forces contributing to reversed-phase liquid chromatographic retention on a β-cyclodextrin bonded phase

Reversed-phase liquid chromatographic (RPLC) capacity factors for a number of organic solutes on a β-cyclodextrin (β-CD) bonded phase column in pure water mobile phase ( k w ) were compared with formation constants of inclusion complexes ( k′ f) between β-CD and the solutes based on the linear solvation energy relationship (LSER) in order to understand the types and relative strengths of various intermolecular forces between CD and the guest solute affecting the stability of inclusion complexes and hence retention in RPLC. A close fit of capacity factors ( k′ w) with complexation constants indicated that inclusion complexation is the major driving force in retention on the β-CD-bonded phase in RPLC. Comparison of LSERs for K f and k′ w showed that an increasing guest molecular size stabilizes the complex by virtue of increasing dispersive interactions between the hydrophobic interior of CD cavity and the guest and hence increases the RPLC retention, and that increasing guest dipolarity and hydrogen bond (HB) acceptor basicity lead to a decrease in the stability of the complex due to the stronger dipolar and HB interactions with water, and hence decrease the retention.

Application of Quantitative Structure-Retention Relationships for Reversed-Phase Liquid Chromatographic Separation of Pesticides.

Quantitative structure-retention relationships (QSRRs) models that predict the reversed-phase liquid chromatographic retention behavior of carboxamides and oxadiazoles are proposed. The intermolecular interaction, isomeric effect and substituent effect were explained by the descriptors determined from calculations with MM+ and AM1 methods. The retention of carboxamides was elucidated by using solvent-accessible surface area and x component of dipole moment. For oxadiazoles, 1-octanol/water partition coefficient (log P) and dipole moment were useful descriptors.

Rule of Intersection Point between Retention of Homologues and Column Temperature in Reversed-Phase Liquid Chromatography

The rule of intersection point between retention of homologues and column temperature in reversed-phase liquid chromatography is reported, based on the thermodynamic theory, with the basic equation of the retention in reversed-phase liquid chromatography, proposed by Vigh and others. We have derived the rule of intersection point between the logarithm of capacity factor and column temperature for homologues in reversed-phase liquid chromatography, and used the experimental data to verify it. The result is satisfactory.

Retention Behavior of Poly(Methyl Methacrylate) and Poly(Ethylene Glycol) in Reversed-Phase Liquid Chromatography

The reversed-phase liquid chromatographic retention behavior of 33,500-Dalton and 75,000-Dalton poly(methyl methacrylate) (PMMA) and 26,000-Dalton to 913,000-Dalton poly(ethylene glycol) (PEG) samples are examined. It is found that isocratic retention can be obtained under carefully controlled experimental conditions, and the retention behavior of PMMAs and PEGs can be explained by conventional chromatographic models. Linear plots of the logarithm of the capacity factor (k') versus volume fraction (Φ) of the strong solvent are obtained for both PMMA and PEG samples. The slope (S) values in these plots are found to be in good agreement with S values calculated from two runs by gradient elution. S values from two gradient runs are used to successfully predict the retention time of the PMMA samples in a third gradient system.

Influence of solvent effects on retention in reversed-phase liquid chromatography and solid-phase extraction using a cyanopropylsiloxane-bonded, silica-based sorbent

The solvation parameter model is used to characterize the retention properties of a cyanopropylsiloxanebonded, silica-based sorbent with methanol, acetonitrile, tetrahydrofuran, and isopropanol in water as mobile phases. The system constants over the composition range 1 to 50 % (v/v) organic solvent indicate that retention occurs because of the relative ease of cavity formation in the solvated stationary phase compared to the same process in the predominantly aqueous mobile phase as well as from more favorable stationary phase interactions with solutes containing π- and n-electrons. The capacity of the solute for dipole-type interactions is not important whereas all hydrogen-bond-type interactions result in reduced retention. Graphing the system constants as a function of mobile phase composition provides a simple mechanism for interpreting the change in capacity of the chromatographic system for retention in terms of changes in the relative weighting of fundamental intermolecular interactions. A comparison is also made with the retention properties of an octadecylsiloxane-bonded, silica-based sorbent with 30 % (v/v) methanol in water as the mobile phase and the extraction characteristics of a porous polymer sorbent with 1 % (v/v) methanol, acetonitrile, tetrahydrofuran, and isopropanol in water as the sample processing solvent. Changes in sorbent selectivity due to selective uptake of the processing solvent are much smaller for the cyanopropylsiloxane-bonded sorbent than the results found for a porous polymer sorbent.

Influence of solvent effects on retention in reversed-phase liquid chromatography and solid-phase extraction using a cyanopropylsiloxane-bonded, silica-based sorbent

Influence of solvent effects on retention in reversed-phase liquid chromatography and solid-phase extraction using a cyanopropylsiloxane-bonded, silica-based sorbent

Quantitative structure-retention relationships of acyclovir esters using immobilised albumin high-performance liquid chromatography and reversed-phase high-performance liquid chromatography

Acyclovir and 18 of its esters have been investigated by systematic measurement of their reversed-phase high-performance liquid chromatographic retention using differing mobile phase compositions. The methanol content of the mobile phase was varied between 5 and 95%. By linear least squares regression of the logarithmic retention factor (log k′) against methanol concentration, the slope ( S)_and intercept (log k′ 0) of the so obtained straight lines were calculated for each compound. The chromatographic hydrophobicity index ( φ 0,MeOH) calculated from the S and log k′ 0 values ( φ 0,MeOH = −log k′ 0/ S) showed significant correlation ( r > 0.96) to the calculated octanol-water partition coefficients (cLog P). The albumin-binding properties of the compounds were characterised by the log k′ HSA values obtained by using an immobilised human serum albumin (HSA) HPLC column and 1% propan-2-ol 99% aqueous 10 m M phosphate buffer pH 7.0 as mobile phase. The measured albumin-binding parameters showed significant correlations to the cLog P, φ 0MeOH, S and log k′ 0 values, establishing the importance of hydrophobic properties to the interaction of the acyclovir derivatives with HSA.