New insights on the retention mechanism of non-polar solutes in reversed-phase liquid chromatographic columns

A clarification of the retention mechanism of non-polar solutes in octadecyl reversed-phase chromatographic columns is attempted based on a systematic comparison of the retention in C18 and C2 columns under the assumption that the retention in C2 columns is due to adsorption. The comparison involves curve fitting procedures and tests based on the properties of special functions suggested in the present paper. For the application of this approach the retention behaviour of six non-polar solutes, benzene, toluene, ethylbenzene, propylbenzene, isopropylbenzene and tert-butylbenzene, is studied from aqueous mobile phases modified with methanol, isopropanol, acetonitrile and tetrahydrofuran using C18 and C2 reversed-phase columns. It was found that the retention mechanism in C18 columns is not the same in the four modifiers. In particular, our results show that the adsorption mechanism has a significant contribution in mobile phases modified by acetonitrile and tetrahydrofuran, the partition mechanism is likely to predominate in isopropanol-water mobile phases provided that the mole fraction of isopropanol is higher than 0.2, whereas the case of MeOH is rather obscure, since the various tests did not give a clear picture about the retention mechanism in methanol-water mobile phases.     

Laterally attached liquid crystalline polymers as stationary phases in reversed-phase high-performance liquid chromatography. V. Study of retention mechanism using linear solvation energy relationships

A linear solvation energy relationship model was used to characterize the retention behavior of a stationary phase based upon a nematic side-on liquid crystalline polymer (SOLCP) in reversed-phase liquid chromatography. The set of solutes was constituted of a high variety of compounds whose molecular sizes were considerably smaller than the mesogenic unit size. The results showed good statistical fits for these retention data in 65:35, 75:25 and 85:15 (v/v) methanol-water mobile phases. Both the cavity term and excess molar refraction are the most important favorable retention-governing parameters, whereas the solute hydrogen bond acceptor basicity is the most unfavorable retention parameter. Hydrophobicity and pi-pi interactions decrease strongly when the percentage of methanol increases, leading to an important retention decrease despite the fact that the hydrogen bond interaction weakens as the organic solvent is added. The shape recognition ability of this side-on liquid crystalline stationary phase on polycyclic aromatic hydrocarbon solutes is partly explained by the solutes' high polarizability due to the presence of pi-electrons. However, the solute polarizability is not sufficient and a stationary phase's "structure effect" must to be taken into account for the shape discrimination observed. The strong interaction between liquid crystal molecules caused likely a adsorption retention mechanism rather than a partition mechanism.     

New approach to linear gradient elution used for optimisation in reversed-phase liquid chromatography

A new mathematical treatment concerning the gradient elution in reversed-phase liquid chromatography when the volume fraction psi of an organic modifier in the water-organic mobile phase varies linearly with time is presented. The experimental ln k versus psi curve, where k is the retention factor under isocratic conditions in a binary mobile phase, is subdivided into a finite number of linear portions and the solute gradient retention time tR is calculated by means of an analytical expression arising from the fundamental equation of gradient elution. The validity of the proposed analytical expression and the methodology followed for the calculation of tR was tested using eight catechol-related solutes with mobile phases modified by methanol or acetonitrile. It was found that in all cases the accuracy of the predicted gradient retention times is very satisfactory because it is the same with the accuracy of the retention times predicted under isocratic conditions. Finally, the above method for estimating gradient retention times was used in an optimisation algorithm, which determines the best variation pattern of psi that leads to the optimum separation of a mixture of solutes at different values of the total elution time.     

Modeling the effect of solvation on solute retention in reversed-phase liquid chromatography

The retention of a homologous series of alkylbenzenes was determined on octyl and octadecyl reversed-phase columns in several polar organic liquids. Free energies of transfer were calculated by the SM5.0R classical solvation model for each organic liquid tested and for several alkanes. The relationships between the measured retention factors and the calculated free energies of transfer were then investigated. Although the natural logarithms of the retention factor and the calculated free energies of transfer were linearly correlated, the obtained free energies of transfer of the solutes did not completely explain the retention behavior of the solutes. Nonetheless, even in these pure organic liquids, the energetics of RPLC retention behaved very similarly to those of partitioning.     

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.     

Prediction of peptide retention times in reversed-phases high-performance liquid chromatography during linear gradient elution

The retention behavior of 100 peptides was studied during high-performance liquid chromatography on a C₁₈ column using aqueous trifluoroacetic acid as the mobile phase and acetonitrile as the mobile phase modifier in a linear gradient elution system. Retention times of the peptides were linearly related to the logarithm of the sum of Rekker's constants (R.F. Rekker, The Hydrophobic Fragmental Constant, Elsevier, Amsterdam, 1977, p. 301) for the constituent amino acid. Assuming this relationship, the best fit constants for this system were computed by non-linear multiple regression analysis. Using the new constants, it is possible to predict retention times for a wide variety of peptides at any slope of linear gradient, if the amino acid composition is known. It also enables accurate prediction of the retention time of peptides, whose amino acid composition in not known, after an analytical run with an alternate gradient.     

Overloaded gradient elution chromatography on heterogeneous adsorbents in reversed-phase liquid chromatography

Overloaded band profiles of phenol were measured on a C18-Kromasil column in gradient elution conditions. The mobile phase used was a mixture of methanol and water. The volume fraction of methanol was allowed to vary between 0 and 0.5. A general adsorption model, which expresses the amount of phenol adsorbed q* as a function of both its concentration C and the composition phi of the organic modifier (methanol) in the mobile phase, was empirically derived from previous independent adsorption experiments based on frontal analysis (FA) and frontal analysis by the characteristic point (FACP). Accordingly, the general model was an extension of the simplest heterogeneous model, the Bilangmuir model, to non-isocratic conditions. The low-energy sites followed the classical linear solvent strength model (LSSM), but not the high-energy sites whose saturation capacity linearly decreased with phi. The general model was validated by comparing the experimental and simulated band profiles in gradient elution conditions, in linear and non-linear conditions, as well. The band profiles were calculated by means of the equilibrium-dispersive model of chromatography with a finite difference algorithm. A very good agreement was observed using steps gradient (delta phi) from 0 to 50% methanol and gradient times t(g) of 20, 25, 30, 40, 60, 80 and 100 min. The agreement was still excellent for steps gradient from 5 to 45% (t(g) = 25 min), 5 to 35% (t(g) = 50 min), 5 to 25% (t(g) = 50 min) and 5 to 15% (t(g) = 50 min). Significative differences appeared between experience and simulation when the slope of the gradient (delta phi/t(g)) became too strong beyond 3.3% methanol per minute. This threshold value probably mirrored the kinetic of arrangement of the G18-bonded chains when the methanol content increased in the mobile phase. It suggested that the chromatographic system was not in a full thermodynamic equilibrium state when very steep mobile phase gradients were applied.     

Comparison between the isocratic and gradient retention behaviour of polypeptides in reversed-phase liquid chromatographic environments.

The isocratic and gradient elution behaviour of beta-endorphin and glucagon, two polypeptides known to exist in amphipathic alpha-helical conformations in lipophilic environments, have been examined under reversed-phase high-performance liquid chromatographic (RP-HPLC) conditions with low pH, aquo-acetonitrile mobile phases. The effects of changes in the volume fraction, psi, of the organic solvent modifier and temperature, T, on the magnitudes of the S and log k(o) values of these two polypeptides, obtained from the plots of logarithmic capacity factor (log k') vs. psi using isocratic elution conditions have been determined. These data have then been compared to the corresponding S and log k(o) values, obtained from the plots of logarithmic median capacity factor (log k) versus the median volume fraction of the organic solvent modifier (psi) derived from the linear gradient elution data, using the same n-butyl silica sorbent and related aquo-acetonitrile mobile phase conditions. As apparent from these studies, substantial differences occur in the temperature-dependent trends and magnitudes of the corresponding S and S values, or the log k(o) and log k(o) values, when these parameters are derived from experimental data acquired by these two different elution methods. Moreover, when gradient elution data for beta-endorphin and glucagon are utilised, the extrapolated values of the intercept and slope of the plots of log k vs. 1/T (corresponding to an apparent change in the median enthalpy of association, deltaH(o)assoc, or an apparent change in the median entropy of association, deltaS(o)assoc) substantially deviated from the values obtained for the thermodynamic parameters, deltaH(o)assoc and deltaS(o)assoc, derived from the log k' vs. 1/T plots using the corresponding isocratic data. These findings thus have important implications for biophysical and thermodynamic investigations when gradient elution data are employed to assess the molecular basis of the interaction of polypeptides with non-polar ligates.     

Prediction of internal standards in reversed-phase liquid chromatography: IV. Correlation and prediction of retention in reversed-phase ion-pair chromatography based on linear solvation energy relationships

This paper describes the results of the evaluation of a new solvation parameter model for reversed-phase ion-pair chromatography by linear gradient elution. This model is described as . The first six terms are the usual solvation parameter equation for neutral solutes, and the seventh term represents the contribution to retention from solute’s ionization. The last term describes the retention increase due to ion-pair effect. Retention times obtained for 60 solutes (neutral, acidic and basic) in acetonitrile/aqueous mobile phases with different ion-pair reagents (phosphoric acid, trifluoroacetic acid, heptafluorobutyric acid, perchloric acid, and hexafluorophosphoric acid) are used to evaluate the capability of the function. It is concluded that the model describes the retention of ionizable/ionized compounds under ion-pair conditions very well. Accordingly, the function extends the application of linear solvation energy relationships (LSERs) to ionizable compounds in ion-pair chromatography, and allows us to easily predict their retention for chromatographic optimization, including selectivity optimization and internal standard selection. Finally, the conclusion can be extended to ioscratic elution.     

Prediction of internal standards in reversed-phase liquid chromatography. 1. Initial study on predicting internal standards for use with neutral samples based on linear solvation energy relationships

This paper describes the results of an initial study on the application of linear solvation energy relationships (LSERs) to the prediction of internal standard compounds in reversed-phase liquid chromatographic (RPLC) method development. Six neutral samples are separated on an Inertsil ODS(3) column by either acetonitrile-water or methanol-water mobile phases under either isocratic or linear gradient conditions. After the separation conditions are optimized, the desired positions for internal standard candidates are selected based on the "open windows" of the chromatograms. The compounds with the desired retention range are then predicted based on LSERs from a database consisting of more than 700 compounds with defined physicochemical properties. The prediction requires the use of LSER coefficients under the separation conditions for each sample. They are determined a priori by performing multivariable linear regression on the retention of 20 reference solutes against their physicochemical properties. It can be concluded from the study that LSER is an excellent approach to the selection of internal standard compounds for RPLC under either isocratic or gradient elution. The average prediction error is usually within 10%, but no more than 20%. Finally, LSER approach is fast and systematic, and will save a significant amount of time and resources during RPLC method development.     

Application of the reversed-phase liquid chromatographic model to describe the retention behaviour of polydisperse macromolecules in gradient and isocratic liquid chromatography

This paper illustrates how conventional models of chromatographic behaviour can be used to predict the separation behaviour of polydisperse macromolecules. Using polystyrene and polymethylmethacrylate homo- and co-polymeric standards, the models were validated by comparing experimental retention behaviour with that predicted by the chromatographic model. The experimental retention time of each of the samples was entered into a spreadsheet application, which calculated the parameters that best described retention (for a given model). When a correlation between the relevant parameters and molecular mass was established, that correlation was used to predict the change in retention behaviour over the molecular-mass range. An expression introduced in a previous paper, to calculate the critical mobile-phase composition of a homopolymer was validated using polystyrene homopolymers. A second expression, which can predict the elution behaviour of copolymers, was also validated. This expression can predict the retention of a copolymer, based solely onthe retention of the homopolymeric units that make up the copolymer.     

Characterization of stationary phases based on monosubstituted benzene retention indices using correspondence factor analysis and linear solvation energy relationships in RPLC

In reversed-phase liquid chromatography (RPLC), the comparison of experimental results obtained from different columns is a complex problem. A correspondence factor analysis (CFA) and a linear solvation energy relationship (LSER) were applied on retention data to characterize second-order intermolecular interactions responsible for retention on a set of RPLC columns. Seven octadecyl-C₁₈ columns with different packing materials are obtained from different manufacturers and one octyl-C₈ column. The retention data were determined under isocratic conditions using a methanol–water (65:35, v/v) mobile phase. The chromatographic retention indices based on alkan-2-ones and alkyl aryl ketones retention index scales are calculated using a multiparametric least-squares regressions iterative method. The CFA and LSER results permitted to highlight that the retention indices were appropriate for studying the second-order retention mechanisms on the eight chromatographic systems investigated and exhibited the best reproducibility. Although many earlier studies have reported the use of chemometric methods to characterize chemical factors affecting retention in RPLC using retention factors as retention parameters, this is the first study based on retention indices.     

Influence of stationary phase solvation on shape selectivity and retention in reversed-phase liquid chromatography

In the past few decades, shape selectivity has drawn a great deal of attention from chromatographers. The chemistry and characteristics of bonded stationary phases such as phase type, length of bonded phase, surface coverage, and silica surface material have an effect on the shape selectivity of the columns. Although the effects of bonded phase shape selectivity are relatively well understood, one remaining question is the effect of intercalated solvent on shape selectivity. The intercalation of organic modifier and water molecules into the stationary phase is believed to introduce more rigidity into bonded alkyl chains in RPLC. The use of gas chromatography (GC) opens a new dimension to approach this question. C18 columns 4 cm in length were prepared in our laboratory and used in both LC and GC experiments. Shape selectivity and thermodynamic constants for the transfer of a solute from the mobile phase to the stationary phase have been determined as a function of monomeric octadecyl stationary phase bonding densities over the range of 1.44-3.43 micromol/m2 and a polymeric phase (nominal surface coverage 4.77 micromol/m2). Comparing LC and GC experiments, we observed: (a) similar relationships between shape and phenyl selectivities with monomerically bonded C18 phase densities; (b) different correlation of thermodynamic quantities (DeltaH degrees , DeltaS degrees , and DeltaG degrees ) versus bonded phase densities. The effects of high temperature and residual silanol groups are sources of difficulty in elucidation of the intercalated mobile phase role in selectivity and retention for GC measurements.