Wayne R. Melander

Solvophobic interactions in liquid chromatography with nonpolar stationary phases

Abstract Solute interaction with nonpolar stationary phases in liquid chromatography is examined on the basis of the solvophobic theory. The chromatographic process is viewed as a reversible association of the solute with the hydrocarbonaceous ligands of bonded phases. A detailed analysis of the effect of the solvent on this process yields an expression for the capacity factor with essentially no adjustable constants. The theory satisfactorily accounts for the factors affecting solute retention under a wide range of experimental conditions. It makes possible the characterization of the solvophobic (eluent) strength of mixed solvents having different composition and the evaluation of the various solvophobic forces representing incremental values of the logarithm of the capacity factor. The wide applicability of nonpolar stationary phases (reversed phases) in liquid chromatography is demonstrated by the rapid separation of biogenic acids and bases on octadecylsilica columns with neat aqueous elements.

Salt effects on hydrophobic interactions in precipitation and chromatography of proteins: An interpretation of the lyotropic series

Abstract The effect of neutral salts on electrostatic and hydrophobic interactions in salting out and chromatography of proteins is treated by a simple theory. The electrostatic energy change in these processes is expressed according to the classical theory. The corresponding hydrophobic energy change is evaluated from the nonpolar contact area of the interacting species and the corrected surface tension of the medium. The property of a salt that affects hydrophobic interactions is quantified by its molal surface tension increment. This parameter is a measure of the increase in surface tension by the salt and forms the basis of a natural lyotropic series. In agreement with the theory, the analysis of solubility and Chromatographic data shows that the overall salt effect can be described by the two antagonistic effects of salts on electrostatic and hydrophobic interactions. The expression derived for the salting-out constant is employed to calculate the relative surface hydrophobicity of proteins. The results suggest that the salt effect is not only useful in the quantitative treatment of hydrophobic interactions but also in the measurement of hydrophobic properties.

Surface silanols in silica-bonded hydrocarbonaceous stationary phases : II. Irregular retention behavior and effect of silanol masking

Abstract Dibenzo-crown ethers and certain peptides with unprotected amino groups do not exhibit the regular linear dependence of the logarithmic retention on the composition of binary hydro-organic eluents on silica-bonded C8 and C18 hydrocarbonaceous stationary phases but the plots have minima. Similar results are found when the stationary phase is “naked” silica having no ligates. The results are interpreted in terms of a dual retention mechanism that postulates solute retention as a result of both solvophobic (hydrophobic) and silanophilic interactions between the eluite and stationary phase even in the case of “naked” silica. Masking of the surface silanols by increasing the water concentration of the eluent or by the addition of a suitable amine, which can also be a buffer component, is shown to attenuate silanophilic interactions so that regular retention behavior is observed with both crown ethers and peptides. Beyond its traditional use, this approach may make possible the measurement of chromatographic data without interference by silanophilic effect and, thus, facilitate the development of a retention index system. On the other hand, silanol groups at the surface of bonded phases may be essential to obtain adequate selectivity as illustrated by reversed-phase chromatography of a peptide mixture. The similarity between the retention data obtained with aqueous eluents on “naked” silica gel and on C8 and C18 alkyl-silicas implies that the relative polarity of the mobile and stationary phase is the chief distinguishing mark for classification of various chromatographic systems. Furthermore, it also suggests that the dual retention mechanism may be a rather common phenomenon which needs to be taken into account for interpretation of retention data and designing chromatographic separations.

Enthalpy—entropy compensation in reversed-phase chromatography

Abstract The enthalpy of hydrophobic interactions, which are believed to account for retention in reversed-phase chromatography, was found to have constant negative values eve at temperatures near the freezing point of water. The analysis of data obtained in reversed-phase chromatography with various octadecylsilicas under a wide range of conditions as far as the eluites** and eluents are concerned, showed a linear correlation between the logarithm of the capacity factors, measured at an appropriate reference temperature, and the corresponding enthalpies for the particular chromatographic process. This behavior is due to enthalpy—entropy compensation, which is a manifestation of free-energy relationships. As the compensation temperatures were indistinguishable, it is concluded that the intrinsic mechanism of the interaction of the eluite with the bonded stationary phase was invariant under the chromatographic conditions examined, although the nature and concentration of the organic solvent in the aqueous eluent varied substantially. Whereas compensation behavior was observed with other chromatographic systems consisting of polar stationary phases and a non-polar eluent, the calculated compensation temperatures were significantly lower than those found in reversed-phase chromatography.

Salt-mediated retention of proteins in hydrophobic-interaction chromatography : Application of solvophobic theory

Retention behavior in hydrophobic-interaction chromatography was examined within the framework of the solvophobic theory. The principal parameters which determine the effect of salt on the retention are salt molality and the molal surface tension increment of the salt. According to the theory, in the absence of special binding effects, increase in salt molality in the mobile phase or change of salt to one of greater molal surface tension increment will result in increased retention of proteins in hydrophobic chromatography. The theory is expanded to treat retention in gradient elution with linear decrease in salt concentration that is equivalent to linear increase in eluent strength. The results of the simple model lead to an expression with two parameters: the adjusted isocratic retention volume of the eluite with the gradient former and the slope of plot of logarithmic adjusted elution volume against salt molality, lambda. The latter parameter is linearly dependent on molal surface tension increment if no specific interactions between the eluite and the stationary phase and/or salt are present. In practice, deviations are to be expected from the predicted behavior due to such effects. The results of calculations are consistent with experimental results obtained with several proteins as the eluites and various salts in the eluent. Although unique values of the critical parameter lambda could not be obtained from the data, the trends showed that lambda is strongly correlated with the value of the molal surface tension increment. The prediction that increase in salt concentration in the initial eluent leads to increase in retention volume was found to be generally true, even when the isocratic retention volumes obtained with use of eluent having low salt concentration were small. Use of NaClO4 in the starting eluent led in some cases to decrease in retention volume with increase in the salt concentration at the beginning of the gradient elution. This effect may be due to specific binding effects.

Stationary phase effects in reversed-phase chromatography : I. Comparison of energetics of retention on alkyl-silica bonded phases

Abstract A large number of retention data on various hydrocarbonaceous bonded phases was examined in order to shed light on the energetics of retention in reversedphase chromatography. Plots of logarithmic retention factors measured on column pairs with different eluites but with the same eluent were used in the analysis that was extended also to retention data obtained at different temperatures. Linear correlation with unit slope indicates identical intrinsic thermodynamic behavior for the two columns, i.e., homoenergetic retention, as predicted by the solvophobic theory. Linear correlation with slope different fromunity suggests similar physico-chemical basis for retention on the two columns that is termed homeonergetic. No correlation is taken as proof that, besides solvophobic interactions with the stationary phase ligates, the eluites interact with residual silanol groups. Such heteroenergetic behavior implies the use of experimental conditions outside of solvophobic chromatography. Most bonded phases with medium - or long-chain alkly ligates showed homoenergetic retention when water-rich eluents and relatively non-polar eluites without amino functions were employed, whereas those packed with stationary phases containing short alkyl chain or adamantyl ligates revealed homeonergetic behavior. In contradistinction, only with eluents rich in organic solvent and/or with basic eluites was heteroenergetic behavior observed. It is concluded therefore that under conditions of solvophobic chromatography the main source of the differences between columns packed with hydrocarbonaceous bonded phases is a difference in phase ratio and possibly pore size distribution. In fact, if several columns exhibit homoenergetic retention behaviour it may indicate that the experimental conditions are those for solvophobic chromatography. Nevertheless the analysis clearly demonstrates that when eluites having polar groups are chromatographed in water-lean eluents the interaction with surface silanols can be significant. The approach presented can serve not only as a diagnostic tool for column and chromatographic conditions but also as a means to obtain information on the relative magnitude of column phaase ratios and to predict retention data in solvophobic chromatography.

Mobile phase effects in reversed-phase chromatography : I. Concomitant dependence of retention on column temperature eluent composition

Abstract Enthalpy-entropy compensation observed in reversed-phase chromatography affords an expression of the free energy change associated with the chromatographic process asd a function of both the eluent composition and temperature. It takes a simple form and reveals the existence of a non-compensating enthalpy residue in the absence of heat capacity effects when the free energy change is linear in slvent composition. Statistical analysis confirmed that the resulting three-parameter equation suffices to transform retention data for a solute obtained at a given temperature and solvent composition to another set of experimental conditions. With arylalkanes investigated so far, the parameters exhibit a linear relationship with the carbon number of the solutes. These findings suggest the possibility of constructing a hydrophobic indix system for characterization of biological substances on the basis of retention data.

Effect of molecular structure and conformational change of proline-containing dipeptides in reversed-phase chromatography

Abstract Peptides that contain proline residues may yield multiple peaks in high-performance liquid chromatography if the proline is not at the N-terminus. The phenomenon is caused by slow kinetics of isomerization that are on the same time-scale as the chromatographic separation with such peptides. In this study, the shape and number of peaks are examined qualitatively in view of the influence of isomerization kinetics on the bandspreading, and as functions of pH, temperature and flow velocity. These effects in the chromatography of alanylproline are shown in detail. Single peaks are obtained for each peptide investigated by proper adjustment of these varible in light of their effect on the pertinent rate and equilibrium constants.

Measurement of association constants for complexes by reversed-phase high-performance liquid chromatography

Abstract Association constants for metal-binding by various nucleotides, crown ethers and nitroso-napththolsulfonic acids in solution were measured by high-performace liquid chromatography employing appropriate eluents and non-polar bonded stationary phases. The results are in excellent agreement with pertinent literature data. The procedure is applicable to the measurement of stability constants of complexes which contain one solute molecule and are formed in the solution used as the eluent, provided species bound to the stationary phase are not involved in the complex formation and the chromatographic system is linear. The concentration of the metal ion in the appropriate mobile phase was varied and small quantities of the above substances were injected into the column. The hyperbolic dependence of the retention factgor, k, on the concentration of Na+, K+, Mg2+ or Zn2+ in the eluent allowed the use of various linear plots for the evaluation of the stability constants for complexes formed by such ions with substances mentioned above. The effect of complexation on retention is conveniently measured by the retention modulus, ν, which is given for the complex, νc, by the ratio of the retention factors of the complex and the uncomplexed solute, both measured under otherwise identical conditions. With nucleotides chromatographic retention increases upon metal binding due to reduction of electronic charge on the molecule and η > 1. On the other hand nitroso-naphtholsulfonates and crown ethers eluted faster in the form of a complex with metal ions so that η

Mechanistic study on ion-pair reversed-phase chromatography

Summary The mechanism of “ion-pair” chromatography that employs a non-polar stationary phase and an amphiphilic ion in the eluent to augment the retention of oppositely charged sample components is investigated. Examination of literature data indicates that neither of the simple limiting mechanisms, “ion pairing” or “dynamic ion exchange”, can account for the observed dependence of the retention factor on the concentration of the amphiphilic ion over a sufficiently wide range of conditions. According to the ion-pairing mechanism the analyte traverses the column in the form of an ion pair whereas the dynamic ion-exchange mechanism implies that the interaction of the analyte with the hydrophobic counter-ion adsorbed on the stationary phase surface is responsible for retention. Results of the present study indicate that a mechanistic model based on the assumption that both phenomena take place concurrently does not give adequate agreement with experimental findings either. Therefore, a so-called dynamic complex exchange model is proposed that assumes a metathetical exchange of the analyte between the amphiphilic ion bound to the stationary phase and the ion pairs formed in the mobile phase. Re-examination: of earlier experimental data and the results of computer simulation suggest that this model appropriately reflects the experimentally observed hyperbolic dependence of the retention factor on the concentration of the “ion-pairing” agent as long as the retention-attenuating effect of micelle formation is negligible.