A study of the enthalpy and entropy contributions of the stationary phase in reversed-phase liquid chromatography

The goal of this study was to elucidate the roles played by the stationary and mobile phases in retention in reversed-phase liquid chromatography (RPLC) in terms of their individual enthalpic and entropic contribution to the Gibbs free energy of retention. The experimental approach involved measuring standard enthalpies of transfer of alkylbenzenes from typical mobile phases used in RPLC (methanol/water and acetonitrile/water mixtures), as well as from n-hexadecane (a simple analogue of the stationary phase) to the gas phase, using high-precision headspace gas chromatography. By combining the measured enthalpies with independently measured free energies of transfer, the entropies of transfer were obtained. This allowed us to examine more fully the contribution that each phase makes to the overall retention. It was found that the standard enthalpy of retention in RPLC (i.e., solute transfer from the mobile phase to the stationary phase) is favorable, due to the large and favorable stationary-phase contribution, which actually overcomes an unfavorable mobile-phase contribution to the enthalpy of retention. Further, the net free energy of retention is favorable due to the favorable enthalpic contribution to retention, which arises from the net interactions in the stationary phase. Entropic contributions to retention are not controlling. Therefore, to a great extent, retention is due to enthalpically dominated lipophilic interaction of nonpolar solutes with the stationary phase and not from solvophobic processes in the mobile phase. Further, our enthalpy data support a "partition-like" mechanism of retention rather than an "adsorption-like" mechanism. These results indicate that the stationary phase plays a very significant role in the overall retention process. Our conclusions are in direct contrast to the solvophobic model that has been used extensively to interpret retention in RPLC.     

Justification of statistical overlap theory in programmed temperature gas chromatography: thermodynamic origin of random distribution of retention times

A specific distribution of compounds' standard-state changes of enthalpy and entropy between mobile and stationary phases in programmed temperature gas chromatography (PTGC) is shown to produce the Poisson distribution of retention times often postulated in statistical-overlap theory (SOT). A three-part model is proposed, in which the enthalpy change is Poisson distributed, the average entropy change depends on the enthalpy change, and the actual entropy change varies in a uniformly random manner about the average entropy change. To test the model, the entropy and enthalpy changes of 350 aliphatic and aromatic hydrocarbons in petroleum were calculated with commercial GC software. These changes are shown to follow the three-part model. The model then was used with Monte Carlo methods to mimic the enthalpy and entropy changes. The substitution of the mimicked enthalpy and entropy changes into an equation for the retention temperature in PTGC is shown to produce a Poisson distribution of retention times that is statistically significant. This finding establishes a scientific link between the thermodynamics governing retention in PTGC and the superficially ad hoc assumption of the Poisson retention time distribution in SOT. Similar thermodynamic distributions are found for flavors and fragrances and for tetrachlorodibenzo-p-dioxins and furans, which follow SOT based on the Poisson distribution, but not for polychloronaphthalenes, which do not follow that SOT.     

Combining the enantioselectivities of L-valine diamide and permethylated beta-cyclodextrin in one gas chromatographic chiral stationary phase

An L-valine diamide chiral selector was attached to a polysiloxane through a long hydrocarbon spacer giving rise to a chiral stationary phase (CSP), Chirasil-Val-C11. The enantioselective properties of this readily accessible diamide CSP under gas chromatographic conditions were found to be similar to that of the commercially available Chirasil-Val CSP prepared by a polymer-analogous route. A new binary CSP, Chirasil-DexVal-C11, was synthesized by means of simultaneous attachment of both the L-valine diamide and permethylated beta-cyclodextrin selectors to a polysiloxane using platinum-catalyzed hydrosilylation, thereby overcoming the immiscibility problem known for Chirasil-Val and Chirasil-Dex. This binary CSP retained both the enantioselectivity of Chirasil-Val-C11 toward alpha-amino acid derivatives and the unsurpassed enantioselectivity of Chirasil-Dex toward underivatized chiral alcohols, ketones, and hydrocarbons. Furthermore, it was shown that the presence of the cyclodextrin selector in Chirasil-Val-C11 significantly improved the enantioseparation of proline, which represented a problematic amino acid on diamide CSPs.     

An alternative procedure to screen mixture combinatorial libraries for selectors for chiral chromatography

An alternative process for the analysis of mixture library components for their potential as selectors for chiral chromatography is described. The procedure involves the immobilization of each enantiomer of the target racemic analyte to silica gel, followed by incubation of each resulting stationary phase with a mixture library. The adsorbed library components on the two stationary phases are then analyzed by reversed-phase liquid chromatography. A comparison of the resulting two chromatograms is made. Any peak of identical retention time but with a significant difference in intensity in the two chromatograms indicates that this component is most likely a chiral selector. Its chemical structure is then determined by LC-MS or LC-MS-MS. This new screening method significantly increases the efficiency of chiral selector determination by eliminating the need for multilibrary syntheses, as opposed to our previous method. This technique should also allow for the screening of much larger libraries as compared to our previous work.     

High-stability ionic liquids. A new class of stationary phases for gas chromatography

Room-temperature ionic liquids are a class of non-molecular ionic solvents with low melting points. Their properties have the potential to be especially useful as stationary phases in gas-liquid chromatography (GLC). A series of common ionic liquids were evaluated as GLC stationary phases. It was found that many of these ionic liquids suffer from low thermal stability and possess unfavorable retention behavior for some classes of molecules. Two new ionic liquids were engineered and synthesized to overcome these drawbacks. The two new ionic liquids (1-benzyl-3-methylimidazolium trifluoromethanesulfonate and 1-(4-methoxyphenyl)-3-methylimidazolium trifluoromethanesulfonate) are based on "bulky" imidazolium cations with trifluoromethanesulfonate anions. Their solvation characteristics were evaluated using the Abraham solvation parameter model and correlations made between the structure of the cation and the degree to which the ionic liquids retain certain analytes. The new ionic liquids have good thermal stability up to 260 degrees C, provide symmetrical peak shapes, and because of their broad range of solvation-type interactions, exhibit dual-nature selectivity behavior. In addition, the ionic liquid stationary phases provided different retention behavior for many analytes compared to a commercial methylphenyl polysiloxane GLC stationary phase. This difference in selectivity is due to the unique solvation characteristics of the ionic liquids and makes them very useful as dualnature GLC stationary phases.     

Characterization and thermodynamic studies of the interactions of two chiral polymeric surfactants with model substances: phenylthiohydantoin amino acids.

Analytical ultracentrifugation is used for determination of the molecular weights and the sedimentation coefficients of poly(sodium undecanoyl-L-valinate) (PSUV) and poly(sodium undecanoyl-L-threoninate) (PSUT) at different temperatures. Plots of absorbance as a function of radius indicates that both PSUV and PSUT are highly monodispersed. A method for evaluating the partial specific volumes using density measurements is presented. The partial specific volumes of PSUV are slightly higher than those of PSUT. In addition, the temperature dependence of the retention factor in electrokinetic chromatography was used to estimate the enthalpy, the entropy, and the Gibbs free energy of the surfactant/analyte complexes. Five phenylthiohydantoin-DL-amino acids were separated and each enantiomeric pair was completely resolved. Comparison of the thermodynamic values obtained with PSUV vs PSUT using a van't Hoff relationship suggests that PSUT, with a less favorable free energy change (i.e., less negative delta (delta G)), generates a more positive entropy change, hence slightly less chiral resolution.     

Influence of analyte overloading on retention in gas-liquid chromatography: a molecular simulation view.

In an attempt to elucidate the molecular basis for concentration (isotherm) effects on retention in gas-liquid chromatography, configurational-bias Monte Carlo simulations in the Gibbs ensemble were carried out to investigate changes in analyte partitioning caused by overloading a model chromatographic system with either an alkane or an alcohol. Squalane was used as the stationary-phase material, and the analytes included n-pentane, n-hexane, n-heptane, 1 -butanol, and 1-pentanol. Three systems were studied that differed in the mobile-phase composition: (i) a helium vapor, (ii) a n-hexane vapor, and (iii) a 1-pentanol-saturated helium vapor. While the amount of helium that partitions into the stationary phase is very small, both n-hexane and 1-pentanol partition strongly into and thereby swell the stationary phase. Although the swelling of the stationary phase leads to a reduction in the partition coefficients for the alkane solutes for both the n-hexane- and 1-pentanol-swollen stationary phases, the effects on the alcohol solutes differ markedly. Whereas saturation by n-hexane causes a decrease of the alcohol partition contants (to an extent similar to that for the alkane solutes), the saturation by 1-pentanol causes a dramatic increase of the alcohol partition coefficients; e.g., the Kovats index of 1-butanol increases by more than 150 Kovats units. The formation of hydrogen-bonded alcohol aggregates in the liquid phase is the microscopic origin for the dramatic effect of 1-pentanol saturation on the retention of alcohols.     

Investigation of Enantiomer Bonding on a Chiral Stationary Phase by FT-Raman Spectrometry

The bonding of serine, phenylalanine, and mandelic acid enantiomers on an N-3,5-dinitrobenzoyl-l-leucine chiral stationary phase (on zeolite A support) has been investigated by FT-Raman spectrometry. It was found that retention is due to hydrogen bonds and π-stacking interactions between the stationary phase and the analyte. The involvement of the two different amide groups (as donor and/or acceptor) in the complexation reaction can be followed based on spectral data. A correlation was found between the ratio of the amide I and the ring stretching (1532 cm(-)(1)) bands and retention data.     

Degree of solute inclusion in native beta-cyclodextrin: chromatographic approach

The reversed-phase liquid chromatography retention and separation of a series of D,L dansyl amino acids were investigated over a wide range of salting-out agent (sucrose) concentrations using native beta-cyclodextrin as a chiral stationary phase. An original treatment was developed to determine the number of sucrose molecules (n) excluded from the solute-beta-cyclodextrin cavity interface when the analyte transfer occurred. Using the n values, the relative degrees of compound inclusion were calculated and correlated to the steric bulkiness of the solute. Thermodynamic parameter variations are discussed in relation to the inclusion degree of the dansyl amino acids. This numerical approach is a valuable tool to explore the steric effects implied in the host-guest complex formation.     

Vancomycin dimerization and chiral recognition studied by high-performance liquid chromatography

The retention and separation of D,L-dansylvaline enantiomers (used as test solutes) were investigated using silica gel as stationary phase and vancomycin as chiral mobile-phase additive. A retention model was developed to describe the mechanistic aspects of the interaction between solute and vancomycin in the chromatographic system. It considered the formation of vancomycin dimers both "free" in the mobile phase and adsorbed on silica. By fitting the model equation to experimental data, it appeared clearly that the approach taking into account the vancomycin dimerization described accurately the retention behavior of the compounds. The examination of the model equation parameters showed that the glycopeptide dimerization increased the enantioselectivity by a factor of approximately 3.7. This study demonstrated the preponderant role of the vancomycin dimerization on the chiral recognition process of D,L-dansylvaline. Also, an additional analysis on a vancomycin chiral stationary phase indicated that the addition of vancomycin in the mobile phase promoted a greater enantioselectivity mediated by the formation of dimers in the stationary phase.     

Selectivity in capillary electrophoresis: application to chiral separations with cyclodextrins

In order to accurately evaluate the performances of any electrolyte medium, a clear concept of selectivity in capillary electrophoresis and related electroseparation techniques is proposed. Selectivity is defined as the ratio of the affinity factors of both analytes for a separating agent (phase, pseudophase, or complexing agent present in the background electrolyte). When in the presence of a complexing agent and if only 1:1 complexation occurs, selectivity corresponds to the ratio of the apparent binding constants and is independent of the concentration of the complexing agent. This concept is illustrated through the separations of neutral and anionic enantiomers in the presence of a cationic cyclodextrin, the mono(6-amino-6-deoxy)-β-cyclodextrin, as a chiral complexing agent. The values obtained for different pairs of enantiomers are discussed with regard to the functional groups that distinguish them. When the analytes have the same mobilities in free solution and in their complexed form, then the resolution equation developed in micellar electrokinetic chromatography may be applied and optimum conditions (affinity factors, chiral agent concentration) can be predicted.     

Molecular dynamics study of chiral recognition for the whelk-O1 chiral stationary phase

In this article, we examine the docking of 10 analytes on the Whelk-O1 stationary phase. A proper representation of analyte flexibility is essential in the docking analysis, and analyte force fields have been developed from a series of B3LYP calculations. Molecular dynamics simulations of a representative Whelk-O1 interface, in the presence of racemic analyte and solvent, form the basis of the analysis of chiral selectivity. The most probable docking arrangements are identified, the energy changes upon docking are evaluated, and separation factors are predicted. From comparisons between the analytes, the mechanism of chiral selectivity is divided into contributions from hydrogen bonding, ring-ring interactions, steric hindrance, and molecular flexibility. We find that both hydrogen bonding and ring-ring interactions are necessary to localize the analyte within the Whelk-O1 cleft region. We also identify one docking mechanism that is often dominant and analyze the conditions that lead to alternate docking modes.     

Preparative enantiomer separation of dichlorprop with a cinchona-derived chiral selector employing centrifugal partition chromatography and high-performance liquid chromatography: a comparative study

A countercurrent chromatography protocol for support-free preparative enantiomer separation of the herbicidal agent 2-(2,4-dichlorphenoxy)propionic acid (dichlorprop) was developed utilizing a purposefully designed, highly enantioselective chiral stationary-phase additive (CSPA) derived from bis-1,4-(dihydroquinidinyl)phthalazine. Guided by liquid-liquid extraction experiments, a solvent system consisting of 10 mM CSPA in methyl tert-butyl ether and 100 mM sodium phosphate buffer (pH 8.0) was identified as a suitable stationary/mobile-phase combination. This solvent system provided an ideal compromise among stationary-phase retention, enantioselectivity, and well-balanced analyte distribution behavior. Using a commercial centrifugal partition chromatography instrument, complete enantiomer separations of up to 366 mg of racemic dichlorprop could be achieved, corresponding to a sample load being equivalent to the molar amount of CSPA employed. Comparison of the preparative performance characteristics of the CPC protocol with that of a HPLC separation using a silica-supported bis-1,4-(dihydroquinidinyl)phthalazine chiral stationary phase CSP revealed comparable loading capacities for both techniques but a significantly lower solvent consumption for CPC. With respect to productivity, HPLC was found to be superior, mainly due to inherent flow rate restrictions of the CPC instrument. Given that further progress in instrumental design and engineering of dedicated, highly enantioselective CSPAs can be achieved, CPC may offer a viable alternative to CSP-based HPLC for preparative-scale enantiomer separation.     

Study of interactions in supercritical fluids and supercritical fluid chromatography by solvatochromic linear solvation energy relationships

Linear solvation energy relationships were used to study the retention process in supercritical fluid chromatography (SFC) and to gain a better understanding of intermolecular interactions in supercritical fluids. Correlation of SFC retention data with a set of solute solvatochromic parameters, which are also applicable to gas and liquid chromatography, yields information regarding the relative contributions of dispersion, cavity formation, dipolar, and hydrogen-bonding processes to retention. Dispersion interactions and cavity formation processes dominate retention on an open tubular poly(dimethylsiloxane) stationary phase with pure carbon dioxide as the mobile phase. Dipolar interactions and hydrogen-bonding interactions are of decidedly less importance but do contribute significantly to retention. Based on prior solvatochromic studies of poly(dimethylsiloxane) and carbon dioxide, the changes in the regression coefficients with temperature and pressure are interpreted chemically. The relative importance of these contributions changes with temperature and pressure. As pressure increases, the carbon dioxide becomes more dense, and dispersion interactions between the solute and the mobile phase increase. A temperature increase at constant pressure decreases dispersion interactions with the stationary phase, as in gas chromatography, but also decreases dispersion interactions with the mobile phase, due to a decrease in carbon dioxide density. On the basis of the solvatochromic coefficients, carbon dioxide acts as both a Lewis base and a Lewis acid. The quality of fit for these correlations is very high and compares favorably with similar studies in gas chromatography and liquid chromatography, permitting the prediction of retention behavior from a solute's solvatochromic parameters.     

Retention mechanism of weak polar solutes in reversed phase liquid chromatography

The retention mechanism of a weak polar solute, a series of 10 benzodiazepines in reversed phase liquid chromatography, was investigated over a wide range of mobile phase compositions. The values of enthalpy (ΔH°) and entropy (ΔS°) of transfer from the mobile to the stationary phases were determined. The method studied each factor (water fraction Φ in the acetonitrile (ACN)/water mixture and column temperature) controlling the retention mechanism. The changes in ΔH° and ΔS° as a function of the water fraction Φ in the ACN/water mixture were examined. These variations are explained using the organization of organic modifier (ACN) in clusters in the ACN/water mixture. A change in the retention mechanism thus indicated when the ACN/water mixture was used instead of the hydrogen-bonded mobile phase such as CH(3)OH/water. Enthalpy-entropy compensation revealed that the retention mechanism was independent of the water fraction Φ but showed that differences between the molecular structures of the benzodiazepines contributed more significantly to changes in the retention process in the CH(3)OH/water mixture than in the ACN/water mixture.     

Normal-phase TLC separation of enantiomers of 1.4-dihydropyridine derivatives

A new TLC-based method was proposed for the separation of enantiomers and mixtures of racemic DHP derivatives differing in the kind of substituent in the phenyl ring. The conditions for the effective determination of the substances involved and the mechanism of their sorption were also studied. For the separation of felodipine, nilvadipine, and isradipine enantiomers, thin-layer chromatography was used, with a chiral stationary phase of the ligand exchange type, and developing phases of a different concentration of methanol (phi) as an organic modifier. The retention coefficient values k' were used to make the plots log k' = f(log phi) and log k' = f(phi). The processes taking place in the chromatographic systems were shown to be described by the Snyder-Soczewiński equation.     

Immobilized ionic liquids as high-selectivity/high-temperature/high-stability gas chromatography stationary phases

Ionic liquids (ILs) are a class of nonmolecular solvents in which the cation/anion combination can be easily tuned to provide desired chemical and physical properties. When used as stationary phases in gas-liquid chromatography, ionic liquids exhibit dual nature retention selectivity. That is, they are able to separate polar molecules such as a polar stationary phase and nonpolar molecules such as a nonpolar stationary phase. However, issues such as optimization of the wetting ability of the ionic liquid on fused-silica capillaries, the maximum operating temperatures of the stationary phases, and nonuniform film thickness on the wall of the capillary at high temperatures have limited their use in gas chromatography. As described in this paper, these limitations are overcome by cross-linking a new class of ionic liquid monomers by free radical reactions to provide a more durable and robust stationary phase. By lightly cross-linking the ionic liquid stationary phase using a small amount of free radical initiator, high-efficiency capillary columns were produced that are able to endure high temperatures with little column bleed. Two types of cross-linked IL stationary phases are developed. A partially cross-linked stationary phase allows for high-efficiency separations up to temperatures of approximately 280 degrees C. However, by creating a more highly cross-linked stationary phase of geminal dicationic ILs, exclusively, an increase in efficiency is observed at high temperatures allowing for its use over 350 degrees C. In addition, through the use of solvation thermodynamics and interaction parameters, it was shown that the cross-linking/immobilization of the ionic liquid does not affect the selectivity of the stationary phase thereby preserving its dual nature retention behavior.     

Classical retention mechanism in ion exchange chromatography. Theory and experiment

The classical model for ion exchange chromatography is characterized by firmly adsorbed driving ions at the surface of the stationary phase in an amount required by electroneutrality and stoichiometric ion exchange between the bulk of the eluent electrolyte and this immobilized Stern layer. Retention equations have been derived for system peaks, labeled eluent ions, and analytes in a system containing only strong electrolytes by strictly respecting this model. It is shown that the classical model described dependence of retention data on concentration and composition of the binary eluent with excellent precision, but the resulting system parameters were not self-consistent. Inconsistency of the results might be due to contributions from another retention mechanism.     

Covalently bound ionene polyelectrolyte-silica gel stationary phases for HPLC

Micelle-mimetic ionene-based stationary phases for high-performance liquid chromatography (HPLC) are prepared by attaching [3,16]- and [3,22]-ionenes to aminopropyl silica through a carbon-nitrogen bond. These [x,y]-ionenes are polyelectrolytic molecules consisting of dimethylammonium charge centers interconnected by alternating alkyl chain segments containing x and y methylene groups, some of which can form aggregate species whose properties mimic those of conventional surfactant micelles. These ionene-bonded stationary phases were characterized using different recommended HPLC test mixtures. Test solute chromatographic behavior on the ionene phases was found to be similar to that of intermediate oligomeric or polymeric C-18 and/or phenyl phases, depending upon the specific test mixture employed. In addition, the phases exhibit significant solute shape recognition ability. The ionene stationary phases were successfully employed for the separation of the components of the recommended ASTM reversed-phase test mixture, as well as for ortho-, meta- and para-disubstituted benzenes and other positional or geometric isomeric compounds. The ionene materials allow for chromatographic separations under either reversed-phase or ion-exchange conditions. The retention mechanism on these multimodal phases can occur by hydrophobic partitioning or electrostatic interactions, depending upon the characteristics of the components of the analyte mixture (neutral or anionic). The effects of alteration of the percent organic modifier, flow rate and temperature of the mobile phase on chromatographic retention and efficiency on these phases were briefly examined.     

Mechanistic aspects of chiral discrimination on modified cellulose

Cellulose and cellulose derivatives are biopolymers which are often used as stationary phases for the separation of enantiomers. Describing the mechanism of such separations is a difficult task due to the complexity of these phases. In the present study, we attempt to elucidate the types of interactions occurring between a diol intermediate for a LTD(4) antagonist and a tris(4-methylbenzoate)-derivatized cellulose stationary phase. Thermodynamic studies indicate that, at low temperatures, the enantioselectivity is entropy driven. At higher temperatures, the separation is enthalpy driven. DSC and IR experiments reveal that the transitions between the enthalpic and the entropic regions of the van't Hoff plots are a result of a change in conformation of the stationary phase. Investigation of chromatographic kinetic parameters reveals that, at low temperature, the second eluted enantiomer undergoes sluggish inclusion interactions. Subtle changes in the structure of the analyte indicates that π-π interactions do not contribute to enantioselectivity. Finally, molecular modeling of (R)- and (S)-diol and the stationary phase suggests that hydrogen bonding is a primary factor in the separation, and the calculated energy values obtained from the molecular modeling correlate well with the chromatographic elution order.