High-performance liquid chromatographic separation of rolipram, bupivacaine and omeprazole using a tartardiamide-based stationary phase influence of flow rate and temperature on the enantioseparation

Chromatographic separation of the chiral drugs rolipram, bupivacaine and omeprazole on a tartardiamide-based stationary phase commercially named Kromasil CHI-TBB is shown in this work. The effect of temperature on the chromatographic separation of the chiral drugs using the Kromasil CHI-TBB stationary phase was determined quantitatively so as to contribute toward the design for the racemic mixtures of the named compound by using chiral columns. A decrease in the retention and selectivity factors was observed, when the column temperature increased. Van't Hoff plots provided the thermodynamic data. The variation of the thermodynamic parameters enthalpy and entropy are clearly negative meaning that the separation is enthalpy controlled.     

Temperature and enantioseparation by macrocyclic glycopeptide chiral stationary phases

Seventy-one chiral compounds were separated on four macrocyclic glycopeptide chiral selectors: teicoplanin, its aglycone, ristocetin A and vancomycin, using three possible separation modes: reversed phase with methanol/buffer mobile phases, normal phase with hexane/ethanol mobile phases and polar ionic mode (PIM) with 100% methanol mobile phase with trace amounts of acid and/or base. These 148 separations were studied in a 5-45 degrees C temperature range. Peak efficiencies always increased with temperature, but in only 17% of the separations studied a small increase of the enantioresolution factor was observed. In the majority (83%) of the cases, the enantioresolution decreased or even vanished when temperature increased. All 148 Van't Hoff plots were linear showing that the selector did not change in the temperature range studied. The calculated enthalpy and entropy variations showed that the interaction of the solute with the stationary phase was always enthalpy driven with normal and reversed mobile phases. It could be enthalpy as well as entropy driven with PIM mobile phases strongly dependent on the solute. The plots of delta(deltaH) versus delta(deltaS) were linear in most cases (enthalpy entropy compensation). This observation cannot be used to give clear information on chiral recognition mechanisms, but it allowed identifying specific stationary phase-solute interactions because the points corresponding to the respective thermodynamic parameters were clearly delineated from the general compensation lines.     

Effects of temperature on retention of chiral compounds on a ristocetin A chiral stationary phase

The isocratic retention of enantiomers of chiral analytes, i.e. tryptophan, 1,2,3,4-tetrahydroisoquinoline and gamma-butyrolac tone analogs, was studied on a ristocetin A chiral stationary phase at different temperatures and with different mobile phase compositions, using the reversed-phase, polar-organic and normal-phase modes. By variation of the both mobile phase composition and the temperature, baseline separations could be achieved for these enantiomers. The retention factors and selectivity factors for the enantiomers of all investigated compounds decreased with increasing temperature. The natural logarithms of the retention factors (ln k) of the investigated compounds depended linearly on the inverse of temperature (1/T). van't Hoff plots afforded thermodynamic parameters, such as the apparent change in enthalpy (deltaH(o)), the apparent change in entropy (deltaS(o)) and the apparent change in Gibbs free energy (deltaG(o) ) for the transfer of analyte from the mobile to the stationary phase. The thermodynamic parameters (deltaH(o), deltaS(o) and deltaG(o)) were calculated in order to promote an understanding of the thermodynamic driving forces for retention in this chromatographic system.     

Effect of phase ratio on van't Hoff analysis in reversed-phase liquid chromatography,and phase-ratio-independent estimation of transfer enthalpy

In analysis of the thermodynamics of the transfer of a solute from the mobile phase to the stationary phase in reversed-phase liquid chromatography, it is nearly always assumed that the phase ratio is constant. This type of analysis is typically performed by applying a form of the van't Hoff equation, which relates the retention factor to temperature via the enthalpy and entropy of transfer. When non-linear van't Hoff plots are observed, it is often assumed that the enthalpy and entropy of transfer change with temperature. However, when the possibility of a change in the phase ratio is considered, it becomes apparent that non-linear van't Hoff behavior may or may not be due to changes in enthalpy or entropy. In this work, we present mathematical evidence that phase ratio changes, if they occur, can cause deviations from linearity in a van't Hoff plot. We also show that the phase ratio influence can be eliminated by considering the molecular difference between two solutes instead of the solutes themselves. The resulting selectivity van't Hoff plots may be linear, even when the van't Hoff plots of the two solutes are non-linear. In such cases, temperature-dependent phase ratio changes, and not necessarily changes in the transfer enthalpy, may be responsible for the curved van't Hoff plots of the individual solutes. In addition, we present chromatographic evidence that different solutes may "see" different thermodynamic phase ratios. It is clear that the concept of a phase ratio in reversed-phase chromatography is not nearly as well defined as a phase ratio in a bulk system like a liquid-liquid extraction.     

Influence of Mobile Phase Composition on the Apparent Thermodynamic Characteristics in Liquid Chromatographic Enantioseparation on a Tartardiamide-Based Stationary Phase

Four amino acid derivatives and two binaphthyl compounds were enantioseparated successfully by HPLC with a chiral stationary phase commercially named Kromasil CHI-DMB, which contains an immobilized network polymer derived from L-tartaric acid. Chromatographic experiments were performed under normal phase conditions. n-Hexane/2-propanol was used as mobile phase. The effects of the content of 2-propanol and the column temperature on the retention and enantioseparation were studied systematically. Associated apparent thermodynamic parameters were calculated from the van’t Hoff plots. It was found that the enantioseparations of these amino acid derivatives were typical enthalpy driven, but for binaphthyl compounds the entropy term was also favorable to enantioselectivity in most cases. From the changes of the thermodynamic parameters and the isoenantioselective temperatures with the variety of mobile phase polarity, it was deduced that the CSP underwent remarkable conformational change when the content of 2-propanol was below 3%.     

Role of the water as a surface tension modifier for the retention of a series of dansylaminoacids on a beta-CD stationary phase

In reversed phase liquid chromatography (RPLC), using a surface tension treatment, the retention and separation of a series of d,l-dansylaminoacids were investigated with native beta-cyclodextrin as a chiral stationary phase. The enantioselectivity thermodynamic parameters were determined from linear van't Hoff plots. An analysis of the experimental variations in the retention factor with different fractions of water in the mobile phase was performed. The number of water molecules, n, excluded from the solute beta-cyclodextrin cavity interface when the analyte transfer occurred, was determined. Using these n values, the relative degrees of compound inclusion were calculated and correlated to both the steric bulkiness of the solute and the thermodynamic data.     

Chromatographic behavior of ion pair enantiomers of dansyl leucine cyclohexylammonium salt on a beta-cyclodextrin stationary phase and the effect of a competitive-binding mobile phase additive

The separation of dansyl leucine enantiomers on a beta-cyclodextrin stationary phase is significantly complicated by the association of the amino acid with its cyclohexylammonium counter ion, in a mobile phase of 80:20 (v/v) methanol-water. This produces very unusual chromatography, with two partially superimposed peaks observed for each enantiomer at lower column temperatures. The peak shape is attributed to the irreversible, oncolumn conversion of the ion pair (I) to the free, protonated (neutral) dansyl amino acid (II+H). Increasing the ionic strength of the mobile phase greatly improves the chromatography by transforming the solute species to enantiomers of II (the anionic, free amino acid). Van't Hoff plots are constructed for both species I and II (under different mobile phase conditions) to provide thermodynamic insight into the major enantioselective driving forces of separation. The chiral discrimination of the stationary phase is found to be primarily enthalpically driven for both solutes. Finally, 1-adamantanecarboxylic acid (ACA) is investigated as a solute-competitive mobile phase additive to intentionally block the hydrophobic cyclodextrin cavities on the stationary phase. By varying the concentration of ACA additive in the mobile phase, control over the retention and chiral recognition of the stationary phase is demonstrated.     

Thermodynamic analysis of the heterogenous binding sites of molecularly imprinted polymers

The thermodynamic interactions of two polymers, one Fmoc-L-Trp-imprinted (MIP), the other one an unimprinted reference (NIP), with the two Fmoc-tryptophan enantiomers were studied by frontal analysis, which allows accurate measurements of the adsorption isotherms. These isotherms were acquired at temperatures of 40, 50, 60, and 70 degrees C, for sample concentrations ranging between 0.005 and 40 mM. The mobile phase used was acetonitrile with one percent acetic acid as an organic modifier. Within the measured concentration ranges, the tri-Langmuir isotherm model accounts best for the isotherm data of both enantiomers on the MIP, the bi-Langmuir model for the isotherm data of Fmoc-L-Trp on the NIP. These isotherm models were selected using three independent processes: statistical tests on the results from regression of the isotherm data to different isotherm models; calculation of the affinity energy distribution from the raw isotherm data; comparison of the experimental and the calculated band profiles. The isotherm parameters obtained from these best selected isotherm models showed that the enantiomeric selectivity does not change significantly with temperature, while the affinity of the substrates for both the MIP and the NIP decrease considerably with increasing temperatures. These temperature effects on the binding performance of the MIP were clarified by considering the thermodynamic functions (i.e., the standard molar Gibbs free energy, the standard molar entropy of adsorption, and the standard molar enthalpy of adsorption) for each identified type of adsorption sites, derived from the Van't Hoff equation. This showed that the entropy of transfer of Fmoc-L-Trp from the mobile to the MIP stationary phase is the dominant driving force for the selective adsorption of Fmoc-L-Trp onto the enantioselective binding sites. This entropy does not change significantly with increasing temperatures from 40 to 70 degrees C.     

Synthesis and evaluation of a chiral stationary phase based on quinine: enantioresolution of dinitrophenyl derivatives of α-amino acids

The natural alkaloid quinine (QN) was immobilized on porous silica particles, and part of the material was subsequently endcapped with n-hexyl hydrocarbon chains. Two synthetic strategies for silanization of the support were first compared. These columns were thoroughly evaluated in order to study the influence of endcapping in the enantiorecognition features. Enantioseparations of twenty N-derivatized 2,4-dinitrophenyl α-amino acids (DNP-amino acids) were studied by changing mobile phase pH, buffer concentration, type of organic solvent in the mobile phase, and column temperature. Maximum retention factors were observed at pH ≈6, at this intermediate pH the tertiary amine of the quinine is protonated to a high degree and therefore available for strong electrostatic interactions with unprotonated anionic DNP-amino acids. The enantioselectivity factors, however, increased as the pH did in the range between 5 and 7. The increase in ionic strength had influence on retention, but not on enantioselectivity, allowing the use of this variable for optimization of retention factors. Finally, the thermodynamic transfer parameters of the enantiomers from the mobile to both CSPs (with and without endcapping, QN-CSP(EC) and QN-CSP, respectively) were estimated from van't Hoff plots within the range of 10-40 °C. Thus, the differences in the transfer enthalpy, Δ(ΔH°), and transfer entropy, Δ(ΔS°), enabled an investigation of the origin of the differences in interaction energies.     

High‐performance liquid chromatography thermodynamic study of new potential antiepileptic compounds on a cholesterol column using isocratic elution with methanol/water and acetonitrile/water eluent systems

Basic thermodynamic functions responsible for retention of new 1,2,4‐triazole derivatives exhibiting varied antiepileptic activity on cholesterol‐based stationary phase were determined. Evaluation of the Gibbs energy change, the change in enthalpy and the change in entropy was based on the van't Hoff relationship representing lnk versus 1/T . A detailed discussion of the van't Hoff equation, exploring the influence of the phase ratio on deviations from linearity in a van't Hoff plot is presented. We show chromatographic evidence to the question of how a varied mobile phase composition may cause different thermodynamic phase ratios. The analysis of data from a differential scanning calorimetry excluded any phase transitions of either the individual solutes or cholesterol stationary phase suspended in the mobile phase components within the studied temperature range.     

高效液相色谱使用两种类型的纤维素-三(对甲基苯甲酸酯)固定相手性拆分非洛地平的比较

以高效液相色谱手性固定相法对非洛地平(FEL)进行手性拆分。分别采用两种类型的纤维素-三(对甲基苯甲酸酯)手性柱Chiralcel OJ-R和Chiralcel OJ-H进行比较实验,以正己烷-异丙醇(90:10, v/v)为流动相,考察了流动相、柱温对保留及手性拆分的影响。实验显示,两柱拆分FEL的van’t Hoff图均发生了转折,在高温区域为焓驱动,在低温区域为熵驱动。两柱在温度升高时拆分FEL的分离度均提高,其中OJ-H的分离度优于OJ-R。两种手性柱对FEL具有相似的拆分机理。     

联苯类对映体在纤维素类手性固定相上的高效液 相色谱法直接拆分

涂敷纤维素-三(3,5-二甲基苯基氨基甲酸酯)于自制的球形氨丙基硅胶上,制备了手性固定相。用该固定相直接拆分了一系列外消旋联苯类保肝药物。考察了柱温和流动相中乙醇含量对样品的保留和立体选择性的影响,升高柱温或增大流动相中乙醇含量都会使对映体的保留因子和分离因子降低。氨考察榈的分离因子的对数与温度的倒数成线性关系。确定了对映体从流动相传递到固定相的吉布斯自由能变之差值、焓变之差值及熵变之差值。     

Retention studies of sulphated glycosaminoglycan disaccharides on porous graphitic carbon capillary columns

Summary The retention behaviour of highly polar and charged disaccharide isomers has been studied on porous graphitic carbon columns and experimental parameters were varied over wide a range, including concentration and type of mobile phase constituents as well as temperature. The hydrophilic and anionic sugar analytes were highly retained on this stationary phase compared to the octadecyl-derivatized silica packings more commonly used. E.g., an increase in retention with polarity of a solute and with temperature was observed. By isotherm measurements and nonlinear fitting of Langmuirian expressions to the experimental data the graphite surface appeared homogeneous with only one kind of active adsorption site for these kinds of compound which was furthermore supported by the linear Van't Hoff plots obtained by varying the temperature. The gain in free energy was found to be entropically driven after determination of the ΔH° and ΔS° values. However, enthalpy-entropy compensation behavior was not met.     

Cyclofructan 6 based stationary phases for hydrophilic interaction liquid chromatography

New stationary phases for hydrophilic interaction liquid chromatography (HILIC) were synthesized by covalently attaching native cyclofructan 6 (CF6) to silica gel. The chromatographic characteristics of the new stationary phases were evaluated and compared to three different types of commercial HILIC columns. The CF6 columns produced considerably different retention and selectivity patterns for various classes of polar analytes, including nucleic acid compounds, xanthines, β-blockers, salicylic acid and its derivatives, and maltooligosaccharides. Univariate optimization approaches were examined including organic modifier (acetonitrile) contents and buffer pH and salt concentration. The thermodynamic characteristic of the CF6 stationary phase was investigated by considering the column temperature effect on retention and utilizing van't Hoff plots. CF6 based stationary phases appear to have exceptionally broad applicability for HILIC mode separations.     

Investigations into the chromatographic behavior of a doxorubicin-peptide conjugate

HPLC impurity profile method development for a doxorubicin-heptapeptide conjugate included significant changes of the separation profile with diluent, eluent and pH. These separation variables were also temperature-dependent with a shift in retention from 35 to 45 degrees C. There was also a direct relationship of temperature with LC retention, and a pH minimum at 5.9. Atypical dependence of the impurity profile on diluent at a k' of 18 led to further investigation. A large change in retention by several minutes was a function of both the organic eluent composition and temperature between 15 and 30 degrees C. Several Van't Hoff temperature studies from 5 to 65 degrees C on several column types resulted in non-linear plots. Analysis of the molecular subunits suggested that the peptide portion of the analyte influenced the non-linear retention behavior. The stationary phase type was not a significant factor causing non-linearity. Circular dichroism-temperature studies indicated a notable transition in ellipticity for the amine regions (198-202 nm) that occurred between 39 and 44 degrees C. This transition temperature range coincided with the results of the Van't Hoff analysis, between 35 and 44 degrees C, to indicate that these effects were not primarily stationary phase induced.     

Effect of ester molecular structure and column temperature on the retention of eight esters in gas chromatography

Summary An experimental design has been used to study the effect of column temperature on the gas chromatographic retention of eightpara-hydroxy benzoic esters. A rapid procedure has been developed to reduce the number of experiments compared with traditional methods. Capacity factors were determined and the enthalpy and entropy of transfer from the mobile phase to the stationary phase, H^o and S^o, respectively, were calculated using the linear Van't Hoff equation (dependence of ln k on 1/T). A retention prediction system (RPS) for these compounds in GC was investigated. The molecular connectivity index was used to describe the quantitative structure relationships. Enthalpy — entropy compensation revealed that the mechanism was similar for all the compounds studied.     

Comparison of performance of Chirobiotic T, T2 and TAG columns in the separation of beta2- and beta3-homoamino acids

The enantiomers of eight unusual beta(2)- and beta(3)-homoamino acids were separated on chiral stationary phases containing the macrocyclic glycopeptide antibiotic teicoplanin (Chirobiotic T or T2) or teicoplanin aglycone (Chirobiotic TAG) as chiral selectors. The effects of the organic modifier, the mobile phase composition, and temperature on the separations were investigated. Linear van't Hoff plots were observed in the studied temperature range, 280-318 K, and the changes in enthalpy, Delta(DeltaH(o)), entropy, Delta(DeltaS(o)), and free energy, Delta(DeltaG(o)) were calculated. The values of the thermodynamic parameters depended on the nature of the selectors, the structures of the analytes, and especially the positions of the substituents on the analytes. A comparison of the separation performances of the macrocyclic glycopeptide stationary phases revealed that the Chirobiotic TAG column exhibited much better selectivity for beta(2)-homoamino acids, while the separation of beta(3)-homoamino acid enantiomers was better on Chirobiotic T or T2. The elution sequence was determined in some cases and was observed to be R < S.     

Determination of thermodynamic parameters for the interaction of a lipid-binding peptide and insulin with a reversed-phase column

The effect of temperature was investigated on the reversed-phase chromatography of a synthetic lipid associating peptide (LAP), with the following sequence (LESFLKSWLSALEQALKA) and on insulin. The LAP was chromatographed on a muBondapak-alkylphenyl column with 1% aq. triethylammonium phosphate, pH 3.2-2-propanol (80:20) as the isocratic mobile phase. The insulin was separated on a Resolve-C18 column with a mobile phase that contained 0.1 M sodium phosphate, pH 2.0-acetonitrile (71.5:28.5). With the LAP the Van't Hoff plot (In k' vs. 1000/T) was linear and the value of enthalpy for association of the peptide with the reversed-phase column was large and negative. The phase ratio was estimated for the column used in the separation and then derivation of the corresponding entropic term demonstrated that the association was enthalpy and not entropy drive. By contrast the corresponding Van't Hoff plot derived or the insulin study was non-linear and with a positive slope. Further study indicated that the formation of the insulin-reversed phase complex could be attributed to a favorable entropy change. It is probable that the non-classical thermodynamics observed during the insulin chromatography could be related to conformational changes in the insulin structure during the chromatographic process.     

Thermodynamic study of retention in liquid exclusion-adsorption chromatography

The retention behaviour of fatty alcohol ethoxylates and fatty acid methyl ester ethoxylates on various reversed-phase columns in acetone-water has been studied in the regime of liquid exclusion-adsorption chromatography at different temperatures. Straight lines were obtained in the van't Hoff plots. The entropy and enthalpy changes were found to be negative (at least in the range of lower oligomers) and showed a dependence of the number of oxyethylene units. For higher oligomers, both entropy and enthalpy changes approach a constant value. This can be explained by the existence of a rather thick layer of organic solvent close to the surface of the stationary phase.     

Retention mechanism of fatty alcohol ethoxylates in reversed-phase liquid chromatography

Fatty alcohol ethoxylates (FAEs) are widely used nonionic surfactants that have distributions in both alkyl and poly(ethylene oxide) (PEO) chain length. Generally, two-dimensional liquid chromatography technique is required for the complete characterization of both distributions. By selecting a proper stationary and mobile phase condition, however, we can obtain fully resolved chromatograms of a FAE sample (Brij 30) with respect to both alkyl and PEO chain length by using a single reversed-phase C18 column and aqueous acetonitrile mobile phase. FAEs show a peculiar reversed-phase liquid chromatography (RPLC) retention behavior with an aqueous-organic mobile phase, the retention mechanism of which has not been fully elucidated. For a fixed alkyl chain length, FAEs with higher-molecular-mass PEO block elutes first and the van't Hoff plot of the retention factor shows a curvature. The unique retention behavior can be understood from the opposite thermodynamic characteristics associated with RPLC retention of PEO block and alkyl chain: the sorption process of PEO to the non-polar stationary phase shows deltaH(o) > 0 and deltaS(o) > 0 while the alkyl chain shows deltaH(o) < 0 and deltaS(o) < 0 in contrast. The relative magnitude of the two contributions can change the elution order of the FAE. Therefore the often found, inverted elution order of FAEs (the early elution of FAEs with longer PEO block) is due to the positive enthalpic interaction of PEO blocks, which is a characteristic of the hydrophobic interaction. And the curvature of the van't Hoff plots was analyzed assuming the temperature dependent thermodynamic variables.