High-performance liquid chromatographic enantioseparation of alpha-substituted glycine analogs on a quinine-based anion-exchanger chiral stationary phase under variable temperature conditions

The retention of enantiomers of chiral analytes, i.e. alpha-substituted glycine analogs, on a quinine-based anion-exchanger chiral stationary phase was studied in the temperature range of 5-70 degrees C and at different mobile phase compositions, using isocratic elution in the reversed-phase mode. By variation of both mobile phase composition and temperature, baseline separations could be achieved for these enantiomers. Separation could be optimized more quickly by adjusting the column temperature rather than the mobile phase composition. The dependence of the natural logarithms of retention and selectivity factors (lnk' and lnalpha) on the inverse of temperature, 1/T (van't Hoff plots) was used to determine thermodynamic data on the enantiomers. Calculated thermodynamic constants (Delta(DeltaH degrees ), Delta(DeltaS degrees ) and Delta(DeltaG degrees )) were applied to promote an understanding of the thermodynamic driving forces for retention in this chromatographic system. The elution sequence of the enantiomers in most cases was determined.     

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.     

高效液相色谱-串联质谱法结合多糖衍生物手性固定相拆分氰戊菊酯

建立了以多糖衍生物为手性固定相的高效液相色谱-串联质谱(HPLC-MS/MS)直接拆分氰戊菊酯对映体的方法。在反相液相色谱条件下,考察了手性固定相的种类、流动相组成、柱温、流速对氰戊菊酯4个立体异构体分离的影响。同时,利用热力学方法对氰戊菊酯的立体异构体与固定相之间的色谱保留和分离的热力学机理进行了探讨。结果表明:采用Lux Cellulose-3(纤维素-三(4-甲基苯甲酸酯))手性色谱柱,在以流动相为乙腈-水(5 mmol/L甲酸铵)=(55:45,V:V)流速0.4 mL/min,柱温30℃的条件下,可在14 mins内实现氰戊菊酯4个立体异构体的基线分离。拓展了HPLC-MS/MS在菊酯类手性农药对映体分离及检测上的应用。     

Retention behavior of basic compounds on alkylphosphonate-modified magnesia-zirconia composite stationary phase in RPHPLC

Summary The chromatographic properties of an alkylphosphonate-modified magnesia-zirconia composite stationary phase have been investigated by reversed-phase high-performance liquid chromatography with basic compounds as probes. The influence of organic modifier composition and mobile phase pH was studied. The new stationary phase, similar to a silica-based reversed-phase stationary phase, has hydrophobic properties, but greater pH stability. Use of the phase results in more symmetric peaks for basic compounds. A possible mechanism of retention of basic solutes on the new stationary phase is discussed. The chromatographic behavior of the basic solutes depends mainly on hydrophobic interactions between the solutes and the hydrophobic moiety of the stationary phase. Br?nsted acidic and basic sites on the surface of the new stationary phase play an important role in the retention of ionized solutes by ion-exchange interaction. Promising separations of some basic compounds have been achieved by use of methanolic TRIS buffer, pH 10.0, as the mobile phase.     

Characterization of C18-bonded liquid chromatographic stationary phases by Raman spectroscopy: the effect of mobile phase composition

Raman spectroscopy is used to examine the effect of mobile phase composition on the orientation of octadecyl-bonded silica-based reversed-phase liquid chromatographic (RPLC) stationary phase ligands. The effect of ligand bonding density is also investigated. The present experimental set-up utilizes a direct, noninvasive, on-column approach to examine the solvent dependent conformational behavior of the bonded ligands under flow-rate and back pressure conditions similar to those used during conventional RPLC measurements. Neat, single-component, mobile phase solvents including water, acetonitrile, methanol and chloroform are used to investigate the hypothesized collapsing and extension of stationary phase ligands with changes in mobile phase composition. No evidence of phase collapse was observed upon changing the mobile phase composition from an organic to an aqueous content. Also, Raman spectroscopic measurements allowed the differentiation between associated and free acetonitrile solvent.     

Evaluation of ternary mobile phases for reversed-phase liquid chromatography: Effect of composition on retention mechanism

The effect of varying mobile phase composition across a ternary space between two binary compositions is examined, on four different reversed-phase stationary phases. Examined stationary phases included endcapped C8 and C18, as well as a phenyl phase and a C18 phase with an embedded polar group (EPG). Mobile phases consisting of 50% water and various fractions of methanol and acetonitrile were evaluated. Retention thermodynamics are assessed via use of the van’t Hoff relationship, and retention mechanism is characterized via LSER analysis, as mobile phase composition was varied from 50/50/0 water/methanol/acetonitrile to 50/0/50 water/methanol acetonitrile. As expected, as the fraction of acetonitrile increases in the mobile phase, retention decreases. In most cases, the driving force for this decrease in retention is a reduction of the enthalpic contribution to retention. The entropic contribution to retention actually increases with acetonitrile content, but not enough to overcome the reduction in the enthalpic contribution. In a similar fashion, as methanol is replaced with acetonitrile, the v, e, and a LSER system constants change to favor elution, while the s and c constants change to favor retention. The b system constant did not show a monotonic change with mobile phase composition. Overall changes in retention across the mobile phase composition range varied, based on the identity of the stationary phase and the composition of the mobile phase.     

Factors Affecting the Interpretation of Selectivity on Synergi Reversed-Phase Columns

The solvation parameter model is used to establish the contribution of cohesion, dipole-type, and hydrogen-bonding interactions to the retention mechanism on Synergi Hydro-RP, Fusion-RP, and Polar-RP reversed-phase columns with methanol–water mobile phases containing from 10–70% (v/v) methanol. Large changes in relative retention on the compared columns can result from steric resistance, differences in the phase ratios, and from dewetting at low methanol compositions while changes in intermolecular interactions are responsible for smaller changes at a fixed mobile phase composition. For Synergi Hydro-RP and Polar-RP changing methanol for acetonirile is more powerful for affecting changes in retention order than changing the stationary phase. The three Synergi columns show useful selectivity differences for method development when compared with 13 other modern reversed-phase columns representing a selection of different stationary phase chemistries. The results from this study indicate the limitations of classifying reversed-phase columns by the retention of prototypical compounds to define specific retention mechanisms.     

Enthalpy-Entropy Compensation of Octylsilica Stationary Phase in Reversed-Phase HPLC

Abstract

Enthalpy-entropy compensation has been investigated in reversed-phase high performance liquid chromatography with octylsilica stationary phase. The compensation temperatures were determined for this system, and the results show that their change with the composition of the mobile phase was almost similar to that with octadecylsilica stationary phase. It is concluded that the retention mechanism of the separation of alkylbenzenes in both systems with the mobile phase exceed 20% water content is the same.     

Reversed-phase retention thermodynamics of pure-water mobile phases at ambient and elevated temperature

The use of pure water at superheated temperatures, between 100 and 200 degrees C, as a mobile phase for reversed-phase separations is explored. The thermodynamics of the retention process at low temperature (15-55 degrees C) are compared to the thermodynamics at elevated temperature (125-175 degrees C). Significant differences in the enthalpy of the retention process are observed between the two temperature ranges. This is possibly due to changes in the hydrogen-bond network of the pure-water mobile phase, which would change the solvation, and therefore retention, of non-polar solutes. The change in thermodynamic values between the two temperature regions invalidates extrapolation of retention as a function of temperature between the two temperature regions for the prediction of room-temperature pure-water retention factors. The thermodynamic changes observed as the temperature is increased are similar to those seen when mobile phase composition is changed (by adding organic modifier) at constant temperature.     

Secondary isotope effects in liquid chromatography behaviour of 2H and 3H labelled solutes and solvents

The separation of solutes that differ only in the extent of isotopic substitution of their hydrogen atoms, using either mixtures of isotopically non-modified or perdeuterated solvents as mobile phases, is described. The occurrence of a secondary isotope effect is demonstrated in reversed-phase liquid chromatography, which is independent of the nature of the stationary phase (different octadecyl-bonded silicas, an embedded alkylamide-bonded silica, as well as one polymeric stationary phase were tested), and the water content and the nature of organic modifier of the mobile phase. The separation of 24 structurally different isotopologue pairs (apolar compounds and polar compounds with exchangeable or non-exchangeable hydrogen atoms) is examined using reversed-phase liquid chromatography. It is found that the greater the number of isotopically substituted hydrogen atoms in a given organic solute, the better is the separation of a particular isotopologue pair. The single secondary isotope effect is shown to be dependent on the number of isotopic substitutions. The greater the number of these substitutions, the smaller is the single isotope effect. The single secondary isotope effect is higher for aromatic hydrocarbons than for aliphatic hydrocarbons. A secondary isotope effect is also observed in chiral chromatography and normal-phase liquid chromatography, as well as on changing the nature of the substituting isotope, i.e.: tritium instead of deuterium. Thus, we have demonstrated that the total secondary isotopic effect for hydrogen/tritium is higher than for hydrogen/deuterium. This isotope effect involves only the consequences of changes in interactions due to nuclear motions. Overall this study confirms the predominance of hydrophobic effects in retention processes in reversed-phase liquid chromatography. In reversed-phase liquid chromatography, a secondary isotope effect related to mobile phase composition is also observed. The behaviour of deuterium oxide and water in mobile phases of the same composition (%, w/w) is compared. Independent of the nature of the organic modifier (methanol, acetonitrile or ethanol), the effect of replacing H2O with 2H2O in the mobile phase, is an increase in the retention factors and an improvement in the chromatographic resolution of isotopologue pairs. This increase in the resolution is not accompanied by a change in the chromatographic selectivity. The measurement of liquid-liquid extraction coefficients proves that the effect is mainly due to the modification of the phase ratio. In general the effect of 2H-labelled solvents (2H2O and C2H3CN) as mobile phase components, compared to their isotopically non-modified isomers, can be rationalized on the basis of their lower polarisabilities. Overall the use of perdeuterated rather than isotopically non-modified solvents as mobile phase components leads to the most efficient separation systems.     

Joint use of cyclodextrin additives in chiral discrimination by reversed-phase high-performance liquid chromatography: temperature effects

The temperature dependence of chiral separations was investigated in combined system of reversed-phase (RP) liquid chromatography using two chiral additives: single or β native cyclodextrins and their permethylated derivatives. The model tested compounds of pharmaceutical interest were: methylphenobarbital, mephenytoin, morsuximide and camphor. Taking the localization of a complexation process as a criterion – the combined system with two selectors has been rationalized as occurring in three stages. The influence of temperature (in narrow range of 20°C) on retention and enantioselectivity was studied in; System I (complexation occurs in the mobile phase), in System II (complexation on the stationary phase) and in System III (complexation in both phases together). In System III (as for System I) it has been found that the model compounds could be classified into three groups based on their retention dependence on temperature: retention decrease with temperature decrease, retention increase with temperature decrease or no influence of temperature on retention. For all the compounds investigated, decrease in temperature increases the selectivity. Standard enthalpy (ΔH⁰) and entropy (ΔS⁰) changes of solute transfer between the mobile and the stationary phase and standard enthalpy (ΔH⁰_CD) and entropy (ΔS⁰_CD) changes of complex formation were also calculated. In Systems I and III, if the complexation in the mobile phase is favored process compared with interaction with the stationary phases (RP or covered by permethylated cyclodextrin), the shortest retention time and the best selectivity is observed at low temperature.     

Thermodynamics and kinetics of solute transfer in reversed-phase liquid chromatography

In this study, the thermodynamic and kinetic behavior of a homologous series of fatty acids is examined using a polymeric octadecylsilica stationary phase and a methanol mobile phase. The zone profiles are evaluated as the temperature is varied from 20 to 60 degrees C and the average pressure from 400 to 4570 p.s.i. (1 p.s.i.=6894.76 Pa). The rate constant for solute transfer from mobile to stationary phase (k(ms)) appears to be relatively constant with carbon number. In contrast, the rate constant from stationary to mobile phase (k(sm)) decreases logarithmically with increasing carbon number. This suggests that the mass transport processes become progressively slower, owing to the smaller diffusion coefficients of the larger solutes in the stationary phase. The activation energy decreases slightly in the mobile phase and increases slightly in the stationary phase with increasing carbon number. The activation energy in the stationary phase ranges from 41.6 to 55.9 kcal/mol, while the thermodynamic change in internal energy ranges from -9.8 to -29.0 kcal/mol for C10 to C22, respectively (1 cal=4.184 J). The activation volume increases with increasing carbon number in both the mobile and stationary phase. The activation volume in the stationary phase ranges from 31.7 to 211 cm3/mol, while the thermodynamic change in molar volume ranges from -27.1 to -104 cm3/mol for C10 to C22, respectively. These large changes in activation energy and volume suggest that the solutes do not enter and leave the stationary phase in a single step, but in a stepwise or progressive manner.     

Retention mechanism of the multifunctional solute on columns with different coverage densities using highly aqueous reversed‐phase conditions

A series of 11 homemade octadecyl bonded phases with different coverage densities were tested to determine the influence of the stationary phase on the retention in highly aqueous mobile phases. The concentrations of the organic modifiers (methanol and ACN) were in the range of 0–20%_v/v. The coverage density of bonded ligands and the presence of the end‐capping have strong influence on the solute retention. Amoxicillin (AMO) was chosen as the test compound. Dual properties of AMO, which contain hydrophobic skeleton and polar groups (amino, hydroxyl and carbonyl), cause irregular changes of the retention over the stationary phase hydrophobicity and silanol activity at given mobile phase composition. Presented data show that application of non‐standard low coverage density C18 phases allow to determine AMO in the RPLC condition with high retention.     

Evaluation of the influence of residual silanol groups on the total retention in reversed-phase chromatography

It was demonstrated that the characteristics of stationary phases for reversed-phase chromatography can be compared by analyzing the retention of several compounds belonging to a homologous series during elution with one and the same composition of mobile phase or the retention of a nonpolar compound during elution with several mobile phases of different compositions. The slope of the corresponding linear correlations can be used to characterize the hydrophobicity of the columns (stationary phases) under study. To analyze the polar interactions between the sorbate and the stationary phase it is advantageous to study the retention of a polar substance in several (at least in two) mobile phases of different compositions. The degree of displacement of the linear correlation relative to that for nonpolar substances can be considered a measure of the influence of residual silanol groups on the total retention characteristics.     

Evaluation of ODS-AQ stationary phase for use in capillary electrochromatography

The aim of this study was to evaluate the applicability of ODS-AQ packing material as a stationary phase in capillary electrochromatography (CEC). The electroosmotic flow created on an ODS-AQ stationary phase was measured at different mobile phase compositions and at different column temperatures. It was observed that the electroosmotic flow generated in the column increased by 50% when the temperature of the system was raised from 20 degrees C to 60 degrees C, while all other conditions were kept constant. The electroosmotic flow produced by the ODS-AQ stationary phase was found to be comparable to the flow generated in a column packed with Nucleosil bare-silica material. In addition, a set of polar compounds (D-lysergic acid diethylamide derivatives) was utilized to determine the influence of temperature and mobile phase composition on their chromatographic behavior on an ODS-AQ stationary phase in a CEC mode. A linear relationship between the solute retention factor and column temperatures was seen over the temperature range studied (20 degrees C to 60 degrees C). A quadratic function was used to describe the changes in the solute retention factors with variation of acetonitrile concentration in the mobile phase.     

High performance liquid chromatography of slightly soluble brominated indigoids from Tyrian purple

Attempts at identification of mollusc species producing Tyrian purple from archaeological material are usually done with high performance liquid chromatography in the reversed-phase system, but the peaks obtained are often wide and asymmetric. This is due to the low solubility of the indigoids and their brominated derivatives in the mobile phase, especially 6,6'-dibromoindigotin, which is soluble in only few, particular solvents. Our study focused on improving both symmetry and peak height for more precise quantification. The influence of various factors was evaluated: stationary phase characteristics, mobile phase composition, elution gradient parameters and temperature on the peak shape of the main components of Tyrian purple. The best results were obtained using highly retentive, but moderately bonded ODS stationary phases (about 2.8 μmol m(-2)), percolated with gradient of acetonitrile with acidified aqueous mobile phases (0.1% strong acid) at elevated temperatures (70°C). The upper quantification limit for 6,6'-dibromoindigotin was improved by over 350%, between standard and optimised systems. Using them, the detection and quantification of trace Tyrian purple components (less than 0.15%) aside from major indigoids becomes possible. Consequently, for the first time, the new analogues of brominated and unbrominated indirubins were found in the shellfish purple from Hexaplex trunculus.     

稠环芳烃在烷基膦酸改性锆镁复合氧化物固定相上的反相液相色谱保留行为

 以稠环芳烃为探针,考察了烷基膦酸改性锆镁复合氧化物材料的反相色谱性能。研究了稠环芳烃类化合物的结构与其保留值的关系,比较了烷基膦酸改性锆镁复合氧化物固定相和十八烷基键合硅胶ZorbaxODS对稠环芳烃异构体的选择性,并对可能的保留机理进行了讨论。以甲醇-水(体积比为75∶25)为流动相,在烷基膦酸改性锆镁复合氧化物固定相上分离了8种稠环芳烃类化合物。     

Mobile phase effects in reversed-phase liquid chromatography: a comparison of acetonitrile/water and methanol/water solvents as studied by molecular simulation

Molecular simulations of water/acetonitrile and water/methanol mobile phases in contact with a C(18) stationary phase were carried out to examine the molecular-level effects of mobile phase composition on structure and retention in reversed-phase liquid chromatography. The simulations indicate that increases in the fraction of organic modifier increase the amount of solvent penetration into the stationary phase and that this intercalated solvent increases chain alignment. This effect is slightly more apparent for acetonitrile containing solvents. The retention mechanism of alkane solutes showed contributions from both partitioning and adsorption. Despite changes in chain structure and solvation, the molecular mechanism of retention for alkane solutes was not affected by solvent composition. The mechanism of retention for alcohol solutes was primarily adsorption at the interface between the mobile and stationary phase, but there were also contributions from interactions with surface silanols. The interaction between the solute and surface silanols become very important at high concentrations of acetonitrile.     

On the chromatography mechanism of reversed-phase liquid chromatography

Summary An equation is derived which can describe how the retention of solutes is influenced by the composition of the mobile phase in reversed-phase liquid chromatography, the retention of solutes in alkyl bonded stationary phase regarded as the complexation between solute molecule and the active sites on the surface of the stationary phase. When the stationary phase is not fully saturated by the organic modifier, the activity of the active sites, the activity coefficient of the adsorbed solute as well as the activity coefficient of the solute in the mobile phase depend on the composition of the mobile phase. However, when the stationary phase is fully saturated, the composition of the mobile phase mainly influences the activity coefficient of the solute in the mobile phase. In addition, the selectivity of retention is discussed in terms of the derived equation.