Fractionation of histones on a metal ion equilibrated cation exchanger. I. Chromatographic profiles on an Amberlite IR-120 (A1 3+) column

Amberlite IR-120, a polystyrene sulphonate type of cation exchanger, equilibrated with A1 3+ ions, has been employed for the fractionation of whole histone. This adsorbent permits the quantitative and reproducible recovery of whole histone in six fractions.     

Measurement of the axial and radial temperature profiles of a chromatographic column. Influence of thermal insulation on column efficiency

The temperatures of the metal wall along a chromatographic column (longitudinal temperature gradients) and of the liquid phase across the outlet section of the column (radial temperature gradients) were measured at different flow rates with the same chromatographic column (250 mm x 4.6 mm). The column was packed with 5 microm C18-bonded silica particles. The measurements were carried out with surface and immersion thermocouples (all junction Type T, +/-0.1 K) that measure the local temperature. The column was either left in a still-air bath (ambient temperature, T(ext) = 295-296 K) or insulated in a packing foam to avoid air convection around its surface. The temperature profiles were measured at several values of the inlet pressure (approximately = 100, 200, 300 and 350 bar) and with two mobile phases, pure methanol and a 2.5:97.5 (v/v, %) methanol:water solution. The experimental results show that the longitudinal temperature gradients never exceeded 8 K for a pressure drop of 350 bars. In the presence of the insulating foam, the longitudinal temperature gradients become quasi-linear and the column temperature increases by +1 and +3 K with a water-rich (heat conductivity approximately = 0.6 W/m/K) and pure methanol (heat conductivity approximately = 0.2 W/m/K), respectively. The radial temperature gradients are maximum with methanol (+1.5 K at 290 bar inlet pressure) and minimum with water (+0.8 K at 290 bar), as predicted by the solution of the heat transfer balance in a chromatographic column. The profile remains parabolic all along the column. Combining the results of these measurements (determination of the boundary conditions on the wall, at column inlet and at column outlet) with calculations using a realistic model of heat dispersion in a porous medium, the temperature inside the column could be assessed for any radial and axial position.     

Effect of temperature on the chromatographic retention of ionizable compounds. III. Modeling retention of pharmaceuticals as a function of eluent pH and column temperature in RPLC

We propose a general simple equation for accurately predicting the retention factors of ionizable compounds upon simultaneous changes in mobile phase pH and column temperature at a given hydroorganic solvent composition. Only four independent experiments provide the input data: retention factors measured in two pH buffered mobile phases at extreme acidic and basic pH values (e. g., at least +/- 2 pH units far from the analyte pK(a)) and at two column temperatures. The equations, derived from the basic thermodynamics of the acid-base equilibria, additionally require the knowledge of the solute pK(a )and enthalpies of acid-base dissociation of both the solute and the buffer components in the hydroorganic solvent mixture. The performance of the predictive model is corroborated with the comparison between theoretical and experimental retention factors of several weak acids and bases of important pharmacological activity, in mobile phases containing different buffer solutions prepared in 25% w/w ACN in water and at several temperatures.     

Combined effect of solvent content, temperature and pH on the chromatographic behaviour of ionisable compounds

The organic solvent content and the pH in the mobile phase are the usual main factors in reversed-phase liquid chromatographic separations, owing to their strong effects on retention and/or selectivity. Temperature is often neglected. However, even in cases where the impact of this factor on selectivity is minor, the reduction in analysis time is still an interesting reason to consider it. In addition, ionisable compounds may exhibit selectivity changes, owing to the interaction of organic solvent and/or temperature with pH. The separation of ionisable compounds (nine diuretics: bendroflumethiazide, benzthiazide, bumetanide, chlorthalidone, furosemide, piretanide, probenecid, trichloromethiazide and xipamide, and two beta-blockers: oxprenolol and propranolol) exhibiting different acid-base behaviour was studied. The compounds were tested in a Zorbax SB C18 column under a wide range of conditions: 25-45% (v/v) acetonitrile, pH 3-7 and 20-50 degrees C. Models considering two factors (organic solvent/pH and temperature/pH), and three factors (organic solvent/temperature/pH) were developed from a previously reported equation, which considers the polarity contributions of solute, stationary and mobile phases. This allowed a comprehensive method to predict the retention of the 11 compounds, the modification of their acid-base behaviour (i.e. determination of protonation constants and shifts of the retention versus pH curves), and the selectivity changes within the studied factor ranges.     

Unusual effect of column temperature on chromatographic enantioseparation of dihydropyrimidinone acid and methyl ester on amylose chiral stationary phase

This paper reports an unusual effect of column temperature on the separation of the enantiomers of dihydropyrimidinone (DHP) acid and its methyl ester on a derivatized amylose stationary phase by normal-phase liquid chromatography. The separation of the DHP acid enantiomers was investigated using both carbamate-derivatized amylose and cellulose stationary phases (Chiralpak AD and Chiralcel OD) with an ethanol-n-hexane (EtOH-n-Hex) mobile phase. On the amylose phase, the van 't Hoff plot of the retention factor of the S-(+)-DHP acid was observed to be non-linear while that of R-(-)-DHP acid was linear. Likewise, the van 't Hoff plot for DHP acid enantioselectivity was non-linear with a transition occurring at approximately 30 degrees C. Furthermore, the van 't Hoff plot for the DHP acid enantioselectivity factor for data taken when heating the column from 5 to 50 degrees C was not superimposable with the same plot prepared with data from the cooling process from 50 to 5 degrees C. This observation suggested that the stationary phase was undergoing a thermally induced irreversible conformational change that altered the separation mechanism between the heating and cooling cycles. Similar phenomena were observed for the separation of the enantiomers of the DHP ester probe compound. The conformational change of the AD phase was shown to depend on the polar component of the mobile phase. When 2-propanol (2-PrOH) was used as the modifier instead of EtOH, the van 't Hoff plots for DHP acid were linear and thermally reversible, suggesting that no such irreversible conformational change occurs with this modifier. Conversely, when the AD phase was pre-conditioned with a more polar methanol (MeOH) or water containing mobile phase, thermal irreversibility of DHP acid enantioselectivity was once again observed. Interestingly, when the stationary phase was changed to its cellulose analogue, the Chiralcel OD, all van 't Hoff plots for the retention and selectivity of DHP acid were thermally reversible for both EtOH-n-Hex and 2-PrOH-n-Hex mobile phases.     

Combined effect of solvent content,temperature and pH on the chromatographic behaviour of ionisable compounds. III: Considerations about robustness

We previously reported a model able to predict the retention time of ionisable compounds as a function of the solvent content, temperature and pH [J. Chromatogr. A 1163 (2007) 49]. The model was applied further, developing an optimisation of the resolution based on the peak purity concept [J. Chromatogr. A 1193 (2008) 117]. However, we left aside an important issue: we did not consider incidental overlaps caused by shifts in the predicted peak positions, owing either to uncertainties in the source data, modelling errors, or the practical implementation in the chromatograph of the optimal mobile phase (or any other). These shifts can ruin the predicted separation, since they can easily amount several peak-width units at pH values close to the logarithm of the solutes’ acid–base constants. A probabilistic optimisation is proposed here, which is able to evaluate the uncertainties associated with the model and the consequences when the optimal mobile phase is implemented in the chromatograph. This approach assumes peak fluctuations in replicated assays obtained through Monte Carlo simulations, which gives rise to a distribution of elementary peak purities. The results yielded by the conventional (i.e. non-robust), derivative-penalised, and probabilistic optimisations were compared, checking the predicted and experimental chromatograms at several critical experimental conditions. Among the three approaches, only the probabilistic one was able to appraise properly the practical difficulties of the separation problem.     

The effect of the pH and solvent concentration on the adsorption chromatographic separation of125I-labelled iodotyrosines

¹²⁵I-labelled 3-iodo- and 3,5-diiodotyrosine were separated by adsorption chromatography using Sephadex LH-20 dextran gel and ethanol-water binary eluent. The effect of the pH on the distribution coefficient vs. ethanol concentration relationship was determined and interpreted.     

Influence of pressure on the properties of chromatographic columns. II. The column hold-up volume

The effect of the local pressure and of the average column pressure on the hold-up column volume was investigated between 1 and 400 bar, from a theoretical and an experimental point of view. Calculations based upon the elasticity of the solids involved (column wall and packing material) and the compressibility of the liquid phase show that the increase of the column hold-up volume with increasing pressure that is observed is correlated with (in order of decreasing importance): (1) the compressibility of the mobile phase (+1 to 5%); (2) in RPLC, the compressibility of the C18-bonded layer on the surface of the silica (+0.5 to 1%); and (3) the expansion of the column tube (<0.001%). These predictions agree well with the results of experimental measurements that were performed on columns packed with the pure Resolve silica (0% carbon), the derivatized Resolve-C18 (10% carbon) and the Symmetry-C18 (20% carbon) adsorbents, using water, methanol, or n-pentane as the mobile phase. These solvents have different compressibilities. However, 1% of the relative increase of the column hold-up volume that was observed when the pressure was raised is not accounted for by the compressibilities of either the solvent or the C18-bonded phase. It is due to the influence of the pressure on the retention behavior of thiourea, the compound used as tracer to measure the hold-up volume.     

Electrochemically modulated liquid chromatographic separation of triazines and the effect of pH on retention

Electrochemically modulated liquid chromatography (EMLC) manipulates analyte retention by changing the potential applied (E_app) to a conductive stationary phase. This paper applies EMLC to the separation of a set of seven triazines which are commonly used but environmentally hazardous herbicides. Experiments herein examine the influence of E_app and the pH of the mobile phase on triazine retention. The results are discussed in terms of: (1) retention of triazines of dissimilar acid strengths and by correlations with the pH of the mobile phase; (2) how changes in E_app and acid–base equilibria modulate elution; (3) qualitative insights into EMLC-based retention; and (4) potential merits of EMLC in realizing the rapid separation of the seven-component triazine mixture.     

The use of mobile phase pH and column temperature to reverse the retention order of enantiomers on chiral-AGP®

Summary Enantiomeric separation of mosapride and a structurally related compound was performed using chiral chromatography and experimental design. Unique effects of mobile phase pH and column temperature made it possible to control the elution order of the enantiomers when using Chiral-AGP as the solid phase. At a low mobile phase pH (<6) the (R)-enantiomer of mosapride elutes before the (S)-form whereas the (S)-enantiomer elutes first at a high mobile phase pH (>6). By using a mobile phase pH around 6, the column temperature could also be used to control the elution order of the enantiomers of mosapride. Similar effects of mobile phase pH and column temperature were obtained for the enantiomers of a structurally related compound, a metabolite (M1). Isocratic chromatographic systems made it possible to determine enantiomeric impurities less than 0.1% in the respective enantiomer of mosapride. The enantiomers of mosapride as well as the enantiomers of M1 could easily be separated simultaneously using Chiral-AGP and a simple gradient elution. Part of this work has been presented as lectures at HPLC'96 in San Francisco USA, at AAPS-96 in Seattle USA and as a poster at HPLC'95 in Innsbruck Austria.