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.     

Immobilized artificial membrane liquid chromatography: proposed guidelines for technical optimization of retention measurements

The objectives of this study were to establish guidelines for the proper measurement of capacity factors (log k(IAMw) on immobilized artificial membrane (IAM) stationary phases. In this context, some aspects related to the extrapolation of log(kIAMw) values, the stability and properties of IAM.PC.DD2 stationary phases and the column-to-column variability are discussed. No significant difference was observed when using either acetonitrile or methanol for the linear extrapolation of log k(IAM) values. However, methanol seems more appropriate when working with ionized compounds. Plotting isocratic capacity factors against the percentage (v/v) of co-solvent instead of the mole fraction leads to more reliable log k(AMW) values. Furthermore, our results with a YMC ODS-AQ and an IAM.PC.DD2 HPLC column indicate that only small differences arise between extrapolated capacity factors when using the (w(w))pH or the (s(w))pH operational scale and correcting or not the ionic strength for dilution caused by the co-solvent. The use of the (s(w))pH scale is recommended when working with ionized compounds in order to avoid parabolic relationships during linear extrapolation. The pH-dependent retention of three ionizable drugs on an IAM.PC.DD2 phase showed that secondary interactions with the charged moieties of the chromatographic surface affect the retention of ionized compounds around physiological pH. Finally, it was shown that column ageing occurs also with IAM.PC.DD2 stationary phases and that it depends on the column as well as on the investigated analyte. The intra-batch variability for IAM.PC.DD2 phases was small, whereas a marked and solute-dependent batch-to-batch variability was apparent.     

pH gradient high-performance liquid chromatography: theory and applications

pH gradient high-performance liquid chromatography (HPLC) is a method of reversed-phase high-performance liquid chromatography suitable for ionogenic substances. It consists in programmed increase during the chromatographic process of the eluting strength of eluent with respect to the analytes separated. On the analogy of the conventional organic modifier gradient reversed-phase HPLC, in the pH gradient approach the eluting strength of the mobile phase increases due to its changing pH: increasing in case of acids or decreasing in case of bases. At the same time the content of organic modifier remains constant. A theory of the pH gradient HPLC has been elaborated. The resulting mathematical model is easily manageable. Its ability to predict changes in retention and separation of analytes following the changes in chromatographic conditions is demonstrated. The pH gradient method is uniquely suitable to determine pKa values of analytes. An equation is presented allowing to calculate pKa values basing on appropriate retention data. The effects on pKa are discussed of the concentration of methanol in the mobile phase. The RP HPLC-derived pKa data correlate to the reference pKa values (w(w)pKa) but are not identical. That may be explained by the effects on the chromatographically determined pKa of the specific interactions of analytes with stationary phases. The proposed pH gradient RP HPLC procedure offers a fast and convenient means to get comparable acidity parameters for larger series of compounds, like drug candidates, also when the analytes are available only in minute amounts and/or as complex mixtures.     

Effect of temperature on the chromatographic retention of ionizable compounds. I. Methanol-water mobile phases

The retention mechanism of acids and bases in reversed-phase liquid chromatography (RPLC) has been experimentally studied by examining the temperature dependence of retention, with emphasis on the role of the buffer ionization equilibria in the retention and selectivity. Retention factors of several ionizable compounds in a typical octadecylsilica column and using buffers dissolved in 50% (w/w) methanol as eluents at three temperatures in the range of 25-50 degrees C were measured. Two pairs of buffer solutions were prepared by a close adjusting of their pH at 25 degrees C; differences in their ionization enthalpies determined a different degree of ionization when temperature was raised and, as a consequence, a different shift in the eluent pH. Predictive equations of retention that take into account the temperature effect on both the transfer and the ionization processes are proposed. This study demonstrates the significant role that the selected buffer would have in retention and selectivity in RPLC at temperatures higher than 25 degrees C, particularly for co-eluted solutes.     

液固色谱中溶质保留规律的研究—多元流动相组成对保留值的影响

根据热力学原理和简单的吸附色谱分子模型。本文提出了一个新的液固色谱保留模型，并导出了相应的保留值基本方程，以三元溶剂系统作为展开剂，用薄层色谱实验数据验证了所得方程的正确性。结果显示，计算Ｒｆ值与实验Ｒｆ值完全一致。     

The theoretical basis of column chromatography in multicomponent separations. Part 2: Peak capacity. Column systems with good selectivity

The selectivity of a column system, S, defined by equation 28, includes a variation coefficient (β) of plate number with capacity ratio which has a large influence on the peak capacity, as shown in Fig. 18. Some typical chromatograms are given. In order to predict S for a column system from Kovat's Index, equations 40 and 41 are given to calculate the constant of the carbon number rule for squalane at different temperatures or for different stationary phases. The specific retention value of heptane on squalane at different temperatures can be calculated from equation 42. The nonpolarity index, defined by equation 43, was used to calculate the retention value of heptane on various stationary phases. In liquid chromatography, the order of elution may be reversed by changing the composition of the eluent on the same chemically bonded silica (manufactured in China). The linear relations between the log retention values of different kinds of solutes or of a single solute on the silicas of different surface areas when using the same eluent are given.     

Effect of ionization and the nature of the mobile phase in quantitative structure-retention relationship studies

The octanol-water distribution constant, commonly called partition coefficient, Po/w, is a parameter often retained as a measure of the hydrophobicity of a molecule. log Po/w, for a given molecule, can be conveniently evaluated constructing correlation lines between standard retention factor logarithms (log k) in reversed-phase liquid chromatography (RPLC) and standard log Po/w values. Many compounds of pharmaceutical interest can be quite hydrophobic and have, simultaneously, basic nitrogen atoms or acidic sulfur containing groups in their structure. This renders them ionizable. The hydrophobicity of the molecular drug form (Po/w value) is completely different from its ionic form (log Po/w(+ or -) value). The actual hydrophobicity of such ionizable molecule depends on the pH. It can be represented by an apparent Papp value that takes into account the amount of compound in its molecular and ionic state combining the Po/w and Po/w(+ or -) values. In this work, log k in RPLC for ionizable as well as non-ionizable pharmaceutical compounds with different therapeutic properties (10 beta-blockers, seven tricyclic antidepressants (TA), eight steroids and 12 sulfonamides) were correlated with log Po/w. Similar correlations were done between log k and the corrected log Papp values at pH 3. Aqueous-organic mobile phases containing acetonitrile (conventional RPLC) and micellar-organic mobile phases (micellar liquid chromatography, MLC), prepared with the anionic surfactant sodium dodecyl sulfate and the organic solvents acetonitrile, propanol or pentanol, were also used to elute the compounds. All mobile phases were buffered at pH 3. Using conventional retention RPLC data, the correlation of log k with log Po/w, was satisfactory for steroids because they cannot ionize. For ionizable beta-blockers and TAs, the use of log Papp values improved the quality of the correlations, but yielded similar results for sulfonamides. In MLC, since an electrostatic interaction is added to hydrophobic forces, poorer correlations were obtained in all cases. The retention data obtained in RPLC also seems to correlate better with the biological activity of the drugs.     

Retention of ionizable compounds on HPLC. 6. pH measurements with the glass electrode in methanol-water mixtures

The relationship, delta values, between the two rigorous pH scales, S(S)pH (pH measured in a methanol-water mixture and referred to the same mixture as standard state) and S(W)pH (pH measured in a methanol-water mixture but referred to water as standard state), in several methanol-water mixtures was determined (delta = S(W)pH-S(S)pH). Delta values were measured using a combined glass electrode and a wide set of buffer solutions. The results are consistent with those obtained with the hydrogen electrode. This confirms the aptness of the glass electrode to achieve rigorous pH measurements in methanol-water mixtures. An equation that relates delta and composition of methanol-water mixtures, and allows delta computation at any composition by interpolation, is proposed. Therefore, S(S)pH can be achieved from the experimental S(W)pH value and delta at any mobile phase composition. S(S)pH (or S(W)pH) values are related to the chromatographic retention of ionizable compounds through their thermodynamic acid-base constants in the methanol-water mixture used as mobile phase. These relationships were tested for the retention variation of several acids and bases with the pH of the mobile phase. Therefore, the optimization of the mobile phase acidity for any analyte can be easily reached avoiding the disturbances observed when W(W)pH is used.     

反相液相色谱中同系物体系实验点与预测方程参数间的匹配研究

以波相色谱中溶质计量置换统一保留模型（SDM-R）的二参数、三参数和四参数方程为基础,研究了在预测同系物保留值时实验点与预测方程参数间的匹配关系。用两个实验点,分别以二、三和四参数方程对反相色谱中同系物保留值进行预测,发现预测值与实验值基本相符,但以二参数方程预测结果为最佳。又与其它保留方程的预测结果进行了比较,证实了上述结论。当实验点数增加到4～5个时,多参数方程预测同系物的准确度增加,且明显高于二参数方程。同时还讨论了方程中各参数与同系物碳数间的线性关系,发现二参数方程中的参数遵循同系物变化规律。     

A new approach to the determination of relative retention in thin-layer liquid chromatography

The relative retention values in chromatography depend on the experimental conditions to a lesser degree than the absolute values. Unfortunately, according to IUPAC recommendations, the relative retention values in planar and column chromatography are different. Therefore, a new equation is proposed to determine relative retention in TLC and a new method and new value are introduced to determine it experimentally. The equation is identical in principle to that used in column chromatography. Using the proposed equation, the relative retention value can be determined with a smaller error.     

Influence of mobile phase acid-base equilibria on the chromatographic behaviour of protolytic compounds

A review about the influence of mobile phase acid-base equilibria on the liquid chromatography retention of protolytic analytes with acid-base properties is presented. The general equations that relate retention to mobile phase pH are derived and the different procedures to measure the pH of the mobile phase are explained. These procedures lead to different pH scales and the relationships between these scales are presented. IUPAC rules for nomenclature of the different pH are also presented. Proposed literature buffers for pH standardization in chromatographic mobile phases are reviewed too. Since relationships between analyte retention and mobile phase pH depends also on the pKa value of the analyte, the solute pKa data in water-organic solvent mixtures more commonly used as chromatographic mobile phase are also reviewed. The solvent properties that produce variation of the pKa values with solvent composition are discussed. Chromatographic examples of the results obtained with the different procedures for pH measurement are presented too. Application to the determination of aqueous pKa values from chromatographic retention data is also critically discussed.     

Prediction of retention behaviour and evaluation of pka values of peptides and quinolones in liquid chromatography.

The present paper examines the effect of the solute ionisation on the retention behaviour in liquid chromatography of a series of peptide and quinolone compounds of biological interest, using acetonitrile-water media as mobile phases and a polymeric-based stationary phase. Polymeric columns with polystyrene-divinylbenzene (PS-DVB) polymer show advantages over silica-based reversed-phase packings since the former are stable in a wide pH range. (s)(s)pKa values have been evaluated using chromatographic data in acetonitrile-water mixtures with acetonitrile percentages of 30, 35, 40 and 50% (v/v) for quinolones and 12.5 and 20% (v/v) for peptides. The quinolones show great retention on PS-DVB phase stationary. It was thus necessary to work with a higher acetonitrile content in the mobile phase than for the less retained peptides. The pH values were measured in the hydroorganic mixtures, used as mobile phases, instead of in water and account was taken of the effect of activity coefficients. The derived equations permit the chromatographic determination of (s)(s)pKa. values of the peptides and quinolones in acetonitrile-water mixtures by fitting it to the experimental data in a nonlinear least-square procedure and also permit the prediction of the effect of (s)(s)pH on their chromatographic behaviour. We have also compared the obtained (s)(s)pKa values with those previously obtained in acetonitrile-water mixtures from potentiometric measurements.     

Evaluating the surface charge of C18 stationary phases

The surface charge of four C18 stationary phases was investigated by measuring the flow induced streaming potential, a well known electrokinetic property of charged surfaces. Three of the stationary phases (Symmetry, Gemini, and Xterra-MS) had significantly positive streaming potentials at both pH 3 and 4.5. The fourth (Zorbax-SB) appeared to be essentially neutral at pH 3 and became negative at pH 4.5. Apparent zeta potentials ranged from approximately +16 to -4 mV. The retention behavior was also investigated using chloride as model anion and glycinamide (in its protonated form) as model cation. When the retention factor (k) of glycinamide was subtracted from k of chloride anion, the resulting delta k values showed very similar trends as apparent zeta potential values, suggesting that the simple chromatographic method could be used to estimate zeta potential values, or that the zeta potential values could be useful for ranking columns according to ion exchange or exclusion behavior. The anion exchange capacity of the Symmetry and Gemini columns was also estimated, using a published chromatographic procedure, and the results suggest about 2 microEq. capacity per gram of packing.     

Adsorption and surface complexation of trimesic acid at the alpha-alumina-electrolyte interface

Adsorption kinetics, adsorption isotherms and surface complexation of trimesic acid onto alpha-alumina surfaces were investigated. Adsorption kinetics of trimesic acid with an initial concentration of 0.5 mM onto alpha-alumina surfaces were carried out in batch method in presence of 0.05 mM NaCl (aq) at pH 6 and 298.15, 303.15 and 313.15 K. Adsorption isotherms were carried out at 298.15 K, pH 5-9, and 0.05 mM NaCl (aq) by varying trimesic acid concentration from 0.01 to 0.6 mM. Three kinetics equations such as pseudo-first-order, pseudo-second-order and Ho equations were used to estimate the kinetics parameters of the adsorption of trimesic acid on the alpha-alumina surfaces. Ho equation fits the experimental kinetics data significantly better and the estimated equilibrium concentration is in excellent agreement with the experimental value. The adsorption data were fitted to Freundlich and Langmuir adsorption model and the later best fits the adsorption isotherms. Comparison of adsorption density of trimesic acid with that of benzoic and phthalic acids follows the sequence: benzoic acid < trimesic acid < phthalic acid. The negative activation energy and the Gibbs free energy for adsorption indicate that the adsorption of trimesic acid onto alpha-alumina is spontaneous and facile. DRIFT spectroscopic studies reveal that trimesate forms outer-sphere complexes with the surface hydroxyl groups that are generated onto alpha-alumina surfaces in the pH range of the study.     

Adsorption characteristics of various organic substances on the surfaces of tantalum, titanium, and zirconium

Adsorption characteristics of carboxylic acids, amines, an octapeptide composed of four L-alanine and four L-aspartic acid residues (Peptide-A4D4), and beta-lactoglobulin (beta-Lg) on tantalum (Ta), titanium (Ti), and zirconium (Zr) particles were examined at 30 degrees C and in some case, were compared with their adsorption onto SUS316L stainless steel particles (S6L). The adsorption isotherms on the Ta, Ti, and Zr particles could usually be expressed either by a Langmuir-type equation for reversible adsorption or by a modified Langmuir-type adsorption equation including terms for both reversible and irreversible adsorption. The adsorption equilibrium of benzoic acid, benzylamine, and m-xylylenediamine on all the metal surfaces followed a Langmuir-type equation, while those of phthalic acid, mellitic acid, and Peptide-A4D4 could be fitted to the modified Langmuir-type adsorption equation. The adsorption characteristics of different adsorbates on the different surfaces were discussed particularly with reference to the pH dependencies of the q(irrev), q(rev), and K values and the electrostatic properties of the oxidized surface of the metal particles. Fourier transform infrared spectroscopic analyses using a reflection/absorption technique (RA-IR) indicated that phthalic acid and mellitic acid are adsorbed in similar adsorption states irrespective of the type of metal. beta-Lg was adsorbed onto the surfaces principally in an irreversible manner. The desorption behavior of beta-Lg from Ta, Ti, and S6L surfaces was examined, in order to evaluate the extent of interaction between beta-Lg and the metals.     

Theoretical evaluation of methods for extracting retention factors and kinetic rate constants in liquid chromatography

A three-dimensional stochastic simulation is used to provide a detailed understanding of mass transfer processes in liquid chromatography. In this simulation, the migration of individual molecules is established through diffusion and laminar convection within the mobile phase. The molecules interact with the stationary phase by a partition (absorption) mechanism. For these studies, the column length, linear velocity, stationary-phase diffusion coefficient, interfacial mass transfer coefficient, and equilibrium constant are varied in a system with a homogeneous surface. Heterogeneous surfaces are also investigated by having multiple types of interaction sites that are equally or unequally distributed. For each simulation, the molecular distribution is examined and characterized at specified times or column lengths. Five individual methods are then applied to extract the thermodynamic and kinetic information for transport between the mobile and stationary phases. In the first method, all of the molecules are initially distributed in the mobile phase and the fraction remaining is monitored as a function of time. These simulation data are fit to a single exponential decay by nonlinear regression to determine the "true" retention factors and rate constants. The other methods rely on evaluating the shape of the zone profiles along the column. The statistical moments of the profiles are used to calculate the mean and the variance, which are related to the retention factors and the rate constants, respectively. The profiles are also fit to the exponentially modified Gaussian equation, the Giddings equation, and the Thomas equation. The fitting parameters from these equations are then used to calculate the retention factors and rate constants. Comparisons of the accuracy relative to the "true" retention factors and "true" rate constants, as well as the advantages and limitations of each method are discussed.     

Modelling of retention behaviour of solutes in micellar liquid chromatography

In micellar liquid chromatography (MLC), the resolution for a given multi-component mixture can be optimized by changing several variables, such as the concentrations of surfactant and organic modifier, the pH and temperature. However, this advantage can only be fully exploited with the development of mathematical models that describe the retention and the separation mechanisms. Several reports have appeared recently on the possibilities of accurately predicting the solute retention in MLC. Although the retention and selectivity may strongly change with varying concentrations of surfactant, organic modifier and/or pH, the observed changes are very regular, and are well described by simple models. This characteristic enables a successful prediction of retention times and compensates the negative effect of the broad and tailed chromatographic peaks obtained for some solutes when micellar eluents are used. An overview of the models proposed in the literature to describe the retention behaviour in pure micellar eluents and micellar eluents containing an organic modifier, at a fixed pH or at varying pH, is given. The equations derived permit the evaluation of the strength of micelle-solute and stationary phase-solute interactions. The prediction of the retention based on molecular properties and the use of neural networks, together with the factors affecting the prediction capability of the models (linearization of the equations, dead time, critical micellar concentration, ionic strength and temperature) are commented on. The strategies used for the optimization of resolution are also given.     

Effect of temperature on the chromatographic retention of ionizable compounds. II. Acetonitrile-water mobile phases

The retentive behavior of weak acids and bases in reversed-phase liquid chromatography (RPLC) upon changes in column temperature has been theoretically and experimentally studied. The study focuses on examining the temperature dependence of the retention of various solutes at eluent pH close to their corresponding pKa values, and on the indirect role exerted by the buffer ionization equilibria on retention and selectivity. Retention factors of several ionizable compounds in a typical octadecylsilica column and using buffer solutions dissolved in 30% (v/v) acetonitrile as eluent at five temperatures in the range from 25 to 50 degrees C were carefully measured. Six buffer solutions were prepared from judiciously chosen conjugated pairs of different chemical nature. Their pKa values in this acetonitrile-water composition and within the range of 15-50 degrees C were determined potentiometrically. These compounds exhibit very different standard ionization enthalpies within this temperature range. Thus, whenever they are used to control mobile phase pH, the column temperature determines their final pH. Predictive equations of retention that take into account the temperature effect on both the transfer and the ionization processes are evaluated. This study demonstrates the significant role that the selected buffer would have on retention and selectivity in RPLC at temperatures higher than 25 degrees C, particularly for solutes that coelute.     

Model and experimental studies for contact angles of surfactant solutions on rough and smooth hydrophobic surfaces

Despite the practical need, no models exist to predict contact angles or wetting mode of surfactant solutions on rough hydrophobic or superhydrophobic surfaces. Using Gibbs' adsorption equation and a literature isotherm, a new model is constructed based on the Wenzel and Cassie equations. Experimental data for aqueous solutions of sodium dodecyl sulfate (SDS) contact angles on smooth Teflon surfaces are fit to estimate values for the adsorption coefficients in the model. Using these coefficients, model predictions for contact angles as a function of topological f (Cassie) and r (Wenzel) factors and SDS concentration are made for different intrinsic contact angles. The model is also used to design/tune surface responses. It is found that: (1) predictions compare favorably to data for SDS solutions on five superhydrophobic surfaces. Further, the model predictions can determine which wetting mode (Wenzel or Cassie) occurred in each experiment. The unpenetrated or partially penetrated Cassie mode was the most common, suggesting that surfactants inhibit the penetration of liquids into rough hydrophobic surfaces. (2) The Wenzel roughness factor, r, amplifies the effect of surfactant adsorption, leading to larger changes in contact angles and promoting total wetting. (3) The Cassie solid area fraction, f, attenuates the lowering of contact angles on rough surfaces. (4) The amplification/attenuation is understood to be due to increased/decreased solid-liquid contact-area.