Dissociation constants of phenols in methanol--water mixtures

A preferential solvation model that relates solute properties with solvent composition in binary mixtures has been applied to the dissociation pKa values of a set of 28 substituted phenols in methanol-water mixtures. The parameters of the model allow estimation of the pKa value of each phenol for any methanol-water composition. Moreover, it is demonstrated that the pKa values of the whole set of phenols at any methanol-water composition are linearly related to the pKa values of the phenols in water. Equations that relate the correlations' slope and intercept values with the solvent composition have been derived and tested with the set of phenols. The general parameters obtained for these equations allow an accurate calculation of the pKa value of any phenol, even of those not included in the original set, at any methanol-water composition solely from the pKa value of the phenol in water. These calculated pKa values can be used for quantitative structure-HPLC retention relationships. The method is tested by comparison of the calculated pKa values with the HPLC determined pKa values of 26 phenols in a polymeric column with a 50% methanol as mobile phase.     

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.     

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.     

Retention of ionisable compounds on high-performance liquid chromatography XVII. Estimation of the pH variation of aqueous buffers with the change of the methanol fraction of the mobile phase

The use of methanol-aqueous buffer mobile phases in HPLC is a common election when performing chromatographic separations of ionisable analytes. The addition of methanol to the aqueous buffer to prepare such a mobile phase changes the buffer capacity and the pH of the solution. In the present work, the variation of these buffer properties is studied for acetic acid-acetate, phosphoric acid-dihydrogenphosphate-hydrogenphosphate, citric acid-dihydrogencitrate-hydrogencitrate-citrate, and ammonium-ammonia buffers. It is well established that the pH change of the buffers depends on the initial concentration and aqueous pH of the buffer, on the percentage of methanol added, and on the particular buffer used. The proposed equations allow the pH estimation of methanol-water buffered mobile phases up to 80% in volume of organic modifier from initial aqueous buffer pH and buffer concentration (before adding methanol) between 0.001 and 0.01 mol L(-1). From both the estimated pH values of the mobile phase and the estimated pKa of the ionisable analytes, it is possible to predict the degree of ionisation of the analytes and therefore, the interpretation of acid-base analytes behaviour in a particular methanol-water buffered mobile phase.     

Retention of ionizable compounds in high-performance liquid chromatography. IX. Modelling retention in reversed-phase liquid chromatography as a function of pH and solvent composition with acetonitrile-water mobile phases

The influence of pH and solvent composition of acetonitrile-water mobile phases on the retention of acids and bases on a polymeric stationary phase is studied. Very good relationships between retention and mobile phase pH are obtained if the pH is measured in the proper pH scale. The fit of retention to pH for a particular solvent composition provides the pKa values of the equilibria between the different acid-base species and the retention parameters of these species at this solvent composition. Several models are tested that relate these parameters to solvent composition and properties in order to propose a general model to predict retention for any mobile phase pH and composition.     

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.     

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.     

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.     

Explanation for the enhanced dissolution of silica column packing in high pH phosphate and carbonate buffers

It has been reported that at high pH, the rate of bonded phase packing degradation in methanol/water mobile phases is greater for carbonate and phosphate buffers than for amine buffers. This conclusion was based on buffer pH determined in the aqueous buffer before dilution with methanol. Changes in buffer species pKa, and therefore buffer pH, upon methanol dilution are consistent with the observed degradation results. Measurements of pH in the methanol/water solutions confirm that the carbonate and phosphate buffers were considerably more basic than the amine buffer, even though all the buffers were pH 10 before dilution with methanol. These results demonstrate that it can be misleading to extrapolate aqueous pH data to partially aqueous solutions. Measurements of pH in the mixed solvent provide more reliable predictions of column and sample stability.     

Effect of temperature on competitive adsorption of the solute and the organic solvent in reversed-phase liquid chromatography

In analysis of the temperature effect on chromatographic separations the influence of the adsorption of organic solvent on the retention properties of solute is generally not taken into account. In fact, adsorption behavior of solutes is strongly affected by competitive adsorption of organic solvents, which is temperature dependent. In this work changes of adsorption equilibrium of an organic solvent as well as a solute with temperature have been analyzed. Data of the excess adsorption of methanol from aqueous solutions on octadecyl-bonded silica have been acquired at different temperature. Experiments have been performed over a relatively narrow temperature range corresponding to typical chromatographic conditions, i.e., 10-50 degrees C. The competitive adsorption equilibria of model solutes (i.e., two homologous compounds: cyclopentanone and cyclohexanone) have been measured at different temperature and composition of the mobile phase. Temperature alterations to the retention properties were found to result from combined effects of changes in adsorption behavior of the organic solvent and of the solute. The influence of temperature on the separation selectivity has been considered.     

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.     

Capillary zone electrophoresis of basic analytes in methanol as non-aqueous solvent mobility and ionisation constant

The electrophoretically relevant properties of monoacidic 21 bases (including common drugs) containing aliphatic or aromatic amino groups were determined in methanol as solvent. These properties are the actual mobilities (that of the fully ionised weak bases), and their pKa values. Actual mobilities were measured in acidic methanolic solutions containing perchloric acid. The ionisation constants of the amines were derived from the dependence of the ionic mobilities on the pH of the background electrolyte solution. The pH scale in methanol was established from acids with known conventional pK*a values in this solvent used as buffers, avoiding thus further adjustment with a pH sensitive electrode that might bias the scale. Actual mobilities in methanol were found larger than in water, and do not correlate well with the solvent's viscosity. The pK*a values of the cation acids, HB-, the corresponding form of the base, B, are higher in methanol, whereas a less pronounced shift was found than for neutral acids of type HA. The mean increase (compared to pure aqueous solution) for aliphatic ammonium type analytes is 1.8, for substituted anilinium 1.1, and for aromatic ammonium from pyridinium type 0.5 units. The interpretation of this shift was undertaken with the concept of the medium effect on the particles involved in the acid-base equilibrium: the proton, the molecular base, B, and the cation HB+.     

Peptide separations and dissociation constants in nonaqueous capillary electrophoresis: comparison of methanol and aqueous buffers

Nonaqueous capillary electrophoresis was evaluated for its potential to separate peptides in methanolic background electrolytes in comparison to aqueous-methanol (50% v/v) and water. Isomeric aspartyl dipeptides and Leu- and Met-enkephalin served as model compounds. pK(a) values were determined in the three solvent systems based on the apparent pH scale and in the case of methanol additionally based on the conventional pH scale. Changing from water to methanol led to an increase of the ionization constants describing the dissociation equilibria of the carboxyl group and the amino group, respectively. The pK(a) shift was more pronounced for the carboxylic acid function leading to a compression of the mobility-pH curve. As reported for aqueous buffers, efficient separations of the peptides were achieved in methanolic background electrolytes including the resolution of the diastereomers of the isomeric alpha- and beta-aspartyl dipeptides. In contrast to aqueous buffers, the separation of Leu- and Met-enkephalin could also be obtained in buffers in methanol at high pH.     

Assignment of standard pH values [pH*(s)] to buffers in 50 mass % methanol + water from 288.15 to 308.15 K

The secondary dissociation constants of o-phthalic and phosphoric acids have been determined in methanol + water (50 mass %) from reversible e.m.f. measurements of the cell of the type: Pt, H(2)(1 atm)|M(2)A(m), MHA(m), MCl|AgCl; Ag at different temperatures (288.15-308.15 K) and at different ionic strengths. To minimize the unsteadiness in potential measurements palladium coated platinum electrodes have been used. The large set of such e.m.f. values has been analyzed in terms of a multi-linear regression method recommended in recent IUPAC documents. The thermodynamic values DeltaG degrees , DeltaH degrees and DeltaS degrees , for the respective equilibria, were estimated. Standard pH values [pH*(s)] have been assigned to buffers in methanol + water (50 mass %) at temperatures between 288.15 and 308.15 K.     

Retention of ionisable compounds on high-performance liquid chromatography XVI. Estimation of retention with acetonitrile/water mobile phases from aqueous buffer pH and analyte pKa

In agreement with our previous studies and those of other authors, it is shown that much better fits of retention time as a function of pH are obtained for acid-base analytes when pH is measured in the mobile phase, than when pH is measured in the aqueous buffer when buffers of different nature are used. However, in some instances it may be more practical to measure the pH in the aqueous buffer before addition of the organic modifier. Thus, an open methodology is presented that allows prediction of chromatographic retention of acid-base analytes from the pH measured in the aqueous buffer. The model presented estimates the pH of the buffer and the pKa of the analyte in a particular acetonitrile/water mobile phase from the pH and pKa values in water. The retention of the analyte can be easily estimated, at a buffer pH close to the solute pKa, from these values and from the retentions of the pure acidic and basic forms of the analyte. Since in many instances, the analyte pKa values in water are not known, the methodology has been also tested by using Internet software, at reach of many chemists, which calculates analyte pKa values from chemical structure. The approach is successfully tested for some pharmaceutical drugs.     

A Rapid Stability-Indicating HPLC Assay for the Arabinosylcytosine Prodrug,Cyclocytidine

Abstract

Hydrolysis of cyclocytidine in aqueous solutions produced arabinosylcytosine which, in some cases, further reacted to form arabinosyluracil. No other degradation products were detected. A rapid isocratic reverse-phase HPLC assay for all three components in mixtures arising from cyclocytidine hydrolysis was developed. The analysis employs a 4.6 cm column together with a low methanol mobile phase containing 1-heptane sulfonic acid at pH 2.9. The ion-paring of cyclo-C, a cation, was independent of pH. However, ion-paring of arabinosylcytosine was controlled by adjusting the pH to 2.9 which is below its pKa of 4.2. The retention time of neutral arabinosyluracil (pKa = 9.2) was not affected by either the pH or the ion-pairing agent. Its separation was achieved by using a primarily aqueous mobile phase with the minimum methanol required for the other components. The time courses for cyclocytidine and its hydrolysis products were successfully defined under a variety of aqueous conditions.     

Multi-wavelength spectrophotometric determination of the protolytic constants of tetracycline hydrochloride in some nonaqueous-water mixed solvents: a solvatochromism study

Annihilation of the contribution of one chemical component from the original data matrix is a general method in rank annihilation factor analysis (RAFA). However, RAFA is not applicable for studying the protonation equilibria of multiprotic acids but in this study two-rank annihilation factor analysis (TRAFA) was used as an efficient chemometrics algorithm for determination of the protolytic constants (pKa) of tetracycline hydrochloride (TCHC) in some nonaqueous-water mixed solvents such as acetonitrile (AN)-water and methanol (MeOH)-water from the spectral pH-absorbance data. The spectral data was obtained from spectrophotometric acid-base titrations of different solutions of TCHC at (25.0±0.10)°C and an ionic strength of 0.10 M. In TRAFA algorithm the pKa values were obtained with relationship between residual standard deviation (R.S.D.) and hypothetical pKa values. In the case of TCHC, the spectra were divided in two consecutive subdivisions according to their pH range having two pKa and TRAFA was run twice. The validity of the obtained pKa values was checked with well-known chemometrics algorithms such as DATAN, EQUSPEC, SPECFIT/32 and SQUAD. The effects of changing solvent composition on the protolytic constants were explained by linear solvation energy relationships (LSER) utilizing solvatochromic parameters.     

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.     

Assignment of reference pH-values to primary standard buffer solutions for standardization of potentiometric sensors in acetonitrile-water mixtures

Standard pH-values pH(PS) for seven primary standard buffer solutions in 0, 10, 30, 40, 50, 70 and 100% (w/w) acetonitrile-water mixed solvents at 298.15 K were determined according to the criteria recently endorsed by IUPAC. The preferential solvation of the ions in acetonitrile-water mixtures was studied in order to clarify the acid-base behaviour of the solutes in such mixtures. The influence of variation in the solvent composition on pH(PS) values was considered with a view to obtaining correlations that can be used to determine the pH(PS) values in any acetonitrile-water mixture up to 70% (w/w) acetonitrile. The pH(PS) values were then correlated with the weight and volume percentages and molar fraction of acetonitrile and with the Kamlet-Taft, pi(*), alpha and beta solvatochromic parameters of the acetonitrile-water mixtures. The equations obtained permit the standardization of potentiometric sensors in these mixtures.     

High-throughput evaluation of lipophilicity and acidity by new gradient HPLC methods

There is a need for fast testing of drug candidates for properties of pharmacokinetics and pharmacodynamics importance, in particular lipophilicity and acidity. These two parameters can conveniently be estimated by gradient reversed-phase HPLC. Appropriate conventional organic solvent gradient and the new pH gradient HPLC procedures are presented. The chromatographic parameter of lipophilicity, log kw, can be determined from two organic solvent gradient runs instead of 6-8 runs necessary in the standard isocratic (polycratic) approach. The newly introduced pH gradient reversed-phase HPLC consists in a programmed increase during the chromatographic run of the eluting power of the mobile phase with regards to ionizable analytes. The eluting strength of the mobile phase increases due to its increasing (in case of acidic analytes) or decreasing (basic analytes) pH, whereas the content of organic modifier remains constant. It has been theoretically and experimentally demonstrated that the pKa and log kw values can be evaluated based on retention data from a pH gradient run, combined with appropriate data from two organic solvent gradient runs. The gradient HPLC-derived log kw parameters correlate well with analogous parameters determined isocratically as well as with reference lipophilicity parameter log P (logarithm of n-octanol/water partition coefficient). Also, the HPLC-derived pKa parameters correlate to the literature pKa values (w(w)pKa), conventionally determined by titrations in water. The approach described allows rapid and high-throughput assessment of log kw and pKa for large series of drugs candidates, also when the analytes are available in a form of mixture, e.g. produced by combinatorial synthesis.