I. Toro

Solvatochromic parameter values and pH in acetonitrile-water mixtures optimization of mobile phase for the separation of peptides by high-performance liquid chromatography☆

The proportion of the organic modifier and the pH of the mobile phase were optimized in order to separate a series of low-molecular-mass peptides by HPLC. The composition of the mobile phase was optimized by establishing relationships between retention parameters and Reichardts ENT scale of solvent polarity, and between retention and the Kamlet-Taft multiparameter solvent scale of the eluent, using linear solvation energy relationships (LSER). The pH of the mobile phase was also successfully optimized by establishing relationships between the retention and the pH measured in the aqueous-organic mixture used as eluent.

Factor analysis applied to the correlation between dissociation constants and solvatochromic parameters in water-acetonitrile mixtures: I. Solvent effects on the dissociation of carboxylic acid groups in some diuretics, quinolones, buffers and peptides

Abstract The dissociation constants in several acetonitrile-water mixtures of the carboxylic acid groups of 19 substances, included into four different sets (diuretics, quinolones, buffers and peptides) were first submitted to factor analysis (FA) in order to find how many factors affect the variations in the data sets. Then target factor analysis (TFA) was performed in order to identify these factors. The influence of solvatochromic parameters in the interactions between carboxylic acid groups and the solvents studied was identified and quantified. The model Kamlet-Taft equation for each substance was established. The general effect of the preferential solvation of electrolytes in acetonitrile-water mixtures on the values of the protonation constants in these media is also described.

Preferential solvation in acetonitrile–water mixtures

Ionization constants of several carboxylic acids in acetonitrile–water mixtures, usually used as mobile phases in liquid chromatography (LC), have been obtained in order to predict the influence of pH on retention and selectivity in LC. In this work, the quasi-lattice quasi chemical (QLQC) theory of preferential solvation was applied to obtain the degree of preferential solvation of acetate ions in these media in order to explain the variation of the pK values obtained over the whole composition range studied. Also, the relationships between pK values and various macroscopic and microscopic properties were used to calculate pK values of the carboxylic acids in any acetonitrile–water mixture up to 70% (w/w) and, thus, to determine the acid–base behaviour of substances in the widely used acetonitrile–water media.

Evaluation of electrophoretic method versus chromatographic, potentiometric and absorptiometric methodologies for determing pKa values of quinolones in hydroorganic mixtures

The advantages of using capillary electrophoresis (CE) over other methodologies for determining pK(a) values of drugs in hydroorganic media are discussed. The focus of the discussion based upon the pK(a) values of a series of quinolones determined in acetonitrile (MeCN)--water mixtures by CE, liquid chromatography, potentiometric, and spectrophotometric methods.

Retention behaviour of peptides, quinolones, diuretics and peptide hormones in liquid chromatography. Influence of ionic strength and pH on chromatographic retention.

Peptides, quinolones, diuretics and peptide hormones are important substances of biomedical interest. In this work a model describing the effect of pH on retention in liquid chromatography (LC) is established and tested for these compounds using an octadecylsilica column. The suggested model uses the pH value measured in the hydro-organic mixture used as mobile phase instead of the pH value in water and takes into account the effect of the activity coefficients. The proposed equations permit the prediction of the pH optimum using a minimum number of measurements and also permit the determination of the acidity constants of the compounds considered in the medium used as mobile phase. Moreover, these equations can be combined with the previously derived equations, that relate the retention with the solvent composition of the mobile phase, to establish a general model that relates the elution behaviour of the solute with the significant mobile phase properties: composition, pH and ionic strength.

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.

Migration behavior of therapeutic peptide hormones: Prediction of optimal separation by capillary electrophoresis

A general equation that relates electrophoretic mobility of polyprotic peptide substances and pH of the running electrolytes is established, taking into account the species in solution and the activity coefficients. Modelling electrophoretic mobility as a function of pH can be simultaneously used for determination of ionization constants and selection of the optimum pH for separation of mixtures of the modelled compounds. The proposed relationships allow an important reduction of the experimental data needed for development of new separation methods. The accuracy of the proposed equations is verified by modelling the migration behavior of a heterogeneous series of polyprotic amphoteric peptide hormones. By calculating the values of predicted resolutions, selection of the optimum pH to perform separation of their mixtures becomes a rapid and simple process.

Chromatographic behavior of ionizable compounds in liquid chromatography. Part 2. Standardization of potentiometric sensors and effect of pH and ionic strength on the retention of analytes using acetonitrile–water mobile phases

In this study several properties of acetonitrile–water (ACN–water) mixtures were examined which have an influence on the liquid chromatography (LC) behavior of ionizable compounds, such as Debye–Huckel parameters, pH scale, LC-useful pH range and solvatochromic parameters. Factor analysis techniques were applied to the correlation between standard pHS values of NIST buffer solutions and solvatochromic parameters in acetonitrile (ACN)–water. The relationships obtained allow the calculation of standard pHS values in any ACN–water mixture and thus permit correct pH measurement in these media. Through correct pH, pKa and activity coefficient values, a model describing the effect of pH on retention in LC is established and tested for several diuretic acids and peptide ampholytes using an octadecylsilica column and ACN–water mixtures of 7, 30, 35, 40, 45 and 50% (v/v) ACN as mobile phases. The proposed model uses the pH values in ACN–water mobile phases instead of in water, and takes into account the effect of activity coefficients. The usefulness of the proposed equations is twofold: they permit the determination of pKa of analytes in the hydroorganic media used as mobile phases, and also permit the prediction of the optimum pH for the separation methodologies using a minimum number of measurements. The proposed equations can be combined with the previously derived equation, which relates the retention to the composition of the mobile phase, in order to establish a general model relating elution behavior of the solutes with the significant mobile phase properties: composition, pH and ionic strength.

Secondary chemical equilibria in high-performance liquid chromatography: influence of ionic strength and pH on retention of peptides

Abstract In this work a model describing the effect of pH on retention in LC is established and tested for two series of peptides using an octadecylsilica column. The suggested model uses the pH value measured in the acetonitrile–water mixture instead of the pH value in water and takes into account the effect of the activity coefficients. The proposed equations permit the prediction of the pH optimum using a minimum number of measurements and can be combined with the previously derived equations, that relate the retention with the solvent composition of the mobile phase, to establish a general model that relates the elution behaviour of the solute with the significant mobile phase properties: composition, pH and ionic strength.

Solvent effects on protonation equilibria of peptides and quinolones by factor analysis applied to the correlation between dissociation constants and solvatochromic parameters in acetonitrile-water mixtures

Abstract Dissociation constant values of series of peptides and quinolones in 5.54, 10, 16.3, 25 and 50% (w/w) acetonitrile-water mixed solvents at 298.15 K were, firstly, submitted to factor analysis in order to obtain the number of factors which affects the variation of the whole data sets and, afterwards, to target factor analysis to identify these factors. The p K values were correlated with the Kamlet and Taft solvatochromic parameters (α, β and π ∗ ) of acetonitrile-water mixtures according to the results obtained through these chemometric techniques. The equation proposed allowed the calculation of p K values of peptides in any acetonitrile-water mixture up to 50% (w/w) and, therefore, the acid-base behaviour of these compounds in widely used acetonitrile-water media is to be known. Further, an overview of the effect of preferential solvation of electrolytes in acetonitrile-water mixtures on the values of the protonation constants in these media was also obtained.