Ionogenic Solutes Research Articles

Practical examination of flow rate effects and influence of the stationary phase water layer on peak shape and retention in Hydrophilic Interaction Liquid Chromatography.

The effect of flow velocity on retention and peak shape of neutral, acidic and basic probe compounds was studied using seven different UHPLC hydrophilic interaction chromatography (HILIC) columns. Surprisingly on some columns, the retention factor k was found to vary somewhat with flow velocity, due to the combined effects of pressure and of frictional heating on retention. The selectivity of different HILIC columns was much greater than typically found with RP columns. The volume of the water layer on the HILIC columns was measured using the toluene exclusion procedure. For the neutral solute uridine, a good correlation was found between the volume of the water layer and retention, indicating the likely domination of a partition mechanism. For the ionogenic solutes, the correlation was generally poor, due to the presence of strong additional mechanisms such as ionic retention and repulsion. Reduced van Deemter plots for uridine showed a negative correlation between the reduced b coefficient and the volume of the water layer, which can be attributed to reduced surface diffusion in this viscous layer. Once again, the behaviour of ionic solutes was complex on some columns making detailed interpretation difficult.

Effects of pH mismatch between the two dimensions of reversed-phase × reversed-phase two-dimensional separations on second dimension separation quality for ionogenic compounds—I. Carboxylic acids

Two persistent impediments to wider adoption of two-dimensional liquid chromatography (2D-LC) are the perceptions that 2D methods are generally less sensitive than 1D ones, and that coupling of certain separation modes in a 2D system is difficult because of the negative impact of the effluent of the first separation on the second separation. In this work we address these problems in the specific case where reversed-phase separations are used in both dimensions of a 2D-LC system, but the pH is varied such that the ionization state of carboxylic acid analytes is different (i.e., neutral or negatively charged, in eluents buffered at pH 2 or 7) in the two columns. We first demonstrate that the effect of first dimension (1D) effluent on the performance of second dimension (2D) separation of ionogenic solutes is much more serious than it is for neutral compounds where the pH of the eluent does not play a role in retention. We have systematically varied the properties of the sample solution injected into the 2D column (i.e., the 1D effluent), as well as the 2D eluent, with the goal of establishing guidelines for conditions that yield acceptable 2D performance. We find that the organic solvent content of the 1D effluent and 2D eluent is not as important as the buffer concentrations in these two solutions, and that the greater the ratio of buffer concentration in the 1D effluent relative to the 2D eluent, the smaller the volume one can inject into the 2D column before dramatic peak splitting occurs. We have then used the information from these simple experiments to guide both 1D experiments that mimic the 2D separation, and actual 2D separations, to demonstrate that online adjustment of the properties of the 1D effluent by dilution with a buffered solvent prior to injection into the 2D column is a very effective solution to the pH mismatch problem. We find that when the buffer capacity of the diluent is high enough to effectively titrate the 1D effluent such that its pH approaches that of the 2D eluent, excellent 2D peak shape is obtained for the carboxylic acid analytes, even when the volume of injected sample solution exceeds the 2D column volume.

Statistical tests for the selection of the optimum parameters set in models describing response surfaces in reversed-phase liquid chromatography

An appropriate procedure based on statistical criteria is suggested for the determination of the optimum set of model parameters for a given chromatographic system. The criteria employed are the t-ratio test, the rate of change in the sum of squares of residuals, the standard error of the fit, the F-test, and the CP-test. The suggested procedure has been evaluated using two different models, one based on partition and the other on adsorption mechanisms, which describe the combined effect of pH and organic modifier content on the retention of ionogenic solutes in reversed-phase liquid chromatography. It is shown that all the criteria give almost converged results and therefore we may simply use the F-test, which seems to be the most sensitive and reliable criterion excluding any personal judgement. It is also found that the retention models tested show a different behavior towards their simplification. In particular, the use of a reduced equation of the partition model, selected on the basis of the suggested procedure, is necessary for the prediction of meaningful retention surfaces, whereas the decrease in the number of the adjustable parameters in the adsorption model offers only noise reduction and fitting simplicity, because no version of this model predicts abnormal retention surfaces.

Non-linear least-squares fitting with excel solver and related routines in HPLC modelling of retention

The usefulness of a non-linear least-squares type algorithm such as the Microsoft Excel Solver for response surface modeling of ionogenic solutes in reversed-phase liquid chromatography has been examined. The retention factors of adenosine, a typical ionogenic solute, in buffered mobile phases of different pH modified with methanol and/or acetonitrile were taken from the literature and fitted to a general 9-parameter equation using adequate initial estimates of the fitting parameters and/or ranges of these parameters estimated by a Monte Carlo technique described previously The validity of each fit was tested with a method suggested in the present communication. In addition, the physical meaning and significance of the fitted coefficients is discussed together with the standard errors calculated previously. The possibility of predicting the retention behaviour of adenosine on the basis of a limited number of data was also explored.

Retention and column efficiency in reversed phase liquid chromatography as a function of pH for optimization purposes

The effects of pH on both the solute retention and the peak shape of ionogenic compounds are studied in order to propose accurate models for pH optimization purposes. Several mathematical models (theoretical and empirical) for describing the variation of the retention factor versus pH are compared within different pH ranges. Limits of such models used for optimizing the pH by requiring only 3 preliminary experimental runs, are discussed in terms of deviations (≤±5%) of predicted retention times from experimental retention times. An original procedure is developed for selecting the most convenient retention model, from a given set of three retention data. This set is also applied to modeling the variation of both peak width and peak asymmetry with mobile phase pH conditions. Such a procedure is demonstrated as helpful for the separation of ionogenic solutes by considering mobile phase pH as an additional variable that can be useful during optimization procedures.

Modelling chromatographic behaviour as a function of pH and solvent composition in RPLC

In this work we establish the basic layout of IONICS, an expert system for optimizing the separation of ionogenic solutes in Reversed-Phase Liquid Chromatography, using the pH and the organic-modifier concentration of the mobile phase as parameters. We also present REMO, a front-end system that automates the retention modelling stage, based on a 9-parameter model. This system uses a scale transformation to suppress several numerical problems previously observed and features a strategy for automatic calculation of an initial approximation to the model optimum. The successful application of this system to a set of seven drugs is described. The final models are accurate and have smaller numerical problems. We also describe the use of a genetic algorithm instead of classical non-linear least-squares for fitting the model to the experimental data. Results indicate that genetic algorithms are a valuable, complementary tool for retention modelling.

Optimizing separations in reversed-phase liquid chromatography by varying pH and solvent composition

An interpretive optimization procedure in which pH can be one of the variables is presented with the emphasis on optimizing separations. When varying the pH in reversed-phase liquid chromatography the retention of ionogenic solutes will change. Thus, the selectivity between ionogenic and neutral solutes or between ionogenic solutes mutually can be optimized. However, pH also greatly affects the efficiency (plate count) and peak shape (asymmetry). Optimum selectivity (i.e. large differences in retention times) may be observed under conditions where peaks are broad and asymmetrical. Thus, it is essential to simultaneously consider retention, peak width and peak shape and their effects on separation (effective resolution) in pH-optimization studies. A procedure in which this is done is presented and applied to optimizing the separation of a synthetic mixture of selected pharmaceuticals. After initial experiments to establish the parameter space (boundaries for pH and binary methanol — water composition), twelve experiments are performed according to a 3×4 experimental design. At each loaction the retention, peak height, peak area and peak symmetry are recorded for each solute. These data are then used to build models for each of the four characteristics and for each solute. From this set of models the response surface, describing the quality of separation as a function of pH and composition, can be calculated. A variety of optimization criteria (quantifying quality of separation) can be used. The optimum corresponds to the highest point on the response surface.

Modelling retention in reversed-phase liquid chromatography as a function of pH and solvent composition

Models describing the concomitant effects of pH and organic modifier concentration on retention in reversed-phase liquid chromatography are established. Two different octadecyl-modified silica columns were used. The retention behaviour of several acidic, basic and neutral solutes were studied, using methanol as the organic modifier. The suggested models accurately describe retention as a function of pH and composition. A unified formalism for retention modelling that is applicable to all ionogenic solutes is also established. This formalism is tested with the modelling of retention for several weak bases. The resulting (general) model describes retention accurately and is applicable to all solutes studied.

Electrophoresis of small particles and fluid globules in weak electrolytes

The electrical double layer plays an essential role in the electrokinetic behavior of both rigid and fluid spheres. One of the assumptions woven into the formulation of the classical balance laws of electrokinetics is that the ionogenic solutes are fully ionized. In aqueous media, common inorganic electrolytes such as KCl and NaOH dissociate completely into their constituent ions; the high dielectric constant of water favors dissociation by lowering the energy required to ionize a solute. Not all ionic solutions are aqueous, however, and electrokinetic effects are important in these media, too. Less polar liquids have a much lower dielectric constant, and so they are unable to sustain a high degree of solute ionization; in fact ionogenic solutes may dissociate less than a percent. Here we examine the influence of partial ionization on the electrophoresis of small particles and fluid globules, with a view toward understanding how, and under what conditions, dissociation-association alters the electrokinetics. We find generally that mass-action, consistent with Le Chatelier's principle, works to minimize disturbances to the electrical double layer, resisting polarization of the diffuse ion cloud. Thus, dissociation-association processes are quantitatively important in cases where double layer polarization and relaxation would otherwise prevail. Consequently, the predicted impact on the electrophoretic mobility is greatest for drops and bubbles, since their surfaces are fluid and convection within the interface is a factor. Mass-action can reduce the mobility of a conducting drop by an order of magnitude, and sizeable decreases (50% and more) in drop mobility are even found at ζ-potentials below 50 mV. Rigid particles are affected less dramatically and quantitative effects rarely exceed 10%; particles are markedly insensitive to partial solute ionization unless the ζ-potential is high (above ca. 100 mV) and aκ > 1. The computation scheme employed applies strictly to situations in which the magnitude of the forcing-field is small. Nevertheless, the results imply that for electrokinetic phenomena driven by strong forcing-fields, dissociation-association processes involving ionogenic solutes may be significant in apolar liquids.

Reversed-Phase Liquid Chromatography with Mixed Eluents: Partition Model of Retention for Ionogenic Solutes

Abstract Basing on the pure partition model of solute retention new theoretical equations for ionogenic solutes are discussed. These equations define dependence of the capacity ratio on the mobile phase composition, which is characteristic for the reversed-phase liquid chromatography.

Solute Dissociation Effects in Reversed Phase Liquid Chromatography with Mixed Eluents

Abstract Basing on the displacement model of liquid chromatography new equations are proposed for the capacity ratio of ionogenic solutes in the mixed mobile phase. Effects of different parameters on the capacity ratio of the monotropic acids are systematically studied for RPLC process.

Physico-chemical factors governing partition behaviour of solutes and particles in aqueous polymeric biphasic systems. : I. Effect of ionic composition on the relative hydrophobicity of the phases

Partition coefficients for a homologous series of dinitrophenylated amino acids with aliphatic side-chains have been determined in aqueous polymeric Dxtran 500—poly(ethylene glycol) 6000 and Ficoll 400—Dextran 70 biphasic systems of various ionic compositions, and in systems formed by n-octanol and the aqueous phases of the above systems. The free energy of transfer of a CH 2 group from one phase to the other in these systems was estimated and used as a measure of the hydrophobic character of an aqueous phase with respect to n-octanol. The results show that the hydrophobic properties of both phases of the aqueous polymeric biphasic systems are greatly affected by the ionic composition. It is demonstrated that solute partition behaviour is governed by the relative hydrophobicity of the phases, although this is not the only factor important for the partition of ionogenic solutes.

Ionisation Effects in the Reversed Phase Liquid Chromatographic Separation of Thyromimetic Iodoamino Acids

Abstract The influence of ionisation equilibria on the retention behaviour of iodoamino acids and related compounds on micro-particulate octadecylsilica supports has been examined. The chromatographic data for these ionogenic solutes have been discussed in terms of current concepts for reversible solvophobic interactions with the hydrocarbonaceous stationary phase. This treatment permits the conditional effects of the mobile phase composition and pH on solute retention to be assessed and the relationship between the molecular surface area of a solute and its retention to a non polar stationary phase evaluated.

Mobile phase effects in reversed-phase chromatography : II. Acidic amine phosphate buffers as eluents

In order to meet the particular requirements with regard to the eluent in liquid chromatography of ionogenic substances on non-polar stationary phases, various buffers composed of phosphate and a vicinal diamine having a p K a value in acidic pH domain were examined and characterized by their physico-chemical properties. The heat of ionization for such amines is generally much greater than that of ac idic compounds having commensurable p K a values. Consequently changing temperaturre may strongly affect the degree of ionization of the buffer and as a result the temperature dependence of retention can be drastically altered for certain sample components. This caloric effect of the buffer on retention in reversed-phase chromatography was theoretically treated by taking into account the enthalpies for all processes involved, the constants of the protonic equilibria in solution and the limiting retentiion factors of both the fully ionized and the neutral of the eluite. Thus thermal analysis of a chromatographic system containing a buffer in the eluent and ionogenic solutes by using Van't Hoff plots of chromatographic data may not yield the enthalpy for solute transfer between mobile and stationary phases due to secondary equilibria. Experimental results did not always agree the theoretical predictions. The aberration was attributed to complex formation between sample molecules and buffer species, that is to a hetaeric effect of the buffer. Some dramatic changes were observed in retention behavior with concomitant improvement in peak shape and chromatographhic efficiency upon replacing sodium phosphate by piperazine or tetramethylethylene- diamine phosphate in the eluent at pH 6.0. The propitious buffer effect, however, was attributed, at least in part, to masking the silanol groups at the stationary phase surface by the weak amine component of the buffer. The results demonstrate that besides their classical static role to maintain the pH of a solution constant, buffers may play a variety of other roles and affect significantly the properties and efficiency of a chromatography system.

Reverse-phase chromatography of polar biological substances: separation of catechol compounds by high-performance liquid chromatography.

Catecholamines and their metabolites have been separated isocratically by reverse-phase chromatography with aqueous (no organic solvent admixed) eluents. Unlike ion-exchange or ion-pair chromatography, mixtures of both acidic and basic substances can be separated in a single chromatographic run, because the retention is governed by hydrophobic interactions between the nonpolar moiety of the solute molecules and the octadecyl-silica stationary phase. The relative retention values strongly depend on the pH of the eluent, which governs the degree of dissociation of ionogenic solutes. The reproducibility of the results and the stability and efficiency of the chromatographic systems make this approach particularly attractive for use in clinical analysis.