Chromatographic models to predict the elution of ionizable analytes by organic modifier gradient in reversed phase liquid chromatography.

The retention of an ionizable analyte under RP-HPLC organic modifier gradient elution is strongly affected by its ionization degree which, in turn, depends on its pK(a) and on the pH of the mobile phase. The values of both parameters change depending on the mobile phase composition and thus retention becomes a parameter quite difficult to predict, particularly when working in gradient mode. In this work, an equation describing the retention of ionizable analytes has been combined with three different models of different complexity, developed for gradient elution of neutral compounds (1, 2, or 3 fitting parameters), in order to predict retention of compounds with acid-base properties with particular buffers. All models have been tested under 16 different gradient patterns (4 linear gradients, 4 concave gradients, 4 convex gradients and 4 combinations between them) for the prediction of the retention time of 12 acid-base compounds (pK(a) values from 4 to 9) in 3 different buffered mobile phases (pH 5, pH 7 and pH 9) with acetonitrile as organic modifier. The agreement between the experimental and calculated retention times is good for all models. The best results are obtained through the model that depends on three parameters and the accuracy of the two-parameter model is slightly lower but very acceptable too. On the other hand, the predictions performed with the one-parameter model are the less accurate, but good enough to become a valid model taking into account that it requires very little experimental work.

Is the general conclusion justified that higher applicable field strength results in shorter analysis time with organic solvents in CE?

In this paper, a widespread opinion in CE with organic solvents for the background electrolyte is critically questioned, namely that in general a shorter analysis time can be achieved due to the higher field strength applicable compared with aqueous electrolyte systems. This view, common in the literature, is based on the supposition that the conductance in organic solvents is lower than in water. Indeed in many organic solvents with higher viscosity than water lower ion mobility is observed, and higher fields can be applied in these cases. However, in this paper the problem is sharper defined and treated two-fold: (i) in all solvents conditions are such that either the same electric power is generated, or (ii) the same temperature increase is taken into account. It was shown that for the same electric power the field strength in the organic solvent can be changed to a less extent than the ionic mobility changes. As a result, the migration velocity of the analytes is lower and the analysis time is longer in most organic solvents compared with water; acetonitrile (MeCN) is an exception (in this solvent the mobilities are higher than in water). The more stringent treatment of the problem takes an equal temperature increase due to Joule heating into account rather than equal electric power. The temperature increase in the capillary depends on the thermal conductivity of the solvent, which is only about one-third of that of water for organic liquids. The consequence is that in none of the organic solvent systems a shorter analysis time can be achieved compared with water (given that the experimental conditions are comparable, e.g. zero EOF). The theoretical predictions were confirmed by measurements with water, methanol, propylenecarbonate, and MeCN as solvents.

Modeling the retention of neutral compounds in gradient elution RP-HPLC by means of polarity parameter models.

A three-parameter expression, which was already employed before for the prediction of the retention time in gradient mode in reversed-phase high-performance liquid chromatography (RP-HPLC) with satisfactory results, has been tested here under a variety of gradient patterns, using methanol and acetonitrile as the organic modifiers. A wide variety of compounds have been employed as test solutes, including some complex ones used as drugs, such as hydrocortisone and estriol. Simplifications of this expression have been made by considering two- and one-parameter expressions based on the p polarity parameter model, which was successfully employed before in isocratic mode to perform predictions of retention time. The advantages that this model gives in isocratic conditions, namely simplicity and less previous experimental work, have been applied with profit in its application to gradient mode. Good correlations between the experimental retention times and the predicted ones have been obtained with both equations in most cases.

Formamide as an organic modifier in MEKC with SDS.

The effect of formamide (FA) as a modifier on the retention in MEKC with SDS as the detergent was investigated. The mobility of a series of alkylphenones and of a zwitterionic fluorescent compound as a function of the FA and the SDS concentration was determined for this purpose. Buffering electrolyte was borate, pH 9.23, with total ionic strength of 50 mM. The dependence of the mobility on the FA content - up to 63% w/w - of the BGE (at 10 mM SDS) allows the conclusion that the micelles are destabilized, and the CMC is shifted to higher values. In the system containing 33% FA or more no micelles are present anymore, and the retention factors of all compounds tend to zero. In an MEKC system with 27% v/v FA the CMC of SDS is increased from 2.4 mM in the aqueous BGE with the same buffer composition to 9.7 mM, a behavior that is in contrast to electrolyte-free FA-water systems. The partition constants of free analytes and the formation constants of the adduct between analyte and detergent monomer (assuming 1:1 stoichiometry) were derived from the dependence of the mobility on the SDS concentration. In addition, the involved equilibria were extended by that from the distribution of the analyte-monomer adduct between aqueous and micellar phase, and the according partition constants were derived as well. A selective change in the extent of partitioning was observed for the zwitterionic compound. In general, all binding constants were decreased upon addition of FA, though to a different extent. Although the binding constants of the analyte-monomer associate were only slightly influenced, the most pronounced decrease is found for their partitioning into the micelles.

An extended description of the effect of detergent monomers on migration in micellar electrokinetic chromatography.

Three equilibria determine the interaction of a neutral analyte with the detergent in micellar electrokinetic chromatography and therefore its migration: (i) that of the free analyte in the aqueous phase with the micelle, (ii) its association with free detergent monomers in the aqueous phase, and (iii) the partition of the associate of analyte and monomer between the aqueous solution and the micelle. For the first equilibrium, non-stoichiometric partitioning between two phases is preferred in the present work over the assumption of complex formation between one molecule of the analyte with one micelle. The second equilibrium is described by the formation of a 1:1 associate of the analyte and monomer. In this paper, thirdly an additional equilibrium is introduced, namely, the distribution of the analyte-monomer associate between the aqueous and the micelle phase; it is expressed by the according partition coefficient. The three equilibrium constants are interrelated. Mobility data for a lipophilic fluorescent compound and a series of n-alkylphenones (differing in chain length) were measured as a function of the SDS concentration below and above the critical micellar concentration. Curve fitting enabled the derivation of the equilibrium constants. It was found that the association constants of the analytes with the detergent monomers are between 2 and 75 M(-1). Interestingly, the partition coefficient of the analyte-monomer associate between the aqueous and micellar phase is by a factor of 5-200 larger than that of the free analyte.

Optimization of the separation of ionizable compounds in micellar electrokinetic chromatography by simultaneous change of pH and SDS concentration.

A method to optimize the separation in micellar EKC (MEKC) of mixtures of acidic compounds as a function of two parameters, pH and concentration of sodium dodecyl sulfate, has been developed. The method considers the prediction of the retention time and the shape of the peaks. The retention time is predicted from the retention factor model and the peak shape by a polynomically modified Gaussian function that considers peak width, asymmetry factor, and height. An algorithm to calculate the global resolution of the separation at any experimental pH and [SDS] has been applied. This algorithm provides a 3-D resolution map to easily detect the areas in which resolution for the separation of the compounds is maximum. Initial experiments to fit the models have been performed with a set of ten phenolic compounds with different hydrophobicities and pK(a) values, and therefore, expected to behave in a different way with changes of pH and surfactant concentration. The experiments encompassed a pH range from 6.7 to 11.1, and a sodium dodecyl sulfate concentration range from 40 to 80 mM. Through the proposed methodology, chromatograms have been simulated at different pH and [SDS] very accurately. Furthermore, the resolution at any experimental point within the studied ranges have been also calculated, giving an optimum resolution value at pH 6.7 and [SDS] = 72 mM.

Comparison of migration models for acidic solutes in micellar electrokinetic chromatography.

The validity of two models that explain the migration of ionisable solutes in micellar electrokinetic chromatography (MEKC), mobility model and retention factor model, has been tested. For this purpose, the mobility (mu) and retention factor (k) of a set of 10 phenolic compounds with different hydrophobicity and pKa values have been determined for several sodium dodecyl sulphate (SDS) concentrations and pH values, and fitted to the models. Results show that in general the retention factor model explains better the retention of ionisable solutes, although for hydrophilic compounds at low SDS concentration, mobility model can give better fits. The different drawbacks pointed out by several authors in relation to both models have been checked, and a deep evaluation of each one has been done. As a result we have observed that, while in the retention factor model the variation of k with pH and [SDS] always follows the same trend, the variation of mu with these variables mainly depends on the value of the binding constant of the neutral form of the solutes to the micelles, KHA(m), which plays a critical role in the fit of the mobility model. Also we provide rules and advices to set up the experimental conditions to apply each model to a particular solute.