Gradient elution in micellar liquid chromatography. I. Micelle concentration gradient.

Gradient elution in micellar liquid chromatography (MLC) is discussed. On the basis of the gradient elution theory, first developed by Snyder, equations were derived for the prediction of gradient retention times in micelle concentration gradient from isocratic data. Likewise, partition coefficients into micelles and stationary phase, and subsequently isocratic retention at different micelle concentrations can be estimated from two gradient runs. However, more studies need to be done to achieve better agreement between isocratic and gradient data. The equations will be useful for efficient development of practical separations by MLC.

Gradient elution in micellar liquid chromatography. II. Organic modifier gradients.

The application of organic modifier gradients in micellar liquid chromatography (MLC) is discussed. The equation derived by Snyder and co-workers describing gradient elution in hydro-organic reversed-phase LC was verified for organic solvent gradients in the presence of micelles. It is also demonstrated that the use of these gradients require little re-equilibration time due to the limited range of organic modifier concentration used in the gradient. This would result in shorter analysis time. Lastly, a practical application of the use of propanol and acetonitrile gradients in MLC is described.

Computer-assisted modeling, prediction, and multifactor optimization in micellar electrokinetic chromatography of ionizable compounds.

Previously, the use of phenomenological models to describe the migration behavior of acidic solutes in micellar electrokinetic chromatography (MEKC) was reported. In this paper, the phenomenological approach is further extended by including both acidic and basic solutes and simultaneously taking two important experimental factors (pH and micelle concentration) into consideration. In addition, a general method is described to model the migration behavior of ionizable (both acidic and basic) solutes in MEKC with anionic and cationic micelles. The practical implication of the phenomenological approaches is that they will provide quantitative relationships between solute migration and experimental factors such that the migration behavior can be predicted on the basis of a few initial experiments and that physicochemical parameters of solutes can also be estimated from model fitting. Through computer-assisted modeling, migration behavior of several acidic and basic solutes over a pH-micelle concentration factor space was successfully predicted on the basis of only five experiments. Furthermore, this phenomenological approach was used to predict the separation of a group of aromatic amines in MEKC with anionic micelles, which resulted in a successful separation of 18 aromatic amines in less than 15 min.

Simultaneous optimization of pH and micelle concentration in micellar liquid chromatography.

A retention model for ionizable compounds in micellar liquid chromatography is derived and verified. The use of the model for the prediction of retention is illustrated and appropriate optimization strategies for the separation of ionizable compounds in Micellar Liquid Chromatography are discussed.

Multiparameter optimizations in micellar liquid chromatography using the iterative regression optimization strategy.

An extension of the iterative regression optimization strategy to multi-parameter optimizations is described and applied to the separation of ionic compounds (amino acids and peptides) by means of micellar liquid chromatography. The parameters examined are the concentration of surfactant, the concentration of 2-propanol and pH. Fairly regular (linear, weakly curved) retention behaviour of the compounds as a function of the parameters results in an efficient optimization using a relatively small number of initial experiments.

Migration behavior of cationic solutes in micellar electrokinetic capillary chromatography.

A phenomenological approach is presented to describe the migration of cationic solutes in micellar electrokinetic capillary chromatography (MECC). The migration behavior of an organic base is complicated by the presence of an acid-base equilibrium, the ion-pairing formation between the conjugated acid of the base and the monomer surfactants, and the interactions of both the base and its conjugated acid with the micellar pseudophase. An equation was derived that allows the calculation of the migration factor of a cationic solute in MECC with anionic micelles. Two limiting cases were considered: first the cationic solute completely associates with the anionic surfactant (ion-pair formation constant, KIP, approaches infinity), and therefore there is no free charged species in the solution; second, the KIP = 0 and the free conjugated acid, BH+ migrates in the aqueous bulk solvent at its own electrophoretic velocity. An estimate for the ion-pair formation constant between cationic solutes and free surfactant can be obtained by using the model.

Influence of operating parameters on reproducibility in capillary electrophoresis.

The reproducibility of two migration parameters (retention time and mobility) of a seven-component test mixture was examined under various operating conditions using laboratory-built capillary electrophoresis systems. It was found that the frequency of rinsing the capillary and the solutions used for rinsing had the greatest effect on migration reproducibility. In addition, it was found that the migration behavior of solutes that interact with micelles is not repeatable unless the proper rinse protocol is applied. Inconsistent migration behavior is linked to inconsistent total current of the system. Preliminary investigations indicate that the fluctuation in total current were associated with non-equilibrium conditions between the buffer and the capillary wall.

Micellar electrokinetic capillary chromatography of acidic solutes: migration behavior and optimization strategies.

Micellar electrokinetic capillary chromatography (MECC) is suitable for the separation of mixtures of uncharged and charged solutes. In this paper, the migration behavior of acidic compounds in MECC is quantitatively described in terms of different models. These equations describe the relationships between the two migration parameters in MECC (retention factor and mobility) and the two important experimental parameters (pH and micelle concentration) that have a great influence on the migration behavior and selectivity. Interestingly, the mobility and retention factor of a given solute could behave differently with the variations in pH. This would raise a question of which parameter actually represents the migration behavior of a solute in MECC: retention factor (a chromatographic parameter) or mobility (an electrophoretic parameter). The consequences of micellar-mediated shifts of ionization constants on selectivity and optimization strategies in MECC are discussed. The mathematical models would allow the prediction of migration behavior of solutes based on a limited number of initial experiments. This would greatly facilitate the method development and optimization of separations of ionizable compounds by MECC and, in addition, important physical and chemical characteristics of solutes such as their apparent ionization constants in micellar media and their partition coefficients into micelles (over a wide range pH values) can be determined. The models were verified, as good agreements were observed between the predicted and the experimentally observed migration behavior. Based on the preliminary results, the pH and micelle concentration are likely to be interactive parameters in many situations. As a result, simultaneous optimization of these two parameters would be the most effective strategy to enhance the MECC separation of acidic solutes.

Quantitative retention-biological activity relationship study by micellar liquid chromatography.

In a previous paper, the usefulness of micellar liquid chromatography (MLC) in predicting octanol-water partition coefficients of organic compounds was reported. This paper is the first successful report of a quantitative retention-activity relationship study using the retention factor in MLC for predicting the biological activity of a group of phenolic compounds. Excellent correlation was obtained between the capacity factor in MLC and the bioactivity (measured as log 1/C, where C is the 50% inhibitory growth concentration) of 26 para-substituted phenols. A single MLC retention parameter is capable of describing the bioactivity of phenols, while three conventional molecular descriptors (log P(ow), pKa, and R) are needed to achieve a similar correlation. This indicates that both hydrophobic and electronic interactions are incorporated in a single MLC retention parameter, which is due to the amphiphilic nature of surfactants in the system. In situations like this, QRAR is a suitable alternative to QSAR since measuring MLC retention is much easier than measuring different molecular descriptors needed to build the QSAR model. Addition of 10% 2-propanol to a micellar system (hybrid system) proved to be the best chromatographic system for the best estimation of the phenols bioactivity. Other chromatographic factors such as pH and stationary phase also showed significant effect on the correlation between capacity factor k' and log 1/C.

Simultaneous optimization of variables influencing selectivity and elution strength in micellar liquid chromatography. Effect of organic modifier and micelle concentration.

Previously, the simultaneous enhancement of separation selectivity with elution strength was reported in micellar liquid chromatography (MLC) using the hybrid eluents of water-organic solvent-micelles. The practical implication of this phenomenon is that better separations can be achieved in shorter analysis times by using the hybrid eluents. Since both micelle concentration and volume fraction of organic modifier influence selectivity and solvent strength, only an investigation of the effects of a simultaneous variation of these parameters will disclose the full separation capability of the method, i.e. the commonly used sequential solvent optimization approach of adjusting the solvent strength first and then improving selectivity in reversed-phase liquid chromatography is inefficient for the case of MLC with the hybrid eluents. This is illustrated in this paper with two examples: the optimization of the selectivity in the separation of a mixture of phenols and the optimization of a resolution-based criterion determined for the separation of a number of amino acids and small peptides. The large number of variables involved in the separation process in MLC necessitates a structured approach in the development of practical applications of this technique. A regular change in retention behavior is observed with the variation of the surfactant concentration and the concentration of organic modifier, which enables a successful prediction of retention times. Consequently interpretive optimization strategies such as the interative regression method are applicable.

Simultaneous enhancement of separation selectivity and solvent strength in reversed-phase liquid chromatography using micelles in hydro-organic solvents.

The role of micelles and organic solvents as the modifiers of the aqueous mobile phase in reversed-phase liquid chromatography (RPLC) in controlling retention and selectivity is discussed. Elution strength increases in RPLC with an increase in organic solvent or micelle concentration. Simultaneous enhancement of separation selectivity with elution strength in the hybrid eluents of water-organic solvent-micelles was observed. This selectivity enhancement occurs systematically, i.e. peak separation increases monotonically with volume fraction of organic solvent added to micellar eluent, and is observed for a large number of ionic and nonionic compounds with different functional groups and for two surfactants (anionic and cationic). For two test mixtures, 13 amino acids/peptides and 15 phenols, it is shown that a better separation and shorter analysis time are observed at stronger hybrid eluents. This selectivity enhancement can be attributed to the competing partitioning equilibria in micellar LC systems and/or to the unique characteristics of micelles to compartmentalize solutes and organic solvents.