Controlling solvent strength and selectivity in micellar liquid chromatography: role of organic modifiers and micelles.

Simultaneous enhancement in elution strength and selectivity which has been previously observed in micellar liquid chromatography (MLC) for a variety of compounds is further investigated. The reasons behind the occurrence of this unique phenomenon are studied, and the influence of micelles and organic solvents on elution strength and selectivity is discussed. A model is developed which explains the dependence of the solvation ability of organic solvents in MLC (represented by the solvent strength parameter, S, of solutes) and the degree of solute interactions with micelles. Whenever the difference in solvent strength parameter values of two solutes in micellar eluents, dS, is positive, maximum selectivity is observed at the weakest eluent strength. When dS less than 0, there exists an inverse relationship between retention and solvent strength parameter so that selectivity monotonically increases with volume fraction of organic solvent in micellar eluents. It is shown that usually there is no direct relationship between the solvent strength parameter in MLC and retention. As a result, selectivity enhancement due to an increase in the concentration of organic modifier (i.e. solvent strength) occurs frequently in MLC. Interestingly, for cases where selectivity decreases with an increase in organic modifier, simultaneous enhancement of selectivity and solvent strength can be observed by increasing micelle concentration. In a sense, the concentrations of organic modifier and micelles complement one another in improving selectivity at higher elution strengths. As a result of this unique phenomenon better separations in shorter analysis times can be observed. The mutual effects of micelles and organic modifier on one another would also require a simultaneous optimization of these two parameters.     

Quantitative retention-structure and retention-activity relationship studies of local anesthetics by micellar liquid chromatography

The retention of compounds in micellar liquid chromatography (MLC) is governed by hydrophobic and electrostatic forces. For ionic compounds, both interactions should be considered. The present report offers a novel retention model that includes the hydrophobicity of compounds and the molar fraction of the charged form of compounds and compares it with other previously reported models. High correlations between the logarithm of capacity factors and these structural parameters were obtained for local anesthetics with different degrees of ionization using a nonionic surfactant solution as mobile phase. Modeling the retention of compounds as a function of physicochemical parameters and experimental variables is established by means of multiple linear regression. In addition, a predictive model for estimating the hydrophobicity of local anesthetics is proposed. Finally, quantitative and qualitative retention-activity relationships in MLC are also investigated for these compounds. An excellent correlation between the capacity factors in MLC and the anesthetic potency of local anesthetics was obtained.     

Prediction of retention in micellar electrokinetic chromatography from solute structure. 1. Sodium dodecyl sulfate micelles.

Like other chromatographic techniques, retention factor, k, in micellar electrokinetic chromatography (MEKC) is directly related to solute partition coefficient and the chromatographic phase ratio as k = Kphi. Unlike conventional chromatography, however, the phase ratio and partition coefficient can be accurately determined in MEKC for a given micellar pseudostationary phase. This means that retention factor in MEKC can be predicted for solutes with known micelle-water partition coefficients without any prior experimentation. In this paper, the use of this simple relationship for prediction of retention behavior in MEKC is examined. The principle of additivity of functional group contribution to partitioning is used to calculate the micelle-water partition coefficient, Kmw, for SDS micellar pseudophase. The micellar substituent constants for 20 functional groups (training set) were determined. Using these substituent constants, the Kmw and retention factors for a group of 80 neutral solutes (test set) were predicted. The linear plot of predicted versus observed log k had an R2 = 0.97 and a slope equal to 1.01. It is shown that the retention times (thus chromatograms) in MEKC can be predicted from the calculated retention factors after only one initial experiment to measure teo and t(mc) under the experimental conditions.     

Ligand-exchange chromatography at zirconium(IV) immobilized on IDA-type chelating polymer gel

Ligand-exchange chromatography of organo-acidic compounds at Zr(IV) immobilized on IDA-type chelating polymer gel using an aqueous mobile phase was studied, to obtain some insight into their retention on zirconia. Zirconium(IV) has no contribution to the retention of phenols, while appreciably enhancing that of benzoic acids and strongly or occasionally irreversibly adsorbing the potentially chelating substances. The presence of two stationary-phase species adsorbing benzoates was confirmed, and the retention over a wide pH range of 3.5-9.5 was quantitatively formulated. The correlation of the retention factor with the Bronsted acidity of samples and its dependence on pH were theoretically explained, and the performance of this chromatographic system was compared with that of zirconia.     

Estimation of biological properties by means of chromatographic systems: evaluation of the factors that contribute to the variance of biological-chromatographic correlations

The performance of chromatographic systems to emulate biological systems is evaluated in terms of the precision that can be achieved. The variance obtained when biological parameters are correlated against physicochemical ones can be decomposed in three terms: the variance of the biological data, the variance of the physicochemical data, and the variance caused by the dissimilarity between the two correlated systems (biological and physicochemical). The three terms contribute to the overall variance observed when measurements in chromatographic systems are correlated with experimental biological properties. The Abraham linear free energy relationships (LFERs) provide a very good approach to characterize biological and physicochemical systems and thus the variance of the analyzed data and the similarity/dissimilarity between them. The contribution of the three variances to the precision of the biological parameter estimated in this way is evaluated from the characterization of the biological and chromatographic systems by means of the Abraham model. The proposed method is able to estimate the goodness of chromatographic systems to predict particular biological properties. In particular, this method is illustrated by comparison of toxicity data (-log LC(50)) for the fish fathead minnow with retention data (log k) in several micellar electrokinetic chromatography (MEKC) systems and also by correlations between retention data (log k) in the sodium taurocholate (STC) MEKC system and data of several biological systems.     

Systematic Approach to Links between Separations in MEKC and Reversed-Phase HPLC

Retention factors and partition coefficients in micellar electrokinetic chromatography (MEKC) and reversed-phase high-performance liquid chromatography (RP-HPLC) are compared for a series of alkylbenzenes and substituted phenols. In both techniques, separations are based on partitioning between an aqueous phase and an alkyl phase. In MEKC, this was an SDS (C12) micellar pseudostationary phase, and in RP-HPLC an ODS 2 (C18) stationary phase. A nonporous silica (Micra 1.5-μm NPS), which has a low carbon loading, was used rather than a standard porous silica to avoid excessive retention in HPLC and to allow identical mobile phase conditions to be used in both separation modes. The average ratio of analyte retention factors, k(MEKC):k(HPLC), was found to be equal to the ratio β(MEKC):β(HPLC), where β is the phase ratio. This implies that partition coefficients, P, are similar in both MEKC and HPLC, since P = k/β, and that the dominant contribution to stability within each alkyl phase arises from hydrophobic interactions which are common to both separation media. Since partition coefficients are similar in MEKC and HPLC under aqueous buffer conditions, information on retention in one technique may be transferred to the other, provided that the phase ratios are known. In MEKC and HPLC, linear correlations of log octanol-water partition coefficients, K(ow), vs log k for the test compounds were transformed, knowing the phase ratio, to give log P values as a function of log K(ow). This allows quantitative links between MEKC and HPLC to be extended to include octanol-water partitioning. The addition of acetonitrile as an organic modifier over the concentration range 0-20% (v/v) was found to have a greater effect on k in HPLC than in MEKC. This could be a result of a decrease in the MEKC phase ratio due to an increase in the critical micelle concentration.     

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.     

Chromatographic Hydrophobicity Index by Fast-Gradient RP-HPLC: A High-Throughput Alternative to log P/log D

A new chromatographic hydrophobicity index (CHI) is described which can be used as part of a protocol for high-throughput (50-100 compounds/day) physicochemical property profiling for rational drug design. The index is derived from retention times (t(R)) observed in a fast gradient reversed-phase HPLC method. The isocratic retention factors (log k') were measured for a series of 76 structurally unrelated compounds by using various concentrations of acetonitrile in the mobile phase. By plotting the log k' as a function of the acetonitrile concentration, the slope (S) and the intercept (log k'(w)) values were calculated. The previously validated index of hydrophobicity φ(0) was calculated as -log k'(w)/S. A good linear correlation was obtained between the gradient retention time values, t(R) and the isocratically determined φ(0) values for the 76 compounds. The constants of this linear correlation can be used to calculate CHI. For most compounds, CHI is between 0 and 100 and in this range it approximates to the percentage (by volume) of acetonitrile required to achieve an equal distribution of compound between the mobile and the stationary phases. CHI values can be measured using acidic, neutral, or slightly basic eluents. Values corresponding to the neutral form of molecules could be measured for 52 of the compounds and showed good correlation (r = 0.851) to the calculated octanol/water partition coefficient (c log P) values.     

The effect of stationary-phase pore size on retention behavior in micellar liquid chromatography

One of the limitations that has restricted the applicability of micellar liquid chromatography (MLC) is the weak eluting power of micellar mobile phases compared to conventional hydro-organic mobile phases used in reversed-phase liquid chromatography. This may be the result of Donnan or steric exclusion of the micelles from the pores of the stationary phase, within which nearly all (> or = 99%) of the stationary phase resides and the analytes spend most of their time. To determine whether wide-pore stationary phases would overcome this limitation in MLC, several C8 and C18 stationary phases ranging from 100 to 4000 A were investigated using a diverse set of test solutes and micellar solutions of anionic, neutral, and cationic surfactants as mobile phases. With the larger pore size stationary phases, the eluting power of the MLC mobile phases was enhanced with all surfactant types, the greatest effect being with the neutral surfactant. Differences in retention behavior were observed between various solute types and between the C8 and C18 stationary phases. These differences appear to be related to the relative hydrophobicity of the solutes and to differences in the surfactant-modified stationary phases. Partitioning behavior of representative solutes on the large-pore C8 and C18 columns was shown to follow the three-phase partitioning model for MLC. Methylene group selectivity data showed only minor differences in the stationary-phase characteristics between the small- and large-pore size C18 columns. The true eluting power of micellar mobile phases was revealed with wide-pore stationary phases and was demonstrated by the separation and elution of an extended series of alkylphenones on C18 columns.     

Evaluating "fast" micellar monolithic liquid chromatography for high-throughput quantitative structure-retention relationship screening

The recently introduced monolithic silica columns were tested for their use in micellar liquid chromatography. Micellar methods are utilized in high-throughput quantitative structure-retention relationships to estimate an indicator of the membrane permeability of drugs, namely, the octanol-water partition coefficient, log P. The monolithic column's ability to function at higher flow rates might be useful to speed up these chromatographic methods estimating the log P. Therefore, the elution behavior of diverse basic pharmaceutical substances was determined on a classical particle-based and a monolithic column, both with and without a micellar medium in the mobile phase. Utilizing among others principal component analysis, the extent to which these methods differ in retention characteristics was examined in the context of high-throughput determination of log P. Results indicate that combining monolithic columns with micellar media leads to faster log P and possibly even better permeability predictions.     

Effect of a variety of organic additives on retention and efficiency in micellar liquid chromatography

The effect of 21 organic additives (alkanols, alkane diols, dipolar aprotic solvents, alkanes) on the chromatographic behavior (retention, elution strength, efficiency) of probe solutes of widely differing hydrophobicity, such as benzene and 2-ethylanthraquinone, have been examined using a C18 stationary phase and sodium dodecyl sulfate (SDS) micellar mobile phases. The mobile-phase elution strength parallels the octanol-water partition coefficients of the additives or their ability to bind to the SDS micellar system, due to the increased solubility in the mobile phase and reduced affinity for the additive-modified surfactant-coated stationary phase. The comparison of the elution strength of micellar mobile phases with that of a reference acetonitrile-water system indicates that the elution strength is lower for micellar systems and depends on the nature of the eluted solute. The displacement of the solute-micelle and solute-stationary phase binding equilibria is quantified for several probe solutes eluted with micellar mobile phases in the presence of 1-propanol, 1-butanol, 1-pentanol, and acetonitrile. A correlation was also observed between the number of theoretical plates and the hydrophobicity of the alcohol additives: the efficiency initially increased steeply and reached a plateau. Compared to benzene, a more hydrophobic additive was needed to attain the maximum efficiency for the more hydrophobic 2-ethylanthraquinone analyte. Dipolar aprotic solvents appear to be somewhat more effective in enhancing the efficiency than alcohols. The results are rationalized in terms of the ability of the organic additives to alter the composition, structure, dynamics, and properties of the micelles and the surfactant-coated stationary phase.     

Estimating octanol-air partition coefficients of nonpolar semivolatile organic compounds from gas chromatographic retention times

Relative gas chromatographic retention times on a non-polar stationary phase can be used to determine the octanol-air partition coefficient (K(OA)) and the energy of phase transfer between octanol and the gas phase (delta(OA)U) for semivolatile, nonpolar organic compounds. The only prerequisites are knowledge of the temperature-dependent K(OA) of a standard reference compound and directly measured K(OA) values at one temperature for a sufficient number of calibration compounds. It is shown that the technique is capable of predicting the K(OA) of polychlorinated benzenes, biphenyls and naphthalenes as well as polybrominated diphenyl ethers within the environmentally relevant temperature range with an average deviation from directly measured values of <0.2 log units.     

Modeling nonspecific toxicity of organic compounds to the fathead minnow fish by means of chromatographic systems

The performance of chromatographic systems to mimic aquatic toxicity to the fathead minnow fish is evaluated taking into account the factors that contribute to the variance of biological-chromatographic correlations. These factors are the precision to measure the fathead minnow toxicity, the precision of the surrogate chromatographic system, and the error from the dissimilarity between the fathead minnow and chromatographic systems. The precisions are estimated through the characterization of the systems by the solvation parameter model. Several chromatographic systems as well as the common reference octanol-water partition system have been selected to test their ability to model the nonspecific toxicity to the fathead minnow by means of the proposed approach. Predictions and experimental tests show that the micellar electrokinetic chromatography system of sodium taurocholate and chromatographic measurements using an immobilized artificial membrane column provide the most precise estimations of this biopartitioning property. The octanol-water partition system, the conventional C18 high-performance liquid chromatography systems, and the micellar electrokinetic chromatography system of sodium dodecylsulfate show worse performances.     

QSARs for monosubstituted phenols and the polar narcosis mechanism of toxicity.

Eighty 2-, 3-, and 4-position monosubstituted phenols representing various substituents were evaluated for relative toxicity, log IGC50(-1), with a short-term static protocol in the Tetrahymena population growth inhibition bioassay. Quantitative structure-activity relationships (QSAR) were examined using the 1-octanol/water partition coefficient (log K(ow)) and ionization constant (pKa) as independent variables. Four derivatives did not elicit the measured response at saturation. Five derivatives revealed altered high-performance liquid chromatography spectra with time. None of these derivatives were included in QSAR development. In addition, the carboxyl and nitroso derivatives were detected as statistical outliers. The model log IGC50(-1) = 0.6655 (log K(ow)) - 0.1464 (pKa) + 0.2206, n = 67, r2 = 0.909, s = 0.212, was found to be an excellent predictor of activity of phenols which elicit their toxic response by the polar narcosis mode of action. For the most part the tested derivatives showed little abiotic loss over the duration of the bioassay.