Adsorption of the anionic surfactant sodium dodecyl sulfate on a C18 column under micellar and high submicellar conditions in reversed‐phase liquid chromatography

Micellar liquid chromatography makes use of aqueous solutions or aqueous‐organic solutions containing a surfactant, at a concentration above its critical micelle concentration. In the mobile phase, the surfactant monomers aggregate to form micelles, whereas on the surface of the nonpolar alkyl‐bonded stationary phases they are significantly adsorbed. If the mobile phase contains a high concentration of organic solvent, micelles break down, and the amount of surfactant adsorbed on the stationary phase is reduced, giving rise to another chromatographic mode named high submicellar liquid chromatography. The presence of a thinner coating of surfactant enhances the selectivity and peak shape, especially for basic compounds. However, the risk of full desorption of surfactant is the main limitation in the high submicellar mode. This study examines the adsorption of the anionic surfactant sodium dodecyl sulfate under micellar and high submicellar conditions on a C₁₈ column, applying two methods. One of them uses a refractive index detector to obtain direct measurements of the adsorbed amount of sodium dodecyl sulfate, whereas the second method is based on the retention and peak shape for a set of cationic basic compounds that indirectly reveal the presence of adsorbed monomers of surfactant on the stationary phase.     

Chromatographic Behavior of Aromatic Diamines in Hydro-Organic, Micellar and Submicellar Reversed Phase Liquid Chromatographic Modes

The chromatographic behavior (retention, elution strength, efficiency, peak asymmetry and selectivity) of some aromatic diamines in the presence of methanol with or without anionic surfactant SDS in the four different reversed phased liquid chromatographic (RPLC) modes, i.e., hydro-organic, micellar (MLC), low submicellar (LSC) and high submicellar (HSC), was investigated. In the three surfactant-mediated modes, the surfactant monomers coat the stationary phase even up to 70 % methanol; this results in the suppression of peak tailing (by masking the silanol groups on the stationary phase). In MLC and HSC, the solute retention decreases by increasing the surfactant concentration, while this situation was reversed in LSC. In the region between MLC and HSC modes (25–50 % methanol), retention of late eluting solutes was increased by increasing methanol content which is seemingly due to disaggregation of SDS micelles. Changes in selectivity were observed after changing the concentrations of SDS and methanol, in a greater extent when concentration of SDS was changed. Among the four studied RPLC modes, HSC showed the best efficiency with nearly symmetrical peaks. Prediction of retention of solutes in HSC based on a mechanistic retention model combined with Pareto-optimality method allowed the full resolution of target diamines in practical analysis times.     

Studies on the Interaction of Surfactants with Cationic Dye by Absorption Spectroscopy

The interaction of methyl violet, a cationic dye, with various surfactants, viz. anionic (SDS), nonionic (Triton X-100), and cationic (CTAB), has been investigated spectrophotometrically in submicellar and micellar concentration range. While in the submicellar concentration region of SDS the higher aggregates of the dye are found, in the micellar concentration region the monomer of the dye predominates. With nonionic surfactant the dye is solubilized primarily as the monomer. CTAB produces no perturbation to the visible spectra of the dye. In the presence of strong electrolytes such as NaNO(3) and NaCl the dye aggregates are formed at a much lower SDS concentrations. Copyright 2000 Academic Press.     

Use of the surfactant 3-(3-cholamidopropyl)-dimethyl-ammoniopropane sulfonate in hydrophobic interaction chromatography of proteins

Isocratic hydrophobic interaction chromatography of five proteins has been carried out using mobile phases containing the surfactant 3-(3-cholamidopropyl)-dimethylammoniopropane sulfonate (CHAPS). Linear relationships were found between log k' and ammonium sulfate concentrations for all the proteins with CHAPS in the submicellar concentration range. The slope of such a plot decreases monotonically as CHAPS concentration is increased. To a first approximation, the effect of CHAPS on protein retention can be explained in terms of a competitive binding model. However, CHAPS does show differential effects on the elution of proteins, substantially altering selectivity. The use of a normalized capacity factor, k'/k'o, proves useful for comparing retention times of different proteins as a function of CHAPS concentration. The magnitudes of k'/k'o were found to be inversely correlated with the slopes of plots of log k' vs. ammonium sulfate concentration in the absence of CHAPS. Adsorption isotherms for CHAPS were determined over the working range of ammonium sulfate. The binding of CHAPS to the SynChropak Propyl stationary phase and its effects on retention were found to be readily reversible. For each protein, plots of k'/k'o vs. surface concentration of CHAPS were superposable for data obtained at different salt concentrations. These findings support a competitive binding model. A simple geometric argument for stationary phase occupancy provides a qualitative explanation for the observed surfactant selectivity.     

Micellar liquid chromatography retention model based on mass-action concept of micelle formation

Mass-action model of surfactant micelle formation has been used to develop a conceptual retention model in micellar liquid chromatography (MLC). The retention model bases on the consideration of the changes of the sorbate microenvironment at its transferring from the mobile phase (hybrid micellar eluent) to the stationary phase (a modified surface of alkyl-bounded sorbent). Principal retention equation contains the characteristics of hybrid micelles (critical micelle concentration, degree of counterion binding, partition coefficient of modifier between aqueous solution and micellar pseudo-phase) as well as three fitting parameters. The fitting parameters are an absolute term and coefficients that are equal to the number of molecules of surfactant and modifier, which are attached/detached by sorbate transferring from a hybrid micellar eluent to a modified surface of the stationary phase. On the MLC separation of five antibiotics of rubomicin derivatives and four esters of 4-hydroxybenzoic acid the model of the change of sorbate microenvironment has been tested. The adequateness of model to experimental data has been shown. A simple three-parameter function connecting log k with log cS and log cR that provides a high goodness-of-fit follows from principal retention equation (cS and cR are the molar concentrations of surfactant and organic modifier in the micellar eluent, respectively).     

Micellar Liquid Chromatography Study of Quantitative Retention–Activity Relationships for Antihypertensive Drugs

Use of micellar mobile phases in reversed-phase liquid chromatography (RPLC) results in hydrophobic and electrostatic sites for interaction. Modified stationary phases in micellar liquid chromatography (MLC) are structurally similar to biomembranes. To confirm this we focused on the effects of the type and concentration of surfactant (Brij 35, SDS, and CTAB) and mobile phase pH on the retention of antihypertensive drugs on modified C₁₈ stationary phases. Quantitative retention-activity relationships are proposed for the drugs and the different surfactants and compared with those obtained using aqueous–organic mobile phases. Finally, a correlation was obtained between the logarithm of retention factors (log k) and the toxicity (LD₅₀) of antihypertensive drugs. Revised: 14 September 2005 and 4 April 2006     

Submicellar and micellar reversed-phase liquid chromatographic modes applied to the separation of β-blockers

The behaviour of a reversed-phase liquid chromatographic (RPLC) system (i.e. elution order, resolution and analysis time), used in the analysis of β-blockers with acetonitrile–water mobile phases, changes drastically upon addition of an anionic surfactant (sodium dodecyl sulphate, SDS). Surfactant monomers cover the alkyl-bonded phase in different extent depending on the concentration of both modifiers, in the ranges 1 × 10⁻³–0.15 M SDS and 5–50% acetonitrile. Meanwhile, the surfactant is dissolved in the mobile phase as free monomers, associated in small clusters or forming micelles. Four characteristic RPLC modes are yielded, with transition regions between them: hydro-organic, micellar, and low and high submicellar. The mobile phases in the two latter modes contain a concentration of SDS below or well above the critical micellar concentration (CMC) in water (i.e. 8 × 10⁻³ M), and more than 30% acetonitrile. High submicellar RPLC appeared as the most promising mode, as it allowed full resolution of the β-blockers in practical times, while these were unresolved or highly retained in the other RPLC modes. The strong attraction of the cationic solutes to the anionic SDS makes a direct transfer mechanism between surfactant molecules in the stationary and mobile phases likely.     

Determination of solute partition behavior with room-temperature ionic liquid based micellar gas-liquid chromatography stationary phases using the pseudophase model

The use of micelles in ionic liquid based gas-chromatography stationary phases was evaluated using equations derived for a "three-phase" model. This model allows the determination of all three partition coefficients involved in the system, and elucidates the micellar contribution to retention and selectivity. Four types of micellar-ionic liquid columns were examined in this study: 1-butyl-3-methylimidazolium chloride with sodium dodecylsulfate or dioctyl sulfosuccinate, and 1-butyl-3-methylimidazolium hexafluorophosphate with polyoxyethylene-100-stearyl ether or polyoxyethylene-23-lauryl ether. The partition coefficients were measured for a wide range of probe molecules capable of a variety of types and magnitudes of interactions. In general, most probe molecules preferentially partitioned to the micellar pseudophase over the bulk ionic liquid component of the stationary phase. Therefore, addition of surfactant to the stationary phase usually resulted in greater solute retention. It is also shown that the selectivity of the stationary phase is significantly altered by the presence of micelles, either by enhancing or lessening the separation. The effects of surfactant on the interaction parameters of the stationary phase are determined using the Abraham solvation parameter model. The addition of sodium dodecylsulfate and dioctyl sulfosuccinate to 1-butyl-3-methylimidazolium chloride stationary phases generally increased the phase's hydrogen bond basicity and increased the level of dispersion interaction. Polyoxyethylene-100-stearyl ether and polyoxyethylene-23-lauryl ether surfactants, however, enhanced the pi-pi/n-pi, polarizability/dipolarity, and hydrogen bond basicity interactions of 1-butyl-3-methylimidazolium hexafluorophosphate to a greater degree than the ionic surfactants with 1-butyl-3-methylimidazolium chloride. However, these nonionic surfactants appeared to hinder the ability of the stationary phase to interact with solutes via dispersion forces. Therefore, it is possible to effectively predict which analytes will be most highly retained by these micellar-ionic liquid stationary phases.     

Prediction of peak shape in hydro-organic and micellar-organic liquid chromatography as a function of mobile phase composition

A simple model is proposed that relates the parameters describing the peak width with the retention time, which can be easily predicted as a function of mobile phase composition. This allows the further prediction of peak shape with global errors below 5%, using a modified Gaussian model with a parabolic variance. The model is useful in the optimisation of chromatographic resolution to assess an eventual overlapping of close peaks. The dependence of peak shape with mobile phase composition was studied for mobile phases containing acetonitrile in the presence and absence of micellised surfactant (micellar-organic and hydro-organic reversed-phase liquid chromatography, RPLC). In micellar RPLC, both modifiers (surfactant and acetonitrile) were observed to decrease or improve the efficiencies in the same percentage, at least in the studied concentration ranges. The study also revealed that the problem of achieving smaller efficiencies in this chromatographic mode, compared to hydro-organic RPLC, is not only related to the presence of surfactant covering the stationary phase, but also to the smaller concentration of organic solvent in the mobile phase.     

Critical compilation of solute-micelle binding constants and related parameters from micellar liquid chromatographic measurements

Over 150 solute-micelle association (binding) constants and free solute retention factors are reported for a variety of neutral compounds and PTH-amino acids. The solute-micelle parameters were calculated from critically evaluated micellar liquid chromatographic data. The potential utility of these parameters for the optimization of micellar liquid chromatography and micellar electrokinetic capillary chromatography, including selection of the surfactant and/or stationary phase and the optimization of surfactant concentration, is briefly discussed.     

阴离子型胶束液相色谱的溶质保留行为

以SDS阴离子表面活性剂作流动相,酸性、中性及两性药物为受试药物,运用三相平衡理论考察影响阴离子型胶束液相色谱（AMLC）溶质保留行为的几个因素。保留由溶质与胶束相及修饰后固定相的综合作用决定。有机调节剂正丙醇的加入改变了溶质从水相到固定相或到胶束相的平衡,保留取决于溶质疏水性和静电性间的平衡。此外对羟基苯甲酸酯类同系物的亲脂性与3种细菌最小抑菌浓度具有显著相关性,提示其抑菌机理主要取决于药物与生物膜的亲和性。     

Retention mechanisms in micellar liquid chromatography

Micellar liquid chromatography (MLC) is a reversed-phase liquid chromatographic (RPLC) mode with mobile phases containing a surfactant (ionic or non-ionic) above its critical micellar concentration (CMC). In these conditions, the stationary phase is modified with an approximately constant amount of surfactant monomers, and the solubilising capability of the mobile phase is altered by the presence of micelles, giving rise to diverse interactions (hydrophobic, ionic and steric) with major implications in retention and selectivity. From its beginnings in 1980, the technique has evolved up to becoming a real alternative in some instances (and a complement in others) to classical RPLC with hydro-organic mixtures, owing to its peculiar features and unique advantages. This review is aimed to describe the retention mechanisms (i.e. solute interactions with both stationary and mobile phases) in an MLC system, revealed in diverse reports where the retention behaviour of solutes of different nature (ionic or neutral exhibiting a wide range of polarities) has been studied in a variety of conditions (with ionic and non-ionic surfactants, added salt and organic solvent, and varying pH). The theory is supported by several mechanistic models that describe satisfactorily the retention behaviour, and allow the measurement of the strength of solute-stationary phase and solute-micelle interactions. Suppression of silanol activity, steric effects in the packing pores, anti-binding behaviour, retention of ionisable compounds, compensating effect on polarity differences among solutes, and the contribution of the solvation parameter model to elucidate the interactions in MLC, are commented.     

Imitation of artificial membrane system via mobile phases with Tween-80 and cholic acid in biopartitioning micellar chromatography

The chromatographic behaviour of compounds of biomedical significance was studied using micellar mobile phases modified with polyoxyethylene (20) sorbitan monooleate (Tween-80). The influence of the surfactant within the 0.75-4% concentration range on the retention factor of model compounds was investigated. The biological surfactant cholic acid was introduced into the mobile phases in order to approach to the structure of natural membranes, viz. erythrocyte and cytoplasmatic membranes. It was found that curves of dependence of retention factor vs concentration of Tween-80 in the absence and presence of cholic acid in the mobile phase considerably diverge with one another, especially in the 2-3% concentration range of Tween-80 using C18-type support. Increasing the concentration of Tween-80 resulted in the increase of retention factors using phenyl-coated stationary phase.     

New Pseudostationary Phases for Electrokinetic Chromatography: A High-Molecular Surfactant and Proteins

To extend the applicability of electrokinetic chromatography (EKC), two new types of pseudostationary phases have been introduced. A high-molecular surfactant, butyl acrylate/butyl methacrylate/methacrylic acid copolymer (BBMA) is employed as a micellar forming surfactant for miccllar electrokinetic chromatography (MEKC). The critical micelle concentration of BBMA is essentially zero, which means the micellar concentration is constant irrespective of temperature and buffer. Some characteristic features of BBMA as the pseudostationary phase for MEKC is investigated in comparison with conventional ionic surfactants. Ovomucoid and avidin, which are proteins isolated from egg white, have been found to be useful chiral selectors in affinity EKC. A few examples of the separation of enantiomers with these proteins are shown.     

Preparation of a silica‐based high‐performance hydrophobic interaction chromatography stationary phase for protein separation and renaturation

In this work, based on the structural characteristics of bio‐membrane molecules, a novel type of high‐performance hydrophobic interaction chromatography stationary phase was prepared using cholesterol as a ligand. Investigating the separation performance of this stationary phase, the effect of pH and salt concentration of the mobile phase on the retention time, the absorption capacity, and the hydrophobic ability revealed that this stationary phase had a high loading capacity and moderate hydrophobic interactions compared with four different hydrophobic interaction chromatography stationary phase ligands. Five types of standard proteins could be baseline separated with a great selection for protein separation. When 3.0 M urea was added to the mobile phase, it could be refolded with simultaneous purification of denatured lysozyme by one‐step chromatography. The mass recovery of lysozyme reached 89.5%, and the active recovery was 96.8%. Compared with traditional hydrophobic interaction chromatography, this new stationary phase has a good hydrophobic ability and a significant refolding efficiency.     

Performance of short-chain alcohols versus acetonitrile in the surfactant-mediated reversed-phase liquid chromatographic separation of β-blockers

Organic solvents are traditionally added to micellar mobile phases to achieve adequate retention times and peak profiles, in a chromatographic mode which has been called micellar liquid chromatography (MLC). The organic solvent content is limited to preserve the formation of micelles. However, at increasing organic solvent contents, the transition to a situation where micelles do not exist is gradual. Also, there is no reason to neglect the potentiality of mobile phases containing only surfactant monomers instead of micelles (high submicellar chromatography, HSC). This is demonstrated here for the analysis of β-blockers. The performance of four organic solvents (methanol, ethanol, 1-propanol, and acetonitrile) was compared in mobile phases containing the anionic surfactant sodium dodecyl sulphate in the MLC and HSC modes. The association of the organic solvent molecules with micelles gives rise to a significant loss in the elution strength of the organic solvent; whereas upon disruption of micelles, it tends to that observed in the hydro-organic mode. The elution behaviour of the β-blockers was modelled to predict the retention times. This allowed the detailed exploration of the selectivity and resolution of the chromatographic systems in relatively wide ranges of concentration of surfactant and organic solvent. The best performance in terms of resolution and analysis time was achieved using HSC with acetonitrile, being able to base-line resolve a mixture of eight β-blockers. Ethanol also provided a good separation performance, significantly improved with respect to methanol and 1-propanol. In contrast, the hydro-organic mode using acetonitrile or any of the short-chain alcohols could not succeed with the separation of the β-blockers, owing to the poorer selectivity and wider peaks.     

Polymeric sulfated surfactants with varied hydrocarbon tail: I. Synthesis, characterization, and application in micellar electrokinetic chromatography

The influence of surfactant hydrocarbon tail on the solute/pseudostationary phase interactions was examined. Four anionic sulfated surfactants with 8-, 9-, 10-, and 11-carbon chains having a polymerizable double bond at the end of the hydrocarbon chain were synthesized and characterized before and after polymerization. The critical micelle concentration (CMC), polarity, and aggregation number of the four sodium alkenyl sulfate (SAIS) surfactants were determined using fluorescence spectroscopy. The partial specific volume of the polymeric SAIS (poly-SAIS) surfactants was estimated by density measurements and capillary electrophoresis (CE) was employed for determination of methylene selectivity as well as for elution window. The CMC of the monomers of SAIS surfactants decrease with increase in chain length and correlated well when fluorescence method was compared to CE. The physicochemical properties (partial specific volume, methylene selectivity, electrophoretic mobility, and elution window) increased with an increase in chain length. However, no direct relationship was found between the aggregation number and the length of hydrophobic tail of poly-SAIS surfactants. These polymeric surfactants were then used as pseudostationary phases in micellar electrokinetic chromatography (MEKC) to study the retention behavior and selectivity factor of 36 benzene derivatives with different chemical characteristics. Although variation in chain length of the polymeric surfactants significantly affects the retention of nonhydrogen bonding (NHB) benzene derivatives, these effects were less pronounced for hydrogen bond acceptor (HBA) and hydrogen bond donor (HBD) benzene derivatives. Therefore, hydrophobicity of poly-SAIS surfactants was found to be a major driving force for retention of NHB derivatives. However, for several benzene derivatives (NHB, HBA, and HBD) significantly higher selectivity factor was observed with longest chain polymeric surfactant (e.g., poly(sodium 10-undecenyl sulfate), poly-SUS) compared to shorter chain polymeric surfactant (e.g., poly(sodium 7-octenyl sulfate), poly-SOcS). In addition, the effect of the surfactant hydrophobic chain was also found to have some impact on migration order of NHB, HBA, and HBD benzene derivatives.     

New Perspectives in Micellar Liquid Chromatography

Abstract

This paper will summarize several new findings obtained in our laboratory on the use of micellar mobile phases in liquid chromatography. The topics to be addressed include (i) stationary phase modification by the mobile phase surfactant in micellar liquid chromatography, (ii) investigation of the retention mechanism in micellar liquid chromatography (MLC) using an alkyl-benzene homologous series, (iii) evaluation of the effects of organic additives upon retention and efficiency in MLC, and (iv) preliminary characterization of several new classes of surfactant molecules for use in MLC. The information gained from these studies provides new insights into the dynamics of MLC and demonstrates their potential usefulness in several new separation applications including the resolution of optical isomers.     

Electrochemical Study of the Micellization of Hexadecylpyridinium Bromide in the Presence of Some Crown Ethers

A membrane electrode selective to hexadecylpyridinium bromide was used to study the micellization of the surfactant in the presence of varying concentrations of crown ethers 15-crown-5, dicyclohexyl-18-crown-6 and dicyclohexyl-24-crown-8 at 27°C. The critical micelle concentration of surfactant was largely influenced by the crown ethers used, indicating their participation in the structure of the resulting micelle. The degree of attachment of crown ethers to the surfactant in the micellar structure was found to be 1. An estimation of the K_f for the 1:1 surfactant-crown interaction in the micellar region is reported. The degree of counter ion dissociation was found to increase with the crown ether concentration, and with the nature of macrocyclic ligands in the order 15-crown-5 < dicyclohexyl- l8-crown-6 < dicyclohexyl-24-crown-8. There was also some evidences for a rather strong interaction between dicyclohexyl-24-crown-8 and the surfactant in the submicellar concentration ranges in solution.     

Widening of the elution window in micellar electrokinetic chromatography with cationic surfactants. II. Cationic additives and modifiers of the electroosmotic flow.

In micellar electrokinetic chromatography (MEKC) with cationic surfactants the migration window is significantly narrower than with anionic surfactants. In order to overcome this disadvantage of cationic surfactants, it is investigated whether it is possible to widen the migration window by reducing the velocity of the aqueous phase while the electrophoretic mobility of the micelles is maintained. Short chain alkylammonium compounds, hexamethonium bromide and hydroxypropylmethylcellulose are tested as additives to the separation electrolyte with the potential to improve the migration window via reducing the velocity of the electroosmotic flow. It will be shown that these modifiers can be successfully used in order to widen the migration window in MEKC with cationic surfactant employing an alkyltrimethylammonium bromide as micelle forming agents. Influence of the modifiers selected on retention of neutral and acidic solutes and on efficiency of the separation system is investigated.