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.     

Simultaneous Determination of Dopamine,Epinephrine and 5-Hydroxytryptamine in Toad Venom and Common Yam Rhizome by MEKC

A micellar electrokinetic chromatography method was developed for the simultaneous determination of dopamine, epinephrine and 5-hydroxytryptamine. Several experimental parameters such as surfactant type and concentration, buffer concentration and pH, type and concentration of organic modifier were evaluated for the analysis of the studied compounds. Among the investigated separation conditions, the composition of micelles, pH and the methanol concentration were the critical parameters. Dopamine, epinephrine and 5-hydroxytryptamine were separated and determined successfully within 7.5 min in Toad venom and Common yam rhizome under the optimum conditions.

     

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).     

High-Performance Liquid Chromatography of T-RNA

Abstract

Fractionation of amino acid specific tRNAs by descending salt gradient on a reversed-phase high-performance liquid chromatography column is demonstrated. Stationary phase variables such as n-alkyl chain length and silica type are discussed in terms of stability and selectivity. Mobile phase parameters including salt concentration, organic modifier, pH, and temperature are investigated. The concentration of organic modifier to adjust elution profiles can be critical in terms of tRNA retention.     

Modelling of retention behaviour of solutes in micellar liquid chromatography

In micellar liquid chromatography (MLC), the resolution for a given multi-component mixture can be optimized by changing several variables, such as the concentrations of surfactant and organic modifier, the pH and temperature. However, this advantage can only be fully exploited with the development of mathematical models that describe the retention and the separation mechanisms. Several reports have appeared recently on the possibilities of accurately predicting the solute retention in MLC. Although the retention and selectivity may strongly change with varying concentrations of surfactant, organic modifier and/or pH, the observed changes are very regular, and are well described by simple models. This characteristic enables a successful prediction of retention times and compensates the negative effect of the broad and tailed chromatographic peaks obtained for some solutes when micellar eluents are used. An overview of the models proposed in the literature to describe the retention behaviour in pure micellar eluents and micellar eluents containing an organic modifier, at a fixed pH or at varying pH, is given. The equations derived permit the evaluation of the strength of micelle-solute and stationary phase-solute interactions. The prediction of the retention based on molecular properties and the use of neural networks, together with the factors affecting the prediction capability of the models (linearization of the equations, dead time, critical micellar concentration, ionic strength and temperature) are commented on. The strategies used for the optimization of resolution are also given.     

The Quantitation of Heroin and Selected Basic Impurities Via Reversed Phase HPLC. I. The Analysis of Unadulterated Heroin Samples

Abstract

Methodology was developed employing reversed phase liquid chromatography for the simultaneous determination of heroin, O³-monoacetylmorphine, o⁶-monoacetylmorphine, acetylcodeine, noscapine and papaverine in unadulterated illicit powders. An HS-5 C18 column was used with a gradient system using methanol and a hexylamine phosphate buffer at pH 2.2. This method, suitable for automated analysis, used a multi-mode detection scheme via the use of a photodiode array detector. In order to arrive at the optimum chromatographic conditions in terms of selectivity and stability, a study was performed on the effect of various mobile phase parameters on log k′ for heroin, various impurities, and common adulterants. The mobile phase parameters included amine concentration, organic modifier type, and eluent pH.     

Separation optimization of quercetin,hesperetin and chrysin in honey by micellar liquid chromatography and experimental design

The chemometrics approach was applied for the separation optimization of flavonoid markers (quercetin, hesperetin and chrysin) in honey using micellar liquid chromatography (MLC). The investigated method combines SPE of flavonoids from honey using C₁₈ cartridge and their separation and quantification by micellar liquid chromatography. A two level full factorial design was carried out to evaluate the effect of four experimental factors including concentration of SDS, alkyl chain length of the alcohol used as the organic modifier (N), volume percentage of the organic modifier (V_m) and volume percentage of acetic acid (AcOH) in mobile phase on analytes retention times. Experiments for analytes retention times modeling and optimization of separation were performed according to central composite design. Multiple linear regression method was used for the construction of the best model based on experimental retention times. Pareto optimal method was used to find suitable compatibility between resolution and analysis time of analytes in honey. The optimum mobile phase composition for separation and determination of analytes in honey were [SDS]=0.124 mol/L; 7.8% v/v ethanol and 5.0% v/v AcOH. Limits of detection and linear range of flavonoid markers were 0.0079–0.0126, 0.05–50.0 mg/L, respectively.     

Separations in Thin Layer and High Performance Liquid Chromatography Using Alkyl Silica Gel Bonded Phases

Abstract

Separations in thin layer (TLC) and high performance liquid chromatography (HPLC) using alkyl bonded phases were carried out under optimum solvent conditions for each of three phases, RP-2, RP-8 and RP-18. The phases were tested for their efficiency and resolving power using three groups of compounds in three binary organic-water mobile phases. The organic solvents were acetonitrile, methanol and tetrahydrofuran, which are widely used as solvent modifiers in reversed phase liquid chromatography.

The results showed that, unlike HPLC, TLC using RP-18 plates was the most, and RP-2 plates the least, efficient. A naphthalene and biphenyl mixture which was resolved by HPLC using any of the three solvents and columns was not resolved by TLC using any plate or solvent combination, unless the plate was prewashed with an organic modifier. The addition of NaCl (1–2% wt/vol) to the solvent for TLC speeded development unless an alcohol was used, but did not greatly affect the separation.     

The Preparative Separation of Synthetic Peptides on Reversed-Phase Silica Packed in Radially Compressed Flexible-Walled Columns.+

Abstract

The rapid and efficient separation of multigram amounts of unprotected synthetic peptides on octadecylsilica, packed in flexible walled polyethylene columns, is described. The mobile phase used was 0.05% trifluoroacetic acid with methanol as the organic modifier. An advantage of this eluant system was that the salt free peptide could be isolated simply by lyophilisation of the sample after chromatography. The following peptides were purified with this system: glycylgly-     

Modelling and prediction of retention in high-performance liquid chromatography by using neural networks

Summary Multi-layer feed-forward neural networks trained with an error back-propagation algorithm have been used to model retention behaviour of liquid chromatography as a function of the composition of the mobile phases. Conventional hydro-organic and micellar mobile phases were considered. Accurate retention modelling and prediction have been achieved using mobile phases defined by two, three and four parameters. With micellar mobile phases, the parameters involved included the concentrations of surfactant and organic modifier, pH and temperature. It is shown that neural networks provide a competitive tool to model varied inherent nonlinear relationships of retention behaviour with respect to the mobile phase parameters. The soft models defined by the weights of the networks are capable of accommodating all types of linear and nonlinear relationships, neural networks being specially useful when the relationships between retention behaviour and the mobile phase parameters are unknown. However, to train neural networks more experimental points than with hard-modelling methods are required, hence the use of the networks is recommended only for those cases where adequate theoretical or empirical models do not exist.     

Use of an Aqueous Micellar Mobile Phase for Separation of Phenols and Polynuclear Aromatic Hydrocarbons via HPLC

Abstract

An aqueous solution of sodium dodecyl sulfate (SDS) micelles is shown to be a novel, highly effective mobile phase in high performance liquid chromatography. Using a reverse phase column, nine phenols and two polynuclear aromatic hydrocarbons were easily separated. The possible advantages and disadvantages of aqueous micellar solutions over traditional organic and mixed solvent mobile phases is discussed.     

High Performance Liquid Chromatography of Uroporphyrin Isomers

Abstract

The separation of uroporphyrin I and III isomers by reversed-phase high performance liquid chromatography on ODS-Hypersil with ammonium acetate buffer-acetonitrile solvent systems is described. The effects of buffer concentration, pH, organic modifier proportion and different organic modifiers on the resolution are studied. The optimum conditions for the separation were 12–13% acetonitrile in 1M ammonium acetate buffer pH 5.10–5.20. The method also separated uroporphyrin I and III from the II isomers but the resolution of uroporphyrin III and IV isomers was not achieved.     

Systematic development and validation of stability indicating micellar electrokinetic chromatography methods for early stage drug candidates

Suitable methods for assessment of purity, potency and stability of new drug substances and drug products are required to be rapidly developed and validated to provide appropriate data for early project development decisions. In order to routinely provide methods of consistent and suitable quality to meet increasingly aggressive timelines, systematic approaches to both develop and validate analytical technologies have been developed. Systematic approaches to evaluate separation parameters such as buffer pH, buffer ionic strength, surfactant concentration, organic modifiers, organic modifier concentration, applied voltage and temperature were evaluated for an early stage drug candidate. Techniques to improve method precision and ruggedness were also examined. Finally, the validation results from the micellar electrokinetic chromatography method utilizing an internal standard were compared against the simultaneously developed high-performance liquid chromatography method.     

Retention modeling in micellar liquid chromatography

The aim of this review is to present the most relevant work on retention modeling in micellar liquid chromatography. First, physico-chemical models explaining the variation of capacity factors with one or more experimental variables (such as micellar concentration, organic modifier concentration, and pH) will be shown. Secondly, studies carried out to model the solute retention in micellar liquid chromatography by means of empirical equations will be presented, and finally new trends in this area will be introduced.     

Quantitative Analysts of 5-Hydroxytryptamtne in Biological Material by High Perfornance Liquid Chromatography and Electrochmical Detection

Abstract

A quantitative method for the analysis of 5-hydroxytryptamine in biological material is described. The method is based on high performance liquid chromatography (HPLC) with electrochemical detection. A simple purification on a weakly acidic ion exchange resin prior to the analysis gives quite clean samples and permits concentration of diluted samples. The chromatographic separation is performed on a reverse phase column with organic modifier added to an aqueous eluent. With this analytical system 25 pg of 5-hydroxytryptamine can be detected.     

Determination of Cytokinins by Ion Suppression-Reverse Phase High Performance Liquid Chromatography

Abstract

A method for the separation of cytokinins has been developed by the use of ion suppression-reverse phase high performance liquid chromatography (IS-RP HPLC) with high efficiency, specificity and low sensitivity. Four reverse phase chromatographic columns were compared for their ability to separate and quantify six cytokinins with respect to signal response, resolution (R_S), capacity factor (k¹) and efficiency, N (number of theoretical plates). The optimal aqueous and organic phase components for the resolution of these cytokinins were determined with respect to pH, ionic strength and the proportion of the organic solvents, methanol and acetonitrile. Heptane sulfonic acid was used as an organic modifier. The optimal HPLC operating conditions were applied to separate and quantify the cytokinins present in a culture supernatant of Azotobacter chroococcum.     

Reversed-Phase Systems for the Separation of Coumarins and Furocoumarins by Thin-Layer and High-Performance Liquid Chromatography

Abstract

The retention behaviour of eleven derivatives of counarin was investigated by reversed-phase (RP) thin-layer and high performance liquid chromatography. The linear relationships between log k'or R_M values and the content of organic modifier in the aqueous mobile phase obtained for wide composition ranges indicate that the plots can be used to determine R_Mw and log k_w values by extrapolation to pure water. The effects of individual substituants on the retention and the correlation between TLC and HPLC data was analysed.     

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