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.     

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.     

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.     

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.     

Liquid chromatographic separations with mobile-phase additives: influence of pressure on coupled equilibria

On the basis of equilibrium thermodynamics, pressure can cause a shift in equilibrium for any interaction that exhibits a change in partial molar volume. This shift in equilibrium can be observed in liquid chromatography as a pressure-dependent shift in solute retention. In this paper, the impact of pressure on liquid chromatographic separations with mobile-phase additives is examined from both theoretical and experimental perspectives. The theoretical development for coupled-equilibria separations shown here is general and can be applied to any separation using mobile-phase additives. Predictions indicate that the coupled nature of these equilibria leads to pressure-induced perturbations in partitioning and complexation that can either compete with or complement one another. Using positional isomers and enantiomers as model solutes, experimental retention observations are fully consistent with these predictions, showing the diminution of individual pressure effects for competing cases and enhanced pressure effects for complementary cases. When pressure-induced changes in capacity or retention factor differ between individual solutes, changes in solute selectivity are predicted and observed. Using a C18 stationary phase with beta-cyclodextrin as the mobile-phase additive, solutes studied here exhibit changes in selectivity ranging from - 7 to + 10% for a change in average pressure of approximately 215 bar. Perhaps the most dramatic change in selectivity is observed for the separation of positional isomers where pressure-induced changes in selectivity actually reverse solute elution order.     

Electrokinetic chromatography using thermodynamically stable vesicles and mixed micelles formed from oppositely charged surfactants

The electrokinetic chromatography (EKC) of a novel mixed surfactant system consisting of oppositely charged surfactants, sodium dodecyl sulfate (SDS) and n-dodecyltrimethylammonium bromide (DTAB), was investigated. The chromatographic characteristics of large liposome-like spontaneous vesicles and rodlike mixed micelles formed from the mixture were explored and compared with those of SDS micelles. Separations of a series of n-alkylphenones showed that the spontaneous vesicles provided about a 2 times wider elution window than SDS micelles. Both vesicle and mixed micelle systems were found to provide larger methylene selectivity than SDS. The different elution order of a group of nitrotoluene geometric isomers with DTAB/SDS spontaneous vesicles and SDS micelles pseudostationary phases suggested the possibility of different separation mechanisms with these two systems. Comparisons of polar group selectivity, retention, and efficiency were made between vesicles, mixed micelles, and SDS micelles. The correlation between the logarithms of the retention factors (log k') and octanol-water partition coefficients (log P(ow)) for a group of 20 neutral compounds was also studied with DTAB/SDS vesicles. Spontaneous vesicles have great potential as a pseudostationary phase in electrokinetic chromatography.     

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.     

Analytical solutions of the ideal model for gradient liquid chromatography

The analytical solutions of the ideal model for gradient elution that ignores the influence of the solute concentration on the retention factor (k) were studied by using the method of characteristics for solving partial differential equations. It is found for any gradient profiles and solvent strength models used that the concentration of the solute will be discontinuous where the mobile-phase composition is. On a given characteristic curve, the product of the concentration and the retention factor is kept constant at the point where the concentration is continuous. At the point where the concentration is discontinuous, the product on the left side of this point is equal to that on the right side. We also discussed the basic equations to predict the retention time in gradient elution and introduced the injection time into them. For linear solvent strength stepwise and linear gradient elution, general expressions were proposed for the prediction and they can be used as the basis to derive others for specific gradient modes such as single linear, stepwise, and ladderlike gradients. For these modes, simple expressions to account for the band compression and the concentration change during the elution were also given.     

Nonaqueous electrophoresis microchip separations: conductivity detection in UV-absorbing solvents

The use of organic solvents in microfabricated capillary electrophoresis (CE) devices is demonstrated in connection with the separation of aliphatic amines in pure dimethylformamide, dimethylacetamide, dimethyl sulfoxide, or propylene carbonate media. Contactless conductivity detection is employed for monitoring the separated solutes in these UV-absorbing solvents. The effect of the physicochemical properties of the organic solvent upon the migration behavior is investigated. The apparent mobility increases nearly linearly with the reciprocal of the solvent viscosity, while the electroosmotic mobility increases in a linear fashion with the dielectric constant/ viscosity ratio. Some deviation from theoretical predictions is observed using propylene carbonate. The nonaqueous CE microchip offers high separation efficiency, reflected in plate numbers ranging from 93,680 to 127,680, using a separation voltage of +3,000 V, a dimethylformamide medium, and a contactless conductivity detection. Experimental parameters affecting the analytical performance of the nonaqueous CE/conductivity microchip are examined. Calibration and precision experiments indicate a linear and reproducible response. Such use of organic solvents can benefit microchip separations through extended scope (toward nonpolar solutes) and tunable selectivity.     

Separation of Neutral Compounds and Basic Drugs by Capillary Electrophoresis in Acidic Solution Using Laurylpoly(oxyethylene) Sulfate as an Additive

Working at pH 2.4 with uncoated silica capillaries has the advantage that electroosmotic flow is virtually eliminated. Excellent separations of protonated organic bases were obtained when ethanesulfonic acid was added to the running electrolyte to coat the capillary surface by a dynamic equilibrium. The effect of adding a new surfactant, sulfonated Brij-30, to the acidic electrolyte was also investigated. Use of this surfactant in acidic organic-aqueous solutions changes the elution order of many organic cations and also permits the separation of neutral organic compounds. Excellent resolution of a mixture of 19 PAHs and similar compounds was obtained in 40% organic solvent in only 20 min. The largest organic compounds form the most stable association complexes with the sulfonated Brij-30 and, thus, have the shortest migration times. It is shown that the type and concentration of surfactant, as well as the composition of the aqueous-organic solution, are conditions that can be varied over a broad range to obtain superior separations of both neutral and cationic organic compounds. The type of organic solvent is yet another condition that can be manipulated advantageously. For example, the use of equal volumes of acetonitrile and 2-propanol in water-organic solutions can give better resolution of neutral organic analytes than either solvent used alone.     

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.     

The role of Lewis acid-base processes in ligand-exchange chromatography of benzoic acid derivatives on zirconium oxide.

Porous microparticulate zirconium oxide shows very different selectivities and pH dependencies for the separation of benzoic acid derivatives than do conventional bonded-phase anion-exchange supports. This results from a very significant ligand-exchange contribution to the retention of hard Lewis bases on the surface of transition-metal oxide supports. We have found that the capacity factors of a wide variety of derivatives of benzoic acid are closely correlated with their Bronsted acidities. The eluent pH is also a critical factor in determining the magnitude of the capacity factor, but it does not have much influence on chromatographic selectivity. The differential selectivity of this phase in comparison to conventional polymeric and bonded-phase anion exchangers can be attributed to complexation and steric effects which profoundly alter the elution patterns of certain solutes.     

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.     

C60 and C70 HPLC retention reversal study using organic modifiers

A novel equation (Guillaume Y. C. et al. Anal. Chem. 1998, 70, 608) modeling the weak polar solute retention in reversed-phase liquid chromatography (RPLC) was applied to fullerene molecules C60 and C70. In RPLC, with an organic modifier (OM)/water mobile phase, the fullerene cluster solvation energies were calculated for OM = methanol, ethanol, propanol, butanol, and pentanol. An enthalpy-entropy compensation revealed that the type of interactions between fullerenes and the stationary phase was independent of both the fullerene and organic modifier structures. The energetics of OM and OM-water cluster exchange processes in the mobile phase were investigated in relation to the carbon atom number of the hydrophobic chain of the OM. Two linear correlations were found between the Gibbs free energy changes in the solvent exchange processes which confirmed that (i) a reversal elution order existed for C60 and C70 when methanol was changed into ethanol, propanol, butanol, pentanol and that (ii) the mobile phase was dominant in governing selectivity changes in nonpolar solutes.     

Lateral transport of solutes in microfluidic channels using electrochemically generated gradients in redox-active surfactants

We report principles for a continuous flow process that can separate solutes based on a driving force for selective transport that is generated by a lateral concentration gradient of a redox-active surfactant across a microfluidic channel. Microfluidic channels fabricated with gold electrodes lining each vertical wall were used to electrochemically generate concentration gradients of the redox-active surfactant 11-ferrocenylundecyl-trimethylammonium bromide (FTMA) in a direction perpendicular to the flow. The interactions of three solutes (a hydrophobic dye, 1-phenylazo-2-naphthylamine (yellow AB), an amphiphilic molecule, 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (BODIPY C(5)-HPC), and an organic salt, 1-methylpyridinium-3-sulfonate (MPS)) with the lateral gradients in surfactant/micelle concentration were shown to drive the formation of solute-specific concentration gradients. Two distinct physical mechanisms were identified to lead to the solute concentration gradients: solubilization of solutes by micelles and differential adsorption of the solutes onto the walls of the microchannels in the presence of the surfactant concentration gradient. These two mechanisms were used to demonstrate delipidation of a mixture of BODIPY C(5)-HPC (lipid) and MPS and purification of BODIPY C(5)-HPC from a mixture of BODIPY C(5)-HPC and yellow AB. Overall, the results of this study demonstrate that lateral concentration gradients of redox-active surfactants formed within microfluidic channels can be used to transport solutes across the microfluidic channels in a solute-dependent manner. The approach employs electrical potentials (<1 V) that are sufficiently small to avoid electrolysis of water, can be performed in solutions having high ionic strength (>0.1M), and offers the basis of continuous processes for the purification or separation of solutes in microscale systems.     

Capillary Electrokinetic Chromatography Employing p-(Carboxyethyl)calix[n]arenes as Running Buffer Additives

Calixarenes, a class of macrocyclic phenolic compounds with a basket-like shape, are used as capillary electrophoresis reagents for separations of native and substituted polycyclic aromatic hydrocarbons. The p-(carboxyethyl)calix[n]arenes reported herein are a series of charged, moderately water soluble macrocyclic molecules that can form complexes with neutral molecules. Electrokinetic chromatographic separations are based on the differential distribution of molecules between a running buffer phase, which is transported by electroosmotic flow, and an electrophoretically mediated calixarene. The size of the calixarene influences separation performance, illustrating the importance of cavity size and geometry in the complexation process. p-(Carboxyethyl)calix[7]arene provides the best efficiency (>10(5) plates/m) and selectivity in these studies. The influences of pH, organic solvent, and field strength on elution range, capacity factors, efficiency, and selectivity are also reported. In general, capacity factors are rather low, but the high charge-to-mass ratios of certain calixarenes produce relatively wide elution ranges. Molecular modeling data and solubility data are used to interpret the observed selectivity.     

Band-trajectory model for temperature-programmed series-coupled column ensembles with pressure-tunable selectivity

A model and a spreadsheet algorithm is described for the prediction of solute-band migration trajectories in a series-coupled combination of two capillary GC columns with pressure-tunable and -programmable selectivity and operated under temperature-programmed conditions. The model takes into account the acceleration of carrier gas in the two columns as a result of decompression effects, the deceleration of carrier gas as a result of the increase in viscosity during temperature programming, the decrease in solute retention factors with increasing temperature during the temperature program, the differences in retention factors for the two columns, and programmed changes in the carrier-gas flow rates in the two columns during selectivity programming. In the model, the 20-meter-long column ensemble is divided into 1-cm-long intervals, and the carrier-gas velocity and column temperature are assummed to be constant in any interval. Migration times for all of the mixture solutes are computed for each column interval, and the solute-band positons in the column ensemble are plotted versus the running sum of these migration times to obtain band trajectory plots. The sum of these migration times for all 2,000 intervals gives the ensemble retention times for the solutes. Isothermal retention factors (k) for all of the mixture components at various column temperatures (Tc) are used as imput to the algorithm. Slope and intercept values of In(k) vs 1/Tc plots are used in the algorithm. General features of the model are tested using a mixture of C12-C24 normal alkanes. A mixture of polar and nonpolar compounds is used to test the utility of the model for the predicition of peak separations and retention times with pressure-tunable and -programmable selectivity. Good agreement is observed in all cases.     

Immobilized-metal affinity and hydroxyapatite chromatography of genetically engineered subtilisin

High-performance immobilized-metal affinity and hydroxyapatite chromatography were employed to investigate the engineered subtilisin S1 binding site microenvironment. Although these methods are classified as affinity techniques, unlike traditional affinity columns, both are capable of probing the entire surface of a molecule. The metal chelate study employed gradient elution to assemble retention maps for a wide range of mobile-phase pH. Resolution of single substitution variants was achieved at the optimum mobile-phase pH. A total of four metals were applied separately to the metal chelate column to investigate ligand specificity with respect to protein retention. Hydroxyapatite chromatography, albeit an established technique, has only recently been developed as a high-performance chromatographic method. Gradient elution separations were performed to determine selectivity. Immobilized-metal affinity chromatography was found to be the more effective method for the separation of site-specific variants.     

Shape selectivity for constrained solutes in reversed-phase liquid chromatography

In reversed-phase liquid chromatography (RPLC), the separation of compound mixtures of similar polarity can present a significant challenge for the analyst. Examples of such compounds include geometric isomers present in environmental samples (e.g., polycyclic aromatic hydrocarbons, polycyclic aromatic sulfur heterocycles, and polychlorinated biphenyl congeners) and compounds of biological significance (e.g., carotenoids and steroids). In general, compounds with rigid, well-defined molecular shape are best separated using a column with enhanced shape selectivity characteristics. This perspective presents an overview of column properties that influence shape selectivity for constrained solutes. Approaches to the characterization of stationary-phase structure are described, and the findings are correlated with chromatographic performance. Finally, retention models of shape discrimination are presented that are consistent with observed retention behavior. An appreciation for shape recognition effects in RPLC will facilitate method development for certain classes of difficult to resolve compounds.