Micellar affinity gradient focusing in a microfluidic chip with integrated bilinear temperature gradients

Micellar affinity gradient focusing (MAGF) is a microfluidic counterflow gradient focusing technique that combines the favorable features of MEKC and temperature gradient focusing. MAGF separates analytes on the basis of a combination of electrophoretic mobility and partitioning with the micellar phase. A temperature gradient is produced along the separation channel containing an analyte/micellar system to create a gradient in interaction strength (retention factor) between the analytes and micelles. Combined with a bulk counterflow, species concentrate at a unique point where their total velocity sums to zero. MAGF can be used in scanning mode by varying the bulk flow so that a large number of analytes can be sequentially focused and passed by a single detection point. In this work, we develop a bilinear temperature gradient along the separation channel that improves separation performance over the conventional linear designs. The temperature profile along the channel consists of a very sharp gradient used to preconcentrate the sample followed by a shallow gradient that increases resolution. We fabricated a hybrid PDMS/glass microfluidic chip with integrated micro heaters that generate the bilinear profile. Performance is characterized by separating several different samples including fluorescent dyes using SDS surfactant and pI markers using both SDS and poly-SUS surfactants as the micellar phase. The new design shows a nearly two times improvement in peak capacity and resolution in comparison to the standard linear temperature gradient.     

Counterflow gradient electrophoresis for focusing and elution

Counterflow gradient electrofocusing uses the bulk flow of a liquid solution to counterbalance the electrophoretic migration of an analyte. When either the bulk velocity or the electrophoretic velocity of the analyte is made to vary across the length of the channel, there exists a unique zero‐velocity point for the analyte. This focusing method enables simultaneous separation and concentration of different analytes. The high resolution and sensitivity achieved are similar to that of isoelectric focusing, which separates analytes based on their isoelectric points, but the key difference is that analytes will instead focus based on their electrophoretic mobility. Dynamically changing the applied voltage or the counterflow rate over time will shift the zero‐velocity point, and therefore allows the focused analytes to pass through a fixed detection point, or elute from the separation channel. Throughout the review, a number of different counterflow gradient techniques will be discussed, along with their recent advancements and potential applications.     

DNA hybridization assays using temperature gradient focusing and peptide nucleic acids

Two types of DNA hybridization assays are demonstrated with temperature gradient focusing (TGF) and peptide nucleic acids (PNAs). In TGF, the application of a controlled temperature gradient along the length of a microchannel filled with an appropriate temperature-dependent buffer results in the formation of a gradient in both the electric field and electrophoretic velocity. Ionic species move in this gradient and concentrate at a unique point where the total velocity sums to zero. The first assay is a mixing assay in which PNA is allowed to flow through spatially focused DNA targets within a capillary. The second assay detects single base pair mutations (SBPM) by monitoring the fluorescence intensity of PNA/DNA duplexes as a function of temperature within the capillary. The SBPM analysis can be performed in less than 5 min with 100-fold more dilute analyte compared to conventional UV melting measurements.     

Ion-exclusion chromatography with mixed micellar mobile phases

Mixed ionic-nonionic surfactant mobile phases for ion-exclusion chromatography of carboxylic acids permitted the analysis time to be reduced by partitioning the analytes could be determined without interference even when they eluted near the void volume. These advantages result from the formation of mixed micelles of the two surfactants. The partition behaviour of both neutral and charged analytes to a nonionic micellar phase was evaluated on the basis of a retention model that included dissociation equilibria of analytes. In addition, the partition coefficients to mixed micellar phases were also determined by using this model.     

胶束溶液体系对毛细管电动色谱酸性化合物分离的影响

以阴离子表面活性剂十二烷基硫酸钠（SDS）、非离子表面活性剂吐温20（ Tween 20）及两者组成的混合胶束体系作为毛细管胶束电动色谱（MECC）的分离介 质，进行4种结构相似的酸性化合物的MECC分离研究，考察了胶束的类型、表面活 性剂的浓度、缓冲溶液的pH值及有机改性剂乙醇对分离的影响。结果表明各因素对 酸性药物的MECC分离有不同的影响规律。SDS胶束体系对溶质的保留值最大， Tween 20体系的保留值最小，二者的分离选择性正好相反，混合胶束体系的分离行 为则介于两者之间；在SDS和Tween 20体系中，表面活性剂浓度增加，溶质的保留 时间均随之递增，混合胶束体系中，总浓度一定，随Tween 20配比的增加，溶质的 保留时间先减少后增加；缓冲溶液的pH值增大，使溶质的分离效果均能变差；有机 改性剂乙醇的加入对容量因子的影响主要与溶质的疏水性有关，并对分离作用机理 进行了探讨。在SDS和Tween 20 MECC体系下，分别进行了实样测定，取得了满意的 结果。     

Temperature gradient focusing in a PDMS/glass hybrid microfluidic chip

This paper reports the application of temperature gradient focusing (TGF) in a PDMS/glass hybrid microfluidic chip. With TGF, by the combination of a temperature gradient along a microchannel, an applied electric field, and a buffer with a temperature-dependent ionic strength, analytes are focused by balancing their electrophoretic velocities against the bulk velocity of the buffer containing the analytes. In this work, Oregon Green 488 carboxylic acid was concentrated approximately 30 times as high as the initial concentration in 45 s at moderate electric strength of 70 V/cm and a temperature gradient of 55 degrees C across the PDMS/glass hybrid microfluidic chip with a 1 cm long capillary.     

Recent innovation in capillary electrokinetic chromatography with replaceable charged pseudostationary phases or additives

Recent developments of separation of neutral analytes in capillary systems with the mobile phase driven by the electroosmotic flow (EOF) and charged additives acting as a pseudostationary phase are reviewed. As pseudostationary phases a number of additives are used. Soluble polymers, either anionic or cationic, were applied as alternatives to micelles. Monomeric charged additives are also intended to form associates with the analytes, leading to selective retention and separation in a similar way as the polymeric pseudostationary phases. Dendrimers, spherical macromolecules with highly branched chains and charged terminal groups, are successfully applied for the separation of lipophilic analytes. Polymers with covalently stabilized structures are introduced in the form of permanent micelles and are therefore insensitive to the mobile phase composition, enlarging the applicability of micellar electrokinetic capillary chromatography (MEKC).     

Vesicle formation from temperature jumps in a nonionic surfactant system

When heating a dilute sample of the binary system of tetraethyleneglycol dodecyl ether (C12E4) and water from the micellar phase (L1) into the two-phase region of a lamellar phase (L(alpha)), and excess water (W) vesicles are formed. During heating, one passes a region of phase separation in the micellar phase (L1' + L1') where the initial micelles rapidly fuse into larger aggregates forming the concentrated L1 phase (L1') with a structure of branched cylindrical micelles, a so-called "living network". The static correlation length of the micelles are increasing with increasing concentration, from ca. 10 nm to 80 nm in the concentration range of 0.0001 g/cm3-0.0035 g/cm3. The overlap concentration was determined to 0.0035 g/cm3. When the temperature reaches the L1' + L(alpha) region the network particles transform into bilayer vesicles with a z-average apparent hydrodynamic radius in the order of 200 nm depending on the composition. The size of the final vesicles depends on the extent of aggregation/fusion in the L1' + L1' region and hence on the rate of heating. The aggregation/fusion in the L1' + L1' is slower than diffusion-limited aggregation, and it is shown that 1/100 of the collisions are sticky results in the fusion event.     

Concentration Gradient Perturbations in Micellar Liquid Chromatography with Thermal Lens Spectrophotometric Detection

Abstract

In HPLC with a sodium dodecyl sulphate micellar mobile phase, a perturbation of the sodium ion equilibrium gives rise to a refractive index gradient associated to a concentration gradient. This produces a loss of sensitivity and an increase of noise at a reproducible value of the retention time when thermal lens spectrometric detection is used. Baseline perturbations as this one produced by retained components of the mobile phase can overlap with the peaks of the analytes.     

Two-step stacking in capillary zone electrophoresis featuring sweeping and micelle to solvent stacking: II. Organic anions

Two-step stacking of organic anions by sweeping and micelle to solvent stacking (MSS) using cationic cetyltrimethylammonium micelles in co-electroosmotic flow (co-EOF) capillary zone electrophoresis (CZE) is described. The co-EOF condition where the direction of the EOF is the same as the test anions was satisfied by positive dynamic coating of a fused silica capillary with hexadimethrine bromide. The strategy was as follows. After conditioning the capillary with the background solution (BGS), a micellar solution (MS) was injected before the sample solution (S). The BGS, MS and S have similar conductivities. Voltage was applied at negative polarity. The analytes in the micelle-free S zone were swept by micelles from the MS. The swept analytes were brought by the micelles to the MSS boundary where the second stacking step was induced by the presence of organic solvent in the BGS. Finally was the separation of concentrated analytes by CZE. The effect of electrolyte concentration in the S, injection time of the MS and the S and surfactant concentration in the MS were studied. A 20-29, 17-33 and 18-21 times increase in peak height sensitivity was obtained for the test hypolipidaemic drugs (gemfibrozil, fluvastatin and atorvastatin), non-steroidal anti-inflammatory drugs (diflunisal, naproxen, ketoprofen, indoprofen and indomethacin), and herbicides (mecoprop and fenoprop), respectively. The LODs (S/N=3) were from 0.05 to 0.55 μg/mL. The intraday and interday repeatabilities (%RSD, n=12) in terms of retention time, corrected peak area, and peak heights was less than 3.6, 8.9, and 10.8%, respectively. The application of sweeping and MSS in co-EOF CZE together with a simple extraction procedure to a waste water sample spiked with the test herbicides was also demonstrated.     

The nature of the micellar stern region as studied by reaction kinetics. 2

The nature of rate-retarding effects of cationic micelles on the water-catalyzed hydrolyses of a series of para-substituted 1-benzoyl-1,2,4-triazoles (1a-f) and 1-benzoyl-3-phenyl-1,2,4-triazole (2) has been studied using kinetic methods. A comparison is drawn between medium effects in the micellar Stern region and in model solutions for the micellar Stern region. Simple model solutions involving concentrated aqueous solutions of a small ionic molecule resembling the surfactant headgroup, as reported before,(1) were improved. New model solutions for alkyltrimethylammonium bromide micelles contain both tetramethylammonium bromide (TMAB), mimicking micellar headgroups, and 1-propanol, mimicking hydrophobic tails. The rate-retarding effect of micelles on the hydrolysis of 1a-f and 2 is caused by the high concentration of headgroups as well as by hydrophobic tails in the Stern region where 1a-f and 2 bind to the micelle. Individual contributions of these interactions are quantified. Rate-retarding effects found for different probes, with different sensitivities for interactions as they occur when the probe binds to the micellar Stern region, as well as the micellar Stern region's micropolarity as reported by the E(T)(30) probe, are satisfactorily reproduced by new model solutions containing both TMAB and 1-propanol.     

Analysis of sulfobutyl ether-beta-cyclodextrin mixtures by ion-spray mass spectrometry and liquid chromatography-ion-spray mass spectrometry

A comparison is made of the retention properties of additives applied as positively charged pseudo-stationary phases for electrokinetic chromatography of neutral analytes. All additives have a quaternary ammonium as functional group. The polymeric additive [poly(N,N,N',N'-tetramethyl-N-trimethylenehexamethylenediammonium), Polybrene] has a concentration of 2% (w/w) in the background electrolyte (acetate, pH 5.2). Monomeric octyltrimethylammonium (OTMA) was used at a concentration below or above its critical micelle concentration (CMC) (140 mmol/l). At a concentration (259 mmol/l) above the CMC the system is that normally used for micellar electrokinetic chromatography with cationic micelles. However, even below the CMC, where OTMA is present as monomer, retention of the neutral analytes is observed as well. In all systems coating of the capillary wall with Polybrene establishes an electroosmotic flow directed towards the anode, counter-migrating to the electrophoretic movement of the additive. Based on the measurement of the mobility of the analytes (15 small, monofunctional aromatic compounds with different functional groups), their capacity factors, k(i), were determined in all systems. Low correlation of the k(i) values is observed between the particular systems, indicating their different selectivity at least for individual pairs of analytes. Based on the log k(i) values, a linear free energy relationship was applied to elucidate the main types of chemical interaction responsible for retention. As a result, cavity formation and n or pi electron interactions were found being significant for the micellar OTMA system, which agrees with findings described in the literature for other (cationic and anionic) micellar systems. For the polymeric system and for the monomeric OTMA system, the significant retention parameter is indicating n and pi electron interactions.     

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。     

Control of separation selectivity in micellar electrokinetic chromatography by modification of the micellar phase with solubilized organic compounds

On the basis of the data on the distribution of various neutral solutes between sodium dodecyl sulfate (SDS) micelles and water, the control of separation selectivity in micellar electrokinetic chromatography (MEKC) by modification of the micellar phase with organic additives has been proposed and applied to the separation of simple model compounds. It was found that the distribution constants between the micelles and water (K_d,mc), which were determined by means of MEKC, of the solutes possessing hydrophilic functional groups are much larger than those between heptane and water (K_d,hep), whereas the K_d,mc values of the solutes possessing no hydropholic groups are comparable to their K_d,hep values. This indicates that the former solutes are preferentially solubilized in the Stern layer of the micelles and that the latter are located in the hydrocarbon core. In MEKC separations of aromatic compounds and metal acetylacetonates, considerable changes in separation selectivity were caused by the addition of compounds possessing both hydrophilic functional groups such as alcohols, phenol and ketones to the SDS micellar solution. The variations of the retention factors of the analytes could be explained in terms of saturation of the solubilization sites in the Stern layer with the modifiers, specific interaction of the modifiers with the analytes via hydrogen bonding in the micelles, and expansion of the core volume with the hydrocarbon parts of the modifiers. Such effects of the micellar modification could improve the resolution as well as the selectivity of MEKC separations.     

Self-assembly of mixed anionic and nonionic surfactants in aqueous solution

We present the phase diagram and the microstructure of the binary surfactant mixture of AOT and C(12)E(4) in D(2)O as characterized by surface tension and small angle neutron scattering. The micellar region is considerably extended in composition and concentration compared to that observed for the pure surfactant systems, and two types of aggregates are formed. Spherical micelles are present for AOT-rich composition, whereas cylindrical micelles with a mean length between 80 and 300 ? are present in the nonionic-rich region. The size of the micelles depends on both concentration and molar ratio of the surfactant mixtures. At higher concentration, a swollen lamellar phase is formed, where electrostatic repulsions dominate over the Helfrich interaction in the mixed bilayers. At intermediate concentrations, a mixed micellar/lamellar phase exists.     

Electric field gradient focusing

Electric field gradient focusing (EFGF) is a relatively new separation technique with promising attributes, particularly for protein analysis. The fundamental fractionation mechanism in EFGF involves a gradient in electric field along the length of a separation column. The electrophoretic force that drives charged analytes in a region of changing electric field is opposed by a constant, pressure-driven bulk fluid flow. When the electrophoretic velocity of a particular moiety is equal and opposite to the velocity of the fluid flow, the analyte focuses into a stationary band. Thus, EFGF can both concentrate and separate charged species according to electrophoretic mobility. To date, the electric field gradients needed for EFGF have been established using a number of different approaches, including channels having changing cross-sectional areas, conductivity gradients caused by the diffusion of buffer ions across a membrane, electrode arrays, and temperature gradients in buffers whose conductivities change as a function of temperature. EFGF has proven particularly effective for sample enrichment, with concentration factors of 10,000 reported. In this article we review advances in EFGF technology and discuss prospects for further improving EFGF for chemical analysis.     

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.     

Formamide as an organic modifier in MEKC with SDS

The effect of formamide (FA) as a modifier on the retention in MEKC with SDS as the detergent was investigated. The mobility of a series of alkylphenones and of a zwitterionic fluorescent compound as a function of the FA and the SDS concentration was determined for this purpose. Buffering electrolyte was borate, pH 9.23, with total ionic strength of 50 mM. The dependence of the mobility on the FA content – up to 63% w/w – of the BGE (at 10 mM SDS) allows the conclusion that the micelles are destabilized, and the CMC is shifted to higher values. In the system containing 33% FA or more no micelles are present anymore, and the retention factors of all compounds tend to zero. In an MEKC system with 27% v/v FA the CMC of SDS is increased from 2.4 mM in the aqueous BGE with the same buffer composition to 9.7 mM, a behavior that is in contrast to electrolyte‐free FA–water systems. The partition constants of free analytes and the formation constants of the adduct between analyte and detergent monomer (assuming 1:1 stoichiometry) were derived from the dependence of the mobility on the SDS concentration. In addition, the involved equilibria were extended by that from the distribution of the analyte–monomer adduct between aqueous and micellar phase, and the according partition constants were derived as well. A selective change in the extent of partitioning was observed for the zwitterionic compound. In general, all binding constants were decreased upon addition of FA, though to a different extent. Although the binding constants of the analyte–monomer associate were only slightly influenced, the most pronounced decrease is found for their partitioning into the micelles.     

Fluorescence from pyrene solubilized in aqueous micelles. A model for quenching by inorganic ions

When pyrene is solubilized within cetyl trimethyl ammonium bromide micelles the lifetime of its fluorescent state is very dependent upon the concentration of iodide ions within the surrounding aqueous phase. Quantitative examination of the kinetic data throws doubt on the existing model for quenching which requires pyrene to diffuse from within the hydrocarbon core to the peripheral region where encounter and quenching can occur. An alternative model is proposed which argues that the water-lipid interface penetrates deep into the micellar body close to the included pyrene such that quencher ions can encounter pyrene without the latter having to move appreciably. The observed effects of ionic strength add support to the new model.     

Shape and Size of Highly Concentrated Micelles in CTAB/NaSal Solutions by Small Angle Neutron Scattering (SANS)

Highly concentrated micelles in CTAB/NaSal solutions with a fixed salt/surfactant ratio of 0.6 have been studied using Small Angle Neutron Scattering (SANS) as a function of temperature and concentration. A worm-like chain model analysis of the SANS data using a combination of a cylindrical form factors for the polydisperse micellar length, circular cross-sectional radius with Gaussian polydispersity, and the structure factor based on a random phase approximation (RPA) suggests that these micelle solutions have a worm-like micellar structure that is independent of the concentration and temperature. The size of the micelle decreases monotonically with increasing temperature and increases with concentration. These observations indicate that large micelles are formed at low temperature and begin to break up to form smaller micelles with increasing temperature.