Micellar affinity gradient focusing: a new method for electrokinetic focusing

This report describes a new method for the concentration and separation of neutral and/or hydrophobic analytes based on a combination of the analytes' electrophoretic mobility, and affinity for partitioning into a micellar phase. Micellar affinity gradient focusing (MAGF) works by creating a gradient in the micellar retention factor. An electric field is applied along the channel to cause the (negatively charged) micelles to move from the region of high retention to the region of low retention, and the mobile phase is forced to move from the region of low retention to the region of high retention. Consequently, the analyte moves into the gradient region from both directions where it is concentrated at a point where its total velocity is zero. Different analytes, which interact differently with the micelles, will have zero total velocity at different points along the gradient, and will thereby be simultaneously concentrated and separated.     

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.     

Bilinear electric field gradient focusing

Electric field gradient focusing (EFGF) uses an electric field gradient and a hydrodynamic counter flow to simultaneously separate and focus charged analytes in a channel. Previously, most EFGF devices were designed to form a linear field gradient in the channel. However, the peak capacity obtained using a linear gradient is not much better than what can be obtained using conventional CE. Dynamic improvement of peak capacity in EFGF can be achieved by using a nonlinear gradient. Numerical simulation results indicate that the peak capacity in a 4-cm long channel can be increased from 20 to 150 when changing from a linear to convex bilinear gradient. To demonstrate the increased capacity experimentally, an EFGF device with convex bilinear gradient was fabricated from poly(ethylene glycol) (PEG)-functionalized acrylic copolymers. The desired gradient profile was confirmed by measuring the focusing positions of a standard protein for different counter flow rates at constant voltage. Dynamically controlled elution of analytes was demonstrated using a monolith-filled bilinear EFGF channel. By increasing the flow rate, stacked proteins that were ordered but not resolved after focusing in the steep gradient segment were moved into the shallow gradient segment, where the analyte peak resolution increased significantly. In this way, the nonlinear field gradient was used to realize a dynamic increase in the peak capacity of the EFGF method.     

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.     

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.     

Rapid concentration of deoxyribonucleic acid via Joule heating induced temperature gradient focusing in poly-dimethylsiloxane microfluidic channel

This paper reports rapid microfluidic electrokinetic concentration of deoxyribonucleic acid (DNA) with the Joule heating induced temperature gradient focusing (TGF) by using our proposed combined AC and DC electric field technique. A peak of 480-fold concentration enhancement of DNA sample is achieved within 40 s in a simple poly-dimethylsiloxane (PDMS) microfluidic channel of a sudden expansion in cross-section. Compared to a sole DC field, the introduction of an AC field can reduce DC field induced back-pressure and produce sufficient Joule heating effects, resulting in higher concentration enhancement. Within such microfluidic channel structure, negative charged DNA analytes can be concentrated at a location where the DNA electrophoretic motion is balanced with the bulk flow driven by DC electroosmosis under an appropriate temperature gradient field. A numerical model accounting for a combined AC and DC field and back-pressure driven flow effects is developed to describe the complex Joule heating induced TGF processes. The experimental observation of DNA concentration phenomena can be explained by the numerical model.     

Analytical equilibrium gradient methods

Analytical equilibrium gradient methods are non-linear separation methods in which the separation mechanism involves a force gradient along the separation channel. These methods can be classified into two categories: those in which the gradient is a field gradient applied along the separation channel (i.e., field gradient), and those in which the channel is subjected to a constant field with a gradient formed in some other property (i.e., constant field). Standard deviation of peak width, resolution and peak capacity are important parameters in characterizing equilibrium gradient methods, and general expressions can be obtained from considering both the point of force acting on the analyte and the basic flux equation. Several successful examples, such as density gradient sedimentation, isoelectric focusing and electromobility focusing are discussed. Based on equilibrium gradient methods in the field gradient category, a method to dynamically improve peak capacity is described. An example of such an approach is given using electromobility focusing.     

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。     

Dynamic analyte introduction and focusing in plastic microfluidic devices for proteomic analysis

Isoelectric focusing (IEF) separations, in general, involve the use of the entire channel filled with a solution mixture containing protein/peptide analytes and carrier ampholytes for the creation of a pH gradient. Thus, the preparative capabilities of IEF are inherently greater than most microfluidics-based electrokinetic separation techniques. To further increase sample loading and therefore the concentrations of focused analytes, a dynamic approach, which is based on electrokinetic injection of proteins/peptides from solution reservoirs, is demonstrated in this study. The proteins/peptides continuously migrate into the plastic microchannel and encounter a pH gradient established by carrier ampholytes originally present in the channel for focusing and separation. Dynamic sample introduction and analyte focusing in plastic microfluidic devices can be directly controlled by various electrokinetic conditions, including the injection time and the applied electric field strength. Differences in the sample loading are contributed by electrokinetic injection bias and are affected by the individual analyte's electrophoretic mobility. Under the influence of 30 min electrokinetic injection at constant electric field strength of 500 V/cm, the sample loading is enhanced by approximately 10-100 fold in comparison with conventional IEF.     

Adjusting selectivity in micellar electrokinetic capillary chromatography with 1,2-hexanediol

In this report, we introduce a new micelle modifier useful to alter selectivity in micellar electrokinetic capillary chromatography (MECC). 1,2-Hexanediol acts as a class I organic modifier in that its effects are on the sodium dodecyl sulfate (SDS) micellar rather than the surrounding aqueous phase. This characteristic allows 1,2-hexanediol to improve resolution when applied at concentrations as low as 20 mM (0.25% v/v) by altering the selectivity observed with SDS alone. The effects of 1,2-hexanediol on the critical micelle concentration of SDS, electroosmotic flow, electrophoretic mobility of the SDS micelle, and reproducibility are presented. 1,2-Hexanediol had little impact on the migration time window at concentrations below 100 mM. Changes in selectivity induced by 1,2-hexanediol for a large set of model compounds are presented. Analytes capable of forming hydrogen bonds tend to decrease their interactions with the micellar phase while nonhydrogen bonding analytes increase their interactions. The usefulness of 1,2-hexanediol was demonstrated by examining its effects on the separation of dansylated amino acids. Eighteen of twenty amino acids could be separated with a resolution greater than 1.6 within 1600 s using a combination of 1,2-hexanediol and isopropanol.     

Characterizing the impact of thermal gels on isotachophoresis in microfluidic devices

Thermally reversible Pluronic gels have been employed as separation matrices in microfluidic devices in the analysis of biological macromolecules. The phase of these gels can be tuned between liquid and solid states using temperature to vary fluidic resistance and alter peak resolution. Although separations in thermal gels have been characterized, their effect on isotachophoresis has not. This study used fluorescein as a model analyte to evaluate isotachophoretic preconcentration as a function of thermal polymer concentration and temperature. Results demonstrated that increasing polymer concentration in microfluidic channels increased the apparent analyte concentration. A critical minimum of 10% (w/v) Pluronic was required to achieve efficient preconcentration with maximum focusing occurring in 20 and 25% polymer gels. Temperature of the thermal gel also impacted analyte focusing. Most efficient focusing was achieved at 25°C with diminishing analyte accumulation at higher and lower temperatures. Under optimal conditions, isotachophoretic preconcentration increased an additional threefold simply by including thermal gels in the system. This approach can be readily implemented in other applications to increase detection sensitivity and measure low-concentration analytes within simple microfluidic devices.     

Separation and selectivity in micellar electrokinetic chromatography using sodium dodecyl sulfate micelles or Tween 20-modified mixed micelles

The separation and selectivity of eight aromatic compounds ranging from hydrophilic to hydrophobic properties in micellar electrokinetic chromatography (MEKC) using sodium dodecyl sulfate (SDS) micelles or Tween 20-modified mixed micelles were investigated. The effect of different operation conditions such as SDS and Tween 20 modifier surfactant concentration, buffer pH, and applied voltage was studied. The resolution and selectivity of analytes could be markedly affected by changing the SDS micelle concentration or Tween 20 content in the mixed micelles. Applied voltage and pH of running buffers were used mainly to shorten the separation time. Complete separation of eight analytes could be achieved with an appropriate choice of the concentration of SDS micelles or Tween 20-modified mixed micelles. Quicker elution and better precision could be obtained with SDS-Tween 20 mixed micelles than with SDS micelles. The mechanisms that migration order of those analytes was mainly based on their structures and solute-micelle interactions, including hydrophobic, electrostatic, and hydrogen bonding interactions, were discussed.     

正离子型疏水改性聚氧乙烯单成相组分双水相系统的相行为

对正离子型疏水改性聚氧乙烯(HM-EO)单成相组分双水相系统的相行为进行了考察,并分析其电荷特性.HM-EO在水溶液中呈现两亲性,可以形成胶束,进而形成带电的胶束簇集体.通过改变溶液的pH值、盐浓度及添加带相反电荷的表面活性剂SDS,可改变胶束簇集体的带电状态,从而影响系统的相行为.增大pH值,有利于系统的分相.盐的添加也可以增大双水相两相区域,正离子影响次序为K+>Na+,负离子次序为SO42->F->Cl->Br->I-.进一步考察了HM-EO和SDS之间的相互作用,结果表明SDS能与HM-EO形成混合胶束簇集体,改变HM-EO双水相系统的带电特性.     

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.     

Flow-induced thermal effects on spatial DNA melting

Continuous-flow temperature gradient microfluidics can be used to perform spatial DNA melting analysis. To accurately characterize the melting behavior of PCR amplicon across a spatial temperature gradient, the temperature distribution along the microfluidic channel must be both stable and known. Although temperature change created by micro-flows is often neglected, flow-induced effects can cause significant local variations in the temperature profile within the fluid and the closely surrounding substrate. In this study, microfluidic flow within a substrate with a quasi-linear temperature gradient has been examined experimentally and numerically. Serpentine geometries consisting of 10 mm long channel sections joined with 90 degrees and/or 180 degrees bends were studied. Infrared thermometry was used to characterize the surface temperature variations and a 3-D conjugate heat transfer model was used to predict interior temperatures for multiple device configurations. The thermal interaction between adjacent counter-flow channel sections, which is related to their spacing and substrate material properties, contributes significantly to the temperature profile within the microchannel and substrate. The volumetric flow rate and axial temperature gradient are directly proportional to the thermal variations within the device, while these flow-induced effects are largely independent of the cross-sectional area of the microchannel. The quantitative results and qualitative trends that are presented in this study are applicable to temperature gradient heating systems as well as other microfluidic thermal systems.     

Micellar and aqueous‐organic liquid chromatography using sub‐2 μm packings for fast separation of natural phenolic compounds

The objective of the present work was to investigate the chromatographic behavior of natural phenolic compounds in micellar and aqueous‐organic LC using a short column packed with 1.8 μm particles. Firstly, the effect of ACN and SDS on elution strength and selectivity was examined by isocratic submicellar (0–30% ACN/5% 1‐butanol/1–6 mM SDS) and micellar (0–30% ACN/5% 1‐butanol/40–60 mM SDS) systems. The varied concentrations of two modifiers in the mobile phases revealed different eluting power. Then, the application of organic modifier gradient was discussed in both submicellar and micellar LC using mobile phases of 4 mM SDS/5% 1‐butanol or 50 mM SDS/5% 1‐butanol containing ACN gradient from 0 to 30%, respectively. For micellar system, the separation was found to be better in gradient than isocratic elution. Additionally, the sensitivity of aqueous‐organic LC was examined. The mobile phase was a mixture of ACN and water employing gradient elution at a flow rate of 0.5 mL/min, with analysis time below 9 min. It was found that separation efficiency was significantly better compared with micellar LC. Besides, the aqueous‐organic LC has been applied to separation of various phenolic compounds in Yangwei granule or Radix Astragali samples.     

DNA mutation detection with chip-based temperature gradient capillary electrophoresis using a slantwise radiative heating system

A simple and robust chip-based temperature gradient capillary electrophoresis (TGCE) system was developed for DNA mutation/single-nucleotide polymorphism (SNP) analysis using a radiative heating system. Reproducible, stable and uniform temperature gradients were established along a 3 cm length of the electrophoretic separation channel using a single thermostated aluminium heater plate. The heater was slightly slanted relative to the plane of the glass chip at 0.2-1.3 degrees by inserting thin spacers between the plate and chip at one end to produce differences in radiative heating that created the temperature gradient. On-chip TGCE analyses of 4 mutant DNA model samples amplified from plasmid templates, each containing a single base substitution, with a wide range of melting temperatures, showed that mutations were successfully detected under a wide temperature gradient of 10 degrees C and within a short gradient region of about 3 cm (3.3 degrees C cm(-1) gradient). The radiative heating system was able to establish stable spatial temperature gradients along short microfluidic separation channels using simple peripheral equipment and manipulation while ensuring good resolution for detecting a wide range of mutations. Effectiveness of the system was demonstrated by the successful detection of K-ras gene mutations in 6 colon cancer cell lines.     

Phase-transfer catalytic determination of phenols as methylated derivatives by gas chromatography with flame ionization and mass-selective detection

A microemulsion electrokinetic chromatographic (MEEKC) method was developed for the separation of six catechins, specific marker phytochemicals of Cistus species. The MEEKC method involved the use of sodium dodecyl sulfate (SDS) as surfactant, heptane as organic solvent and butan-1-ol as co-solvent. In order to have a better stability of the studied catechins, the separation was performed under acidic conditions (pH 2.5 phosphate buffer). The effects of SDS concentration and of the amount of organic solvent and co-solvent on the analyte resolution were evaluated. The optimized conditions (heptane 1.36% (w/v), SDS 2.31% (w/v), butan-1-ol 9.72% (w/v) and 50 mM sodium phosphate buffer (pH 2.5) 86.61% (w/v)) allowed a useful and reproducible separation of the studied analytes to be achieved. These conditions provided a different separation profile compared to that obtained under conventional micellar electrokinetic chromatography (MECK) using SDS. The method was validated and applied to the determination of catechin and gallocatechin in lyophilized extracts of Cistus incanus and Cistus monspeliensis.     

Separation of alkamides from Echinacea purpurea extracts by cyclodextrin-modified micellar electrokinetic chromatography

Separation of nine important alkyl methylbutyl- and isobutylamides (known as alkamides) obtained from Echinacea purpurea extracts was investigated by using cyclodextrin-modified micellar electrokinetic chromatography (CD-MEKC). Hydrophobic alkamides interact strongly with the micelles from the most common surfactants used in MEKC and this lead to predominant partition of the analytes into the micellar phase, resulting in poor resolution. The addition of neutral CDs to the alkaline (10 mM phosphate buffer pH 8.0) micellar system of sodium dodecyl sulfate (SDS), sodium cholate (SC) and sodium deoxycholate (SDC) was found to improve the separation of the studied alkamides. Among the several combinations surfactant/CD, three different systems showed to be particularly effective: SDS/hydroxypropyl-beta-CD (110 mM/100 mM) and SC/heptakis (2, 3, 6-tri-O-methyl)-beta-CD (200 mM/40 mM) which provided a complete separation of the studied compounds, and SDC/heptakis (2, 6-di-O-methyl)-beta-CD. The importance of appropriate surfactant vs. CD concentration ratio as well as that of total concentration of both surfactant and CD was considered. The optimization of the separation was performed by focussing the need for a rapid separation of nine alkamides diagnostically useful to define the fingerprint of Echinacea species.