Free Zone Electrophoresis Research Articles

Free flow electrophoresis allows preparation of extracellular vesicles with high purity

Background & Aim Currently, it remains a challenge to prepare extracellular vesicles (EVs) to high purity. Neither fractionation by density nor by size alone is sufficient to separate EVs from all contaminants e.g. high and low density lipoprotein (HDL/LDL) and adherent proteins. For now, a time consuming combination of two methods (density and size separation) is required to enrich EVs to high purity at the expense of time and low recovery. A fast and efficient alternative is represented by Free Flow Electrophoresis (FFE). It is a well-established (semi-) preparative method to separate analytes with inherent difference of charge density and/or difference of pI-value. Methods, Results & Conclusion A Free Flow Interval Zone Electrophoresis (FF-IZE) method has been developed, for the purification and isolation of EVs as well DNA and RNA from human plasma samples, using a set of buffer media of different pH-values ranging from pH 8.0 to pH 4.8. Upon processing supernatants of mesenchymal stem/stromal cells (MSCs) cultured in the presence of 10% human platelet lysate, EVs are recovered in a small number of FFE fractions lacking most proteins of the conditioned media. Currently, we characterize the identified fractions in more detail. Prepared EVs are quantified by Nano Particle Tracking Analysis (NTA) and imaging flow cytometry. Furthermore, the presence of several EV markers and the absence of contaminants are analyzed by Western Blot and mass spectrometry-based proteomics. We conclude FFE is a feasible and quick method to highly purify EVs in an accurate manner. Currently, it remains a challenge to prepare extracellular vesicles (EVs) to high purity. Neither fractionation by density nor by size alone is sufficient to separate EVs from all contaminants e.g. high and low density lipoprotein (HDL/LDL) and adherent proteins. For now, a time consuming combination of two methods (density and size separation) is required to enrich EVs to high purity at the expense of time and low recovery. A fast and efficient alternative is represented by Free Flow Electrophoresis (FFE). It is a well-established (semi-) preparative method to separate analytes with inherent difference of charge density and/or difference of pI-value. A Free Flow Interval Zone Electrophoresis (FF-IZE) method has been developed, for the purification and isolation of EVs as well DNA and RNA from human plasma samples, using a set of buffer media of different pH-values ranging from pH 8.0 to pH 4.8. Upon processing supernatants of mesenchymal stem/stromal cells (MSCs) cultured in the presence of 10% human platelet lysate, EVs are recovered in a small number of FFE fractions lacking most proteins of the conditioned media. Currently, we characterize the identified fractions in more detail. Prepared EVs are quantified by Nano Particle Tracking Analysis (NTA) and imaging flow cytometry. Furthermore, the presence of several EV markers and the absence of contaminants are analyzed by Western Blot and mass spectrometry-based proteomics. We conclude FFE is a feasible and quick method to highly purify EVs in an accurate manner.

Microcapillary electrophoresis chip with a bypass channel for autonomous compensation of hydrostatic pressure flow

The application of chip-based microcapillary electrophoresis (µCE) to determine the electrophoretic mobility of molecules and particles has been intensively studied in the last two decades. Balancing the hydrostatic pressure between both ends of the microchannel is essential for free-zone electrophoresis and highly accurate measurement. This balancing operation appears simple on a macroscale (e.g., > 10−3 m); however, on a microscale (e.g., 10−6–10−3 m), it is not straightforward because of the complexity of the interface dynamics at the meniscus. The hydrostatic pressure flow is unstable because of the small size of the microchannel, which is smaller than a single droplet of water. In this study, a µCE chip design was proposed by adding an extra bypass channel to balance the fluid level of the two open reservoirs and inhibit the generation of hydrostatic pressure flow within the microchannel. The fluid behaviors in the microchannel and current and voltage (I–V) characterization were experimentally studied. In addition, a numerical simulation of the electroosmotic flow and hydrostatic flow in the µCE chip was performed. The comparison between the µCE chip with and without the bypass channel showed that the bypass channel did not produce a disturbance in the microchannel for the electrophoretic measurement. The simple microchannel design enabled autonomous compensation of the hydrostatic pressure from the instability of the meniscus, and thus improved the usability of the chip-based µCE chip and the accuracy in the electrophoretic measurement.

On-chip single column transient isotachophoresis with free zone electrophoresis for preconcentration and separation of α-lactalbumin and β-lactoglobulin

Isotachophoresis (ITP) coupled to zone electrophoresis (ZE), either free zone electrophoresis (FZE) or gel electrophoresis (GE), carried out mainly in capillaries and, although less frequently, also in microchips, is a powerful preconcentration and separation technique which has been successfully used for the study of many low molecular-weight analytes. However, this analytical technique has been scarcely applied for proteins separation. In this work, an on-chip transient ITP coupled to free zone electrophoresis (t-ITP-MFZE) mode with LIF detection is developed for the preconcentration and separation of the proteins α-lactalbumin and β-lactoglobulin.Firstly, for LIF detection, the proteins were off-chip fluorescently labeled with the fluorogenic reagent Chromeo P503. Then, several separation parameters in t-ITP-MFZE mode such as leading electrolyte, terminating electrolyte, separation voltage, and injection time were optimized to achieve the maximum sensitivity while maintaining an adequate resolution between α-lactalbumin and β-lactoglobulin in a single column configuration t-ITP. Using the optimized electrolytes (50mM imidazole/HCl pH=8 as leading electrolyte and 100mM imidazole/12mM HEPES pH=8 as terminating electrolyte) separation of both proteins was achieved in less than 4min with peak resolution of 1.5. The LODs were 55nM and 380nM, for α-lactalbumin and β-lactoglobulin, respectively, which are adequate for some food allergenicity studies.Finally, comparison of the optimized t-ITP-MFZE method to the equivalent MFZE method, carried out also in microchips but without the isotachophoretic preconcentration step, provided preconcentration indexes for both proteins around 10.

Physico-chemical characterization of polymeric micelles loaded with platinum derivatives by capillary electrophoresis and related methods

(1,2-diamino-cyclohexane)Platinum(II) ((DACH)Pt) loaded polymeric micelles of poly(ethylene glycol-b-sodium glutamate) (PEG-b-PGlu) are currently studied as a potential candidate to replace oxaliplatin in the treatment of cancers with the aim to reduce side effects like cumulative peripheral distal neurotoxicity and acute dysesthesias. As for all synthetic polymeric drug delivery systems, the characterization of the (co)polymer precursors and of the final drug delivery system (polymeric micelles) is crucial to control the repeatability of the different batches and to get correlation between physico-chemical structure and biological activity. In this work, the use of capillary electrophoresis (CE) and related methods for the characterization of (DACH)Pt-loaded polymeric micelles and their precursor (PEG-b-PGlu copolymer) has been investigated in detail. The separation and quantification of residual PGlu homopolymer in the PEG-b-PGlu sample were performed by free solution capillary zone electrophoresis mode. This mode brought also information on the PEG-b-PGlu copolymer composition and polydispersity. It also permitted to monitor the decomposition of polymeric micelles in the presence of NaCl at room temperature. Interactions between PEG-b-PGlu unimers, on one hand, and polymeric micelles or surfactants, on the other hand, were studied by using the Micellar Electrokinetic Chromatography and Frontal Analysis Capillary Electrophoresis modes. Finally, weight-average hydrodynamic radii of the loaded polymeric micelles and of the PEG-b-PGlu unimers were determined by Taylor Dispersion Analysis (an absolute size determination method that can be easily implemented on CE apparatus).

Microscale separation methods for enzyme kinetics assays

Miniaturization continues to be one of the leading trends in analytical chemistry and one that brings advantages that can be particularly beneficial in biochemical research. Use of a miniaturized scale enables efficient analysis in a short time and requires very small amounts of samples, solvents, and reagents. This can result in a remarkable decrease in costs of enzyme kinetics studies, especially when expensive or rare enzymes and/or substrates are involved. Free zone electrophoresis is without a doubt the most common microscale separation technique for capillary and on-chip enzyme assays. Progress and applications in this field are reviewed frequently whereas other modes of separation, although successfully applied, receive only marginal interest in such publications. This review summarizes applications of less common modes of separation in capillary or chip formats, namely micellar electrokinetic chromatography, liquid chromatography, gel electrophoresis, isoelectric focusing, and isotachophoresis. Because these techniques are based on separation mechanisms different from those of free zone electrophoresis, they can be, and have been, successfully used in cases where zone electrophoresis fails. Advantages and drawbacks of these alternative separation techniques are discussed, as also are the difficulties encountered most often and solutions proposed by different research groups.

General Approach for Certain Quantitative Calculations for Instance of the Variance of Reversible Adsorption to the Capillary Wall in CE

Miniaturization of analytical separation methods offers several advantages, including short run times, high resolution, and high recovery of the sample constituents. To optimize these parameters, the reversible adsorption (to minimize loss in resolution), as well as the irreversible adsorption (to minimize loss of analytes) must be quantified.However, no useful equation is available for the calculation of the variance of reversible adsorption. Therefore,we have taken another approach to quantify the reversible interaction. The method is unique and important since no equation for calculation of this variance is required. Instead, two experiments are required, which are run under such conditions that the variance of a certain parameter has the same numerical value in the two experiments (one with and without EOF), except for the variance of reversible adsorption. The approach is universal in the sense that it can be used for many different mathematical concepts and be modified to also cover certain functions other than a sum of parameters.We have also introduced a simple expression for their reversible adsorption, which shows that the hydrophobic interaction from only two methyl groups in the coating gives rise to as much as 40-50% loss of protein, and the width of the zones in the capillary with this coating was 8-15% larger compared to the zone width in the polyacrylamide-coated capillaries. The reproducibility in migration time, peak area, and peak width in two consecutive runs in capillaries with two methyl groups in the coating was very low, but in EOF-free polyacrylamide-coated capillaries extremely high, indicating that the reversible and irreversible adsorption of proteins to this coating is negligible. The scanning detector, frequently used in free zone electrophoresis in the 1960s-1970s, gives true separation parameters and is, therefore, much preferable to the stationary detector used in most CE experiments, because this detector gives apparent separation parameters.

Oats protein isolate: Thermal, rheological, surface and functional properties

Abstract Oat protein isolate (OPI) was extracted in 0.015 N NaOH and acetylated or succinylated. The thermal analysis of the isolate showed a glass transition ( T g ) at 43.4 °C and Δ C p of 0.102 J/g/°C. The positive net charge of OPI and the positive or neutral charge of the modified OPI were apparent from the free capillary zone electrophoresis (FZCE) profiles. Acetylation significantly lowered foaming and emulsifying properties of OPI, while succinylation showed the highest foaming capacity, foam stability, and emulsion stability. Acetylated OPI showed the highest surface hydrophobicity compared to the other samples, while OPI was the most soluble of all. The water holding capacity of all samples analyzed was the same except for acetylated-crosslinked (ACXL). The surface tension test confirmed that unmodified and modified OPI possessed surface activity and the equilibrium surface tensions decreased sharply with increasing protein concentration and leveled off to a constant value. The elastic modulus, G ′, for the acetylated OPI suspension exhibited the highest value, while the G ′ of the crosslinked (XLOPI) had the lowest. The plateau of G ′, was 2961 Pa, 920 Pa, 223 Pa, 41 Pa, and 1.8 Pa for the ACOPI, ACXL, SOPI, and XL, respectively.

Cover Picture: Electrophoresis 8/2008

AbstractRegular issues provide a wide range of research and review articles covering all aspects of electrophoresis. Here you will find cutting‐edge articles on methods and theory, instrumentation, nucleic acids, CE and CEC, miniaturization and microfluidics, proteomics and two‐dimensional electrophoresis.On April 2 Professor Hjertén celebrated his 80th birthday, and it is an honor to take this opportunity to congratulate him on this special occasion and at the same time on his fruitful work. Stellan Hjertén's distinguished personality in research and life makes this celebration very special. It is therefore appropriate to devote a separate laudation in ELECTROPHORESIS to his achievements through which he has attained renown within the separation science community: indeed, he is considered undoubtedly to be the “Father of Capillary Electrophoresis”. Professor Hjertén's preliminary work with Arne Tiselius motivated him to commit his career to electrophoresis: the development of free zone electrophoresis certainly revolutionized separation science, and since the construction of the first “capillary electrophoresis” equipment, one of the most cited works in this field carries his name. His friends were very keen to contribute manuscripts to this Issue, covering almost all areas in which Professor Hjertén has worked in his distinguished career.

Universal method for synthesis of artificial gel antibodies by the imprinting approach combined with a unique electrophoresis technique for detection of minute structural differences of proteins, viruses and cells (bacteria). Ib. Gel antibodies against proteins (hemoglobins)

Using the molecular imprinting approach, we have shown that polyacrylamide-based artificial antibodies against human and bovine hemoglobin have a very high selectivity, as revealed by the free-zone electrophoresis in a revolving capillary. By the same technique we have previously synthesized gel antibodies not only against proteins but also against viruses and bacteria. The synthesis is thus universal, i.e., it has the great advantage of not requiring a modification - or only a slight one - for each particular antigen. The combination synthesis of artificial gel antibodies and electrophoretic analysis reveals small discrepancies in shape and chemical composition not only of proteins, as shown here and in paper Ia, but also of viruses and bacteria, to be illustrated in papers II and III in this series. Upon rehydration, the freeze-dried gel antibodies, selective for human hemoglobin, regain their selectivity. The gel antibodies can repeatedly be used following the removal of the antigen (protein in this study) from the complex gel antibody/antigen by an SDS washing or an enzymatic degradation.

Universal method for synthesis of artificial gel antibodies by the imprinting approach combined with a unique electrophoresis technique for detection of minute structural differences of proteins, viruses, and cells (bacteria): II. Gel antibodies against virus (Semliki Forest Virus)

Artificial and highly selective antibodies (in the form of gel granules) against proteins can easily be synthesized by a simple, cost-effective imprinting technique [Liao, J.-L. et al., Chromatographia 1996, 42, 259-262]. Using the same method for synthesis of gel antibodies against viruses in combination with analysis by free zone electrophoresis in a rotating narrow bore tube we have shown that artificial gel antibodies against Semliki Forest Virus (wild type) can sense the difference between this virus and a mutant, although they differ in their chemical composition only by three amino acids in one of the three proteins on the surface of the virus particle. The reason for this extremely high resolution is explained by the fact that we use three types of selectivity: (i) shape selectivity (created by the close fit between the antigen and its imprint in the gel), (ii) bond selectivity in the contact area between the antigen and its imprint in the gel antibody, and (iii) charge selectivity, originating from slightly different structures or/and conformations of the antigens.

Universal method for synthesis of artificial gel antibodies by the imprinting approach combined with a unique electrophoresis technique for detection of minute structural differences of proteins, viruses, and cells (bacteria): Ia. Gel antibodies against proteins (transferrins)

Artificial antibodies in the form of gel granules were prepared by the molecular imprinting technique from the monomers acrylamide and N,N'-methylene-bis-acrylamide. Gel granules, freed from the selectively adsorbed protein (the antigen), are neutral and, accordingly, do not migrate in an electrical field. However, upon selective interaction with the antigen at a pH different from its pI, the granules become charged. The selectivity of the gel antibodies was studied by free zone electrophoresis in a tube with inside diameter larger than the size of the granules. Such electrophoretic analyses showed that gel antibodies against iron-free transferrin had a high selectivity for this protein, although some crossreaction took place with iron-saturated transferrin, indicating that these artificial antibodies can easily distinguish the minute differences in the 3-D structure of the transferrins. Analogously, gel antibodies against iron-saturated transferrin were highly selective for this protein with some crossreaction with iron-free transferrin. The mobilities of iron-free and iron-saturated transferrin are very similar, and, therefore, capillary free zone electrophoresis cannot distinguish between these structurally related proteins. However, significant differences in the mobilities of the selective gel granules can be observed depending on their interaction with iron-free or iron-saturated transferrin, i.e., the artificial gel antibodies may become powerful analytical tools.

Probing affinity via capillary electrophoresis: advances in 2003–2004

This review addresses recent advances in capillary electrophoresis of biological-based molecular interaction from a broader perspective, based on applications reported during the period 2003-2004. These capillary electrophoresis-based studies of molecular interactions include affinity capillary electrophoresis, electrokinetic chromatography, and free zone electrophoresis. The review is written as a general synopsis of applications and does not cover the theory or protocol involved in the implementation of the analyses.

Determination of meningococcal polysaccharides by capillary zone electrophoresis

Meningococcal polysaccharides are medically important molecules and are the active components of vaccines against Neisseria meningiditis serogroups A, C, W135, and Y. This study demonstrates that free solution capillary zone electrophoresis (CZE) using simple phosphate/borate separation buffers is capable of separating intact, native polysaccharides from these four serogroups. Separation appeared to be robust with respect to variations in test conditions and behaved in expected ways with respect to changes in temperature, ionic strength, and addition of an organic modifier. Serogroups W135 and Y are composed of sialic acid residues alternating with either galactose or glucose, respectively. Separation of these serogroups could be achieved using phosphate buffer and was therefore not dependent on differential complexation with borate. Addition of sodium dodecyl sulfate to the separation buffer (i.e., MEKC) resulted in peak splitting for all four serogroups. Changes in polysaccharide size did not affect migration time for the size range examined, but serogroup C polysaccharide (a sialic acid homopolymer) was separable from sialic acid monosaccharide. CZE quantification of multiple lots of each of the four serogroups was compared to wet chemical determination by phosphorus or sialic acid measurement. Results from CZE determination showed good agreement with the wet chemical methods.

Influence of ignored and well-known zone distortions on the separation performance of proteins in capillary free zone electrophoresis with special reference to analysis in polyacrylamide-coated fused silica capillaries in various buffers☆I. Theoretical studies

Influence of ignored and well-known zone distortions on the separation performance of proteins in capillary free zone electrophoresis with special reference to analysis in polyacrylamide-coated fused silica capillaries in various buffers☆I. Theoretical studies

Influence of ignored and well-known zone distortions on the separation performance of proteins in capillary free zone electrophoresis with special reference to analysis in polyacrylamide-coated fused silica capillaries in various buffers☆II. Experimental studies at acidic pH with on-line enrichment

The separation of acidic and basic model proteins was studied in capillary free zone electrophoresis in a polyacrylamide-coated, electroosmosis-free capillary at pH below their isoelectric points (pI) using various buffers at pH 2.7-4.8 with UV detection at 200 nm. The separation performance was significantly dependent on the coating quality, which may even differ within the same batch of capillaries. In addition, a washing step with 2 M HCl and the storage of the capillary in distilled water was essential for the performance. For high efficiency and resolution the choice of buffer constituents was extremely important which is discussed in quantitative terms in Part I. The most promising buffers were ammonium acetate and ammonium hydroxyacetate at pH 4 (ionic strengths: 0.12 and 0.15 M, respectively) with plate numbers up to 1,700,000 plates/m, corresponding to a zone width (2sigma) of only 1 mm in a capillary with 40 cm effective length, when the injected samples were dissolved in a 10-fold diluted background electrolyte (BGE), a zone even narrower than those obtained in polyacrylamide gel electrophoresis, the characteristic feature of which is remarkably thin zones. In the experiment giving this plate number, the calculated variance for longitudinal diffusion was larger than all the other calculated variances (those for the width of the starting zone, Joule heating, sedimentation and the curvature of the capillary). Interestingly, the effect of capillary curvature was significant. In addition, the sum of all other imaginable variances (corresponding to various types of slow on/off kinetics and hyper-sharp peaks) was in the most successful experiments only 28-50% of the variance for longitudinal diffusion. One hundred- to two hundred-fold dilution of the BGE improved the detection limits and provided high precision in both migration times and peak areas with ammonium hydroxyacetate and ammonium acetate as background electrolytes. However, that high dilution increased the variance 140-400% for these buffers, respectively, at least partly due to conductivity or pH differences between the sample and buffer zones (hyper-sharp peaks). Sedimentation of the enriched sample, a factor that has not previously been treated theoretically or experimentally, was probably another reason for our finding that peak heights did not increase when the sample was dissolved in a buffer diluted more than 200-fold, although pH changes and in some cases thermal expansion in the capillary also may contribute. Loss of protein may occur at the ionic strength 0.01 and lower due to precipitation. Limits of detection were in the range 4-17 pmol of proteins with ammonium acetate as BGE. No indication of denaturation of proteins at pH 4 was observed. However, the separation performance at pH 3 was not satisfactory and loss of proteins was observed, possibly indicating such problems. The protein mobilities decreased unexpectedly from pH 4 to 3--a further indication of conformation changes.

Influence of ignored and well-known zone distortions on the separation performance of proteins in capillary free zone electrophoresis with special reference to analysis in polyacrylamide-coated fused silica capillaries in various buffers: I. Theoretical studies

Influence of ignored and well-known zone distortions on the separation performance of proteins in capillary free zone electrophoresis with special reference to analysis in polyacrylamide-coated fused silica capillaries in various buffers: I. Theoretical studies

Influence of ignored and well-known zone distortions on the separation performance of proteins in capillary free zone electrophoresis with special reference to analysis in polyacrylamide-coated fused silica capillaries in various buffers: II. Experimental studies at acidic pH with on-line enrichment

The separation of acidic and basic model proteins was studied in capillary free zone electrophoresis in a polyacrylamide-coated, electroosmosis-free capillary at pH below their isoelectric points (p I) using various buffers at pH 2.7–4.8 with UV detection at 200 nm. The separation performance was significantly dependent on the coating quality, which may even differ within the same batch of capillaries. In addition, a washing step with 2 M HCl and the storage of the capillary in distilled water was essential for the performance. For high efficiency and resolution the choice of buffer constituents was extremely important which is discussed in quantitative terms in Part I. The most promising buffers were ammonium acetate and ammonium hydroxyacetate at pH 4 (ionic strengths: 0.12 and 0.15 M, respectively) with plate numbers up to 1 700 000 plates/m, corresponding to a zone width (2 σ) of only 1 mm in a capillary with 40 cm effective length, when the injected samples were dissolved in a 10-fold diluted background electrolyte (BGE), a zone even narrower than those obtained in polyacrylamide gel electrophoresis, the characteristic feature of which is remarkably thin zones. In the experiment giving this plate number, the calculated variance for longitudinal diffusion was larger than all the other calculated variances (those for the width of the starting zone, Joule heating, sedimentation and the curvature of the capillary). Interestingly, the effect of capillary curvature was significant. In addition, the sum of all other imaginable variances (corresponding to various types of slow on/off kinetics and hyper-sharp peaks) was in the most successful experiments only 28–50% of the variance for longitudinal diffusion. One hundred- to two hundred-fold dilution of the BGE improved the detection limits and provided high precision in both migration times and peak areas with ammonium hydroxyacetate and ammonium acetate as background electrolytes. However, that high dilution increased the variance 140–400% for these buffers, respectively, at least partly due to conductivity or pH differences between the sample and buffer zones (hyper-sharp peaks). Sedimentation of the enriched sample, a factor that has not previously been treated theoretically or experimentally, was probably another reason for our finding that peak heights did not increase when the sample was dissolved in a buffer diluted more than 200-fold, although pH changes and in same cases thermal expansion in the capillary also may contribute. Loss of protein may occur at the ionic strength 0.01 and lower due to precipitation. Limits of detection were in the range 4–17 pmol of proteins with ammonium acetate as BGE. No indication of denaturation of proteins at pH 4 was observed. However, the separation performance at pH 3 was not satisfactory and loss of proteins was observed, possibly indicating such problems. The protein mobilities decreased unexpectedly from pH 4 to 3—a further indication of conformation changes.

Fluid mechanics of electroosmotic flow and its effect on band broadening in capillary electrophoresis.

Electroosmotic flow (EOF) usually accompanies electrophoretic migration of charged species in capillary electrophoresis unless special precautions are taken to suppress it. The presence of the EOF provides certain advantages in separations. It is an alternative to mechanical pumps, which are inefficient and difficult to build at small scales, for transporting reagents and analytes on microfluidic chips. The downside is that any imperfection that distorts the EOF profile reduces the separation efficiency. In this paper, the basic facts about EOF are reviewed from the perspective of fluid mechanics and its effect on separations in free solution capillary zone electrophoresis is discussed in the light of recent advances.

Hybrid microdevice electrophoresis of peptides, proteins, DNA, viruses, and bacteria in various separation media, using UV-detection.

Recently we described the design of a hybrid microdevice for micro(nano)electrophoresis and electrochromatography, discussed its advantages and disadvantages compared to conventional microdevices and presented a few applications with low-molecular-weight samples. In this paper, we demonstrate the broad application range of this device using UV-based analyses of (i) peptides by free-zone electrophoresis and electrophoresis in a recently introduced gel (polyacrylamide cross-linked with allyl-beta-cyclodextrin), (ii) proteins by electrophoretic molecular-sieving in a polymer solution supplemented with SDS, (iii) DNA fragments by electrophoresis in the above gel, (iv) virus particles in this gel, as well as in free buffer and (v) bacteria in free buffer. To illustrate the advantages of the hybrid microdevice we can mention that electrophoresis of proteins in a polymer-containing buffer, supplemented with sodium dodecyl sulfate (SDS), in a 4.30 (2.75) cm long channel gave a resolution similar to that in conventional capillary electrophoresis in a 23.5 (18.6) cm long capillary and analysis times which were 15-fold shorter.

Capillary electrophoretic analysis of the derivatives and isomers of benzoate and phthalate

A capillary electrophoretic method for the analysis of 12 commonly found derivatives and isomers of benzoate and phthalate, including p-toluic acid, p-acetamido and p-hydroxy derivatives of benzoic acid, salicylic acid and its acetyl ester, 2- and 4-isomers of carboxybenzaldehyde, meta-, para-, and ortho-isomers of phthalic acid, and monomethyl terephthalic acid was developed. Capillary electrophoresis (CE) was performed in the free zone electrophoresis mode. Performing CE in 10 m M phosphate buffer, pH 7.0 could separate most of the benzoic acid derivatives except the structural or positional isomers. The positional isomers of phthalic acids could be completely separated with co-addition of α- and β-cyclodextrins. Addition of poly(ethylene glycol) 600 (4%) could further resolve some structural isomers. The CE method developed here is rapid, i.e. complete separation could be achieved in less than 8 min for the nine monoanionic benzoate derivatives and in less than 14 min for the three dianionic phthalate isomers. The new method has good precision and linearity and can be readily applied to real samples for quantitative analysis. It is sensitive and can detect sub-ppm (w/w) level of impurity in real terephthalic samples.