Ernst Kenndler

Organic solvents in CE.

In this contribution some fundamental aspects are discussed serving for a critical reflection and elucidation of the role of organic solvents in CE. The implications of the solvent on the parameters governing peak resolution are discussed based on the concepts describing migration and zone broadening in capillary zone electrophoresis. This discussion includes the solvent‐dependent influence of the ionic strength on the mobility. The role of the solvent on the plate number in case of the inevitable diffusional peak dispersion is outlined, and its effect on other peak broadening contributions is briefly examined. This paper also deals with the problems of conductance, applicable voltage and analysis time upon application of organic solvents, and tries to clarify some misunderstandings common in the literature.

Recent developments in capillary and chip electrophoresis of bioparticles: Viruses, organelles, and cells.

In appropriate aqueous buffer solutions, biological particles usually exhibit a particular electric surface charge due to exposed charged or chargeable functional groups (amino acid residues, acidic carbohydrate moieties, etc.). Consequently, these bioparticles can migrate in solution under the influence of an electric field allowing separation according to their electrophoretic mobilities or their pI values. Based on these properties, electromigration methods are of eminent interest for the characterization, separation, and detection of such particles. The present review discusses the research papers published between 2008 and 2010 dealing with isoelectric focusing and zone electrophoresis of viruses, organelles and microorganisms (bacteria and yeast cells) in the capillary and the chip format.

Determination of duloxetine in human plasma by capillary electrophoresis with laser-induced fluorescence detection

A method based on capillary electrophoresis has been developed for the analysis of the novel antidepressant drug duloxetine in human plasma. The method makes use of laser-induced fluorescence detection after derivatisation of the analyte with 5-(4,6-dichlorotriazinyl)aminofluorescein at pH 11. A single step liquid/liquid extraction procedure with a mixture of hexane/2-propanol allows the sample clean-up with extraction yields always >or=84% and interference removal. The electrophoretic separation is achieved using uncoated fused silica capillaries (60.0 cm effective length, 75.0 cm total length, 50 microm internal diameter) and a background electrolyte composed of borate buffer (40 mM, pH 10.3), tetrabutylammonium bromide (10 mM), and acetone (10%, v/v). The applied voltage is 20 kV; the samples are injected by pressure (50 mbar x 8 s). The method has been fully validated in terms of linearity range (2.5-150 ng mL(-1)), LOD and LOQ (1.0 and 2.5 ng mL(-1), respectively), precision (R.S.D.<6.7%) and accuracy (recovery >78%). Application to samples obtained from patients under treatment with duloxetine gave good results. The method represents the first application of capillary electrophoresis to the analysis of duloxetine in human plasma.

Virus analysis using electromigration techniques

We discuss the progress during the last 4 years in the analysis of viruses by electrophoresis in capillaries and microfluidic devices. The paper is the continuation of a review published in this journal in 2005 [Kremser, L., Blaas, D., Kenndler, E., Electrophoresis 2004, 25, 2282–2291]. Eighteen papers on the topic have appeared since; the majority deals with zone electrophoresis and three reports are on IEF. These methods have been applied to human rhinoviruses, poliovirus Semliki Forest virus, norovirus‐like particles, and the two bacteriophages MS2 and T5. A main finding was that addition of detergents and salts to the BGEs are essential for the robustness of the CE analysis. Analyte detection was usually via UV absorbance but there are some examples where the viruses were rendered fluorescent via modification of the capsid proteins with reactive dyes and/or by non‐covalent attachment of intercalating fluorescent compounds to the nucleic acids making up the viral genome. Interestingly, some viruses are permeable to small molecular mass components; this allows fluorescent dyes to diffuse into the intact virus where they attach to the nucleic acid. Release of a viral genome upon heating was also monitored by using similar methodologies. Interactions of viruses and subviral particles with antibodies, receptors, and receptor‐decorated liposomes were investigated with CE methods, all by using a non‐equilibrium approach (i.e. co‐incubation of the components prior to CE separation). Viruses are multivalent (i.e. possess many identical surface‐exposed patches) and most of them are composed of defined numbers of identical subunits. The high resolution of CE has been most remarkably demonstrated by the separation of stoichiometric complexes between virus and a distinct number of soluble recombinant receptors and revealed their concentration‐dependent distribution.

Mimicking virus attachment to host cells employing liposomes: Analysis by chip electrophoresis

Electrophoresis on a chip increasingly replaces electrophoresis in the capillary format because of its speed and containment of the sample within a disposable cartridge. In this paper we demonstrate its utility in the analysis of the interaction between a virus and a liposome‐anchored receptor, mimicking viral attachment to host cells. This became possible because detergents, obligatory constituents of the BGE for capillary electrophoretic separation of the virus, were not necessary in the chip format. Separations were carried out in sodium borate buffer, pH 8.3. Liposomes and virus were both labeled for laser‐induced fluorescence detection at λex/λem 630/680 nm. Free virus and virus‐receptor complexes were resolved from virus attached to receptor‐decorated liposomes in the absence of additives or sieving matrices within about 30 s on commercially available microfluidic chips.

Characterization of rhinovirus subviral A particles via capillary electrophoresis, electron microscopy and gas-phase electrophoretic mobility molecular analysis: Part I.

During infection, enteroviruses, such as human rhinoviruses (HRVs), convert from the native, infective form with a sedimentation coefficient of 150S to empty subviral particles sedimenting at 80S (B particles). B particles lack viral capsid protein 4 (VP4) and the single‐stranded RNA genome. On the way to this end stage, a metastable intermediate particle is observed in the cell early after infection. This subviral A particle still contains the RNA but lacks VP4 and sediments at 135S. Native (150S) HRV serotype 2 (HRV2) as well as its empty (80S) capsid have been well characterized by capillary electrophoresis. In the present paper, we demonstrate separation of at least two forms of subviral A particles on the midway between native virions and empty 80S capsids by CE. For one of these intermediates, we established a reproducible way for its preparation and characterized this particle in terms of its electrophoretic mobility and its appearance in transmission electron microscopy (TEM). Furthermore, the conversion of this intermediate to 80S particles was investigated. Gas‐phase electrophoretic mobility molecular analysis (GEMMA) yielded additional insights into sample composition. More data on particle characterization including its protein composition and RNA content (for unambiguous identification of the detected intermediate as subviral A particle) will be presented in the second part of the publication.

Fast analysis of amino acids in wine by capillary electrophoresis with laser-induced fluorescence detection

A fast analytical method has been developed for the determination of nine amino acids, together with serotonin, in wine samples of different origin and vintage. The method is based on capillary electrophoresis coupled to laser‐induced fluorescence detection. Separation was obtained by using a fused‐silica capillary (75 μm id, 74.0 cm total length, 60.0 cm length to detector) and a background electrolyte composed of carbonate buffer (20 mM, pH 9.2), applying a 20 kV voltage. Direct hydrodynamic injection of wine samples was made after an original microwave‐assisted derivatisation step with 5‐(4,6‐dichlorotriazinyl)aminofluorescein. Fluorescence was induced by an Ar‐Ion laser, exciting at 488 nm. Good linearity (r2>0.9990) was obtained for all considered analytes and sensitivity was also good, with limits of detection in the 7–50 ng/mL range. The method was successfully applied for the analysis of commercial Italian wines and thus seems to be suitable for the determination of the relevant amino acids and serotonin, providing good results in terms of accuracy and precision, together with the advantage of a very fast, microwave‐assisted derivatisation procedure. Future applications of the method are planned to check for wine adulterations and commercial frauds.

Determination of topiramate in human plasma by capillary electrophoresis with indirect UV detection.

A rapid capillary zone electrophoresis method with indirect UV detection for the determination of topiramate in human plasma was developed and validated. The analyses were carried out with a background electrolyte composed of 10mM sulfamethoxazole as chromophore in phosphate buffer (25 mM, pH 12.0); gabapentin was selected as the internal standard. Application of a voltage of +15 kV led to an analysis time shorter than 5 min; indirect UV detection was operated at 256 nm. Isolation of topiramate from plasma was accomplished by a carefully implemented solid-phase extraction procedure on C18 cartridges. The method provided a linear response over the concentration range of 2-60 microg of topiramate per mL of plasma. The limit of detection (LOD) was 0.8 microg mL(-1) and the limit of quantitation (LOQ) was 2.0 microg mL(-1). Precision, expressed as relative standard deviation, was always lower than 7.3%, extraction yields were always greater than 92%. The results obtained analysing plasma samples from epileptic patients undergoing therapy with topiramate were satisfactory in terms of precision and selectivity.

Chip electrophoretic characterization of liposomes with biological lipid composition: Coming closer to a model for viral infection

In first attempts at elucidating the transfer of the RNA genome of a human Rhinovirus through lipid membranes in vitro we made use of liposomes decorated with recombinant receptors. This model system was characterized previously by CE but suffered from the requirement for inclusion of polyethylene glycol‐modified lipids for reliable separations [Weiss, V. U., Bilek, G., Pickl‐Herk, A., Blaas, D., Kenndler, E., Electrophoresis 2009, 30, 2123–2128.]. We here report the analysis of liposomes with a lipid composition much more similar to that of biological lipid bilayers. We found that vesicles containing and lacking this non‐physiologic lipid differ significantly in their electrophoretic mobility (by factor 2) although the concentration of charge‐bearing lipids in their bilayers is the same. We demonstrate that binding of a human Rhinovirus to the latter liposomes decorated with a cognate receptor can be analysed via electrophoresis on microchips; we support our results with transmission electron microscopy.

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.