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Monitoring analytes during the transfer step from the first to the second dimension in multidimensional electrophoretic separations is crucial to determine and control the optimal time point for sample transfer and thus to avoid band broadening or unwanted splitting of the sample band with consequent sample loss. A spatially resolved intermediate on-chip LED-induced fluorescence detection system was successfully implemented for a hybrid capillary-chip glass interface. The setup includes a high-power 455-nm LED prototype as an excitation light source and a linear light fiber array consisting of 23 light fibers with a diameter of 100 μm for spatially resolved fluorescence detection in combination with a push-broom imager for hyperspectral detection. Using a basic FITC solution, the linear working range was determined to be 0.125 to 25 μg/ml for a single light guide and the absolute detection limit was 0.04 fmol at a signal-to-noise ratio of 4. With the setup presented here, labeled β-lactoglobulin focused via capillary isoelectric focusing was detectable on-chip with a sufficient intensity to monitor the analyte band transfer in the glass-chip interface demonstrating its applicability for full or intermediate on-chip detection. Fil: Sydes, Daniel. Eberhard Karls Universität Tübingen; Alemania Fil: Kler, Pablo Alejandro. Eberhard Karls Universität Tübingen; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Centro de Investigaciones en Métodos Computacionales. Universidad Nacional del Litoral. Centro de Investigaciones en Métodos Computacionales; Argentina Fil: Meyer, Hans. J&M Analytik AG; Alemania Fil: Zipfl, Peter. University of Applied Sciences Aalen. Optoelectronics and Laser Technology Department; Alemania Fil: Lutz, Daniel. CalvaSens GmbH; Alemania Fil: Huhn, Carolin. Eberhard Karls Universität Tübingen; Alemania

Here we present the study on the sample transfer characteristics of two different microfluidic interfaces for two–dimensional capillary electrophoresis. These interfaces were manufactured using two different microfabrication technologies: one was obtained via the classical photolithography - wet etching - anodic bonding process; and the other was obtained via the selective laser-induced etching process. The comparison of the two interfaces, and an intact capillary as a reference, was made via the CE separation of amino acids (arginine and lysine) under different bulk flow conditions, with and without applying bias potential to the secondary channels. The influence on peak shapes, migration times and repeatabiliy were evaluated.

One of the greatest challenges in multi-channel networks for electromigrative separation techniques is the control of the leakage of sample constituents and band broadening at the channel intersections in microfluidic devices or capillary-chip interfaces, which can be achieved using fixed bias or pullback potentials. These may be implemented as the first separation dimension in a 2D setup, where the electric potential at the interface to the second dimension changes with time. Thus, a dynamic control via on-line potential measurement in combination with a feedback system is needed to control electromigration into the side channels. We here present for the first time a prototype for in-channel potential measurements using a low working current in combination with a Si3N4 passivated Ti/Pt electrode at the intersection of the channels in a microfluidic interface. Exemplarily we chose capillary electrophoresis and isotachophoresis as model applications with constant vs. dynamic potential. Parallel on-chip intersection potential measurements were successfully conducted without disturbing capillary zone electrophoretic and isotachophoretic analysis regarding separation and peak performance of amino acids chosen as model analytes. This was possible due to a Si3N4 passivation layer, but also due to an ad-hoc developed high impedance instrumentation, resulting in a very low measuring electric current. Simulations of the detected isotachophoretic cross-section potential allowed a deeper understanding of the potential development during the separation. Fil: Sydes, Daniel. Iptc - Karls Eberhardt Universität Tübingen; Alemania Fil: Kler, Pablo Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Centro de Investigaciones en Métodos Computacionales. Universidad Nacional del Litoral. Centro de Investigaciones en Métodos Computacionales; Argentina Fil: Zipfl, P.. Fachhochschule Aalen; Alemania Fil: Lutz, D.. Fachhochschule Aalen; Alemania Fil: Bouwes, Hans. Ix-factory Gmbh; Alemania Fil: Huhn, Carolin. Iptc - Karls Eberhardt Universität Tübingen; Alemania

Multidimensional electrophoretic separations represent one of the most common strategies for dealing with the analysis of complex samples. In recent years we have been witnessing the explosive growth of separation techniques for the analysis of complex samples in applications ranging from life sciences to industry. In this sense, electrophoretic separations offer several strategic advantages such as excellent separation efficiency, different methods with a broad range of separation mechanisms, and low liquid consumption generating less waste effluents and lower costs per analysis, among others. Despite their impressive separation efficiency, multidimensional electrophoretic separations present some drawbacks that have delayed their extensive use: the volumes of the columns, and consequently of the injected sample, are significantly smaller compared to other analytical techniques, thus the coupling interfaces between two separations components must be very efficient in terms of providing geometrical precision with low dead volume. Likewise, very sensitive detection systems are required. Additionally, in electrophoretic separation techniques, the surface properties of the columns play a fundamental role for electroosmosis as well as the unwanted adsorption of proteins or other complex biomolecules. In this sense the requirements for an efficient coupling for electrophoretic separation techniques involve several aspects related to microfluidics and physicochemical interactions of the electrolyte solutions and the solid capillary walls. It is interesting to see how these multidimensional electrophoretic separation techniques have been used jointly with different detection techniques, for intermediate detection as well as for final identification and quantification, particularly important in the case of mass spectrometry. In this work we present a critical review about the different strategies for coupling two or more electrophoretic separation techniques and the different intermediate and final detection methods implemented for such separations.

We present the study on the sample transfer characteristics of two different microfluidic interfaces for 2D-CE . These interfaces were manufactured using two different microfabrication technologies: one was obtained via the classical photolithography-wet etching-anodic-bonding process; and the other was obtained via the selective laser-induced etching process. The comparison of the two interfaces, and an intact capillary as a reference, was made via the CE separation of amino acids (arginine and lysine) under different bulk flow conditions, with and without applying bias potential to the secondary channels. The influence on peak shapes, migration times, and repeatabiliy were evaluated.

One of the broadest German-speaking analytical conferences—ANAKON—was held from March 23 to March 26, 2015, at the University of Graz (Austria). Located for the first time in Austria, ANAKON 2015 was hosted by the local organization committee consisting of Walter Gossler (University of Graz) and Erich Leitner and Harald Wiltsche (Graz University of Technology). Almost 500 participants traveled to Styria, the green heart of Austria.

In this study, we characterized unexpected side-products in a commercially synthesized peptide with the sequence RPRTRLHTHRNR. This so-called peptide D3 was selected by mirror phage display against low molecular weight amyloid-β-peptide (Aβ) associated with Alzheimer's disease. Capillary electrophoresis (CE) was the method of choice for structure analysis because the extreme hydrophilicity of the peptide did not allow reversed-phase liquid chromatography (RPLC) and hydrophilic interaction stationary phases (HILIC). CE-MS analysis, applying a strongly acidic background electrolyte and different statically adsorbed capillary coatings, provided fast and efficient analysis and revealed that D3 unexpectedly showed strong ion-pairing with sulfuric acid. Moreover, covalent O-sulfonation at one or two threonine residues was identified as a result of a side reaction during peptide synthesis, and deamidation was found at either the asparagine residue or at the C-terminus. In total, more than 10 different species with different m/z values were observed. Tandem-MS analysis with collision induced dissociation (CID) using a CE-quadrupole-time-of-flight (QTOF) setup predominantly resulted in sulfate losses and did not yield any further characteristic fragment ions at high collision energies. Therefore, direct infusion Fourier transform ion cyclotron resonance (FT-ICR) MS was employed to identify the covalent modification and discriminate O-sulfonation from possible O-phosphorylation by using an accurate mass analysis. Electron transfer dissociation (ETD) was used for the identification of the threonine O-sulfation sites. In this work, it is shown that the combination of CE-MS and FT-ICR-MS with ETD fragmentation was essential for the full characterization of this extremely basic peptide with labile modifications.

Mycelium of the basidiomycete Pleurotus sapidus known to contain a novel dioxygenase was used for the bioconversion of car-3-ene [I]. After 4 h of incubation 25.3 mg L−1 car-3-en-5-one [V], 5.4 mg L−1 car-3-en-2-one [VII], and 7.3 mg L−1 car-2-en-4-one [XV] accumulated as major oxidation products. The identity of the respective carenones and their corresponding alcohols was confirmed by comparison with MS and NMR spectral data obtained for synthesized authentic compounds. The peak areas of oxidation products were at least five times higher as compared with autoxidation. A radical mechanism similar to lipoxygenase catalysis was proposed and substantiated with detailed product analyses. The reduction of assumed car-3-ene hydroperoxides to the corresponding alcohols evidenced the radical initiated formation of hydroperoxides and confirmed the regio- and stereo-selectivity of the dioxygenase. The introduction of molecular oxygen into the bicyclic car-3-ene [I] molecule occurred at allylic positions of a cyclic isopentenyl moiety with a pronounced preference for the position adjacent to the non-substituted carbon atom of the C–C-double bond. This co-factor independent selective oxygenation presents an alternative to P450 mono-oxygenase based approaches for the production of terpene derived flavor compounds, pharmaceuticals and other fine chemicals.

This work deals with simultaneous determination of ammonium, calcium, magnesium, sodium and potassium in drinking waters by capillary zone electrophoresis (CZE) on a column-coupling (CC) chip with suppressed hydrodynamic and electroosmotic transports. CZE separations were carried out in a propionate background electrolyte at a low pH (3.2) containing 18-crown-6-ether (18-crown-6) to reach a complete resolution of the cations. In addition, triethylenetetramine (TETA) coated the inner wall surface of the chip channels. The concentration limits of detection (cLOD) for the studied cations ranged from 4.9 to 11.5 μg/l concentrations using a 900 nl volume of the sample injection channel. 93–106% recoveries of the cations in drinking waters indicate a good predisposition of the present method to provide accurate analytical results.