Detlev Belder

Cross-linked poly(vinyl alcohol) as permanent hydrophilic column coating for capillary electrophoresis

A fast method for the generation of permanent hydrophilic capillary coatings for capillary electrophoresis (CE) is presented. Such interior coating is effected by treating the surface to be coated with a solution of glutaraldehyde as cross‐linking agent followed by a solution of poly(vinyl alcohol) (PVA), which results in an immobilization of the polymer on the capillary surface. Applied for capillary zone electrophoresis (CZE) such capillaries coated with cross‐linked PVA exhibit excellent separation performance of adsorptive analytes like basic proteins due to the reduction of analyte‐wall interactions. The long‐term stability of cross‐linked PVA coatings could be proved in very long series of CZE separations. More than 1000 repetitive CE separations of basic proteins were performed with stable absolute migration times relative standard deviation (RSD > 1.2%) and without loss of separation efficiency. Cross‐linked PVA coatings exhibit a suppressed electroosmotic flow and excellent stability over a wide pH range.

General approach for the analysis of various alkaloid classes using capillary electrophoresis and capillary electrophoresis-mass spectrometry

Abstract The analysis of various alkaloid classes employing capillary electrophoresis (CE) and on-line combined CE-mass spectrometry (CE-MS) is described. A CE method is presented for the analysis of alkaloids without derivatisation or purification. The separation of four different groups of alkaloids consisting of monoterpenoid indole alkaloids, protoberberines/benzophenanthridines, β-carboline alkaloids, and isoquinolines from poppy by free zone capillary electrophoresis has been obtained using a 1:1 mixture of 100 mmol 1 −1 ammonium acetate (pH 3.1) and acetonitrile. The influence of alkaloid structure on the electrophoretic mobility is discussed. The CE-MS reconstructed total ion current (RIC) of the indole- and the opium-type standard alkaloids shows a decreased signal-to-noise ratio compared to CE using only UV detection. As expected the single-ion traces (or individual mass traces) of the [M+H] + ions show higher signal-to-noise ratios than the RIC. The electrospray MS data of the alkaloids are dominated by the protonated molecules and the Na + -, and K + -adducts. They display the typical pattern resulting from cluster formation or doubly charged species.

Asymmetric organocatalysis and analysis on a single microfluidic nanospray chip.

The miniaturization of chemical processes onto so-called labon-a-chip devices has gained significant importance in different fields of chemistry. Besides the advantages of enhanced portability, reduced reagent consumption, and improved safety, a characteristic feature of miniaturized platforms is the possibility to achieve higher reaction and analysis rates. From their roots in analytical sciences, microfluidic systems have received much attention over the past decade. At present, microfluidics is becoming increasingly popular in inorganic and organic chemistry, as syntheses are performed on chip-based microreactors or capillary-based microflow reactors. While diverse reactions have been performed in microfluidic chip devices with impressive results, the analytical characterization is, however, usually carried out off-chip by conventional macroscopic instruments. However, this approach does not exploit the promise and the full potential of chip technology, namely, the integration of different functionalities such as chemical synthesis and analysis on one single device. Thus, it is desirable to develop, in analogy to microelectronics, integrated chemical circuits as new chemical tools, for example, for catalyst screening or for online monitoring of biological processes. In previous work we demonstrated a first approach for integrating chemical reactions and analysis on a single microchip for the screening of enantioselective biocatalysts. Nevertheless, this system was limited to aqueous media and native fluorescent molecules. Therefore we intended to develop an advanced chip system with a wider applicability in synthetic chemistry, including the utilization of non-aqueous reaction media and a more general detection system. In this context, the coupling to mass spectrometry appears to be very attractive, as it provides additional structural information for substance identification. To demonstrate our concept, we focused on organocatalysis, which has been one of the most innovative research fields in synthetic chemistry over the past years. Recently, Odedra and Seeberger described a first miniaturized approach, performing organocatalysis in a microfluidic flow reactor chip. The reaction products were, however, analyzed offline by traditional HPLC. Herein we present the, to our knowledge, first asymmetric organocatalytic reaction on a single chip with integrated analysis. As a model system we chose the enantioselective vinylogous Mannich reaction published in 2008, which is catalyzed by chiral phosphoric acid. The alcoholic solvent mixture and the nonfluorescent reaction products in this synthesis are quite challenging for chip integration. Thus, it was necessary to adapt the reaction media to the aqueous separation electrolyte on-chip to enable an undistorted electrophoretic analysis of the reaction products. Therefore, we developed a new microfluidic chip design containing different functionalities, including a reaction structure for the organic synthesis, a structure for aqueous dilution of the reaction mixture, a cross section for injection, and a separation channel for chip electrophoresis. Furthermore, the chip layout includes an integrated nanoelectrospray emitter with makeup-flow channels for dead-volume free coupling to mass spectrometry at the end of the separation channel. A schematic drawing of the chip with 50 mm wide channels and a photo is shown in Figure 1. Reaction and analysis processes on the single chip were carried out as follows: initially, the reaction structure was filled with the alcoholic solvent mixture for synthesis; then the remaining structure was replenished with the aqueous separation electrolyte. Afterwards, each reactant solution

Label-free fluorescence detection in capillary and microchip electrophoresis

Herein, we summarize the current status of native fluorescence detection in microchannel electrophoresis, with a strong focus on chip-based systems. Fluorescence detection is a powerful technique with unsurpassed sensitivity down to the single-molecule level. Accordingly fluorescence detection is attractive in combination with miniaturised separation techniques. A drawback is, however, the need to derivatize most analytes prior to analysis. This can often be circumvented by utilising excitation light in the UV spectral range in order to excite intrinsic fluorescence. As sensitive absorbance detection is challenging in chip-based systems, deep-UV fluorescence detection is currently one of the most general optical detection techniques in microchip electrophoresis, which is especially attractive for the detection of unlabelled proteins. This review gives an overview of research on native fluorescence detection in capillary (CE) and microchip electrophoresis (MCE) between 1998 and 2008. It discusses material aspects of native fluorescence detection and the instrumentation used, with particular focus on the detector design. Newer developments, featured techniques, and their prospects in the future are also included. In the last section, applications in bioanalysis, drug determination, and environmental analysis are reviewed with regard to limits of detection.

Chiral separations of basic and acidic compounds in modified capillaries using cyclodextrin-modified capillary zone electrophoresis

Chiral separations of basic drugs and organic acids can be achieved using cyclodextrins or cyclodextrin derivatives as chiral selectors in surface-modified capillaries. The influence of temperature and selector concentration on separation performance was investigated in specially modified capillaries. Separations of basic compounds at acidic pH were performed in capillaries modified by either dynamic or permanent coating by adsorption of non-ionic hydroxylic polymers. Separations of acidic compounds could be significantly improved by use of thermally immobilized poly(vinyl alcohol) or adsorbed cationic polymers as surface coatings. The influence of non-ionic and cationic coatings on separations of acidic compounds was compared with regard to resolution, separation efficiency and analysis time.

Chip-based separation devices coupled to mass spectrometry

The hyphenation of miniaturized separation techniques like chip electrophoresis or chip chromatography to mass spectrometry (MS) is a highly active research area in modern separation science. Such methods are particularly attractive for comprehensive analysis of complex biological samples. They can handle extremely low sample amounts, with low solvent consumption. Furthermore they provide unsurpassed analysis speed together with the prospect of integrating several functional elements on a single multifunctional platform. In this article we review the latest developments in this emerging field of technology and summarize recent trends to face current and future challenges in chip-based biochemical analysis.

Poly(vinyl alcohol)-coated microfluidic devices for high- performance microchip electrophoresis

The channels of microfluidic glass chips have been coated with poly(vinyl alcohol) (PVA). Applied for microchip electrophoresis, the coated devices exhibited a suppressed electroosmotic flow and improved separation performance. The superior performance of PVA‐coated channels could be demonstrated by electrophoretic separations of labeled amines and by video microscopy. While a distorted sample zone is injected using uncoated channels the application of PVA‐coated channels results in an improved shape of the sample zone with less band broadening. Applying PVA‐coated microchips for the separation of amines labeled with Alexa Fluor 350™ even sub‐second separations, utilizing a separation length of only 650 νm, could be obtained, while this was not possible using uncoated devices. By using PVA‐coated devices rather than an uncoated chip a threefold increase in separation efficiencies could be observed. As the electroosmotic flow (EOF) was suppressed, the anionic compounds were detected at the anode whereas the dominant EOF in uncoated devices resulted in an effective mobility to the cathode. Besides improved separation performance another important feature of the PVA‐coated channels was the suppressed adsorption of fluorescent compounds in repetitive runs which results in an improved robustness and detection sensitivity. Applying PVA‐coated channels, rinsing or etching steps could be omitted while this was necessary for a reliable operation of uncoated devices.

Spray Performance of Microfluidic Glass Devices with Integrated Pulled Nanoelectrospray Emitters

The performance of microfluidic glass devices with a monolithically integrated nanospray tip have been evaluated. The nanospray tip is generated directly on the edge of a microfluidic glass chip by a pulling step followed by HF-etching for directed formation of a sharp tip with defined emitter area. For a fair judgment of the MS-detection sensitivity, we compared the detection performance with commercial nanospray needles. For that purpose the effect of the emitter opening on the sensitivity was studied in detail for different microfluidic chips as well as for commercial nanospray needles. A comparison of the chip-nanospray device with commercial nanospray needles revealed that a comparable spray performance is obtained at similar emitter diameters. A stable electrospray could be generated at such tapered tips without any need for hydrodynamic or electroosmotic pumping. The nanospray chips were successfully applied for coupling microchip electrophoresis and mass spectrometry. For improved performance, the separation channel of the microdevice was flushed with hydroxypropylmethylcellulose (HPMC) in order to reduce analyte-wall interactions and electroosmotic flow.

Chip-Based High-Performance Liquid Chromatography for High-Speed Enantioseparations

In this work, the first high-performance chiral liquid chromatography in packed microfluidic chips is presented. The chromatographic separation was performed on a column integrated into the microfluidic glass chip and packed with the particulate chiral stationary phase. Cellulose tris(3,5-dimethylphenylcarbamate) coated on 5-μm fully porous silica was used as chiral stationary phase material. Several racemic analytes including pharmaceutical products were baseline separated into their corresponding enantiomers under reversed-phase, polar organic and normal-phase conditions, demonstrating the versatility of the glass chip in the field of chiral separations. Van Deemter plots revealed a reduced plate height of 2.2 and a trend to enhanced mass transfer processes for solutes under low retention conditions. The utilization of very short column lengths of down to 12 mm led to ultrafast separations of enantiomers within 5 s.

Towards an Integrated Chemical Circuit

Everything on a chip: Recent developments in microfluidics enable the combination of droplet microfluidics with continuous-flow systems (see picture). This is a promising step towards the development of integrated, complex synthesis and analysis laboratories on chips. For example, a building-block principle could be used to integrate a multistep reaction with purification and analysis.