Two-photon excited fluorescence detection at 420 nm for label-free detection of small aromatics and proteins in microchip electrophoresis

Two photon excited (TPE) fluorescence detection was applied to native fluorescence detection of aromatics in microchip electrophoresis (MCE). This technique was evaluated as an alternative to common one photon excitation in the deep UV spectral range. TPE enables fluorescence detection of unlabeled aromatic compounds, even in non-deep UV-transparent microfluidic chips. In this study, we demonstrate the proof of concept of native TPE fluorescence detection of small aromatics in commercial microfluidic glass chips. Label-free TPE fluorescence detection of native proteins and small aromatics in MCE was achieved within the micromolar concentration range, utilising 420 nm excitation light.     

Monitoring on-chip Pictet-Spengler reactions by integrated analytical separation and label-free time-resolved fluorescence

High-throughput screening for optimal reaction conditions and the search for efficient catalysts is of eminent importance in the development of chemical processes and for expanding the spectrum of synthetic methodologies in chemistry. In this context we report a novel approach for a microfluidic chemical laboratory integrating organic synthesis, separation and time-resolved fluorescence detection on a single microchip. The feasibility of our integrated laboratory is demonstrated by monitoring the formation of tetrahydroisoquinoline derivatives by Pictet-Spengler condensation. After on-chip reaction the products and residual starting material were separated enantioselectively on the same chip. On-chip deep UV laser-induced fluorescence detection with time-correlated single photon counting was applied for compound assignment. The system was utilized to screen reaction conditions and various substrates for Pictet-Spengler reactions on-chip. Finally, the microlab was successfully applied to investigate enantioselective reactions using BINOL-based phosphoric acids as organocatalysts.     

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 microm, 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.     

Modification of silica surfaces for CZE by adsorption of non-ionic hydrophilic polymers or use of radial electric fields

Polyvinylalcohols (PVA) and hydroxyethylcelluloses (HEC) have been used as additives in cyclodextrin-modified capillary zone electrophoretic chiral separations of aromatic amines such as tocainide and its analogs in unpretreated 50 μm i.d. fused silica capillaries. The additives were used at low concentrations (<0.05%) in common buffers, together with the γ-cyclodextrin as chiral selector. They reduce the electroosmotic flow, i.e. increase the migration times of the analytes in these chiral separations, and, moreover, considerably improve both peak symmetry and the widths of the peaks relative to migration time. In terms of the chromatographic theory of efficiency, more than 200000 theoretical plates can be achieved with unpretreated fused silica capillaries. This enhancement of efficiency arises because adsorptive “dynamic” coating with the hydroxylic modifier molecules suppresses adsorption of the analyte molecules by the capillary walls. The influence of field strength and buffer composition on the separation efficiency attainable with and without modifier in the buffers has also been investigated. Alternative experiments on the influence of analyte adsorption on efficiency have been performed by superimposing radial electric fields on the capillary to modify the ζ potentials. Although the EOF could be freely adjusted, it was not possible to obtain an improvement in efficiency comparable with that furnished by coating the adsorptive surface with PVA or HEC.     

A new weakly basic amino‐reactive fluorescent label for use in isoelectric focusing and chip electrophoresis

In this study, we present a novel amino‐reactive fluorescence marker (referred to as UR‐431), which is well suited for electrophoretic techniques. A main feature of this marker is its weakly basic behavior when conjugated to analytes. Labeled primary amines exhibit a positive net charge and accordingly a cathodic mobility below a pH of 2.4. The label features a pH‐independent fluorescence emission and is thus very interesting for electrophoretic applications such as IEF. The absorption maximum of this yellow daylight chromophore is at 431 nm, whereas fluorescence emission peaks at 537 nm (quantum yield≈0.1). The label was successfully conjugated to amines, peptides and proteins and separated via CE and MCE. The on‐chip detection limit of labeled lysine using a mercury‐lamp‐based fluorescence microscope was determined as 12 nM. An important feature of the new label is that it effects only a subtle change of the pI of proteins compared with common anionic labels, e.g. FITC. pI values of proteins were investigated by comparing native proteins and labeled proteins in CIEF. UR‐431 was also applied to sensitive detection of amines and peptides in MCE.     

Design and performance of a microchip electrophoresis instrument with sensitive variable-wavelength fluorescence detection

A modular instrument for high-speed microchip electrophoresis (MCE) equipped with a sensitive variable-wavelength fluorescence detection system was developed and evaluated. The experimental setup consists mainly of a lamp-based epifluorescence microscope for variable-wavelength fluorescence detection and imaging and a programmable four-channel bipolar high-voltage source capable of delivering up to +/- 10 kV per channel. The optical unit was equipped with a high-sensitivity photomultiplier tube and an adjustable aperture. The system was applied to MCE separations of flurescein isothiocyanate (FITC)-labelled amines utilizing blue light (450-480 nm) for excitation as well as for the separation of rhodamines utilizing excitation light in the green spectral region (531-560 nm). At optimized conditions baseline separation of four FITC-labelled amines could be obtained in less than 50 s at a detection limit of 460 ppt (1 nM) with a signal-to-noise ratio of 3:1. Three rhodamines could be baseline-separated in less than 6 s at a detection limit of 240 ppt (500 pM). The relative standard deviations of absolute migration times determined in repetitive MCE separations of FITC-labelled amines were below 2.5% (n= 25). By the application of cyclodextrin-modified electrolytes, chiral separation of FITC-labelled amines could be performed in seconds demonstrating the potential of microchip electrophoresis for chiral high-throughput screening.     

Influence of pH*-value of methanolic electrolytes on electroosmotic flow in hydrophilic coated capillaries

The dependency of EOF on the H+-concentration and the related so called pH* value of methanolic electrolytes has been examined with poly(ethylene glycol) (PEG), poly(vinyl alcohol) (PVA) and uncoated capillaries. These results were compared with the pH dependency of EOF of these capillaries using aqueous buffers. In uncoated capillaries the dependency of EOF on the pH(*)-value is very similar for aqueous and methanolic electrolytes. The EOF increases with increasing H+-concentration and pH-hysteresis is observed. In PVA coated capillaries the EOF is strongly reduced over wide pH* or pH ranges for both methanolic electrolytes and aqueous buffers. The EOF in PEG coated capillaries is surprisingly directed to the anode with methanolic electrolytes whereas a reduced cathodic EOF is observed in aqueous electrolytes. The anodic EOF of PEG-coated capillaries in methanolic electrolytes is independent of the pH*-value. The usefulness of PEG- and PVA-coated capillaries for adjusting the EOF in non-aqueous electrolytes for the analysis of isomeric organic acids was demonstrated.