A free flow electrophoresis (FFE) device was developed for continuous electrophoretic separation of charged compounds and implemented in a continuous flow biochemical detection (BCD) system. These continuous separation characteristics make FFE well suitable for online implementation in a chromatographic or flow injection analysis system, in which an additional separation step of charged compounds is desired. In a heterogeneous biochemical flow assay for the determination of biotin, an analyte zone reacts with an excess of an affinity protein. Subsequently, the free binding sites of the affinity protein react with an excess of fluorescein-labeled ligand. Free and affinity protein-bound label are separated on the FFE device prior to fluorescence detection of the separated fractions. Biotin and streptavidin were chosen as, respectively, model ligand and affinity protein. Since all the compounds that are involved possess different electrophoretic properties, quantitative analysis is performed after completely separating the fluorescent affinity complex and labeled biotin in the FFE device within 2 min. Since the device is optically transparent, the separated zones can be detected in the separation compartment, using laser-induced fluorescence. The applicability of the BCD-FFE system in combination with a HPLC separation is demonstrated in the bioanalysis of biotin in human urine at the micromole per liter level.
A novel microelectrospray interface for the on-line coupling of capillary-zone electrophoresis (CZE) and mass spectrometry is described. The outlet of the fused-silica CZE capillary is tapered and serves as the microspray tip. The spray does not contain a sheath flow nor does the capillary tip contain an electrode or a conductive coating. The capillary tip is placed directly in front of the mass spectrometer inlet. The electrical field for the CZE separation as well as for the electrospray is delivered by the CZE high-voltage power supply. The electrical contact at the capillary outlet is established through the air between the spray tip and the inlet of the mass spectrometer, which is at ground potential.The performance of this interface is investigated using several -agonists, which are low-molecular-weight, cationic drugs, as test compounds. The combination of capillary electrophoresis (CZE) and mass spectrometry offers high separation power with a highly compound-selective and universally applicable detection technique.1,2 CZE/MS interfacing is generally performed using electrospray ionization (ESI) and its application has been successfully demonstrated for a broad range of compounds. 3,4 Since the introduction of ESI as a CZE/MS interface, 5 the interface has mainly been operated with a coaxial sheath liquid to establish the electrical contact and adjustment of the spray and ionization conditions. However, the presence of additional ions in the sheath flow can reduce the detectability of the analytes. 6 The sheath liquid is delivered with a flow of several microlitres per minute via a stainless steel capillary, which surrounds the CZE capillary terminus. A different approach has been presented, in which a liquid junction was used to establish the electrical contact and to add a make-up flow before the capillary terminus. The diameter of the formed droplets depends on the flow of the sheath liquid 8,9 and influences the evaporation efficiency of the droplets and, therefore, affects the amount of ions entering the mass spectrometer. Reduction of the flow can be obtained by removing the sheath flow and spraying the CZE capillary content directly into the mass spectrometer as has already been demonstrated. [10][11][12][13][14][15][16] In this approach, the electrical contact is made either via a metal coating at the capillary tip [10][11][12][13][14][15] or by inserting a narrow metal wire into the outlet of the capillary. 16 Although a metal coating functions properly as an electrode, a coating apparatus is necessary and in several cases flaking of the coating is reported during electrospraying. The use of a metal wire as an outlet electrode, although reported to be successful by Zare et al., 16 in our experience does not always result in a stable spray.Micro-or nanospray systems have gained recently a lot of interest, because of the low flow rate and the high electrospray efficiency. 8,9,17,18 Moreover, a microspray is very stable due to the small surface from which the spray originates and allows the use of...
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