H. Michael Widmer

Continuous separation of high molecular weight compounds using a microliter volume free-flow electrophoresis microstructure

A microliter volume free-flow electrophoresis microstructure (μ-FFE) was used to perform a continuous separation of high molecular weight compounds. The μ-FFE microstructure had a separation bed volume of 25 μL and was fabricated from silicon using standard micromachining technology. Laser-induced fluorescence was used to detect the sample components, which were labeled with fluorescein isothiocyanate (FITC) prior to analysis. The continuous separation of human serum albumin (HSA), bradykinin, and ribonuclease A demonstrated that only 25 V/cm was required to isolate HSA from bradykinin and ribonuclease A, while 100 V/cm was needed for the separation of bradykinin from ribonuclease A. Comparison of the observed band broadening with the theoretical variance indicated that dispersion due to the initial bandwidth, diffusion, and hydrodynamic broadening were the main contributors to the band broadening of HSA and bradykinin. However, the band broadening for ribonuclease A could not be sufficiently accounted for using the above contributors. Adsorption was found to be a possible contributor to the overall variance for ribonuclease A. Calculation of the theoretical variance due to Joule heating indicated that broadening due to Joule heating effects was insignificant. This was likely due to the narrow cross-sectional area of the device, which facilitated efficient cooling. Electrohydrodynamic distortion was observed for HSA as it migrated toward the side bed. Studies of the resolution of bradykinin and ribonuclease A as a function of field strength at various sample and carrier flow rates indicated that, for maximum throughput, high field strengths and high flow rates were required. However, no optimal conditions were found. The μ-FFE device has a peak capacity of ∼8 bands/cm, while for a typical separation of proteins using a commercial system, a peak capacity of 10 bands/cm is obtained. Thus, the resolving power of the μ-FFE device is similar to those of conventional systems. The continuous separation of tryptic digests of mellitin and cytochrome c demonstrated the ability to continuously separate more complex mixtures. Finally, modifications were made to the microstructure to facilitate fraction collection, and the fractionation of whole rat plasma was performed. Off-line analysis of the resulting fractions indicated that the complete isolation of serum albumin and globulins was possible using a field strength of 25 V/cm.

Design and development of a miniaturised total chemical analysis system for on-line lactate and glucose monitoring in biological samples

A miniaturised Total chemical Analysis System (μTAS) for glucose and lactate measurement in biological samples constructed based on an integrated microdialysis sampling and detection system. The complete system incorporates a microdialysis probe for intravascular monitoring in an ex vivo mini-shunt arrangement, and a silicon micromachined stack with incorporated miniaturised flow cell/sensor array. The prototype device has been developed based on state-of-the-art membrane and printed circuit board technology. The flow-through detection system is based on a three-dimensional flow circuit incorporating silicon chips with stacked micromachined channels. An integrated biosensor array (comprising enzyme sensors specific for glucose and lactate) is placed at the base of the stack allowing the detector to be incorporated within the μTAS assembly. These glucose and lactate biosensors are prepared using photolithographic techniques, with measurement based on the detection of hydrogen peroxide at glucose oxidase and lactate oxidase modified platinum electrodes. The resulting amperometric current (at 500 mV vs, Ag/AgCl) is proportional to the concentration of analyte in the sample. All instrumentation is under computer control and the complete unit allows continuous on-line monitoring of glucose and lactate, with fast stable signals over the relevant physiological range for both analytes. The microdialysis system provides 100% sampling efficiency. Sensor performance studies undertaken include optimisation of sensitivity, linearity, operational stability, background current, storage stability and hydration time. The total system (sampling and detection) response time is of the order of 4 min, with sensor sensitivity 1-5 nA mM-1 for glucose and lactate over the range 0.1-33 and 0.05-15 mM, respectively.

Rapid separation of fluorescein derivatives using a micromachined capillary eletrophoresis system

Abstract Using micromachining techniques eletrophoresis systems consisting of sample injectors and separation capillaries have been fabricated in planar glass structures. A device with a total capillary channel length of 13.9 cm was used to inject and separate fluorescein and fluorescein sulfonate dyes within 3 min, with an injector to detector distance of 6.0 cm, and applied field of 520 V/cm. A similar device with a capillary length of 1.6 cm effected injection and separation within 4 s for an injector to detector length of 0.75 cm at an applied field of 1875 V/cm. Injected sample volumes of 60 pl or less readily injected and analyte detected at 10 μM concentrations.

µ-TAS: Miniaturized Total Chemical Analysis Systems

The miniaturized total chemical analysis system is a concept for on-line monitoring combining classical analytical techniques and photolithographically defined micro structures. Examples of silicon and glass micro structures for flow-injection analysis, capillary liquid chromatography and capillary electrophoresis are given. The results obtained indicate faster separations, dramatically reduced reagent consumption, and access to novel types of analysis techniques.

Planar chip technology for capillary electrophoresis

Miniaturization of separation columns implies equally reduced volumes of injectors, detectors and the connecting channels. Planar chip technology provides a powerful means for the fabrication of micron sized structures such as channels. This is demonstrated with three examples. An optical absorbance detector chip exhibits the expected behavior of a 1 mm optical pathlength cell despite its volume of 4 nL. A capillary electrophoresis device allows for integrated injections of 100 pL samples, for efficiencies of 70 000 to 160 000 theoretical plates in 10 to 20 seconds, and for external laser-induced fluorescence detection at any capillary length of choice between 5 and 50 mm. A system for synchronized cyclic capillary electrophoresis is also presented in which plate numbers per volt can be dramatically increased.

Characterization of yeast cultivations by steric sedimentation field-flow fractionation

The characterization and quantification of biomass is often time consuming and dependent on the cultivation media and gives no detailed information between cell size and shape and their productivity. By monitoring the bioprocess with steric sedimentation field-flow fractionation (Sd/StFFF) in combination with laser light scattering, not only cell growth, but also the variation of cell size and shape during the cultivation, can be observed. In this work, the feasibility of separating and characterizing cell populations by steric sedimentation field-flow fractionation is demonstrated by its application to three different yeast cultivation broths. For this purpose samples which were collected at different cultivation times were injected into an FFF system. Fractograms were obtained in less than 4 min. Due to the relatively high resolution of the method, a cell sample could be fractionated in several subpopulations differing in their size as well as in their number of buds.

Thermo-optical absorbance detection of native proteins separated by capillary electrophoresis in 10 μm I.D. tubes

Abstract Thermo-optical absorption (TOA) detection of native proteins separated by capillary electrophoresis is demonstrated in 10 μm I.D. tubes. The eluents were on-column pumped at 257 nm and probed by a hologram-based refractive index detector. The use of 10 μm capillaries allowed fast 2-min separations. Slower separations in wider tubes led to limits of detection (LODs) of 7·10−9 M for bovine serum albumin. These LODs are comparable to those obtained with laser induced fluorescence and two orders of magnitude lower than in absorbance detection. Since native fluorescence of proteins is rare, TOA detection appears as a more universal detection scheme and thus suitable for other proteins having smaller amounts of fluorescent amino acids.

Theoretical considerations on the design of cylindrical flow cells utilizing optical fibres

Abstract The performance of microbore cylindrical flow cells featuring optical fibres depends on the geometry and materials of the components. The results of an algorithm which optimizes cell transmittance and minimizes inherent refractive index effects are presented. Optimum fibres and capillary tube geometries are expressed in terms of three reduced parameters for general application.

Automated analysis of light hydrocarbons in ambient air using the commercially available system AAR-KA 5890A : technical adaptations and experience from field measurements

Abstract An automated analytical system is used for the analysis of light hydrocarbons in ambient air. Sample pretreatment is performed with the instrument AAR-KA 5890A, manufactured by AMA(FRG). Sorbent materials (Tenax TA, Carbosieve S II, Molecular sieve) are used for sampling and enrichment of the hydrocarbons, which are analyzed by capillary gas chromatography with FID detection. In addition, an instrument was developed in our laboratories, in which the dehydration of the air samples and the thermal regeneration of the drying agent (K2CO3) were automated. The experience from field measurements, in which nine light hydrocarbons from ambient air were quantified, is used to discuss the performance of the instrument.

Why use a Delicate Biosensor for Monitoring? Alternative Routes by Miniaturizing and Speeding up the Classic Analytical Techniques

Glass and silicon microstructures for flow injection analysis and capillary electrophoresis have been manufactured. Maximum voltages of up to 600 V/cm gave efficient electrophoretic separations of several amino acids within 40 to 110 s .