Precolumn Reactions with Electrophoretic Analysis Integrated on a Microchip

Abstract

A glass microchip was constructed to perform chemical reactions and capillary electrophoresis sequentially. The channel manifold on the glass substrate was fabricated using standard photolithographic, etching, and deposition techniques. The microchip has a reaction chamber with a 1 nL reaction volume and a separation column with a 15.4 mm separation length. Electrical control of the buffer, analyte, and reagent streams made possible the precise manipulation of the fluids within the channel manifold. The microchip was operated under a continuous reaction mode with gated injections to introduce the reaction product onto the separation column with high reproducibility (< 1.8% rsd in peak area). The reaction and separation performances were evaluated by reacting amino acids with o-phthaldialdehyde to generate a fluorescent product which was detected by laser-induced fluorescence. Control of the reaction and separation conditions was sacient to measure reaction kinetics and variation of detection limits with reaction time. Ha-times of reaction of 5.1 and 6.2 s and detection limits of 0.55 and 0.83 fmol were measured for arginine and glycine, respectively.The microfabrication of analytical instrumentation will potentially enable the laboratory to be transported to the samples rather than vice versa. Miniature chemical instrumentation can be based on conventional laboratory approaches to chemical measurement problems. Many laboratory-based procedures require sample manipulation prior to the actual measurement, and many analyses are fully automated to circumvent operator bias. Similarly, a portable instrument should have these sample preparatory steps incorporated into the design and function of the instrument. In addition, micromachined chemical instruments as opposed to single-analyte chemical sensors should be able to identify and quantify all members of a desired class of compounds using a single device. The approach taken is to micromachine a channel manifold in a monolithic device that includes all desired fluid manipulation for complete chemical analysis. Chemical separation techniques including capillary electrophoresis,1-6 free-flow elec-(1) Harrison, D. J.; Manz, A.; Fan, Z.; Liidi, H.; Widmer, H. M. Anal. Chem. 1992, 64, 1926. (2) Manz, A; Harrison, J.; Verpoorte, E. M. J.; Fettinger, J. C.; Paulus, A; Liidi, H.; Widmer, H. M.trophore~is,~ open channel electrochromatography,s and capillary electrophoresis with postcolumn derivati~ation~ have been demonstrated using microfabricated devices.To demonstrate a reactor/analyzer microchip, we chose the relatively simple reaction of amino acids with o-phthaldialdehyde (OPA), which yields a fluorescent product.'O For capillary electrophoresis, both pre-and postseparation labeling using OPA have been studied.ll The reaction is moderately fast (a half-time of reaction for alanine at room temperature of x4 s l2 ), but the fluorescent product can be short lived (x10 min for glycineI3 1.This reaction is fast enough to perform postcolumn labeling when the analysis time is lo...

New advances in microchip fabrication for electrochromatography

“…Multichannel structures for parallel processing were introduced with straight [31,40] as well as with radial channels [78]. For postcolumn derivatization chip configuration, an extra side channel was connected to the end of the separation column [79]. 2-D separation was presented with multichannel structure as shown in Fig.…”

Study of injection bias in a simple hydrodynamic injection in microchip CE

“…The common modes are pinched [8], floating [8], gated [9], and dynamic [10] injections. Pinched injection provides an accurate, precise and well-defined volume of sample solution with high separation efficiency, but the injection volume is limited by the dimensions of the microchip intersection, and the tight focus of the sample may be detrimental to detection sensitivity due to dilution and dispersion effects [11].…”

Injection by hydrostatic pressure in conjunction with electrokinetic force on a microfluidic chip

“…In the loading step, the sample fluid is transported or driven through the cross region from the sample reservoir by pressure or electrokinetic forces. After the cross-region is filled with the sample, the dispensing step begins when a plug of sample is pushed into the separation channel, usually by an electroosmotic pump [1][2][3]. According to the forces employed in these two steps, three injection methods can be used: electrokinetic injection, pressure injection, and pressure in conjunction with electrokinetic injection.…”

“…The first method, which was extensively investigated through experiments [1][2][3][4][5][6] and numerical simulation [7,8], can be divided into the use of different types of valves based on the dispensing strategy: the floating valve [9], the pinched valve [1,2], or the gated valve [5,6]. Electrokinetic force, including electroosmosis flow and electrophoresis migration, can be simply induced by an applied high voltage, which provides a well-defined sample plug.…”