Experimental optimization of flow distributors for pressure-driven separations and reactions in flat-rectangular microchannels.

We report on the results of an experimental study established to optimize the design of microfabricated flow distributors for use in pressure-driven separations and reactions in flat-rectangular channels. For this purpose, the performance of a wide variety of possible flow distributor designs etched in glass/silicon wafers was compared, using CCD camera detection to study the shape and variance of the bands eluting from them. The best performance was obtained with radially interconnected distributors with a diverging inlet section and filled with diamond-shaped pillars, oriented perpendicular to the main flow direction and with a high transversal over axial aspect ratio. It was found that the best distributor designs start with a diverging section containing some 10-12 subsequent rows of high aspect ratio pillars (with a transversal width making up 10-15% of the final channel width) and with a divergence angle selected such that the sloped side-walls run parallel with the sides of the diamond-shaped pillars. After this zone, one or more regions with pillars with a smaller aspect ratio should be provided to increase the number of exit points. To prevent the formation of dead zones in these subsequent zones, so-called distributor wedges can be used to prevent the formation of any dead zones in the wake of the large aspect ratio pillars of the preceding section.

Design and evaluation of flow distributors for microfabricated pillar array columns.

Five different flow distributors have been compared as a function of the flow rate for their ability to distribute small sample volumes over the entire width of flat rectangular microfabricated pillar array columns. The investigated designs can be divided in two major categories: (1) bifurcating, radially non-interconnecting distributors and (2) radially interconnecting distributors consisting of diamond-shaped pillars, elongated in the direction perpendicular to the flow, providing a high ratio of radial permeability over axial permeability. The quality of the flow distribution was evaluated experimentally by injecting equal volumes of fluorescent tracer into each of the tested designs and calculating the obtained peak variances using the method of moments. Purely bifurcating distributors perform less well than the best possible radially interconnected distributors, because the former inevitably require the use of wide open channels (d > 10 microm), wherein a lot of band broadening can occur. By doubling the aspect ratio of the radially stretched pillars from 5 to 10, the measured peak variance drops to 1/8 of the original value. The best results were obtained with a distributor in which the flow is distributed by a bed of anisotropic pillars with an aspect ratio of 10, but our results indicate that a substantial improvement can still be made by increasing the aspect ratio and adding gradually diverging sidewalls to the inlet.

Fabrication and chromatographic performance of porous-shell pillar-array columns.

We report on a new approach to obtain highly homogeneous silica-monolithic columns, applying a sol-gel fabrication process inside a rectangular pillar-array column (1 mm in width, 29 microm in height and 33.75 mm in length) having a cross-sectional area comparable to that of a 200 microm diameter circular capillary. Starting from a silicon-based pillar array and working under high phase-separation-tendency conditions (low poly(ethylene glycol) (PEG)-concentration), highly regular silica-based chromatographic systems with an external porosity in the order of 66-68% were obtained. The pillars, 2.4 microm in diameter, were typically clad with a 0.5 microm shell layer of silica, thus creating a 3.4 microm total outer pillar diameter and leaving a minimal through-pore size of 2.2 microm. After mesopore creation by hydrothermal treatment and column derivatization with octyldimethylchlorosilane, the monolithic column was used for chip-based liquid-chromatographic separations of coumarin dyes. Minimal plate heights ranging between 3.9 microm (nonretaining conditions) and 6 mum (for a retention factor of 6.5) were obtained, corresponding to domain-size-reduced plate heights ranging between 0.7 and 1.2. The column permeability was in the order of 1.3 x 10(13) m(2), lower than theoretically expected, but this is probably due to obstructions induced by the sol-gel process in the supply channels.

Micron-sized pillars for ion-pair reversed-phase DNA separations.

In the present paper, the feasibility to construct micron-sized silicon pillar channels to be used in HPLC is studied. For this, a channel with flow-through pores of 1 µm and with critical sidewall dimensions below 1 µm was constructed using advanced deep-UV lithographic equipment. Integrating a 3-nL injection system on the chip directly in front of the separation channel and using elongated distribution structures, a very controlled and high aspect ratio sample definition across the relatively wide separation channel was accomplished. The system was evaluated in isocratic ion-pair RP mode, allowing the separation of a mixture of two components with, respectively, 300 and 400 base pairs in 5 s only.

High-speed shear-driven flows through microstructured 1D-nanochannels.

A new flow type for the conduction of rapid chromatographic and macro-molecular separations in 1D nanochannels is reported. It combines the pressure-drop-less operation of shear-driven flows with the meandering flow paths that are present in ordered arrays of micro- and nanopillars. Similar to shear-driven flows in open channels, the achievable fluid velocity is quasi unlimited and is not affected by a pressure- or voltage-drop, while the axial dispersion in the microstructured pillar arrays remains surprisingly low. In the present paper, we report on a series of flow resistance and band broadening experiments that have been conducted to characterize the hydrodynamical properties of this new flow type. In addition, theoretical computational fluid dynamics (CFD) simulations have been performed to explain the observations. Good agreement between theory and experiment was obtained.

Purification and crystallization of Vibrio fischeri CcdB and its complexes with fragments of gyrase and CcdA.

The ccd toxin-antitoxin module from the Escherichia coli F plasmid has a homologue on the Vibrio fischeri integron. The homologue of the toxin (CcdB(Vfi)) was crystallized in two different crystal forms. The first form belongs to space group I23 or I2(1)3, with unit-cell parameter a = 84.5 A, and diffracts to 1.5 A resolution. The second crystal form belongs to space group C2, with unit-cell parameters a = 58.5, b = 43.6, c = 37.5 A, beta = 110.0 degrees, and diffracts to 1.7 A resolution. The complex of CcdB(Vfi) with the GyrA14(Vfi) fragment of V. fischeri gyrase crystallizes in space group P2(1)2(1)2(1), with unit-cell parameters a = 53.5, b = 94.6, c = 58.1 A, and diffracts to 2.2 A resolution. The corresponding mixed complex with E. coli GyrA14(Ec) crystallizes in space group C2, with unit-cell parameters a = 130.1, b = 90.8, c = 58.1 A, beta = 102.6 degrees, and diffracts to 1.95 A. Finally, a complex between CcdB(Vfi) and part of the F-plasmid antitoxin CcdA(F) crystallizes in space group P2(1)2(1)2(1), with unit-cell parameters a = 46.9, b = 62.6, c = 82.0 A, and diffracts to 1.9 A resolution.

Computer aided design optimisation of microfluidic flow distributors.

This study reports on a quantitative study of the influence of the most important geometrical design parameters for micro-machined flow distributors with uniform cross-section and filled with diamond-shaped pillars having their longest dimension oriented perpendicular to the axial flow direction. It was found that the shape of the bands eluting from the distributor improves with increasing aspect ratio (AR) of the pillars, both in terms of global warp and local axial dispersion. Increasing the AR from 5 to 25 reduces the distributor length needed to bring the maximal transversal velocity difference below 5% from 170 µm to 15 µm when using pillars with axial width of 5 µm. To solve the problem that high AR pillar distributors only have a limited number of exit points, and therefore produce bands with a strong local warp, one can conceive mixed size distributors, wherein a zone filled with several rows of very high AR pillars is followed by one or more zones consisting of pillars with a smaller AR. With such a design, the variance of the eluting bands can be reduced to only 30% of the variance of a single size distributor.

Theoretical optimisation of the side-wall of micropillar array columns using computational fluid dynamics.

The present study provides an overview of the ideal side-wall position in micro-pillar array columns for the case of semi-embedded side-walls. The position has been determined using computational fluid dynamics simulations of the flow field in flow domains with different side-wall shifts. Optimal side-wall shift values are presented for a wide range of shapes (cylinders, and diamonds and hexagons with different aspect ratios) and packing densities. Simple linear correlations that allow calculating the optimised side-wall geometries for the different considered variety of shapes and packing densities could be established. Interestingly, only two correlations are needed to represent all investigated cases: one correlation for all diamonds, and one correlation for the cylinders and all hexagons. Compared to the case of a flat side-wall, the minimal feature size on the mask can be increased by a factor of 2.5 in the case of cylindrical pillar bed with external porosity ɛ=0.4, implying that that much smaller pillar diameters can be used in the bulk of the bed before the minimal feature size on the mask falls below the lithography resolution.

Hydrodynamic chromatography of polystyrene microparticles in micropillar array columns.

We report on the possibility to perform HDC in micropillar array columns and the potential advantages of such a system. The HDC performance of a pillar array column with pillar diameter = 5 microm and an interpillar distance of 2.5 microm has been characterized using both a low MW tracer (FITC) and differently sized polystyrene bead samples (100, 200 and 500 nm). The reduced plate height curves that were obtained for the different investigated markers all overlapped very well, and attained a minimum value of about h(min)=0.3 (reduction based on the pillar diameter), corresponding to 1.6 microm in absolute value and giving good prospects for high efficiency separations. The obtained reduced retention time values were in fair agreement with that predicted by the Di Marzio and Guttman model for a flow between flat plates, using the minimal interpillar distance as characteristic interplate distance.

Pressure-driven reverse-phase liquid chromatography separations in ordered nonporous pillar array columns.

Building upon the micromachined column idea proposed by the group of Regnier in 1998, we report on the first high-resolution reversed-phase separations in micromachined pillar array columns under pressure-driven LC conditions. A three component mixture could be separated in 3 s using arrays of nonporous silicon pillars with a diameter of approximately 4.3 microm and an external porosity of 55%. Under slightly retained component conditions (retention factor k' = 0.65-1.2), plate heights of about H = 4 microm were obtained at a mobile phase velocity around u = 0.5 mm/s. In reduced terms, such plate heights are as low as hmin = 1. Also, since the flow resistance of the column is much smaller than in a packed column (mainly because of the higher external porosity of the pillar array), the separation impedance of the array was as small as E = 150, i.e., of the same order as the best currently existing monolithic columns. At pH = 3, yielding very low retention factors (k' = 0.13 and 0.23), plate heights as low as H = 2 microm were realized, yielding a separation of the three component mixture with an efficiency of N = 4000-5000 plates over a column length of 1 cm. At higher retention factors, significantly larger plate heights were obtained. More experimental work is needed to investigate this more in depth. The study is completed with a discussion of the performance limits of the pillar array column concept in the frame of the current state-of-the-art in microfabrication precision.