Solving advanced micromachining problems for ultra-rapid and ultra-high resolution on-chip liquid chromatography

High-performance liquid chromatography (HPLC) is one of the most versatile separation techniques available for the analysis of complex samples that are typically encountered in fields such as environmental monitoring, biology, pharmacy, biochemistry, etc. A distinction between different HPLC formats can occur in the shape of the stationary phase, which necessarily displays a selective interaction with the present analytes in order to establish a separation. The most prevailing format is the packed particulate bed, which generally consists of functionalized porous spherical particles that are randomly packed in a capillary. Monolithic media (polymeric or silica) have recently become popular because of the high permeability combined with reasonable mass transfer characteristics. In this frame it is important to stress that it is theoretically expected that disorder restrains the performance of a column. When conceiving the ideal chromatographic format, the achievement of more order is therefore an interesting route to pursue. A practical realization of this approach was first put forward in 1998 by prof. Fred Regnier, making use of a microfabricated array of pillars in glass in capillary electro-chromatography mode, replacing the random particles by very accurately positioned pillars. Recognizing the potential of the technique, Desmet and coworkers performed a number of computational fluid dynamics (CFD) to study the fluidic behaviour of such a pillar array in pressure-driven mode. As the flow-through pores can be chosen independently of the pillar diameter, optimal designs can be tailored to provide the optimal combination of flow resistance and plate height. A first device containing non-porous silicon pillars was then characterized by De Pra et al. in 2005 under non-retaining conditions, achieving a minimal reduced plate height of 0.2 in a 40 % porosity pillar bed consisting of 10 μm pillars, in agreement with the CFD predictions. Even though this work was an important trigger in generating interest in this novel format, no separations were demonstrated, hence keeping the more relevant fluidic behaviour under retentive conditions in the dark

Visualization and quantification of the onset and the extent of viscous fingering in micro-pillar array columns

New experimental data of the viscous fingering (VF) process have been generated by studying the VF process in perfectly ordered pillar array columns instead of in the traditionally employed packed bed columns. A detailed quantitative analysis of the contribution of VF to the observed band broadening could be made by following the injected species bands using a fluorescence microscope equipped with a CCD-camera. For a viscosity contrast of 0.16 cP, a plate height increase of about 1 μm can be observed, while for a contrast of respectively 0.5 cP and 1 cP, additional plate height contributions of the order of 5–20 μm were observed. Citing these values is however futile without noting that they also depend extremely strongly on the injection volume of injected sample. It was found that, for a given viscosity contrast of 0.314 cP, the maximal plate height increase varied between 0.5 μm and 18 μm if the injection volume was varied between 3.0 nl and 32.7 nl. These values furthermore also strongly vary with the distance along the column axis

Ultra-rapid separation of an angiotensin mixture in nanochannels using shear-driven chromatography

The present paper reports on the separation of a mixture of fluorescein isothiocyanate-labeled angiotensin I and II peptides in a shear-driven nanochannel with a C-18-coating and using an eluent consisting of 5% acetonitrile in 0.02 M aqueous phosphate buffer at pH 6.5. The flat-rectangular nanochannel in fused silica consisted of an etched structure in combination with a flat moving wall. The very fast separation kinetics that can be achieved in a nanochannel allowed to separate the angiotensin peptides in less then 0.2 s in a distance of only 1.8 mm. Plate heights as small as 0.4 mu m were calculated after substraction of the injection effect. (c) 2006 Elsevier B.V. All rights reserved. \u

Exploring the speed limits of liqui chromatography using shear-driven flows through 45 and 85 nm deep nano-channels

We explored the possibility to perform high speed and high efficiency liquid chromatographic separations in channels with a sub-100 nm depth. The mobile phase flow through these nano-channels was generated using the shear-driven flow principle to generate high speed flows which were the equivalent of a 12000 bar pressure-driven flow. It was found that the ultra-fast mass transfer kinetics prevailing in this range of small channel depths allow to drastically reduce the C-term contribution to band broadening, at least up to the upper speed limit of our current set-up (7 mm s−1 mobile phase velocity), leaving the inescapable molecular diffusion (i.e., B-term band broadening) as the sole detectable source of band broadening. Due to the greatly reduced mass transfer limitations, 50000 to 100000 theoretical plates could be generated in the span of 1 to 1.5 seconds. This is nearly two orders of magnitude faster than the best performing commercial pressure-driven UHPLC-systems. With the employed channel depths, we appear to have struck a practical lower limit for the channel miniaturization of shear-driven flows. Despite the use of channel substrates with the highest grades of optical flatness, the overall substrate waviness (on the order of some 5 to 10 nm) can no longer be neglected compared to the etched channel depth, which in turn significantly influenced the local retention factor and band broadening

Experimental investigation of the band broadening originating from the top and bottom wall in micromachined non-porous pillar array columns

We report on the experimental investigation of the effect of the top and bottom wall plates in micromachined nonporous pillar array columns. It has been found that their presence yields an additional c-term type of band broadening that can make up a significant fraction of the total band broadening (at least if considering nonporous pillars and a nonretained tracer). Their presence also induces a clear (downward) shift of the optimal velocity. These observations are, however in excellent quantitative agreement with the theoretical expectations obtained from a computational fluid dynamics study. The presently obtained experimental results, hence, demonstrate that the employed high aspect ratio Bosch etching process can be used to fabricate micromachined pillar arrays that are sufficiently refined to achieve the theoretical performance limit