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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., (Copyright 2010 Elsevier B.V. All rights reserved.)

An experimental study of a micromachined non-porous pillar array column performance under non-retentive conditions is presented. The same pillar structure has been fabricated in cyclo-olefin polymer (COP) chips with three different depths via hot embossing and pressure-assisted thermal bonding. The influence of the depth on the band broadening along with the already known contribution arising from the top and bottom cover plates has been studied. The experimental results exhibit reduced plate heights as low as 0.2, which are in agreement with the previous experimental work. Moreover, the constant values of the reduced Van Deemter expression are also in accordance with the previous studies. A more exhaustive study of the C-term band broadening is also presented, showing that comparing the space between the pillars with different open tubular rectangular channels offers a good estimation of the C-term band broadening that is obtained experimentally. These experimental results, hence, confirm that micromachined pillar array columns fabricated in COP can achieve the same performance as the ones fabricated in silicon for the presently studied pillar channel design., (Copyright 2010 Elsevier B.V. All rights reserved.)

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.

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.

Aerobic batch cultivations of Candida utilis were carried out in two micro bioreactors with a working volume of 100 muL operated in parallel. The dimensions of the micro bioreactors were similar as the wells in a 96-well microtiter plate, to preserve compatibility with the current high-throughput cultivation systems. Each micro bioreactor was equipped with an electrochemical sensor array for the online measurement of temperature, pH, dissolved oxygen, and viable biomass concentration. Furthermore, the CO(2) production rate was obtained from the online measurement of cumulative CO(2) production during the cultivation. The online data obtained by the sensor array and the CO(2) production measurements appeared to be very reproducible for all batch cultivations performed and were highly comparable to measurement results obtained during a similar aerobic batch cultivation carried out in a conventional 4L bench-scale bioreactor. Although the sensor chip certainly needs further improvement on some points, this work clearly shows the applicability of electrochemical sensor arrays for the monitoring of parallel micro-scale fermentations, e.g. using the 96-well microtiterplate format.

In this work a novel room-temperature bonding technique based on chemically activated Fluorinated Ethylene Propylene (FEP) sheet as an intermediate between chemically activated substrates is presented. Surfaces of silicon and glass substrates are chemically modified with APTES bearing amine terminal groups, while FEP sheet surfaces are treated to form carboxyl groups and subsequently activated by means of EDC-NHS chemistry. The activation procedures of silicon, glass and FEP sheet are characterized by contact angle measurements and XPS. Robust bonds are created at room-temperature by simply pressing two amine-terminated substrates together with activated FEP sheet in between. Average tensile strengths of 5.9 MPa and 5.2 MPa are achieved for silicon-silicon and glass-glass bonds, respectively, and the average fluidic pressure that can be operated is 10.2 bar. Moreover, it is demonstrated that FEP-bonded microfluidic chips can handle mild organic solvents at elevated pressures without leakage problems. This versatile room-temperature intermediate layer bonding technique has a high potential for bonding, packaging, and assembly of various (bio-) chemical microfluidic systems and MEMS devices.

We report on the first separation of a complex biomixture in pressure-driven mode using perfectly ordered pillar array columns. The separations were conducted in the reversed-phase mode using a highly aqueous mobile phase, while the outer surface of the non-porous pillars was chemically functionalized with a hydrophobic C8-layer. The samples originated from two different bacterial strains (Pseudomonas aeruginosa PAO1 and Pseudomonas sp. W15Feb38) of fluorescent pseudomonads. These produce fluorescent yellow-green pyoverdines that serve as siderophores to shuttle iron inside the cell. The pyoverdines of both strains were prepared from the supernatant through a crude solid phase extraction without any further purification step. In case of the PAO1 mixture, a separation of 15 components within a column length of 2.5 cm could be observed through the transparent cover glass of the chip. For the W15Feb38 mixture, a separation of eight components could be observed within the same distance. These fast chromatographic separations were compared with those obtained via iso-electrofocusing (IEF), which is the traditionally employed fingerprinting method to characterize pseudomonad strains based on their pyoverdine profiles (siderotyping). With this technique, and despite the injection of a 10,000 times larger sample mass, only nine bands were maximally observed for the PAO1 mixture, whereas maximally six bands were observed in case of the W15Feb38 mixture. The chromatographic pillar array method, yielding a separation in less than 1 min, was also significantly faster than the IEF method, which typically needs 1.5h. The present system can therefore be considered as a potential alternative fingerprinting tool for the fast identification of different strains of fluorescent pseudomonads, including as diagnostic tool for typing strains of the important opportunistic pathogen P. aeruginosa.

To explore the applicability of a laminar fluid diffusion interface (LFDI) for the controlled feeding of microbioreactors, glucose diffusion experiments were carried out in a rounded H-shaped microstructure etched in a glass substrate. The diffusion channel of the microstructure had a length of 4 mm and a depth of 50 microm with a trapezoidal cross section with a width of 100 microm at the bottom and 200 microm at the surface of the channel. The microchannel was operated at residence times of less than 1 s ensuring high-mass-transfer rates. It was confirmed, both by microscopic observations as well as computational fluid dynamics (CFD) studies that the flow characteristics in the microchannel were fully laminar. Special attention was paid to flow splitting at the end of the channel, because the CFD simulations indicated that the performance of the device was sensitive to unequal flow splitting. The difference in outflow volume of the two streams was measured to be small (1.25% +/- 0.6%). The measured glucose concentration in both exit ports at a fixed residence time was found to be stable in time and reproducible in multiple experiments. CFD simulation was shown to be a powerful tool for estimating the mass transfer in the LFDI, even at very short residence times. The results obtained in this work show the applicability of LFDI for the controlled diffusive supply of a solute to a water stream, with as possible application substrate and/or precursor feeding to microreactors., ((c) 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009.)

We report on the synthesis of siloxane-based monoliths in the presence of a two-dimensional, perfectly ordered array of micro-pillars. Both methyltrimethoxysilane- and tetramethoxysilane-based monoliths were considered. The obtained structures were analyzed using scanning-electron microscopy and can be explained from the general theory of surface-directed phase separation in confined spaces. The formed structures are to a large extent nearly exclusively determined by the ratio between the bulk domain size of the monolith on the one hand and the distance between the micro-pillars on the other hand. When this ratio is small, the presence of the pillars has nearly no effect on the morphology of the produced monoliths. However, when the ratio approaches unity and ascends above it, some new types of monolith morphologies are induced, two of which appear to have interesting properties for use as novel chromatographic supports. One of these structures (obtained when the domain size/inter-pillar distance ratio is around unity) is a 3D network of linear interconnections between the pillars, organized such that all skeleton branches are oriented perpendicular to the micro-pillar surface. A second interesting structure is obtained at even higher values of the domain size/inter-pillar distance ratio. In this case, each individual micro-pillar is uniformly coated with a mesoporous shell.

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 microm 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 microm 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 microm and 18 microm 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.

The current paper describes the development and characterization of a pillar array chip that is constructed out of a sandwich of cyclo olefin polymer (COP) sheets. The silicon master of a 5 cm long pillar array was embossed into the COP, yielding 4.3 microm deep pillars of 15.3 microm diameter with an external porosity of 43 % and a well designed sidewall region to avoid side wall induced band broadening. A closed channel configuration was obtained by pressure assisted thermal bonding to a non-processed COP lid. Injection of coumarin dye plugs and detection with a fluorescence microscope showed very close agreement of this channel configuration to theoretical expectations in terms of band broadening. This agreement is due to the low taper, the optimized sidewall region and the excellent bonding quality between the two polymer sheets, even at the pillar area. Under non-retained conditions (pure methanol as mobile phase), plate heights as low as 4 microm were obtained. Under retained conditions, using the native hydrophobic properties of the COP channel (in 70/30 v/v water/methanol mixture as mobile phase), a minimum plate height of 6 microm was obtained. A 4 component separation was successfully achieved, demonstrating that COP is a cheap and efficient alternative for silicon and silica based liquid chromatography formats.

The present paper provides a detailed analysis of the analyte-wall adsorption effects in nanochannels, including a random walk study of the analyte-wall collision frequency, and uses these insights to estimate wall desorption times from chromatographic experiments in nanochannels. Using coumarin dye analytes and using a methanol/water mixture buffered at pH 3 in 120-nm deep channels, the surface desorption times on naked fused-silica glass were found to be maximally of the order of 60 to 150 mus, while they were found to be on the order of 100 to 500 mus on a hydrophobically coated wall. These nonzero adsorption and desorption times lead to an additional band broadening when conducting chromatographic separations. Shear-driven flows, requiring a noncoated moving wall and a stationary coated wall, intrinsically turn out to be more prone to this effect than pressure-driven or electro-driven flows for example. The present study also shows that, interestingly, the number of analyte-wall collisions increases with the inverse of the channel depth and not with its second power, as would be expected from the Einstein-Smoluchowski relationship for molecular diffusion.

We developed and applied a conductometric method for the quantitative online measurement of the carbon dioxide (CO(2)) production during batch cultivations of Candida utilis on a 100-microL scale. The applied method for the CO(2) measurement consisted of absorption of the produced CO(2) from the exhaust gas of the microbioreactor in an alkali solution, of which the conductivity was measured on-line. The measured conductivity change of the alkali solution showed a linear relation with the total amount of CO(2) absorbed. After calibration of the CO(2) measurement system, it was connected to a well of a 96-well microtiter plate. The mixing in the well was achieved by a magnetic stirrer. Using online measurement of the CO(2) production during the cultivation, we show reproducible exponential batch growth of C. utilis on a 100-microL scale. The CO(2) production measurements obtained from the microcultivation were compared with the CO(2) production measurement in a 4-L bioreactor equipped with a conventional off-gas analyzer. The measurements showed that on-line measurement of the CO(2) production rate in microbioreactors can provide essential data for quantitative physiological studies and provide better understanding of microscale cultivations., (2009 American Institute of Chemical Engineers)

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.

The interesting possibilities offered by micropillar array columns to investigate the relation between bed heterogeneity and band broadening in chromatographic columns are illustrated and investigated. The perfect control over the microscopic bed structure offered by the photolithographic fabrication technique could be used to produce a heterogeneous bed displaying a short-range interchannel heterogeneity and having exactly the same particle size and external porosity as the perfectly ordered bed that was used as the reference system. According to this approach, any observed difference in band broadening could be directly owed to the effect of the bed heterogeneity. Knowing that the only difference between the investigated beds was their degree of heterogeneity, the obtained plate height measurements provided a unique test to distinguish among the abilities to recognize short-range interchannel heterogeneity of the different existing plate height models. Interpreting the data with the classical van Deemter and Knox models, it was found that the increased bed heterogeneity leads not only to an increase in the A-term constant but also to a clear increase in the C-term constant.

Experimental measurements of the retention capacity and the band broadening in perfectly ordered porous shell pillar array columns are presented for a wide range of retention conditions and layer thicknesses. The porous silicon shells were obtained using electrochemical anodization of the solid silicon pillars obtained using deep reactive ion etching. Using 10-microm-wide pillars, minimal reduced plate height values of the order of h(min) = 0.4-0.5 were obtained under nonretained conditions, even in cases where the outer shell made up 20% of the total diameter. Under retained conditions, minimal plate heights around h(min) = 0.9 were obtained, even at retention factors up to k' = 12. Using a model based on Giddings nonequilibrium theory, and using a newly calculated value for the stationary zone configuration factor for the case of porous shell cylinders, a plate height model describing the band broadening in porous shell pillar arrays has been established. The validity of this model is demonstrated by showing that the geometrical parameters appearing in the model and fitted using band-broadening measurements under nonretained conditions can be used to relatively accurately predict the band broadening under retained component conditions. Using this model, some speculations on the ultimate performance of porous pillar array columns could be made.

The present study reports on the exploration of the separation speed limits of RPLC chromatography in open-tubular channels. Applying the shear-driven chromatography principle in a 120-nm deep open channel, and using an improved detection set-up, the separation of three coumarin dyes was detectable 8mm downstream of the injection point. At this distance, separation efficiencies of N = 17,900 - 24,100 plates were obtained at a velocity of 10 mm s(-1), corresponding to a plate generation velocity of 21,100 to 28,300 plates per second for the most and least retained component, respectively.

This article shows the development and testing of a microchip with integrated electrochemical sensors for measurement of pH, temperature, dissolved oxygen and viable biomass concentration under yeast cultivation conditions. Measurements were done both under dynamic batch conditions as well as under prolonged continuous cultivation conditions. The response of the sensors compared well with conventional measurement techniques. The biomass sensor was based on impedance spectroscopy. The results of the biomass sensor matched very well with dry weight measurements and showed a limit of detection of approximately 1 g/L. The dissolved oxygen concentration was monitored amperometrically using an ultra-microelectrode array, which showed an accuracy of approximately 0.2 mg/L and negligible drift. pH was monitored using an ISFET with an accuracy well below 0.1 pH unit. The platinum thin-film temperature resistor followed temperature changes with approximately 0.1 degrees C accuracy. The dimensions of the multi sensor chip are chosen as such that it is compatible with the 96-well plate format., (Copyright 2007 Wiley Periodicals, Inc.)

The present paper describes a method for the production of partly porous micro-pillars in columns suitable for use in liquid chromatography. These layers increase the available surface at least two orders of magnitude without destroying the huge benefits of the ordered nature of the system. A process flow was developed that enabled us to create a 550 nm thick porous layer on the pillar array in a sealed channel configuration, withstanding pressures up to at least 70 bar. Measuring band broadening under non-retained conditions, only a modest increase in plate height was observed in the porous pillar array as compared to that in a non-porous pillar array. The homogeneity of the layers was demonstrated using an optical microscope and SEM pictures and by monitoring peak velocities at constant pressures. The internal porosity was determined using particles with a diameter larger than the mesopores in combination with a dye that could penetrate into the pores.

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.

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.

The present paper reports on the experimental difficulties encountered when trying to realize the full potential of shear-driven chromatography in nanochannels. While it theoretically offers the possibility to yield over 10,000 plates per centimetre in a few seconds, the practical realization of this potential requires a detector miniaturisation that is carried to the extreme combined with very high sampling rates. In the present study, a charge coupled device camera and a photomultiplier tube combined with pinhole were tested as detector. Despite the fact that the photomultiplier tube could offer a higher sampling rate and a better sensitivity, the charge coupled device turned out to be better suited for the current set-up because of inevitable problems with the stray-light transported through the glass channel wall. The chemistry of the separation surface was additionally studied getting more homogenous coating, thus higher separation efficiency. Having also carried out a number of mechanical improvements, it is now possible to measure separations at a distance of 8mm downstream from the injection point. This is four times further downstream than ever before while realizing a four components mixture separation in less than 1.5s, with a plate generation velocity of about 2000-7000 plates per second depending on the sample.

The present paper reports on an experimental study of the possibility to use a micro-machined detection groove to enhance the detection sensitivity in flat-rectangular nano-channels for ultra-rapid liquid chromatography separations. Transversally running detection grooves with three different axial widths (respectively, 2, 4 and 6 microm) and one depth (4.75 microm) were tested in glass and silicon channels for the whole range of detectable fluorescein isothiocyanate isomer I, FITC, concentrations. The groove with the most square-like cross-section (i.e., 4 microm wide and 4.75 microm deep) yielded the best combination of detection gain and minimal additional band broadening. In a 1cm long channel, the effective plate loss caused by the 4 microm wide groove would only be of the order of 20%, while the gain in S/N-ratio was of the order of a factor of 5. The detection groove concept yields larger gains in silicon channel substrates than in glass channel substrates, due to the larger stray light losses occurring in the latter.

This paper describes a method to automatically and reproducibly inject sharply delimited sample plugs in the shallow (i.e., sub-micron) channels typically used in shear driven chromatography. The formation of asymmetric plugs, which typically occurs during loading of the sample in wide channels, is circumvented by etching a slit in the middle of the channel that is connected to a micro-well and a vacuum system with syringes for the supply of both the analyte and the mobile phase. The design of the injection slit was supported by a series of CFD simulations to optimize its shape and that of the corresponding injection well. The system was intensively tested experimentally and showed good reproducibility, both for the width and the area of the injected peaks (relative standard deviations are max. 4 and 6%, respectively). The concentration of the injected plug was found to be approximately 80% of the original sample concentration. It was also observed that with the current setup the lower limit of the peak width was about 120 microm. This is a consequence of the fact that the peak width originating from the convection filling step becomes negligible to the contribution of diffusion during the filling and flushing time. Being fully automated and perfectly closed, the presently proposed injection system also paves the way to integrate other functionalities in shear driven chromatography, i.e. gradient elution and parallelization.