Liquid chromatography on-chip: progression towards a μ-total analysis system

Abstract

After a brief introduction to the area of miniaturised analytical separation systems, this paper details the fabrication of components for a miniaturised liquid chromatography system. To date, inlet and outlet connection ports, separation μ-channels (20–200 μm in width, 0.5–10 μm in depth, 15–50 cm in length), and an intersection for picolitre injection have been etched on a silicon wafer and then sealed with a Pyrex cover plate on which platinum electrodes for on-chip detection have been machined. In addition the μ-channel walls have been chemically modified with n-octyltriethoxysilane and the reverse phase retention of the drug caffeine was observed using off-chip injection (20 nl) and UV detection (3 nl flow cell). The on-chip platinum electrodes have been used for the amperometric detection of phenol. In addition, using external valving, initial results indicated a successful mode for a partially integrated on-chip injector which should allow further progression towards a μ-chromatographic analysis system.

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Introduction

In recent years there has been a growing interest and activity in the area of miniaturised analytical systems, with gas chromatography, capillary electrophoresis, liquid chromatography and ion chromatography systems all been miniaturised on silicon or Pyrex substrate with varying degrees of success.^1,2 The microfabrication technologies employed were initially developed for the microelectronics industry, where they allow the precise and reproducible construction of structures ranging from simple channels to pumps with moving parts on silicon and silicon compatible materials. The main incentives in miniaturisation include a reduction in the consumption of reagents and sample, increased analytical performance, shorter analysis time, and applicability to process and field analysis. The overall goal is progression towards a μ-total-analysis system, a system whereby chemical information is periodically transformed into an electronic or optical signal; analysis is carried out on a micrometer scale using centimeter-sized glass, silicon or plastic chips.³ While tremendous advances have been made in the miniaturisation of capillary electrophoretic separation systems, the challenges of microfabrication of an efficient μ-LC system remain.^1,2,4 By exploiting micromachining technologies it is relatively easy to fabricate a large number of columns reproducibly; however there are a number of problems, in particular the sealing of the μ-channels to allow the mobile phase to be pumped through the chip at pressures exceeding 2000 psi. Also, there are many difficulties associated with coupling to external instrumentation without leakage and without the introduction of dead volumes. In 1990 Manz et al.⁵ published research on the design of an open tubular liquid chromatography column using silicon chip technology and their results concluded that such a device could be competitive with an array of chemical sensors. Research at the Laboratory of the Government Chemists⁶ showed on-chip separation with amperometric detection. In 1995 Verpoote et al.⁷ slurry packed rectangular channels on chip using a microfabricated frit to retain the particles. Further developments were made by Regnier et al.⁸ whereby collocated monolith support structures were micromachined within the microchannels, therefore circumventing the need for packing particles in the column. They concluded that this could provide a route to highly efficient micro- and nanovolume columns.

Despite the contributions of these papers, significant research work is still necessary in order to achieve the ultimate goal of μ-TAS incorporating liquid chromatography on-chip. In this paper, we describe the fabrication of the components of the miniaturised liquid chromatography system, the reversed phase retention of a drug substance on a coated μ-channel, the on-chip amperometric detection of phenol, and a mode for on-chip injection, as a significant step in the progression towards a μ-chromatographic analysis system.

Experimental

Materials and reagents

Methanol and acetonitrile (HPLC Grade) were obtained from Labscan (Dublin, Ireland). Caffeine, PEEK tubing, PEEK sleeves and zero dead volume fittings were obtained from Sigma Aldrich Ltd. (Dublin, Ireland). The derivatization agent, n-octyltriethoxysilane was purchased from Fluorochem (Derbyshire, UK). Potassium dihydrogen orthophosphate, disodium hydrogen orthophosphate, and phenol (analytical grade) were obtained from BDH (Poole, UK). Sodium chloride was purchased from Merck (Germany). Fused silica capillary (50 μm id, Type TSP050150) was purchased from Composite Metals Ltd. (Hallow, Worcester, UK). Gold wire (0.125 mm id) was obtained from Goodfellow (Cambridge Ltd., UK).

Instrumentation

An Ultra Plus micro-pump system (Micro-Tech Scientific, USA) together with a 20 nl manual injector (Presearch Ltd., UK) was employed for off-chip pumping and injection. A Shimadzu SPD 6A UV detector (Shimadzu, Japan) fitted with a 3 nl flow cell (Presearch Ltd.) was used for spectrophotometric detection. An LC-4B and CC-4 (Biotech Instruments Ltd., UK) provided the electronics for amperometric detection. Two platinum electrodes on-chip and a Ag/AgCl reference electrode, immersed in a small vial with the external connecting capillary from the chip, constituted the three-electrode amperometric detection system. Both the UV detection and amperometric detection outputs were recorded by an HP3392A integrator (Hewlett-Packard, USA). Connections were realized using PEEK sleeves and standard LC zero dead volume fittings. For testing on-chip injection, electropneumatic valves (Ireland Valve & Fittings, Dublin, Ireland) were employed.

Fabrication

The miniaturised liquid chromatography chips were fabricated using standard photolithography, wet and dry chemical etching, and bonding techniques described previously.⁴ Chip layout was designed on a Sun Sparc workstation at the National Microelectronics Research Centre (Cork, Ireland). The separation channels and intersection for injection (Fig. 1) were micromachined on 4 inches <100> orientation silicon wafers. These channels were isotropically etched to depths of 0.5–10 μm using the dry etchant SF₆. Using a second photolithography stage and anisotropic etching, V groove connection ports were micromachined on the same wafers, into which connecting capillaries could be inserted. The platinum electrodes (Fig. 2) for electrochemical detection were micromachined on a Pyrex cover plate. Finally, the patterned glass silicon wafer was aligned to the patterned Pyrex cover plate and anodically bonded to form a seal between the silicon and glass (Fig. 3). After diamond sawing to reveal the V grooves, fused silica capillaries were inserted into the grooves and held in place using a non-conductive epoxy. Standard LC fittings were then used to connect the capillaries to the pump, injector, and detector. Finally, the electrodes were accessed by inserted gold wires (0.125 mm id) into the grooves and held in place using a conductive epoxy to ensure electric contact.

Fig. 1 Micromachined channels and intersection for on-chip injection.

Fig. 2 Platinum electrodes on-chip.

Fig. 3 Components of a liquid chromatography system on-chip.

Results and discussion

Separation and retention of components with on-chip coated μ-channels

Previous results from this laboratory have shown the on-chip separation of the test solutes benzamide and biphenyl in less than two minutes in μ-channels coated with n-octyltriethoxysilane. The chromatographic efficiency was low, due to a number of factors including the volume of injection.⁴ In this more recent work the on-chip retention of a pharmaceutical drug substance, caffeine, is demonstrated. The separation μ-channels (100 μm in width, 5.3 μm in depth, 16 cm in length) were chemically modified with an n-octyltriethoxysilane phase (0.5 g of n-octyltriethoxysilane in 5 ml of toluene) and then interfaced with the nano-LC system (micro-pump and UV detector with 3 nl flow cell). The measured flow rate and injection volume were 400 nl min⁻¹ and 20 nl, respectively, and the off-chip UV detectior was set at 254 nm. Using a mobile phase of 0.025 mol l⁻¹ phosphate buffer at pH 2.5 and acetonitrile (98 + 2), the reversed phase retention of caffeine was obtained (Fig. 4). While the unretained component eluted after 39 s, caffeine was retained and eluted after 111 s. The chromatography of caffeine, salicylamide, and salicyclic acid has also been studied, but baseline separation of these components has not yet been achieved. This may be attributed to the nature of the stationary phase coating on the μ-channel walls and the relatively large sample loading. To solve this problem, either a reduction in the injection volume, or improved coating with sol–gel technology⁹ or packing⁷ may be necessary in the future. The results, which have been obtained so far, demonstrate the feasibility of a miniaturised system; however the use of off-chip injection and detection contribute significantly to band broadening. According to the scaling factor for nanoscale liquid chromatography, the injection and detection volumes should be in the low nanoliter to picoliter range for this system.¹⁰

Fig. 4 Chromatogram showing retention of caffeine (t_R = 111 s) on a coated μ-channel. Mobile phase: phosphate buffer pH 2.5–acetonitrile (98 + 2); injection volume: 20 nl; UV detection: 254 nm.

On-chip electrochemical detection

In order to reduce detector volume greatly, on-chip detection is required. Electrochemical detection offers a cheap, small, and sensitive alternative to the currently employed laser induced fluorescence (LIF) detection.¹¹ With on-chip microelectrodes, conductometric detection of sodium nitrate has previously been shown.⁴ However, amperometric detection is also possible with the on-chip electrodes. Amperometric detection was performed in the three-electrode mode with the upstream electrode as the working electrode and the electrode closer to the exit as counter electrode, respectively, together with an external Ag/AgCl reference electrode. Phenol standards with different concentrations (5–100 mmol l⁻¹) were injected (20 nl) and amperometrically detected in an FIA mode with 50 mmol l⁻¹ NaCl as the carrier stream. The potential applied was +1.0 V. The preliminary results (Fig. 5) show both the feasibility of on-chip amperometric detection and a good linearity between peak current and concentration (R = 0.989). As the electrode area is 100 × 80 μm² each, the corresponding detector volume or cell volume is as low as 0.052 nl or 52 pl (channel depth is 6.5 μm). This on-chip amperometric detector will be applied later in conjunction with chromatography on-chip and improved efficiency is expected.

Fig. 5 Amperometric detection of phenol in a μ-channel with on-chip microelectrodes. Potential: +1.0 V vs. Ag/AgCl; injection volume: 20 nl; carrier stream: 50 mmol l⁻¹ NaCl; concentrations of phenol: 10, 25, and 50 mmol l⁻¹.

On-chip injection

As with the injection volume, a corresponding reduction in the injection volume is required; an on-chip injector was therefore designed and tested. The injection volume is dependent on the μ-channel depth, width, and also the intersection length, and ranges from the low nanolitres to picolitres. For testing, the chip was connected to four electropneumatic valves (Fig. 6),whose opening and closing were controlled by the application of a voltage, further controlled via a Labview program. Opening and closing the valves in sequence can control both the sample flow and carrier flow in the μ-channels. Initial program sequencing involves filling the μ-channel with mobile phase, the ‘carrier in valve’ was then closed and sample solution introduced. After the sample was loaded, the injection was made by a further valve switching to direct the sample plug through the μ-channel.This mode of injection has been followed using colored indicator solutions and well defined sample plugs were successfully injected. Further research goals involve combining on-chip injection, separation and electrochemical detection for the integrated μ-liquid chromatographic analysis system.

Fig. 6 Schematic of on-chip injection set-up.

Conclusion

Important components for μ-LC, such as injector, separation column, detector, and connection ports have been fabricated on-chip and tested separately. The preliminary results have already shown the feasibility of the endeavor. Even though much work is required to combine all the functional parts to work simultaneously in a chromatographic mode, the achievements made so far definitely represent a significant step in the progression toward the objective of a μ-liquid chromatographic analysis system.

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Footnotes

† Presented at the SAC 99 Meeting Dublin, Ireland, July 25–30, 1999.

‡ On leave from the Department of Chemistry, Central South University of Technology, Changsha 410083, China.

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