Electrophoretic Separations

Much of the literature available on chip-based elec-trophoretic separations features capillary zone elec-

trophoretic (CZE) separations; however, there are many other types of separation possible, such as iso-tachophoresis and electrokinetic focusing.

Perhaps the simplest applications are based on CZE within silica microchannels. Here EOF and elec-trophoretic mobility can be utilized, the EOF for injection and bulk flow of solutions through the capillary, and electrophoretic mobility for the actual separation process. A typical separation capillary would be 50 mm long, 45 |im wide and 8 |im in depth, with an applied potential in the range 600-1200 V along the 50 mm length. The types of samples that can be separated by this technique are extensive (not surprising, given the diversity of the applications for conventional CZE) but include small anions and cations, monoclonal antibodies, theophylline and DNA fragments. There are a number of potential detectors, but those based on optical or electrochemical methods are the most frequently used.

Electrochemical detection can easily be incorporated on to a microchip, but requires the detector to be located after the high voltage section of the channel. This is necessary to prevent the high voltage causing interference with the detection. This can be achieved in such a system as described above, by locating the electrochemical detector in the channel just after the ground electrode. The EOF occurring in the channel would pump the fluid along the channel from the ground electrode to the detector electrodes. Over this short region, band broadening should not pose a significant problem. It is similar in principle to the porous junction technique widely used in conventional CE. It is possible to achieve limits of detection of micromolar levels or better with electrochemical detection.

Spectroscopic methods fall into two main classes - absorbance and fluorescence. Absorbance measurements are simple to effect, but commonly suffer from relatively low sensitivity. This is primarily due to the channel dimensions resulting in a very small path length. Measurements across a 50 | m channel would give rise to a very small absorbance, since absorbance is proportional to path length. It is possible to increase the path length (Figure 5), but absorbance measurements do not have the sensitivity of fluorescence measurements, although they are generally applicable to a wider range of analytes. In addition, for practical reasons, dual-channel systems are not easily set up and this can lead to instability in the detector signal.

Fluorescence measurements can provide limits of detection in the picomolar range (varying from around 2pmolL~1 upwards), and have even been reported for counting single chromophore molecules. Generally, the excitation source is directed along the

Figure 5 UV detection can be made more sensitive by increasing the path length of the measurement. When the absorbance of the analytes is measured across the channel at point x, the path length is equal to the channel width (typically 50 |im). By making the measurement at point y the path length is equal to the channel length (typically 3-5 mm).

Figure 5 UV detection can be made more sensitive by increasing the path length of the measurement. When the absorbance of the analytes is measured across the channel at point x, the path length is equal to the channel width (typically 50 |im). By making the measurement at point y the path length is equal to the channel length (typically 3-5 mm).

occur at potentials of 100-200 V cm"1, and can be used to drive fluids through channels, and indeed physical objects such as cells, e.g. Escherichia coli. It is possible to transport whole cells around the channels on a microchip. In addition to the EOF, there will also be electrophoretic separations occurring, but in practice, these are small compared to the EOF on uncoated silica surfaces. To be of practical use, it is necessary to have the ability to make meaningful measurements on the contents of the cells. This can be most easily achieved by lysis of the cells with detergent. It would then be possible to measure compounds, which would otherwise have been trapped within the cell wall. Since the volume of the channels is small, the released compounds will not be extensively diluted, and the time to analysis will be very short; this is particularly important if the aim is to study rates of reaction or unstable compounds.

EOF serves to deliver the sample beyond the high voltage area if it is intended to use off-chip detection. For example, to transfer the separated compounds from a separation chip to a mass spectrometer, EOF can be used to deliver the compounds to an electrospray interface. Indeed, it is possible to generate the electrospray between the terminal end of the capillary and a suitably located conductor, without the need to apply a conductive coating to the end of the chip.

channel, in order to minimize the scatter from the walls of the channels. By careful alignment, it is possible to minimize the background, and obtain very sensitive measurements. There are many other detection methods, including optical waveguide sensors and chemiluminescence, but fluorescence detection currently offers the most sensitive analysis.

Similar results can be obtained with channels produced from polymeric support materials. There is one issue that must be addressed with certain materials, e.g. plastics; that is, the background fluorescence that is frequently observed. This can be due to the actual substrate, or the adhesive used to seal the chip. Careful selection of materials helps to reduce the problem. However, it is the prospect of the mass production of thousands of chips with hundreds of channels per chip from just one master template that is particularly attractive. Once mass production is achieved, the devices will become truly disposable.

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