Liquid Chromatographic Separations with Solvent Programming on Microfabricated Devices

For many analytical tasks, such as environmental monitoring or industrial process control, a range of neutral analytes are present. These samples require both, pretreatment and separation and chromatography-based methods are widely accepted means of analysis. There are several different options to realize liquid chromatographic systems in microchips. Micellar electrokinetic chromatography (MEKC), which has been demonstrated on microchips by this group [1], makes use of partitioning of analytes between a micellar, hydrophic phase and the hydrophilic buffer. Separation is based on the different partition coefficients and the resulting differences in migration times. Another possibility uses a true stationary phase consisting of a long hydrocarbon chain chemically bonded to the channel walls. This technique is dubbed open channel electrochromatography (OCEC), and has also been demonstrated on microchips [2]. Similar to MEKC, the separation can be attributed to different partitioning between the hydrophobic layer at the wall and the hydrophilic buffer.

In both techniques, the buffer (mobile phase) is transported through the channel manifold using the electroosmotic flow (EOF). The primary interface to control this flow is through the adjustment of potentials applied to the reservoirs at the end of the channels. Together with the ease of manufacturing interconnected channels (where the connections are virtually dead-volume-free) this opens the possibilities to implement different types of fluidic manipulation protocols on microchips. One such protocol which is very advantageous and powerful for chromatographic separations is solvent programming.

The partitioning equilibrium of an anlyte between the stationary phase and the mobile phase can be influenced by a number of parameters, the most important of which is the amount of organic modifier (e. g., methanol or acetonitrile) which is present in the mobile phase. Variations in the contents of organic modifier affect the chromatographic performance as indicated by resolution, selectivity, plate numbers, and analysis time. A stepwise, discontinuous change in the solvent strength or elution strength of the buffer can already lead to the desired improvement in one or more of the above mentioned indicator parameters. More often, however, resolution is gained for the sacrifice of analysis time or vice versa. A continuous, gradual change in the solvent strength allows more fine-tuning of the chromatographic conditions, resulting in optimized separations. We have developed a microchip device which allows both, the discontinuous adjustment of the elution strength for every run (isocratic mode) as well as the continuous change during a run (gradient mode).

Figure 1 depicts a typical solvent programming microchip which features a mixing tee at one side of a basic cross. The voltages which were applied to the reservoirs have been calculated using Ohm's Law and Kirchhoff's Rules. During a gradient run, only the voltages at the two solvent reservoirs need to be adjusted - all other potentials are held constant to ensure a constant separation field strength in the separation channel. Figure 2 shows a separation of a coumarin dye mixture (5 components) using MEKC [3]. A linear gradient ranging from 14% acetonitrile to 30% over 10 seconds was employed. A similar coumarin dye mixture (4 components) was separated in OCEC with a linear gradient ranging from 29% to 50% acetonitrile over just 5 seconds (see Figure 3) [4]. In both cases the separations are performed in under 25 seconds with excellent efficiencies. The plate numbers for the later eluting peaks were determined to 99,900 (C480, Fig. 2), and 49,200 (C480, Fig. 3), respectively. Fast reconditioning times after a gradient run allow new injections after just about one minute. In combination with fast and easy adjustments in the gradient parameters (slope, shape, initial and final conditions) this approach is very conducive to optimizations of analytical separation problems.

More information can be found in the following papers:

[1] Moore Jr., A. W.; Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 1995, 67, 4184-4189.

[2] Jacobson, S. C.; Hergenröder, R.; Koutny, L. B.; Ramsey, J. M. Anal. Chem. 1994, 66, 2369-2373.

[3] Kutter, J. P.; Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 1997, 69, 5165-5171.

[4] Kutter, J. P.; Jacobson, S. C.; Matsubara, N.; Ramsey, J. M. Anal. Chem. 1998, 70, 3291-3297.

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