Hydrodynamic injection with pneumatic valving for microchip electrophoresis with total analyte utilization

Abstract

A novel hydrodynamic injector that is directly controlled by a pneumatic valve has been developed for reproducible microchip CE separations. The PDMS devices used for the evaluation comprise a separation channel, a side channel for sample introduction, and a pneumatic valve aligned at the intersection of the channels. A low pressure (≤ 3 psi) applied to the sample reservoir is sufficient to drive sample into the separation channel. The rapidly actuated pneumatic valve enables injection of discrete sample plugs as small as ~ 100 pL for CE separation. The injection volume can be easily controlled by adjusting the intersection geometry, the solution back pressure, and the valve actuation time. Sample injection could be reliably operated at different frequencies (< 0.1 Hz to > 2 Hz) with good reproducibility (peak height relative standard deviation ≤ 3.6%) and no sampling biases associated with the conventional electrokinetic injections. The separation channel was dynamically coated with a cationic polymer, and FITC-labeled amino acids were employed to evaluate the CE separation. Highly efficient (≥ 7.0 × 10³ theoretical plates for the ~2.4-cm-long channel) and reproducible CE separations were obtained. The demonstrated method has numerous advantages compared with the conventional techniques, including repeatable and unbiased injections, little sample waste, high duty cycle, controllable injected sample volume, and fewer electrodes with no need for voltage switching. The prospects of implementing this injection method for coupling multidimensional separations for multiplexing CE separations and for sample-limited bioanalyses are discussed.

Figure 1

Illustration of the microchip design and fabrication. A) Design of the microchip including flow channel (solid line) and control channel (dashed line). B) Schematic of the microchip fabrication. (a) Flow layer containing the sample injection and separation channels. (b) Control layer containing the valving channel on a thin PDMS film. (c) Unpatterned bottom layer to enclose the control channel. C) Template for flow layer fabricated with positive photoresist. D) Template for control layer fabricated with SU-8. E) Photograph of the final PDMS microchip. F) Photograph of the T intersection assembled with a pneumatic valve.

Figure 2

Schematic of the experimental setup for microchip CE separation using hydrodynamic injection method with pneumatic valving.

Figure 3

Micrograph sequences depicting a cycle of sample injection controlled by a pneumatic valve. The microchannel width was 100 μm. The valve was integrated at the interface with an area of 100 × 100 μm². The tested sample was fluorescein solution.

Figure 4

Fluorescence intensity of the multiple injected fluorescein samples at different frequencies indicated in each figure. Other operation conditions were described in section 3.2.

Figure 4

Fluorescence intensity of the multiple injected fluorescein samples at different frequencies indicated in each figure. Other operation conditions were described in section 3.2.

Figure 4

Fluorescence intensity of the multiple injected fluorescein samples at different frequencies indicated in each figure. Other operation conditions were described in section 3.2.

Figure 4

Fluorescence intensity of the multiple injected fluorescein samples at different frequencies indicated in each figure. Other operation conditions were described in section 3.2.

Figure 5

Characterization of hydrodynamic injection controlled by a pneumatic valve. A) Injected sample peak width versus valve actuation time, B) peak width versus sample injection pressure, C) peak width versus valve control pressure, and D) peak width versus valve actuation frequency.

Figure 5

Characterization of hydrodynamic injection controlled by a pneumatic valve. A) Injected sample peak width versus valve actuation time, B) peak width versus sample injection pressure, C) peak width versus valve control pressure, and D) peak width versus valve actuation frequency.

Figure 5

Characterization of hydrodynamic injection controlled by a pneumatic valve. A) Injected sample peak width versus valve actuation time, B) peak width versus sample injection pressure, C) peak width versus valve control pressure, and D) peak width versus valve actuation frequency.

Figure 5

Characterization of hydrodynamic injection controlled by a pneumatic valve. A) Injected sample peak width versus valve actuation time, B) peak width versus sample injection pressure, C) peak width versus valve control pressure, and D) peak width versus valve actuation frequency.

Figure 6

Microchip electrophoresis of four FITC-labeled amino acids. Peak identifications: (1) Asp, (2) Gly, (3) Phe, and (4) Arg. Applied electric field: 1kV/cm; valve control pressure: 40 psi; sample injection pressure: 3 psi; valve actuation time: 67 ms; valve actuation frequency: ~0.1 Hz.

Figure 7

Repeated microchip CE of three FITC-labeled amino acids. Peak identifications: (1) Gly, (2) Phe, and (3) Arg. The operation conditions were the same as those shown in Figure 6.