Gradient Elution Isotachophoresis (GEITP)

Elizabeth A. Strychalski and David Ross
National Institute of Standards and Technology, Gaithersburg, MD, 20899, USA

Gradient elution isotachophoresis (GEITP) is a fluid-phase electroseparation technique that combines the ability of isotachophoresis (ITP) to concentrate analytes with a pressure-driven counterflow for improved control and selectivity in the focusing process.1-3 Demonstrated originally with dye molecules, amino acids, proteins, and nucleic acids,2 GEITP uses a simple device geometry consisting of two reservoirs connected by a microfluidic capillary and, like ITP, requires two different buffer solutions (Fig. 1). A leading electrolyte (LE) solution is composed of electrophoretically fast ions, and a trailing electrolyte (TE) solution is composed of electrophoretically slow ions. One reservoir and the capillary are filled with LE solution, while the sample is dissolved or suspended in TE solution. The electrophoretic mobilities of the LE and TE ions bracket that of the analytes in the sample. Using a constant electric field applied through the fluid reservoirs, the analytes are driven from the sample reservoir toward the capillary, where they focus at the interface between the LE and TE solutions. With conventional ITP, this interface moves through the capillary toward the LE solution reservoir with a constant velocity that depends on the electrophoretic mobilities of the LE and TE ions and the magnitude of the applied electric field.4 Unlike conventional ITP, GEITP uses a controlled, variable, pressure-driven counterflow, which enables effective exclusion of contaminants from the capillary and control over the position of the focusing interface.

This ability to prevent contaminants from interfering with analysis is a central feature of GEITP. The variable pressure that controls the counterflow is applied to the headspace of the LE reservoir, while the TE reservoir is kept at ambient pressure. At the beginning of an analysis, the pressure is relatively large, so that fluid flows from the LE solution reservoir, fills the capillary, and pushes the ITP focusing interface into the TE solution reservoir. During analysis, the pressure is reduced to allow the focusing interface to enter the capillary and transport focused analytes past any detectors, such as a capacitively-coupled contactless conductivity detector or laser induced fluorescence detector. During this process, particulates, molecules, or other contaminants in the sample are excluded from the capillary, both by their relatively slow electrophoretic mobilities and their hydrodynamic transport from the counterflow.5, 6 In this way, much like in the related analytical technique gradient elution moving boundary electrophoresis (GEMBE),7, 8 these contaminants are not able to foul or clog the capillary and do not focus with the analytes of interest. The counterflow confers the additional benefit of enabling control over the location of the focusing interface in the TE solution reservoir or capillary, which can help tailor the amount of an analyte that is focused and allow the delivery of the focused analyte for subsequent analysis.

Figure 1. Device schematic and working principle for GEITP. (a) A typical device for GEITP consists of two fluid reservoirs connected by a microfluidic capillary. High voltage (HV) drives ITP transport from the TE solution reservoir containing the sample and analytes of interest towards the LE solution reservoir, while a pressure controller at the LE solution reservoir controls a hydrodynamic counterflow towards the TE solution reservoir. A microscope objective or other detection hardware can be situated along the capillary. (b) As for conventional ITP, GEITP uses LE and TE solutions and a constant voltage to focus analytes. The controlled, variable pressure-driven counterflow in GEITP can control the location of the focusing interface, for example, outside the capillary entrance in the TE solution reservoir, as shown here schematically. (Adapted from Fig. 1 of Shackman et al.2)

GEITP was developed with simplicity as a guiding principle, which has important consequences for the potential utility and impact of this analytical technique. GEITP features a simple device geometry, the absence of a sample injection step, and the ability to adjust analysis parameters in real time, which results in a rapid, robust, and miniaturizable analysis. These qualities, along with the ability to analyze crude samples, render GEITP amenable to implementation for practical applications either as a sample preparation method at the front end of a more comprehensive analytical system or outside a laboratory setting as a stand alone instrument.

References
  1. Mamunooru, M.; Jenkins, R. J.; Davis, N. I.; Shackman, J. G., Journal of Chromatography A, 2008, 1202 (2), 203-211.
  2. Shackman, J. G.; Ross, D., Analytical Chemistry, 2007, 79 (17), 6641-6649.
  3. Strychalski, E. A.; Konek, C.; Butts, E. L. R.; Vallone, P. M.; Henry, A. C.; Ross, D., Electrophoresis, 2013, 34 (17), 2522-2530.
  4. Goet, G.; Baier, T.; Hardt, S., Biomicrofluidics, 2011, 5 (1), 014109.
  5. Strychalski, E. A.; Henry, A. C.; Ross, D., Analytical Chemistry, 2009, 81 (24), 10201-10207.
  6. Strychalski, E. A.; Henry, A. C.; Ross, D., Analytical Chemistry, 2011, 83 (16), 6316-6322.
  7. Shackman, J. G.; Munson, M. S.; Ross, D., Analytical Chemistry, 2007, 79 (2), 565-571.
  8. Vyas, C. A.; Flanigan, P. M.; Shackman, J. G., Bioanalysis, 2010, 2 (4), 815-827