pH Gradients

Development of Radial pH Gradients within Micro-Capillaries using Electric Fields


Researcher

Aytug Gencoglu

Description and Motivation

The development of pH gradients can be observed in electrolytic solutions flowing through 20 micron ID capillaries using fluorescence video microscopy. The pH gradients are induced by electrolysis reactions at the electrodes in a linear electric field and are a function of electrolyte concentration. If this phenomenon can be quantified, microdevices can be easily screened to detect ion gradients and thus avoid unexpected behaviors in new device designs.

Fluorophores can be dissolved in the electrolytic solution which is electroosmotically driven through the capillary. A high powered fluorescent microscope with a pseudo-confocal attachment can be used to spatially detect the fluorescence intensity down the length of the capillary. The fluorophore chosen is a pH indicator called SNARF, a molecule having pH sensitive dual emission when excited near 600 nm. Another fluorophore that can be used is a Lysosensor probe, also pH-dependent. Typically, for both fluorophores, higher emission of light corresponds to higher concentrations of H+. These dyes are used to image the radial and axial pH gradient down the length of the capillary. As the pH rises, zeta potential, the charge distribution between the solution and the wall of the capillary, increases causing the electroendoosmotic flow (flow within a capillary induced by an electric field) to vary.

If you would like to learn more, we suggest the following reading in the literature:

  • Electroosmotic Capillary Flow with Nonuniform Zeta Potential. A.E. Herr, J.I. Molho, J.G. Santiago, M.G. Mungal, and T.W. Kenny. Stanford University. This article shows the study of electroosmotic flow in cylindrical capillaries with nonuniform charge distributions on the walls causing the flow to be altered.
  • Electroosmotic Flow in Channels with Step Changes in Zeta Potential and Cross Section. Brotherton C.M., Davis R.H. This article analyzes the effects that step changes in zeta-potential and cross section have on electroosmotic flow.
  • Electroosmotic Flow Control of Fluids on a Capillary Electrophoresis Microdevice Using an Applied External Voltage. Polson N.A., Hayes M.A. This article analyzes separation techniques such as capillary zone electrophoresis through electroosmosis.
  • Minerick, A.R., A. Ostafin, and H.-C. Chang. "Electrokinetic Transport of Red Blood Cells in Micro-Capillaries," Electrophoresis, 23, 2165-2173, 2002.

    • Data Collected

    To perform the experiment a microdevice was created by attaching two electrodes to wells on each end of a capillary. Platinum wire 100µm in diameter was used for electrodes while 20µm fused silica capillaries were employed. An electric field ranging from 12 to 50 V/cm was induced by a direct current generator over a capillary length of approximately 3 cm. As the experiment progressed, a pH gradient developed between the anode and cathode of the microdevice. A Zeiss Axiovert 200M inverted light microscope was used to take images down the length of the capillary. Images were taken at each end of the capillary near the microdevice wells as well as in the center of the capillary. Pixel values were analyzed to discern the differences in fluorescent intensities within the capillary. The differences in intensities were used to determine the pH at the different locations within the capillary.

    The pH gradient influences a voltage difference, called zeta-potential, between the walls of the capillary and the fluid. This has a tremendous impact on electroendoosmotic flow (EOF-the flow along a charged surface induced by an electric field). As pH drops, the negative charge of the fused silica wall decreases, thus decreasing the zeta potential between the wall and the electrolyte solution. As a result, the local electroosmotic flow decreases. As pH increases near the cathode well, the zeta potential increases, thus increasing local EOF. The net flow flux through the capillary is the same at all locations in the capillary due to mass balance kinematics. This causes the flow profile to be flat near the anode and curved due to a pressure driven backflow at the anode end of the capillary. Please see the attached figure borrowed from [4] above.

    Two-dimensional flow profiles showing development of pressure driven back flow opposing the electroendosmotic flow in a capillary.

    • Classic electroendosmotic flat velocity profile determined by the Smoluchowski slip velocity at the capillary wall.
    • Zeta potential begins to drop at the anode end of the capillary. A pressure driven backflow develops in the region of high zeta potential. Flow rate is controlled by the ζ (anode) region of the capillary.
    • Pressure driven back flow increases to the point where the center axis velocity is zero (l). At this point, ζ min (anode) = 1/2 ζ max (cathode).
    • Anode end surpasses the isoelectric point and EOF is now toward the anode. ζ max is still the same and a large back flow is present.

    Potential Impact

    As the use of microfluidic devices is increasing, the knowledge of behaviors of biological fluids and other electrolytes in electrical field modified channels is growing. However, the extremely small volumes lead to situations where ion concentration is limiting and complex fluid, wall, sample interactions occur. Quantifying and imaging gradients in pH are the first step in screening microdevices for unexpected affects on zeta-potential and thus electroosmotic flow. Prediction of results will influence future microdevice design.

    Funding

    SGER (Small Grants for Exploratory Research): Exploration and Quantification of Ion Gradients in a Capillary Microdevice, National Science Foundation: CBET 0636254, $50,000 (PI: Minerick), 9/1/06 – 2/28/08.

    • REU supplement, $5,999, 5/1/07 – 2/28/08