Procedure for Microchip Electrophoresis with C4D with the ET121

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Equipment

How to Fill the Channel with BGE

Simply pipetting the background electrolyte (BGE) into reservoir of the ChipShop chip does NOT fill the main channel by capillary action! You must use syringe to push the BGE to fill main channel; this can be observed by looking at C4D signal and seeing a change in conductivity as the air is replaced with the BGE.

Here is a procedure:

  1. Have PowerChrom software running with 2V range, C4D settings: 100 % and 1000 kHz , signal zeroed
  2. Place 30 µL of BGE in reservoir 4 (50 µL is too much as it will overflow when the pipette is pushed into reservoir luer); capillary action alone doesn’t fill channel
  3. Place the syringe filled with BGE (make sure there is absolutely no air in the syringe or its luer neck) into reservoir 4 and push down gently so it makes seal with luer; this should fill the chip's channel with BGE, which can be observed by big drop in C4D signal (about 300 mV drop)
  4. Push on syringe while looking at reservoirs 1, 2 and 3 with magnifying glass; make sure you see BGE rising from the hole inside EACH reservoir; this ensures all the channels are filled, with no air bubbles.
  5. Remove BGE from all reservoirs
  6. Add 50 µL BGE to each reservoir
  7. Push the high voltage electrodes into each reservoir
  8. Use a magnifying glass to check each electrode is immersed in liquid, but is NOT at the very bottom of the reservoir inside the hole leading to the chip's channel.
  9. Apply 500 V at reservoir 2 and ground at reservoir 4; check there is a current, which shows there is no air gap; should see slight change in C4D signal when applying voltage along the channel; see note below about positioning of high voltage electrodes
  10. Increase the current on reservoir 2 to 1000V to check the current increases
  11. Apply the voltage across reservoirs 1 and 3 to check there is current flowing through these reservoirs as well.
  12. Check the currents are stable, not fluctuating.


Positioning of the High Voltage Electrodes

Figure . Gas bubbles forming inside the reservoir

Applying a high voltage to a solution can cause the formation of gas bubbles at the electrodes. This can prevent the flow of electrical current. It can also result in an electric arc which produces high temperatures and can damage the chip. You should avoid positioning the high voltage electrodes inside the hole at the very bottom of reservoir, that leads to the chip's channel. The formation of a gas bubble inside this hold could prevent the flow of current through the channel.

Chip Preparation

Unlike glass microfluidic chips, plastic chips don’t normally require to be preconditioned before use. Researchers using plastic chips such as PMMA poly(methylmethacrylate) have reported they simply flushed the chip's channel with deionized water and then background electrolyte for a few minutes each. At the end of a working day, the microchips should be rinsed with deionized water in order to prevent the formation of salts which may clog the channels.

Selection of C4D Frequency and Peak Shape

Selecting an unsuitable C4D frequency can greatly change the shape of the analyte peaks. The electropherograms below show the same analysis recorded using different C4D frequencies. When measuring potassium, sodium and lithium in acetic acid, negative peaks are expected. The first run at a C4D frequency of 1000 kHz produced what looked like three triplet peaks. When the frequency was reduced to 700 kHz, the negative peaks became more obvious. The optimum frequency was found to be at 250 kHz.

Figure . 1000 kHz: three triplet peaks
Figure . 700 kHz: negative peaks are more obvious
Figure . 250 kHz: optimum frequency