Difference between revisions of "Procedure for Microchip Electrophoresis with C4D with the ET121"
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# Remove BGE from all reservoirs. | # Remove BGE from all reservoirs. | ||
# Add 50 µL BGE to each reservoir. | # Add 50 µL BGE to each reservoir. | ||
− | # | + | # Position the high voltage electrodes into each reservoir (see note below about positioning of high voltage electrodes). |
# 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. | # 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. | ||
− | # Apply 500 V at reservoir 2 and ground at reservoir 4; check there is a current | + | # Apply 500 V at reservoir 2 and ground at reservoir 4; check there is a current flowing along the channel of the chip in QuadSequencer software. A flowing current shows there is no air gap. You should also see slight change in C4D signal (in PowerChrom software) when applying a voltage along the channel. |
− | # Increase the current on reservoir 2 to 1000V to check the current increases. | + | # Increase the current on reservoir 2 to 1000V to check in QuadSequencer software that the current increases. |
# Apply the voltage across reservoirs 1 and 3 to check there is current flowing through these reservoirs as well. | # Apply the voltage across reservoirs 1 and 3 to check there is current flowing through these reservoirs as well. | ||
# Check the currents are stable, not fluctuating. | # Check the currents are stable, not fluctuating. |
Revision as of 17:35, 24 May 2017
Contents
Equipment
- ER225 C4D Data System
- ET121
- ER430 High Voltage Sequencer
- EC230 High Voltage Cables
- Computer with ** PowerChrom and Sequencer softwares
- EC20 Standard Test Solutions:
- BGE = 0.5M acetic acid
- Sample = 1mM LiCl, KNO3 and Na2SO4 in deionised water
- EC20 Standard Test Solutions:
- Chips
- start with ChipShop 02-0772-0202-05, Zeonor material (a type of Cyclic Olefin Copolymer (COC)), with 100 µm base
- then use custom made chip with 60 µm base (might be similar to 02-0772-0202-05)
- the channel has a square cross section (not circular); Main channel width, depth, length= 75 µm, 75 µm, 87 mm; 0.49 µL main channel volume; dimensions of chip File:1407 chip for rereleased ET121.pdf (68 KB PDF)
- One 20 - 200 µL pipette, with pipette tips
- One syringe 5 mL
- Deionised water
- Lint-free tissues
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:
- Have PowerChrom software running with 2V range, C4D settings: 100 % and 1000 kHz , signal zeroed.
- 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.
- 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).
- 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.
- Remove BGE from all reservoirs.
- Add 50 µL BGE to each reservoir.
- Position the high voltage electrodes into each reservoir (see note below about positioning of high voltage electrodes).
- 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.
- Apply 500 V at reservoir 2 and ground at reservoir 4; check there is a current flowing along the channel of the chip in QuadSequencer software. A flowing current shows there is no air gap. You should also see slight change in C4D signal (in PowerChrom software) when applying a voltage along the channel.
- Increase the current on reservoir 2 to 1000V to check in QuadSequencer software that the current increases.
- Apply the voltage across reservoirs 1 and 3 to check there is current flowing through these reservoirs as well.
- Check the currents are stable, not fluctuating.
Positioning of the High Voltage Electrodes
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.