Difference between revisions of "Procedure for Microchip Electrophoresis with C4D with the ET121"

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(Positioning of the High Voltage Electrodes)
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=== Positioning of the High Voltage Electrodes ===
 
=== Positioning of the High Voltage Electrodes ===
 
[[File:Gas bubble in chip reservoir.jpg|300px|thumb|right|Gas bubbles forming inside the reservoir]]
 
[[File:Gas bubble in chip reservoir.jpg|300px|thumb|right|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.
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Applying a high voltage to a solution can cause the formation of gas bubbles at the electrodes. Species like OH<sup>-</sup>, H<sup>+</sup>, H<sub>2</sub> and O<sub>2</sub> can be produced, which can produce changes in the pH of the solution in the reservoirs. 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 ===
 
=== Chip Preparation ===

Revision as of 16:33, 23 August 2017

Equipment

Preparing the Hardware

The Microchip Electrophoresis Kit contains many components and cables. Please follow the instructions below in the order listed.

  1. Check you have all the components listed in the packing lists.
  2. Install the QuadSequencer software and PowerChrom software on the computer. Ensure you have installed the latest versions of softwares from https://www.edaq.com/software-downloads
  3. Connect the High Voltage Sequencer (HVS) to the computer with a USB cable, as described in the instruction manual. Connect the power cable. Do not turn it on yet.
  4. Connect the C4D Data System to the computer with a USB cable, as described in the instruction manual. Connect the power cable. Do not turn it on yet.
  5. Connect the Chip Platform to the front of the C4D Data System.
  6. Connect the coloured EC230 High Voltage Cables to the HVS as follows: blue cable to Output 1, red cable to Output 2, yellow cable to Output 3, and black cable to Output 4.
    • You will need to create an interlock system for the HVS. Connect the interlock system between between the Chip Platform and the HVS. Warning: the interlock is designed to disarm the HVS if the interlock is open. Do not try to bypass the interlock.
  7. Grounding is important. Use a green ground cable to connect the at the back of the C4D Data System, to the green connector at the back of the HVS.
    How to set up the HV Sequencer to Trigger PowerChrom Software
  8. The HVS can trigger the PowerChrom software to start recording. Use the red and black EC201 trigger cable to connect the “CTL1 +” at the back of the HVS to the “TRIG +” at the back of the C4D Data System. Connect the HVS “CTL1 –” to the C4D “TRIG –”. Make sure you push the connection port block fully into each hardware unit. See the video (The software screen shots may look a bit different).
  9. Turn on the HVS. The driver will be installed if this is the first use.
  10. Open the QuadSequencer software. Move and resize the window so it occupies the left half of the screen.
  11. Turn on the C4D Data System. The driver will be installed if this is the first use.
  12. Open the PowerChrom software. Ensure the software has setup the hardware unit; you should see the Easy Access window, not the Hardware Unavailable window. Move and resize the PowerChrom software screen so it occupies the right half of the screen.
  13. Setup the trigger commands in the software packages as shown below:
    • QuadSequencer software: in the menu Setup,Hardware, for the Digital Outputs, select Contact Closure.
    • PowerChrom software: in the menu Edit, Preferences, Digital IO Settings, select External Trigger Mode as Voltage Level (TTL).
    • PowerChrom software: in the Manual Sampling window, Inject Settings, select Wait for Inject.
    • Sequencer software: in the later step, when you are setting up the sequence, remember to select High/Closed, under Digital Output 1, during the separation step, as this will send the trigger command. Also remember to select Low/Open in the very last step (after the separation is complete).
  14. Use gloves to place the empty microchip on the Chip Platform. If you look at the chip closely, you may see the channel running along the chip. Place the channel above the white line on the platform. This will ensure that the channel sits on top of the C4D electrodes, which is essential for the C4D to detect analytes in the channel. Position the chip so that the reservoir at the end of the chip is about 1 cm away from the gold C4D electrodes.
  15. Secure the chip to the platform using the rectangular block with screws. Check the chip's reservoir is on top of the gold C4D electrodes.

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. Position the high voltage electrodes into each reservoir (see note below about positioning of high voltage electrodes).
  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 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.
  10. Increase the current on reservoir 2 to 1000V to check in QuadSequencer software that 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

Gas bubbles forming inside the reservoir

Applying a high voltage to a solution can cause the formation of gas bubbles at the electrodes. Species like OH-, H+, H2 and O2 can be produced, which can produce changes in the pH of the solution in the reservoirs. 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.

The overshoot or “shoulders” on either side of each peak has been observed and report when using C4D with capillary electrophoresis, in research papers such as this one. The authors state this overshoot is “due to complicated vectorial sum of complex currents flowing in the spatially distributed RC network of the cell.”

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

Positioning the chip on the ET121

It is important that the microfluidic chip is accurately positioned on the ET121 platform. The channel inside the chip must sit directly above the golden C4D electrodes on the platform. The channel can be difficult to see with the naked eye.

One technique is to fill the channel with BGE or distilled water, and slide the chip while observing the C4D signal in PowerChrom software. Slide the chip until the max C4D signal is obtained, and then turn the two screws to secure the chip in that place

Hints and Tips

  • Make sure current is flowing! The cross display in QuadSequencer Software shows the current flowing through each reservoir; you should make sure there is current flowing where expected. Air bubbles in the channel can prevent the current from flowing and cause arcing which can result in high temperatures. If no current is flowing, you won't get any peaks! With 1000V across chip during injection, with acetic acid BGE in channel, you should get about 100 µA ; With 1000V applied along channel during separation, you should measure about 5-10 µA (or 10-20 µA when applying 2000V). If the voltage or current numbers are in red, there is an issue; see the QuadSequencer Software manual
  • You can increase high voltage (to 3000V for example) after the analyte peaks have been measured, to flush out the EOF more quickly. Note that this may cause Joule heating resulting in sloping baseline.
  • Don’t use C4D Profiler to try to obtain best C4D settings; the C4D Profiler gives C4D settings that give heights signal, but not best peak shape