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

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(Preparing the Hardware)
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#* PowerChrom software: in the Manual Sampling window, Inject Settings, select Wait for Inject.
 
#* 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). [[File:Figure 2. A microchip in the platform.JPG|300px|thumb|right|Figure 2. A microchip in the platform]]
 
#* 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). [[File:Figure 2. A microchip in the platform.JPG|300px|thumb|right|Figure 2. A microchip in the platform]]
# Use gloves to place the empty microchip on the Chip Platform.
+
# 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.
# Secure the chip to the platform using the rectangular block with screws.
+
# 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 ===
 
=== How to Fill the Channel with BGE ===

Revision as of 11:07, 25 May 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. 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. 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.
    Video 2. 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 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 –”. See Video 2.
  9. Turn on the HVS. The driver will be installed if this is the first use.
  10. Open the QuadSequencer software and ensure you are able to connect to the HVS by clicking on the Online button at the top of the screen. You may need to go into the File, Preferences menu to select the correct Connection Serial Port for the HVS. You can use the Serial Port Monitor software (from the eDAQ website) to see which COM port the HVS is connected to. Move and resize the Sequencer software screen 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).
      Figure 2. A microchip in the platform
  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

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