It is important to establish a baseline prior to undertaking actual experiments because absorbance measurements are used to detect analyte. Establishing a baseline ensures that the UV-vis aborbance detector is working properly and conditions inside the capillary are conducive to successful analyte detection. In this section a baseline will be established for use in Capillary Electrophoresis (CE). Because the HV Power Supply will be used during this process, please review the Safety Precautions for using high voltage prior to turning on the power supply.
The data collection software used in Learning Modules I – IV is IGOR Pro 5.0 with NIDAQ Tools by Wavemetrics, Inc. All software references in this section pertain to IGOR Pro. If you are using different data collection software, please refer to the user’s manuals accompaning that software.
- Make sure the black control switch located on the three-way valve is turned towards the solenoid (pressure only introduced via the electronically actuated solenoid valve) while establishing a baseline.
- Check to see that the power source is properly grounded. Make sure the anode and cathode vials contain equivalent volumes of the same buffer, and the electrodes and capillary are adequately immersed in buffer.
- Turn on the voltmeter and set it to read the resistance. You will convert resistance readings to current (v = iR). Adjust the current and voltage control knobs so that they both read zero initially.
- Turn on the power supply (the anode reservoir box lid needs to be closed to complete the circuit). Set the voltmeter to read current. Adjust the current control knob on the power supply (See a Picture of the HV Power Supply) so that it reads 100 μA. Peg this value as the maximum allowed current by throwing the black switch on the current control knob. Next, carefully adjust the voltage knob until the voltage dial and scale reads the correct separation voltage (typically above 10 kV, but not to exceed 20 kV).
- The separation current should be between 3 and 30 μA when the power is on. For standard free zone capillary electrophores is, the polarity switch located next to the voltage control knob should be set to “positive” or “+”. Turn off the power supply. The power source configuration is now set for capillary electrophoresis analyses.
- To being establing a baseline, simultaneously turn on the power source and click <Start> on the Scan Control window of IGOR Pro. If you cannot do this comfortably, click start on IGOR Pro first, and then turn on the power supply and hit the Event button on the lower right corner of the UV-vis detector. This will produce a small peak (~ 0.2) on the graph which will mark the actual start time of the separation.
- After approximately 3 seconds, hit the <Zero> button on the UV-visible absorbance detector. Return to IGOR Pro and set the y-axis range to -1, 1.
- Monitor the baseline. The baseline may drift slightly, but should not go outside of -0.15 to 0.15 on the y-axis, nor should any sudden shifts (> 0.05) appear for a period of 5 minutes (300 sec).
- Make note of the voltage reading on the voltmeter (that you convert to current), it should not fluctuate more than +/- 0.001V during the 5 minute period. Once a steady baseline is established, it is time for sample injection.
Notes: If the baseline drifts outside the prescribed range, or shifts suddenly and does not, turn off the power supply and click <Stop> on the Scan Control window of IGOR Pro. Some trouble shooting tips are provided below.
- Check to make sure the anode and cathode vials contain equal volumes of buffer and that the electrodes are equally immersed in buffer. If extreme baseline drift continues or sudden baseline shifting reoccurs, try using new vials of room temperature, filtered, degassed buffer.
- Disturbing the lab jack, anode reservoir box, anode vial holder, capillary or UV-visible absorbance detector while conducting a run may result in sudden shifts of the baseline.
- If the Range on the UV detector is set to a very sensitive setting (<0.001), the baseline drift will appear to be greater, but should not exceed a range of -0.2 to 0.2 on the y-axis.
- If the baseline seems to oscillate, you may have a ground loop in your system. The power source may need to be grounded using a ground connection to a cold-water tap. Make sure than random metal objects (pens, paper clips, spare parts, etc.) are not in contact with any of the electrical wiring connecting the power source to the voltmeter or the anode reservoir box.