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 Flow Injection Analysis (FIA). Flow Injection Analysis is a technique used to determine the presence of analyte in a sample. After injection, pressure is used to push the injection plug passed the detection window. This is not a separation technique as all sample components reach the detector at the same time. FIA is used solely to ensure analyte is present in the sample and that the response remains onscale with respect to the range setting of the UV-vis absorbance detector.
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.
- To establish a baseline for FIA, first make sure the black control switch located on the three-way valve is pointed towards the solenoid.
- Next examine the pressure reading on the pressure gauge (See Diagram of the Homebuilt CE System). Adjust to desired run pressure by using the black control valve located on the regulator on the gas cylinder.
- Next pressurize the capillary by turning the black control switch from the solenoid to the gas cylinder while simultaneously clicking on the <Start> button located in the Scan Control window of IGOR Pro. If you cannot do this comfortably, click start on IGOR Pro first, and then turn on the pressure 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.
- At this point the graph screen should be displaying data real-time. To zero the UV-visible absorbance detector, press the <Zero> button located on the front lower right portion of the panel. 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 there be any sudden shifts (>0.05) for a period of 5 minutes (300 sec).
- If the baseline is not steady, performing a quick flush, such as 5 min NaOH, 3 min deionized water, and 5 min run buffer all at 20psi may stabilize the baseline. See System Preparation for more detail on flushing.
- Once a steady baseline is established proceed to sample injection protocol. If the baseline drifts significantly or there are sudden shifts in the baseline the following troubleshooting steps may be useful:
- Examine the pressure gauge to make sure the pressure is at the desired level. Adjust this value as appropriate.
- Examine the anode and cathode vials to make sure they contain equivalent volumes of buffer and be sure the capillary is equally immersed in both vials. Try to establish a new baseline. If drift continues, try using new vials of room temperature, filtered, degassed buffer.
- Physically jarring or 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. It is possible to cause vibrations, which changes the orientation of the capillary with respect to the optics. If this occurs, consider tightening or realigning the capillary in the optical flow cell.
- If the range on the UV-visible absorbance detector is set to a very sensitive setting (<0.001), and the baseline drift will appear to be greater.