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. When first learning Capillary Electrophoresis it is a useful practice to perform a FIA prior to performing CE.
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.
- From the start menu find IGOR pro under programs. Under IGOR pro, click on the Igor icon. Once the application starts, go to the file menu. Open the file with your preferences. The most recently used file will be under recent experiments. This will bring up a table for the number of samples to collect, the channel selector, and the graph for the data collected.
- Check that the channel selector is set to the correct channel. The channel you should select depends on what inputs you have used and how they are configured in the break-out box.
- Set the numbers of data points that you need to collect. Remember that the scan control panel is set to scan at intervals of every 0.1 sec. So if you want to let a sample run for 6000 sec, which is 10 minutes, you need to have number of samples set to 60000.
- With the buffer vial in place at the injection end (anode), turn on the pressure and hit the <Start> button on the bottom of the scan control on the computer program. Let the buffer run through the capillary until you have a steady baseline. If the absorbance on the UV-visible absorbance detector is not at zero, hit the <Zero> button on the UV-visible absorbance detector.
- Once baseline is steady, turn off pressure. Remove the waste vial at the detection end of the capillary (cathode) and replace it with a vial containing run buffer. Remove the vial at the injection end (anode) and replace it with sample.
- Set the timer box to the correct injection time and turn it on. Make sure that the capillary is submerged in the sample (but not the Teflon-pressure tubing).
- Remove the sample vial and replace it with a vial containing running buffer.
- Turn on pressure and simultaneously hit the <Start> button to collect data. If you cannot do this comfortably, click start on IGOR Pro first, 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 FIA.
- To adjust the data graph, for example to expand the first 500 data points, click the cursor on the bottom line and type “setaxis bottom 0, 500”. You can also do the same with the left axis, for example by typing, “setaxis left –1, 1”.
- After observing the analyte signal, stop data collection by clicking the <Stop> button of the scan control window. Turn off the pressure.
- Click on the graph and then go to <Graph> on the menu bar. Click <Show info> and <Show tools>. The graph will now have two icons in the upper left corner. Click on the bottom icon and select the “T” icon, which stands for text. Then click on the graph. This will allow you to type information about the run (buffer, sample, run name, injection time, sample name, pressure, etc) directly on the graph.
- Next, you will need to click on the top icon (graph icon) and select the circle cursor at the bottom of the graph. Drag this circle cursor to the left of where the peak starts. Then drag the square cursor to the end of where the peak stops. Both of these cursors need to be placed on the baseline before and after the peak you want to fit.
- On the menu, click analysis and go to curve fittings. Select “gauss” under the “Function and Data” tab and select cursors under Data Options. Click <DO IT>. The peak will be fit with a Gaussian curve at the peak cursors. Following the fit, the program will report a “w-coefficient”, which will provide the following information: baseline (k0), peak height (k1), migration time (k2), peak width (k3). It is important that the peak height falls between 0.1 and 1.0 on the full scale due to sensitivity issues with the UV-vis abosorbance detector. To bring the signal onscale adjust the range of the detector or change the sample injection conditions.
- Down at the bottom of the screen is preferences box. This box will contain the w-coefficient. Copy the w coefficient numbers and paste them into the data graph.
- SAVING DATA: After each run you need to save the data. Click on “Data” tab on the menu bar. Go to <save waves> and then <save Igor binary>. Click on the input that is associated with the channel connection (for example, input 2 is recorded at channel 2) and then click <DO IT>. Select <new folder> and name the folder for the date you are collecting data. Then name the run (for example “100μM ibuprofen FIA run 1”).