The following section describes how to analyze peaks obtained from a CE run. The peaks will be analyzed using a Gaussian fit. Peak height, migration time, and sigma value will be the primary data values determined.
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 accompanying that software.
- The first step after collecting data is to save the run data. This is done by first clicking on the graph window. Next click Data from the toolbar at the top of the IGOR Pro window. Select Save Waves. Select Save IGOR Binary. Select the appropriate input, for example Input 2, from the list under Waves. Click <Do It>.
- A file directory screen should appear. Select the appropriate data folder to store your run or create a new one by moving the cursor to the directory window, right clicking with the mouse, and selecting New and the selecting Folder.
- Double left click on the name of the folder to change the folder name. Double left click on the folder icon to open the folder. Click on the appropriate wave, for example Input 2, in the file name line and change to desired file name to something more informative. Click on <Save>.
- After saving the data, return to the main IGOR Pro window. Click on the graph. Now begin analyzing the data. First select Graph from the toolbar, and then select <Show Info>. Select Graph again from the toolbar and then select <Show Tools>.
- Two icons will appear in the upper left corner of the graph window. Click the [T] icon, which stands for text. Next, click on the graph. A screen should appear that will allow you to type the run parameters (such as buffer, run name, injection time, sample, pressure, etc.) directly onto the graph.
- After typing this information, click on the graph icon located next to the [T] icon in the graph window. Two lines should appear at the bottom of the graph window. These contain movable cursors to designate the region of the graph to be fit. First, expand the region of interest in the graph, by moving the cursor to the graph, holding down the left mouse button and drawing a box around the peak. Right click and select Expand.
- Select the circle cursor from the bottom two lines appearing under the graph. Drag the circle cursor to the graph and place it on the baseline as close to the beginning of the peak as possible.
- Next, drag the square cursor to the graph and place it on the baseline as close to the end of the peak as possible.
- Select Analysis from the toolbar. Select Curve Fitting. A new window should open. Select <Function and Data>. Make sure “Gauss” is selected from the Function list and the appropriate wave is selected from Y-Data list, for example Input 2, and “_Calculated_” from the X-Data list. Select <Data Options>. Click the <Cursors> button from under the Range box. This should set the peak range to the square and circle cursors placed on the peak. Click <Do It>.
- A fitted curve should appear on the graph. A rectangular box should open that provides four parameters (peak height, sigma, time, and y-initial). Click <Ok> to close the window. Next go the text command box.
- The curve fitting data should automatically be displayed as the last entry. Scroll up in the text command box to find the w-coefficient line. The four values listed are (from left to right): y-initial, peak height, migration time, and sigma (4σ defined as width of the peak base).
- After the values are recorded or printed, close the graph by hitting the [X] in the upper right corner of the graph window. For a new run, go to the menu at the top of the tool bar. Select <New Graph> and a window should pop up. Select the appropriate wave, for example “Input 2”, from the list under Y Waves and select “calculated” from the list under X Waves. Click on the <Do It> button at the bottom left corner of the window.