First, you need to generate a black and white picture of the chromatogram. You may scan the chromatogram scan your chromatogram at high contrast, and save it in any graphics program.
Open your graphics (or Exel) file. Convert the graph into black and while, making sure that the lines are solid black (you may need to change the color and weight of the lines in Exel).
Open NIH Image, paste your inmage into a new file. Check under "Analyze"->"options" that "area" is selected.
Image can measure any completely enclosed area; thus, you have to make sure that every peak is completely surrounded by an uniterrupted, black line. Use the line draw tool to draw a baseline and other lines, if necessary.
Next, use the "wand" tool and click within the peak you wish to measure. "Analyze"->"Measure" (or "command 1") measures the area and shows the results in the "results" window (press "command 2" to open it).You can label the peak with the integrated value by selecting the "text" icon in the tools bar, moving the cursor in the desired position, and holding down the option key while pression the mouse.
