I used DAD to analyze the IAA（indole-3-acetic acid）standard sample at different concentration. Oddly, I got two peaks at low concentration and one peak at high concentration. If I added the area of the two peaks, I got a good linear relation.
How can I explain the two peaks at low concentration?
Sen Li: Observing a "linear relation" as you say does not in fact show any indication that you have a valid HPLC method of analysis at all. When no chromatography takes place (as may be the case in your example when the sample(s) elute at or near the column void volume or with low K prime values), the area counts often show near perfect r2 values. *Adding unknown peak areas together that you do not understand is not the approach that should be taken.
- Using HPLC for the first time without training or supervision always leads to false conclusions.
To put into context what your chromatogram may imply, the actual LC chromatography method information must be shared (required). The details of the HPLC method should be provided. These include all of the common parameters and settings which describe the method used (e.g. Column name, type, dimensions, particle size; flow rate, detailed mobile phase composition (and if applicable, gradient time table); temperature, injection volume, injection solution, detector type and settings used (e,g, for UV/VIS this would include wavelength and bandwidth), sample concentration etc.
For the most practical assistance with this project, please contact someone at your school who has practical experience using HPLC for direct help. Learning the basics of HPLC takes many years of full-time, hands-on work so working with an experienced local chromatographer will improve your chances of collecting valid data.
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