Hello.using HPLC device,I tested a mixture of 12 standards(all in 1000 ppm concentration).I got 12 peaks.but they are not at same size (peak area or peak height).is it normal?Thanks
The relationship between peak area and concentration is called the response factor (RF) and is unique for each analyte in your mix. RF is the proportionality constant under given instrumental conditions and can be determined as the concentration divided by the area. If RFs for all analytes in the mix are reproducible from run to run, you are fine.
The response of ELENA ROMANOVA is very logical and satisfactory. Indeed the response factor is unique to each analyte but if the response facotr is reproducible then your analysis is fine.
Which detector do you use? Just an example: For UV/VIS detectors, you can set certain wavelengths (e.g. 278 nm for Trp (W) in peptides and proteins). At the end, if your components absorb differently at the certain wavelength, you'll get different peak heights. Try different wavelngths or record the whole UV spectrum during your run, so that you can follow the intensities.
differeent compound absorb UV light at different wavelength, when you monitor at a fixed wavelength, you will have different absorbance intensity, therefore, different peak height at the same concentration.
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