Waters 600 HPLC - Estimation of concentration for an unkowon peak (Sep/07/2007 )
HI to all ...
Well thanks in advance. I am new to HPLC I have injected one unkown sample. i got two peaks with 0.0112 AU . How to calculate the concentration from these AU. Please tell me some useful analysis caculations and tips with HPLC chromatogram. Please iam in need of your help , help me out to learn something..
You need to use a standard for comparison of your unknown. The standard should be a close derivative of your target compound (e.g a carboxy when you are analysing a methyl).
Once you have your target compound peak height (or area) you work out the ratio of this to the standard's height (or area). Knowing the standard's concentration and peak height, you can easily calculate the unkbown concentration since you have it's peak height.
The two peaks sound like at least one metabolite. What's the retention time? Is there sufficient resolution from the solvent front to your peaks? And how good is the resolution between these two peaks that you get? (Do you get baseline in between them?)
Hope this helps.