Hello everyone, I am experiencing difficulties in the HPLC analysis of adipic acid. I have tried Atlantis C18, mobile phase phosphoric acid solution PH 2.65, flow rate 0.7 ml/min, column temp 35 degrees at 210 nm. I have extracted the sample in the mobile phase also. I got an acceptable calibration curve once but never got the same results again. Is something wrong with my column? Should I equilibrate between injections? What recommendation and advice you have for me? Please help!
What is your matrix and are you applying gradient or isocratic elution?
Is your calibration curve plotted as matrix-matched or is it an example of external calibration? It is better to evaluate handle the problem if you can share the chromatograms and elution strategy such as mobile phase composition and elution profile. Since you choose a reverse-phase interaction, there must be an organic ingredient in your phases like ACN or MeOH to break the Van Der Waals interactions between the analyte and column.
I don't think that problem comes from the analytical column. Related to your question, that's for sure you should equilibrate your column and system before the second injection.
What recommendation and advice you have for me?
Read and understand how the column behaves with respect to the interaction with the analyte. Also you should understand especiation of your analyte. At low pH it is likely (depending on its concentration) that your analyte precipitates.
@ İsmail Emir Akyildiz, thank you sir for helping. That's an external calibration. Mobile phase composition is 0.1% phosphoric acid solution ph 2.65 (100% aqueous phase ). Thank you for the recommendation. I will try to add 5-10% of ACN and I will try equilibrating between injections.
@ Omar Gonzalez-Ortega. Thank you for help sir. I read we need to use PH between 2.1 to a maximum of 2.65 so that we keep organic acid unionized
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