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Carry over seen in RP-HPLC Amino Acid Analysis with application 5991-7694

Question asked by marcus.jones on Aug 27, 2020

I am running amino acid analysis using the AdvancedBio AA 2.7um column. Needle wash is 50:50 mobile phase A with 0.4% phosphoric acid : mobile phase B (mobile phase A is 10mM di-sodium phosphate anhydrous, 10mM sodium tetraborate anhydrous pH 8.2, mobile phase B is 45: 45: 10 MeCN: MeOH: H2O).

Seeing quite significant carryover in a blank after injection of a 250 pmol/mL amino acid standard - see attached. Starting composition of the gradient is 98% A, not sure if there is another cure for this apart from a stronger needle wash which may affect peak shape due to the difference in solvent strength between diluent and mobile phase?

 

Also, other users have experienced poor peak shape with this application and I was tasked with regenerating the column. When I came to run it, the chromatography looks great. Please see attached the last run with this same column. Any idea what could be causing this, system not set up correctly, blockage in the column that has now cleared?

 

I have noticed that the FMOC and OPA reagents have been stored in snap top vials, which the application not states to avoid due to volatility, however not sure how much affect that would have on the peak shape if they are stored at 5C.

 

Thanks,

Marcus

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