Low Reproducibility in vitamins peaks

Ruthvn 1 month ago

Hi,

I am trying do develop/validate a test method for water soluble vitamins (B group ) and I am facing very low reproducibility.

I am using a 1200 system and my current mobile phases are 25 mM NH4H2PO4 and methanol (gradient program).

The problem is the method works perfectly fine the first 15-20 injections and then some of the vitamins start to co-elute and show some tailing. Even after column washing.

I am using an Agilent HC-C18 Column 150 x 4.6 mm, 5 um.

Please help

0 fgunawan 13 days ago

Hello Ruthvn
Please share some photos of your chromatograms and check for void volumes in capillary connections.
Once the issue occurs, does it keep occurring for subsequent injections or do the resolution and acceptable peak shape come back in later injections?

Are you using a guard column and is there a change to the pressure across different injections?
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