Decreasing retention time

[vanblanes]

Hi everybody!

I'm validating a general method for the quantification of different APIs by HPLC, and I'm finding for one particular case that the retention time shifts 2 minutes from one sequence to the other. However, the retention time remains stable in every sequence.

In order to solve the problem I went to different changes like rinse the solvent inlet filters in isopropanol and try a brand new column but it hasn't solved the problem. Any ideas?

Thanks very much in advance!

[chromatographer1]

What is different about the two 'sequences'?

Care to elaborate?

best wishes,

Rod

[Anthony Crawshaw]

How much validation of the method have you done so far?

Some questions that may help answer:

Are the two sequences being run on the same system?
Is the same mobile phase being used in each case?
Has the preparation of the mobile phase been validated ie Robustness of organic content, pH or buffer concentration?
Is the mobile phase possibly evaporating?
Has the mobile phase been mixed well enough?
Is the column temperature consistant?
How old was the original column? If a different/new column has been used it may not have been "bedded" in and so still have active sites in the silica. This may lead to retention changes.

Maybe just a start
Anthony