Chromatography Forum

I am working on an Ion exchange method for monoclonal antibody
The current method has following criteria
Column:- TSK CM STAT column 4.6*100 mm column
Mobile phase: A 0.02M MES pH 6.75 and mobile Phase: B 0.02M MES+0.5M NaCl pH 6.75
The samples were injected at a condition of 10% B (which ran for a period of 2 mins) and then eluted by linear gradient of 1% B/min
When I increased the initial equilibration/loading time from 2 minutes to 8 minutes, the resolution of my peaks increased.
Why a simple increase in initial equilibration/loading time caused an increase in peak resolution???

Why a simple increase in initial equilibration/loading time caused an increase in peak resolution???

Off the top of my head, I would suspect that the column was not fully equilibrated after 2 minutes. In our Method Development course we suggest 10 X the column dead time (plus the system dwell time) as a good "first guess" as to equilibration time.