Chromatography Forum

Hi,
I am working with 20 mM ammonium acetate buffer: acetonitrile (60 :40 ,v/v) on Synergi Hydro-RP (250 x 4.6 mm, 4 µm). During all runs, the pressure is constant (around 1550). At the end of my work every day I follow this washing procedure:
Composition Duration (min)
Water: acetonitrile (95: 5) 60
Water: acetonitrile (50: 50) 10
Water: acetonitrile (70: 30) 15
Acetonitrile (100) 10
Since two days, I realized gradual increase in column back pressure during washing as follows:
Composition Normal P New P
Water: acetonitrile (95: 5) 1550 1800 (flow 0.8 )
Water: acetonitrile (50: 50) 1550 1850
Water: acetonitrile (70: 30) 1280 1450
Acetonitrile (100) 650 1200

What could be the problem and how to return the column to its normal pressure?
N.B:
The buffer (pH 5.8 ) is freshly prepared, then mixed manually with acetonitrile and filtered before use. The solution remains completely clear without any precipitation till the end of work.

Is the pressure really increasing "gradually" or did it jump to the new values and stay there?

If it was a sudden jump and the pressure is not stable at the new values, my guess would be that something from a sample or mobile phase clogged the column. In that case, I would just keep using the column at the new higher pressures.

If the pressure is increasing gradually (i.e., it gets a bit higher each day), then you'll have to figure out whether the problem is sample- or mobile phase-related. Easiest way is to run the column through several wash cycles and see if the pressure increases (i.e., without injecting samples). If it does, then you may have to consider filtering your mobile phases through a 0.2 micron filter.