Chromatography Forum

I am running a method with a mobile phase of A: 70%MeOH:30% 28mM TBAA pH 6.5 and B: 100% MeOH. The column is a C8 rapid res column, 3.5um at 60C. I have been having issues in that after every injection the pressure climbs a little higher rather than settling at a baseline pressure (e.g. 125, 130 145, 155, 200 bar etc.). I know this method has worked on the Agilent 1100series system but I cant seem to get it up and going on the Agilent 1200 RRLC with Binary pump. I have tried several different columns and all do the same thing. Has anyone else had this issue or have any ideas as to why this is occurring?

Assuming the pressure increase only occurs for each injection, my first guess would be sample insolubility, either fine particles in sample/blank, or an inappropriate sample solvent ( partially immiscible - or affecting TBAA solubility at 60C).

Are you using a suitable wash solvent?.

If the increase also occurs with injection of blanks of mobile phase, you could try flushing the column with a suitable mobile phase to ascertain if the pressure then reverts to normal.

You could also inject larger volumes and ascertain if the pressure increase is greater.

You could also put a restrictor in place of the column and ensure that the problem does not occur - which would eliminate most hardware issues.

You could also leave the TBAA out of the mobile phase and inject sample solvent blanks ( no sample ) and see if the pressure still increases.

You have to systematically exclude components until the pressure increase no longer occurs, but I would start with the injection sample/solvents.

Bruce Hamilton

Can you tell us what particle size the column is packed with? Is it a 1.8-micron RRHT column?

It is a 3.5um column, Zorbax Eclipse C8. Actually today I will be trying something similar to what was just mentioned. I shall try running the method that is described in the column insert (80%MeOH:20%H20) as the mobile phase. I am going to try multiple injections of mobile phase as a blank as see if I observe the same pressure profile. I have been performing a wash with MeOH at the end of the day.

There is the usual inrease in pressure after injection when the sample hits the head of the column. But after every sample that pressure is a oittle higher and never seems to stabilize at the end of the run. It should stabilize at the starting pressure (i.e. when I first start up the instrument) at 125 bar. I go through a gruelling process in the morning, incrementally bringing up the column from MeOH to the TBAA solvent.

Thanks!