Chromatography Forum

Hi all, I've had trouble conditioning a diphenyl restek reverse phase column for some time now. The retention times are drastically increased when using a new column which seems to tell me that the analytes are adhering more strongly to a new column and taking longer to elute out compared to a column that has been used and conditioned. One would think that you could just stick the new column on right out of the box and run it but it doesn't happen that way, there seems to be a need for a conditioning step and a change at the molecular level.

Do any of you use restek? Dephenyl? Even if you use other reverse phase columns, how do you guys condition a brand new column? Or is there no need for this at all?
my mobile phase is water and acetonitrile at various flow rates (from mostly water , to 50/50, to mostly acetonitrile), the column was originally stored as new in 70/30 methanol/water.

Some online sources say to wash it with 20 mLs of acetonitrile. I tried this and it didn't help. Another thing is that I initially washed the column out with mostly water, 1 mL/min for 10 minutes, I'm hoping that this didn't alter the column, perhaps " wet " the column.

http://chromatographyonline.findanalyti ... ?id=542752

There should not be much "conditioning" required, just sufficient to equilibrate in your mobile phase. I would double check the catalog # and see if this is really the same column as you had previously. Your earlier column might have been a phenyl instead of biphenyl. Also the silica may be different, which would give you different retention characteristics right off the bat. The Allure is more retentive than the Ultra. There are also some slight differences between the Ultra II and Ultra. If this is still not making sense, you should contact Technical Services.

checked the catalog number and they are the same. I'm going to try to do some high volume repeat injections of a standard and hopefully that retention time shifts , then maybe check to see if the acetonitrile pump is working by measuring out the volume in accordance with the flow rate. Again, you would think that just plugging in the new column would be enough - last run with old column showed no problems whatsoever - but it's just not happening. Tried another brand new one which showed the same, late , retention time.

Two possibilities come to mind here:

1. Restek has made some (seemingly) minor change to their manufacturing process, to which your method is very sensitive.
2. Your old column had already been damaged in use (extreme pH? very high temperature? dirty samples?) to change its chemistry.

Go back in your records and see what the retention looked like on your old column when it was new. That should give the answer.

As far as conditioning goes, aside from ion-pair methods (which are notorious for long equilibration times), the only cases I've seen where extensive "conditioning" of the column was required involved the use of end-capped columns at low pH. The "conditioning" essentially amounted to removing the end-capping!

Really appreciate the advice, thanks. I called Restek and the technician claimed that no changes were implemented to the columns.

It turns out that the fittings to the degasser were in bad condition, replaced the water fitting to the degasser which seemed to resolve flow rate for the water. However, the acetonitrile flow rate from the volume tests are still below that of expected, in fact, close to half the volume expected from the set flow rate.

Investigation ensues.

edit: kept purging and purging, much more purging then usual, and then checked the flow rate by measuring volume of the purged eluent, volume matched expected values from the flow rate and time ran. Then checked the volume through the system and surprisingly it matched. Will now run standards to see if issue has been resolved.

A long shot,

If you have analysing negatively charged compounds (i.e. acids above the pKa), they may elute earlier when the columns gets older due to the fact that the endcapping is slowly removed. That exposes the silanol groups that then can push out the negatively charged compounds by ion-exclusion. You will not see any decrease of plate count, or retention shift of neutral compounds.

But I would first check the pump (as you are doing now)...

It turns out that the acetonitrile pump was being stubborn for some reason - purged it more then five times at high flow rate and finally it came into tune. The fitting to the degasser from the water was in bad condition. Not sure how both of these things happened upon installing a new column, because it worked fine the other day, anyways, strange coincidence. It's working fine now.

I know things are working OK now, but I was curious.. Are you running a gradient? If not- I would mix the ACN and Buffer and use one pump.

I know things are working OK now, but I was curious.. Are you running a gradient? If not- I would mix the ACN and Buffer and use one pump.

yeah, running a gradient with water and acetonitrile. Thought about using buffer, even though at this point it's unnecessary, since it improves the peaks but not sure it's worth it with all of the things it can do to the pumps and lines.