Chromatography Forum

Hi everybody, i'm new on this forum that i just found today, i'm sure you all guys can help me and i'm thanking you by advance.

I'm currently working on a HPLC device using 2 colums REZEX ROA organic acid H+ in line. Since several weeks, we are experiencing increasements on the up to 82bars (68 bars are the theoric maximum pressure we should have in those kind of columns).

We decided to apply a cleaning protocol in order to reduce this pressure (reversed flowing 50% Methanol for 22 hours, then equilibration with eluent for 27h), but Nothing changed. However, by trying to check where the high pressure problem come from, i noticed that by inverting the first and second column, pressure is almost back to normal.

Here is my question: do you think that one column can flow both of the side without changing anything on the retention times, etc..? I actually putted the second column first, and the exit became the entrance, and the same for the first column (which also became the second).

Thanks by advance for your time, and sorry for my bad English
Thanks !

Most HPLC columns can, in fact, be reversed. There are some exceptions so it's best to check with the manufacturer before trying it. In this case, it looks like it worked.

In a past life, I developed a method that actually called for the column to be reversed at the end of every work day. That was easier than changing the sample workup to avoid the precipitating material that was causing the pressure increase.

Thanks for your answer Tom,
After i inverted the column and try to pass a representative sample, it looks like everythig is working fine (my methods are still working) and the pressure dropped down by 15 bars.

This kind of column can also be inverted!

I have a question for next time we'll have a problem : Is it possible/Wise to pass methanol 100% as a washing agent for columns ? Won't it damage it (constructor tells to use 5% methanol solution, which is totally uneffective) ?

Thanks again,

8% crosslinked PS-DVB cation exchangers don't like methanol (actually, I'm surprised that you didn't kill it with 50%). Using 100% MeOH potentially will shrink the resin beads and disrupt the packing bed.

OH ... Seems like i am Lucky then...
Do you know which could be the better option for me to wash my colums then ?

Thanks

First of all I would not clean both columns inline, not in the normal direction and not in the reversed direction since you would flush dirt from one column to the other. Maybe the frit of your first column is clogged. If you are experienced enough you can replace the frit with a new frit. That would be my first choice.
Tom is right, you are lucky that you did not killed your column with 50% MeOH. 100% MeOH definitively will kill the column.
Do you use a guard column? That would be highly recommended.
What temperature you are running the columns?

Hi, first of it all thank you for your answers.
I already replaced the frit with no effect on the pressure. I washed both columns in line (same order than the normal running order, but by counterflowing with methanol 5% first, then 50%)

I do use a guard-columnn, and I also changed the small filter inside (with no effect on the pressure) before starting the cleaning process.
The columns are running at 50-51°C.

Is there another compouns than methanol that i could use to clean my columns ?

Please take the column instruction guide to your hand, it should be shipped together with the column, and look for the COA of the column. Use the initial conditions to run the QC test and redo this QC test. You should get similar values as stated on the COA. If not it maybe that the column is already killed.
Only one recommendation left - time to purchase 2 new columns. Sorry