Chromatography Forum

I have installed a new C18 3um 4.6x150 column on my system. after the second injected sample the pressure rose 3 times (above the working value). My eluents are Water and Methanol spiked with HCOOH. I didnot have this problem with the previous column (from the same manifacturer but 5um). My attempts to clean it were 95%AcN and 100% THF - no result. If anybody has experienced this and has a solution it'll be nice to share it. Or maybe I should contact the salesman A.S.A.P.?

thanks

Definitely call the sales person. But first:

Have you confirmed that the problem is, in fact, the column and not a blockage elsewhere in the system (connecting tube, injection valve, etc.). If you haven't already done it, try disconnecting tubing starting from the outlet of the system and moving back upstream. Note when the pressure drops. The component immediately downstream of that last fitting is blocked.

If it is, indeed, the column, try back-washing (reverse-flow). Check with the vendor first to be sure that back-wash is OK.

Cleaning the column involves flushing it with something that's a good solvent for whatever is contaminating the column. You have to make a judgement based on what you know about the sample(s) that did the damage.

Yes I have verified, that the pressure comes from the column, It is possible that I have some retained lipids in there. I did back-wash - a long one and with different solvents:
1. 50/50 Methanol Water, spicked with HCOOH
2. MetOH
3. MetCN
4. Isopropanol
5. Hexane
And then back to methanol at 0.2ml/min each for 10 column volumes.
This managed to lower the pressure by 20% but it is still higher than the recommendations, which depicts a heavy clugging or "dead"column.
Should I continiue back-washing it. Should I try only Water: Anyone has Idea.

Thaanks

I don't think at the end there you can go back to methanol directly from hexane. I'm not sure they're miscible in all proportions.

I meant the same steps backwards (through isopropanol)...

Hi,

The column wash depends on your samples, so what kind of samples did you inject?

Also, maybe its the inlet frit of your column which is blocked, so it may be possible to replace this as well.

If you inject very dirty samples (containing lipids for example), it's advisable to use a guard column, which takes most of the dirt and is easy to replace.

Ace

Maybe the sample was dirty, even though it underwent centrifugation 30 min 30 000xg and 2um filtration. Its a liver homogenizate. ist' it dagerous to remove the frit? and ist there a way to clean it.

thanks

Maybe the sample was dirty, even though it underwent centrifugation 30 min 30 000xg and 2um filtration. Its a liver homogenizate. ist' it dagerous to remove the frit? and ist there a way to clean it.

thanks

dangerrous to remove the frit? Depends from which manufacturer.
The way to clean it is to backflush your column (don't couple to your detector!)

Another idea is to wash with acidic and basic mobile phase (take care of the limitations of your column if silica)

Ace

Maybe the sample was dirty, even though it underwent centrifugation 30 min 30 000xg and 2um filtration. Its a liver homogenizate. ist' it dagerous to remove the frit? and ist there a way to clean it.

thanks

I would not recommend removing the frit on a "newer" column such as yours. these columns are packed under considerable pressure and the staionary phase may exude if the column is opened.

okay man thank you, I'll try the pH variation, but I've never opened a column to change a frit and I'm a little bit afraid

thanks again and I'll let you know if this gave a result

By the way: Backflushing leads to higher and faster increasing pressure than the normal flow - does it mean that the problem is in the end of the column...

Most of the time the problem is at the beginning of the column, as this is the first contact with the sample.

But it's not unusual that you have an increased pressure when backwards flushing.

Ace

It looks like your column is badly crudded with proteins and a bunch of other macromolecules. To get this out you should not use any pure organic solvents early on, but rather some chaotropes or even detergents, maybe even antioxidants. This sort of thing has been discussed many times here.

Hello Everybody,

Being a new forum-visitor, im not sure whether it is the best topic to write in, but i hope u can give some help.
I have a HyperClone C18 ODS (150*3,2*3) column, and it has worked so far very well. Some days ago it looked to have contaminants; i made a blackflow with the following protocol: ACN-water 5/95, then THF, and after ACN/water 95/5. After this it looked to be ok and work. I made 2 run (ACN / water + AA; from ~20%-100%; i had been using this column such system before). Now i changed ACN to methanol (20% metanol), and the pressure flied into the sky. I tried to wash with 100% ACN, then 100% methanol and blackflow, too, it was all right, but next time with the system mentioned previously pressure has gone up again.
This is the problem. Any advice...?

Methanol-containing mobile phases will nornally run at higher pressures (due to higher viscosities of methanol-aqueous mixtures). Try raising the column temperature to 40C, slowing flow rate, etc.