Chromatography Forum

Dea all,
I am running an HPLC method with quite dirty sample (plasma). Several days ago, I realized that the retention time of my peak keep increasing a bit (from first injection to 30th injectioon, it increases about 2-3%). From "HPLC troubleshooting", I decided to wash my column with a range of strong solvents :MeOH-dichloromethane-hexane-dichloromethane-MeOH, with the hope to remove the dirts building-up in my column. After washing, I run my mobile phase and found out, the pressure ofmy column increases 40bars-from 60 bar to 100bar! I don't know why, I checked everywhere and couldn't find anything. I injected several samples and everything seems OK, the retention time, the peak area, the separation are as before.
Do you have any suggestion about the reason of pressure increasing? Did any dirts ome out and blok my column? Do I have to worry about that? What can I do to get back my normal pressure?
Thanks in advance!

Maybe you could change the frit at the outlet of your column?

regards Bert

Are you sure the column is the culprit? Do not forget to troubleshoot the system for blockages.

Most modern HPLC columns do not generally react well to "changing the frits", if you want to totally trash a column crack the fittings and look inside.

TOT,

Are you pretreating your plasma (i.e. protein precipitation) before injection to your analytical column? Increasing backpressures is not a good sign in general, but the usability of your column would be defined from your chromatography. Sometimes inversion of the column and cleaning at low flow rates with solvents such as the ones that you mentioned can bring your backpressures back to normal.

As your retention times are the same as before I would propably start working again without further cleaning the column. However you might want to find out the cause of this retention time shift and maybe use a more effective plasma pretreatment.

Another question, was this a new or an already used column?

Bert, I think that is unlike that any dirt would reach the outlet frit of the column as the stationary phase itself works as a (huge) filter. Maybe the inlet frit, although I tend to believe that is maybe plugging of the pores of the stationary phase close to the column inlet (proteins in general is the first suspect...).

Do not take your column apart - reverse it and flush w/ a series of solvents to clean out suspected frit blocking compounds.

To prevent blockage, use a guard column.

To assess the situation with changing retention times, more information is needed - mobile phase composition(s) in particular.

Sounds like you caked out some remaining (probably harmless) poteins with CH2Cl2, hexane, or even the MeOH. These should be avoided unless you are sure there is no protein left on the column. If a reverse flush with MeOH (iso-propanol is sometimes better water mixtures of the that, or MeOH/H2O) does not work you may have to use chaotropes (urea), etc. This has been discussed before.
In our hands such a problem was almost always due to protein precipitation at the inlet frit. Pre-cleaning or even a two step chromatography may not eliminate the protein problem entirely. Again in short: As mentioned, pre-cleaning, a guard column or even a guard frit is quite helpful. We found it absolutely necessary to wash the column with mobile phase then with organic/H2O of a composition near that of the mobile phase (or a little bit higher in organic) at the end of a session. We completely came off using pure organics.

We've had some samples where gelling agents and thickeners kicked out of solution after injection and gradually built up pressures. We reverse-flowed the columns to clean them, but the real solution was to precipitate/remove the thickeners first so we could run numerous samples automated. I think your problem is similar as suggested above, proteins kicking out of solution, gradually clogging the frit and increasing pressure.

Thank you all for great suggestions! I did clean-up step for my plasma sample (extraction with cyclohexane-isopropanol 9:1) and used guard column as well. However, as HW Mueller suggested, there is still protein remaining. I will try to reverse my column and wash with organic solvents to see how pressuren is. My column is not new one but the separation and shape of the peak is very good. My mobile phase is ACN: acetate buffer pH 4 (4:1). I hope after reverse washing my column, I will get rid of retention time increasing because I found out with increasing of retention time, the reproducibility is worse.
Thanks again and all of your further suggestions are highly appreciated!