Chromatography Forum

Hi all,

I have a problem with a column degradation and the problem come from the eluant which is Methanol / water / pH 6 buffer solution : 250 / 500 / 250.

After 200 injection all my peaks are splitted.

Pressure is stable (200bars), I work in isocratic mode, column is Nuclesosil 100-C18 5um, 125mm, 4.6mm and injection volume is 10ul.

When my column split my peaks, I can return it and work 20 injections.

DO you have idea?

PS: it is my first post

1) what is your buffer?
2) by "return", do you mean you turn it around - install it backwards?
3) what kind of samples are you running and how concentrated are they?
4) Have you tried a "clean out" gradient (run isocratically to collect your data, then increase the methanol concentration to flush the column, then back to your isocratic conditions)?

Thanks for answering!

1) Buffer is pH6 (phosphate)
2) Yes we connect the column in the wrong way and sometimes (30%), we can use the column more time than in the good way.
3) We try to add cleanup with MeOH/Water (80/20) between injection without good effect and also try to add 1hour cleaning column with MeOH/Water 20/80 and 1 hour MeOH/Water 80/20 without good effect.

Thanks!

even you say the pressure is stable but for me this sounds like microbial contamination and partial restriction of the inlet frit.

- How good is your water purity?

- Do you filter your buffer with 0.2µm and keeping the bottle really closed by dedicated caps or just are they just capped with the old fashioned caps?

- How old is the buffer, how often do you replace it, incl. cleaning of the bottle (not just filling up the used bottle)

- How do you perform the isocratic mixing? Online or is the eluent premixed in the bottle? Premixing may be superior in that 25% MeOH may prevent/slow down the microbial growth.

- the use of a precolumn/crude catcher/inline filter may be indicated, where you change the cartdridge about every 180 injection (if the issue is correlated to the numbers of injections)

- try to clean your system with some strong solvents and replace the bottle sinker frits.