HPLC Nucleodur C-18 Degradation

[Loys74]

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

[DR]

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)?

[Loys74]

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!

[Hollow]

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.