Chromatography Forum

Hello everybody,

i have a problem relating the stability of column.

Now i'm using the C18 column to analyze caffeine and 1-naphthol. But recently, it is not stable. The repeatability is not good.

I tried making many different methods but it also doesn't improve such as:

+ I rinse it by water for 5h, then by ACN for 1 h. After that, i let it be stable for 1 day.
+ I use dichlomethane to rinse it to remove the contaminant

Please give me some advice to make it stable again !

Thank you for your reply !

Repeatability of what? areas? retention times?
Is the repeatability problem random variations, or systematic changes during the day?
what is your mobile phase? column dimensions & flow rate? isocratic or gradient?

Repeatability of both areas and retention times is not good.

Mobile phase: ACN : H2O = 40:60, I also tried using ACN:H2O = 20:80 and Methanol : H2O = 30:70, but the phenomenon is similar.

Column: C18 10u 250mm, Phenomenex,
Flow rate : 5ml/minute, isocractic

I think this problem doesn't relate to "systematic changes during the day" because i measure two continuous samples which are the same concentration , the repeatability is also not good.

Thanks for tom jupille !

People recommend never going to 100% aqueous, as the column has a hard time being "re-wet" with organic. Why would you need to go to 100% water anyway?

I have a problem relating the stability of column. I tried making many different methods but it also doesn't improve such as:

+ I rinse it by water for 5h, then by ACN for 1 h. After that, i let it be stable for 1 day.
+ I use dichlomethane to rinse it to remove the contaminant

Please give me some advice to make it stable again !

Did you contact the manufacturer as to instructions for cleaning/regeneration of the column - doesn't sound like it !!!

Using 100% aqueous is a BAD idea, already mentioned. For most, if one wanted to clean with dichloromethane, then one would first run an intermediate polarity solvent through, like acetonitrile or IPA, then dichloromethane, then back to intermediate solvent, then back to desired mobile phase.

"If you didn't follow the manufacturer's advice, then don't blame the manufacturer !!!" - Yogi Berra or some other chemist

Go back and look at your data. If your dead time (t0) and retention times change by the same percentage, the problem is with your flow rate, not your column.

If t0 remains constant when retention times change, then you have a chemistry problem. In that case, are you "pre-mixing" your mobile phase (manual preparation) or does your system mix "on-line"? If the latter, check the proportioning system accuracy. Your pump service manual should have detailed instructions for this.

Thanks for your reply !!!