Chromatography Forum

Hi all,

I have some yellowish E. coli supernatant samples that mess up my HPLC analysis.

If I add 2 parts of acetonitrile (ACN) and 1 part of sample, the 'yellow rubbish' precipitates and analysis is fine.
I am using a Rezex ROA organic acid, and the manual says to do not exceed 10% ACN. Phenomenex revised this number in the last edition (01 2013) to 30%. I can get down to 30% ACN, but not to 10%.

Which number should I trust as max ACN limit?
Is ACN going to interfere with analysis or kill my column? Are the standard made in water still good?
Is anyone aware of alternative methods for sample prep before analysis (eg get rid of crap?).


Thanks for reading and any help!

Miche

These IEX materials are based on PolystyroleDivinylbenzene and with changing amount of organic solvent will schrink or swell. I trust that your column will withstand 30% ACN under standard conditions.
I would recommend to use SPE cartridges, maybe a C18 will work to clean up your sample. You can rinse with water and afterwards evaporate your sample to get rid of the ACN. Manufacturers of SPE cartridges have a lot of protocolls available and will provide sample cartridges to test.
Good luck

Thanks for your reply, Gerhard.

I have something like 100 samples to run, I hope the column will survive.
Actually, the ACN mixing was part of the cleaning protocol with a C18 cartridge, but when mixing ACN I noticed the yellow stuff precipitating, and the trick was done (at least for my first batch of trial samples).

I was trying to avoid extensive handling of my samples, since I have hundreds per run. The simple addition of ACN seems to work well so far, as long as the column will tolerate ACN on a regular basis.
Does anyone have any comment on this?

Before ACN I had huge peak drifting, with peaks impossible to quantify. Now the problem seems gone, but I wonder what the yellow stuff is.

Many thanks for the support,

Miche

If you end up having trouble with the acetonitrile (even though I believe it will be able to withstand it), couldn't you evaporate the acetonitrile after the precipitation step and re-dissolve in whatever solvent you used before adding acetonitrile? 100 samples seems like a pain to evaporate though, but perhaps it's necessary. Just saying you know

Which number should I trust as max ACN limit?

Those limits are for ACN content in the mobile phase, not the sample. You are only injecting microliters of sample, which is quickly diluted out by the mobile phase. I would worry more about other junk in the sample than I would about the ACN. As far as that goes, with dirty samples, I would use a guard cartridge (same packing as the analytical column) just to be on the safe side.

Thanks everyone for their replies.

Yes, Tom your explanation makes sense, indeed the numbers refer to the mobile phase. Would the people in the analytic dept understand it?

Thanks again.

All the best,

Michele

Would the people in the analytic dept understand it?

I suppose it depends on how you write it. Then again, there's no point trying to make things foolproof, because fools are very ingenious!

You right. Better to be objective. Clearly people in the analytic dept do not want to risk their columns and I want my data.

I have to correct the column type, it is RezexRHM, instead of ROA as stated before, but the chemistry is the same.

If anyone has anyother comment please let me know.
Will keep posted about whether the column survived 100 samples over the weekend.

Thanks again,

Michele

I don't have much to add column or chromatography-wise, but lipopolysaccharides can be faintly yellow, precipitated with alcohol, and are going to be quite abundant in a pure bacterial culture.