Chromatography Forum

try 70%organic solvent alongwith 40'c column oven during washing for 1hr

Sneha, I think it is better if you use column volume than time unit in column washing procedure.

Syx,

I and my colleague were both drafted onto other projects after my last post, so could not follow up on suggestions immediately.

"100 mM buffer/ MeOH (1:1)" was mentioned in an old training course manual, and the use of ammonium salts as additives in SFC (CO2 with MeOH) to elute aryl sulfonates was described in a recent paper.

Therefore, I spent some time in the lab. yesterday p.m. (but did not have access to acetone at that time) using LC/MS to monitor elution of SDS at m/z 265 (-ve ion mode) with mixtures of aq. amm. acetate/MeOH.

Seems to work well, except for prolonged tailing of the SDS (albeit at low levels) so still needs some improvement.

Cannot use phosphate or other non-volatile buffers due to required use of LC/MS for the desired application.

Any thoughts from the chromatogaphers ???

Thanks,

JMB

Further runs revealed that the prolonged low-level tailing of the SDS elution profile (which in retrospect was quite similar for all elution conditions) was due to SDS deposition/desorption from the LC/MS interface; it was not a chromatographic effect.

Thanks to all for the input,

JMB

Mark, is your cleaning method effective to other ion-pair reagents?

I have found it works satisfactorily for Triton X-100 (not an IP agent). I have not formally evaluated cleaning of other IP agents.

Hope you will let us know the result.

A regeneration procedure I used for anionic ion pairing reagents is the following (for a 4.6 mm column) : methanol (50 mL), acetonitrile (50 mL), tetrahydrofuran (30 mL), then methanol (50 mL). I used this succesfully to get rid of long perfluorinated carboxylic acids from the column. The method is described in Petritis et al. J. Chromatogr. A. 833, 1999, 147-155.

I also wondered what kind of test could guarantee me that all the ion-pairing reagent was removed from the column (or else how am I sure that my column has not been modified). I thought that one of the tests from Tanaka and co-workers paper (J. Chromatogr. Sci. 27, 1989, 721) that uses the separation factor between caffeine and phenol should do the job. The test was conceived to measure qualitativly the presence or absence of residual silanols but I think that could help to identify if all the ion-pairing reagent was removed as the residual ion-pairing would retain caffeine and or modify the presence of residual silanols (especially in the case of cationic ion-pairing reagents).

In general I was measuring the separation factor of the new columns or columns before I use an ion-pairing reagent and then I was measuring it again after regeneration of the column from the ion-pairing reagent (and expecting it to be similar, if not identical).

A regeneration procedure I used for anionic ion pairing reagents is the following (for a 4.6 mm column) : methanol (50 mL), acetonitrile (50 mL), tetrahydrofuran (30 mL), then methanol (50 mL). I used this succesfully to get rid of long perfluorinated carboxylic acids from the column. The method is described in Petritis et al. J. Chromatogr. A. 833, 1999, 147-155.

Pure tetrahydrofuran?

Indeed!

Actually it is pretty commonly used in the regeneration procedures of reversed phase columns...

Somebody had told me that pure THF is not compatible with PEEK. If we have PEEK parts and tubing in the system, what alternative will you recommend us to do?

Syx,

Compatibility of THF with PEEK has been discussed previously in this forum... and yes THF and PEEK are compatible... Actually maybe the discussion is old enough that is in archieves by now...

Hmmm... I went on and searched for it and here you are:

http://www.lcresources.com/discus/messa ... 20020814pm

You'll see that I advice not to use 100% THF with PEEK tubing but they are compatible... The only problem is PEEK's physical swelling and lower burst pressures, so you might want to use lower flow rates when you are pumping 100% THF...

WOW, I just realized that the discussion had taken place 3 years ago and all the usual suspects participated in that discussion (HW Mueller, Chris Pohl and Uwe Neue)...

I'm impressed...

I find the selectivity (alpha) of an acid and a base at pH 7 to be very sensitive to ion-exchange effects. I have used 4-butylbenzoic acid and acebutolol for that.