Chromatography Forum

Dear all,
I have to separate 3-Pyridylacetic acid from it's isomers 2-Pyridylacetic acid and 4-Pyridylacetic acid.

To check structure please visit: http://www.sigmaaldrich.com/catalog/sea ... ICH/P65800

Does anyone have any ideas?
I have tried C8 column and citric acid buffer at pH 3.7; flow 1 ml/min; at 263 nm. At those parameters I can separate 4 and 3, but 2 and 3 are overlapping. After a while column collapses due to 100% aquaeous mobile phase. I have tried to regenerate column with 30/70 ACN/water. Is there any better way of regenerating columns?

Thanks,
Gordan

Hi Gordan,

We are working right now on separation of quinolinic/picolinic acids for one of our customers. We are using mixed mode approach to separate these compounds. I will update you when we have a method. If you are willing to send your compounds (three pyridilacetic acids) we will develop a method for you.

Here is an example on how you can se mixed mode chromatography for separation of closely related isomers of dihydroxybenzoic acids:

http://www.sielc.com/compound_035.html
Contact us directly if you have questions:
http://www.sielc.com/Services_MethodDevelopment.html

regards,

Vlad

So if I am correct, you sugest a Primesep B column.
Only thing that is somewhat unclear is how do you prepare your mobile phase. What is composition of A and B mobile phase.
Thanks once again,

Gordan

Gordan,

Your compounds are rather zwitter ionic then acidic. Depending on pH you can use different columns. If you go below pH 4 you will suppress acidic fragments of your acids and can separate it a weak basic molecule on Primesep 100 or Primesep A column. In your case you should be able to do it with isocratic conditions. With Primesep columns you don't need to go to 100% aqueous (although you can). You will retain compounds based on reverse phase and ion-exchange mechanisms. At lower pH your compounds will retain similar to pyridine:
http://www.sielc.com/compound_079.html

Another example of this approach is separation of toluidine isomers (developed by us for chromforum user):

http://www.sepsci.com/chromforum/viewto ... =toluidine

Do you know pKa values of these compounds?

I checked our lab supplies and we don't have these three compounds. Let me know if you are willing to send a sample (it cost around $100 fro all three at Aldrich and I'd rather request it from you). I think that be can develop a method in a day.

Vlad

Gordan, did you try slightly basic mobile phases? . . . just wonder whether these are still poorly retained by rp (when the pyridine is not ionized).
(I am a bit surprised that the competition of the above has not mentioned columns which do not de-wet in an aqu phases, or HILIC)

There are several asnwers to your problem.

The dewetting can be prevented, if you add pressure to the column post-column. A longer than normal piece of 9/1000 tubing could do the job.

The column can be rewetted in less than a minute with a large slug (1 to 2 mL) of organic solvent. It takes no time to do this.

Following HW recommendation that I shall open my mouth about columns that do not dewet, you could use an column Atlantis dC18 column from Waters. Chances are high that you get MORE retention from this column than from the one that you are using now.

Gordan,

I already ordered your compounds fro Aldrich. I will update you as soon as we have method for you.
what are your requirements for resolution, retention and total run time?

Regards,

Vlad

P.S. I would be glad if somebody will run this on Atlantis (like Uwe suggested)-want to compare results.

several comments:
I think every column mfg. makes one or more columns designed to work in 100% water. And many that are not specifically designed for 100% water Silwork fine too.

Forget the complication of citrate buffer. use eluant of 0.05% phosphoric acid.

This would be an easy GC problem. Silylate it and shoot.

Gordan,

Please provide me with your email address and I will send you method for 2-Pyridinecarboxylic acid, 3-Pyridinecarboxylic acid and 4-Pyridinecarboxylic acid we just developed for one of our customers. Isomers are well separated (3-5 minutes between peaks) with good peak symmetry and plate count. Column-Primesep, mobile phase ACN/water/sulfuric acid. Your pyridylacetic acids will retain similary (by combination of reverse phase and cation exchange mechanisms.

We will post it on the web next week (if you don't want to provide us with email address)

Regards,

Vlad