HPLC method suggestion needed.

[rrm1987]

I would like to develop a method for Patulin.pl give your suggestions.
i have schimadzu binary gradient system with PDA and flurosence detectors.C18 phenomenex 5micron column.

[Mattias]

Google?

It appears to have been done by both HILIC and RPLC. Since you have a C18 column at hand, a simple gradient from e.g. 0 - 20% acetonitrile in water should work as a starting point. The molecule has no charges, so pH should not be important. This should be straighforward unless you have a complicated sample matrix.

[rrm1987]

thanks.I worked on it.intially no tailing was observed but after some tailing was observed .what could be the reason?

[rrm1987]

can you tell how came to know there is no charge on the patulin molecule.

[Mattias]

There are no acidic (e.g. COOH) or basic groups (e.g NH2) attached to the molecule. The molecule has no possibility to accept or get rid of a proton = neutral molecule.

That being said, the OH group is slightly acidic and will loose the proton at about pH 10-11 (guess). This pH is anyhow not realistic to use in the mobile phase.

[rrm1987]

Thank you.can you tell me whether acidic pH mobile phase is required for Cyclopiazonic acid determination by HPLC

[Mattias]

Cyclopiazonic acid contains a secondary amine (NH) which is quite basic. I would use 0.1% TFA in the mobile phase for this molecule to reduce tailing.

[JMcK]

Cyclopiazonic acid has a pKa of 3. You usually want to work 2 pH units away from the pKa. An acetate buffer could keep you at pH 5.

[Mattias]

It is indeed called an acid, so I guess it is acidic... It is quite strange when you see the structure on Wikipedia, no obvious acidic groups and a couple of quite basic ones. My knowledge in organic chemistry is not great and this is more complex than my basic courses.

However, I would still go for acidic mobile phase, in order to protonate the acid and reduce the silanol effects of the column.

[rrm1987]

Thanks a lot for your suggestions.

[MestizoJoe]

There is only one weakly basic nitrogen on the molecule which should have a pka around 4 - 5 (similar to pyridine, another aromatic amine). The other nitrogen is part of an amide group which is not basic. I see no acidic functional group. Could it be the wrong structure in wikipedia or maybe it is incorrectly called an acid?

If you get a higher retention time at pH 7 than at low pH it may be evidence that the molecule is nuetral at pH 7 and has no group with significant acidity.

see also http://www.chemicaldictionary.org/dic/C ... d_180.html

[Vlad Orlovsky]

At lower pH 91.8-2.5) your compound will behave as weakly basic, at pH 3-5 it will be zwitter-ionic, and at higher pH it will become acidic. At lower pH you might observe tailing due to intercation of your amine with residual silanols (some of them have low pKa due to impurity inclusions). One of the choices would be to do this at lower pH on Primesep 100 column 9reversed-phase cation-exchange column). Should behave similar to amoxicillin:
http://www.sielc.com/Application-Separa ... illin.html