Chromatography Forum

well hi,

I'm developing a hlpc method to analyse a mix of natural products. i got some trials and i notice that using a classical column C18 (luna) i can't separate the pool of compounds. Observing the chemistry of the compounds of interest i notice a difference in polar groups.
i ask you if there's a column with polar groups that surely can separate a polar compound containing nitrogen (N of pyridin and amide) from a pool of compounds containing oxigen (OH and OMe). Is it possible to gain this kind of selettivity?
I try to vary wavelenght during the run and mobile phase so, before trying a lot of columns, i 'd like to razionalise the problem using a probably suitable one.
thank you for your help

Hi Vicentexxx,

There are lots and lots of the kind. Here are some brands, but there are many more: Waters Atlantis, Phenomenex Synergi Polar and MetaChem Polaris.
By the way, I don’t know what your expectations were, when you varied the wavelength, but if I were you I wouldn’t expect changes in separation. Trying different pH values is a much better approach. Start with a relatively low pH - f. x. 2.5 or something like that.

Good luck

hi danko and thank you,
i tried to vary wave lenght becouse there's a pool of compounds (anthocyanidins family) i'm not intersted in, that overlaps with my compound (pyridin derivate). So, if i use a polar ether or something else instead of a polaris amide or can i eliminate the overlapping?
or Am i a dreamer?

Hi Vicentexxx,

If a pyridine derivate is the compound of your interest, you might like to try a phenyl column.
Both Waters and Phenomenex can supply you with one. But don’t forget the power of pH in connection with selectivity. Also you can try some ion parrying agents, but this is the last resort.

Good luck

You may get more selectivity changes (read separation) also by either 1) changing your organic mobile phase or 2) changing your gradient conditions (assuming you are using these). If it appears to be a very difficult separation try slowing down the gradient time/steepness, changing your initial and end % organic etc.

to me, it seems you will not have short cut. A systematic approach must come in unless some else provide a ready method. Get a list of parameters you can change(text book) and write down a list of the change you are planning to try. setup up these conditions in your LC or mutilple LC and let it(them) run, you can have hundreds of conditions done over one weekend. you should be able to get one decent in one week. Good luck.

I think that you can try mixed mode columns. Your pyridine based compounds will retain based on hydrophobic and ion-exchange interactions and you can use such mobile phase that you pyridine containing compounds will retain much longer due to ion-exchange interaction. At low buffer concentration (or no buffer at all) pyridine will stay longer on the column then your neutral species. You can operate it in almost SPE type of mode. All your compounds are slightly different in terms of ionic and hydrophobic properties and you can use this to your benefits. Here is how it works:

http://www.sielc.com/Technology_2D_Properties.html

Also check these applications of pyridine containing compounds-with mix mode approach you can separate position isomers of substituted pyridines (and other basic molecules):

http://www.sielc.com/compound_228.html
http://www.sielc.com/compound_079.html
http://www.sielc.com/compound_214.html
http://www.sielc.com/compound_219.html

Regards,

Vlad