Sepration of two drugs with opposite Physicochemical Prperty

[Lucifer]

hi i am working on separation of two drugs both having entirely different physicochemical property,one is having log p -1.95(x) and other is having log p 2.5(y), i tried most of the ph range from 2-7 and different proportion of ACN and Phosphate buffer. on varying the percentage of ACN Rt of drug y is shifting but drug x is eluting with mobile phase at 2.6 (i tried on C18,C8and Phenyl column).On phenyl column both x and y have same Rt and on C8 drug y is not eluting.Can anyone please suggest me what to do?

[HPLCaddict]

In other words, drug x is very hydrophilic and you don't get it retained on RP phases.
Some more information would be helpful.
Do you know the pka values of those two drugs? Did you try gradient elution or only isocratic? Can you reveal which drugs we're talking about?

[Lucifer]

drug x is famotidine and drug y is rabeprazole and pka values are 9.29 and 8.38 for fammotidine and 9.35 and 4.2 for rabeprazole i have to use isocratic elution only as directed by group leader, thank you for your help.
and famotidine is eluting at 2.6 minute

[tristanewalters]

What is your group leader's reasoning for not allowing a gradient method? Since the drugs are so different in terms of hydrophobicity and you are wanting to run RP, gradient seems like the best way to go. A mixed mode functionality column may work for this too to be able to retain the hydrophillic compound longer, but your run time would probably be pretty long to elute the hydrophobic compound

[Lucifer]

i am sorry sir i cant ask him.. as i am new ..well i can try what u said thanks a lot sir.but i will be grateful if you could suggest me which column will be better 150cm or 250 cm with particle size 5 micron.

[tristanewalters]

Have you told him what you have found so far? Let the data you have obtained speak for itself.

Are you premixing your mobile phase or are you letting the instrument mix? If you are letting the instrument mix, you might as well run a quick gradient 5%-95% B over 20 minutes and see how that looks.

As for whether to use 150 or 250mm column? It doesn't matter a whole lot because you are either not retaining one compound or you the column is holding on to the other depending on your conditions.

Columns that may hold on to your hydrophillic compound better could be the Waters XBridge Shield columns or perhaps Coresep columns as they have some polar groups in the stationary phase. If that is not possible, I would try the gradient.

[sepscientologist]

if you can run your pH high enough to deprotonate all of those amines on Famotidine then I suspect
it might be possible. I would guess their logP are not that different in their non-charged states.

[Lucifer]

ok sir i will try gradient elution once on c18 column and today i showed him the chromatogram, i asked him whether i cna try gradient, he agreed.and i am not mixing the mobile phase i am allowing instrument to mix and i should start with more of buffer initially and increase the organic phase gradually.
thank you everyone for your suggestions means a lot to me.

[DJ]

logP values apply only to the neutral form of a compound. Since you are separating cationic forms of these solutes, forget about what their clogP is supposed to be. It's meaningless.

Get an appropriate column (Gemini, Xbridge, or a polymeric reversed phase-C18) and do your separation at pH 11.

[Vlad Orlovsky]

Mixed-mode allows to transfer practically any gradient method to isocartic, particularly in cases when compounds are totally different in properties. If you have highly hydrophobic compound and hydrophilic ionic compound, you set up method to minimize hydrophobic interaction and enhance ionic interaction. In a lot of cases you can change order of elution for compounds. Here are couple of good examples:
http://www.sielc.com/Application-Separa ... sep-C.html
http://www.sielc.com/Application-HPLC-S ... edies.html
http://www.sielc.com/Application-HPLC-S ... Sinus.html

Methods for above mixtures on traditional RP columns will be at least 3 times longer.

If you can send me both samples we can show you how this can be done under 8 minutes in isocratic mode.

contact me if you have question