I am trying to resolve cis/trans isomers of a theophylline derivative by RPHPLC, I have tried many gradient and isocratic separations using 150x4.6 C18 3u phenomenex with 0.05% TFA in MeCN and ditto H2O. I cannot resolve these isomers and am running out of ideas. I have no access to a column oven so temperature cannot be used as a factor (although this would be beneficial yes?, I will try a 250x4.6 column aswell to try to pull these peaks apart. Are there any tricks for sepearting such isomers by RPHPLC? or do I have to convert my system to NP to stand a chance of getting good resolution?

Kio,
I have found that the addition of THF generally hepls with the resolution of samples such as these. Might I suggest the addition of about 10% THF to the acetonitrile.

Good luck,
Mark

P.S. An alternative column might be a Hypercarb (graphitic carbon, these work wonders on isomers.)

Can you give me some more info on hypercarb columns - cost/best supplier/reliability/stability to what solvents?
I know they change polarity to RP so my polar theophylline is more strongly retained so what would be a comparable column to my current C18?
Thanks

Kio- where is the cis/trans isomerism in
theophylline?

it is a theophylline derivative as i said

Hi, Kio
information about Hypercarb column are avalaible from manufacturer at www.thermohypersil.com.
With a hypercarb column (100*3 mm - 5 µm) I have obtained a separation of rotational isomers of metolachlor (herbicide) in RP chromatography.
Good luck.

I recommend trying methanol as your organic modifier in place of acetonitrile. This will often provide different selectivity on the same column. Just be aware that your backpressure will increase. If that doesn't work, try a different RP stationary phase. Someone else recommended Hypercarb. I've found that a phenyl phase, "polar endcapped" C18 phase, or "polar embedded" phase may provide the selectivity you need where an ordinary C18 fails. Try each phase with methanol and acetonitrile as the organic modifier.

I am sure TFA acts as ion pair with the compounds you are analysing (TFA likes to do such nasty things...).
Before buing a Hypercarb (this column isn't cheap) check out phosphoric or acetic (acid and buffer) to see if you can get some seperation. Maybe a change in pH can help too.

The Hypercarb is a very different column compared to C18. You have to spend a lot of time with method adjustment. But I agree with Anon July 31 - 05:49, it could work (I expect theophylline to retain on a hypercarb quiet fine).
BTW: With the Hypercarb the "disadvantage" of TFA is working as an extra seperation tool.

Cyclodextrin columns e.g. ChiraDex are very useful to separate for enantiomers or other isomers.

Kio- since you aren’t having any luck w RP can you
try NP-say silica gel column, mp =hex/ipa or etoh?
This type of system works well with c/t
separations - for example many (and there are
many) of the c/t isomers of Vitamin A.