Hi everyone

I tried very hard to separate a cis isomer of the drug on the reversed phase which I finally was able to do but the problem is resolution which is about 1.2 Is it acceptable in the worst cases???

What is better to separate isomers (not enantiomers) like constitutional isomers or other stereoisomers- Reversed phase (Any suggestions on particular brands please) or Normal phase (Are cyano, amino and phenyl considered normal phase??)
Any particular brands or types for normal phase which can give good separation of isomers???

Thanks a lot for your insight

Sean

I would try normal phase on good old-fasioned silica.

If you want to use reverse phase, I would suggest this:

http://www.zirchrom.com/CARB.asp

Sean,

Without knowing what is being separated this is just a general suggestion. I have separated several of these compounds by reversed phase quite well, the secret almost always seems to be to add some THF to the mobile phase, this has made very large differences in some of these separations. Another choice is related to the previous suggestion, I have used Hypercarb columns also to separate these types of compounds also. This is a graphitic carbon column similar to the one suggested in the previous message.

Regards,
Mark

We have also had better luck separating isomers (pesticides) by reverse phase using AN-THF than just AN. Methanol also seems to work better than AN. To answer your other question, I believe phenyl is considered reverse phase and cyano and amino columns are generally considered normal phase. However, cyano and amino columns can be used as reverse phase columns (aqueous mobile phase). I believe amino columns are also used as ion exchange columns.

Recently I have had good success with YMC Basic column (5 micron) column in separating positional isomers by just using CH3CN and Ammonium acetate buffer(just to control pH) in reversed phase.

Sean,

I refer you back to the first "anonymous" response. Although you may succeed in this case, as in most others, by varying the mobile phase or even the column, it has long been known that structural isomers are often best separated by adsorption, rather than partition, chromatography.

Silica gel, alumina, and elemental carbon materials are the classical adsorption stationary phases, but there may be other, more modern ones available as well.

The composition of the mobile phase will still matter, because the behavoir of these adsorption phases towards your analytes may vary a lot depending upon the mobile phase. A classic example is the level of water in a normal-phase separation on silica. Other examples include the need to provide a MP component to compete with the analyte for the solid support, in order to elute the analyte in reasonable time and with good peak shape.

For the benefit of newbies:
Most modern HPLC is partition chromatography, wherein the solutes partition between two "liquid" phases. That is to say, a solute is dissolved either in the mobile phase or in the stationary phase. Usually the stationary phase is bonded, so not strictly speaking a liquid, but it behaves rather like one. (Sometimes the mobile phase is a supercritical fluid, so not strictly a liquid either, but that's another issue.) Partition chromatography works principally on the basis of relative solubilities. Solid support surface effects do come into play, but mostly as bad actors, causing tailing, etc.

In contrast, adsorption chromatography is based upon a completely different principle. In this form of chromatography, the solid surface is active, and adsorbs the solute. There is normally no stationary liquid (or bonded) phase. The upshot of this is that it is not solubility that is the driving force for the separation, but the "shape" and polarity of the molecule. Accordingly, this form of chromatography can be tremendously more powerful than partition chromatography for the separation of isomers. In fact, some "chiral" columns can be consider to be highly sophistocated adsorption columns, even though they are otherwise like partition columns.

This is not to say that you won't still be able to perform 90% of your separations on ODS!

You can use cyclodextrin additive in the mobile phase. Cyclodextrins are used in reversed phase chromatography as mobile phase additive for separation of isomers or enantiomers.
The best way to separate enantiomers is capillary electrophoresis using chiral selectors in the background electrolyte.

How much of Cyclodextrin you noramlly add to the MP. Can it be 0.02 M?

Lower cyclodextrin conc. are usually used between 0.001-0.01 M. The use of cyclodextrin derivatives (e.g. hydroxypropyl or randomly methylated beta or gamma cyclodextrin) is advisable instead of parent cyclodextrins.

Thank you for the information. I could only find alpha, beta and gamma cyclodextrin in Aldrich. They are cyclohexaamylose, cycloheptaamylose, and cyclooctaamylose. Aldrich says that alpha cyclodextrin is used in chromatographic separation. Should I have to try that first? or is there any other place where I can get hydroxypropyl or randomly methylated beta or gamma cyclodextrin. PLEASE LET ME KNOW ASAP.

Alpha cyclodextrin has a small cavity, and inclusion complexation seems to be very important for isomer separation.
Beta cyclodextrin, one of the most effective, has low solubility, so much better if you find a supplier for partially hydroxilated Beta cyclodextrin (hydroxipropyl).
Try to get some information in www.cyclodextrin.com

You can find more information of cyclodextrins and Product and price list on the available cyclodextrins in www.cyclolab.hu

try to talk to Beckman instruments. they promotes using beta-CD on CE to do chiral separation

Hi,

Recently we separated geometrical isomers with a Zorbax SB-Aq column using 100% aqueous phase (ammonium acetate buffer) You can try your luck with that.

Zorbax SB-Aq is a reversed phase column designed speciifically to withstand high aqueous condition and it is made by Agilent Technologies