HI,

I was recently ask by a fellow chemist to determine the enantiomer excess of a solution. The compound is very polar and only small resolution was obtained when using a CHIROBIOTIC T column in reversed phase mode. Any clues that could help increase the resolution.

Also, what types of additives could be added to reduce band broadening??

Dear anonymus;

I do not believe there is a "magic" additive that you can add to increase column efficiency.

Try changing the temperature, sometimes that improves resolution. I would try first decreasing it.

If your samples have acid or basic groups try different pH values introducing a buffer. Just make sure to stay within the recommended limits. Another change you may want to try is to use a different buffer salt.

Also, you may have to try a different chiral column, perhaps Chirobiotic V.


Good luck;

Benjamin

Dear Anon,

I'm sure you have already thought of this, but the Chirobiotic V phase can sometimes have dramatically different selectivity for compounds versus the Chirobiotic T phase. Also I believe there is a Chirobiotic R phase available now but I don't have any experience with that phase yet.

Regards,
Mark

Thanks all for your quick answers,

the problem with changing the column is that I'm a Co-op student so I can't really tell my supervisors how to spend their money, but I'll try.

As for the temperature I'll try that today.

Hi,

I did try the suggested tips and got a sligthly better resolution of my two isomers. My problem is that I am still far from a baseline resolution.

Does anybody knows if the chirobiotic T column is efficient in normal phase for polar compounds? I would prefer not to have to transfer a HPLC to normal phase, but it seems to be the last possibility that I haven't tried.

Thank you all for your time

The chirobiotic T is supposed to be good for underivitized amino acids, FMOC, CBZ, carboxcylic acids, phenols, small peptides, neutral aromatics and cyclic aromatic and aliphatic amines. Since we don't know what your compound is, we're not sure if this buffer will work to separate polar compounds, but we use 10 mM ammonium formate in water adjusted to pH 3.8 with formic acid (solvent A) The other solvent (solvent B) is ACN. It runs on a reverse phase C18 4.6 mm x 250 mm column. Gradient = 10% B to 95% B in 15 minutes with a 3 minute hold at 95% + a 5 minute post to re-equal. We get baseline separation with a 30 minute equalization time before hand. Using that solvent system could give you baseline separation of the enantiomers. If you run normal phase, you have to run isocratic. You can't run a gradient in normal phase.

The chirobiotic T is good for separating underivitized amino acids, FMOC, CBZ, carboxcylic acids, phenols, small peptides, neutral aromatics and cyclic aromatic and aliphatic amines. Since you did not include a structure for your compound, we're not sure if this buffer will separate your polar compound, but we use 10 mM ammonium formate in water adjusted to pH 3.8 with formic acid (solvent A) The other solvent (solvent B) is pure ACN. It runs on a reverse phase C18 4.6 mm x 250 mm column. Gradient = 10% B to 95% B in 15 minutes with a 3 minute hold at 95% + a 5 minute post to re-equal. We get baseline separation with a 30 minute equalization time before hand. Using this solvent system could give you baseline separation of the enantiomers on the Chirobiotic T. If you run normal phase I have only had luck with isocratic methods to separate enantomers. Your best bet is to purchase a Chirobiotic V because the Chirobiotic V is "complementary stereoselectivity" to the Chirobiotic T. "If after optimization on the T doesn't resolve the analyte to baseline, using the Chirobiotic V column in the same mobile phase results in complete resolution. This also works in reverse." This is quoted from the Chirobiotic web site www.astecusa.com. which might be useful to you. Good Luck