We are determining the enantiomeric excess by NP-HPLC (using Chiralcel OD-H column employing Hexane:IPA solvent system). The product of racemic reaction show two peaks at Retention Time 18 and 20 min (each have 50% area). When we analyzed the same products (obtained from the reaction, in the presence of chiral catalyst) we obtained peaks with significant change in Retention Time (say at 23 and 25 min, having area 80% and 20%). Is this possible?
you can prepare a mixture containing the racemate and reaction products and inject into the HPLC.
If you see four peaks, then it is quite clear the reaction products are different from the racemate.
If you see two peaks, you may need to consider what else is present in your mixture as mentioned by Larissa.
As both suggested, I think the change in retention time might be due to changed conditions of the sample between your first and second experiment. (can it be that in the longer r.t. times situation, your sample is dissolved in a different solvent?)
Also, chiral chromatography is not my forte, but remember that a chiral catalyst might have different stereo-specificity , leading to the 20% 80% ratio.
Check also if you have any possibilities of diasteroisomers.
Hope this helps
As already mentioned before, you should not see significant changes in retention times if you use exact the same conditions. Chiral columns, on the other hand, are very sensitive to any changes in mobile phase composition and overloading. Did you "clean" your column between runs? If so, how long did you equilibrate it before the next injection? In any case, I would expect shorter rather than longer r.t. if the problem happens to be small amounts of more polar solvents... As suggested by Eugene, I would spike the mixture of your reaction products with your racemate to rule out the possibility of any matrix effect. Another thing to consider is the identity of your reaction products. That could be done by running a quick 1H NMR. Finally, depending on the ration of your chiral catalyst to your staring materials and products you could end up with some sort of cluster that would be favoured in this apolar environment, and would behave completely different inside the chiral column.
I am not familiar with the acronym NP-HPLC... please explain.
However, as for the applications of chiral columns, including cellulose based ones, there is a marked sensitivity to water content in the solvents (I hope you are not using a gradient in a chiral application on this column). For example, if previously your IPA had 0.1% H2O, and more recently it had 0.3% H2O (these numbers are based on some our KF analysis of HPLC grade IPA batches), then differences like that could occur.
Also, if your compounds do not have a good chromophore, but the catalyst absorbs light very strongly, in such case you may be much more strongly detecting a complex of your compounds with the catalyst. This sometimes happens.
thanks for your kind response, sir. NP-HPLC stands for Normal Phase High Performance Liquid Chromatography
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