I want to detect the presence of a compound in a sample through HPLC. I do not know the orientation of the compound. I have a reference standard which is a (-) isomer of the test compound. How do I know whether the orientation of the test compound is (+) or (-)?
As with all of these things, it depends on the compound and what you want from it.
Amphetamine by HPLC cannot discriminate between (+) and (-) and you can only tell the difference with either a chiral mobile phase modifier/derivatising agent or with a chiral stationery phase or a chiral detector. Some other substances have significant conformational interactions or restrictions which would change the bulk structure of the molecule and therefore potentially affect the retention time. For a simple compound where it does not matter to you whether the compound is + or - but you just need the concentration, then simple HPLC should be suitable. If it matters to you whether your measured compound is + or - then you will probably need to try one of these alternatives. If your compound is biological then it is likely that there will only be one isomer present anyway.
The short answer is no. A chiral molecule can only be separated in a chiral environment in HPLC or capillary electrophoresis. What is your molecule?
In terms of quantification - yes. If you will obtain separation via chiral HPLC you can use calibr. parameters from one of isomers to each of analyte- they will have the same answer.
regards
G
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