ABSTRACTOlean is the chiral spiroacetal sex pheromone of the female olive fruit fly Bactrocera oleae. Laboratory tests have demonstrated that the (R)‐(−)‐olean enantiomer is active on males, whereas females respond to (S)‐(+)‐olean. Here we present the first HPLC enantioseparation of olean using polysaccharide derivatives as chiral stationary phases and a polarimetric detector equipped with a micro‐flow cell capable of detecting optical rotation at six different wavelengths. Unlike blind UV detection, the polarimetric detector allows the chiral spiroacetal, which lacks a chromophore, to be detected as bisegnate peaks, indicating the opposite sign of the optical rotation. The HPLC enantioseparation was optimized on the coated type amylose‐based Chiralpak AS‐H CSP using a mobile phase consisting of n‐hexane‐2‐propanol 99.99:0.01 (v/v). These conditions were scaled up to a semi‐preparative level and allowed resolution of 2.5 mg of racemic sample in 5 min. Multiwavelength optical rotation detection during HPLC enantioseparation of racemic samples provides a direct readout of the stereochemistry of olean and allows tracking of virtual optical rotation dispersion curves without the need for preliminary collection of enantiomeric samples by semi‐preparative HPLC.
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