Determination of the optical purity of threonine and hydroxyproline by capillary gas chromatography on a chiral stationary phase

Summary

Experimental conditions for the derivatization and resolution by GLC of all stereoisomers of threonine and 4-hydroxyproline are reported. Threonine was in two steps converted toN,O-bisisobutoxycarbonyl 2,2,2-trifluoroethyl ester derivatives, the second of which was performed under anhydrous conditions. As such the enantiomers could pairwise be separated by capillary gas chromatography on a Chirasil-Val column. SinceL- andD-threonine eluted much earlier than the corresponding allo forms, quantitative determination of the allothreonine content inD- orL-threonine down to the one percent level could be simply accomplished but also enantiomeric impurities could be determined. Unlike for threonine, the corresponding 4-hydroxyproline isomers could not all be resolved asN,O-bisisobutoxycarbonyl 2,2,2-trifluoroethyl esters on this column. Although diastereomers could still be separated, the allo pair cochromatographed and the resolution for theL- andD-isomers was low. Complete separation of the 4-hydroxyproline isomers could be accomplished asN,O-bisprotected isobutyl amides, the formation of which required three derivatization steps. These were used for the determination of allohydroxyproline.