B. Fransson et U. Ragnarsson, Determination of the optical purity of threonine and hydroxyproline by capillary gas chromatography on a chiral stationary phase, AMINO ACIDS, 17(3), 1999, pp. 293-300
Experimental conditions for the derivatization and resolution by GLC of all
stereoisomers of threonine and LC-hydroxyproline are reported. Threonine w
as in two steps converted to N,O-bisisobutoxycarbonyl 2,2,2-trifluoroethyl
ester derivatives, the second of which was performed under anhydrous condit
ions. As such the enantiomers could pairwise be separated by capillary gas
chromatography on a Chirasil-Val column. Since L- and D-threonine eluted mu
ch earlier than the corresponding allo forms, quantitative determination of
the allothreonine content in D- or L-threonine down to the one percent lev
el could be simply accomplished but also enantiomeric impurities could be d
etermined. Unlike for threonine, the corresponding 4-hydroxyproline isomers
could not all be resolved as N,O-bisisobutoxycarbonyl 2,2,2-trifluoroethyl
esters on this column. Although diastereomers could still be separated, th
e allo pair cochromatographed and the resolution for the L- and D-isomers w
as low. Complete separation of the 4-hydroxyproline isomers could be accomp
lished as N,O-bisprotected isobutyl amides, the formation of which required
three derivatization steps. These were used for the determination of alloh
ydroxyproline.