APPLICATION OF DIODE-ARRAY DETECTION WITH A C-30 REVERSED-PHASE COLUMN FOR THE SEPARATION AND IDENTIFICATION OF SAPONIFIED ORANGE JUICE CAROTENOIDS

Thirty-nine carotenoid pigments in saponified orange (Citrus sinensis) juice were separated using a water, methanol, methyl-tert-butyl ether gradient on a non-endcapped C-30 reversed phase column. Pigments were extracted using the International Fruit Juice Union method for orange juice carotenoids, which employs precipitation with Carrez reagent an d resolubilizing with acetone. Chromatographic resolution, R(s), betwe en lutein and zeaxanthin was 2.9. Pigments were identified on the basi s of diode array spectral characteristics, retention times, and relati ve elution order compared to authentic standards and literature values . An examination of the diode array data from the chromatographically resolved peaks indicated that the most useful information could be obt ained from monitoring the chromatographic effluent at 350, 430, and 48 6 nm. More carotenoid peaks were detected at 430 nm, but greater selec tivity for several carotenoids was obtained at 350 and 486 nm. At 430 nm the six largest carotenoid peaks observed in orange juice consist o f auroxanthin A, mutatoxanthin A, mutatoxanthin B, lutein, zeaxanthin, and isolutein. Whereas open column and thin-layer chromatography requ ired days, the separation and identification of saponified orange juic e carotenoids can now be accomplished within 40 min.