Chromatography method optimization for HPLC determination of carotenoids.

This thesis deals with searching for optimal conditions for determination of carotenoids lutein, betacarotene and zeaxanthin. High performance liquid chromatography - HPLC was used for determination. During searching for optimal conditions many various columns and mobile phases were tested. Primary information about carotenoids separation was obtained from scientific literature. There were tested both types of chromatography - normal phases chromatography and reversed phases chromatography. The most frequently tested columns were C18 columns from different producers. The biggest problem for HPLC determination and separation of these three substance were position isomers of lutein and zeaxanthin. Separation of these substances is relatively difficult and it wasn't completely solved in this thesis. In spite of it new metod for determination and separation of three carotenoids was developed. This method used isocratic elution at column Zorbax SB C18 (50 x 4,6 mm, 2 μm), UV detection at 450 nm, mobile phase of methylene chloride:hexan:acetonitrile (2,5:2,5:95), injected volume 1 μl and flow rate at 1 ml/min