Page 31 - Application Notebook - Solution for Food Development
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LAAN-A-LC-E268







            Application                  High Performance Liquid Chromatography

            News                         Comprehensive 2D Separation of Carotenoids in


                                         Red Chili Pepper by the Nexera-e System
            No.L491





            Carotenoids are naturally occurring organic pigments that are   n Comprehensive Separation of Carotenoids
            divided into two classes, carotenes, consisting only of carbon   Detected by the Photodiode Array Detector
            and hydrogen, and xanthophylls, which contain oxygen.   Use of the Nexera-e with a photodiode array detector
            Carotenoids are rich in double bonds, and therefore have   (PDA) permits the separation of complex coexisting
            received much attention in recent years as antioxidants,   substances and detection at the optimal wavelength in a
            which are known for their disease preventive properties,   single analysis. Fig. 1 shows a comprehensive two-
            including lifestyle-related diseases.              dimensional representation of the separation pattern
            The extensive range of carotenoids found in foods makes it   (absorption wavelength = 450 nm) generated using the
            difficult to conduct simultaneous separation and quantitation   specialized two-dimensional analysis software,
            by conventional HPLC. However, the Nexera-e comprehensive   ChromSquare.
            two-dimensional LC is particularly suited for such analyses.   By combining the first-dimension cyano column and the
            Here, carotenoids extracted from red chili pepper were   second-dimension ODS column, 10 groups of substances,
            subjected to two-dimensional analysis, in which micro-scale   including hydrocarbons, monool esters, diol diesters, diol
            separation was conducted in the first stage using normal   monoketo diesters, diol diketo diesters, diol mono epoxide
            phase conditions, and separation using reversed phase   monoesters, free monools, diol monoketo monoesters, diol
            conditions was tried in the second dimension. For detection,   diketo mono esters, and polyoxygenated free xanthophylls
            a combination of a photodiode array (PDA) connected to the   were separated according to class based on molecular
            LCMS-8030 triple quadrupole mass spectrometer was used.   polarity, and the component separation was verified based
            Because the separation modes, normal and reversed phases,   on the hydrophobicity of their respective fatty acid
            differ in the first and second dimensions, this might be   residues.
            considered a two-dimensional LC method by which the
            greatest orthogonality possible is obtained.



                    RP-LC, min   2




                                                 4
                                         3               5
                                                                   8
                                 1


                                                 6
                                                                                       9







                                                          7
                   1: Hydrocarbons
                   2: Monool esters
                   3: Diol diesters
                   4: Diol monoketo diesters                                                       10
                   5: Diol diketo diesters
                   6: Diol monoepoxide monoesters
                   7: Free monools
                   8: Diol monoketo monoesters
                   9: Diol diketo monoesters
                  10: Polyoxygenated free xanthophylls                                         NP-LC, min



                                        Fig. 1  2D Plot of Carotenoids Using ChromSquare Software
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