Page 31 - Application Notebook - Solution for Food Development

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