Purification and use of carotenoid standards to quantify cis-trans geometrical carotenoid isomers in plant tissues

Sidra Anwar, Jwalit J. Nayak, Yagiz Alagoz, Dominika Wojtalewicz, Christopher I. Cazzonelli

Research output: Contribution to journalArticlepeer-review

8 Citations (Scopus)

Abstract

Reverse-phase high-performance liquid chromatography (HPLC) is a preferred method used to identify and quantify carotenoids. Here, we describe a straightforward, reliable, and cost-effective protocol to purify and develop individual carotenoid standards for absolute quantification of carotenoids, including selected cis-trans (geometric) isomers. Analytical techniques to extract, purify and collect individual carotenoids using an HPLC system equipped with a Diode Array Detector (DAD) and fraction collector are described. Carotenoids were separated and identified by their characteristic ultraviolet-visible (UV–Vis) absorption spectra and individually isolated based on their retention times using a C30 column. This chapter outlines how to prepare standard calibration curves using known quantities of purified and/or commercially available carotenoids. A series of molar extinction and slope coefficients for phytoene, phytofluene, ζ-carotene, neurosporene, pro-lycopene, all trans-lycopene, lutein, β-carotene, zeaxanthin, antheraxanthin, violaxanthin, neoxanthin, capsanthin, capsorubin and β-cryptoxanthin are defined to enable absolute quantification of their abundance in plant, animal, and bacterial tissues. Different approaches for reporting carotenoid abundance by absolute concentration, relative composition, and/or using ratios of different pigments are provided as a convenient resource for carotenoid researchers.
Original languageEnglish
Pages (from-to)57-85
Number of pages29
JournalMethods in Enzymology
Volume670
Publication statusPublished - 2022

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