TY - JOUR
T1 - Purification and use of carotenoid standards to quantify cis-trans geometrical carotenoid isomers in plant tissues
AU - Anwar, Sidra
AU - Nayak, Jwalit J.
AU - Alagoz, Yagiz
AU - Wojtalewicz, Dominika
AU - Cazzonelli, Christopher I.
PY - 2022
Y1 - 2022
N2 - 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.
AB - 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.
UR - https://hdl.handle.net/1959.7/uws:68925
M3 - Article
SN - 0076-6879
VL - 670
SP - 57
EP - 85
JO - Methods in Enzymology
JF - Methods in Enzymology
ER -