Evaluation of the extraction and stability of chlorophyll-rich extracts by supercritical fluid chromatography
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RESEARCH PAPER
Evaluation of the extraction and stability of chlorophyll-rich extracts by supercritical fluid chromatography Thibault Lefebvre 1 & Emilie Destandau 1 & Eric Lesellier 1 Received: 2 June 2020 / Revised: 17 July 2020 / Accepted: 3 August 2020 # Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract In this study, a rapid (less than 10 min) analytical method by reverse-phase supercritical fluid chromatography was developed with an isocratic mobile phase, enabling the separation of 11 compounds, chlorophyll a and b, pheophytin a and numerous allomers or epimers. This method was used to examine the stability of chlorophyll pigments of plant extracts obtained with various extraction methods including microwave-assisted extraction (MAE), supercritical fluid extraction (SFE), pressurized liquid extraction (PLE) and ultrasound-assisted extraction (UAE), with ethanol as solvent or modifier. The effect of storage was studied for both liquid and dried extracts. Irrespective of the extraction method, the evaporation of the extracts induced partial degradation of the chlorophyll pigments. It was found that liquid extracts could be stored at 4 °C for 3 weeks without a dramatic change in allomer forms of chlorophylls. However, during this storage period, epimerization appears to be important, leading to a significant decrease in the chlorophyll b native form. Keywords Chlorophyll . Supercritical fluid chromatography . Plant extraction . Sample stability
Introduction Chlorophyll pigments are responsible for the green color of external parts of plants, which is a quality attribute of vegetable products. Post-harvest treatments can be responsible for pigment degradation leading to color changes. Many studies have investigated the effects of various methods, including (i) plant drying, done to improve the duration of plant storage [1–3]; (ii) blanching, applied to inactivate enzymes [4] during specific maturation treatments, for instance maturation of tea leaves [5, 6]; (iii) the ripening of grape berries [7, 8], fruits [9] and olives [10]; and (iv) during many other manufacturing processes such as canning [11], freeze-drying [12], pasteurization [13] or microwave cooking [14]. Beyond the many parameters acting on pigment stability during these processes, the increase of temperature is often reported to play a major role in the chemical change of native chlorophylls a and b, either by replacing the magnesium cation of the porphyric group by two hydrogen atoms, leading to
* Eric Lesellier [email protected] 1
ICOA, CNRS UMR 7311, Université d’Orléans, rue de Chartres, BP 6759, 45067 Orléans Cedex 2, France
pheophytin forms, or by inducing epimerization on carbon 132, leading to a′ and b′ forms, both for chlorophylls and pheophytins. High thermal treatment also yields the loss of carboxy groups (decarbomethoxylation) on carbon 132, leading to pyropheophytin derivatives [14]. Endogenous enzymatic activity can also be promoted by processing conditions leading to dephytylated chlorophyll deriv
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