The development of a method for the extraction of carotenoids and chlorophylls from grapevine leaves and berries for HPLC profiling
Abstract:Abstract Background and Aims:
Carotenoids and chlorophylls perform a number of essential roles in plants making their accurate quantification important to a variety of studies. We aimed to develop an extraction protocol to accurately determine the photosynthetic pigments in grapevine leaf and berry tissue, specifically focusing on limiting the degradation of these pigments. Methods and Results:
An extraction protocol for grapevine leaf and berry tissue was systematically optimised by identifying a number of critical parameters. Extracted pigments were analysed using Reversed Phase-High Performance Liquid Chromatography (RP-HPLC). Specific parameters that were optimised included avoiding freeze-drying the material; the volume of acetone and the time required to extract all the pigments from the tissue; the addition of 0.1% (v/v) N-ethyldiisopropylamine to berry extracts to minimise pigment degradation during the extraction procedure; and avoiding concentration of the extracts that otherwise resulted in differential degradation of pigments. Additionally, the method of extraction and normalisation with an internal standard was adapted and improved for accuracy. The optimised protocol was validated using authentic standards and its utility shown by analysing the pigment content of berries and leaves at different growth stages. Conclusions:
A method has been developed that is able to extract and accurately quantify, by means of HPLC profiling, the levels of photosynthetic pigments from grape berries and leaves. The method avoided any degradation of the pigments during the extraction and was applicable to both berries and leaves in different stages of growth and development, indicating its general usefulness to vegetative and reproductive organs, even if their metabolic states are very different. Significance of Study:
The divergence of methods used for photosynthetic pigment analysis in plants, each with specific advantages and disadvantages were considered and used to optimise a number of parameters in a single method that proved to be applicable to plant organs in different developmental stages. The method is fast, applicable to vegetative and reproductive grapevine tissues, avoids degradation of pigments and ensures maximum accuracy when quantifying these important pigments.
Document Type: Research Article
Publication date: June 1, 2010