Natural rubber quantification in sunflower using an automated solvent extractor

Leaves of sunflower (Helianthus annuus) produce a small amount of low molecular weight natural rubber (NR) and this species has potential as a rubber-producing crop plant. Quantifying NR in plant tissue has traditionally been accomplished using Soxhlet or gravimetric methodologies. Accelerated solvent extraction (ASE) provides faster sample extraction, excellent repeatability, and lower labor costs for analysis than Soxhlet. Other features of ASE, such as low bench space requirement, limited exposure to solvents, flexibility in selecting operating conditions and sample cells, and automated operation create additional incentives for using ASE for NR determination. The objective of this research was to develop a protocol for extracting and quantifying NR in sunflower using ASE technology. Acetone was used to extract resins, methanol to extract chlorophyll and other contaminants, and hexane to extract NR from sunflower tissues in 11-ml extraction cells. Temperature, extraction time, and number of extractions were evaluated for the three solvents. Given that 90+% of the NR in sunflower tissue is low molecular weight (65,000–75,000 g/mol), we recommend two, 16-min acetone extractions at 40 °C; five, 5-min methanol extractions at 60 °C; and finally, two, 16-min hexane extractions at 40 °C for extraction of NR. The methodology includes mixing inert sand with the tissue sample and preparing a tissue sample of suitable mass size by grinding. These methods should be suitable for other low molecular weight rubber-producing plant species.