Chloroform–Methanol Extraction of Chlorophylla

Abstract

Chloroform–methanol (2:1, v/v) extracts significantly more (P < 0.001) chlorophyll a from algal cultures and from freshwater and seawater microcosms than does dimethyl sulfoxide, methanol, absolute methanol with hydrogen sulfide, or 90% acetone. For chlorophyte cultures it yields 97% or more of the chlorophyll a within a 4-h steeping period without grinding. It can be used with both calibrated spectrophotometry and fluorometric instrumentation. Sample filtration onto MgCO₃-coated filters is recommended to ensure buffering of the extraction mixture. Holding the filters in chloroform–methanol at 4 °C or room temperature in the dark prevents loss of chlorophyll a for at least 10 d. More refined analyses of phaeophytin and other chlorophylls require the use of chloroform–methanol–water (2:2:1.8, by volume) and placement in a separatory funnel. After 24 h the lower (chloroform) layer contains all of the chlorophyll. Strict pH control is required for pheophytin determinations.