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Microcystins are an increasingly important group of bioactive compounds produced by a number of mainly planktonic cyanobacteria. They are a family of cyclic heptapeptides that cause both acute and chronic toxicity. Purified microcystins are utilised in a range of research applications including toxicological and biochemical studies, development of detection systems and the investigation of water treatment strategies. The commercial availability of purified microcystins is still relatively limited and for many projects the cost of their purchase prohibitive. The purification of microcystins from both bloom material and laboratory cultures is reviewed including a discussion on extraction, separation, and the determination of purity and yield.

Two closely eluting hydrophobic peptides, microcystin-LW and -LF had proved in the past to be particularly difficult to purify by reversed-phase HPLC. Initial extraction by reversed-phase flash chromatography provided a good method of concentrating microcystins and also partially purifying them, although the separation of closely eluting variants was not possible. The use of normal-phase flash chromatography after initial reversed-phase extraction was found to be a suitable method for achieving high purity and gave a good yield of both microcystins. However, a final reversed-phase flash step was necessary to eliminate trace contaminants. The method described provides a simple three step flash chromatography extraction and purification eliminating the need to use preparative HPLC.

Microcystins were extracted from 7 1 (equivalent to 313 g dry weight) of cyanobacterial scum collected from Rutland Water in Leicestershire, UK in 1989. The resulting aqueous extract was rapidly concentrated on a C18 flash chromatography cartridge and microcystins were eluted using a step gradient. Fractions were collected manually and monitored by UV spectrophotometer and analytical HPLC. Fractions containing microcystins of similar polarity were pooled to give three fractions. Simple isocratic methods for separating each fraction were developed on an analytical column and scaled up to a 15×7.5 cm I.D. column. Closed-loop recycling was used to maximise yield and purity of two hydrophobic microcystins.

A simplified method for the concentration and purification of microcystins was developed. A 3-1 volume of cyanobacterial scum (equivalent to 124 g dry mass of cells) collected from Rutland and Water, Leicestershire, in 1989 was extracted in methanol and centrifuged and the supernatant was retained. This was diluted to ca. 20% aqueous methanol and applied to a reversed-phase flash chromatography cartridge. Microcystins were eluted with 70% aqueous methanol then reduced in vacuo. Gradient separation of the extract was optimized on an analytical column and scaled up to a 15 cm × 7.5 cm I.D. column with subsequent purification of individual compounds.