Separation scheme using Sephadex

Sephadex LH-20 gel column with isopropanol followed by acetone. About 90% of the basic mutagenic activity is recovered in the acetone subfraction, which comprises approximately 0.5 wt% of the crude oil. Development of this separation scheme (Figure 5) was made possible by use of microbial mutagenesis assays as the detector during exploratory liquid chromatographic separations. Table 7 lists some of the preliminary data from these studies. 

Many schemes for fractionating nucleotides, nucleosides and bases on sulphonated polystyrene resins have been published. The main difficulty with these methods is variation between resin batches (e.g. Anderson et al. 1963). Nucleotide separations can be achieved on DEAE-cellulose (Whatman Data Sheet 13, 1967) and DEAE-Sephadex (Piers et al. 1965b) but these media do not seem to be widely used. Gel filtration columns will separate some nucleotide components. Ligand exchange chromatography and partition chromatography of nucleosides are useful for minor components. 

In the isolation of spongistatin 4 (Scheme 31), the CH2CI2 fraction from 2409 kg of Spirastrella spinispirulifera was initially separated by HPLC employing a pilot scale HPLC system on a silica-gel column, 3 x 0.15 m, operated at 150 psi. This was followed by a series of Sephadex LH-20 separations and multiple HPLC steps using the following columns Merck RP-2, Prepex- RP-8, and LiChrospher 100 RP-18 to yield 10.7 mg of spongistatin 4 (53). 

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