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Then this residue was subjected on reverse-phased C18 column eluted with gradient mixture of MeOH and H2O (30:70–100:0, v/v).
Fraction C (23.8 g) was separated by MPLC with gradient mixture of MeOH and H2O (20:80–100:0, v/v) to afford ten sub-fractions (C1 C10).
To a solution of the intermediate in a solvent mixture of MeOH (5 mL) and H2O (5 mL) was added excessive Et3N.
Sub-fraction L was isolated by reverse-phased C18 column eluted with mixture of MeOH and H2O (45:55, v/v), then purified by Sephadex LH-20 CC (Me2CO) to yield compound 1 (1.4 mg).
Sub-fraction A, isolated by Sephadex LH-20 CC (MeOH) and reverse-phased C18 column eluted with mixture of MeOH and H2O (30 70, v/v) to yield compound 4 (0.7 mg).
Fraction B (1.8 g) was separated by reverse-phased CC eluted with gradient mixture of MeOH and H2O (30:70–100:0, v/v) to afford five sub-fractions (B1 B5).
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Fraction VHE4.3 Fraction VHE4.4 (500 mg) was subjected to Sephadex eluted with a mixture of MeOH-H2O (2:1) to give 4 (23 mg) and 5 (40 mg).
Subfraction II-6 was subjected to C18 MPLC eluted with mixtures of MeOH and water (60 40 90 10, v/v).
Moreover, for rich mixtures of MeOH (70 80 vol%) the transition between the charge transfer and the mass-transfer limitations is shifted towards the higher values of TCTFE concentration with rising concentration of NH4Cl.
After that, the separation of products was performed by silica gel preparative TLC using a mixture of CHCl3 : MeOH (93 : 7 v/v) as eluent.
The second follows the Folch procedure using a mixture of chloroform: MeOH (2 1) [ 42] and the third, from Hara et al. [ 43], uses a mixture based on n-hexane:isopropanol (3:2).
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