Fig. 2

Separation of compounds using liquid extractions and liquid chromatography. a Disk diffusion assay using five extracts from spent medium of RIT 623 against Bacillus subtilis BGSC 168. From the top left (50 µl each), (1) Methanol (negative control) (2) Hexanes (3) Toluene (4) Diethyl ether (5) Dichloromethane (6) Ethyl acetate. Only the diethyl ether is active as seen in the clear inhibition zone (yellow arrow). b Chromatogram of ether extract from the five step extraction (refer to methods) of RIT 623 spent medium using a reverse phase chromatography on a C18 semi-preparative scale column. The elution profile is based on the collection of a 3 mL fraction every minute at a flow rate of 3 mL/min. c The activity of fractions 1–8 pooled and concentrated from four runs of the chromatography experiment in b) are shown. The active compound eluted in fractions 5 and 6 (yellow arrows). There was no activity in the pooled fractions 8–42 (data not shown)