Estimation of Magnesium and Purification of Chlorophyll from Chlorobium thiosulphatophilum

Abstract

Chlorobium thiosulphatophilum was grown under conditions similar to those described by Larsen¹. ‘Chlorobium chlorophyll’ (bacterioviridin) was extracted from the cells with 80 per cent methanol, transferred to ether, washed with water and dried over anhydrous sodium sulphate. The volume of the solution was reduced by evaporation under nitrogen, and the bulk of the chlorophyll precipitated by the addition of petroleum ether (b.p. 60–90). The chlorophyll was collected on talcum and washed with petroleum ether (b.p. 30–60) until the washings were colourless. The chlorophyll was removed from the talcum with acetone, transferred to ether, and washed with water. After drying, the solution was concentrated under vacuum, an equal volume of petroleum ether (b.p. 60–90) containing 4 per cent tetrahydrofuran added, and the solution adsorbed on to a sucrose column. The tetrahydrofuran was employed as a means of preventing precipitation of the chlorophyll in the column². The column was developed by stepwise elution with isopropyl alcohol (0.75–2.0 per cent) in petroleum ether (b.p. 60–90 °) containing 2 per cent tetrahydrofuran. The chlorophyll zone was cut out, dissolved in ether, and chromatographed as before. The absorption spectrum of the chlorophyll in ether was obtained on a Cary recording spectrophotometer. Peaks at 650, 575, 530, 425, and 406 mµ, indicated that this chlorophyll was the ‘650’ type described by Stanier and Smith³, and that the organism studied was similar to or identical with Chlorobium thiosulphatophilum, strain L.