Then, 1 ml of 5% calcium chloride was added and the mixture was allowed to stand for 3 h after which it was centrifuged (IEC Centra GP8)at 140.868 g for 15 minutes.
The supernatant was discarded while the precipitate was washed thrice with hot water, with thorough mixing and centrifuging each time.
Then, to each tube, 2 ml of 3 N H2SO4 was added and the precipitate was dissolved by warming in a water bath at 70oC - 80oC for 30 min.
The content of each tube was titrated with freshly prepared 0.01N potassium permanganate solution.
Titration was done at room temperature (29oC) until the first pink colour appeared throughout the solution. The solution was allowed to stand until it was colourless.
It was warmed to 70oC - 80 oC and titration continued until a pink colour persisted for at least 30 s.