y = (Reading of sample + standard tube 2) – (Reading of
sample + standard blank)
Determination of Niacin by Colorimetric Method.
It involves a reaction between niacin (nicotinic acid) and
cyanogens bromide, which under proper conditions forms a
coloured compound with an intensity proportional to the
niacin concentration. The method used in the determination
of niacin is as described by Chopra and Kanwar
[11].
Exactly
5g of sample was weighed (in calcium hydroxide) and it
was saponified for 45mins in ethanolic KOH solution
containing sodium ascorbate and cooled rapidly. It was
transferred into separatory funnel with sequential use of
H2O, EtOH and ether and shaken vigorously. The aqueous
phase was transferred to second separatory funnel and
extracted with 25/100cm3
mixture of EtOH/pentane. The
pentane fraction was placed into the first separatory funnel
and aqueous into a third separatory funnel. The unit was
washed with 10cm3
pentane portions and this portion was
added to the first separatory funnel, the aqueous phase
was washed with 10cm3
EtOH/pentane mixture, and
pentane fraction was added to the first separatory funnel,
then the pentane fraction was washed with 3% KOH in 10%
EtOH portions followed by H2O portion until neutral pH was
obtained. H2O traces are removed by adding filter paper to
separatory funnel with shaking. This was transferred to a
round bottom flask, 1cm3
of 0.1% BTH hexane was added
and evaporated to dryness. The residue was dissolved in a
few cm3
5/95 toluene/hexane with the later containing
0.35% n-amyl alcohol and re-evaporate to dryness at room
temperature under a normal stream, then 2.0 cm3 was
dissolved in the same solvent mixture. The mixture was
cleaned up by passing 200µl through a 250 x 46mm column
packed with 10µm sil-60D-10CN, using n-hexane