December, 1953 ]
M.
L.
MATHUR
245
materials and food stuffs and found that its accuracy was not affected by the presence
of non-phytic substances in plant extracts. This method involved the titration of
phytic acid in 0.6 per cent HCl, ammonium thiocyanate being used as internal
indicator. Heubner and Stadler recommended that the end point was reached when
a flesh pink colour persisting for five minutes was obtained. The disadvantage of
this method was that the end point was not sharp owing to the presence of colloidal
precipitation of ferric phytate, which was formed during the titration. To avoid
that difficulty various modifications had been suggested by Knowles and Watkin
[1932], Andrews and Bailey [1933], Harris and Mosher [1934] and Young (1936).
In the present metabolic experiments the feeding stuffs and the faeces had to be
analysed. The feeding stuffs could be analysed by the modified method of Andrews
and Bailey [1933]. Since the faeces gave coloured extracts with HCl, this procedure
could not be applied. Knowles and Watkin [1932] used ether to remove colouring
materials ; but in the present experiments ether was not found to remove the colour-
ing materials completely. Experiments with adsorbents such as kaolin, alumina,
Fuller's earth, norite charcoal and activated charcoal, prepared from paddy husk in
this laboratory, showed that phytic acid was partially adsorbed on these substances,
though the colouring matter was removed. The method suggested by Young [1936]
was used with slight modification.
Determination of phytic acid in faeces
FeCl 3 in N/HCl : A solution containing 1.5 mg. per c.c.
N/2 HCl solution
5 N and N/2 NaOH solutions.
Eight grams of oven-dried faeces were extracted with 200 c.c. of two per cent
HCl solution in a glass stoppered bottle for three hours. The extract was filtered
on a Buchner funnel, through double layer of hardened filter paper. 25 c.c. aliquot
(containing not more than 4 mg. phytic acid) was taken in a 50 c.c. graduated
centrifuge tube, neutralized with 5 N. NaOH, made just acidic to litmus with HCl.
After taking the reading in the tube, one fifth of its volume of FeCl 3 HCl solution was
added. The tube and its contents were heated in a rack, in boiling water bath, for
15 minutes, with level of water above that of the contents of the tube, then cooled
in a bath of cold water for 15 minutes and centrifuged for 10 minutes at about 3,000
rounds per minute. The supernatent liquid was decanted and the tube allowed to
drain. The precipitate was suspended in 10 c.c. of hot water in the centrifuge tube
and heated in boiling water bath for two minutes. To the hot suspension, 2 c.c.
of N/2 NaOH were added with stirring and heating in the water bath was continued
with occasional stirring. The precipitate of ferric hydroxide was filtered on a 7 cm.
whatman filter paper (No. 31) and washed with hot water. The filtrate and wash-
ings were taken in a graduated cylinder, allowed to cool and made just acidic to
litmus. N/2 HCl was then added to bring the resultant acidity to 0.6 per cent and
the volume made up. Fifty c.c. aliquot was taken in a beaker followed by 10 c.c. of 0.3
per cent ammonium thiocyanate and standard ferric chloride solution was added
from a microburette. When the end point had nearly reached, the titration was
stopped and the solution allowed to stand for five minutes or heated on a water
bath for 15 minutes, cooled and then filtered. The titration was concluded in the
filtrate.