June 1948.]
S. K. TALAPATRA, S. C. BAY AND K. C. SEN 101
extraction is therefore carried out with an ammonical solution so as to prevent the formation of free
acids.
The details of extraction consist of as follows: A suitable quantity, 2 to 5 grams, of finely ground
sample is boiled with an ammonical solution (1:10) under reflux for one hour. At the end of the
period, the reflux is removed and the boiling is continued for another 15 minutes. The content is
cooled and diluted with distilled water to make the extract approximately 200 c.c. After a thorough
shaking, the content is allowed to rest so that the solid may settle at the bottom. The supernatant
liquid is then filtered through a quantitative filter paper to eliminate the possibility of fine crystals
of insoluble oxalate passing into the filtrate. The rest of the procedure is the same as described for
the total oxalate estimation.
EXPERIMENTAL TRIALS
In establishing the method as described above, the fulfilment of the following conditions is
essential:
(a) The conversion of insoluble oxalates into soluble oxalates by metathesis with sodium
carbonate should be quantitative.
(b) The cooking processes should guarantee complete extraction of salts of oxalic acid from
biological materials.
(c) The method should yield reproducible values and effect the desired recoveries of the added
pure salts of oxalate.
The conversion of insoluble oxalate into soluble oxalate. The insoluble oxalates in biological
sample occur mainly as calcium oxalate. In small quantities, oxalates of other alkaline earth metals
and perhaps in some cases micro quantities of manganese oxalate may also occur. To test the
completeness of metathesis, a pure salt of calcium oxalate monohydrate (C 2 O 4 Ca, H 2 O) was prepared
in the laboratory. Duplicate weighings of the salt were directly titrated with 0.1N. permanganate
solution after dissolving in dilute sulphuric acid. Another set of four duplicate weighings of this
salt were first boiled with 4 per cent solution of sodium carbonate for 5, 10, 20 and 30 minutes res-
pectively. The precipitate of calcium carbonate formed in an excess of sodium carbonate solution
according to the equation C 2 O 4 Ca+Na 2 CO 3 =CaCO 3 +Na 2 C 2 O 4 was filtered off and washed. The
mixture of the combined filtrate and wash water was then titrated in the usual way with the standard
permanganate solution. The amounts of permanganate solution required in the cases where no
metathesis was carried and those where the expedient was used are shown in Table I.
It is apparent from the data presented above, that the estimated value is slightly higher than
the calculated value but well within the experimental error. It may also be noted that the complete
metathesis is brought about within such a short period as 5 minutes inspite of the fact that the
quantities of calcium oxalate taken are much in excess of what can be expected in a biological sample.
Similar results were also obtained when oxalates of magnesium, barium and manganese were tested.
The extraction of oxalates in biological samples. To determine the minimum time required for
complete extraction of oxalates, a sample of finely ground paddy straw was taken. Three to five
grams of the sample were first cooked in water for half hour, followed by second cooking in sodium
carbonate solution for 15, 30, 60 and 120 minutes according to the technique described. The values
of oxalic acid estimated from the extracts obtained from the different periods of cooking in sodium
carbonate solution are given in Table II.
The data in Table II, show that there is practically no difference in the value of oxalic acid
estimated in the extracts obtained between 15 minutes and 120 minutes of cooking. The closely
similar values, moreover, suggest that the complete extraction of oxalates is assured if the digestion
with sodium carbonate is fixed for half hour only. Various other biological samples such as, hays,
straws from other cereals and millets, cattle faeces, etc., were also tried and the results were
uniformly satisfactory.
H2