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ORIGINAL ARTICLES
THE ANALYSIS OF MINERAL CONSTITUENTS IN
BIOLOGICAL MATERIALS
PART I. ESTIMATION OF PHOSPHORUS, CHLORINE, CALCIUM,
MAGNESIUM, SODIUM AND POTASSIUM IN FOOD-STUFFS
BY
S. K. TALAPATRA, B.Sc.
S. C. RAY, M.Sc., PH.D., A.I.C.
AND
K. C. SEN, D.Sc.
Animal Nutrition Section, Imperial Veterinary Research Institute, Izatnagar
(Received for publication on 21 August 1939)
INTRODUCTION
I N view of the important bearing of different minerals on the health and
productive power of various animals, the accurate estimation of many
inorganic constituents of feeding stuffs is of great interest. It is now well
known that minute quantities of several minerals such as iodine, manganese,
copper, cobalt etc. exert a powerful effect on the general metabolism of the
animal body. There are certain other substances such as phosphorus, calcium,
magnesium, sodium, potassium, chlorine, etc. which are required in much
greater quantities, and are indispensable not only individually but in a well
balanced mixture for the proper functioning of the animal body. As all these
materials are supplied to the animals in their food, it is often discovered that
animals suffer from malnutrition due either to the deficiency of some of these
materials or due to their unbalanced distribution in the ordinary food-stuffs
available. It is necessary, therefore, to know exactly the amount of various
minerals present in the diets of animals and with a view to study these aspects
of the nutritional problem in this country, the Imperial Council of Agricultural
Research has been financing several schemes for the study of the composition
of animal food-stuffs in different parts of India. A difficulty, however, arose
in the interpretation of the data supplied by different workers in that the
method used for analysis varied considerably from place to place and, probably
due to this reason, the results of analysis of identical samples in different
places did not agree well. Recently the Imperial Council of Agricultural
Research, having considered the position, has made tentative recommendations
for some methods of analysis to be followed by workers in this country. These
methods are based on (1) official and tentative methods of analysis of the
Association of Official Agricultural Chemists, U. S. A. (A. O. A. C.), (2)
Technical Communication No. 9 of the Imperial Bureau of Animal Nutrition
(Rowett method) and (3) the experience of some of the Indian laboratories
including our own.
( 243 )
246 Analysis of Mineral Constituents in Biological Materials
(2) (NH 4 ) 3 PO 4 . 12MoO 3 + 23 NaOH = HNa 2 MoO 4 + (NH 4 ) 2 MoO 4
+ NaNH 4 HPO 4 + 11 H 2 O (Kolthoff).
(3) 2(NH 4 ) 3 PO 4 . 24MoO 3 + 52NaOH = 24Na 2 MoO 4 + 2 Na 2 HPO 4
+ 6NH 3 + 28 H 2 O (Richard and Godden).
In calculating the factor it is apparent that neither Pemberton nor Kol-
thoff took into consideration the alkali necessary to replace the ammonia, as
it was not removed by boiling. Richard and Godden on the other hand provid-
ed sufficient alkali to replace the ammonia which was removed by boiling. Thus
according to the first two authors, the molecular ratio of P 2 O 5 : NaOH is as
1 : 46, i.e. 1 c.c. of 0.5 N NaOH. is equivalent to 0.001543 gram of P 2 O 5 ,
whereas according to the latter authors the molecular ratio of P 2 O 5 : NaOH
is 1 : 52 and the factor, 1 c.c. of 0.5 N NaOH = 0.001365 gram of P 2 O 5 .
Richard and Godden further advocate that after boiling with standard alkali,
a slight excess of standard acid should be added and the boiling should continue
for another half hour to expel CO 2 , the solution being finally titrated back when
cold, with the standard alkali.
PROPOSED METHOD
The method which has been exhaustively studied in this laboratory is
based on the following principles :—To a nitric acid extract of phosphate is
added 10 c.c. of strong HNO 3 and 10 c.c. of 20 per cent ammonium molybdate
solution. The acid and molybdate solution before being added is mixed in a
separate vessel and at once poured into the phosphate solution. The heat
generated during mixing, which is about 35°C, is utilised for complete precipi-
tation of phosphate. The final volume of the mixture should be restricted to
within 100 c. c. The precipitate is filtered, washed with two per cent HNO 3
and then with three per cent solution of KNO 3 till no acid reaction is shown by
the litmus. The phosphomolybdate precipitate is now dissolved in N/ NaOH
and the excess of NaOH is titrated back in the usual way. The reaction is
identical with that of Kolthoff, but because of the different concentration of
NaOH used, the conversion factor becomes 1 c.c. N/7 NaOH = 0.000441
grams of P 2 O 5 . Details of the method are given in the following paragraph.
DETERMINATION OF P 2 O 5
A sufficient amount (the amount varies with the phosphate and chlorine
content) of the material is weighed out into a vitreosil basin and about 0.5 gram
of precipitated calcium oxide added, the whole mixed thoroughly after moisten-
ing with a little distilled water, dried on a water bath and gently ignited to a
greyish white ash. The ash is thoroughly extracted with hot 2 N nitric acid,
filtered and washed into a graduated flask and the volume made up to the mark
with distilled water.
A suitable aliquot is measured into a beaker and to this is added 20 c.c. of
molybdate mixture which is always prepared just before use, by mixing
equal volumes of concentrated HNO 3 (sp. gr. 1.42) and ammonium molybdate
solution. The mixture is gently stirred with a glass rod without touching the
walls of the beaker and allowed to stand over night, (The precipitate can be
S. K. TALAPATRA, S. C. RAY AND K. C. SEN 247
filtered off after two hours without affecting the result.) The yellow precipi-
tate is filtered off through a disc of filter paper in a Gooch crucible by suction.
As far as possible the precipitate is not transferred to the crucible, the super-
natant liquid alone is passed through. The precipitate is washed twice with two
per cent HNO 3 (by decantation) and then with three per cent solution of KNO 3
until the wash water is acid free (tested by litmus). The precipitate with the
filter paper is then transferred back to the beaker with CO 2 free water and
dissolved completely in a sufficient known volume of N/7 NaOH. A few drops
(not more than five) of a one per cent alcoholic solution of phenolpthalein are
added and the solution is titrated back with N/7 HNO 3 adding a few drops in
excess. The titration is completed finally by adding standard alkali to the
first faint pink colour.
The ammonium molybdate solution to be used for the analysis.—200 grams
of ammonium molybdate are dissolved in 800 c.c. distilled H 2 O in a graduated
measuring cylinder. Ammonia (25 per cent NH 4 OH) is now added until
the mixture is faintly alkaline and clear. The volume is made up to 1000 c.c.
and the mixture well shaken, filtered and used.
Experimental.—One sample of mustard cake and one of grass were taken
for studying the method. Large quantities of these materials were ashed at a
time with lime as the fixing agent and then extracted with hot 2 N HNO 3 .
The extract was finally made up to a volume. Another phosphate solution of
known strength was also made by carefully weighing out a suitable quantity
of KH 2 PO 4 and dissolved in slightly acidulated distilled water and made up
to a volume.
The amount of phosphate was estimated by the proposed method accord¬
ing to the following scheme.
(a) The P 2 O 5 content was estimated in three different aliquots of (i) the
acid extract of the ash of the biological sample and (ii) the stand-
ard phosphate solution.
(b) Known amounts of standard phosphate solution were added in vary-
ing quantities to an aliquot of acid extract of the ash of a biologi-
cal sample and recoveries of P 2 O 5 were observed.
(c) The P 2 O 5 content was estimated in the standard solution by precipi
tating the phosphates in presence of excess of ammonium nitrate
with a view to determine if the addition of NH 1 NO 3 is necessary
for complete precipitation or not.
(d) Estimating phosphate in the solution by allowing the precipitate to
settle for two hours and for 24 hours, in order to find out the
minimum time that is necessary before the filtration of the preci-
pitate can be made.
RESULTS AND DISCUSSION
The result of the experiments as outlined in the schemes (a), (b), (c) and (d)
are shown in Tables I, II, III and IV.
The data shown in Tables I and II are quite convincing as regards the
accuracy of the proposed method.
248 Analysis of Mineral Constituents in Biological Materials
TABLE I
Estimation of phosphate (1) in the acid extracts of the ash of grass and mustard
cake and (2) in the standard solution of KH 2 PO 4 .
Grass solution
Aliquot taken
25 c.c. . .
20 c.c. . .
10 c.c. . .
Amount of
P 2 O 5 (mgm)
found
7.36
7.36
7.35
5.90
5.95
5.90
2.90
2.90
Mustard cake solution
No. 1.
Aliquot
taken
25 c. c.
...
...
A mount of
P 2 O 5 (mgm)
found
13.03
13.01
12.97
...
...
Mustard cake solution
No. 2.
Aliquot
taken
20 c. c.
10 c. c.
5 c.c.
Amount of
P 2 O 5 (mgm)
found
9.08
9.13
9.08
4.59
4.56
4.56
2.29
2.25
Standard phosphate
solution
Aliquot
taken
25 c. c.
20 c. c.
10 c.c.
Amount of
P 2 O 5 (mgm)
found
13.27
13.24
13.24
10.65
10.65
10.61
5.30
5.35
5.30
TABLE II
Percentage recoveries of added P 2 O 5
Grass solution
P 2 O 5 (mgm)
present in the
acid extract
7.35
7.35
7.35
7.35
P 2 O 5 (mgm)
added to the
acid
extract
2.64
5.28
7.92
10.56
Total P 2 O 5
(mgm)
observed
10.00
12.66
15.22
17.99
Per cent re-
covery of
added P 2 O 5
100.40
100.57
99.37
100.76
Mustard cake solution
P 2 O 5 (mgm.)
present in
the acid
extract
13.00
13.00
13.00
13.00
P 2 O 5 (mgm)
added to
the acid
extract
2.64
5.28
7.92
10.56
Total P 2 O 5
(mgm)
observed
15.65
18.40
21.08
23.89
Per cent re-
covery of
added P 2 O 5
100.40
102.27
102.02
103.12
It may be noted (Table III) that the introduction of ammonium nit-
rate before the precipitation of phosphate definitely introduces an error.
While washing the precipitate by decantation with KNO 3 it is also noticed
that the supernatant liquid is not clear and a little turbidity always persists
throughout the column of the liquid. No such difficulty is encountered when
the addition of ammonium nitrate is omitted. Addition of NH 4 NO 3 , however,
hastens the precipitation of ammonium-phospho-molybdate.
S. K. TALAPATRA, S. C. RAY AND K. C. SEN 249
TABLE III
The effect of the addition of 25 c.c. of 50 per cent solution of ammonium nitrate
on the precipitation of ammonium-phospho-molybdate
Analysis of the standard phosphate solution
Without ammonium nitrate
Observed
mgm
10.65
10.65
10.61
13.28
13.28
13.28
Calculated
mgm
10.56
10.56
10.56
13.20
13.20
13.20
Error
+0.85
+0.85
+0.57
+0.60
+0.60
+0.60
With ammonium nitrate
Observed
mg
10.74
10.65
10.65
10.68
10.68
10.68
Calculated
mg
10.56
10.56
10.56
10.56
10.56
10.56
Error
+1.70
+0.85
+0.85
+1.13
+1.13
+1.13
In the Rowett method the precipitate can be filtered off within an hour
which is a distinct advantage. In our proposed method the precipitate can be
filtered off within two hours without affecting the results as can be seen from
the data recorded in Table IV.
TABLE IV
Time interval for complete precipitation of ammonium-phospho-molybdate
Filtered after two hours
P 2 O 5 (mgm)
present in the
acid extract
2.28
4.56
9.13
9.13
9.13
P 2 O 5 (mgm)
added to the
acid extract
5.81
5.81
5.81
11.61
17.42
Total P 2 O 5
(mgm)
observed
8.47
10.36
14.95
20.77
26.42
Per cent
recovery of
the added
P 2 O 5
99.66
99.83
100.17
100.25
99.28
Filtered after keeping overnight
P 2 O 5 (mgm)
present in
the acid
extract
2.28
4.56
9.13
9.13
9.13
P 2 O 5 (mgm)
added to the
acid
extract
5.81
5.81
5.81
11.61
17.42
Total P 2 O 5
(mgm)
observed
8.05
10.41
14.95
20.79
26.42
Percent
recovery of
the added
99.31
100.69
100.17
100.25
99.28
It may be noticed from the results recorded in Tables I to IV that by
following the identical course of procedure it is possible to estimate P 2 O 5
within such wide range as 2.27 mgm. to 26.42 mgm. Unlike the A. O A. C.
method there is moreover no prejudice regarding the amount of molybdate
250 Analysis of_ Mineral Constituents in Biological Materials
mixture to be added. The same volume of molybdate mixture has been used
between 2.27 mgm. of P 2 O 5 to 26.42 mgm. of P 2 O 5 without affecting the
accuracy of the result.
ESTIMATION OF CHLORINE
There is practically no disagreement in the standard method (Technical
Communication No. 9 of the Imperial Bureau of Animal Nutrition) of
estimating chlorine in biological materials. However Caldwell and Moyer
[1935] give a method in which the silver chloride need not be filtered off before
titrating the excess of silver nitrate with thiocyanate, if 1 c.c. of nitrobenzene
for each 0.5 gram Cl-ion is added. This modification thus has a distinct
advantage as the filtration of the AgCl precipitate can be avoided.
Experimental.—The trial was done with pure salt according to the follow-
ing schemes.
(1) AgCl precipitate was filtered off and the excess of silver nitrate was
titrated back with standard thiocyanate solution using ferric
alum as indicator.
(2) Nitro-benzene (1 c.c.) was added before the precipitation of AgCl
and the excess of silver nitrate was titrated back with standard
thiocyanate solution without filtering the AgCl precipitate.
The results of the experiments, recorded in Table V, show that the differ-
ent modes of treatment yield practically the same results.
TABLE V
Titration of excess silver nitrate in presence of nitrobenzene
Added 1 c.c. nitrobenzene, titrated back the
excess of AgNO 3 by thiocyanate without
filtering off the AgCl precipitate.
Cl 2 -Observed
(mgm)
12.3
12.3
12.4
31.6
31.6
31.6
Cl 2 -Calculated
(mgm)
12.4
12.4
12.4
31.9
31.9
31.9
Filtered off the AgCl precipitate, the
excess of AgNO 3 in the aliquot titrate
back by thiocyanate.
Cl 2 -Observed
(mgm)
31.8
31.7
31.8
19.3
19.3
19.3
Cl 2 -Calculated
(mgm)
31.9
31.9
31.9
19.5
19.5
19.5
In working with biological materials, the chloride is precipitated in an
aliquot of the nitric acid extract of the ash as prepared for the determination
of the phosphoric acid with lime as fixing agent which has been described
earlier.
252 Analysis of Mineral Constituents in Biological Materials
titration is reached as soon as the solution becomes faintly pink. The con-
version factor is :—One c.c. of N/20 KMnO 4 = 0.0014 grm. CaO.
Experimental—A suitable quantity of the materials (grass and mustard
cake) was ashed slowly in a vitreosil basin, the ash was then extracted with
2-N HCl, filtered, washed free of acid and made up to a volume. Another
calcium solution of known strength was made by dissolving a weighed
quantity of calspar in HCl solution and made up to a volume. The amount
calcium was estimated by the proposed method according to the following
of scheme.
(a) The CaO-content was estimated in three different aliquots of (i) the
acid extract of the ash of the biological samples and (ii) the
standard calspar solution.
(b) Known amounts of standard calcium solution were added in varying
quantities to an aliquot of the acid extract of the ash of the
biological sample and the recoveries of CaO were observed.
(c) The CaO-content was estimated in a few synthetic solutions which
approximate the mineral composition of a number of typical
food-stuffs. (Table VI). The synthetic solutions were prepared
from salts of known purity.
TABLE VI
Synthetic solutions—the amount of minerals present in 250 c.c. of dilute HCl
solution.
Composition
Fe 2 O 3
. .
AL 2 O 3
. .
P 2 O 5 . .
Mn 3 O 4
. .
CaO . .
MgO . .
No. 1
(mgm)
23.50
32.70
26.60
13.30
56.1
30.8
No. 2
(mgm)
23.5
87.2
199.5
6.7
56.1
81.6
No. 3
(mgm)
75.50
16.35
199.8
15.3
98.0
100.1
No. 4
(mgm)
75.5
65.4
13.30
15.3
56.1
30.6
No. 5
(mgm)
7.8
65.4
53.2
6.7
14.0
30.60
The composition of No. 1 represents the approximate inorganic composition of
10 grm. of rice straw.
The composition of No. 2 represents the approximate inorganic composition of
10 grm. of linseed cake.
The composition of No. 3 represents the approximate inorganic composition of
10 grm. of mustard cake.
The composition of No. 4 represents the approximate inorganic composition of
10 grm. of rice straw containing high iron and manganese.
The composition of No. 5 represents the approximate inorganic composition of
10 grm. of mixed bran.
S. K. TALAPATRA, S. C. RAY AND K. C. SEN 253
RESULTS AND DISCUSSION
The results of the experiments conducted according to the schemes (a), (b)
and (c) are recorded in Tables VII, VIII and IX.
The data in Tables VII, VIII and IX definitely establish the fact that the
method is accurate, even under varied composition of the interfering substances
in the solution for test.
TABLE VII
Estimation of calcium in the acid extract of the ash of grass and mustard cake
and in standard solution of calspar
Grass solution
Aliquot
taken
100 c.c.
50 c.c.
25 c.c.
Amount of
CaO (mgm)
found
(1)43.26
(2)42.98
(3)42.98
(1)21.56
(2)21.28
(1) 10.64
(2) 10.85
(3) 10.85
Mustard cake solution
Aliquot
taken
100 c. c.
50 c c.
25 c. c
Amount of
CaO (mgm)
found
(1) 43.96
(2) 43.96
(3) 43.82
(1)21.84
(2)21.84
(1)10.78
(2)10.64
(3)10.78
Standard calspar solution
Aliquot
taken
20 c. c.
10 c. c.
. .
. .
. .
Amount of
CaO (mgm)
found
(1)28.00
(2)28.00
(3)27.93
(1)14.00
(2)13.93
(3)14.00
. .
. .
. .
TABLE VIII
Percentage recoveries of added CaO
Grass solution
CaO (mgm)
present in
the acid
extract
21.42
21.42
21.42
CaO (mgm)
added to the
acid extract
14.0
35.0
28.0
Total CaO
(mgm)
observed
35.30
56.00
40.28
Per cent
recovery of
the added
CaO
99.11
99.00
99.50
Cake solution
CaO (mgm)
present in
the acid
extract
21.84
21.84
21.84
CaO (mgm)
added to the
acid extract
14.0
14.0
28.0
Total CaO
(mgm)
observed
35.8O
35.70
50.04
Per cent
recovery of
the added
CaO
100.00
99.09
100.71
254 Analysis of Mineral Constituents in Biological Materials
TABLE IX
Estimation of calcium in synthetic solutions
Samples
Synthetic solution No. 1
Synthetic solution No. 2
Synthetic solution No. 3
Synthetic solution No. 4
Synthetic solution No. 5
CaO present
mgm
56.1
50.1
98.0
56.1
14.0
CaO found
mgm
56.3
50.3
98.4
56.6
13.83
Error
per cent
+ 0.4
+0.4
+ 0.4
+ 0.9
—1.2
In the Rowett method for precipitating calcium it has been found that the
pH at which the litmus paper turns just red is not favourable for the precipi-
tation of calcium in the samples like cakes which contain relatively much more
phosphoric acid. Further in the Rowett method, if the precipitates of iron and
aluminium do not disappear after the addition of ammonium oxalate, the alter-
native choice has been given to filter off the precipitate of hydroxide of iron and
aluminium before the precipitation of calcium by ammonium oxalate. As the
pH at which calcium is precipitated in the Rowett method is definitely higher
than five, the removal of the precipitates formed after the adjustment of the
acidity of the medium with acetic acid also entails the loss of calcium phosphate
which is formed at that pH. This is especially true in the case of those samples
in which the phosphate-content preponderates over the calcium content.
In the present method no such risk is involved as the solution is buffered
with ammonium oxalate at the beginning and the required pH can be control-
led. Moreover the excess of NH 4 C1 present at the particular pH prevents the
co-precipitation of magnesium even when present in large amount
(Table IX). In the same table it may be observed that the estimation of
calcium in the presence of various interfering elements can be quite
accurately made even when it is present in very small amounts. It may be
pointed out here that the results are most satisfactory when the titration by
N/20 KMnO 4 lies between 10 c.c. to 20 c.c.
The method described here is somewhat similar in principle to that given
in A. O. A. C. method of analysis (4th Edn., 1935, p. 123). The latter proce-
dure, however, is complicated by the introduction of the method for separating
soluble silica previous to the precipitation of calcium. In our procedure, since
soluble silica is not separated, the technique of calcium estimation is simpler.
It may be mentioned here that there are several food-stuffs like rice bran,
wheat bran, linseed cake, cotton seed cake, etc. in which the amount of MgO
is two to twelve times more than CaO. With our method we can accurately
estimate calcium when the MgO present is about five times that of CaO, or
when the MgO content in the aliquot taken for calcium estimation does not
exceed 100 mgm. But when MgO present is more than 100 mgm (as it is in the
S. K. TALAPATRA, S. C. RAY AND K. C. SEN 255
case of rice bran and certain cattle urine), the first precipitate of calcium
oxalate (contaminated with some magnesium oxalate) should be again
dissolved in HCl and re-precipitated according to our procedure.
ESTIMATION OF MAGNESIUM
The combined filtrate and washing from the calcium determination are
evaporated to dryness on water bath. If NH 4 C1, equivalent to 5 c.c. of
strong HCl, is present then 30 c.c. of concentrated HNO 3 should be used but
if more, then 40 c.c. of HNO 3 might be necessary to drive off the ammonium
salts. The remaining procedure is the same as that of A. O. A. C. excepting that
10 c.c. sodium citrate should be used instead of 5 c.c., otherwise the
magnesium ammonium phosphate precipitate will be contaminated with iron,
and a brownish tint is always invariably found.
ESTIMATION OF SODIUM
The routine analysis of sodium in the ash of food-stuff is generally made
indirectly from the weight of mixed chloride or sulphate of sodium and
potassium obtained after a thorough separation and purification process.
The success of an indirect method of estimating sodium naturally depends
upon the rapidity and at the same time the efficiency with which the method
can lead to the complete removal of the foreign elements, so that finally
sodium and potassium alone in the unknown solution can be weighed as mixed
chloride or sulphate.
The indirect method as advocated by A. O. A. C. is not only a lengthy
procedure but also the efficiency of the method as regards the complete removal
of all the heavy metals and alkaline-earth metals has been questioned by
Wright [1938]. Husband and Godden [1927] have undoubtedly shortened the
procedure by using the alcoholic ammonium carbonate of Gooch and Eddy
[1908] for the removal of the alkaline-earth metals, but it is felt that the conver-
sion of the mixed chloride into sulphate and the subsequent removal of free
H 2 SO 4 are not only tedious but superfluous as well. Instead of weighing the
alkali metals as sulphate, they can be weighed as chloride with some manipu-
lative care. Moreover from the mixed chloride, it is easily possible to estimate
potassium by the simple perchloric acid method.
It has already been pointed out that the indirect method will only give
reliable results when all the foreign elements could completely be removed.
In the proposed method we have achieved this by successfully utilising some of
the procedures outlined in the Technical Communication No. 9 of the Imperial
Bureau of Animal Nutrition and the A. O. A. C. method. The important
points which have been considered are as follows :—
(i) Iron, aluminium, sulphates, etc. are removed by Ba (OH) 2 . Excess
of this reagent is carefully avoided.
(ii) Barium, calcium and magnesium are removed by using alcoholic
(NH 4 ) 2 CO 3 (as given in the Rowett bulletin).
(iii) The alkali metals are weighed as chloride, not as sulphate.
(iv) Potash is estimated from the mixed chloride as KClO 4 .
(v) Vitreosil basins are used instead of platinum in heating the mixed
chloride. (Vide A. O. A. C. 1935).
B