IS : 7874 ( Part I ) • 1975

2. Q,UALITY OF REAGENTS
2.1 Unless specified otherwise, pure chemicals and distilled water (see
IS: 1070-1960· ) shall be employed in tests,
NOTE - C Pure chemicals t shall mean chemicals that do not contain impurities which
affect the test results.

3. PREPARATION OF THE SAMPLE

3.1 Grind about 100 g of "the sample so that it passes through I·OO-mm
IS Sieve (sel IS: 460-1962t). Alternatively, ASTM Sieve 18 or BS Sieve
16 or Tyler Sieve 16 may be used. Transfer this prepared sample to a
well-stoppered glass bottle.

4. DETERMINATION OF MOISTURE

~.1 Procedure - Weigh accurately about 5 g of the prepared sample

( see 3.1) in a tared aluminium dish with a cover, having a diameter of at
least 50 mm and a depth of about 40 rom. Shake the dish until the
contents are evenly distributed. With cover removed place the dish and
cover in an air-oven maintained at 135 ± 2°C and dry for at least 2 hours.
Place cover on dish, cool in a desiccator and weigh. Repeat the process
of heating, cooling and weighing until the difference in mass between two
successive weighings is less than one milligram.
4.2 CalcaiadoD

4.2.1 Moisture, percent by mass == 100k~~ ~M, )

where
M, -= mass in g. of the dish with the material before drying,
M: == mass in g of the dish with the material after drying, and
M -= mass in g of the empty dish.

5. DETERMINATION OF CRUDE PROTEIN

5.1 Prlaciple - The percentage of crude protein is ascertained by multi-
plying the percentage of nitrogen other than ammoniacal nitrogen, by a
factor. The quantity of ammoniacal nitrogen is separately determined and
deducted from total nitrogen.

·Specification Cor water, distilled quality (ffviMJ).

tSpecification (or teat aieva (,mull).
 IS z 7874 ( Part I ) • 1975
5.1.1 For animal feeds and feeding stuffs the factor to be used will be
6·25, except in case of wheat and its products for which it will be 5·70.
5.2 Apparatus
5.2.1 Kj·eldahl Flask - 500 ml capacity.
5.2.2 Distillation Assembly - The assembly consists of a round-bottom flask
of a 1 000 ml capacity fitted with a rubber stopper through which passes one
end of the connecting bulb tube. The other end of the bulb tube is con-
nected to the condenser which is attached by means of a rubber tube to a
dip tube which dips into a known quantity of standard sulphuric acid or
boric acid solution contained in a conical flask of 500 ml capacity, to which
3 to 4 drops of methyl red indicator solution have been added.
5.3 Reagents
5.3.1 Potassium Sulphate or Anhydrous Sodium Sulphate
5.3.2 Copper .~ulphate

5.3.3 Concentrated Sulphuric Acid- r.d, 1-84 (see IS: 266-1961*).

5.3.4 Sodium Hydroxide Solution - Dissolve about 450 g of sodium hydro..
xide in 1 000 ml of water,
5.3.5 Standard Sulphllrie Acid - 0'5 N.
5.3.6 Standard Sodium Hydroxid» Solution - 0·25 N.
5.3.7 Methyl R,d Indicator Solution - Dissolve 1 g of methyl red in 200 ml
of rectified spirit (see IS: 323-1959t ), 95 percent by volume.
5.3.8 Boric Acid Solution - Saturated. Dissolve 60 g of boric acid in 1 litre
of hot water; cool and allow to mature for 3 days before decanting the
clear liquid.
5.3.' Magnesium O%ide - carbonate free, freshly ignited.
5.4 Procedare
5 •••1 Total Nitrogen - Transfer carefully about 2 g of the prepared
sample (sel 3.1 ), accurately weighed, to the Kjeldahl flask, Add about 10
g of potassium sulphate or anhydrous sodium sulphate, about 0-5 g of
copper sulphate and 25 ml or more, if necessary, of concentrated sulphuric
acid. Place the flask in an inclined position, and heat below the boiling
point of the acid until frothing ceases. Increase heat until the acid boils
vigorously and digest for a time after the mixture is clear or until oxidation
is complete (about 2 hours ). Cool the contents of the flask. Transfer
·Specification for sulphuric acid (,.isltl).
f5pecification for rectified apirit ( "";$,d).
lS I 7874 ( Part I ) - 1975
quantitatively to the round-bottom flask with water, the total quantity of
water used being about 200 ml. Add a few pieces of pumice stone to
prevent bumping. Add carefully the sodium hydroxide solution in quantity
which is sufficient to make the solution alkaline by the side of the flask so
that it does not mix at once with the acid solution but forms a layer below
the. acid layer. Assemble the apparatus taking care that the tip of the dip-
tube extends below the surface of the standard sulphuric acid solution in
the receiver. Mix the contents of the flask by shaking and distil until all
ammonia has passed over into the standard sulphuric acid solution. Titrate
with standard sodium hydroxide solution,
[Link] Carry out a blank determination using all reagents in the same
quantities but without the material to be tested.
5.4.2 Alternatively, the ammonia evolved by distillation shall be
absorbed in boric acid. Carry out digestion as prescribed in 5.4.1. Transfer
completely the contents of the digestion flask into the round-bottom
flask through the separating funnel. Rinse the separating funnel with water.
The total volume of liquid in the distillation flask should not exceed
half the capacity of the flask otherwise frothing may occur. Add excess of
sodium hydroxide solution to make the solution alkaline. Connect immedia-
tely the round-bottom flask to steam trap and condenser. The condenser
should be arranged to dip the dip-tube in 50 ml of boric acid which is kept
cool in the conical flask. Add 2 or 3 drops of the mixed indicator. Distil
about one-third of total volume of the solution in the flask. Cool and
dismantle the distillation assembly. Rinse the tip of the condenser and the
dip-tube with water, collecting the washings in the receiver. Titrate the
ammonia present in the distillate with sulphuric acid until the grass-green
colour changes to steel-grey, an additional drop then giving the purple
colour.
5.4.3 Ammoniacal Nitrogen - Weigh accurately 2 to 4 g of the prepared
sample (see 3.1). Shake it with water and filter. Wash the residue thoro-
ughly with water. Transfer the filtrate to the distillation flask and dilute
to about 200 ml with water. Add about 5 g of magnesium oxide. Connect
the flask to the condenser by means of the connecting bulb tube and distil
about 100 ml of liquid into the receiver containing standard sulphuric acid
and methyl red indicator solution. Titrate the contents of the receiver
with standard sodium hydroxide solution.
[Link] Carry out a blank determination using all reagents in the same
quantities but without the material to be tested.
5.5 CalculatioD
5.5.1 Total nitrogen ( on moisture-free basis ),
percent by mass 140 (B - A) N
-=-~~--....;....-
.. ( 100 - M)
6
 18 : 1874 ( Part I ) • 1975
where
B lIB
.
volume in ml of the standard sodium hydroxide solution
used to neutralize the acid in blank determination
(sle [Link]),
A == volume in ml of the standard sodium hydroxide solution
used to neutralize the excess acid in the test with the
material (see 5.4.1),
.N = normality of the standard sodium hydroxide solution,
m == mass in g of the material taken for the test (see 5.4..1 ),
and
M = moisture percentage (see 4.2.1 ).
5.5.2 When boric acid solution has been used for absorption, calculation
of total nitrogen shall be as given below:
Total nitrogen (on moisture-free basis ), 140 V N
percent by mass = m ( 100 _ M)
where
v= volume in ml of standard sulphuric acid used in titration
( see 5.4.2 ),
N normality of the standard sulphuric acid,
=:I

m = mass in g of the material taken tor the test (see 5.4.1),

and
M = moisture percentage ( see 4.2.1 ).
5.5.3 Ammoniacal nitrogen (on moisture- 140 ( b - a) N
free basis), percent by mass
m (100 - M)
where
b =
volume in ml of the standard SOdiUDl hydroxide solution
used to neutralize the acid in blank determination
( se« [Link] ),
a == volume in ml of the standard sodium hydroxide solution
used to neutralize the excess acid in the test with the
material (set 5.4.3 ),
N -= normality of the standard sodium hydroxide solution,
m .. mass in g of the material taken for the test (see 5.4.3),
and
M -= moisture percentage (SII 4.2.1 ).
7
III 7874 ( Part I ) • 1975
5.5.4 Crude protein (on moisture-free basis ),
percent by mass =z: 6·25 (X - Y)
where
X = percent by mass of total nitrogen (SI' 5.5.1 or 5.5.2 ),
and
r == percent by mass of ammoniacal nitrogen (s" 5.5.3).

&.1 Prladple - The percentage of urea nitrogen is ascertained by deduc-

ting the percentage of ammoniacal nitrogen from percent total ammoniacal
nitrogen ( SI' 5.5.3) which includes both ammoniacal and the urea nitro-
gen. However t a suitable qualitative test for detecting the presence of
urea nitrogen should precede the quantitative determination as outlined
in this method.
&.2 Apparataa
6.2.1 Kj,ldahl Flask - 500 ml capacity.
6.2.2 Distillation Assnnbly - The assembly consists of a round-bottom flask
of 1 000 ml capacity, fitted with a rubber stopper through which passes one
end of the connecting bulb tube. The other end of the bulb tube is
connected to the condenser which is attached by means of a rubber tube to
a dip-tube which dips into a known quantity of standard sulphuric acid
contained in a conical flask of 500 ml capacity, to which 3 to 4 drops of
methyl red indicator solution have been added.
6.3 Reageat.
6.3.1 DejOaming Solution - Dissolve 50 g diglycol stearate in 375 ml of
benzene, 75 ml of alcohol, and 250 ml of dibutyl phthalate. Warm, if
necessary.
6.3.2 U"tJS, Solution - Dissolve standardized urease in water 10 that each
10 ml neutralized solution will convert the nitrogen of at least 0·1 g pure
urea. Prepare fresh urease solution for every determination.
[Link] Standardization of U'~1IS6 solution - To determine alkalinity of
commercial urease preparation dissolve 0·1 g in 50 ml of water and titrate
with 0·1 N hydrochloric acid using methyl red indicator. Add the same
quantity of 0·1 N hydrochloric acid to each 0-1 g of urease in preparing the
urease solution. To determine the enzyme activity, prepare about 50 ml of
a neutralized one percent solution of urease. Add different quantities of
solution to 0·1 g samples of pure urea and follow with the' enzyme digestion
and distillation as directed In thedetermination, Calculate activity of the
urease preparation frc,m quantity of the urease solution that converted the
..area, thereby permitting complete recovery of the nitrogen ~y [Link].

8
IS I 7874 ( Part I ) · "1975
6.5.2 Urea nitrogen -= a - b
where
a = percent by mass of total ammoniacal nitrogen (se, 6.5.1),
and
b = percent by mass of ammoniacal nitrogen ( see 5.5.3 ).

7. DETERMINATION OF CRUDE FAT

7.1 Reagents
7.1.1 Petroleum Ether - of boiling range 40°C to 60 0 e.
7.1.2 Hexane, Food Grade - conforming to IS: 3470-1966*.
7.2 Procedure - Weigh accurately about 2·5 g of the prepared sample
( se,'3.1 ), dried as described under 4.1 and extract with petroleum ether or
hexane, food grade, in a Soxhlet or other suitable extractor. The extraction
period. may vary from 4 hours at a condensation rate of 5 to 6 drops per
second to 16 hours at 2 to 3 drops per second. Dry the extract on a
steam-bath for 30 minutes, cool in a desiccator and weigh. Continue at 30-
minutes intervals this alternate drying and weighing until the difference
between two successive weighings is less than one mg. Note the lowest
mass.
NOTE - If necessary preserve the fat..free material in a desiccator for the determi-
nation of crude..fi bre conten t ( s" 8 )_

7.3 CalcalatioD
7.3.1 Crude fat ( on moisture-free basis ),
percent by mass 100 (M1 - M.,.l
m
where
M 1 == mass in g of the extraction flask with dried extract,
M a =-= mass in g of extraction flask, and
'" == mass in g of the dried sample ( s" 7.2 ) taken for the test.

8. DETERMINATION OF CRUDE FIBRE

8.1 Reageat.
8.1.1 SulplwricAcid - 0·255 N [1-25 percent i mlu )], accurately prepared.

·SpecificatioD Cor hexane, food ,rade.

10
 IS I 7874 ( Part 1 ) • 1975
8.1.2 Sodium Hydroxide Solulioll- 0·313 N [1·25 percent ( m]» )], accu-
rately prepared.
8.2 Procedure - Weigh accurately about 2 g of the dried material (see 4.1)
and extract the fat for about 8 hours with petroleum ether or hexane, food
grade, using a Soxhlet or other suitable extractor or use the residue from
the crude fat determination (see Note under 7.2). Transfer the fat-free
dry residue to a one-litre conical flask. Take 200 ml of dilute sulphuric acid
in a beaker and bring to the boil. Transfe-r the whole of the boiling acid
to the flask containing the fat-free material and immediately connect the
flask with a reflux water condenser and heat, so that the contents of the
flask begin to boil within one minute. Rotate the flask frequently, taking
care to keep the material from remaining on the sides of the flask out of
contact with the acid. Continue boiling for exactly 30 minutes. Remove
the flask and filter through fine linen ( about 18 threads to the centimetre)
held in a funnel, and wash with boiling water until the washings are no
longer acid to litmus. Bring to the boil some quantity ofsodium hydroxide
solution under a reflux condenser, Wash the residue on the linen into the
flask with 200 ml of the boiling sodium hydroxide solution. Immediately
connect the flask with the reflux condenser and boil for exactly 30 minutes.
Remove the flask and imrnediatcly filter through the filtering cloth.
Thoroughly wash the residue with boiling water and transfer to a Gooch
crucible prepared with a thin but compact layer of ignited asbestos. Wash
the residue thoroughly first with hot water and then with about 15 ml of 95
percent ( by volume) ethyl alcohol. Dry the Gooch crucible and contents
at 105 ± lOC in the air-oven to constant mass. Cool and weigh. Incine-
rate the contents of the Gooch crucible at 600 4- 20°(: in a muffle furnace
until all the carbonaceous matter is burnt. Cool the Gooch crucible con-
taining the ash in a desiccator and weigh.
NOTE - Alternatively, instead of the conical flask and the reflux condenser, use a tall-
form spoutless beaker of 600 to 800 III 1 capacity and cover it with a round-bottomed
flask filled with cold water, which acts as a condenser. If the water in the flask becomes
hot, it may be replaced by another flask containing cold water.

8.3 CalealadoD
8.3.1 Crude fibre ( on moisture-free basis) J
100(M1 - M . )
percent by mass = m

where
M1 == mass in g of Gooch crucible and contents before ashing,
M. - mass in g of Gooch crucible containing asbestos and
ash, and
III - mass in g of the dried material taken for the test.

11
IS I 7874 ( Part I) • 1975
8.3.2 When the residue from fat determination is used:
Crude fibre ( on moisture-free basis ),
percent by mass == (~_M_l_- _M--=I~_=__
) ( 100 - f)
_ _....;;;....:;.-.
ml
where
M 1 == inass in g of Gooch crucible and contents before ashing,
M. = mass in g of Gooch crucible containing asbestos and ash,
f == crude fat (on moisture-free basis), percent by mass
(SI' 7.3.1), and
ml :=I,mass in gofthe fat-free material taken for the test.

9. DETERMINATION OF TOTAL ASH

9.1 Procedare - Weigh accurately about 2 g of the dried material
( SII 4.1) in a tared porcelain, silica or platinum dish. Ignite with the
flame of a Meker burner for about one hour. Complete the ignition by
keeping in a muffle furnace at 550 ± 20°C until grey ash results, Cool in
a desiccator and weigh. Ignite the dish again in the muffle furnace for
30 minutes, cool and weigh. Repeat this process until the difference in mass
between two·successive weighings is less than 1 mg. Note the lowest mass.
NOTE - Preserve the dish containing this ash for the determination of acid insoluble
ash (s" 10).

9.2 CalCalatlOD
9.2.1 Total ash ( on moisture-free basis ),
percent by mass

where
M. =- the lowest mass in g of the dish with the ash,
M c=a mass in g of the empty dish, and
M 1 . . mass in g of the dish with the dried material taken for
the test.

10. DETERMINATION OF ACID INSOLUBLE ASH

10.1 :RealeDt
. ~

10.1.1 Dil"," Hydrochloric Acid- approximate1y 5 N. prepared from

concentrated hydrochloric acid (s" IS : 265-1962* ).
• SpecificatioD Cor hydrochloric acid (m1iutl).

12
 IS I 7874 ( Pa.. I ) • 1975

10.2 Procedure - Weigh accurately 2 g of the dried material (SI' 4:.1 )

in a tared porcelain, silica or platinum dish. Ignite with a Meker burner
for about one hour. Complete the ignition by keeping in a muffle furnace
at 550 0 ± 20°C until qrey ash results. Moisten with concentrated hydro-
chloric acid and evaporate to dryness. Keep in an electric air-oven
maintained at 135 ± 2°C for about 3 hours. Cool and add 25 ml of dilute
hydrochloric acid, cover with a watch-glass and heat on a water-bath for
10 minutes, Cool and filter through Whatman filter paper No. 42 or its
equivalent. Wash the residue with hot water until tile washings are free
from chlorides as tested with silver nitrate solution and return the filter
paper and residue to the dish. Ignite it in a muffle furnace at 550° ± 20°0
for one hour. Cool in a desiccator and weigh. Ignite the dish again for
30 minutes, cool and weigh, Repeat this process till the difference
between two successive weighings is less than one milligram. Note the
lowest mass.
10.3 CalculatioD
10.3.1 Acid insoluble ash (on moisture-free 100 ( lvI 2 - M)
basis), percent by mass -
4~11 - M
where
Ms = the lowest mass in g of the dish with the acid insoluble
ash,
M = mass in g of the empty dish, and
M1 = mass in g of the dish with dried material (see M 1
in 9.2.1 ).

11. DETERMINATION OF CASTOR HUSK

11.1 Principle - The method is based on the fact that castor husk is not
bleached under the conditions which cause the bleaching of almost all other
materials of vegetable origin likely to be present in animal feeds or feeding
stuffs. The method consists of the treatment of the material with dilute
alkali and acid solutions followed by treatment with bleaching powder
solution and the isolation of the unbleached castor husk.

11.2 Apparat••
11.2.1 White Pholog,apllic DisJ,
11.3 ReaseDt.
11.3.1 Sodium Hydroxid. Solution - 5 percent (mlv).
11.3.2 Diluu Hydrochloric Acid - 5 percent ( mlv)
13