1971L0393 — EN — 15.12.1972 — 001.001 — 1
This document is meant purely as a documentation tool and the institutions do not assume any liability for its contents
►B SECOND COMMISSION DIRECTIVE
of 18 November 1971
establishing Community methods of analysis for the official control of feedingstuffs
(71/393/EEC)
(OJ L 279, 20.12.1971, p. 7)
Amended by:
Official Journal
No page date
►M1Commission Directive 73/47/EEC of 5 December 1972 L 83 35 30.3.1973
▼B
SECOND COMMISSION DIRECTIVE
of 18 November 1971
establishing Community methods of analysis for the official control
of feedingstuffs
(71/393/EEC)
THE COMMISSION OF THE EUROPEAN COMMUNITIES,
Having regard to the Treaty establishing the European Economic
Community;
Having regard to the Council Directive of 20 July 1970(1) on the intro-
duction of Community methods of sampling and analysis for the
official control of feedingstuffs, and in particular Article 2 thereof;
Whereas that Directive requires that official controls of feedingstuffs
be carried out using Community methods of sampling and analysis forthe purpose of checking compliance with requirements arising underthe provisions laid down by law, regulation or administrative actionconcerning the quality and composition of feedingstuffs;
Whereas Commission Directive No 71/250/EEC of 15 June 1971(
2)ha s
already established a number of Community methods of analysis;
whereas the progress of work since then makes it advisable to adopt asecond set of methods;
Whereas the measures provided for in this Directive are in accordance
with the Opinion of the Standing Committee for Feedingstuffs;
HAS ADOPTED THIS DIRECTIVE:
Article 1
The Member States shall require that analyses for official controls of
feedingstuffs as regards their contents of moisture, volatile nitrogenousbases, total phosphorus and crude oils and fats be carried out according
to the methods described in the Annex to this Directive.
The general provisions set out in Part I (Introduction) of the Annex to
the First Commission Directive No 71/250/EEC of 15 June 1971 estab-lishing Community methods of analysis for the official control offeedingstuffs shall be applicable to the methods described in the Annexto this Directive.
Article 2
The Member States shall, not later than 1 January 1973, bring into
force the laws, regulations or administrative provisions necessary to
comply with this Directive. They shall forthwith inform the Commis-
sion thereof.
Article 3
This Directive is addressed to the Member States.1971L0393 — EN — 15.12.1972 — 001.001 — 2
(1) OJ No L 170, 3.8.1970, p. 2.
(2) OJ No L 155, 12.7.1971, p. 13.
▼B
ANNEX
I.DETERMINATION OF MOISTURE
1.Purpose and Scope
This method makes it possible to determine the moisture content of feeding-
stuffs.►M1It does not cover the analysis of milk products as straight
feedingstuffs, the analysis of mineral substances and mixtures composed
predominantly of mineral substances, the analysis of animal and vegetable
fats and oils or the analysis of the oil seeds and oleaginous fruit defined in
Council Regulation No 136/66/EEC(1) of 22 September 1966 on the estab-
lishment of a common organization of the market in oils and fats. ◄
The determination of the moisture content of oil seeds and oleaginous fruit is
described in Annex III to Commission Regulation (EEC) No 1470/68(2)o f
23 September 1968 on the drawing and reduction of samples and the deter-
mination of the oil content, impurities and moisture in oil seeds.
2.Principle
The sample is desiccated under specified conditions which vary according to
the nature of the feedingstuff. The loss in mass is determined by weighing. It
is necessary to carry out preliminary drying when dealing with solid feeding-
stuffs which have a high moisture content.
3.Apparatus
3.1. Crusher of non moisture-absorbing material which is easy to clean,
allows rapid, even crushing without producing any appreciable heating,
prevents contact with the outside air as far as possible and meets the
requirements laid down in 4.1.1. and 4.1.2. (e.g. hammer or water-
cooled micro-crushers, collapsible cone mills, slow motion or cog-
wheeled crushers).
3.2. Analytical balance, accurate to 0·5 mg.3.3. Dry containers of non-corrodible metal or of glass withlids ensuring
airtight closure; working surface allowing the test sample to be spread
at about 0·3 g/cm
2.
3.4. Electrically heated isothermal oven (± 1 ºC) properly ventilated and
ensuring rapid temperature regulation(3).
3.5. Adjustable electrically heated vacuum oven fitted with an oil pump and
either a mechanism for introducing hot dried air or a drying agent (e.g.
calcium oxide).
3.6. Desiccator witha th ick perforated metal or porcelain plate, containing
an efficient drying agent.
4.Procedure
N.B.: The operations described in this section must be carried out immedi-
ately after opening the packages of samples. Analysis must be carried
out at least in duplicate.
4.1.Preparation
4.1.1.Feedingstuffs other than those coming under 4.1.2 and 4.1.3
Take at least 50 g of the sample. If necessary, crush or divide in
sucha way as to avoid any variation in moisture content (see 6).
4.1.2.Cereals and groats
Take at least 50 g of the sample. Grind into particles of which at
least 50% will pass through a 0·5 mm mesh sieve and will leave
no more than 10% reject on a 1 mm round-meshed sieve.1971L0393 — EN — 15.12.1972 — 001.001 — 3
(1) OJ No 127, 30.9.1966, p. 3025/66.
(2) OJ No L 239, 28.9.1968, p. 2.
(3) For the drying of cereals, flour, groats and meal, the oven must have a thermal capacity
such that, when pre-set at 131 ºC, it will return to that temperature in less than 45minutes after the maximum number of test samples have been placed inside to drysimultaneously. Ventilation must be such that, when as many samples of commonwheat as it can contain are dried for two hours, the results differ from those obtained
after four hours of drying by less than 0·15%.
▼B
4.1.3.Feedingstuffs in liquid or paste form, feedingstuffs predominantly
composed of oils and fats
Take about 25 g of the sample, weigh to the nearest 10 mg, add
an appropriate quantity of anhydrous sand weighed to the nearest
10 mg and mix until a homogeneous product is obtained.
4.2.Drying
4.2.1.Feedingstuffs other than those coming under 4.2.2 and 4.2.3
Weigha container (3.3) withits lid to th e nearest 0·5 mg. Weigh
into the weighed container, to the nearest 1 mg, about 5 g of the
sample and spread evenly. Place the container, without its lid, in
the oven preheated to 103 ºC. To prevent the oven temperature
from falling unduly, introduce the container as rapidly as
possible. Leave to dry for four hours reckoned from the time
when the oven temperature returns to 103 ºC. Replace the lid on
the container, remove the latter from the oven, leave to cool for
30 to 45 minutes in the desiccator (3.6) and weigh to the nearest
1 mg.
For feedingstuffs composed predominantly of oils and fats, dry in
the oven for an additional 30 minutes at 130 ºC. The difference
between the two weighings must not exceed 0·1% of moisture.
4.2.2.Cereals, flour, groats and meal
Weigha container (3.3) withits lid to th e nearest 0·5 mg. Weigh
into the weighed container, to the nearest 1 mg, about 5 g of the
crushed sample and spread evenly. Place the container, without
its lid, in the oven preheated to 130 ºC. To prevent the oven
temperature from falling unduly, introduce the container as
rapidly as possible. Leave to dry for two hours reckoned from
the time when the oven temperature returns to 130 ºC. Replace
the lid on the container, remove the latter from the oven, leave
to cool for 30 to 45 minutes in the desiccator (3.6) and weigh to
the nearest 1 mg.
4.2.3.Compound feedingstuffs containing more than 4% of sucrose or
lactose: straight feedingstuffs such as locust beans, hydrolized
cereal products, malt seeds, dried beet chips, fish and sugar solu-
bles; compound feedingstuffs containing more than 25% of
mineral salts including water of crystallization.
Weigha container (3.3) withits lid to th e nearest 0·5 mg. Weigh
into the weighed container, to the nearest 1 mg, about 5 g of the
sample and spread evenly. Place the container, without its lid, in
the vacuum oven (3.5) preheated to between 80 ºC and 85 ºC. To
prevent the oven temperature from falling unduly, introduce the
container as rapidly as possible.
Bring the pressure up to 100 Torr and leave to dry for four hours
at this pressure, either in a current of hot, dry air or using a
drying agent (about 300 g for 20 samples). In the latter instance,
disconnect the vacuum pump when the prescribed pressure has
been reached. Reckon drying time from the moment when the
oven temperature returns to 80 ºC to 85 ºC. Carefully bring the
oven back to atmospheric pressure. Open the oven, place the lid
on the container immediately, remove the container from the
oven, leave to cool for 30 to 45 minutes in the desiccator (3.6)
and weighto th e nearest 1 mg. Dry for an additional 30 minutes
in the vacuum oven at 80 ºC to 85 ºC and reweigh. The differ-
ence between the two weighings must not exceed 0·1% of
moisture.
4.3.Preliminary drying
4.3.1.Feedingstuffs other than those coming under 4.3.2
Solid feedingstuffs with a high moisture content which makes
crushing difficult must be subjected to preliminary drying as
follows:
Weigh50 g of uncrushed sample to the nearest 10 mg
(compressed or agglomerated feedingstuffs may be roughly
divided if necessary) in a suitable container (e.g. a 20 × 12 cm
aluminium plate witha 0·5 cm rim). Leave to dry in an oven
from 60 ºC to 70 ºC until the moisture content has been reduced
to between8% and 12%. Remove from the oven, leave to cool
uncovered in the laboratory for one hour and weigh to the nearest1971L0393 — EN — 15.12.1972 — 001.001 — 4
▼B
10 mg. Crushimmediately as indicated in 4.1.1 and dry as indi-
cated in 4.2.1 or 4.2.3 according to the nature of the feedingstuff.
4.3.2.Cereals
Grain witha moisture content of over 17% must be subjected to
preliminary drying as follows:
Weigh50 g of unground grain to the nearest 10 mg in a suitable
container (e.g. a 20 × 12 cm aluminium plate witha 0·5 cm rim).
Leave to dry for 5 to 7 minutes in an oven at 130 ºC. Remove
from the oven, leave to cool uncovered in the laboratory for two
hours and weigh to the nearest 10 mg. Grind immediately as indi-
cated in 4.1.2 and dry as indicated in 4.2.2.
5.Calculation of results
The moisture content, as a percentage of the sample, is calculated by using
the following formulae:
5.1.Drying without preliminary drying
E/C0m ðÞ /C1100
E
where:
E = initial mass, in grammes, of the test sample,
m = mass, in grammes, of the dry test sample.
5.2.Drying with preliminary drying
M0/C0m ðÞ M
M0 þE/C0M/C20/C21
/C1100
E¼100 1 /C0Mm
EM0/C18/C19
where:
E = initial mass, in grammes, of the test sample,
M = mass, in grammes, of the test sample after preliminary drying,M′= mass, in grammes, of the test sample after crushing or grinding,
m = mass, in grammes, of the dry test sample.
5.3.Repeatibility
The difference between the results of two parallel determinations carried
out on the same sample should not exceed 0·2% of moisture.
6.Observation
If crushing proves necessary and if this is seen to alter the moisture content
of the product, the results of the analysis of the components of the feeding-
stuff must be corrected on the basis of the moisture content of the sample in
its initial state.
II.DETERMINATION OF VOLATILE NITROGENOUS BASES
A. MICRODIFFUSION
1.Purpose and Scope
This method makes it possible to determine the content of volatile nitro-
genous bases, expressed as ammonia, in feedingstuffs.
2.Principle
The sample is extracted with water and the solution clarified and filtered.
The volatile nitrogenous bases are displaced by microdiffusion using a solu-
tion of potassium carbonate, collected in a solution of boric acid and titrated
withsulph uric acid.1971L0393 — EN — 15.12.1972 — 001.001 — 5
▼B
3.Reagents
3.1. 20% (w/v) solution of trichloroacetic acid.
3.2. Indicator: dissolve 33 mg of bromocresol green and 65 mg of methyl
red in 100 ml of 95%—96% (v/v) of ethanol.
3.3. Boric acid solution: in a 1 litre graduated flask dissolve 10 g of boric
acid A.R. in 200 ml of 95%—96% (v/v) ethanol and 700 ml of water.
Add 10 ml of indicator (3.2.). Mix and, if necessary, adjust the colour of
the solution to light red by adding a solution of sodium hydroxide. 1 ml
of this solution will fix a maximum of 300 µg of NH3.
3.4. Saturated potassium carbonate solution: dissolve 10 Og of potassium
carbonate A.R. in 100 ml of boiling water. Leave to cool, filter.
3.5. Sulphuric acid 0·02 N.
4.Apparatus
4.1. Mixer (tumbler): approximately 35 to 40 r.p.m.
4.2. Glass or plastic Conway cells (see diagram).
4.3. Microburettes graduated in 1/100 ml.
5.Procedure
Weigh10 g of sample to th e nearest 1 mg and place with100 ml of water in
a 200 ml graduated flask. Mix in the tumbler for 30 minutes. Add 50 ml of
trichloroacetic acid solution (3.1), make up to volume with water, shake
vigorously and filter through a pleated filter.
Using a pipette, introduce 1 ml of boric acid solution (3.3) into the central
part of the Conway cell and 1 ml of the sample filtrate into the crown of
the cell. Cover partially with the greased lid. Drop 1 ml of saturated potas-
sium carbonate solution (3.4) quickly into the crown and close the lid so that
the cell is airtight. Turn the cell carefully rotating it in a horizontal plane so
that the two reagents are mixed. Leave to incubate either for at least four
hours at room temperature or for one hour at 40 ºC.
Using a microburette (4.3), titrate the volatile bases in the boric acid solution
withsulph uric acid 0·02 N (3.5).
Carry out a blank test using the same procedure but without a sample to be
analysed.
6.Calculation of results
1m lo fH2SO40·02 N corresponds to 0·34 mg of ammonia.
Express the result as a precentage of the sample.
Repeatibility
The difference between the results of two parallel determinations carried out
on the same sample should not exceed:
10%, in relative value, for ammonia contents of less than 1·0%;
0·1%, in absolute value, for ammonia contents of 1·0% or more.
7.Observation
If the ammonia content of the sample exceeds 0·6%, dilute the initial filtrate.1971L0393 — EN — 15.12.1972 — 001.001 — 6
▼B
CONWAY CELL
Scale 1/1
1971L0393 — EN — 15.12.1972 — 001.001 — 7
▼B
B. BY DISTILLATION
1.Purpose and Scope
This method makes it possible to determine the content of volatile nitro-
genous bases, expressed as ammonia, in fish-meal containing practically
no urea. It is applicable only to ammonia contents of less than 0·25%.
2.Principle
The sample is extracted with water and the solution clarified and filtered.
The volatile nitrogenous bases are displaced at boiling point by adding
magnesium oxide and collected in a specific quantity of sulphuric acid,
the excess of which is back-titrated with a solution of sodium hydroxide.
3.Reagents
3.1. 20% (w/v) solution of trichloroacetic acid.3.2. Magnesium oxide A.R.3.3. Anti-foaming emulsion (e.g. silicone).3.4. Sulphuric acid 0·1 N.
3.5. Sodium hydroxide solution 0·1 N.
3.6. 0·3% (w/v) solution of methyl red in 95%—96% (v/v) ethanol.
4.Apparatus
4.1. Mixer (tumbler): approximately 35 to 40 r.p.m.4.2. Distilling apparatus of the Kjeldahl type.
5.Procedure
Weigh 10 g of the sample to the nearest 1 mg and place with 100 ml of
water in a 200 ml graduated flask. Mix in the tumbler for 30 minutes. Add
50 ml of trichloroacetic acid solution (3.1), make up to volume with
water, shake vigorously and filter through a pleated filter.
Take a quantity of clear filtrate appropriate for the presumed content of
volatile nitrogenous bases (100 ml is usually suitable). Dilute to 200 ml
and add 2 g of magnesium oxide (3.2) and a few drops of anti-foaming
emulsion (3.3).The solution should be alkaline to litmus paper; otherwise
add some magnesium oxide (3.2). Distil about 150 ml of the solution in
the Kjeldahl apparatus and collect the distillate in an Erlenmeyer flask
containing an accurately measured volume (25 to 50 ml) of sulphuric
acid 0·1 N (3.4). While distilling, avoid overheating of the sides. Boil
the sulphuric acid solution for two minutes, cool it and back-titrate theexcess sulphuric acid with the sodium hydroxide solution 0·1 N (3.5) in
the presence of the methyl red indicator (3.6).
Carry out a blanktest using the same procedure but without a sample to
be analysed.
6.Calculation of results
1m lo fH
2SO40·1 N corresponds to 1·7 mg of ammonia.
Express the result as a percentage of the sample.
Repeatibility
The difference between the results of two parallel determinations carried
out on the same sample should not exceed, in relative value, 10% of
ammonia.
III.DETERMINATION OF TOTAL PHOSPHORUS
PHOTOMETRIC METHOD
1.Purpose and Scope
This method makes it possible to determine the content of total phosphorus
in feedingstuffs. It is particularly appropriate for the analysis of products low
in phosphorus. In certain cases (product rich in phosphorus), a gravimetric
method may be used.1971L0393 — EN — 15.12.1972 — 001.001 — 8
▼B
2.Principle
The sample is mineralised, either by dry combustion (in the case of organic
feedingstuffs) or by acid digestion (in the case of mineral compounds and
liquid feedingstuffs), and placed in an acid solution. The solution is treated
with molybdovanadate reagent. The optical density of the yellow solution
thus formed is measured in a spectrophotometer at 430 nm.
3.Reagents
3.1. Calcium carbonate A.R.
3.2. Hydrochloric acid A.R., d: 1·1 (approx 6 N).
3.3. Nitric acid A.R., d: 1·045.
3.4. Nitric acid A.R., d: 1·38 to 1·42.
3.5. Sulphuric acid A.R., d: 1·84.
3.6. Molybdovanadate reagent: mix 200 ml of ammonium heptamolybdate
solution (3.6.1), 200 ml of ammonium monovanadate solution (3.6.2)
and 134 ml of nitric acid (3.4) in a 1 litre graduated flask. Make up to
volume withwater.
3.6.1. Ammonium heptamolybdate solution: dissolve in hot water 100 g
of ammonium heptamolybdate A.R.
(NH4)6Mo7O24.4H2O. Add 10 ml of ammonia (d: 0·91) and make
up to 1 litre withwater.
3.6.2. Ammonium monovanadate solution: 2·35 g of ammonium mono-
vanadate A.R. NH4VO3dissolve in 400 ml of hot water. Stirring
constantly, slowly add 20 ml of dilute nitric acid (7 ml of HNO3(3.4) + 13 ml of H2O and make up to 1 litre withwater.
3.7. Standard solution of 1 mg phosphorus per ml: dissolve 4·387 g of potas-
sium dihydrogen phosphate A.R. KG2PO4in water. Make up to 1 litre
withwater.
4.Apparatus
4.1. Silica or porcelain ashing crucibles.
4.2. Electric muffle-furnace withth ermostat set at 550 ºC.
4.3. 250 ml Kjeldahl flask.
4.4. Graduated flasks and precision pipettes.
4.5. Spectrophotometer.
4.6. Test tubes about 16 mm in diameter, withstoppers graded to a diameter
of 14·5 mm; capacity: 25 to 30 ml.
5.Procedure
5.1.Preparation of the solution
According to the nature of the sample, prepare a solution as indicated in
5.1.1 or 5.1.2.
5.1.1.Usual procedure
Weigh 1 g or more of the sample to the nearest 1 mg. Place the
test sample in a Kjeldahl flask, add 20 ml of sulphuric acid (3.5),
shake to impregnate the substancecompletely with acid and to
prevent it from sticking to the sides of the flask, heat and keep
at boiling point for 10 minutes. Leave to cool slightly, add 2 ml
of nitric acid (3.4), heat gently, leave to cool slightly, add a little
more nitric acid (3.4) and bring back to boiling point. Repeat this
procedure until a colourless solution is obtained. Cool, add a little
water, decant the liquid into a 500 ml graduated flask, rinsing the
Kjeldahl flask with hot water. Leave to cool, make up to volume
withwater, h omogenise and filter.
5.1.2.Samples containing organic substances and free from calcium
and magnesium dihydrogen phosphates
Weigh about 2·5 g of the sample to the nearest 1 mg in an ashing
crucible. Mix the test sample until completely merged with 1 g of
calcium carbonate (3.1). Ashin th e oven at 550 ºC ± 5 ºC until
white or grey ash is obtained (a little charcoal does not matter).1971L0393 — EN — 15.12.1972 — 001.001 — 9
▼B
Transfer the ash into a 250 ml beaker. Add 20 ml of water and
hydrochloric acid (3.2) until effervescence ceases. Add a further
10 ml of hydrochloric acid (3.2). Place the beaker on a sand
bathand evaporate until dry to make th e silica insoluble. Redis-
solve the residue in 10 ml of nitric acid (3.3) and boil on the sand
bath for 5 minutes without evaporating until dry. Decant the
liquid into a 500 ml graduated flask, rinsing the beaker several
times withh ot water. Leave to cool, make up to volume with
water, homogenise and filter.
5.2.Development of coloration and measurement of optical density
Dilute an aliquot part of the filtrate obtained by 5.1.1 or 5.1.2 to obtain
a phosphorus concentration of not more than 40 µg/ml. Place 10 ml of
this solution in a test tube (4.6) and add 10 ml of molybdovanadate
reagent (3.6). Homogenise and leave to stand for at least 10 minutes at
20 ºC. Measure the optical density in a spectrophotometer at 430 nm
against a solution obtained by adding 10 ml of the molybdovanadate
reagent (3.6) to 10 ml of water.
5.3.Calibration curve
From the standard solution (3.7) prepare solutions containing respec-
tively 5, 10, 20, 30 and 40 µg of phosphorus per ml. Take 10 ml of
each of these solutions and add thereto 10 ml of molybdovanadate
reagent (3.6). Homogenise and leave to stand for at least 10 minutes at
20 ºC. Measure the optical density as indicated in 5.2.
Trace the calibration curve by plotting the optical densities against the
corresponding quantities of phosphorus. For concentrations between 0
and 40 µg/ml, the curve will be linear.
6.Calculation of results
Determine the amount of phosphorus in the test sample by using the calibra-
tion curve.
Express the result as a percentage of the sample.
Repeatibility
The difference between the results of two parallel determinations carried out
on the same sample should not exceed:
3%, in relative value, for phosphorus contents of less than 5%;
0·15%, in absolute value, for phosphorus contents of 5% or more.
IV.DETERMINATION OF CRUDE OILS AND FATS
1.Purpose and Scope
This method makes it possible to determine the content of crude oils and fats
in feedingstuffs. It does not cover the analysis of the oil seeds and oleaginous
fruit defined in Council Regulation 136/66/EEC of 22 September 1966. The
determination of the oil content of those products is described in Annex V to
Commission Regulation (EEC) No 1470/68 of 23 September 1968.
Either one of two methods may be used, depending on the nature of the
feedingstuff.
1.1.Method A (extraction by ether): applicable to all feedingstuffs other than
those coming under 1.2
1.2.Method B: applicable to feedingstuffs from which oils and fats cannot
be totally extracted with diethyl either without prior hydrolysis, to
feedingstuffs of animal origin, glutens, dried potato pulps, dried brewing
and distilling dreys and waste, dried yeasts, waste from biscuits, bread
and cooked foods, milk products and feedingstuffs containing a high
proportion of suchproducts (at least 40%) and to compound feeding-
stuffs enriched with fats.
2.Principle
2.1.Method A: The oils and fats are extracted with diethyl ether. The solvent
is distilled off and the residue dried and weighed.
2.2.Method B: The sample is hydrolised when hot with hydrochloric acid.
The solution is cooled and filtered. The residue is washed and dried
and extracted with diethyl ether using Method A.1971L0393 — EN — 15.12.1972 — 001.001 — 10
▼B
3.Reagents
3.1. Anhydrous diethyl ether, d: 0·720, B.P.: 34·5 ºC, virtually free from
peroxides
3.2. Anhydrous sodium sulphate A.R.
3.3. Hydrochloric acid 3N.
3.4. Filtration aid, e.g. Kieselgur, Hyflo-supercel.3.5. Carbon tetrachloride A.R.
4.Apparatus
4.1. Soxhlet-type extractor or equivalent apparatus.4.2. Explosion-proof heating apparatus with temperature control.4.3. Vacuum drying oven (less than 100 Torr).
5.Procedure
5.1.Method A: (see 7.1)
Weigh 5 g of the sample to the nearest 1 mg and mix with 2 g to 3 g (or
more, if necessary) of anhydrous sodium sulphate (3.2). Place the
mixture in an extraction thimble free from oils and fats and cover with
a fat-free wad of cottonwool. (The mixing may be carried out in the
thimble.)
Place the thimble in an extracter (4.1) and extract for six hours with
diethyl ether (3.1). If a Soxhlet-type extractor is used, regulate the
heating to obtain at least 15 siphonings per hour. Collect the ether
extract in a dry, weighed flask containing fragments of pumice stone(
1).
Distil off the ether and dry the evaporation residue for one and a half
hours in the vacuum drying oven (4.3) at 75 ºC. Cool in a desiccator
and weigh. Dry again for 30 minutes to ensure that the weight of the
oil and fat remains constant (loss in weight must be less than 1 mg).
5.2.Method B
Weigh 2·5 g of the sample to the nearest 1 mg (see 7.2) and place in a
400 ml beaker or a 300 ml Erlenmeyer flask. Add 100 ml of hydro-
chloric acid 3 N (3.3) and fragments of pumice stone. Cover the
beaker witha watchglass or fit th e Erlenmeyer flask witha reflux
condenser. Bring the mixture to a gentle boil over a low flame or a hot
plate and keep it there for one hour. Do not allow the product to stick to
the sides of the container.
Cool and add a quantity of filtration aid (3.4) sufficient to prevent any
loss of oil and fat during filtration. Filter through a moistened, fat-free,
double filter paper. Wash the residue in cold water until the acid reac-
tion has ceased. Check that the filtrate does not contain any oil or fats.
Their presence in the filtrate indicates that the sample must be extracted
with diethyl ether, using the method given in 5.1, before hydrolysis.
Place the double filter paper containing the residue on a watch glass and
dry for one and a half hours in the oven at 95 ºC to 98 ºC.
Place the double filter paper and the dry residue in an extraction
thimble, extract with diethyl ether and proceed as indicated in the
second paragraphof 5.1.
6.Calculation of results
Express the result as a percentage of the sample.
Repeatibility
The difference between the results of two parallel determinations carried out
on the same sample should not exceed 0·3% of oil or fat.1971L0393 — EN — 15.12.1972 — 001.001 — 11
(1) Where the oil or fat has to undergo subsequent quality tests, replace the fragments of
pumice stone by glass beads.
▼B
7.Observations
7.1. For products witha h ighcontent of oils and fats and wh ichare difficult
to crushor unsuitable for drawing a h omogeneous reduced test sample,
proceed as follows. Weigh 20 g of the sample to the nearest 1 mg and
mix with 10 g or more of anhydrous sodium sulphate (3.2). Extract with
diethyl ether (3.1) as indicated in 5.1. Make up the extract obtained to
500 ml with carbon tetrachloride (3.5) and homogenise. Take 50 ml of
the solution and place in a small, dry, weighed flask containing frag-
ments of pumice stone(1). Distil off the solvent, dry and proceed as
indicated in the last paragraph of 5.1. Eliminate the solvent from the
extraction residue left in the thimble and crush the residue to a fineness
of 1 mm. Return the product to the extraction thimble (do not add
sodium sulphate), extract with diethyl ether and proceed as indicated in
the second and third paragraphs of 5.1.
Calculate the result as a percentage of the sample, taking into account
the aliquot part used for the first extraction, by using the following
formula:
(10 a + b) · 5
where:
a = ether extract, in grammes, of the aliquot part after the first extrac-
tion;
b = ether extract, in grammes, after the second extraction.
7.2. For products low in oils and fats the test sample may be increased to 5 g.1971L0393 — EN — 15.12.1972 — 001.001 — 12
(1) Where the oil or fat has to undergo subsequent quality tests, replace the fragments of
pumice stone by glass beads.