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Mazdoor Kisan Shakti Sangathan Jawaharlal Nehru
“The Right to Information, The Right to Live” “Step Out From the Old to the New”

IS 11762 (2005): Evaporated milk and sweetened condensed

milk - Determination of fat content - Gravimetric Method
(Reference Method) [FAD 19: Dairy Products and Equipment]

“!ान $ एक न' भारत का +नम-ण”

Satyanarayan Gangaram Pitroda
“Invent a New India Using Knowledge”

“!ान एक ऐसा खजाना > जो कभी च0राया नहB जा सकता ह”

है”
ह
Bhartṛhari—Nītiśatakam
“Knowledge is such a treasure which cannot be stolen”
 ( Ww7 p%-m)
Indian Standard
EVAPORATED MILK AND SWEETENED
CONDENSED MILK — DETERMINATION OF
FAT CONTENT — GRAVIMETRIC METHOD
(REFERENCE METHOD)
( First Revision)

ICS 67.100.10

@ BIS 2005
BUREAU OF INDIAN STANDARDS
MANAK BHAVAN, 9 BAHADUR SHAH ZAFAR MARG
NEW DELHI 110002

November 2005
Price Group 7
Dairy Products and Equipment Sectional Committee, FAD 19

NATIONAL FOREWORD

This Indian Standard (First Revision) which is identical with ISO 1737:1999 ‘Evaporated milk and
sweetened condensed milk — Determination of fat content — Gravimetric method (Reference method)’
issued by the International Organization for Standardization (ISO) was adopted by the Bureau of Indian
Standards on the recommendations of the Dairy Products and Equipment Sectional Committee and
approval of the Food and Agriculture Division Council.

This standard was first published in 1986 based on the earlier version of the International Standard,
namely, ISO 1737:1985 under dual numbering. The first revision of this standard is being brought out
to align it with the latest edition of the ISO Standard.

The text of the ISO Standard has been approved as suitable for publication as an Indian Standard
without deviations. Certain terminology and conventions are, however, not identical to those used in
Indian Standards. Attention is particularly drawn to the following:

a) Wherever the words ‘International Standard’ appear referring to this standard, they should
be read as ‘Indian Standard’.

b) Comma (,) has been used as a decimal marker while in Indian Standards, the current practice
is to use a point (.) as the decimal marker.

The technical committee responsible for the preparation of this standard has reviewed the provisions of
the following International Standard and has decided that it”isacceptable for use in conjunct”kmwiththis
standard:

International Standard Title

ISO 3889:1977 Milk and milk products — Determination of fat content — Mojonnier-type fat
extraction flasks

In reporting the results of a test or analysis made in accordance with this standard, if the final value,
observed or calculated, is to be rounded off, it shall be done in accordance with IS 2:1960 ‘Rules for
rounding off numerical values (revised)’.

.
 IS 11762:2005
ISO1737:1999

/nciian Standard
EVAPORATED MILK AND SWEETENED
CONDENSED MILK — DETERMINATION OF
FAT CONTENT — GRAVIMETRIC METHOD
(REFERENCE METHOD)
( First Revision)

WARNING — The use of this international Standard may involve hazardous materials, operations and
equipment. This standard does not purport to address all the safety problems associated with its use. It is
the responsibility of the user of this standard to establish safety and health practices and determine the
applicability of regulatory limitations prior to use.

1 Scope

This International Standard specifies the reference method for the determination of the fat content of all types of
evaporated milk and sweetened condensed milk (liquid sweetened and unsweetened concentrated milk).

2 Normative reference

The following normative document contain provisions which, through reference in this text, constitute provisions of
this International Standard. For dated references, subsequent amendments to, or revisions of, this publication do
not apply. However, parties to agreement based on this International Standard are encouraged to investigate the
possibility of applying the most recent edition of the standard indicated below. For undated references, the latest
edition of the normative document referred to applies. Members of ISO and IEC maintain registers of currently valid
International Standards.

ISO 3889, Milk and milk products — Determination of fat content — Mojonnier-type fat extraction flasks.

3 Term and definition

For the purposes of ibis International Standard the following term and definition apply.

3.1
fat content of evaporated milk and sweetened condensed milk
mass fraction of substances determined by the procedure specified in this International Standard

NOTE The fat content is expressed” as a mass fraction, in percent [formerly given as% (m/m)].

4 Principle

An ammoniacal ethanolic solution of a test portion is extracted with diethyl ether and light petroleum. The solvents
are removed by distillation or evaporation. The mass of the substances extracted is determined.

NOTE This is usually known as the Rose-Gottlieb principle.

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 IS 11762:2005
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5 Reagents

> Use only reagents of recognized analytical grade, unless otherwise specified, and distilled or demineralized water or
water of equivalent purity.

The reagents shall leave no appreciable residue when the determination is carried out by the method specified (see
9.2.2),

5.1 Ammonia solution, containing a mass fraction of NH30f approximately 25 Y. (p20 = 910 g/1).

NOTE If ammonia solution of this concentration is not available, a more concentrated solution of known concentration may
t?e used (see 9.4.2).

5.2 Ethanol (C2H50H), or ethanol denatured by methanol, containing a volume fraction of ethanol of at least 94 ?4..

(See A.5.)

5.3 Congo red solution

Dissolve 1 g of Congo red in water in a 100 ml one-mark volumetric flask (6.14). Dilute to the mark with water.

NOTE The use of this solution, which allows the interface between the solvent and aqueous layers to be seen more
clearly, is optional (see 9.4.3). Other aqueous colour solutions may be used provided that they do not affect the result of the
determination.

5:4 Diethyl ether (CzH@CzHs), free from peroxides (see A.3), containing no more than 2 mg/kg of antioxidants,
and complying with the requirements for the blank test (see 9.2.2, A.1 and A.4).

NOTE The use of diethyl ether could lead to hazardous situations. Due to expected changes in safety regulations studies
are ongoing to replace diethyl ether by another reagent provided that it does not affect the end result of the determination.

5.5 Light petroleum, with any boiling range between 30 “C and 60 “C or, as equivalent, pentane (CH3[CH2]SCHS)
with a boiling point of 36 “C and complying with the requirements for the blank test (see 9.2.2, A.1 and A.4).

NOTE The use of pentane is recommended because of its higher purity and constant quality

5.6 Mixed solvent

Shortly before use, mix equal volumes of diethyl ether (5.4) and light petroleum (5.5).

6 Apparatus

WARNING — Since the determination involves the use of volatile flammable solvents, all electrical
apparatus employed shall comply with legislation relating to the hazards in using such solvents.

Usual laboratory equipment and, in particular, the following.

6.1 Analytical balance, capable of weighing to the nearest 1 mg, with a readability of 0,1 mg.

6.2 Centrifuge, capable of holding the fat-extraction flasks or tubes (6.6) and capable of spinning at a rotational
frequency of 500 rein-l to 600 rein-l to produce a radial acceleration of 80 g to 90 g at the outer end of the flasks or
tubes.

NOTE The use of the centrifuge is optional but recommended (see 9.4.6).

6.3 Distillation or evaporation apparatus, for distilling the solvents and ethanol from the boiling or conical flasks,
or evaporating from beakers and dishes (see 9.4.13) at a temperature not exceeding 100 ‘C.

6.4 Drying oven, electrically heated, with ventilation port(s) fully open, capable of being maintained at a
temperature of 102 “C f 2 ‘C throughout its working space.

The oven shall be fitted with a suitable thermometer.

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 IS 11762: “2005
1s01737:1999

‘6.5 Water baths, capable of being maintained at a temperature of between 30 ‘C and 40 “C, and 40 ‘C and 60 “C.

6.6 Mojonnier-type fat-extraction flasks, as specified in ISO 3889.

NOTE It is also possible to use fat-extraction tubes, with siphon or wash-bottle fittings, but then the procedure is different.
The alternative procedure is given in annex B.

The fat-extraction flasks shall be provided with good quality bark corks or stoppers of other material [e.g. silicone
rubber or polytetrafluoroethy lene (PTFE)] unaffected by the reagents used. Bark corks shall be extracted with the
diethyl ether (5.4), kept in water at a temperature of 60 “C or more for at least 15 rein, and shall then be allowed to
cool in the water so that they are saturated when used.

6.7 Rack, for holding the fat-extraction flasks (or tubes) (6.6).

6.8 Wash bottle, suitable-for use with the mixed solvent (5.6).

A plastics wash bottle shall not be used.

6.9 Fat-collecting vessels, such as boiling flasks (flat-bottomed), of capacities 125 ml to 250 ml, conical flasks, of
capacity 250 ml, or metal dishes.

If metal dishes are used, they shall be of stainless steel, flat-bottomed with a diameter of 80 mm to 100 mm and a
height of approximately 50 mm.

6.10 Boiling aids, fat-free, of non-porous porcelain or silicon carbide (optional when metal dishes are used),

6.11 Measuring cylinders, of capacities 5 ml and 25 ml.

6.12 Pipettes, graduated, of capacity 10 ml.

6.13 Tongs, made of metal, for holding flasks, beakers or dishes.

6.14 Volumetric flask, one-mark, of capacity 100 ml.

7 Sampling

Sampling is not part of the method specified in this International Standard. A recommended sampling method is
given in ISO 707.

It is important that the Iaboratoty receive a sample which is truly representative and has not been damaged or
changed during transport or storage.

Store the samples at a temperature of between 2 “C and 6 ‘C from the time of sampling to the time of commencing
the procedure. In the case of samples in sealed cans, store the closed cans at a temperature below 20 “C.

8 Preparation of test sample

8.1 Evaporated milk

Shake and invert the sample container. Open the sample container and pour the sample slowly into a second
sample container (provided with an airtight lid). Mix by repeated transfer, taking care to incorporate in the sample
any fat or other constituent adhering to the wall and ends of the first container. Finally, transfer the product as
completely as possible to the second container.

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IS 11762:2005
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If necessary in the case of samples in sealed cans, condition the unopened container in the water bath (6.5) set at a
temperature of between 40 ‘C and 60 ‘C. Remove and shake the can vigorously every 15 min. After 2 h, remove
the can and allow it to cool to room temperature.

Remove the lid entirely and thoroughly mix the sample by stirring with a spoon or spatula. (If fat separates, do not
test the sample.)

8.2 Sweetened condensed milk

Open the sample container and mix thoroughly with a spoon or spatula. Use an up-and-down rotary movement in
such a way that the top layers and the content of the lower corners of the container are moved and mixed. Take
care to incorporate in the sample any milk adhering to the wall and ends of the container. Transfer the sample as
completely as possible to a second sample container (provided with an airtight lid). Close the second container.

If necessary, in the case of samples in sealed cans, cmndition the unopened can in the water bath (6.5) at a
temperature of between 30 “C and 40 “C. Open the can, scrape out all milk adhering to the interior of the can,
transfer to a dish large enough to permit stirring thoroughly and mix until the whole mass is homogeneous.

In the case of a sample in a collapsible tube, open the tube and transk?r the contents to a jar. Then cut open the
tube and scrape out all material adhering to the interior and add to the contents of the jar.

9 Procedure

NOTE An alternative procedure using fat-extraction tubes with siphon or wash-bottle fittings (see note in 6.6) is given in
annex B.

9.1 Test portion

Mix the test sample (clause 8), by stirring in the case of sweetened condensed milk, or by gently inverting the bottle
three or four times in the case of evaporated milk. Immediately weigh to the nearest 1 mg, directly or by difference,
4 g or 5 g of the test sample of evaporated milk, or 2,0 g to 2,5 g of the test sample of sweetened condensed milk in
a fat-extraction flask (6.6).

Transfer the test portion.as completely as possible into the lower (small) bulb of the fat-extraction flask.

9.2 Blank tests

9.2.1 Blank test for method

Carry out a blank test simultaneously with the determination using the same procedure and same reagents, but
replacing the test portion by 10 ml of water (see A.2).

If the value obtained in the blank test regularly exceeds 1,0 mg, check the reagents if this has not been recently
done (9.2.2). Corrections of more than 2,5 mg should be mentioned in the test report.

9.2.2 Blank test for reagents

To test the quality of the reagents, carry out a blank test as specified in 9.2.1. Additionally use an empty fat-
collecting vessel, prepared as specified in 9.3, for mass control purposes. The reagents shall leave no residue
greater than 1,0 mg (see A.1 ).

If the residue of the complete reagent blank test is greater than 1,0 mg, determine the residue of the solvents
separately by distilling 100 ml of the diethyl ether (5.4) and light petroleum (5.5), respectively. Use an empty fat-
collecting vessel, prepared for control purposes as described above, to obtain the real mass of residue which shall
not exceed 1,0 mg.

Very occasionally, the solvents may contain volatile matter which is strongly retained in fat. If there are indications of
the presence of such substances, carry out blank tests on all the reagents and for each solvent using a fat-

4
 IS 11762:2005
1s01737:1999

collecting vessel with about 1 g of anhydrous butterfat. If necessary, redistil solvents in the presence of 1 g of
anhydrous butterfat per 100 ml of solvent. Use the solvents only shortly after the redistillation.

. Replace unsatisfactory reagents, solvents, or redistil solvents.

9.3 Preparation of fat-collecting vessel

Dry a fat-collecting vessel (6.9) with a few boiling aids (6.10) in the oven (6.4) set at 102 ‘C for 1 h.

NOTE 1 Boiling aids are desirable to promote gentle boiling during the -subsequent removal of solvents, especially when
using glass fat-collecting vessels; their use is optional with metal dishes.

Protect the fat-collecting vessel from dust and allow it to cool to the temperature of the weighing room (glass fat-
collecting vessel for at least 1 h, metal dish for at least 30 rein).

NOTE 2 To avoid insufficient cooling or unduly long cooling times, the fat-collecting vessel should not be placed in a
desiccator.

Use tongs to place the fat-collecting vessel on the balance. Weigh the fat-collecting vessel to the nearest 1,0 mg.

NOTE 3 Tongs should preferably be used to avoid, in particular, temperature variations.

9.4 Determination

9.4.1 Carry outthe determination without delay.

Add water at a temperature of about 50 “C to the test portion in the fat-extraction flask (9.1) to obtain a total volume
of 10 ml to 11 ml. Use the water to wash the test portion on to the bottom of the flask. Shake gently with slight
warming at about 50 “C in the waterbath (6.5) until the test portion is completely dispersed. Cool in running water to
room temperature.

9.4.2 Add 2 ml of ammonia solution (5.1) to the dispersed test portion in the fat-extraction flask (9.4.1), or an
equivalent volume of a more concentrated ammonia solution (see note in 5.1). Mix thoroughly with the test portion in
the small bulb of the fat-extraction flask.

9.4.3 Add 10 ml of ethanol (5.2). Mix gently but thoroughly by allowing the contents of the fat-extraction flask to
flow backwards and forwards between the small and large bulb. Avoid bringing the liquid too near to the neck of the
flask. If desired, add 2 drops of the Congo red solution (5.3).

9.4.4 Add 25 ml of diethyl ether (5.4). Close the fat-extraction flask with a cork saturated with water or with a
stopper of other material wetted with water (6.6). Shake the flask vigorously, but not excessively, for 1 min to avoid
the formation of persistent emulsions.

While shaking, keep the fat-extraction flask in a horizontal position with the small bulb extending upwards,
periodically allowing the liquid to run from the large bulb into the small bulb. If necessary, cool the flask in running
water to about room temperature. Carefully remove the cork or stopper and rinse it-and the neck of the flask with a
little mixed solvent (5.6). Use the wash bottle (6.8) so that the rinsings run into the flask.

9.4.5 Add 25 ml of the light petroleum (5.5). Close the fat-extraction flask with the rewetted (by dipping into water)
cork or stopper. Shake the flask gently again for 30 s as described in 9.4.3. Proceed with shaking as described in
9.4.4.

9.4.6 Centrifuge the closed fat-extraction flask for between 1 min and 5 min at a radial acceleration of 80 g to 90 g.
If a centrifuge is not available, allow the closed flask to stand in the rack (6.7) for at least 30 min until the
supernatant layer is clear and distinctly separated from the aqueous layer. If necessary, cool the flask in running
water to room temperature.

9.4.7 Carefully remove the cork or stopper and rinse it and the inside of the neck of the fat-extraction flaskwith a
little mixed solvent (5.6). Use the wash bottle (6.8) so that the rinsings run into the flask. If the interface is below the

5
IS 11762: 2CI05
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bottom of the Stem of the flask, raise it slightly above this level by gently adding water down the side of the flask
(see Figure 1) to facilitate the decanting of solvent.

NOTE In Figures 1 and 2, one of the three types of fat-extraction flasks as specified in ISO 3889 has been chosen, but this
does not imply any preference over other types.

9.4.8 Hold the fat-extraction flask by the small bulb and carefully decant as much as possible of the supernatant
layer into the prepared fat-collecting vessel (see 9.3) containing a few boiling aids (6.10) in the case of a boiling or
conical flask (optional with metal dishes). Avoid decanting any of the aqueous layer (see Figure 2).

9.4.9 Rinse the outside of the neck of the fat-extraction flask with a little mixed solvent (5.6). Collect the rinsings in
the fat-collecting vessel. Take care that the mixed solvent does not spread over the outside of the fat-extraction
flask. If desired, remove the solvent or a part of it from the fat-collecting vessel by distillation or evaporation as
described in 9.4.13.

9.4.10 Add 5 ml of ethanol (5.2) to the contents of the fat-extraction flask. Using the ethanol, rinse the inside of the
neck of the flask and mix as described in 9.4.3.

L.___/l

—— ——
— ——
——

5 —— ——
3

Key Key
1 Solvent 1 At second and third extraction
2 At second and third extraction 2 At first extraction
3 At first extraction 3 Aqueous layer
4 Interface 4 Interface
,6 Aqueous layer

Figure 1 — Before decanting Figure 2 — After decanting

9.4.11 Carry out a second extraction by repeating the operations described in 9.4.4 to 9.4.8 inclusive. Instead of
25 ml, use only 15 ml of diethyl ether (5.4) and 15 ml of light petroleum (5.5). Using the diethyl ether, rinse the inside
of the neck of the fat-extraction flask too.

If necessary, raise the interface slightly to the middle of the stem of the flask by gently adding water down the side
of the flask (see Figure 1) to enable the final decanting of solvent to be as complete as possible (see Figure 2).

9.4.12 Carry out a third extraction without addition of ethanol by again repeating the operations described in 9.4.4
to 9.4.8 inclusive. Again, use only 15 ml of diethyl ether (5.4) and 15 ml of light petroleum (5.5). Using the diethyl
ether, rinse the inside of the neck of the fat-extraction flask again.

6
 IS 11762:2005
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If necessary, raise the interface slightly to the middle of the stem of the flask by gently adding water down the side
of the flask (see Figure 1) to enable the final decanting of solvent to be as complete as possible (see Figure 2).

NOTE The third extraction may be omitted for evaporated milk and sweetened condensed milk with a fat content of less
than 1 Y..

9.4.13 Remove the solvents (including the ethanol) as completely as possible from the fat-collecting vessel, by
distillation if using a boiling or conical flask, or by evaporation if using a beaker or dish (6.3). Rinse the inside of the
neck of the conical flask with a little mixed solvent (5.6) before commencing the distillation.

9.4.14 Heat the fat-collecting vessel, with the boiling or conical flask placed on its side to allow solvent vapour to
escape, for 1 h in the drying oven (6.4) set at 102 ‘C. Remove the fat-collecting vessel from the oven and
immediately verify whether or not the fat is clear. If the fat is not clear, fatty extraneous matter is presumed to be
present and the whole procedure shall be repeated. If the fat is clear, protect the fat-collecting vessel from dust and
allow the fat-collecting vessel to cool (preferably not in a desiccator) to the temperature of the weighing room
(a glass fat-collecting vessel for at least 1 h, a metal dish for at least 30 rein).

Do not wipe the fat-collecting vessel immediately before weighing. Use tongs to place the fat-collecting vessel on
the balance. Weigh the fat-collecting vessel to the nearest 1,0 mg.

9.4.15 Heat the fat-collecting vessel, with the boiling or conical flask placed on its side to allow solvent vapour to
escape, for a further 30 min in the drying oven (6.4) set at 102 “C. Cool and reweigh as described in 9.4.14. If
necessary, repeat the heating and weighing procedures until the mass of the fat-collecting vessel decreases by
1,0 mg or less, or increases between two successive weighings. Record the minimum mass as the mass of the fat-
collecting vessel and extracted matter.

10 Calculation and expression of results

10.1 Calculation

Calculate the fat content of the sample using the following equation:

,t,f = (m, - rrl*) - (ms - m~) ~ ,00 ~,.

mo

where

Wf is the mass fraction of fat in the sample, in percent;

m. is the mass of the test portion (9.1), in grams;

ml is the mass of the fat-collecting vessel and extracted matter, determined in 9.4.15, in grams;

m2 is the mass of the prepared fat-collecting vessel (9.3), in grams;

m3 is the mass of the ‘fat-collecting vessel used in the blank test (9.2) and any extracted matter determined in
9.4.15, in grams;

m4 is the mass of the fat-collecting vessel (9.3) used in the blank test (9.2), in grams.

1-0.2 Expression of results

Round the result to two decimal places.

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 IS 11762:2005
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11 Precision

> 41.1 Interlaboratory test

Details of an interlaboratory test in accordance with ISO 5725’ J on the precision of the method have been published
(see reference [5]).

The values for repeatability and reproducibility limits are expressed for the 95 ?4. probability level and may not be
applicable to concentration ranges and matrices other than those given.

11.2 Repeatability .

The absolute difference between two independent single test results, obtained using the same method on identical
test material in the same laboratory by the same operator using the same equipment within a short interval of time,
will in not more than 5 % of cases be greater than a mass fraction of fat of:

— 0,02 % for products with a fat content <1 %;

— 0,03 O/. for products with a fat content from >1 % to 4 O/.;

— 0,04 Y. for products with a fat content from >4 ?!. to <10 Y.;

— 0,50 O/. of the proportion of fat in the test sample for products with a fat content = 107..

11.3 Reproducibility

The absolute difference between two independent single test results, obtained using the same method on identical
test material in different laboratories with different operators using different equipment, will in not more than 5 YO of
cases be greater than a mass fraction of fat of:

— 0,03 % for products with a fat content <1 %;

— 0,04 ‘!. for products with a fat content from >1 ‘/. to 4%;

— 0,06 Y. for products with a fat content from >4 % to <10 Y.;

— 1 7. of the proportion of fat in the test sample for products with a fat content >10 %.

12 Test report

The test report shall specify:

— all information necessary for the complete identification of the sample;

— the sampling method used, if known;

— the test method used, together with reference to this International Standard;

— all operating details not specified in this International Standard, or regarded as optional, together with details of
any incidents which may have influenced the test result(s);

— the corrections made, if a value of more than 2,5 mg is obtained in the blank test for the method;

— the test result(s) obtained; or if the repeatability has been checked, the final qwated result obtained.

1J ISO 5725:1986 (now withdrawn) was used to obtain the precision data.

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 [S 11762:2005
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Annex A
(informative)

Notes on procedures

A.1 Blank test to check the reagents (see 9.2.2)

In this blank test, a fat-collecting vessel for mass control purposes has to be used in order that changes in the
atmospheric condition of the balance room or temperature effects of the fat-collecting vessel will not falsely suggest
the presence or absence of non-volatile matter in the extract of the reagents. This fat-collecting vessel may be used
as a counterweight vessel in the case of a two-pan balance. Otherwise deviations of the apparent mass (tn3 - m4
in 10.1) of the fat-collecting vessel for control purposes should be considered when checking the mass of the fat-
collecting vessel used for the blank test. Hence, the change in apparent mass of the fat-collecting vessel, corrected
for the apparent change in mass of the fat-collecting vessel for control purposes, shall show no increase in mass
greater than 1,0 mg.

Very occasionally, the solvents may contain volatile matter which is strongly retained in fat. If there are indications of
the presence of such substances, carry out blank tests on all the reagents and for each solvent using a fat-
collecting vessel with about 1 g of anhydrous butterfat. If necessary, redistil solvents in the -presence of 1 g of
anhydrous butterfat per 100 ml of solvent. Use the solvents only shortly after redistillation.

A.2 Blank test carried out simultaneously with the determination (see 9.2.1)

The value obtained in the blank test, carried out simultaneously with the determination, enables the apparent mass
of substances extracted from a test portion (nIl – mz) to be corrected for the presence of any non-volatile matter
derived from the reagents and also for any change of atmospheric conditions of the balance room and some
temperature difference between the fat-collecting vessel and the balance room at the two weighings (9.4.15 and
93).

Under favorable conditions (low value in the blank test on reagents, equable temperature of the balance room,
sufficient cooling time for fat-collecting vessel), the value will usually be less than 1,0 mg and can then be neglected
in the calculation in the case of routine determinations. Slightly higher values (positive-and negative) up to 2,5 mg
are also often encountered. After correction for these values, the results will still be accurate. When corrections of
more than 2,5 mg are applied, it should be mentioned in the test report (clause 12).

If the value obtained in this blank test regularly exceeds 1,0 mg, the reagents should be checked, if no recent check
has been made. Any impure reagent or reagents traced should be replaced or purified (see 9.2.2 and A.1 ).

A.3 Test for peroxides

To test for peroxides, add 1 ml of a freshly prepared 100 g/1 potassium iodide solution to 10 ml of the diethyl ether in
a small glass-stoppered cylinder which has been previously rinsed with the ether. Shake the cylinder and allow to
stand for 1 min. No yellow colour should be observed in the diethyl ether layer.

Other suitable methods of testing for peroxides maybe used.

To ensure that the diethyl ether is free, and is maintained free, from peroxides, treat the diethyl ether at least 3 days
before it is to be used as follows.
 IS 11762:2005
1s01737:1999

Cut zinc foil into strips that will reach at least half-way up the bottle containing the diethyl ether, using approximately
80 cm2 of foil per Iitre of diethyl ether.

. Before use, completely immerse the strips of foil for 1 min in a solution containing 10 g of copper(n) sulfate
pentahydrate (CUS04.5H20) and 2 ml/1 of concentrated [987. (rTtaSS fraction)] sulfuricacid.

Wash the strips gently but thoroughly with water, place the wet copper-plated strips in the bottle containing the
diethyl ether, and leave the strips in the bottle.

Other methods may be used provided that they do-not affect the result of the determination.

A.4 Diethyl ether containing antioxidants

Diethyl ether containing about 1 mg of antioxidants per kilogram is available in some countries, especially for fat
determinations. This content does not exclude its use for reference purposes.

In other countries, diethyl ether with higher antioxidant contents, for example up to 7 mg/kg, is available. Such ether
should only -be used for routine determinations with an obligatory blank test carried out simultaneously with the
determination(s) to correct for systematic errors due to the antioxidant residue. For reference purposes, such diethyl
ether shall always be distilled before use.

A.5 Ethanol

Ethanol denatured otherwise than by the addition of methanol may be used provided that the denaturant does not
affect the result of the determination.

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 IS 11762:2005
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Annex B
(informative)
.

Alternative procedure using fat-extraction tubes with siphon or wash-bottle

fittings

B.1 General

If fat-extraction tubes with siphon or wash-bottle fittings are to be used, use the procedure specified in this annex.
The tubes shall be provided with good quality bark corks or stoppers as specified for the flasks in 6.6 (see
Figure B.1 as an example).

B.2 Procedure

B.2.1 Preparation of test sample

See clause 8.

B.2.2 Test portion

Proceed as specified in 9:1 but using the fat-extraction tubes (see note in 6.6 and Figure B. 1). The test portion shall
be delivered as completely as possible to the bottom of the fat-extraction tube.

B.2.3 Blank test

See 9.2 and A.2.

B.2.4 Preparation of fat-collecting vessel

See 9.3.

B.2.5 Determination

B.2.5.1 Carry out the determination without delay

Add water at about 50 “C to the test portion in the fat-extraction tube (B.2.2) to obtain a total volume of 10 ml to
11 ml. Use the water to wash the test portion onto the bottom of the tube. Shake gently with slight warming at about
50 ‘C in the waterbath (6.5) until the test portion is completely dispersed. Cool in running water to room
temperature.

B.2.5.2 Add 2 ml of ammonia solution (5.1) to the dispersed test portion in the fat-extraction tube (B.2.5.1 ), or an
equivalent volume of a more concentrated ammonia solution (see note in 5.1). Mix thoroughly with the pretreated
test portion at the bottom of the fat-extraction tube.

B.2.5.3 Add 10 ml of ethanol (5.2). Mix gently but thoroughly at the bottom of the fat-extraction tube. If desired, add
2 drops of the Congo red solution (5.3).

B.2.5.4 Add 25 ml of .diethyl ether (5.4). Close the fat-extraction tube with a cork saturated with water or with a
stopper of other materiil wetted with water (6.6). Shake the tube vigorously, but not excessively, with repeated
inversions for 1 rein, to avoid the formation of persistent emulsions. If necessary, cool the tube in running water.
Carefully remove the cork or stopper and rinse it and the neck of the tube with a little mixed solvent (5.6). Use the
wash bottle (6.8) so that the rinsings run into the tube.

11
 IS 11762:2005
1s01737:1999

6.2.5.5 Add25ml of thelight petroleum (5.5) .Closethe fat-extraction tubewith thewwetied (bydipping inwater)
cork or stopper. Shake the tube gently for 30s, as described in B.2.5.4.
. 6.2.5.6 Centrifuge the closed fat-extraction tube for 1 min to 5 min at a radial acceleration of 80 g to 90 g. If a
centrifuge is not available, allow the closed tube to stand in the rack (6.7) for at least 30 min until the supematant
layer is clear and distinctly separated from the aqueous layer. If necessafy, cool the tube in running ”water to room
temperature.

6.2.5.7 Carefully remove the cork or stopper and rinse it and the neck of the fat-extraction tube with a little mixed
solvent (5.6). Use the wash bottle (6.8) so that the rinsings run into the tube.

6.2.5.8 Insert a siphon fitting or a wash-bottle fitting into the fat-extraction tube. Push down the long inner limb of
the fitting until the inlet is approximately 4 mm above the interface between the layers. The inner limb of the fitting
shall be parallel to the axis of the fat-extraction tube.

Carefully transfer the supernatant layer out of the fat-extraction tube into the fat-collecting vessel (see 9.3)
containing a few boiling aids (6.1 O) in the case of boiling or conical flasks (optional with metal dishes). Avoid the
transfer of any of the aqueous layer. Rinse the outlet of the fitting with a little mixed solvent, collecting the rinsings in
the fat-collecting vessel.

NOTE The supernatant layer can be transferred out of the fat extraction-tube by using, for example, a rubber bulb attached
to the shoti.stem to apply pressure.

.6.2.5.9 Loosen the fittingfrom the neck of the fat-extraction tube. Slightly raise the fitting and rinse the lower part
of its long inner limb with a little mixed solvent (5.6). Lower and re-inserl the fitting and transfer the rinsings to the
fat-collecting vessel.

Rinse the outlet of the fitting with a little mixed solvent again, collecting the rinsings in the fat-collecting vessel. If
desired, remove the solvent or a part of it from the fat-collecting vessel by distillation or evaporation as described in
9.4.13.

6.2.5.10 Again loosen the fitting from the neck. Slightly raise the fitting and add 5 ml of ethanol to the contents of
the fat-extraction tube. Use the ethanol to rinse the long inner limb of the fitting. Mix as described in B.2.5.3,

6.2.5.11 Carry out a second extraction by repeating the operations described in B.2.5.4 to B.2.5.1 O. Instead of
25 ml, use only 15 ml of diethyl ether (5.4) and 15 ml of light petroieum (5.5). Using the diethyl ether, rinse the long
inner limb of the fitting during the removal of the fitting from the fat-extraction tube after the previous extraction.

6:2.5.12 Carry out a third extractionwithout the addition of ethanol by again repeating the operations described in
B.2.5.4 to B.2.5.1O. Again, use only 15 ml of diethyl ether and 15 ml of light petroleum. Using the diethyl ether, rinse
the long inner limb of the fitting as described in B.2.5.11.

NOTE The third extraction may be omitted for evaporated milk and sweetened condensed milk with a fat content of less
than 1 7..

6:2.5.13 Proceed as described in 9.4.13 to 9.4.15.

12
 IS 11762:2005
1s01737:1999

Dimensions in millimetres

\e

‘“o
\ -r

— —
—l—

m
+1 —2—
0
0

0 int, 26 ~1 0 int. 26 tl
I ,

a) With siphon fitting b) With wash-bottle fitting

Key

1 Capacity to this level, with fittings removed, 105 ml + 5 ml.

2 Wall thickness 1,5 mm ~ 0,5 mm.

Figure B.1 — Examples of fat-extraction tubes

13
 IS 11762:2005
1s0 1737:1999

Bibliography

[1] ISO 707, Milk and mi/k products — Guidance on sampling.

[2] ISO 5725:1986, Precision of test methods — Determination of repeatability and reproducibility for a standard
test method by inter-laboratory tests.

[3] ISO 5725-1:1994, Accuracy (trueness and precision) of measurement methods and results — Part 1: General
principles and definitions.

[4] ISO 5725-2:1994, Accuracy (trueness and precision) of measurement methods and results — Pati.2: A basic
method for the determination of repeatability and reproducibility of a standard measurement method.

[5] International Dairy Federation. Interlaboratory Collaborative Studies, Second series. Bull. /nt. Dairy Fed.,
No. 235, 1988.

14
 #

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