BS EN ISO 660:2009

INTERNATIONAL STANDARD ISO 660:2009(E)

Animal and vegetable fats and oils - Determination of acid

value and acidity

1 Scope

This International Standard specifies three methods (two titrimetric and one potentiometric) for the
determination of the acidity in animal and vegetable fats and oils, hereinafter referred to as fats. The acidity is
expressed preferably as acid value, or alternatively as acidity calculated conventionally.

This International Standard is applicable to refined and crude vegetable or animal fats and oils, soap stock
fatty acids or technical fatty acids. The methods are not applicable to waxes.

Since the methods are completely non-specific, they cannot be used to differentiate between mineral acids,
free fatty acids, and other organic acids. The acid value, therefore, also includes any mineral acids that may
be present.

2 Normative references

The following referenced documents are indispensable for the application of this document. For dated
references, only the edition cited applies. For undated references, the latest edition of the referenced
document applies.

ISO 661, Animal and vegetable fats and oils - Preparation of test sample

ISO 3696, Water for analytical laboratory use - Specification and test methods

3 Terms and definitions

For the purposes of this document, the following terms and definitions apply.

3.1
acid value
number of milligrams of potassium hydroxide required to neutralize the free fatty acids present in 1 g of fat,
when determined in accordance with the procedure specified in this International Standard

NOTE The acid value is expressed in milligrams per gram.

3.2
acidity
content of free fatty acids determined according to the procedure specified in this International Standard

NOTE The acidity is expressed as a percentage by mass. If the result of the determination is reported as acidity
without further explanation, this is, by convention, the acidity based on the oleic acid content.

© ISO 2009 - All rights reserved 1

BS EN ISO 660:2009
ISO 660:2009(E)

4 Principle

The sample is dissolved in a suitable solvent mixture, and the acids present are titrated with an ethanolic or
methanolic solution of potassium or sodium hydroxide.

The methods specified in 9.1 and 9.2 are reference methods.

5 Reagents

WARNING - Attention is drawn to the regulations which specify the handling of hazardous
substances. Technical, organizational and personal safety measures shall be followed.

Use only reagents of recognized analytical grade, unless otherwise specified.

5.1 Solvent A for solvent mixture (5.3): ethanol, volume fraction, rp::::, 96 %.

As a replacement, propan-Z-ol, volume fraction, rp::::, 99 %, can be used.

5.2 Solvent 8 for solvent mixture (5.3): diethyl ether, peroxide-free.

As a replacement, tert-butyl methyl ether, light petroleum (boiling range 40 °C to 60 °C) or toluene can be
used.

WARNING - Diethyl ether is very flammable and may form explosive peroxides. Use with great
caution.

5.3 Solvent mixture, mix equal volumes of solvent A and B, (e.g. rpA = 50 mll100 ml and
rpB = 50 ml/100 ml).
For hard or animal fats, a solvent mixture of one volume of solvent A (e.g. 25 ml) and three volumes of tert-
butyl methyl ether or toluene (e.g. 75 ml) is recommended.

Neutralize, just before use, by adding potassium hydroxide solution in the presence of 0,3 ml of the
phenolphthalein solution per 100 ml of solvent mixture.

For the titration with aqueous KOH, the solvent propan-Z-ol can be used.

5.4 Ethanol or methanol, of minimum volume fraction, rp = 95 %.

5.5 Sodium hydroxide or potassium hydroxide, ethanolic or methanolic standard volumetric solutions,
amount of substance concentration c(NaOH) or c(KOH) = 0,1 molll and 0,5 rnol/l. The concentration shall be
checked with a standard volumetric HCI solution.

NOTE The ethanolic/methanolic sodium/potassium hydroxide solution may be replaced by an aqueous

sodium/potassium hydroxide solution, but only if the volume of water introduced does not lead to phase separation.

5.6 Phenolphthalein, solution in ethanol, mass concentration, p = 1 g/100 rnl.

5.7 Thymolphthalein, solution in ethanol, mass concentration, p = 2 g/100 rnl.

5.8 Alkali blue 68, solution in ethanol, mass concentration, p = 2 g/100 rnl.
For dark-coloured fats, alkali blue or thymolphthalein shall be used.

5.9 Water in accordance with ISO 3696, grade 3.

2 © ISO 2009 - All rights reserved

 BS EN ISO 660:2009
ISO 660:2009(E)

6 Apparatus

Usual laboratory equipment and, in particular, the following.

6.1 Burette, capacity 10 ml, graduated in 0,02 ml, ISO 385(1)class A.

6.2 Burette, capacity 25 ml, graduated in 0,05 ml, ISO 385(1)class A.

6.3 Analytical balance, capable of being read to the nearest 0,001 g.

6.4 Automatic titration apparatus (based on potentiometric electrode) or potentiometer.

6.5 Combined pH electrode for non-aqueous acid/base titrations.

6.6 Graduated volumetric flasks, volume 1 000 ml, ISO 1042(2)class A.

7 Sampling

A representative sample should have been sent to the laboratory. It should not have been damaged or
changed during transport or storage.

Sampling is not part of the method specified in this International Standard. A recommended sampling method
is given in ISO 5555 (3).

8 Preparationof test sample

Prepare the test sample in accordance with ISO 661, except that if the sample contains volatile fatty acids, the
test sample shall not be heated and filtered.

9 Procedure

9.1 Cold solvent method using indicator (Referencemethod)

9.1.1 Depending on the expected magnitude of the acid value, select the test portion mass and alkali
concentration from Table 1.

9.1.2 According to Table 1 weigh the test portion into a 250 ml conical flask.

9.1.3 Add 50 ml to 100 ml of the neutralized solvent mixture (5.3) and dissolve the test portion if necessary
with gentle warming.

For high melting point samples, use an ethanol-toluene mixture.

9.1.4 After the addition of an indicator (5.6, 5.7 or 5.8), titrate with constant swirling using standard
potassium hydroxide solution (5.5). The endpoint of the titration is reached when the addition of a single drop
of alkali produces a slight but definite colour change persisting for at least 15 s.

© ISO 2009 - All rights reserved 3

BS EN ISO 660:2009
ISO 660:2009(E)

Table 1 - Test portion masses and alkali concentrations

Accuracy of
Product group Acid value Mass of test Concentration of
weighing of the
(examples) approx. portion KOH
test portion
g molll g

Refined vegetable oils

Animal fats
o to 1 20 0,1 0,05

Crude vegetable oils 1 to 4 10 0,1 0,02

Technical grade animal fats 4 to 15 2,5 0,1 0,01
0,5 0,1
Soap stock fatty acids 15 to 75 0,001
3,0 0,5
0,2 0,1
Technical fatty acids > 75 0,001
1,0 0,5

9.2 Cold solvent method using potentiometric titration (Referencemethod)

9.2.1 According to Table 1, weigh the test portion into a 150 ml beaker.

9.2.2 Add 50 ml to 100 ml of the neutralized solvent mixture (5.3) and dissolve the sample, if necessary with
gentle warming.

For high melting point samples, use an ethanol-toluene mixture.

9.2.3 Introduce the combined electrode in the solvent mixture and connect it with the automatic titration
apparatus.

9.2.4 Start the stirrer for at least 30 s and then titrate with constant swirling using standard potassium
hydroxide solution (5.5).

9.2.5 As soon as the equivalence point is reached, record the amount of standard solution used.

9.3 Hot ethanol method using indicator

9.3.1 Under the conditions specified in this method, short-chain fatty acids, if present, are volatile.

9.3.2 Weigh into a flask a sufficient mass of the test sample as shown in Table 1, according to the colour
and expected acid value.

9.3.3 Heat to boiling 50 ml of the ethanol containing 0,5 ml of the phenolphthalein indicator in a second
flask. While the temperature of the ethanol is still above 70°C, neutralize it carefully with a solution of 0,1 molll
sodium or potassium hydroxide.

The endpoint of the titration is reached when the addition of a single drop of alkali produces a slight but
definite colour change persisting for at least 15 s.

Larger volumes of ethanol and indicator may be necessary for dark-coloured fats. Moreover, for dark-coloured
fats, alkali blue or thymolphthalein shall be used.

9.3.4 Add the neutralized ethanol to the test portion in the first flask and mix thoroughly. Bring the contents
to the boil and titrate with the sodium or potassium hydroxide solution, agitating the flask contents vigorously
during the titration.

4 © ISO 2009 - All rights reserved

 BS EN ISO 660:2009
ISO 660:2009(E)

10 Calculation

The acid value, WAY' or the free fatty acid content, wFFA' is reported as follows:

a) to two decimal places for values between 0 up to and including 1;

b) to one decimal place for values between 1 up to and including 100;

c) as a whole number for values> 100.

In addition to the following calculations, the approximate free fatty acid content (acidity) is calculated from:

WFFA = 0,5 x wAY

10.1 Acid value

The acid value, wAY' expressed as a mass fraction, is equal to

56,1 xcV
wAY =
m

where

c is the exact concentration, in moles per litre, of the standard volumetric sodium or potassium
hydroxide solution used;

V is the volume, in millilitres, of standard volumetric sodium or potassium hydroxide solution used;

m is the mass, in grams, of the test portion.

10.2 Acidity or free fatty acid content

The acidity or free fatty acid content, wFFA' expressed as a percentage mass fraction, and according to fat
type (see Table 2), is equal to

V c Mx 100
wFFA = -1-0-0-0-x-m-

where

V is the volume, in millilitres, of the standard volumetric sodium or potassium hydroxide solution used;

c is the concentration, in moles per litre, of the standard volumetric sodium or potassium hydroxide
solution used;

M is the molar mass, in grams per mole, of the acid chosen for expression of the result (see Table 2)
according to the fat type;

m is the mass, in grams, of the test portion.

© ISO 2009 - All rights reserved 5

BS EN ISO 660:2009
ISO 660:2009(E)

Table 2 - Choice of fatty acid for expression of acidity

Molar mass
Type of fat Expressed as
g/mol

Coconut oil
Palm kernel oil Lauric acid 200
and similar oils

Palm oil Palmitic acid 256

Oils from certain
Cruciferae a
Erucic acid 338

All other fats Oleic acid 282

a In the case of rapeseed oil having a maximum erucic acid content of 5 %, the
acidity shall be expressed as oleic acid.

NOTE If the result is reported simply as "acidity", without further definition,

this is, by convention, expressed as oleic acid. If the sample contains mineral
acids, these are, by convention, determined as fatty acids.

11 Precision
Details of interlaboratory tests are given in Annex A. The values derived from these tests may not be
applicable to concentration ranges and matrices other than those given.

11.1 Repeatability

The absolute difference between two independent single test results, obtained with 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, shall in not more than 5 % of cases exceed the values given in Tables A.1 to A.3.

11.2 Reproducibility

The absolute difference between two single test results, obtained with the same method on identical test
material in different laboratories by different operators using different equipment, shall in not more than 5 % of
cases exceed the values given in Tables A.1 to A.3.

12 Test report

The test report shall contain at least the following information:

a) all information necessary for the complete identification of the sample;

b) a reference to this International Standard;

c) the result obtained, indicating clearly the method of expression used;

d) any operating conditions not specified in this International Standard, or regarded as optional.

6 © ISO 2009 - All rights reserved

 BS EN ISO 660:2009
ISO 660:2009(E)

Annex A
(informative)

Results of interlaboratory tests

The precision of the method is the result of interlaboratory studies on an international basis. The results are
given in Table A.1 for the reference methods in 9.1 and 9.2, and in Tables A.2 and A.3 for the hot ethanol
method (9.3).

A series of interlaboratory tests, carried out by a different number of laboratories, using the methods described
in 9.1 to 9.3, gave the statistical results [evaluated in accordance with ISO 5725: 1986[4] and ISO 5725 (ali
parts)[5]]given in Tables A.1 to A.3.

Table A.1 - Summary of statistical results (acid value, expressed as mg KOH/g fat)

Cold
Refined Crude Lampante
pressed Technical
Sample rapeseed Lard sunflower virgin olive
wheat germ fatty acids
oil seed oil oil
oil
Number of participating laboratories,
26 26 26 26 26 26
N
Number of laboratories retained after
25 24 26 24 23 24
eliminating outliers, n
Number of individual test results of
50 48 52 48 46 48
all laboratories on each sample, flz

Mean value, wAV, mg/g a 0,080 0,381 1,39 5,48 7,48 128,1

Repeatability standard deviation, s.,

0,003 0,006 0,04 0,07 0,08 0,6
mg/g a
Repeatability coefficient of variation,
3,6 1,7 2,6 1,2 1,1 0,4
CV(r}, %

Repeatability limit, r
0,008 0,018 0,10 0,19 0,23 1,6
(sr x 2,8), mg/g a

Reproducibility standard deviation,

0,018 0,019 0,05 0,15 0,40 2,6
mg/g a
SR,

Reproducibility coefficient of
22,2 5,0 3,6 2,7 5,3 2,1
variation, CV(R}, %

Reproducibility limit, R
0,049 0,053 0,14 0,41 1,12 7,4
(SR x 2,8), mg/g a

a The precisiondatafor the acidityas a percentageof freefattyacidscanbe calculatedby dividingthe correspondingvaluesfor the
acidvalueby 1,99.

© ISO2009- All rightsreserved 7

BS EN ISO 660:2009
ISO 660:2009(E)

Table A.2 - Summary of statistical results (acidity, expressed as a percentage mass fraction)

Extra virgin Lampante virgin Sunflower

Sample Olive oil Coconut oil
olive oil olive oil seed oil
Number of participating laboratories, N 39 28 28 37 23 25
Number of laboratories retained after
37 26 24 37 22 25
eliminating outliers, n
Number of individual test results of all
74 52 48 74 44 50
laboratories on each sample, nz

Mean value, wFFA' % mass fraction 0,343 3,80 19,55 0,604 0,830 1,49

Repeatability standard deviation, Sf' % 0,007 0,03 0,09 0,012 0,009 0,009
Repeatability coefficient of variation,
1,9 0,8 0,5 1,9 1,1 0,6
CV(r), %
Repeatability limit, r
0,018 0,07 0,26 0,033 0,025 0,025
(s, x 2,8), %

Reproducibility standard deviation, SR, % 0,019 0,12 0,60 0,035 0,027 0,027
Reproducibility coefficient of variation
5,5 3,2 3,1 5,8 3,3 1,8
CV(R), %

Reproducibility limit, R
0,053 0,33 1,67 0,098 0,075 0,075
(SR x 2,8), %

Table A.3 - Summary of statistical results (acidity, expressed as a percentage mass fraction)

Sample Palm oil Palm kernel fat

Number of participating laboratories, IV 12 27 41 41 23

Number of laboratories retained after

10 27 39 40 22
eliminating outliers, n

Number of individual test results of all

20 54 78 80 44
laboratories on each sample, nz

Mean value, wFFA' % mass fraction 3,11 4,09 6,46 1,72 7,26

Repeatability standard deviation, s., % 0,01 0,021 0,03 0,02 0,03

Repeatability coefficient of variation,

0,4 0,5 0,4 0,1 0,3
CV(r), %

Repeatability limit, r
0,03 0,06 0,07 0,06 0,07
(s, x 2,8), %

Reproducibility standard deviation,

0,161 0,064 0,082 0,07 0,085
sR, %

Reproducibility coefficient of variation

5,2 1,6 1,3 4,1 1,2
CV(R), %

Reproducibility limit, R
0,45 0,18 0,23 0,20 0,24
(SR x 2,8), %

8 © ISO 2009 - All rights reserved

 BS EN ISO 660:2009
ISO 660:2009(E)

Bibliography

[1] ISO 385, Laboratory glassware - Burettes

[2] ISO 1042, Laboratory glassware - One-mark volumetric flasks

[3] ISO 5555, Animal and vegetable fats and oils - Sampling

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

[5] ISO 5725 (all parts), Accuracy (trueness and precision) of measurement methods and results

[6] IUPAC method 2.201, Determination of the acid value (A.V.) and the acidity

© ISO 2009 - All rights reserved 9