TEST METHODS

CONTENTS

Page
1. M1-Determination of Anisidine Value 1
2. M2-Determination of Bleachability Test 3
3. M3-Determination of Cloud Point 5
4. M4-Determination of Colour 6
5. M5-Determination of Deterioration of Bleachability Index ( DOBI ) 8
6. M6-Determination of Free Fatty Acid 9
7. M7-Determination of Free Fatty Acid for Dark Coloured Oils 11
8. M8-Determination of Impurities 13
9. M9-Determination of Iodine Value ( Cyclohexane Method ) 15
10. M10-Determination of Iodine Value ( Wijs Method ) 19
11. M11-Determination of Peroxide Value 22
12. M12-Determination of Phosphorus 25
13. M13-Determination of Saponification Value 27
14. M14-Determination of Slip Point 29
15. M15-Determination of Soap Content 31
16. M16-Determination of Unsaponifiable Matter 33
17. M17-Determination of Volatile Matter 35
 METHODS OF TEST FOR
PALM OIL AND PALM OIL PRODUCTS

M1 - Determination of Anisidine Value

1. Definition
The Anisidine Value is defined by convention as 100 times the optical density measured in a 1 cm cell
resulting from the reaction of 1 g of oil with 100 ml of a mixture of solvent and anisidine reagent.

The method determines the amount of aldehydes (principally 2 alkenals) in oils and fats.

2. Principle
Spectrophotometric measurement of the absorbance at 350 run resulting from the reaction of the
aldehydic compounds in an oil and p - anisidine in the presence of acetic acid.

3. Reagents
3.1. Iso-octane (2,2,4 - trimethylpentane) or n-hexane, optically clear. The absorbance of the solvent,
measured in a 1 cm cell between 300 and 380 nm must be nil or nearly nil. The commercial
product can be freed from absorptive material by percolating it through a glass column (3 - 5 cm
internal diameter, and 100 cm long) filled with silica gel.

3.2. Glacial acetic acid

The moisture content must be below 0.10%. The presence of water tends to decrease the anisidine
value since the reaction involves the formation of water. Check the moisture content by Karl
Fischer titration. If it exceeds 0.1%, discard the acetic acid.

3.3. p~Anisidine (Note 1)

Prepare 0.25% (w/v) solution of the reagent in glacial acetic acid, (Note 2).

4. Apparatus
4.1. Spectrophotometer suitable for use at 350 nm.

4.2. 2 glass cells of 1 cm light path.

4.3. 10 ml test tubes, with ground-glass stoppers.

4.4. 25 ml volumetric flasks.

4.5. 5 ml and 1 ml pipettes.

4.6. Automatic pipettes or 1 ml pipette with rubber bulb to dispense the anisidine reagent.

5. Preparation of sample for analysis

Melt the sample at 10°C above the melting point of the sample and homogenise thoroughly before
taking a test portion. The sample should be clear and dry.

1
 6. Procedure
Weigh 1.5 g of the sample, to the nearest 0.0001 g into a 25 ml volumetric flask. Dissolve and make
up to volume with solvent.

Zero the instrument at 350 run according to the manufacturer's instruction using the solvent.

Measure the absorbance (Ab) of the sample solution at 350 run in the sample cell, using the reference
cell filled with solvent as a blank.

Pipette exactly 5 ml of the fat solution into one test tube and exactly 5 ml of the solvent into a second
test tube. By means of an automatic pipette, add exactly 1 ml of the p-anisidine reagent to each tube.
Shake the tubes to homogenise the solution and reagent.

After exactly 10 minutes, measure the absorbance (As) at 350 nm of the solution in the first test-tube in
the sample cell, using the solution from the second test tube as a blank in the reference cell.

7. Expression of results
The anisidine value is given by

25 x (1.2 As - Ab)
AV =
W
where As is the absorbance of the sample solution after reaction with the p-anisidine reagent.

Ab is the absorbance of the sample solution.

W is the weight, in grams, of the test sample.

Note 1: In storage, p-anisidine tends to darken as a result of oxidation. The commercial product which is discoloured can be
purified by one of two methods:

(a) By molecular distillation using a small still with a cold finger under a pressure
of 1 mm Hg.

(b) By recrystallization
Dissolve 40 g of p-anisidine in 1 litre of water at 75°C. Add 2 g of sodium sulphite and 20 g activated carbon.
Stir for five minutes and then filter through a fast filter paper. If carbon passes through, repeat the filtration using a double
filter paper.

Cool the filtrate to about 0°C and allow to stand for at least 4 hours or preferably overnight. Filter off the crystals and
wash with a small amount of water at a temperature around 0°C. Dry the crystals in a vacuum desiccator and then store
in a brown glass bottle.

If stored in the dark and at low temperature, the crystals obtained should not darken appreciably for 1 year.

Note 2: p-Anisidine reagent solutions having an absorbance greater than 0.200 when measured in a 1.00 cm cell at 350 nm against
the solvent as a blank should be discarded.

The solution should be handled with rubber gloves and the reagents dispensed with an automatic pipette or a rubber bulb.

Acknowledgement : By the kind permission of PORIM for allowing us to use this method of analysis from their publication in the
book “ PORIM TEST METHODS”

2
 METHODS FOR TEST FOR
PALM OIL AND PALM OIL PRODUCTS

M2 - Determination of Bleachability Test

1 . Definition
This method determines the colour of palm oil products after laboratory refining.

2. Principle
A two stage Bleachability test involving preliminary degumming step, followed by earth
bleaching and heat treatment.

3. Scope
Applicable to crude palm oil products.

4. Apparatus
4.1. The suggested equipment is a quick fit FR 250/3S/22A flask with a thermometer inserted in one
of the small (19/26) necks and stirrer inserted through one of the other necks. The remaining
neck can be used to introduce a flow of nitrogen into the flask during the earth bleaching stage.
Filtration should be carried out using a 7 cm Hartley funnel and an FB 500/3 Buchner flask. As
an alternative, a 7 cm Buchner funnel is acceptable but great care should be taken to ensure that
earth particles do not pass the filter during the initial and final stages of filtration.

4.2. For the heat bleach stage of the test, the same basic apparatus is employed except that the stirrer is
replaced by an SH1/33 recovery bend, connected to an FC2/13 as air condenser, connected to an
FB500/3 Buchner flask by an RA9/33 receiver adaptor.

4.3. It is recommended that the vacuum supply should be from an Edward type high vacuum pump,
controlled by a suitable Vacustat to between 1 and 3 millimetres of mercury.

4.4. For heating purpose an electric mantle is suggested, controlled by a thermocouple operated
controller.

5. Procedure
5.1. Carefully melt a sample of crude palm oil and weigh 100 g, into the reaction vessel. Heat the oil,
with agitation, under a nitrogen blanket to a temperature of 90°C. Add 0.5 ml of a 20% w/w
solution of reagent grade citric acid in distilled water, and maintain at 90°C for 10 minutes.

5.2. Add 1% of bleaching earth, (Note 1) raise the temperature to 105°C and maintain under nitrogen
blanket for 15 minutes.

3
 5.3. As soon as possible, filter the hot oil/ earth mixture into a preheated 500 ml Buchner flask through
the appropriate size of Hartley filter using a Whatman No. 1 filter paper. At this stage, the oil is
at a high temperature and therefore prone to oxidation, so delays must be minimised.

5.4. Transfer 90 ± 1 g of the filtrate to the heat bleach apparatus flask, a few anti-bump granules and
draw the appropriate vacuum (1-3 millimetres of mercury). Heat the mixture, without agitation,
to 260°C ± 5°C within 10 minutes of starting and maintain for a further 20 minutes.

5.5. Whilst maintaining vacuum, cool the oil as rapidly and safely as possible, to a temperature of
60°C.

5.6. When cooled to 60°C, quickly release the vacuum and transfer the oil without contaminating it
with condensed fatty acids, into a 5 1/4" Lovibond cell. Use the method in page 6 to read the
colour in the 5 1/4" cell, transfer the oil quickly to a 1 inch cell and repeat the readings.

5.7. As in normal laboratory practice, safety screens and/or safety glasses should be used whenever
high vacuum is applied to glassware.

5.8. The test must be completed within 2 ½ hours of starting to degum.

Note l: Tonsil standard FF is recommended in the Scopa Test .Any bleaching earth may be used depending on the objective of the
experiment.

Reference: Seed Crushers and Oil Processors Association, UK (SCOPA) Test for bleaching palm oil.

Acknowledgement : By the kind permission of PORIM for allowing us to use this method of analysis from their publication in the
book “ PORIM TEST METHODS”

4
 METHODS OF TEST FOR
PALM OIL AND PALM OIL PRODUCTS

M3 - Determination of Cloud Point

1. Definition
The cloud point is that temperature at which, under the conditions of this test, a cloud is induced in the
sample caused by the first stage of crystallisation.

2. Scope
Applicable to palm oil, palm oleins.

3. Apparatus

3.1 Sample bottle (Beatson), 120 ml, external diameter 50 mm and wall thickness of 2 mm or glass
container of equivalent dimensions.

3.2 Thermometer, -5°C to 50°C, 0.1°C, calibrated.

3.3 Water bath, thermostatically controlled at 5°C below the expected cloud point.

4. Procedure
4.1 The sample must be completely dry before making the test. Filter 60 - 75 g of molten oil using a
Whatman No. 1 filter paper. Heat the filtered oils to 130°C for 5 minutes immediately before
making the test. Pour about 45 ml of the heated oil into a sample bottle.

4.2 Begin to cool the bottle and content in the waterbath, stirring to keep the temperature uniform.
When the sample has reached a temperature about 10°C above the cloud point, begin stirring rapidly
in a circular motion so as to prevent supercooling and solidification of fat crystals on the side or
bottom of the bottle.

4.3 Do not remove the thermometer from the sample, since to do so may introduce air bubbles which will
interfere with the test. The test bottle is maintained in such a position that the upper level of sample
in the bottle and the water level in the bath are about the same.

4.4 Remove the bottle from the bath and inspect regularly. The cloud point is the temperature at that
portion of the thermometer immersed in the oil is no longer visible when viewed horizontally through
the bottle and sample.

4.5 Repeat the analysis using a water bath temperature set at 5°C below the cloud point value obtained in
the first determination.

5. Record the cloud point obtained from 4.5, in degree Centigrade, expressed to one decimal place.
6. Repeatability standard deviation. Results of repeated determinations within a single laboratory by a
single operator should not differ by more than 0.6°C.

Reference: AOCS Cc 6-25 (1989).

Acknowledgement : By the kind permission of PORIM for allowing us to use this method of analysis from their publication in the
book “ PORIM TEST METHODS”

5
 METHODS OF TEST FOR
PALM OIL AND PALM OIL PRODUCTS

M4 - Determination of Colour

l., Definition
The colour of a sample is the colour in Lovibond Units as measured on a Lovibond Tintometer.

2. Principle
The method consists of matching the colour of the light transmitted through a specified depth of oil to
the colour of the light, originating from the same source, transmitted through standard colour slides.

3, Scope
Applicable to oils and fats.

4. Apparatus
4.1. Lovibond Tintometer, equipped with standard colour slides (Note 1).

4.2. Glass cells, with lengths of light path 1 inch (25.4 mm) and 5 1/4 inch (133.4 mm).

5. Selection of operators
All operators shall satisfy the requirements of a colour vision test and shall be retested at five yearly
intervals.

Note 1.
It is preferred that the colour vision test be carried out by a qualified optician.

Note 2.
Operators who normally wear spectacles or contact lenses may continue to wear them.

Tinted or light sensitive spectacles or lenses shall not be worn.

The Tintometer shall be maintained in good clean condition in accordance with the manufacturer's
recommendations.

Lighting cabinet, consisting of two 60 W pearl (non coated) lamps, operated at the correct mains
voltage and each illuminating the sample and white reference field at 45°. The lamps shall be
positioned at either side of the viewing tube.

The lamps shall not be used for longer than 100 h. A suitable logging system shall be employed to
register lamp life. Lamps shall be changed in pairs.

The viewing tube shall have a field of view subtending 4° to the eye and contain a daylight correction
filter.

The lighting cabinet shall be such that it enables the sample and white reference field to be viewed at
90° to normal.

6
 The lighting cabinet shall be inspected at frequent intervals for any dirt particles, which shall be
removed, and for ageing of the paint. The cabinet shall be repainted with a matt white paint when the
colour becomes darker than Munsell Notation 5Y 9/1 (obtainable from the Tintometer).

Colour racks, consisting of Red, Yellow, Blue and Neutral racks with colour readings as follows, and
fitted with colourless compensating slides.
Red 0.1 to 0.9; 1.0 to 9.0; 10.0 and 20.0
Yellow 0.1 to 0.9; 1.0 to 9.0; 10.0 to 70.0
Blue 0.1 to 0.9; 1.0 to 9.0
Neutral 0.1 to 0.9; 1.0 to 3.0

6. Procedure
6.1. If the prepared sample is not liquid at room temperature heat it to a temperature of 60 ± 5°C and
pour into the cell. The sample should be clear and bright when determination is made.

For crude and bleached oils, choose a 1 inch (25.4 mm) cell. For refined oils, a 5 1/4 inch
(133.4 mm) cell is suitable.

6.2. Using the Lovibond Tintometer, immediately determine the colour of the sample by achieving the
best possible match with the standard colour slide.

Since the onset of eye fatigue is rapid (i.e. after about 10 secs) it is preferable, if a colour
match cannot be quickly obtained, to repeat the procedure after a few minutes. It is also
desirable that the test be carried out by trained operators.

If the red, yellow and neutral units do not form a possible match of the colour of the oil, use a
minimum of blue units to obtain the suitable match.

7. Expression
Express the results in terms of

a) the number of Red, Yellow, Neutral or Blue units needed to obtain the match and

b) the cell used and the Lovibond Model Number.

8. Reproducibility
The difference between the results obtained by two trained operators carrying out the procedure
described using the same apparatus, in the same laboratory, simultaneously or in rapid succession shall
not exceed the values given in Table 1.

Table 1. Acceptable Colour Reading Differences

Reading Acceptable differences

Up to 0.9 Red 0.2 Red
From 1.0 Red to 2.9 Red 0.4 Red
From 3.0 Red to 4.0 Red 0.5 Red
From 4.1 Red to 12 Red 1.0 Red
Note 1: Model AF900 or AF702 conforms to the requirements of this method. If AF model 905 E is used, higher colour readings are
obtained. The instruments are obtained from The Tintometer, Ltd. Water Road, Salisbury, Wiltshire

Reference: BS 684 Section 1.14: 1987.

Acknowledgement : By the kind permission of PORIM for allowing us to use this method of analysis from their publication in the
book “ PORIM TEST METHODS”

7
 METHODS OF TEST FOR
PALM OIL AND PALM OIL PRODUCTS

M5 - Determination of the Deterioration of Bleachability Index (DOBI)

of Crude Palm Oil

l Definition
The Deterioration of Bleachability Index (DOBI) is the numerical ratio of the spectrophotometric
absorbance at 446 nm to absorbance at 269 nm.

2. Principle
The analysis involves the spectrophotometric measurement of a solution of oil sample in iso-octane or
n-hexane (0.5 to 1% concentration) against the solvent. The absorbance values are not corrected in
any other way.

3. Scope
Applicable to crude palm oil.

4. Reagents
2,2,4-trimethylpentane (iso-octane) or n-hexane of spectroscopic or equivalent grade.

5. Apparatus
5.1. Spectrophotometer suitable for use at 269 nm and 446 nm.
5.2. 10 mm quartz cuvettes
5.3. 25 ml volumetric flasks
5.4. 10 ml volumetric flasks
5.5. 2 ml pipettes.

6. Procedure
Weigh to the nearest 0. 1 mg, about 0. 1 g of a completely melted and homogenised oil sample into a
25 ml volumetric flask. Dissolve and make up to volume. Fill a cuvette with the oil solution and
Measure its absorbances at 269 nm and 446 nm against pure solvent.

If the solution is too concentrated, dilute it by pipetting two millilitres of it into a 10 ml volumetric flask
and making up to volume. Measure the absorbances of this solution at 269 nm and 446 nm against pure
solvent. Further dilution may be necessary for very high DOBI values.

7. Calculation

Absorbance at 446 nm
DOBI =
Absorbance at 269 nm

(Express the result to two decimal places)

Reference: Swoboda, P.A.T. " UV-VIS Spectrophotometric Assays for Palm Oil Quality", paper presented at International Conference
on "Palm Oil Product Technology in the Eighties', 22-24 June, 1981, Kuala Lumpur.

Acknowledgement : By the kind permission of PORIM for allowing us to use this method of analysis from their publication in the
book “ PORIM TEST METHODS”

8
 METHODS OF TEST FOR
PALM OIL AND PALM OIL PRODUCTS

M6 - Determination of Free Fatty Acid

1. Definition
The acid value is the number of milligrams of potassium hydroxide necessary to neutralise the free
acids in 1 g of sample.

The acidity is the content of free fatty acid conventionally expressed as a percentage (m/m) of palmitic
acid, or any relevant acid as indicated in Item 8.

2. Principle
A known mass of the fat is dissolved in neutralised isopropanol and the free fatty acids are neutralised
with standard alkali.

3. Scope
Applicable to all normal oils and fats.

4. Reagents
4.1. Standard potassium or sodium hydroxide, 0.1N for crude oils and
0.02 N for refined oils (Note 1).

4.2. Phenolphthalein indicator solution, 1.0% in iso-propanol.

4.3. Neutralised isopropanol. Place 50 ml isopropanol in a flask and bring the solution to the boil
over a hot plate. Add about 0.5 ml of phenolphthalein and neutralise by drop wise addition of
0.1N potassium hydroxide till a faint, but permanent pink colour is obtained, (Note 2).

5. Apparatus
5.1. Burette, 25 ml with graduation in 0.05 ml divisions.
5.2. Conical flasks of 250 ml and 500 ml.
5.3. Hot plate with temperature control.
5.4. Analytical balance.

6. Preparation of sample for analysis

Melt the sample at 60°C - 70°C and thoroughly homogenise it before sampling.

7. Procedure
Determine the size of sample from the following table:-

Acidity Wt. of sample Weighing accuracy

(+ 10%), grams (grams)

0 to 1 20 0.05
1 to4 10 0.02
4 to 15 5 0.01
15 to 75 2.5 0.01
75 and over 0.5 0.001

9
 Weigh the specified amount of sample into an Erlenmeyer flask. Add 50 ml of the neutralised solvent.
Place the flask on the hot plate and regulate the temperature to about 40°C. Shake the sample gently
while titrating with standard alkali to the first permanent pink colour. The colour must persist for 30
seconds.

8. Expression of results

FFA% as palmitic acid = 25.6 x N x V

(for palm oil and fractions) W

FFA% as lauric acid = 20.O x N x V

(for coconut oil, palm kernel oil W
and fractions)

FFA% as oleic acid = 28.2 x N x V

(for corn oil, soyabean oils W
and other liquid oils)

where N = normality of NaOH solution

V = volume of NaOH solution used in ml
W = weight of sample

Express the FFA to 3 decimal places for FFA below 0.15% and 2 decimal places for FFA above 0.15%.

9. Repeatability
The difference between the results of two determinations on the same test sample, carried out
simultaneously or in rapid succession by the same analyst, should not exceed 0.02% for free fatty acid
contents between 1.5% and 5.0% and 0.004% for free fatty acid contents less than 0.1%.

Note 1 Potassium or sodium hydroxide of approximate 0. 1 Normality should be standardised with potassium hydrogen phthalate as
follows :-
Dry potassium hydrogen phthalate in an oven at 120,C for 2 hrs. and allow to cool in a desiccator before use. Weigh out 0.4 ±
0.02 to 0.1 mg of the potassium hydrogen phthalate directly into a conical flask. Add 50 ml water and phenolphthalein
indicator. Place on a hot plate and swirl till the salt has completely dissolved and titrate with potassium or sodium hydroxide to
the first appearance of a permanent pink colour.

W X 101 Normality of the alkali = t x 204.2

where W is the weight of phthalate taken

t is the volume of potassium or sodium hydroxide in ml

Express the result to nearest 0.01 unit. Carry out duplicate determinations. The concentration of the hydroxide should be
checked prior to it being used.

Note 2: Ethanol 95% (v /v) may also be used.

Reference: AOCS 5a - 40 (1989).

Acknowledgement : By the kind permission of PORIM for allowing us to use this method of analysis from their publication in the
book “ PORIM TEST METHODS”

10
M7 - Determination of Free Fatty Acid for Dark Coloured Oils

Definition

This method is to determine the amount of acid components calculated as free fatty acids.

Application

This method is applicable also to the titration of dark oils where it would be difficult to observe the colour
changes of phenolphthalein.

Apparatus

Burette, 10 or 25 ml. graduated in 0.1 ml.

Erlenmeyer flask, 300ml.

Reagents

NaOH solution : 0.1 N ( in water )

Solvent with indicator : One part of toluene and 3 parts of isopropanol ( isopropyl alcohol, 99 % ). Add
about 0.25 g Alkali Blue 6B per litre of the solvent mixture, dissolving the indicator in the isopropanol and then
adding the toluene and mix.

Recommended stock solution : 1250 ml toluene

3750 ml isopropanol
1.25 g Alkali Blue 6B

Procedure

A carefully mixed sample ( melted in the case of solid fats ) is weighed or pipetted into a 300 ml Erlenmeyer
flask.

Adequate weight of sample used : crude oil 15 % FFA ; refined oil 20 g or more.

Add 50 - 75 ml of neutralised solvent and titrate with NaOH under vigorous stirring until the colour of the
indicator changes to red ( change is gradual from blue through violet to red ). Generally, a single drop of
NaOH solution is sufficient to bring about the change from violet to red.

11
Calculation

FFA ( in % ) = VxNxM
10 G

Acid no. = V x N x 56.1 mg KOH / g oil

G

where V = ml of NaOH solution used

N = normality of NaOH solution
G = weight of sample ( in g )
M = average molecular weight of the fatty acids

Standard values being

Coconut oil 205

Palm kernel oil 214
Palm oil 263
Rapeseed oil 308
Other oils ( linseed, soybean, groundnut, cottonseed,
sunflower seed, rice bran, lard and fish oil) 282

The acidity of an oil is often expressed by the acid number, i.e. the number of mg of KOH required for
neutralising 1 g of the oil, thus

acid number = V x N x 56.1 mg KOH / g oil

G

12
 METHODS OF TEST FOR
PALM OIL AND PALM OIL PRODUCTS

M8 - Determination of Impurities

1. Definition
Impurities are defined as those materials insoluble in n-hexane or light petroleum.

2. Principle
The oil dissolved in the solution is filtered and the residue further washed with solvent. The residue is
then dried and weighed.

3. Scope
Applicable to all normal fats and oils.

4. Reagents
4.1. Petroleum ether (60/80°C) or n-hexane. The solvent must be filtered through a Whatman glass
fibre filter GF/B or its equivalent.

5. Apparatus
5.1. Porcelain Gooch crucible of internal base diameter 20 mm.
5.2. Whatman glass fibre filter GF/B or its equivalent.
5.3. Electric oven set at 103 ± 2°C.
5.4. Vacuum filter flask, 1 litre, with adaptor and ring for the Gooch crucible.
5.5. Conical flask, 250 ml, flat bottom
5.6. Desiccator with efficient desiccant.

6. Preparation of sample for analysis

Samples must be heated at 60 - 70°C till completely melted and homogenised before taking a test
portion.

7. Procedure
Place a glass fibre filter paper on a Gooch crucible. Wash the assembly with ca. 10 ml petroleum ether
or hexane under a slight vacuum and dry for 30 minutes at 103°C. Cool the crucible with filter paper in
a desiccator and weigh to the nearest 0.1 mg.

Weigh into a conical flask, about 20 g of oil and add 100 ml of solvent. Warm and swirl to achieve
complete homogenisation. Carefully filter the solution through the Gooch crucible under slight vacuum.
Use fresh solvent (several 10 ml portions) to transfer all the oil and insoluble matter into the Gooch
crucible and wash with several portions of 10 ml solvent until all the oil is removed.

Wipe clean with tissue paper and dry the crucible and contents in the oven at 103°C for 30 minutes. Cool
in the desiccator to room temperature.

Weigh the Gooch crucible and contents to the nearest 0.1 mg. Carry out two determinations on the same
sample.

13
8. Expression of results
The insoluble impurities expressed as percentage by weight is

W2 - Wl x 100
W

where W is the weight of sample

W1 the weight of Gooch crucible plus filter paper.
and W2 the weight of Gooch crucible plus filter paper plus impurities.

Results should be expressed to 3 decimal places.

9. Repeatability
The difference between the results of two determinations carried out in rapid succession by the same
analyst shall not exceed 0.01% for insoluble impurities within 0.15%.

Reference: MOPGC Test Methods

Acknowledgement : By the kind permission of PORIM for allowing us to use this method of analysis from their publication in the
book “ PORIM TEST METHODS”

14
 METHODS OF TEST FOR
PALM OIL AND PALM OIL PRODUCTS

M9 - Determination of Iodine Value (Cyclohexane Method)

1. Definition
The iodine value is a measure of the unsaturation of fats and oils. It is expressed as the number of
grams of iodine absorbed by 100g of the fat under the test conditions used.

2. Principle
Addition of a solution of iodine monochloride in a mixture of acetic acid and cyclohexane to a test
portion. Reduction of the excess of iodine monochloride after a specified reaction time by adding
potassium iodide solution and water, and titration of the liberated iodine with standard sodium
thiosulphate solution.

3. Scope
This method is applicable to all normal fats and oils not containing conjugated double bonds.

4. Reagents
4.1 Wijs solution - accurately standardized (see Notes, Caution and 2).

4.2 Potassium iodide (KI) solution - 15%, prepared by dissolving 15 g of reagent-grade KI in 100 ml of
distilled water.

4.3 Cyclohexane - reagent grade (see Notes, 3 and 4).

4.4 Soluble starch - tested for sensitivity (see Notes, 5). Make a paste with 1 g of starch and a small
amount of cold distilled water. Add, while stirring, 200 ml of boiling water. Place 5 ml of this
solution in 100 ml of water and add 0.05 ml of 0.1 N iodine solution. The deep blue colour
produced must be discharged by 0.05 ml of 0.1 N sodium thiosulfate.

4.5 Potassium dichromate - reagent grade. The potassium dichromate is finely ground and dried to
constant weight at about 110°C before using.

4.6 Hydrochloric acid-reagent grade, concentrated, sp. gr. l.19 (see Notes, Caution).

4.7 Sodium thiosulphate (Na2S2O3.5H2O) - 0.1 N, accurately standardized, prepared from reagent-grade
Na2S2O3.5H2O (see Notes, 6).

Standardisation of sodium thiosulphate - Weigh 0. 16 - 0.22 g of finely ground and dried potassium
dichromate into a 500 ml flask or bottle by difference from a weighing bottle. Dissolve in 25 ml of water,
add 5 ml of concentrated hydrochloric acid, 20 ml of potassium iodine solution and rotate to mix. Allow
to stand for 5 min, and then add 100 ml of distilled water. Titrate with sodium thiosulphate solution,
shaking continuously until the yellow colour has almost disappeared. The strength of the sodium
thiosulphate solution is expressed in terms of its normality.

Normality of Na2S2O3 solution = 20.394 x wt of K2Cr2O7

ml of sodium thiosulphate

15
5. Apparatus
5.1 Glass weighing scoops or vial of 2 to 4 ml capacity for the test portion.

5.2 Wide-necked glass bottles or flasks with ground glass Stoppers (e.g. iodine flasks) capacity 250 or
500 ml.

5.3 Burettes, 50 ml graduated in 0.1 ml.

5.4 Pipettes, 20 and 25 ml,

6. Preparation of Sample for Analysis

Melt the sample at 60 to 70°C and thoroughly homogenise before taking a test portion.

7. Procedure
Weigh the sample accurately to the nearest 0.0001 g in the glass weighing scoop or vial. The weight of
the sample to be used varies according to its expected iodine value. Use the table below as a guide:

Product Expected iodine Sample weight

value g

Hydrogenated palm <5 3.00

kernel oil, olein, stearin

Palm kernel oil, olein, stearin 5 - 25 1.00

Special high IV olein 25 - 60 0.4

Liquid oils, soya bean oil etc. 60 - 100 0.3

Liquid oils, soya bean oil etc. > 100 0.15

Place the scoop in a 500 ml flask. Add 20 ml of cyclohexane to dissolve the fat. If necessary, warm the
flask slightly to facilitate dissolution of the fat. Add exactly 25 ml of the Wijs solution, insert the stopper,
shake gently and place the bottle in the dark for 1 hour. High IV oils (e.g. > 150) may require a longer
period of 2 hours.

After standing, add 20 ml of the potassium iodide solution and 100 ml of water. Titrate with the sodium
thiosulphate solution until the yellow colour due to iodine has almost disappeared. Add 1 to 2 ml of the
starch indicator solution and continue the titration until the blue colour just disappears after very vigorous
shaking. Carry out two determinations on the same test sample. Carry out a blank test simultaneously
under the same conditions.

16
8. Expression of Results

Iodine value = 12.69N (V2 - Vl)

where N is the exact normality of the sodium thiosulphate solution used:

V2 is the column, in millilitres, of the sodium thiosulphate solution used for the blank test;
Vl is the volume, in millilitres, of the sodium thiosulphate
solution used for the determination;
W is the weight, in grams, of the test portion.

Express the result to one decimal place.

9. Repeatability

The difference between the results of two determinations carried out simultaneously or in rapid succession
by the same analyst shall not exceed 0.5 unit of iodine value.

Notes

Caution

Cyclohexane is flammable and a dangerous fire risk. It is moderately toxic by inhalation and skin contact. The TLV in air is 300 ppm.

Wijs solution causes severe burns, and the vapours can cause lung and eye damage. Use of a fume hood is recommended. Wijs solution
without carbon tetrachloride is available commercially.

Hydrochloric acid is a strong acid and will cause severe burns. Protective clothing should be worn when working with this acid. It is
toxic by ingestion and inhalation and is a strong irritant to eyes and skin. The use of a properly operating fume hood is recommended.
When diluting the acid, always add the acid to the water, never the reverse.

Numbered Notes

1. When the iodine value is determined on materials having conjugated systems, the result is not a measure of total unsaturation, but
rather is an empirical value indicative of the amount of unsaturation present. Reproducible results are obtained which afford a
comparison of total unsaturation. When the iodine value is required on fatty acids, the preparation and separation are performed as
directed in AOCS Official Method Cd 6 - 38.

2. Because the preparation of the Wijs solution is time-consuming and involves the use of both hazardous and toxic chemicals, this
solution may be purchased from a chemical supplier. Solutions are available which contain no carbon tetrachloride, and such
solutions should be used. All Wijs solutions are sensitive to temperature, moisture and light. Store in a cool and dark place, and
never allow to come to a temperature above 25 - 30°C.

3. Fresh cyclohexane should be used. Erratic results may be obtained if old cyclohexane is used.

4. The AOCS study reported in J. Am. Oil Chem. Soc. (Reference No. 1) used cyclohexane alone. When using cyclohexane alone,
the 30 min reaction time specified in the former AOCS version of the Wijs method is insufficient for samples having an IV > 100,
especially fish oils, as reported in Reference No. 1. A study completed by FOSFA International in 1992 (Reference No. 2), showed
that the ISO 3961 (1989) and AOCS Cd lb-87 (1990) methods for determining IV do not give rise to values that are statistically
different, and that the use of cyclohexane (alone) in place of carbon tetrachloride as the solvent does not affect the observed IV for
titration of triglyceride oils having iodine values between 18 and 52 (the range of iodine values encountered in the study). Variable
results were obtained in the application of the method to fish oils; however, further studies carried out by the International
Association of Fish Meal Manufacturers (IAFMM) indicated that reproducible IV results could be obtained if the reaction time of the
Wijs reagent and fish oil was extended.

5. 1% starch solution may be purchased from a chemical supplier.

17
6. The sodium thiosulphate solution may be purchased from a chemical supplier. However, it still must be accurately standardized in
the laboratory.

7. The weight of the sample must be such that there will be an excess of Wijs solution of 50 - 60% of the amount added; i.e. 100 -
150% of the amount absorbed.

8. If the reaction is not terminated within 3 min past the reaction time, the sample must be discarded.

9. The sample must be titrated within 30 min of reaction termination, after which the analysis is invalid.

10. Mechanical stirring is recommended for agitation during the addition of thiosulphate.

References
1. AOCS collaborative study results using cyclohexane alone appear in J. Am. Oil Chem. Soc. 65: 745 (1988).

2. INFORM 3,1992.

3. AOCS Cd 1b - 87 (1992).

Acknowledgement : By the kind permission of PORIM for allowing us to use this method of analysis from their publication in the
book “ PORIM TEST METHODS”

18
 METHODS OF TEST FOR
PALM OIL AND PALM OIL PRODUCTS

M10 - Determination of Iodine Value (Wijs Methods)

1. Definition
The iodine value is a measure of the unsaturation of fats and oils. It is expressed as the number of
grams of iodine absorbed by 100 g of the fat under the test conditions used.

2. Principle
Addition of a solution of iodine monochloride in a mixture of acetic acid and carbon tetrachloride to a
test portion. Reduction of the excess of iodine monochloride after a specified reaction time by adding
potassium iodide solution and water, and titration of the liberated iodine with standard sodium
thiosulphate solution.

3. Scope
This method is applicable to all normal fats and oils not containing conjugated double bonds.

4. Reagents
4.1. Carbon tetrachloride, free from oxidizable matter (Note 1).

4.2. Glacial acetic acid, free from ethanol and oxidizable matter (Note 1).

4.3. Potassium iodide 10% (w/v) aqueous solution free from iodine or iodate.

4.4. Starch solution 1% (w/v) aqueous dispersion, freshly boiled for 3 minutes and allowed to cool.

4.5. Sodium thiosulphate, 0.1 N (Note 2)

Dissolve 24.8 g sodium thiosulphate pentahydrate in distilled water and dilute to 1 litre.

4.6. Potassium dichromate solution, 0.1N

Dissolve 4.9035 g of dried potassium dichromate in distilled water and make up to 1 litre in a
volumetric flask.

4.7. Wijs reagent (Iodine monochloride solution in acetic acid carbon tetrachloride mixture).

The reagent is available commercially. It may be prepared from iodine trichloride as follows :-

Weigh 9 g of iodine trichloride into a brown glass bottle of 1500 ml capacity. Dissolve in a
mixture of 700 ml glacial acetic acid and 300 ml carbon tetrachloride.

Take 5 ml of the solution and add 5 ml of potassium iodide solution and 30 ml of water. Titrate
the liberated iodine with 0.1N sodium thiosulphate solution in the presence of a few drops of starch
indicator.

19
 Add 10 g of pure re-sublimed iodine to the bulk of the reagent and dissolved by shaking. Titrate
the free iodine as above. The titre should now equal one and a half time that of the first
determination. If this is not the case, add a small quantity of iodine until the content slightly
exceeds the limit of one and half times. It is important that no trace whatever of iodine trichloride
should remain as it would cause secondary reactions.

Let the solution stand. Decant the clear liquid into a brown glass bottle. If stored in a well-
stoppered bottle away from light, the solution can be used for months.

5. Apparatus
5.1. Glass weighing scoops or vial of 2 to 4 ml capacity for the test portion.

5.2. Wide-necked glass bottles or flasks with ground glass stoppers (e.g. iodine flasks) capacity 250 or
500 ml.

5.3. Burettes, 50ml graduated in 0.1 ml.

5.4. Pipettes, 20 and 25 ml,

6. Preparation of Sample for Analysis

Melt the sample at 60°C to 70°C and thoroughly homogenise before taking a test portion.

7. Procedure
Weigh the sample accurately to the nearest 0.0001 g in the glass weighing scoop or vial. The weight of
the sample to be used varies according to its expected iodine value. Use the table below as a guide:

Product Expected iodine value Sample weight gm

Hydrogenated palm <5 3.00

kernel oil, olein, stearin

Palm kernel oil, 5 - 25 1.00

olein, stearin

Palm oil, olein, stearin 25 - 60 0.4

Special high IV olein 60 - 100 0.3

Liquid oils, soya bean > 100 0.15

oil etc.

Place the scoop in a 500 ml flask. Add 15 ml of the carbon tetrachloride to dissolve the fat. If necessary,
warm the flask slightly to facilitate dissolution of the fat before addition of the carbon tetrachloride solution.

20
Add exactly 25 ml of the Wijs solution, insert the stopper, shake gently and place the bottle in the dark for 1
hour.

After standing, add 20 ml of the potassium iodide solution and 150 ml of water. Titrate with the sodium
thiosulphate solution until the yellow colour due to iodine has almost disappeared. Add 1 to 2 ml of the starch
indicator solution and continue the titration until the blue colour just disappears after very vigorous shaking.

Carry out two determinations on the same test sample. Carry out a blank test simultaneously under the same
conditions.

8. Expression of Results Iodine value = 12.69N (V2 - Vl )

where N is the exact normality of the sodium thiosulphate solution used;

V2 is the volume, in millilitres, of the sodium thiosulphate solution used for the blank test;
V1 is the volume, in millilitres, of the sodium thiosulphate solution used for the determination;
W is the weight, in grams, of the test portion.

Express the result to one decimal place.

9. Repeatability
The difference between the results of two determinations carried out simultaneously or in rapid
succession by the same analyst shall not exceed 0.5 unit of iodine value.

Note 1: Check the absence of oxidizable matter in glacial acetic acid and carbon tetrachloride by shaking 10 ml of the reagent with 1
ml of saturated potassium dichromate solution and 2 ml of concentrated sulphuric acid. No green colouration should appear.

Note 2: Standardisation of sodium thiosulphate solution

Pipette 25 ml of the standard potassium dichromate solution into a conical or Erlenmeyer flask. Add 5 ml of concentrated
hydrochloric acid, 10 ml of potassium iodide solution and rotate to mix. Allow to stand for 5 minutes and then add 100 ml
distilled water. Titrate with sodium thiosulphate solution, shaking continuously, until the yellow colour has almost
disappeared. Add 1 to 2 ml of starch indicator and continue titration until the blue colour has just disappeared.

Normality of sodium thiosulphate = 25 x N

V

where V is the volume (ml) of sodium thiosulphate solution used and

N is the exact normality of the standard potassium dichromate solution

Reference: British Standards BS 684: Section 2.13:1976

Acknowledgement : By the kind permission of PORIM for allowing us to use this method of analysis from their publication in the
book “ PORIM TEST METHODS”

21
 METHODS OF TEST FOR
PALM OIL AND PALM OIL PRODUCTS

M11 - Determination of Peroxide Value

1. Definition
The peroxide value is a measure of those substances in a sample, expressed in terms of
milliequivalents of active oxygen per kilogram which oxidise potassium iodide under the conditions of
the test.

2. Principle
Treatment of a test portion in a solution of acetic acid and chloroform with a solution of potassium
iodide. The freed iodine is titrated against a solution of sodium thiosulphate.

3. Scope
Applicable to all normal oils and fats.

4. Reagents
All the reagents and water shall be free from dissolved oxygen. This can be achieved by passing a
stream of pure, dry inert gas (carbon dioxide or nitrogen) through the individual reagents.

4.1. Acetic acid-chloroform solution

Mix three parts by volume of glacial acetic acid with two parts by volume of chloroform.

4.2. Potassium iodide, a saturated solution prepared in recently boiled water. The solution must
remain saturated.

4.3. Sodium thiosulphate (Na2 S2O3.5H2O), 0.0IN standard volumetric solution, standardised
before use.

4.4. Starch solution

Mix 5 g of soluble starch in 30 ml of water, add this mixture to 1000 ml of boiling water and
leave boiling for 3 min.

5. Apparatus

5.1. Pipette 1 ml, graduated in 0.01 ml division.

5.2. Conical flask, glass stopper, 250 ml.

5.3. Volumetric flask, 500 ml, 100 ml.

5.4. Burette, 25 ml graduated in 0.05 ml divisions.

6. Sample Preparation
All samples should be analysed as soon as possible or should be kept in a cool, dark place before
analysis.

22
 7. Procedure
Weigh to the nearest 0.1 mg, 5.00 ± 0.05 g (or as given in the table below) of the sample into the
250 ml flask,

Expected peroxide value Mass of test portion

milliequivalents / kg g

0 - 12 5.0 to 2.0
12 - 20 2.0 to 1.2
20 - 30 1.2 to 0.8
30 - 50 0.8 to 0.5
50 - 90 0.5 to 0.3

Add 30 ml of the acetic acid-chloroform solution. Swirl the flask until the sample is dissolved in the solution.
Add 0.5ml of saturated potassium iodide with a graduated pipette. Swirl the solution for 1 minute and then
add 30 ml of distilled water. For freshly produced oils, add a few drops of starch solution.

Titrate with 0.01 N sodium thiosulphate solution, adding it gradually and with constant and vigorous shaking.
Continue the titration, shaking the flask vigorously near the end-point to liberate all the iodine from the
chloroform layers. Add the thiosulphate solution drop wise until the blue colour just disappears.

For samples with high peroxides, titrate with 0.01N until the yellow iodine colour has almost disappeared.
Add 0.5 ml of starch indicator and continue titration until the blue colour just disappears.

Carry out a blank test in parallel with the determination. The blank titration must not exceed 0.1 ml of the
0.01 N sodium thiosulphate solution.

8. Expression of results
The peroxide value, expressed in milliequivalents of active oxygen per kilogram of sample is

(Vs - Vb)N x 1000

where VS is the volume in millilitres, of the sodium thiosulphate solution of normality

N, used for the determination;

Vb is the volume, in millilitres, of the sodium thiosulphate solution used for the blank test;

W is the weight, in grams, of the test portion;

N is the normality of the sodium thiosulphate solution.

Express the result to one decimal place.

23
9. Repeatability
The difference between the results of two determinations carried out simultaneously or in rapid
succession by the same operator on the same sample shall not exceed the following values:

Peroxide value (meq / kg) Repeatability

less than 1 0.1

1 to 6 0.2
6 to 12 0.5
greater than 12 1

Note 1: Test daily the saturated potassium iodide solution by adding 2 drops of starch indicator to 0.5 ml of the solution in 30 ml of
acetic acid-chloroform solution. If a blue colour is formed which requires more than 1 drop of 0.01 N sodium thiosulphate
solution to discharge, discard the iodide solution and prepare a fresh batch.

Note 2: Standardisation of sodium thiosulphate solution, 0.1N

Dissolve 4.9035 g of dried potassium dichromate in distilled water and make up to 1 litre in a volumetric flask.
Pipette 25 ml of the standard dichromate solution into a conical flask. Add 5 ml of concentrated sulphuric acid: (S.G. 1.19),
10 ml of potassium iodide solution (10% w/v) and rotate to mix. Allow to stand for 5 minutes and then add 10 ml of
distilled water. Titrate with the sodium thiosulphate solution, shaking continuously, until the yellow colour has almost
disappeared. Add 1 ml of starch indicator and continue titration until the blue colour has just disappeared.

Normality of sodium thiosulphate = 25 x N

V
where V is the volume (ml) of sodium thiosulphate solution used.

and N is exact normality of the standard potassium dichromate solution.

For standardisation of 0.01N sodium thiosulphate, use a 0.0IN standard solution of potassium dichromate which is prepared by
dissolving 0.4935 g in distilled water and making up to 1 litre in a volumetric flask. Proceed as above.

Note 3: Alternatively standardisation of 0.01 N sodium thiosulphate may be carried out as follows:-

Weigh 0.01 + 0.002 g of potassium dichromate in a conical flask. Weigh in 0.5 g of potassium iodide and add 10 ml distilled
water.

Add 10 ml of 10% hydrogen chloride solution and mix thoroughly for 1 minute. Titrate with sodium thiosulphate until light
yellow and add starch indicator. Continue titration till the blue colour has just disappeared.

N = 1000 x K
49 x T

where K is the weight of potassium dichromate

T is the volume of sodium thiosulphate used

Reference: AOCS Cd 8-53 (1989) ISO 3960 (1977)

Acknowledgement : By the kind permission of PORIM for allowing us to use this method of analysis from their publication in the
book “ PORIM TEST METHODS”

24
 METHODS OF TEST FOR
PALM OIL AND PALM OIL PRODUCTS

M12 - Determination of Phosphorus

1. Definition
The method determines total phosphorus content by charring and ashing in the presence of magnesium
oxide followed by colorimetric measurement as phosphovanadomolybdic complex.

2. Principle
Ignition of the test portion, then nitric acid digestion of the ashes. Formation of a yellow
phosphovanadomolybdic complex between phosphoric, vanadic and molybdic ions, then determination
of the colour so obtained.

3. Scope
Applicable to all palm oil products.

4. Reagents
4.1. Magnesium oxide, light, pure, free from phosphorus.
4.2. Nitric acid, approx. 6 N aqueous solution.
4.3. Ammonium molybdate (NH4)6MO7O24.4H2O), AR grade, 50 g/1 aqueous solution.
4.4. Ammonium vanadate (NH4VO3), acid aqueous solution: dissolve about 2.5 g of ammonium
vanadate in about 500 ml of hot water. Cool, and then add about 20 ml nitric acid ( ρ = 1.33 g/ml).
Make up to 1 litre with distilled water.
4.5. Standardised phosphate stock solution containing 1 mg of phosphorus per ml : dissolve 1.156 g of
disodium hydrogen phosphate (Na2HPO4.12H2O) or 0.4393 g of mono potassium hydrogen
phosphate (KH2PO4) in 1 litre of water.

5. Apparatus
5.1. 5- and 20- ml calibrated pipettes.
5.2. 40-50 ml crucible or porcelain evaporating dish.
5.3. Colorimetric or spectrophotometer suitable for observations at 400 run.
5.4. Furnace, giving a temperature of 800°C to 900°C.

6. Preparation of sample for analysis

Melt the sample at 60°C to 70°C for palm oil or at 10°C above the melting point of the oil.
Thoroughly homogenise before taking a test portion.

25
7. Procedure
7.1. Calibration
Prepare 4, 8, 12, 16 µg/ml solutions by pipetting 1, 2, 3, 4 ml of the phosphate stock solution,
followed by 20 ml of the ammonium molybdate-vanadate mixture and make up to 25 ml with
6N HNO3. Utilise these solution in order to plot the calibration curve in such a way that this
curve expresses the calibration factor as

µg / ml
Absorbance

7.2. Determination
Weigh to within 1 mg exactly 0.1 g of magnesium oxide (4.1) into the crucible or porcelain dish
(5.2). Weigh to within 1 mg about 5 g of fat into the same crucible (or according to its
presumed phosphorus content).

Burn off the fat (if necessary with the assistance of a folded, ashless filter paper). Ignite to a
white ash in the furnace (5.4) at 800°C to 900°C for 2 hours.

Dissolve the magnesium-containing ash in exactly 5 ml of the aqueous nitric acid solution (4.2)
with the aid of a 5 ml pipette (5.1). Add exactly 20 ml of a mixture of 10 ml of the aqueous
ammonium vanadate solution (4.3) and 10 ml of the acidic aqueous ammonium
molybdate solution (4.4). Mix and allow to stand for 20 minutes.

Prepare a blank test, not containing fat, under exactly the same conditions.

Transfer the test solution into the cell of the apparatus (5.3). Measure the extinction at 400 nm
against the blank solution.

Read the absorbance (or other indications on the scale of the calorimeter).

8. Expression of results
With the aid of the calibration curve obtained according to 7.1 calculate the ppm phosphorus in the fat as
follows

ppm P = CF x 25 x Abs
Wt. of oil (g)
where CF is calibration factor obtained earlier (7.1)

Reference: IUPAC Seventh Edition: 2.421

Acknowledgement : By the kind permission of PORIM for allowing us to use this method of analysis from their publication in the
book “ PORIM TEST METHODS”

26
 METHODS OF TEST FOR
PALM OIL AND PALM OIL PRODUCTS

M13 - Determination of Saponification Value

1. Definition
The saponification value is the number of milligrams of potassium hydroxide required to saponify 1 g of
fat under the conditions specified. It is a measure of the average molecular weight of all the fatty acids
present.

2. Principle
The glycerides present are split by alcoholic alkali and any free fatty acids are neutralised. Excess alkali
is back-titrated with hydrochloric acid in the presence of an indicator.

3. Scope
Applicable to all normal oils and fats.

4. Reagents
4.1. Potassium hydroxide, approximately 0.5 N solution in 95% (v/v) ethanol.
The solution should be colourless or straw yellow (Note 1).
4.2. Hydrochloric acid, 0.5N, accurately standardised.
4.3. Phenolphthalein solution, 1% (v/v) in 95% ethanol.
4.4. Boiling aids e.g. pumice stone.

5. Apparatus
5.1. Conical flask, 250 ml capacity, made of alkali resistant glass, with round neck.
5.2. Reflux condenser, with ground glass joint to fit the flask.
5.3. Water bath or a hot-plate with variable heat control.
5.4. Burette, 50 ml capacity graduated in 0.1 ml divisions.
5.5. Pipette, 25 ml.

6. Preparation of Sample for Analysis

Melt and homogenise the sample at 60°C - 70°C before taking a test portion. If necessary, filter
through a dry filter paper to remove any impurities.

7. Procedure
Weigh, to the nearest 0.005 g, a test portion into a conical flask such that the volume of hydrochloric acid
required for the determination will be about half that required for the blank determination. This weight will
usually be around 2 g.

Add 25 ml of the ethanolic potassium hydroxide solution and some boiling aids.

Connect the reflux condenser and boil gently for at least 60 minutes, swirling the contents of the flask from
time to time.

27
 Allow the flask and condenser to cool slightly before washing the inside of the condenser with a little
distilled water. Add 1 ml of phenolphthalein solution and titrate with the 0.5 N hydrochloric acid until
the pink colour of the indicator just disappears.

Conduct a blank determination simultaneously with the test sample using the same procedure.

8. Expression of Results
The saponifiable value is given by

SV = 56.1 N(Vb - V,)

W

where Vb is the volume, in millilitres of the hydrochloric acid solution used for the blank;
Vs is the volume, in millilitres of the hydrochloric acid used for the determination of the
sample;
N is the normality of the hydrochloric acid;
W is the weight, in grams of the test portion

Express the result to the nearest unit.

9. Repeatability
The difference between the results of two determinations on the same test sample, carried out
simultaneously or in rapid succession by the same analyst, should not exceed 0.5% of their mean value.

Note 1 : A stable, colourless solution of potassium hydroxide can be prepared as follows

Either (a) Reflux 1 litre of ethanol with 8 g of potassium hydroxide and 5 g of aluminium pellets for 1 hr, then distil immediately.
Dissolve potassium hydroxide in the distillate to give a solution containing approximately 28 g per litre.

or (b) Add 4 g aluminium tert-butoxide to 1 litre of ethanol and allow the mixture to stand for several days. Decant the supernatant
liquid and dissolve potassium hydroxide in it to give a solution containing approximately 28 g per litre.

Store the solution in a brown glass bottle fitted with a rubber stopper. Decant the solution before use.

Reference : British standards BS 684 2.6: 1977.

Acknowledgement : By the kind permission of PORIM for allowing us to use this method of analysis from their publication in the
book “ PORIM TEST METHODS”

28
 METHODS OF TEST FOR
PALM OIL AND PALM OIL PRODUCTS

M14 - Determination of slip melting point (slip point)

1. Definition
The slip melting point (slip point) is the temperature at which a column of fat, of specified length, rises
in an open capillary tube under the specified conditions of the test.

2. Principle
A prepared capillary tube containing a column of the fat is immersed in a bath of water which is
warmed at specified rate until the melting point is reached,

3. Scope
This method is applicable to all normal fats and vegetable oils which are solid at ambient temperature,
in particular to palm oil and palm oil products.

4. Apparatus
4.1. Capillary glass tubes, uniformly thin-walled, open at both ends with an internal diameter of 1.1 to
1.3 mm, external diameter of 1.4 to 1.7 mm and a length of 50 to 60 mm.

4.2. Thermometer, subdivided at every 0.2°C or less and with a suitable range, calibrated prior to
usage.

4.3. Glass beaker, 500 ml to 600 ml capacity.

4.4. Heat source - electrical hot plate with rheostat and stirring controls.

4.5. Magnetic stirrer.

4.6. Test-tube of 8 mm internal diameter and 1 mm thickness.

4.7. Thermostat water-bath or incubator set at 10 ±1°C

A refrigerator set at this temperature may be used but care should be exercised to avoid opening
it frequently once the samples are being tempered in it.

5. Preparation of sample
Melt the sample and filter through a filter paper. Conduct the filtration in an oven set at 60°C to avoid
any crystallisation of the sample. Leave the filtered sample in the oven for 10 minutes till it is free of air
bubbles.

6. Procedure
Dip at least three clean capillary tubes into the completely liquid sample so that columns of fat ca. 10 mm
high are obtained in the tubes. Chill the fat column at once by holding and rolling the ends of the
tubes containing the sample pressed against a piece of ice, until the fat has solidified. Do not allow the
open end of the tube to touch the ice. Wipe the tubes against a piece of tissue paper as quickly as
possible. Place the tubes in a test-tube which is held in a beaker of water that has been equilibrated at 10
± 1°C in a thermostated water-bath. Transfer the beaker to the water bath and hold for 16 hours at 10
± 1°C.

29
 Remove the capillary tubes from the test-tube and attach with a rubber band to a thermometer such that
the lower ends of the tubes are level with the bottom of the mercury bulb of the thermometer.

Suspend the thermometer in the beaker containing 400 ml of boiled distilled water. The bottom of the
thermometer is immersed in the water to a depth of ca. 30 mm.

Adjust the starting temperature of the bath to 8 to 10°C below the expected slip point of the sample.
Agitate the water bath with a magnetic stirrer and apply heat so as to increase the temperature at a rate
of 1°C per minute, slowing down to 0.5°C per minute as the slip point is reached.

Continue heating until the fat column rises in each tube. Observed the temperature of the water at which
each column rises and calculate the average of all tubes. The difference between the three values should
not be more than 0.3°C.

7. Expression of results
Record the average value of two sets of triplicate results as the slip melting point, expressed to one
decimal place.

8. Repeatability
The difference between values of two determinations carried by the same operator using the same
apparatus on the same test sample, shall not exceed 0.8°C for palm oil, 0.5°C for palm olein and
stearin.

Note: Rapid Method for Quality Control Purposes

For a quick test, the 16 hours may be reduced to 2 hours or 1 hour. The result should be reported as
slip point (2 hours) or (1 hour). For most fats the slip point (2 hours) is about 0.2°C lower than
standard slip point (16 hours). For palm olein the difference is greater, about 1.2°C.

Reference: AOCS Cc 3-25 (1989).

Acknowledgement : By the kind permission of PORIM for allowing us to use this method of analysis from their publication in the
book “ PORIM TEST METHODS”

30
 METHODS OF THE TEST
FOR PALM OIL AND PALM OIL PRODUCTS

M15 - Determination of Soap Content

1. Definition The method determines soap as sodium palmitate in palm oil products.

2. Principle of Method
Volumetric titration with hydrochloric acid using bromophenol blue as indicator.

3, Scope
Applicable to oils and fats.

4. Reagents
The reagents used shall be of recognised analytical reagent quality.
4.1. Distilled acetone, containing 2% of added water.

4.2. Hydrochloric acid, 0.01N, accurately standardised.

4.3. Bromophenol blue indicator, 1% solution in 95% (v/v) ethanol.

5. Apparatus
5.1. Test tubes, approximately 150 x 40 mm of borosilicate glass fitted with ground glass stoppers and
flattened at their ends.

5.2. Microburette, 5 ml.

6. Procedure
6.1. Prepare the test solution by adding 0.5 ml of the bromophenol blue indicator to each 100 ml of the
aqueous acetone just before use and titrating with 0.01 N acid or alkali until it is just yellow in
colour.

6.2. Weigh 40 g of the oil or fat to be tested into the test tube which shall have been previously well
rinsed with the test solution.

6.3. Add 1 ml of water, warm on the steam bath and shake vigorously. Add 50 ml of the neutralised
aqueous acetone and, after warming on the steam bath, shake the test tube well and allow the
contents to stand until they separate into two layers. If soap is present in the oil or fat, the upper
layer will be coloured green to blue.

6.4. Then add 0.01N acid, preferably from a Microburette, until the yellow colour is restored. Continue
the process of warming and shaking until the yellow colour of the upper layer remains permanent.

31
 7. Calculation and Expression of Results

For palm oil products, soap is expressed as sodium palmitate, percent by mass

0.278 T

where T = volume in ml of 0.01N acid required, and

M = mass in g of sample taken

For lauric fats, soap is expressed as sodium laurate,

= 0.222 T
M

For other oils, soap is expressed as sodium oleate,

= 0.304 T
M

Note 1: The above method is suitable for the determination of soap in oils up to 0.05%. At higher
concentrations it is better to take 4 g of oil and use 0.01N acid.

Reference
British Standard 684 2.5: 1977.

Acknowledgement : By the kind permission of PORIM for allowing us to use this method of analysis from their publication in the
book “ PORIM TEST METHODS”

32
 METHODS OF TEST FOR
PALM OIL AND PALM OIL PRODUCTS

M16 - Determination of Unsaponifiable Matter

1. Definition
The unsaponifiable matter is the total quantity of substances present in an oil or fat which after
saponification with an alkaline hydroxide are insoluble in water but soluble in the solvent used for the
determination.

It includes hydrocarbons, higher aliphatic alcohols, sterols tocopherols/ tocotrienols and pigments as well
as any foreign organic matter non-volatile at 103°C (e.g. mineral oil or added antioxidants) which may
be present.

2. Principle
Saponification of the fat by an ethanolic alkaline hydroxide solution, dilution, then extraction of the
unsaponifiable matter with petroleum ether (40°C - 60°C) or n-hexane.

3. Scope
Applicable to normal animal and vegetable fats and oils, soaps and soap products.

4. Reagents
4.1. Ethanol 95% (v/v).
4.2. Petroleum ether (40°C - 60°C) or n-hexane. Both solvents should be free of residue.
4.3. Sodium hydroxide solution, 0.02N, accurately standardised.
4.4. Phenolphthalein 1% (w/v) in ethanol 95% (v/v).
4.5. Aqueous potassium hydroxide solution, 50% KOH by weight,

5. Apparatus
5.1. Round-bottomed flasks with ground joints, 250 ml capacity.
5.2. Reflux condenser to fit the flask.
5.3. Separating funnels, 500 ml capacity.
5.4. Water-bath.
5.5. Vacuum oven
5.6. Rotary evaporator

6. Preparation of sample for analysis

Melt the sample at 60°C - 70°C. Homogenise thoroughly before taking a test portion.

7. Procedure
Weigh accurately about 5 g of the well-mixed sample into the round-bottomed flask.

Add 30 ml of ethanol and 5 ml of aqueous potassium hydroxide solution.

Attach the reflux condenser and boil gently for 1 hour. Stop heating. Transfer the reaction mixture
into the separating funnel. Rinse the flask with 10 ml ethanol followed by 20 ml warm and then 20 ml
cold distilled water, transferring all the washings to the separating funnel. Wash out the flask with 10
ml petroleum ether and add to the separating funnel.

33
 Allow the contents of the separating funnel to cool to room temperature before adding 50 ml of
petroleum ether. Stopper the separating funnel and shake vigorously for 1 minute. Allow to stand
until there is complete separation of the two phases. Draw off the soap solution as completely as
possible into a second separating funnel. Extract the soap solution with a further portion of 50 ml
petroleum ether, transferring the soap solution to a third separating funnel and the solvent extract to the
first funnel.

Repeat the extractions using 50 ml portions of petroleum ether each time, at least five more times,
shaking vigorously with each extraction.

Wash the combined extracts in the separating funnel three times with 25 ml portions of 10% (v/v)
ethanol, shaking vigorously and drawing off the ethanol layer after each wash. Be careful not to
remove any of the petroleum ether layer.

Transfer the petroleum ether extract quantitatively a little at a time into a 250 ml round-bottomed flask
previously dried and weighed to the nearest 0.0001 g.

Evaporate the petroleum ether to dryness under vacuum using a rotary evaporator. Complete the drying
to constant weight in a vacuum oven at 75°C - 80°C and internal pressure of not more than 200 mm of
mercury (Note 1). Cool in a desiccator and weigh. The loss of weight between two successive
weighings should be less than 0.0015 g. After weighing, dissolve the residue in 50 ml of warm (ca. 50°C)
95% ethanol previously neutralised to a faint pink colour using phenolphthalein indicator. Titrate with
0.02 N sodium hydroxide solution to the same final colour. Correct the weight of the residue for the
free fatty acid content.

Wt. of fatty acids in the extract = vol. of 0.02 NaOH x 0.0056

8. Expression of results
The amount of unsaponifiable matter expressed as percentage by weight of the sample is given by

100 (W1 - Wt. of fatty acids)

where W is the weight in grams, of the test portion

W1 is the weight in grams, of the residue

Note 1: In the absence of a vacuum oven, dry the residue in an electric oven regulated at 103 ± 2°C for fifteen minutes, placing the flask in an
almost horizontal position. Cool in a desiccator and weigh to the nearest 0.0001 g. Repeat the drying procedure until the difference
between two successive weighings is less than 0.0015 g.

Reference: AOCS Ca 6a - 40 (1989).

Acknowledgement : By the kind permission of PORIM for allowing us to use this method of analysis from their publication in the
book “ PORIM TEST METHODS”

34
 METHODS OF TEST FOR
PALM OIL AND PALM OIL PRODUCTS

M17 - Determination of Volatile Matter

1. Definition
The volatile matter is defined as the loss in weight of the oil when heated under the test conditions
specified.

2. Principle
The method consists of heating the oil sample in an oven at 103 ± 2°C and fitted with a suitable
thermometer.

3. Scope
Applicable to crude and refined palm oil products.

4. Apparatus
4.1. Electric oven, regulated at 103 ± 2°C and fitted with a suitable thermometer.

4.2. Petri dishes, glass crystallising dishes (25 ml) of diameter 5.5 - 7.0 cm.

4.3. Desiccator with efficient desiccant.

5. Preparation of sample for analysis

Determination of volatile matter should be the first test to be performed on a sample.

6. Procedure
Dry a cleaned petri dish in the oven at 103°C for at least 15 minutes and allow to cool in a desiccator.
Weigh the dish to the nearest 0.1 mg.

Introduce ca 10 ± 1.0 g of the molten oil into the dish. Return the dish to the desiccator until the oil has
thoroughly cooled. Weigh the dish plus the oil to the nearest 0. 1 mg and place the dish in the middle
shelf of the oven at 103°C for exactly 2.5 hours.

Remove the dish and allow it to cool to room temperature in the desiccator (30 - 45 minutes) before
reweighing to the nearest 0.1 mg.

If the volatile matter of the oil exceeds 0.3%, continued drying (at 30 minutes interval) to constant weight
is recommended. In this case, the difference between two successive weighings should not exceed 0.002
g.

35
7. Expression of results
The volatile matter is expressed as a percentage by weight using the formula :

Wb - Wd
% volatile matter = x 100
Wb - W

where W weight of dish

Wb weight of dish and oil
Wd weight of dish and oil after drying

The results are to be expressed to 3 decimal places,

8. Repeatability
The difference between the results of two determinations carried out simultaneously or in rapid
succession by the same analyst should not exceed 0.003% for contents of volatile matter between 0.04%
and 0.300%.

Note 1: It is important to completely cool the dish to balance temperature since even a small deviation from
this temperature can affect the precision and accuracy of weighing. Glass dishes take an
appreciable time to cool especially if large numbers are place in the desiccator at the same time. As
much as one hour may be required for a full desiccator load to cool completely.

Note 2: The performance of the oven must be checked periodically. This is done using a thermometer
placed in a dish filled with the same amount of oil and placed at the same level in the oven as the
sample being tested.

Note 3: The oven door must be kept closed during the entire test. The ventilation hoods should not be
operated once a temperature of 103°C has been established. The oven should not be used for other
purposes during a test.

Note 4: It is convenient to transfer the dishes by hand using a clean cotton glove for protection.

Reference: MOPGC Test Methods.

Acknowledgement : By the kind permission of PORIM for allowing us to use this method of analysis from their publication in the
book “ PORIM TEST METHODS”

36