1991R2568 — EN — 21.07.1992 — 004.001 — 1
This document is meant purely as a documentation tool and the institutions do not assume any liability for its contents
►B COMMISSIONREGULATION(EEC)No2568/91
of11July1991
onthecharacteristics ofolive oilandolive-residue oilandontherelevantmethodsofanalysis
(OJ L 248, 5.9.1991, p. 1)
Amended by:
Official Journal
No page date
►M1CommissionRegulation(EEC) No 3682/91 of 17 December 1991 L 349 36 18.12.1991
►M2CommissionRegulation(EEC) No 1429/92 of 26 May 1992 L 150 17 2.6.1992
►M3CommissionRegulation(EEC) No 1683/92 of 29 Jun e 1992 L 176 27 30.6.1992
►M4CommissionRegulation(EEC) No 1996/92 of 15 July 1992 L 199 18 18.7.1992
Corrected by:
►C1Corrigendum, OJ L 347, 28.11.1992, p. 69 (2568/91)
▼B
COMMISSIONREGULATION(EEC)No2568/91
of11July1991
on the characteristics of olive oil and olive-residue oil and on the
relevantmethodsofanalysis
THE COMMISSION OF THE EUROPEAN COMMUNITIES,
Having regard to the Treaty establishing the European EconomicCommunity,
Having regard to Council Regulation No 136/66/EEC of 22 September
1966 on the establishment of a common organization of the market inoils and fats(
1), as last amended by Regulation (EEC) No 3577/90(2),
and in particular Article 35a thereof,
Whereas the Annex to Regulation No 136/66/EEC contains the descrip-
tions and definitions of olive oil and olive-residue oil marketed withineach Member State, in intra-Community trade and in trade with thirdcountries;
Whereas, for the purpose of differentiating between the various types
of oil, the physical and chemical characteristics of each of them andthe organoleptic characteristics of virgin oil should be defined, in order
to guarantee the purity and quality of the products concerned, without
prejudice to other existing provisions;
Whereas the presence of the characteristics of the different types of oil
should be determined uniformly throughout the Community; whereas,
to that end, Community methods of chemical analysis and organolepticevaluationshould be established; whereas the use should be permitted,for a transitional period, of other methods of analysis applied in theMember States provided that where there is a difference in the results,
those obtained using the common method will be decisive;
Whereas the definition of the physical and chemical characteristics of
olive oil and of the methods of analysis entails the amendment of the
additional notes to Chapter 15 of the combined nomenclature;
Whereas the method of evaluating the organoleptic characteristics of
virgin oil includes the setting up of panels of selected and trained
tasters; whereas the period necessary for establishing such a structure
should therefore be fixed; whereas inview of the difficulties thatsome Member States will encounter in setting up panels of tasters, theuse of panels in other Member States should be authorized;
Whereas, inorder to en sure that the system of levies applicable to
imports of olive residues functions correctly, a single method for thedetermination of the oil content of these products should be laid down;
Whereas, inorder n ot to harm trade, provisionshould be made for oil
packaged prior to the entry into force of this Regulation to be disposedof during a limited period;
Whereas it is necessary to repeal Commission Regulation (EEC)
No 1058/77(
3), as last amended by Regulation (EEC) No 1858/88(4);
Whereas the Management Committee for Oils and Fats has not deliv-
ered an opinion within the time limit set by its chairman,
HAS ADOPTED THIS REGULATION:
Article 1
1. Oils, the characteristics of which comply with those set out in
points 1, 2 and 3 of Annex I to this Regulation, shall be deemed to be1991R2568 — EN — 21.07.1992 — 004.001 — 2
(1) OJ No 172, 30. 9. 1966, p. 3025/66.
(2) OJ No L 353, 17. 12. 1990, p. 23.
(3) OJ No L 128, 24. 5. 1977, p. 6.
(4) OJ No L 166, 1. 7. 1988, p. 10.
▼B
virgin olive oil within the meaning of point 1 (a), (b) and (c) of the
Annex to Regulation No 136/66/EEC.
2. Oil, the characteristics of which comply with those set out in
point 4 of Annex I to this Regulation, shall be deemed to be lampante
virgin olive oil within the meaning of point 1 (d) of the Annex to
RegulationNo 136/66/EEC.
3. Oil, the characteristics of which comply with those set out in
point 5 of Annex I to this Regulation, shall be deemed to be refinedolive oil within the meaning of point 2 of the Annex to RegulationNo 136/66/EEC.
4. Oil, the characteristics of which comply with those set out in
point 6 of Annex I to this Regulation, shall be deemed to be pure oliveoil within the meaning of point 3 of the Annex to Regulation No 136/66/EEC.
5. Oil, the characteristics of which comply with those set out in
point 7 of Annex I to this Regulation, shall be deemed to be olive-
residue oil within the meaning of point 4 of the Annex to RegulationNo 136/66/EEC.
6. Oil, the characteristics of which comply with those set out in
point 8 of Annex I to this Regulation, shall be deemed to be refinedolive-residue oil within the meaning of point 5 of the Annex to Regu-lationNo 136/66/EEC.
7. Oil, the characteristics of which comply with those set out in
point 9 of Annex I to this Regulation, shall be deemed to be olive-residue oil within the meaning of point 6 of the Annex to Regulation
No 136/66/EEC.
Article 2
1. The characteristics of the oils laid down in Annex I shall be
determined in accordance with the methods of analysis set out below:
— for the determination of the free fatty acids, expressed as the
percentage of oleic acid, the method set out in Annex II,
— for the determination of the peroxide index, the method set out in
Annex III,
— for the determination of aliphatic alcohols, the method set out in
Annex IV,
— for the determination of the sterol content, the method set out in
Annex V,
— for the determination of erythrodiol and uvaol, the method set out
in Annex VI,
— for the determination of the saturated fatty acids in position 2 of the
triglyceride, the method set out in Annex VII,
— for the determination of the trilinolein content, the method set out
in Annex VIII,
— for spectrophotometric analysis, the method set out in Annex IX,
— for the determination of the fatty acid composition, the method set
out in Annex X A and X B,
— for the determination of the volatile halogenated solvents, the
method set out in Annex XI,
— for the evaluationof the organ oleptic characteristics of virginolive
oil, the method set out in Annex XII,
— for proof that refining has taken place, the method set out in Annex
XIII.
2. Evaluationof the organ oleptic characteristics shall be carried out
by►
M3an analyst specializing in sensory analysis or assisted by
specialists ◄, according to the procedure described in the tasting notes
referred to in Annex XII. Where analysis shows different characteris-tics to those resulting from the description of the product, the samplemust be examined by a panel of tasters in accordance with the provi-sions of Annex XII.1991R2568 — EN — 21.07.1992 — 004.001 — 3
▼B
Any second analysis shall be carried out by the panel according to the
said provisions.
In order to ascertain the organoleptic characteristics in connection with
operations relating to the intervention system, the panel of tasters willcarry out this evaluation in accordance with the provisions of AnnexXII.
Article 3
Until 31 October 1992, the introduction of the analysis methods
provided for inArticle 2 shall n ot impede the use by the MemberStates of other tested and scientifically valid methods, provided that
products recognized as complying with the rules in force governing
Community methods shall be allowed to move freely. Before usingother methods, the Member States concerned shall notify them to theCommission.
Where one of the other methods produces a result different from that
produced by the commonmethod, the result obtain ed by the lattermethod shall be determinant.
Article 4
1. For the purpose of assessing organoleptic characteristics, the
Member States shall set up panels of trained and selected tasters inaccordance with the rules laid down by the method set out in AnnexXII.
2. Where a Member State encounters difficulties in setting up a
panel in its territory, it may use the services of a panel operating inanother Member State.
Article 5
The additional notes 2, 3 and 4 to Chapter 15 of the combined nomen-
clature are replaced by those contained in Annex XIV.
Article 6
1. The oil content of oil cake and other residues resulting from the
extractionof olive oil (CN codes 2306 9011 an d 230690 19) shall bedetermined using the method set out in Annex XV.
2. The oil content referred to in paragraph 1 shall be expressed as a
percentage of the weight of oil to the weight of dry matter.
Article 7
The Community provisions concerning the presence of undesirable
substances, other than those referred to in Annex XI, shall apply.
Article 8
1. Member States shall notify the Commission of the measures taken
to implement this Regulation.
2. Member States shall send the Commission, at the beginning of
each half-year, a statement of the analytical data relating to the tests
carried out during the previous half-year.
The results shall be considered by the Management Committee for Oils
and Fats in accordance with the procedure laid down in Article 39 of
RegulationNo 136/66/EEC.
Article 9
Regulation(EEC) No 1058/77 is hereby repealed.
Article 10
1. This Regulation shall enter into force on the third day following
its publicationinthe Official Journal of the European Communities .1991R2568 — EN — 21.07.1992 — 004.001 — 4
▼B
However, the method set out in Annex XII shall apply from ►M11
November 1992 ◄,except in so far as operations relating to the inter-
vention system are concerned .
2. This Regulation shall not apply to olive oil and olive-residue oil
packaged before the entry into force of this Regulation and marketedup to 31 October 1992.
This Regulation shall be binding in its entirety and directly applicable
inall Member States.1991R2568 — EN — 21.07.1992 — 004.001 — 5
▼B
ANNEXES
Summary
Annex I: Characteristics of olive oil …
Annex II: Determination of the free fatty acids …
Annex III: Determination of the peroxide value …Annex IV: Determination of aliphatic alcohols content by capillary gas
chromatography …
Annex V: Determination of the composition and content of sterols by
capillary-columngas chromatography …
Annex VI: Determination of erythrodiol and uvaol …Annex VII: Determination of the saturated fatty acids in position 2 of the
triglyceride …
Annex VIII: Determination of composition of trilinolein …Annex IX: Spectrophotometric investigation in the ultraviolet …
Annex XA: Analysis by gas chromatography of methyl esters of fatty acids
…
Annex XB: Preparation of methyl esters of fatty acids …
Annex XI: Determination of the volatile halogenated solvents of olive oil
…
Annex XII: Organoleptic assessment of virgin olive oil …
Annex XIII: Proof that refining has taken place …Annex XIV: Additional Notes 2, 3 and 4 to Chapter 15 of the combined
nomenclature …
Annex XV: Oil content of olive residue …
Annex XVI: Determination of iodine value …1991R2568 — EN — 21.07.1992 — 004.001 — 6
ANNEX I
CHARACTERISTICSOFOLIVEOIL
Type►C1
Acidity
%
◄Peroxide
value
meq/O2/kgHaloge-
nated
solvents
mg/kg (1)Aliphatic
alcohols
mg/kgSaturated
fatty acids
inposition
2 of trigly-
ceride
%Erythiodiol
+ Uvaol
%Trilinolein
%Cholesterol
%Brassicas-
terol
%Campes-
terol
%Stigmas-
terol
%Beta-sitos-
terol
%(2)Delta-7-
stigmas-
terol
%Total
sterols
mg/kg
1. Extra virginolive
oil M 1,0 M 20 M 0,20 M 300 M 1,3 M 4,5 M 0,5 M 0,5 M 0,2 M 4,0 < Camp. m 93,0 M 0,5 m 1 000
2. Virginolive oil M 2,0 M 20 M 0,20 M 300 M 1,3 M 4,5 M 0,5 M 0,5 M 0,2 M 4,0 < Camp. m 93,0 M 0,5 m 1 0003. Ordinary virgin
olive oil M 3,3 M 20 M 0,20 M 300 M 1,3 M 4,5 M 0,5 M 0,5 M 0,2 M 4,0 < Camp. m 93,0 M 0,5 m 1 000
4. Virginlampan te
olive oil > 3,3 > 20 > 0,20 M 400 M 1,3 M 4,5 M 0,5 M 0,5 M 0,2 M 4,0 — m 93,0 M 0,5 m 1 000
5. Refined olive oil M 0,5 M 10 M 0,20 M 350 M 1,5 M 4,5 M 0,5 M 0,5 M 0,2 M 4,0 < Camp. m 93,0 M 0,5 m 1 000
6. Olive oil M 1,5 M 15 M 0,20 M 350 M 1,5 M 4,5 M 0,5 M 0,5 M 0,2 M 4,0 < Camp. m 93,0 M 0,5 m 1 000
7. Crude olive residue
oil m 2,0 — — — M 1,8 m 12 M 0,5 M 0,5 M 0,2 M 4,0 — m 93,0 M 0,5 m 2 500
8. Refined olive
residue oil M 0,5 M 10 M 0,20 — M 2,0 m 12 M 0,5 M 0,5 M 0,2 M 4,0 < Camp. m 93,0 M 0,5 m 1 800
9. Olive residue oil M 1,5 M 15 M 0,20 — M 2,0 > 4,5 M 0,5 M 0,5 M 0,2 M 4,0 < Camp. m 93,0 M 0,5 m 1 800
M = maximum, m = minimum.
(1) Overall upper limit for compounds detected by electron capture detector. For components detected individually the upper limit is 0,10 mg/kg.
(2) Delta-5-23- ►C1stigmastadienol + chlerosterol ◄+ sitosterol + sitostanol + delta-5-avenosterol + delta-5-24 stigmastadienol.
Note:
An oil is to be rejected if any one of its characteristics lies outside the limit laid down.▼B1991R2568 — EN — 21.07.1992 — 004.001 — 7
TypeAcid composition
Sum (of
the) transo-
leic isomers
%Sum (of
the) transli-
noleic and
translino-
lenic
isomers
%K232K270K270 with
aluminium
oxide +Delta K Panel test
Myristic
%Linolenic
%Arachidic
%Eicosanoic
%Behenic
%Lignoceric
%
1. Extra virginolive oil M 0,1 M 0,9 M 0,7 M 0,5 M 0,3 M 0,5 < 0,03 < 0,03 M 2,40 M 0,20 M 0,10 M 0,01 ≥6,5
2. Virginolive oil M 0,1 M 0,9 M 0,7 M 0,5 M 0,3 M 0,5 < 0,03 < 0,03 M 2,50 M 0,25 M 0,10 M 0,01 ≥5,5
3. Ordinary virgin olive
oil M 0,1 M 0,9 M 0,7 M 0,5 M 0,3 M 0,5 < 0,03 < 0,03 M 2,50 M 0,25 M 0,10 M 0,01 ≥3,5
4. Virginlampan te olive
oil M 0,1 M 0,9 M 0,7 M 0,5 M 0,3 M 0,5 < 0,10 < 0,10 M 3,70 > 0,25 M 0,11 — < 3,5
5. Refined olive oil M 0,1 M 0,9 M 0,7 M 0,5 M 0,3 M 0,5 < 0,20 < 0,30 M 3,40 M 1,20 — M 0,16 —6. Olive oil M 0,1 M 0,9 M 0,7 M 0,5 M 0,3 M 0,5 < 0,20 < 0,30 M 3,30 M 1,00 — M 0,13 —7. Crude oil residue oil M 0,1 M 0,9 M 0,7 M 0,5 M 0,3 M 0,5 < 0,20 < 0,10 —————
8. Refined olive residue
oil M 0,1 M 0,9 M 0,7 M 0,5 M 0,3 M 0,5 < 0,40 < 0,35 M 5,50 M 2,50 — M 0,25 —
9. Olive residue oil M 0,1 M 0,9 M 0,7 M 0,5 M 0,3 M 0,5 < 0,40 < 0,35 M 5,30 M 2,00 — M 0,20 —▼M21991R2568 — EN — 21.07.1992 — 004.001 — 8
▼B
ANNEX II
DETERMINATIONOFTHEFREEFATTYACIDS
1. DETERMINATION OF ACIDITY
The determination of free fatty acids in olive oils. The content of free
fatty acids is expressed as acidity calculated conventionally.
1.1.Principle
A sample is dissolved in a mixture of solvents and the free fatty acids
present titrated using an ethanolic solution of potassium hydroxide.
1.2.Reagents
All the reagents should be of recognized analytical quality and the water
used either distilled or of equivalent purity.
1.2.1.►C1Diethyl ether ◄; 95 % ethanol (v/v), mixture of equal parts by
volume.
Note:►C1Diethyl ether ◄is highly inflammable and may form
explosive peroxides. Special care should be takeninits use.
Neutralize precisely at the moment of use with the potassium hydroxide
solution(1.2.2), with the additionof 0,3 ml of the phen olpthaleinsolution
(1.2.3) per 100 ml of mixture.
Note:If it is not possible to use ►C1diethyl ether ◄, a mixture of
solvents containing ethanol and toluene may be used. If necessary,
ethanol may be replaced by propanol-2.
1.2.2. Potassium hydroxide, titrated ethanolic solution, c(KOH) about 0,1 mol/l
or, if necessary, c(KOH) about 0,5 mol/l.
The exact concentration of the ethanolic solution of potassium hydroxide
must be known and checked immediately prior to use. Use a solution
prepared at least five days before use and decanted into a brown glass
bottle with a rubber stopper. The solutionshould be colourless or straw
coloured.
Note:A stable colourless solutionof potassium hydroxide may be
prepared as follows. Bring to the boil 1 000 ml of ethanol with 8 g
of potassium hydroxide and 0,5 g of aluminium shavings and
continue boiling under reflux for one hour. Distill immediately.
Dissolve inthe distillate the required quan tity of potassium hydro-
xide. Leave for several days and decant the clear supernatant liquid
from the precipitate of potassium carbonate.
The solutionmay also be prepared without distillationas follows:
to 1 000 ml of ethanol add 4 ml of aluminium butylate and leave
the mixture for several days. Decant the supernatant liquid and
dissolve the required quantity of potassium hydroxide. The solu-
tionis ready for use.
1.2.3. Phenolphthalein, 10 g/l solution in 95 to 96 % ethanol (v/v) or alkaline
blue, (inthe case of stron gly coloured fats) 20 g/l solutionin95 to 96 %
ethanol (v/v).
1.3.Apparatus
Usual laboratory equipment including:
1.3.1. analytical balance;1.3.2. 250 ml conical flask;1.3.3. 10 ml burette, graduated in0,05 ml.
1.4.Procedure
1.4.1. Preparationof the specimenfor testin g
(Carry out the test onthe filtered sample. Where moisture an d impurities
together are less than1 %, use the specimenwithout further treatmen t;
where they exceed 1 %, it should be filtered.)
1.4.2. Taking the sample1991R2568 — EN — 21.07.1992 — 004.001 — 9
▼B
Take a sample depending on the presumed acid number in accordance
with the following table:
Expected acid valueMass of sample
(g)Weighing accuracy
(g)
< 1 20 0,05
1 to 4 10 0,024 to 15 2,5 0,01
15 to 75 0,5 0,001
> 75 0,1 0,0002
Weigh the sample inthe con ical flask (1.3.2).
1.4.3. Determination
Dissolve the sample (1.4.2) in50 to 150 ml of the previously n eutralized
mixture of diethyl ►
C1ether◄and ethanol (1.2.1).
Titrate while stirring with the 0,1 mol/l solution of potassium hydroxide
(1.2.2) (see Note 2) until the indicator changes (the pink colour of the
phenolphtalein persists for at least 10 seconds).
Note 1.The titrated ethanolic solution of potassium hydroxide (1.2.2)
may be replaced by anaqueous solutionof potassium or sodium
hydroxide provided that the volume of water introduced does not
induce phase separation.
Note 2.If the quantity of 0,1 mol/l potassium hydroxide solution required
exceeds 10 ml, use the 0,5 mol/l solution.
Note 3.If the solution becomes cloudy during titration, add enough of
the solvents (1.2.1) to give a clear solution.
1.5.Acidity:expressedaspercentageofoleic acid
Acidity as a percentage by weight is equal to:
V/C2c/C2M
1000/C2100
m¼V/C2c/C2M
10/C2m
where:
V = the volume of titrated potassium hydroxide solutionused, inmilli-
litres;
c = the exact concentration in moles per litre of the titrated solution of
potassium hydroxide used;
M = the molar weight ingrams per mole of the acid used to express the
result (= 282);
m = the weight ingrams of the sample.
Take as the result, the arithmetic mean of the two determinations carried
out.1991R2568 — EN — 21.07.1992 — 004.001 — 10
▼C1
▼B
ANNEX III
DETERMINATIONOFPEROXIDEVALUE
1. SCOPE
This Standard describes a method for the determination of the peroxide
value of oils and fats.
2. FIELD OF APPLICATION
This Standard is applicable to animal and vegetable oils and fats.
3. DEFINITION
The peroxide value is the quantity of those substances in the sample,
expressed interms of milliequivalen ts of active oxygenper kilogram,
which oxidize potassium iodide under the operating conditions described.
4. PRINCIPLE
Treatment of the test portion, in solution in acetic acid and chloroform, by
a solution of potassium iodide. Titration of the liberated iodine with stan-
dardized sodium thiosulphate solution.
5. APPARATUS
All the equipment used shall be free from reducing or oxidizing substances.
Note:Do not grease ground surfaces.
5.1. 3 ml glass scoop.5.2. Flasks, with ground necks and stoppers, of about 250 ml capacity, dried
beforehand and filled with a pure, dry inert gas (nitrogen or, preferably,
carbondioxide).
5.3. 25- or 50-ml burette, graduated in0,1 ml.
6. REAGENTS
6.1. Chloroform, analytical reagent quality, freed from oxygen by bubbling a
current of pure, dry inert gas through it.
6.2. Glacial acetic acid, analytical reagent quality, freed from oxygen by
bubbling a current of pure, ►
C1dry inert gas ◄through it.
6.3. Potassium iodide, saturated aqueous solution, recently prepared, free from
iodine and iodates.
6.4. Sodium thiosulphate, 0,01 or 0,002 ►C1Mol/L accurately ◄standar-
dized aqueous solution, standardized just before use.
6.5. Starch solution, 10 g/l aqueous dispersion, recently prepared from natural
soluble starch.
7. SAMPLE
Take care that the sample is takenan d stored away from the light, kept
cold and contained in completely filled glass containers, hermetically
sealed with ground-glass or cork stoppers.
8. PROCEDURE
The test shall be carried out indiffuse daylight or inartificial light. Weigh
in a glass scoop (5.1) or, failing this, in a flask (5.2), to the nearest 0,001 g,
a mass of the sample in accordance with the following table, according to
the expected peroxide value:1991R2568 — EN — 21.07.1992 — 004.001 — 11
▼B
Expected peroxide value
(meq)Weight of test portion
(g)
0 to 12 5,0 to 2,0
12 to 20 2,0 to 1,220 to 30 1,2 to 0,830 to 50 0,8 to 0,5
50 to 90 0,5 to 0,3
Unstopper a flask (5.2) and introduce the glass scoop containing the test
portion. Add 10 ml of chloroform (6.1). Dissolve the test portion rapidly
by stirring. Add 15 ml of acetic acid (6.2), then 1 ml of potassium iodide
solution (6.3). Insert the stopper quickly, shake for one minute, and leave
for exactly five minutes away from the light at a temperature from 15 to
25 ºC.
Add about 75 ml of distilled water. Titrate the liberated iodine with the
sodium thiosulphate solution ►
C1(6.4) (0,002 Mol/L solutionfor
expected values less than12, an d 0,01 Mol/L solution ◄for expected
values above 12) shaking vigorously, using starch solution (6.5) as indi-
cator.
Carry out two determinations on the same test sample.
Carry out simultaneously a blank test. If the result of the blank exceeds
0,05 ml of ►C10,01 mol/L sodium ◄thiosulphate solution(6.4), replace
the impure reagents.
9. EXPRESSION OF RESULTS
The peroxide value (PV), expressed inmilliequivalen ts of active oxygen
per kilogram, is givenby the formula:
PV¼V/C2T/C21000
m
where:
V = the number of ml of the standardized sodium thiosulphate solution
(6.4) used for the test, corrected to take into account the blank test;
T=►C1the exact molarity ◄of the sodium thiosulphate solution
(6.4) used;
m = the weight ing, of the test portion .
Take as the result the arithmetic mean of the two determinations carried
out.1991R2568 — EN — 21.07.1992 — 004.001 — 12
▼B
ANNEX IV
DETERMINATION OF ALIPHATIC ALCOHOLS CONTENT BY
CAPILLARYGASCHROMATOGRAPHY
1. OBJECT
The procedure describes a method for the determination ►C1of
aliphatic ◄alcohols content in oils and fats.
2. PRINCIPLE OF THE METHOD
The fatty substance, with 1-eicosanol added as internal ►C1standard,
is saponified with ethanolic potassium hydroxide and then the unsapo-
nifiable matter extracted with diethyl ether. ◄
The alcoholic fraction is separated from the unsaponifiable matter by
chromatography ona layer of potassium hydroxide impregn ated silica
gel; the alcohols recovered from the silica gel are transformed into
trimethylsilyl ethers and analyzed by capillary gas chromatography.
3. APPARATUS
3.1. 250 ml round-bottom flask with reflux condenser and ground glass
joints.
3.2. Separating funnels of 500 ml capacity.3.3. Flasks of 250 ml capacity.3.4. Chromatographic tank for thin-layer chromatographic analysis, for glass
plates of dimensions 20 × 20 cm.
3.5. UV light ►
C1of wavelength ◄366 or 254 nm, for examination of
TLC plates.
3.6. Microsyringe to deliver 100 µl and 500 µl.3.7. Sintered glass filtering funnel with porous frit G 3 ►
C1(porosity 15 to
40 µm) ◄of approximate diameter 2 cm and approximate height 5 cm
suitable for filtering under vacuum and 12/21 ground male joint.
3.8. Vacuum flask of 50 ml capacity with 12/21 ground glass female joint
for use with filter funnel (3.7).
3.9. Test tube of 10 ml capacity with conical bottom and plug.3.10. Gas chromatograph for use with a capillary column, and provided with
a splitting system composed of:
3.10.1. thermostatic chamber for columns (column oven) to hold the tempera-
ture desired with a precisionof ± 1 ºC;
3.10.2. thermostatic vaporizationassembly (in jectionport) with
►
C1silanized ◄coated glass;
3.10.3. flame ionization detector, and converter amplifier;
3.10.4. recorder-integrator for operation with the converter-amplifier (3.10.3),
with response time not exceeding one second and with variable paper-
speed.
3.11. Glass or fused silica capillary column, of length 20 to 30 m, internal
diameter 0,25 to 0,32 mm, with SE-52 or SE-54 liquid phase or equiva-
lent, with a film thickness between 0,10 and 0,30 µm.
3.12. Microsyringe for gas chromatography, of 10 µl capacity with hardened
needle.
4. REAGENTS
4.1. Potassiumhydroxide,approximately ►C12mol/Lethanolicsolution ◄:
130 g potassium hydroxide (minimum concentration 85 %) is dissolved,
with cooling, in 200 ml distilled water and then made up to one litre
with ethanol. The solution should be stored in a well-stoppered dark
coloured glass bottle.
4.2. ►C1Diethyl ether ◄, pure for analysis.
4.3. Anhydrous sodium sulphate, pure for analysis.1991R2568 — EN — 21.07.1992 — 004.001 — 13
▼B
4.4. Silica gel TLC glass plates, ►C1without fluorescence ◄indicator, of
thickness 0,25 mm (may be obtained commercially prepared).
4.5. Potassium hydroxide, approximately ►C10,2 mol/L ethanolic
solution ◄; 13 g of potassium hydroxide are dissolved in20 ml of
distilled water and made up to one litre with ethanol.
4.6. Benzene, for chromatography. (see 5.2.2).
4.7. Acetone, for chromatography. (see 5.2.2).4.8. Hexane, for chromatography. (see 5.2.2).
4.9. ►
C1Diethyl ether ◄, for chromatography. (see 5.2.2).
4.10. Chloroform, for chromatography.4.11. Reference solution for thin layer chromatography: mixture of 5 % C
20to
C28alcohols inchloroform.
4.12. 0,2 % solutionof 2,7-dichlorofluoresceininethan ol. This is made
slightly basic by adding a few drops of ►C12 mol/L potassium hydro-
xide◄solution.
4.13. Anhydrous pyridine, for chromatography.
4.14. Hexamethyldisilazane.
4.15. Trimethylchlorosilane.4.16. Standard solutions of trimethylsilyl ethers of aliphatic alcohols from C
20to C28. They may be prepared from mixtures of pure alcohols at the
time they are required for use.
4.17. A 0,1 % (m/v) solutionof 1-eicosan ol inCHCl3(internal standard).
4.18. Carrier gases: hydrogenan d helium, pure for gas chromatography.4.19. Auxiliary gases:
— hydrogen, pure for gas chromatography,
— air, pure for gas chromatography.
5. PROCEDURE
5.1. Preparation of unsaponifiable matter.
5.1.1. Using a 500 µl microsyringe place, into a 250 ml round-bottom flask, a
volume of 0,1 % 1-eicosanol ►
C1(1-heneicosanol ◄may also be
used) solution (4.17) containing a quantity of 1-eicosanol approximately
equal to 10 % of the aliphatic alcohol content in that portion of sample
to be takenfor an alysis. For example, to 5 g of sample add 250 µl of
the 0,1 % 1-eicosanol solution if olive oil or ►C1seed oil is being
analysed, 1 500 µl if the sample is olive residue oil. Evaporate the
internal standard solution to dryness under N2.◄
Quantitatively weigh into the flask approximately 5 g of dry, filtered
sample.
5.1.2. 50 ml of ►C1mol/L potassium hydroxide ◄ethanolic solution are
added, reflux condenser is fitted and the apparatus ►C1heated to
gently boiling ◄on steam bath and stirred continuously throughout
the heating process until saponification has taken place (the solution
becomes clear). Heating is continued for a further 20 minutes and then
50 ml of distilled water are added through the condenser, the condenser
is►C1then disconnected ◄and the flask is cooled to approximately
30 ºC.
5.1.3. The contents of the flask are quantitatively transferred to a separating
funnel of ►C1500 ml capacity, and the flask rinsed with 2 x 25 ml
distilled water. Approximately 80 ml of diethyl ether are added, the
whole shaken vigorously for 30 seconds and the two phases left to
separate (Note 1). ◄
The lower aqueous phase is transferred to a second funnel. Two further
extractions are effected on the aqueous phase, in the same manner,using 60 to 70 ml diethyl ether each time.
Note 1.Emulsions may be eliminated by adding, using as a spray,
small quantities of ethyl alcohol or methyl alcohol.
5.1.4. ►
C1The diethyl ether ◄extracts are combined in a separating funnel
and washed with distilled water (50 ml at a time) until the washing
water gives a neutral reaction.1991R2568 — EN — 21.07.1992 — 004.001 — 14
▼C1
▼B
▼B
►C1Discard the aqueous phase, dry the ether phase with ◄anhy-
drous sodium sulphate and filter, into a flask of 250 ml capacity
which has been weighed beforehand, the funnel and filter being washed
with small quantities of ►C1diethyl ether ◄which are added to the
total.
5.1.5. The ether is evaporated with ►C1gentle heating ◄to a few ml and
then dried under a slight vacuum or under a current of nitrogen; drying
is finished in an oven at 100 ºC for approximately 15 minutes and the
residue weighed after cooling in ►C1a desiccator. ◄
5.2. Separationof alcoholic fraction s.
5.2.1. Preparationof basic ►C1TLC plates (4.4). The plates are immersed
completely, inO,2 mol/L potassium hydroxide solution(4.5) for 10
seconds, and then left to dry under a hood for two hours, and finally
placed◄inanovenat 100 ºC for on e hour.
The plates are takenfrom the ovenan d stored ina ►C1calcium
chloride desiccator ◄until required for use. The plates thus treated
must be used withintwo weeks.
Note 2.Whenbasic silica gel plates are used for separationof the alco-
holic fraction, there is no need for the unsaponifiable
substances to be treated ►C1with A1203◄. It follows that
all acid compounds (fatty acids and others) are retained at the
origin thereby obtaining both aliphatic alcohol and terpenic
alcohol bands which are both separated distinctly from the
sterol band.
5.2.2. A solution of 95:5 by volume of benzene and acetone is introduced into
the development chamber to an approximate depth of 1 cm. Alterna-
tively, a 65:35 by volume mixture ►C1of hexane and diethyl
ether◄may be used. The tank is closed and left for not less than
half anhour to allow equilibrationbetweenvapour an d liquid. Strips
of filter paper dipping into the eluent may be affixed to the inside
surfaces of the tank to reduce the development time by approximately
one-third and obtain more uniform, regular elution of the components.
Note 3.The developing solution must be replaced for each analysis in
order to obtain reproducible developing conditions.
5.2.3. An approximately 5 % solution of unsaponifiable matter (5.1.5) in
chloroform is prepared ►C1and 300 µl of the solution is streaked as
a uniform strip of minimum thickness, by means of the microsyringe of
100 µl capacity, ona TLC plate approximately 2 cm from the bottom of
the TLC plate. Aligend with the strip, 2 to 3 µl of the aliphatic alcohol
reference solution (4.11) are spotted ◄for the identification of the
aliphatic alcohol band after development has been completed.
5.2.4. The plate is placed inside the development tank as stated in 5.2.2. The
temperature shall be kept between 15 and 20 ºC. The tank is closed
immediately, and the sample is left to elute until the front of the solvent
has reached a distance 1 cm from the top of the plate. The plate is then
removed from the development tank and the solvent evaporated under a
hot air current or the plate is left for a while under the hood.
5.2.5. The plate is sprayed lightly and evenly with the solution of 2,7-dichlor-
ofluoresceinwhenthe plate is observed un der ultra violet light, the
aliphatic alcohol band is identified by comparison to the aliphatic alco-
hols in the band immediately above that, which is the triterpenic
alcohol band, are outlined together.
Note 4.The requirement ►C1for the removal of the ◄aliphatic
alcohol band and the triterpenic alcohol band together is deter-
mined by the possible migration of some aliphatic alcohols into
the triterpenic alcohol band.
5.2.6. The silica gel enclosed ►C1inthe area outlin ed is scraped off with a
metal spatula. The material removed is broken into fine fragments, and
introduced into a filter funnel (3.7), 10 ml of hot chloroform are added.
The contents are mixed ◄thoroughly with the metal spatula and
filtered under vacuum, the filtrate being collected in the flask (3.8)
which is connected to the filter funnel.
The residue inside the funnel is washed 3 × 10 ml with ethyl ether
collecting the filtrate in the same flask fitted to the funnel. The filtrate
is evaporated to a volume of approximately 4 to 5 ml and the residual
►C1solution is transferred into a test tube of 10 ml capacity (3.9)
which has beenweighed beforehan d; the test tube is dried by light
heating under a gentle nitrogen current. The residue is redissolved
with a few drops of acetone, dried again, the placed in an oven at 1051991R2568 — EN — 21.07.1992 — 004.001 — 15
▼B
ºC for 10 minutes, removed and cooled in the desiccator and
weighed. ◄
The residue inside the test tube is composed of the alcoholic fraction.
5.3. Preparationof ►C1trimethylsilyl ◄ethers.
5.3.1. The reagent for silylation, consisting of a mixture of 9:3:1 by volume
(Note 5) of pyridine-hexamethyldisilazane-trimethylchlorosilane in the
proportionof 50 µl for each milligram of alcohols, is added to the test
tube containing the alcoholic fraction, avoiding all absorption of
moisture (Note 6).
Note 5.Solutions ready for use are commercially available; silanizing
reagents such as N, 0-bis (trimethylsilyl) trifluoroacetamide
+ 1 % trimethylchlorosilane for mixing with the same volume
of anhydrous pyridine.
5.3.2. The test tube is stoppered and shaken carefully without overturning
until the alcohols have been ►C1dissolved ◄. It is thenleft for at
least 15 minutes at room temperature and subsequently centrifuged for
some minutes; the clear solution is ready for gas chromatographic
analysis.
Note 6.Any formation of slight opalescence is normal and causes no
interference. The formation of a white flocculate or the appear-
ance of a pink colouring are signs of the presence of moisture
or the deteriorationof the reagen t. Inthis case the test shall be
repeated.
5.4. Gas chromatography analysis.5.4.1. Preliminary operations and conditioning of the capillary column.
5.4.1.1. The capillary columnis fitted in side the gas chromatograph by
connecting the beginning of the column to the evaporator which is
connected to the splitting system and the end of the column to the
detector.
General checking of the gas chromatography assembly is performed
(tightness of gas fittings, efficiency of the detector, efficiency of the
splitting system and of the recording system, etc.).
5.4.1.2. Capillary columns being used for the first time should be conditioned.
A little carrier gas is caused to flow through the capillary columnan d
thenthe gas chromatography assembly is switched onan d ►
C1heated
gradually until ◄a temperature not less than 20 ºC above the oper-
ating temperature (see Note 7) is attained. That temperature is held for
not less than two hours and then the assembly is brought to the oper-
ating conditions (regulation of gas flow, split flame ignition,
connection to the electronic recorder, adjustment of the temperature of
the capillary column oven, the detector and the injector, etc.) and the
signal is adjusted to a sensitivity not less than twice the highest level
contemplated for the execution of the analysis. The base line tracing
shall be linear, devoid of spikes of whatever nature and shall show no
signs of drift.
Negative rectilinear drift is indicative of imperfect tightness of column
connections whereas a positive drift is indicative of insufficient condi-
tioning of the column.
Note 7.The temperature of conditioning shall be at least 20 ºC less
thanthe maximum temperature con templated for the liquid
phase employed.
5.4.2. Selection of operating conditions.
5.4.2.1. General operating conditions are as follows:
— columntemperature: the in itial isotherm is set at 180 ºC for eight
minutes and then programmed at 5 ºC/minute to 260 ºC and a
further 15 minutes at 260 ºC,
— temperature of evaporator: 280 ºC,— temperature of detector: 290 ºC,— linear velocity of carrier gas: helium 20 to 35 cm/s, hydrogen 30 to
50 cm/s,
— splitting ratio: 1:50 at 1:100,
— sensitivity of instrument: 4 to 16 times the minimum attenuation,
— sensitivity of recording: 1 to 2 mV fs,— paper speed: 30 to 60 cm/h,— quantity of substance injected: 0,5 to 1 µl of TMSE solution.1991R2568 — EN — 21.07.1992 — 004.001 — 16
▼B
The above conditions may be modified according to the characteristics
of the columnan d of the gas chromatograph inorder that chromato-
grams satisfying the following conditions be obtained:
— alcohol retention time C26shall be 18 ± 5 minutes,
— the alcohol C22peak shall be 80 ± 20 % of the full scale value for
olive oil and 40 ± 20 % of the full scale value for seed oil.
5.4.2.2. The aforesaid requirements are checked by repeated injection of the
standard TMSE mixture of alcohols and the operating conditions are
adjusted to yield the best possible results.
5.4.2.3. The parameters for the integration of peaks shall be set so that a correct
appraisal of the areas of the peaks considered is obtained.
5.4.3. Executionof an alysis.5.4.3.1. Using the microsyringe of 10 µl capacity draw in 1 µl of hexane
followed by 0,5 µl of air and subsequently 0,5 to 1 µl of the sample
solution; the plunger of the microsyringe is raised to empty the needle.
The needle is introduced through the septum of the injection assembly
and after one to two seconds the solution is injected rapidly and the
needle extracted slowly after approximately five seconds.
5.4.3.2. Recording is effected until the TMSE of the alcohols present have been
eluted completely. The base line shall always correspond to the require-
ments of 5.4.1.2.
5.4.4. Identification of peaks.
The identification of individual peaks is effected according to the reten-
tion times and by comparison with the standard TMSE mixture,
analyzed under the same conditions.
A chromatogram of the alcoholic fractionof a virginolive oil is shown
inFigure 1.
5.4.5. Quantitative appraisal.5.4.5.1. The peak areas of 1-eicosanol and of the aliphatic alcohols C
22to C28are calculated by electronic integration.
5.4.5.2. The contents of each alcohol, expressed in mg/100 g fatty substance are
calculated as follows:
alcohol x =Ax·ms· 100
As·m
where:
Ax= area of the alcohol peak x, insquare millimetres;
As= area of 1-eicosanol in square millimetres;
ms= mass of 1-eicosanol in milligrams;
m = mass of sample drawn for determination, in grams.
6. EXPRESSION OF RESULTS
The contents of the individual aliphatic alcohols in mg/100 g of fatty
substance and the sum of the ‘total aliphatic alcohols’ are reported.1991R2568 — EN — 21.07.1992 — 004.001 — 17
▼B
APPENDIX
Determination of the linear velocity of the gas
1 to 3 µl of methane or propane are injected into the gas chromatograph set at
normal operating conditions and the time taken for the methane or propane to
flow through the column from the instant of injection to the instant the peak
elutes (tM) is measured using a stop clock.
The linear velocity, in cm/s is given by L/tM, where L is the length of the
column, in centimetres, and tMis the time, insecon ds, measured by the stop
clock.
Figure1 Chromatogram of the alcoholic fraction of a virgin olive oil.
1 = Eicosanol (SI)
►C12 = Docosanol, ◄
3 = Tricosanol,
4 = Tetracosanol,5 = Pentacosanol,6 = Hexacosanol,
7 = Heptacosanol,
8 = Octacosanol.1991R2568 — EN — 21.07.1992 — 004.001 — 18
▼B
ANNEX V
DETERMINATION OF THE COMPOSITION AND CONTENT OF
STEROLSBYCAPILLARY-COLUMNGASCHROMATOGRAPHY
1. SCOPE
The method describes a procedure for determining the individual and
total sterols content of fatty substances.
2. PRINCIPLE OF THE METHOD
The fatty substance, with added α-cholestanol as an internal standard, is
saponified with potassium hydroxide in ethanolic solution and the unsa-
ponifiables are then extracted with ►C1diethyl ether ◄.
The sterol fraction is separated from the unsaponifiable extract by chro-
matography ona basic silica gel plate. The sterols recovered from the
silica gel are transformed into trimethyl-silyl ethers and are analysed by
capillary-columngas chromatography.
3. APPARATUS
3.1. 250 ml flask fitted with a reflux condenser having ground-glass joints.3.2. 500 ml separating funnels.
3.3. 250 ml flasks.
3.4. Complete apparatus for analysis by thin-layer chromatography using
20 × 20 cm glass plates.
3.5. Ultraviolet lamp having a wavelength of 366 or 254 nm.
3.6. 100 µl and 500 µl microsyringes.3.7. A cylindrical filter funnel with a G3 porous septum (porosity 15 to
40 µm) of diameter approximately 2 cm and a depth of some 5 cm,
with an attachment suitable for filtration under vacuum and a 12/21
male ground glass joint.
3.8. 50 ml vacuum conical flask with a 12/21 ground-glass female joint
which can be fitted to the filter funnel (3.7).
3.9. A 10 ml test tube with a tapering bottom and a sealing stopper.
3.10. Gas chromatograph suitable for use with a capillary column, provided
with a splitting system consisting of:
3.10.1. a thermostatic chamber for columns capable of maintaining the desired
temperature with anaccuracy of ± 1 ºC;
3.10.2. a temperature-adjustable vaporization unit with a persilanized glass
vapourizing element;
3.10.3. a flame ionization detector and converter-amplifier;3.10.4. an integrator-recorder suitable for use with the converter-amplifier
(3.10.3) having a response time of not more than one second and a vari-
able paper speed.
3.11. A glass or fused-silica capillary column of length 20 to 30 m, internal
diameter 0,25 to 0,32 mm, entirely coated with SE-52 or SE-54 liquid
or equivalent in a uniform thickness between 0,10 and 0,30 µm.
3.12. A 10 µl gas chromatography microsyringe with a hardened needle.
4. REAGENTS
4.1. Potassiumhydroxide, ►
C1approximately2mol/Lethanolicsolution. ◄
Dissolve 130 g of potassium hydroxide ►C1(minimum concentration
85 %)◄with cooling in 200 ml of distilled water and then make up to
one litre with ethanol. Keep the solution in well-stoppered dark glass
bottles.
4.2. ►C1Diethyl ether ◄, analytical purity.
4.3. Anhydrous sodium sulphate, analytical purity.
4.4. Glass plates coated with silica gel, without fluorescence indicator,
thickness 0,25 mm (commercially available ready for use).1991R2568 — EN — 21.07.1992 — 004.001 — 19
▼B
4.5. Potassium hydroxide, ►C10,2 mol/L ◄ethanolic solution. Dissolve
13 g of potassium hydroxide in20 ml of distilled water an d make up
to one litre with ethanol.
4.6. Benzene, for chromatography. (See 5.2.2)
4.7. Acetone, for chromatography. (See 5.2.2)
4.8. Hexane, for chromatography. (See 5.2.2)
4.9. ►C1Diethyl ether ◄, for chromatography. (See 5.2.2)
4.10. Chloroform, analytical purity. (See 5.2.2)
4.11. Reference solution for thin-layer chromatography: cholesterol or
phytosterols, 5 % solutioninchloroform.
4.12. 2,7-dichlorofluorescein, 0,2 % ethanolic solution. Make slightly basic
by adding a few drops of ►C12 mol/L ◄alcoholic potassium hydro-
xide solution.
4.13. Anhydrous pyridine, for chromatography.
4.14. Hexamethyl disilazane.
4.15. Trimethylchlorosilane.
4.16. Reference solutions of sterol trimethylsilyl ethers. To be prepared at the
time of use from pure sterols or mixtures of sterols obtained from oils
containing them.
4.17. ►C1β-cholestanol ◄, 0,2 % solution (m/V) in chloroform (internal
standard).
4.18. Carrier gas: hydrogenor helium, gas-chromatographic purity.
4.19. Auxiliary gases:
— hydrogen, gas-chromatographic purity,
— air, gas-chromatographic purity.
5. PROCEDURE
5.1. Preparation of the unsaponifiables.
5.1.1. Using the 500 µl microsyringe ►C1introduce, into a 250 ml flask a
volume of 0,2 % β-cholestanol solution in chloroform (4.17) containing
an amount of cholestanol corresponding to approximately 10 % of the
sterol content of the sample aliquot to be taken for the determination. ◄
For example, for 5 g of sample add 500 µl of the 0,2 % α-cholestanol
solutioninthe case of anolive oil an d 1 500 µl for seed oils or olive-
pomaca oil.
Evaporate to ►C1dryness in a current ◄of nitrogen and then weigh
accurately 5 g of the dry filtered sample into the same flask.
Animal or vegetable oils and fats containing appreciable quantities of
cholesterol may show a peak having a retention time identical to
cholestanol. If this occurs the sterol fraction will have to be analyzed
in duplicate with and without internal standard.
5.1.2. Add 50 ml of ►C12 mol/L ◄ethanolic potassium hydroxide solu-
tion, fit the reflux condenser and heat to gentle boiling on a water
bath with continuous vigorous stirring until saponification takes place
(the solution becomes clear). Continue heating for a further 20 minutes,
thenadd 50 ml of ►C1distilled water to the ◄top of the condenser,
detach the condenser and cool the flask to approximately 30 ºC.
5.1.3. Transfer the contents of the flask quantitatively into a 500 ml separating
funnel using several rinses of distilled water, amounting in all to about
50 ml. Add approximately 80 ml of ►C1diethyl ether ◄, shake
vigorously for approximately 30 seconds and allow to settle (Note 1).
Separate off the lower aqueous phase collecting it in a second separ-
ating funnel. Perform two further extractions on the aqueous phase in
the same way using 60 to 70 ml of ethyl ether on each occasion.
Note 1.Any emulsion can be destroyed by adding small quantities of
ethyl or methyl alcohol by means of a spray.
5.1.4. Pool the ether extracts into a single separating funnel and wash with
distilled water (50 ml at a time) until the wash water gives a neutral
reaction.1991R2568 — EN — 21.07.1992 — 004.001 — 20
▼B
Whenthe wash water has beenremoved, dry with an hydrous sodium
sulphate and ►C1filter through anhydrous ◄sodium sulphate into a
previously weighed 250 ml flask, washing the funnel and filter with
small quantities of ►C1diethyl ether. ◄
5.1.5. Distil the ether down to a few ml, then bring to dryness under a slight
vacuum or in a current of nitrogen, completing drying in a stove at
100 ºC for approximately a quarter of anhour, an d thenweigh after
cooling in a desiccator.
5.2. Separationof the sterol fraction .5.2.1. Preparationof the basic plates. Immerse the silica gel plates (4.4)
completely inthe ►
C10,2 mol/L ◄ethanolic potassium hydroxide
solution(4.5) for 10 secon ds, thenallow to dry ina fume cupboard
for two hours and finally place ►C1inanovenat ◄100 ºC for one
hour.
Remove from the stove and keep in a calcium chloride desiccator until
required for use (plates treated inthis way must be used within15
days).
Note 2.Whenbasic silica gel plates are used to separate the sterol frac-
tion there is no need to treat the unsaponifiables with alumina.
In this way all compounds of an acid nature (fatty acids and
others) are retained on the spotting line and the sterols band is
clearly separated from the aliphatic and triterpene alcohols
band.
5.2.2. Place a 95:5 (v/v) benzene/acetone ►C1mixture into the ◄plate-
developing chamber to a depth of approximately 1 cm. As an alterna-
tive a 65:35 (v/v) hexane/ethyl ether mixture may be used. Close the
chamber with the appropriate cover and leave thus for approximately
half anhour so that liquid-vapour equilibrium is established. Strips of
filter paper dipping into the eluent may be placed on the internal
surfaces of the chamber. This reduces developing time by approxi-
mately one-third and brings about more uniform and regular elution of
the components.
Note 3.The developing mixture should be replaced for every test in
order to achieve perfectly reproducible elution conditions.
5.2.3. Prepare an approximately 5 % solution of the unsaponifiables (5.1.5) in
chloroform and, using the 100 µl microsyringe, streak a chromato-
graphic plate ►C1(5.2.1) with 300 µl ◄approximately 2 cm from
one end in a streak which is as thin and as uniform as possible. In line
with the streak place 2 to 3 µl of the sterol reference solution (4.11) at
one end of the plate so that the sterol band can be identified after devel-
oping.
5.2.4. Place the plate inthe developin g chamber prepared as specified in
5.2.2. The ambient temperature should be maintained between 15 and
20 ºC. Immediately close the chamber with the cover and allow to elute
until the solvent front reaches approximately 1 cm from the upper edge
of the plate. Remove the plate from the developing chamber and evapo-
rate the solvent in a flow of hot air or by leaving the plate for a short
while under a hood.
5.2.5. Spray the plate lightly and uniformly with the 2,7-dichlorofluoroscein
solution. When the plate is observed under ultraviolet light the sterol
band can be identified through being aligned with the stain obtained
from the reference solution. Mark the limits of the band along the edges
of the fluorescence with a black pencil.
5.2.6. Using a metal spatula scrape off the silica gel in the marked area. Place
the finely comminuted material removed into the filter funnel (3.7).
Add 10 ml of hot chloroform, mix carefully with the metal spatula
and filter under vacuum, collecting the filtrate in the conical flask
(3.8) attached to the filter funnel.
Wash the residue ►C1in the funnel three times with diethyl ether ◄
(approximately 10 ml each time) collecting the filtrate in the same flask
attached to the funnel. Evaporate the filtrate to a volume of 4 to 5 ml,
transfer the residual solution to the previously weighed 10 ml test tube
(3.9), evaporate to dryness by mild heating in a gentle flow of nitrogen,
make up again using a few drops of acetone, evaporate again to
dryness, place in a stove at 105 ºC for approximately 10 minutes and
thenallow to cool ina desiccator an d weigh.
The residue contained in the test tube consists of the sterol fraction.
5.3. Preparationof the trimethylsilyl ethers.1991R2568 — EN — 21.07.1992 — 004.001 — 21
▼B
5.3.1. Add the silylation reagent, consisting of a 9:3:1 (v/v/v) mixture of pyri-
dine/hexamethyl disilazane/trimethyl chlorosilane (Note 4) in the ratio
of 50 µl for every milligram of sterols to the test tube containing the
sterol fraction, avoiding any uptake of moisture (Note 5).
Note 4.Solutions which are ready for use are available commercially.
Other silanizing reagents such as, for example, bis-trimethyl-
silyl, trifluor acetamide + 1 % trimethyl chlorosilane, which
has to be diluted with an equal volume of anhydrous pyridine,
are also available.
5.3.2. Stopper the test tube, shake carefully (without overturning) until the
sterols are completely dissolved. Stand for at least 15 minutes at
ambient temperature and then centrifuge for a few minutes. The clear
solutionis ready for gas chromatographic an alysis.
Note 5.The slight opalescence which may form is normal and does not
cause any interference. The formation of a white floc or the
appearance of a pink colour are indicative of the presence of
moisture or deteriorationof the reagen t. If these occur the test
must be repeated.
5.4. Gas chromatographic analysis.
5.4.1. Preliminary operations, column packing.5.4.1.1. Fit the column in the gas chromatograph, attaching the inlet end to the
evaporator connected to the splitting system and the outlet end to the
detector.
Carry out general checks on the gas chromatograph unit (leaks from the
gas circuits, detector efficiency, efficiency of the splitting system and
recording system, etc.).
5.4.1.2. If the column is being used for the first time it is recommended that it
should be subjected to conditioning. Pass a gentle flow of gas through
the column and then switch on the gas chromatography unit and begin
gradual heating up to a temperature of at least 20 ºC above the oper-
ating temperature (Note 6). Hold this temperature for at least two
hours, then place the entire unit in operating mode (adjustment of gas
flows and splitting, ignition of the flame, connection with the electronic
recorder, adjustment of the column chamber, detector and injector
temperature, etc.) and then record the signal with a sensitivity at least
two times greater than that intended for the analysis. The course of the
base line must be linear, without peaks of any kind, and must not drift.
A negative straight-line drift indicates leakage from the column connec-
tions; a positive drift indicates inadequate conditioning of the column.
Note 6.The conditioning temperature must always be at least 20 ºC
less thanthe maximum temperature specified for the station ary
phase used.
5.4.2. Choice of operating conditions.5.4.2.1. The guideline operating conditions are as follows:
— columntemperature: 260 ± 5 ºC,
— evaporator temperature: 280 ºC,
— detector temperature: 290 ºC,
— linear velocity of the carrier gas: helium 20 to 35 cm/s, hydrogen 30
to 50 cm/s,
— splitting ratio: from 1:50 to 1:100,
— instrument sensitivity: from 4 to 16 times the minimum attenuation,
— recording sensitivity: 1 to 2 mV f.s.,
— paper speed: 30 to 60 cm/hour,— amount of substance injected: 0,5 to 1 µl of TMSE solution.
These conditions may be varied in the light of column and gas-chroma-
tograph characteristics so as to obtainchromatograms which meet the
following requirements:
— the retention time for β-sitosterol should be 20 ± 5 minutes,
— the campesterol peak should be: for olive oil (mean content 3 %)
15 ± 5 % of full scale; for soya oil (mean content 20 %) 80 ± 10 %
of full scale,
— all the sterols present must be separated. In addition to being sepa-
rated the peaks must also be completely resolved, i.e. the peak trace
should return to the base line before leaving for the next peak.
Incomplete resolution is however tolerated provided that the peak
at TRR 1,02 can be quantified using the perpendicular.1991R2568 — EN — 21.07.1992 — 004.001 — 22
▼B
5.4.3. Analytical procedure.
5.4.3.1. Using the 10 µl microsyringe take 1 µl of hexane, draw in 0,5 µl of air
and then 0,5 to 1 µl of the sample solution. Raise the plunger of the
syringe further so the needle is emptied. Push the needle through the
membrane of the injection unit and after one to two seconds inject
rapidly, then slowly remove the needle after some five seconds.
5.4.3.2. Continue recording until the TMSE of the sterols present are comple-
tely eluted
The base line must continue to meet the requirements (5.4.1.2).
5.4.4. Peak identification.
Identify individual peaks on the basis of retention times and by compar-
ison with mixtures of sterol TMSE analysed under the same conditions.
The sterols are eluted inthe followin g order: cholesterol, brassicasterol,
24-methylene cholesterol, campesterol, campestanol, stigmasterol, ∆7-
campesterol, ∆ 5,23-stigmastadienol, ►C1chlerosterol ◄,β-sistos-
terol, sitostanol, ∆5-avenasterol, ∆ 5,24-stigmastadienol ►C1,∆7-
stigmasterol, ◄∆7-avenasterol.
The retention times for sitosterol for SE-52 and SE-54 columns are
showninTable 1.
Figures 1 and 2 illustrate typical chromatograms for some oils.
5.4.5. Quantitative evaluation.
5.4.5.1. Calculate the areas of the ►C1β-cholestanol ◄and the sterol peaks
using the integrator. Ignore peaks for any compounds which are not
included among those listed in Table 1. The response coefficient for
►C1β-cholestanol ◄is to be equal to 1.
5.4.5.2. Calculate the concentration of each individual sterol in mg/100 g of
fatty material as follows:
sterol x =Ax·ms· 100
As·m
where:
Ax= peak area for sterol x, insquare millimetres;
As= area of the ►C1β-cholestanol ◄peak, insquare millimetres;
ms= mass of ►C1β-cholestanol ◄added, im milligrams;
m = mass of the sample used for determination, in grams.
6. EXPRESSION OF THE RESULTS
6.1 Record individual sterol concentrations as mg/100 g of fatty material
and their sum as ‘total sterols’.
6.2 Calculate the percentage of each individual sterol from the ratio of the
relevant peak area to the total peak area for sterols.
% of sterol x =Ax
ΣA· 100
where:
Ax= peak area for x;
ΣA = total peak area for sterols.1991R2568 — EN — 21.07.1992 — 004.001 — 23
▼B
APPENDIX
Determination of the linear velocity of the gas
With the gas chromatograph set to normal operating conditions inject 1 to 3 µl
of methane (or propane) and measure the time taken by the gas to pass through
the columnfrom the time of in jectionto the time at which the peak appears (tM).
The linear velocity in cm/s is given by L/tM, where L is the length of the column
in centimetres and tMis the measured time insecon ds.
TableI
Relative retention times for sterols
Pe-
akIdentificationRelative retention
time
SE 54
columnSE 52
column
1 cholesterol ∆-5-cholesten-3 β-ol 0,67 0,63
2 cholestanol 5 α-cholestan-3 β-ol 0,68 0,64
3 brassicasterol [24S]-24-methyl- ∆-5,22-cholestadien-3 β-ol 0,73 0,71
4 24-methylene-cholesterol 24-methylene- ∆-5,24-cholesten-3 β-ol 0,82 0,80
5 campesterol [24R]-24-methyl- ∆-5-cholesten-3 β-ol 0,83 0,81
6 campestanol [24R]-24-methyl-cholestan-3 β-ol 0,85 0,82
7 stigmasterol [24R]-24-ethyl- ∆-5,22-cholestadien-3 β-ol 0,88 0,87
8 ∆-7-campesterol [24R]-24-methyl- ∆-7-cholesten-3 β-ol 0,93 0,92
9 ∆-5,23-stigmastadienol [24R,S]-24-ethyl- ∆-5,23-cholestadien-3 β-ol 0,95 0,95
10 chlerosterol [24S]-24-ethyl- ∆-5,25-cholastadien-3 β-ol 0,96 0,96
11 β-sitosterol [24R]-24-ethyl- ∆-5-cholestan-3 β-ol 1,00 1,00
12 sitostanol 24-ethyl-cholestan-3 β-ol 1,02 1,02
13 ∆-5-avenasterol [24Z]-24-ethylidene-5-cholesten-3 β-ol 1,03 1,03
14 ∆-5,24-stigmastadienol [24R,S]-24-ethyl- ∆-5,24-cholestadien-3 β-ol 1,08 1,08
15 ∆-7-stigmastenol [24R,S]-24-Ethyl- ∆-7,24-cholestadien-3 β-ol 1,12 1,12
16 ∆-7-avenasterol [24Z]-24-ethyliden- ∆-7-cholesten-3 β-ol 1,16 1,161991R2568 — EN — 21.07.1992 — 004.001 — 24
▼B
Figure1
Gas chromatogram of the sterol fraction of an unrefined olive oil
1991R2568 — EN — 21.07.1992 — 004.001 — 25
▼B
Figure2
Gas chromatogram of the sterol fraction of a refined olive oil
1991R2568 — EN — 21.07.1992 — 004.001 — 26
▼B
ANNEX VI
DETERMINATIONOFERYTHRODIOLANDUVAOL
INTRODUCTION
Erythrodiol (commonly understood as the glycols erythrodiol and uvaol
together) is a constituent of the unsaponifiable fraction, characteristic of
some types of fatty substances. It is found at considerably higher concen-
trations in solvent-extracted olive oil than in other oils, such as pressed
olive oil and grape pip oil, which also contain it, and so its presence
may demonstrate the presence of solvent-extract olive oil.
1. SCOPE
The method describes a procedure for detecting erythrodiol in fatty
substances.
2. PRINCIPLE OF THE METHOD
The fatty substance is saponified with potassium ►C1hydroxide inetha-
nolic solution. The unsaponifiable fraction is then extracted with diethyl
ether◄and purified by passage over a column of alumina.
The unsaponifiables are subjected to thin-layer chromatography on a
silica gel plate until the bands corresponding to the sterol and erythrodiol
fractions are separated. The sterols and the erythrodiol recovered from the
plate are transformed into trimethylsilyl ethers and the mixture is
analysed by gas chromatography.
The result is expressed as the percentage of erythrodiol in the mixture of
erythrodiol and sterols.
3. APPARATUS
3.1. The apparatus described in Annex V (determination of the content of
sterols).
4. REAGENTS
4.1. The reagents described in Annex V (determination of the content of
sterols).
4.2. Reference solution of erythrodiol, 0,5 % solution in chloroform.
5. PROCEDURE5.1.Preparationoftheunsaponifiables.
As described at paragraph 5.1.2 of Annex V.
5.2.Separationoferythrodiolandthesterols.
5.2.1. See paragraph 5.2.1 of Annex V.
5.2.2. See paragraph 5.2.2 of Annex V.
5.2.3. Prepare a 5 % solution of the unsaponifiables in chloroform.
Using the 0,1 ml microsyringe, streak a chromatographic plate with 0,3 ml
of solutionapproximately 1,5 cm from the lower edge ina streak which
is as thin and uniform as possible.
At one end of the plate place a few microlitres of the solutions of choles-
terol and erythrodiol to serve as a reference.
5.2.4. Place the plate inthe developin g chamber prepared as specified in5.2.1.
The ambient temperature should be about 20 ºC. Immediately close the
chamber with the cover and allow to elute until the solvent front reaches
approximately 1 cm from the upper edge of the plate. Remove the plate
from the developing chamber and evaporate the solvent in a flow of hot
air.
5.2.5. Spray the plate lightly and uniformly with the alcoholic 2,7-dichlorofluor-
oscein solution. When the plate is observed under ultralviolet light the
sterol and erythrodiol bands can be identified through being aligned with
the references. Mark with a spot just outside the edges of the fluores-
cence.1991R2568 — EN — 21.07.1992 — 004.001 — 27
▼B
5.2.6. Using a metal spatula scrape off the silica gel in the marked areas. Place
the material from the plate ina 50 ml flask. Add 15 ml of hot chloro-
form, shake well and filter through a funnel with a sintered glass disc so
that the silica gel is transferred to the filter. Wash three times with hot
chloroform (10 ml each time) collecting the filtrate in a 100 ml flask.
Evaporate the filtrate to a volume of 4 to 5 ml, transfer to a calibrated
10 ml conical-bottomed centrifuge tube, dry by gently heating in a
current of nitrogen and weigh.
5.3.Preparationofthetrimethylsily esters
As described in paragraph 5.3 of Annex V.
5.4.Gaschromatographicanalysis
As described in paragraph 5.4 of the above method. The operating condi-
tions of the gas chromatograph in analysis must be such as to perform the
sterol analysis and separate the TMSE from the erythrodiol and uvaol.
Once the sample has been injected, continue recording until the sterols
present, the erythrodiol and the uvaol have been eluted. Then identify
the peaks (the retention times for erythrodiol and uvaol relative to β-
sitosterol are about 1,45 and 1,55 respectively) and calculate the areas
as for the sterols.
6. EXPRESSION OF THE RESULTS
Erythrodiol % =A1+A2
A1+A2+ΣAsterols× 100
where:
A1= peak area for erythrodiol insquare millimetres;
A2= peak area for uvaol insquare millimetres;
ΣAsterols= total peak area for sterols insquare millimetres.
The result is expressed to one decimal place.1991R2568 — EN — 21.07.1992 — 004.001 — 28
▼B
ANNEX VII
DETERMINATION OF FATTY ACIDS IN THE 2-POSITION IN THE
TRIGLYCERIDESOFOILSANDFATS
1. SCOPE
This Standard describes a method for the determination of the composi-
tionof that fractionof the fatty acids of anoil or fat which is esterified
at the 2-position (or internal position) of the glycerol.
2. FIELD OF APPLICATION
This Standard is applicable to oils and fats having a melting point below
45 ºC, owing to the peculiarities of the action of pancreatic lipase.
It is not applicable unreservedly to oils and fats containing substantial
amounts of: fatty acids with 12 or fewer carbon atoms (coconut and palm-
kernel oils, butterfat), or highly unsaturated fatty acids (with more than
four double bounds) containing 20 or more carbon atoms (fish and marine
animals oils), or fatty acids containing oxygenated groups, other than the
acid group.
3. PRINCIPLE
Possible neutralization of acid oils and fats in a solvent. Purification by
passing onto an alumina column. Partial hydrolysis of triglycerides by
pancreatic lipase during a determined time. Separation of the formed
monoglycerides by thin-layer chromatography and methanolysis of these
monoglycerides. Analysis of these methyl esters by gas-liquid chromato-
graphy.
4. APPARATUS
4.1. 100 ml round-bottomed flask.4.2. 25 ml round bottomed flask, with ground joint.4.3. 1 m-long air condenser, to fit the flask 4.2.4.4. 250 ml conical flask.4.5. 50 ml beaker.4.6. 500 ml separating funnel.4.7. Chromatographic glass column, 13 mm internal diameter, 400 mm in
length, fitted with a fritted glass disc and a tap.
4.8. 10 ml centrifuge tube, with ground glass stopper.
4.9. 5 ml burette, graduated in0,05 ml.4.10. 1 ml hypodermic syringe, fitted with a thin needle.4.11. Microsyringe, to deliver drops of 3 to 4 µl.4.12. Spreader for thin-layer chromatography.4.13. Glass plates for thin-layer chromatography, 20 × 20 cm.4.14. Glass developing tank for thin-layer chromatography, with ground-glass
lid, suitable for the 20 × 20 plates.
4.15. Spray for thin-layer chromatography.4.16. Ovenregulated at 103 ± 2 ºC.
4.17. Thermostat regulatable between30 an d 45 ºC to within0,5 ºC.
4.18. Rotary evaporator.4.19. Vibrating electric shaker, allowing vigorous agitation of the centrifuge
tube.
4.20. Ultraviolet lamp for the examination of the thin-layer plates.
For the control of the lipase activity:
4.21. pH meter.
4.22. Spiral stirer.1991R2568 — EN — 21.07.1992 — 004.001 — 29
▼B
4.23. 5 ml burette.
4.24. Stop-watch.
For the possible preparation of the lipase:
4.25. Laboratory stirrer, suitable for the dispersionan d mixture of heteroge-
neous materials.
5. REAGENTS
5.1.n-hexane, or, failing this, light petroleum (bp 30 to 50 ºC), chromato-
graphic quality.
5.2. 2-propanol, or ethanol, 95 % (v/v), analytical reagent quality.
5.3. 2-propanol, or ethanol, 1/1 aqueous solution.5.4. Diethyl ether, free from peroxides.
5.5. Acetone.
5.6. Formic acid, at least 98 % (m/m).
5.7. Developing solvent: mixture of n-hexane (5.1), diethyl ether (5.4) and
formic acid (5.6) inproportion s 70/30/1 (v/v/v).
5.8. Activated alumina for chromatography, neutral, grade Brockmann I.
5.9. Silica powder, with binder, of suitable quality for thin-layer chromato-
graphy.
5.10. Pancreatic lipase of suitable quality (Notes 1 and 2).
5.11. Sodium hydroxide, 120 g/l aqueous solution.
5.12. Hydrochloric acid, aqueous ►
C1solution6 Mol/l. ◄
5.13. Calcium chloride (CaCl2), 220 g/l aqueous solution.
5.14. Sodium cholate (enzymatic quality), 1 g/l aqueous solution.
5.15. Buffer solution: 1 M aqueous solution of tris-hydroxymethylamino-
methane bring to pH 8 by addition of hydrochloric acid (5.12) (check by
potentiometer).
5.16. Phenolphthalein, 10 g/l solution in 95 % (v/v) ethanol.
5.17. 2′,7′-dichlorofluorescein, 2 g/l solution in 95 % (v/v) ethanol, rendered
slightly alkaline by the addition of one drop of 1 N sodium hydroxide
solutionper 100 ml.
For the control of the lipase activity:
5.18. Neutralized oil.
5.19. Sodium ►C1hydroxide, 0,1 M ◄aqueous solution.
5.20. Sodium cholate (enzymatic quality), 200 g/l aqueous solution.
5.21. Gum arabic, 100 g/l aqueous solution.
6. PREPARATION OF THE SAMPLE
If the sample has an acidity below 3 %, determined according to Annex
II, purify directly over alumina according to 6.2.
If the sample has an acidity over 3 %, determined according to Annex II,
neutralize by alkali in the presence of a solvent according to 6.1, then pass
over alumina according to 6.2.
6.1. Neutralization by alkali in the presence of solvent
Into a separating funnel (4.6) introduce about 10 g of the crude oil and
add 100 ml of hexane (5.1), 50 ml of 2-propanol (5.2), a few drops of
phenolphthalein solution (5.16), and an amount of the sodium hydroxide
solution (5.11) corresponding to the free acidity of the oil plus 0,3 %
excess. Shake vigorously for one minute, add 50 ml of distilled water,
shake againan d leave to settle.
After separation, remove the bottom soap layer. Also remove any inter-
mediate layers (mucilage, insoluble matter). Wash the hexane solution of
the neutralized oil with successive 25 to 30 ml portions of the 2-propanol
solution (5.3) until the pink colour of the phenolphthalein disappears.1991R2568 — EN — 21.07.1992 — 004.001 — 30
▼B
Remove most of the hexane by distillation under vacuum in the rotary
evaporator (4.18), dry the oil at 30 to 40 ºC under vaccum with the help
of a current of pure nitrogen until the hexane has been removed comple-
tely.
6.2. Purificationthrough alumin a
Prepare a suspension of 15 g of activated alumina (5.8) in 50 ml hexane
(5.1) and pour it, while stirring, onto the chromatographic column (4.7).
Let the alumina settle evenly, and allow the solvent level to fall to within
1 to 2 mm above the absorbent. Carefully pour onto the column a solution
of 5 g of oil in 25 ml of hexane (5.1); collect the whole of the effluent
from the columnina roun d-bottomed flask (4.1).
7. Preparationof the chromatographic plates
Thoroughly clean the glass plates (4.13) with ethanol, light petroleum and
acetone to eliminate any trace of fatty matter.
Ina con ical flask (4.4) place 30 g of silica powder (5.9). Add 60 ml of
distilled water. Stopper and shake vigorously for one minute. Transfer
the slurry immediately to the spreader (4.12) and coat the clean plates
with a layer 0,25 mm thick.
Dry the plates in the air for 15 minutes and then for an hour in the oven
(4.16) at 103 ± 2 ºC. Cool the plates ina desiccator to room temperature
before use.
Prepared plates are available incommerce.
8. PROCEDURE
8.1. Hydrolysis with pancreatic lipase.
Into the centrifuge tube (4.8) weigh about 0,1 g of the prepared sample, if
the sample is a liquid oil proceed directly as below.
Add 20 mg of lipase (5.10) and 2 ml of the buffer solution (5.15). Shake
well, but carefully, and then add 0,5 ml of the sodium cholate solution
(5.14) and 0,2 ml of the calcium chloride solution (5.13). Close the tube
with the ground stopper, shake cautiously (avoid wetting the stopper) and
put the tube immediately in the thermostat (4.17) maintained at 40 ± 0,5 ºC
and shake by hand for exactly one minute.
Remove the tube from the thermostat, and agitate vigorously by means of
the electric shaker (4.19) for exactly two minutes.
Cool immediately in running water; add 1 ml of hydrochloric acid (5.12)
and 1 ml of diethyl ether (5.4). Stopper and mix vigorously by means of
the electric shaker. Allow to stand and remove the organic layer by means
of the syringe (4.10), if necessary after centrifuging.
8.2. Separation of the monoglycerides by thin-layer chromatography
Apply the extract to the chromatographic plate with the microsyringe
(4.11), about 1,5 cm from the bottom edge, in a thin, uniform line, as
narrow as possible. Set the plate in the well-saturated developing tank
(4.14) and develop with the developing solvent (5.7) at about 20 ºC, up
to about 1 cm from the top edge of the plate.
Dry the plate in the air at the temperature of the tank, and spray it with
the 2′,7′-dichlorofluorescein solution (5.17). Identify the monoglyceride
band (Rfabout 0,035) under ultraviolet light (4.20).
8.3. Analysis of the monoglycerides by gas-liquid chromatography
Remove the band obtained in 8.2 with the help of a spatula (avoid
removing components remaining on the base line) and transfer in the
methylationflask (4.2).
Treat the collected silica directly by the methods described in Annex X B
alternative so as to convert the monoglycerides into methyl esters, and
then examine the esters by gas chromatography as described in Annex X
A.
9. EXPRESSION OF RESULTS
Calculate the fatty acid compositioninthe 2-positionto on e decimal place
(Note 3).
10. NOTES
Note 1:Checking the activity of the lipase1991R2568 — EN — 21.07.1992 — 004.001 — 31
▼B
Prepare anoil emulsionby shakin g a mixture of 165 ml of the
solutionof gum arabic (5.21), 15 g of crushed ice an d 20 ml of
a neutralized oil (5.18) in a suitable agitator.
Ina beaker (4.5) place 10 ml of this emulsion , followed succes-
sively by 0,3 ml of the sodium cholate solution (5.20) and and
20 ml of distilled water.
Put the beaker into a thermostat maintained at 37 ± 0,5 ºC (Note
4); insert the electrodes of a pH meter (4.21) and a spiral stirrer
(4.22).
By means of a burette (4.23) add dropwise the sodium hydroxide
solution(5.19) un til the pH reaches 8,5.
Add sufficient of an aqueous suspension of the lipase (see below).
►C1As soonas ◄the pH meter indicates a pH of 8,3, start the
stop-watch (4.24) and drip in the sodium hydroxide solution
(5.19) at such a rate as to maintain the pH at 8,3. Read off the
volume of alkali solution consumed every minute.
Record the observations in the form of a graph, using the time
readings as abscissae and the ml of alkali solution required to
maintain the pH constant as ordinates. A linear graph should be
obtained.
The lipase suspension mentioned above is a 1 per thousand (m/m)
suspension in water. For each test sufficient of this suspension
should be used so that about 1 ml of the alkali solutionis
consumed in four to five minutes. Usually about 1 to 5 mg of
the powder is required.
The lipase unit is defined as the amount of enzyme which will
liberate 10 µ-equivalents of acid per minute. Then the activity A
of the powder used, measured inlipase un its per mg, is givenby
the formula:
A¼V/C210
m
where V is the number of the sodium hydroxide solution (5.19)
consumed per minute, calculated from the graph, m is the mass
inmg, of the test portionof the powder.
Note 2:Preparationof the lipase
Lipases having satisfactory lipase activity are available commer-
cially. But it is also possible to prepare them inthe laboratory as
follows:
Chill 5 kg of fresh pig pancreas to 0 ºC; remove the surrounding
solid fat and connective tissue and triturate in a blender so as to
obtaina pasty fluid. Stir this paste with the stirrer (4.25) for four
to six hours with 2,5 l of anhydrous acetone and centrifuge.
Extract the residue thrice more with the same volume of acetone,
then twice with 1/1 (V/V) mixture of acetone and diethyl ether,
and twice with diethyl ether.
Dry the residue in vacuo for 48 hours to obtaina stable powder,
which should be stored ina refrigerator.
Note 3:Inevery case it is advisable to determin e the compositionof the
total fatty acids of the same sample, since the comparison with
that of the acids inthe 2-positionwill help inthe in terpretation
of the figures obtained.
Note 4:The temperature of hydrolysis is set at 37 ºC, as a liquid oil is
being used. However, it is set at 40 ºC for the test sample, so as
to allow the examination of fats with melting points up to 45 ºC.1991R2568 — EN — 21.07.1992 — 004.001 — 32
▼B
ANNEX VIII
DETERMINATIONOFTRILINOLEINCONTENT
1. SCOPE
Determination of composition of triglyceride in olive oils ►C1interms of
their equivalent ◄carbon number by high-performance liquid chromato-
graphy.
►C1The present method ◄describes a method of separation and quanti-
tative determination of the triglyceride composition of vegetable oils in
terms of their molecular weight and degree of unsaturation as a function
of their equivalent carbon number (see Note 1).
2. FIELD OF APPLICATION
►C1This method is ◄applicable to all vegetable oils containing trigly-
cerides of long-chain fatty acids. The method is especially appliable to the
detection of the presence of small quantities of semi-drying oils (rich in
linoleic acid) in vegetable oils containing oleic acid as the predominant
unsaturated fatty acid, such as olive oil.
3. PRINCIPLE
Separation of triglycerides according to their equivalent carbon number by
high-performance liquid chromatography (reversed phase polarity) and
interpretation of the chromatograms.
4. APPARATUS
4.1. High-performance liquid chromatograph, allowing thermostatic control of
columntemperature.
4.2. Injection unit for 10 µl delivery.4.3. Detector: differential refractometer. The full-scale sensitivity should be at
least 10
−4unit of refractive index.
4.4. Column: stainless steel tube 250 mm in length and of internal diameter
4,5 mm, packed with 5 µm diameter particles of silica with 22 to 23 %
carboninthe form of octadecylsilan e (Note 2).
4.5. Recorder and/or integrator.
5. REAGENTS
The reagents should be of analytical purity. Elution solvents should be de-
gassed, and may be recycled several times without effect on the separa-
tions.
5.1. Chloroform.5.2. Acetone.
5.3. Acetonitrile.
5.4. Elution solvent: acetonitrile + acetone (proportions to be adjusted to obtain
the desired separation; begin with 50:50 mixture).
5.5. Solubilization solvent: acetone or 1:1 acetone-chloroform mixture.5.6. Reference triglycerides: either commercial triglycerides (tripalmitin, trio-
lein, etc.) may be used and the retention times thence plotted in
accordance with the equivalent carbon number, or alternatively a reference
chromatogram obtained from soya oil (see Notes 3 and 4 and Figures 1 and
2).
6. PREPARATION OF SAMPLES
A 5 % solution of the samples to be analysed is prepared by weighing
0,5 ± 0,001 g of the sample into a 10 ml graduated flask and making up
to 10 ml with the solubilizationsolven t (5.5).1991R2568 — EN — 21.07.1992 — 004.001 — 33
▼C1
▼B
▼B
7. PROCEDURE
7.1. Set up the chromatographic system. Pump elutionsolven t (5.4) at a rate of
1,5 ml/mm to purge the entire system. Wait until a stable base line is
obtained.
Inject 10 µl of the sample prepared as in 6.
8. CALCULATION AND EXPRESSION OF RESULTS
Use the internal standardization method, i.e. assume that the sum of the
areas of the peaks corresponding to the various triglycerides is equal to
100 %. Calculate the relative percentage of each triglyceride using the
formula:
% triglyceride =area of peak
sum of peak areas× 100
The result to be givento on e decimal place.
Note 1.The elution order can be determined by calculating the equivalent
carbon numbers, often defined by the relation ECN = CN − 2n,
where CN is the carbon number and n is the number of double
bonds; it can be calculated much more precisely by taking into
account the origin of the double bond. If no,n1and n1nare the
numbers of double bonds attributable to oleic, linoleic and lino-
lenic acids respectively, the equivalent carbon number can be
calculated by means of a relation of the formula:
ECN = CN − dono−d1n1−d1nn1n
where the coefficients do,d1and d1ncanbe calculated by mean s of
the reference triglycerides. Under the conditions specified in this
method the relationobtain ed will be close to:
ECN = CN − [2,60 no] − [2,35 n1] − [2,17 n1n]
Note 2.Examples: Lichrosorb (Merck) RP18 Art 50333;
Lichrosphere or equivalent (Merck) 100 CH18 Art
50377.
Note 3.With several reference triglycerides it is also possible to calculate
the resolutionwith respect to triolein ,
α=R T′/RT′olein
by use of the reduced retention time RT ′=R T−R Tsolvent.
The graph of log αagainst f (number of double bonds) enables the
retention values to be determined for all the triglycerides of fatty
acids contained in the reference triglycerides — see Figure 2.
Note 4.The efficiency of the column should permit clear separation of the
peak of trilinolein from the peaks of the triglycerides with an adja-
cent RT.
Note 5:Inorder to permit clear separationof the peak of trillon leinfrom
the adjacent peaks, crude olive-residue oil should be purified
beforehand in accordance with point 6.2 of Annex VII or, alterna-
tively:
200 µl of undiluted oil are abosorbed in a silica column for liquid-
solid extraction(type SEP PAC silica cartridge-waters port. No
51900).
The triglycerides are eluted with 20 ml anhydrous hexane for
HPLC.
The eluted product is dried in a nitrogen flow and placed in
Isopropanol or acetone (5 ml). 10-20 µl are injected into HPLC.
For these two purificationmethods, the fatty acid compositionof
the oil must be checked to ensure that it is the same before and
after purification. If it is not the same, the absorbent quality used
should be progressively reduced.1991R2568 — EN — 21.07.1992 — 004.001 — 34
▼M4
▼B
Figure 1
Chromatogram of a sample of soya oil
P = palmitic acid
L = linoleic acid
St = stearic acid
ln = linolenic acidO = oleic acid1991R2568 — EN — 21.07.1992 — 004.001 — 35
▼B
Figure 2
Graphof log αagainst f (number of double bonds)
La = lauric acid
St = stearic acid
Ln= lin oleic acidMy = myristic acidO = oleic acid
P = palmitic acid
L = linoleic acid1991R2568 — EN — 21.07.1992 — 004.001 — 36
▼B
ANNEX IX
SPECTROPHOTOMETRICINVESTIGATIONINTHEULTRAVIOLET
FOREWORD
Spectrophotometric examination in the ultraviolet can provide information
on the quality of a fat, its state of preservation and changes brought about
init by techn ological processes.
The absorptionat the wavelen gths specified inthe method is due to the
presence of conjugated diene and triene systems. These absorptions are
expressed as specific extinctions E1%
1c m(the extinction of 1 % solution of
the fat in the specified solvent, in a thickness of 1 cm) conventionally indi-
cated by K (also referred to as ‘extinction coefficient’).
1. SCOPE
The method describes the procedure for performing a spectrophotometric
examination ►C1of olive oil in ◄the ultraviolet.
2. PRINCIPLE OF THE METHOD
The fat in question is dissolved in the required solvent and the extinction of
the solution is then determined at the specified wavelengths with reference
to pure solvent. Specific extinctions are calculated from the spectrophot-
ometer readings.
3. EQUIPMENT
3.1. A spectrophotometer for measuring extinction in the ultraviolet between
220 and 360 nm, with the possibility of reading individual nanometric
units.
3.2. Rectangular quartz cuvettes, with covers, having an optical length of 1 cm.
When filled with water or other suitable solvent the cuvettes should not
show differences between them of more than 0,01 extinction units.
3.3. 25 ml graduated flasks.
3.4. Chromatography column having a length of 450 mm and a diameter of
35 mm with a discharge tube of diameter approximately 10 mm.
4. REAGENTS
4.1. Spectrophotometrically pure iso-octane (2,2,4-trimethylpentane). With
reference to distilled water this should have a transmittance of not less
than 60 % at 220 nm and not less than 95 % at 250 nm, or
— spectrophotometrically pure cyclohexane: with reference to distilled
water this should have a transmittance of not less than 40 % at 220 nm
and not less than 95 % at 250 nm, or
— another suitable solvent capable of completely dissolving the fat (e.g.
ethyl alcohol for castor oil).
4.2. Basic alumina for column chromatography prepared and checked as
described inAppen dix I.
4.3. n-hexane, for chromatography.
5. PROCEDURE
5.1. The sample in question must be perfectly homogeneous and without
suspected impurities. Oils which are liquid at ambient temperature are to
be filtered through paper at a temperature of approximately 30 ºC, hard
fats are to be homogenized and filtered at a temperature of not more than
10 ºC above the melting point.
5.2. Weigh accurately approximately 0,25 g of the sample so prepared into a
25 ml graduated flask, make up to the mark with the solvent specified and
homogenize. The resulting solution must be perfectly clear. If opalescence
or turbidity is present filter quickly through paper.
5.3. Fill a cuvette with the solution obtained and measure the extinctions at an
appropriate wavelength between 232 and 276 nm, using the solvent used as
a reference.1991R2568 — EN — 21.07.1992 — 004.001 — 37
▼B
The extinction values recorded must lie within the range 0,1 to 0,8. If not
the measurements must be repeated using more concentrated or more dilute
solutions as appropriate.
5.4. When a determination of specific extinction is required after passage over
alumina, proceed as follows. Place 30 g of basic alumina in suspension in
hexane in the chromatography column. After the adsorbent has settled
remove the excess hexane down to approximately 1 cm above the top of
the alumina.
Dissolve 10 g of the fat, homogenized and filtered as described in 5.1, in
100 ml of hexane and pour the solution into the column. Collect the eluate
and evaporate off all the solvent under vacuum at a temperature below
25 ºC.
Proceed immediately as specified in 5.2 using the fat so obtained.
6. EXPRESSION OF THE RESULTS
6.1. Record the specific extinctions (extinction coefficients) at the various
wavelengths calculated as follows:
Kλ¼Eλ
c/C1s
where:
Kλ= specific extinction at wavelengthλ;
Eλ= extinction measured at wavelength λ;
c = concentration of the solution in g/100 ml;
s = thickness of the cuvette in cm.
The results are to be expressed to two decimal places.
6.2. Spectrophotometric analysis of olive oil in accordance with the official
method in the EEC regulations specifies determination of the specific
extinction in iso-octane solution at wavelengths of 232 and 270 nm and
the determination K, which is given by:
∆K¼Km/C0Km/C04þKmþ4
2
where Kmis the specific extinction at wavelength m, the wavelength for
maximum absorption around 270 nm.1991R2568 — EN — 21.07.1992 — 004.001 — 38
▼B
APPENDIX I
Preparation of the alumina and testing its activity
A.1.1. Preperationof the alumin a
Place alumina which has been previously desiccated in a furnace at 380
to 400 ºC for three hours into a hermetically sealed container, add
distilled water inthe ratio of 5 ml per 100 g of alumin a, immediately
close the container, shake repeatedly, and then allow to rest for at least
12 hours before use.
A.1.2. Checking the activity of the alumina
Prepare a chromatographic column with 30 g of alumina. Working as
described in paragraph 5.4 pass a mixture consisting of:
— 95 % virgin olive oil having a specific extinction of less than 0,18 at
268 nm,
— 5 % ground-nut oil treated with earth in the refining process, having
a specific extinction of not less than 4 at 268 nm
through the column.
If after passage through the columnthe mixture has a specific extin ction
of more than 0,11 at 268 nm the alumina is acceptable, if not the level of
dehydrationmust be in creased.1991R2568 — EN — 21.07.1992 — 004.001 — 39
▼B
APPENDIX II
Calibration of the spectrophotometer
A.2. The equipment must be checked at intervals (at least every six months)
for both wavelength response and the accuracy of the response.
A.2.1. The wavelength may be checked using a mercury vapour lamp or by
means of suitable filters.
A.2.2. In order to check the response of the photocell and the photomultiplier
proceed as follows: weigh 0,2000 g of pure potassium chromate for spec-
trophotometry an d dissolve in0,05 N potassium hydroxide solutionina
1 000 ml graduated flask and make up to the mark. Take precisely 25 ml
of the solution obtained, transfer to a 500 ml graduated flask and dilute
up to the mark using the same potassium hydroxide solution.
Measure the extinction of the solution so obtained at 275 nm, using the
potassium hydroxide solution as a reference. The extinction measured
using a 1 cm cuvette should be 0,200 ± 0,005.1991R2568 — EN — 21.07.1992 — 004.001 — 40
▼B
ANNEX X A
ANALYSIS BY GAS CHROMATOGRAPHY OF METHYL ESTERS OF
FATTYACIDS
1. SCOPE
This method gives general guidance for the application of gas chroma-
tography, using packed or capillary columns, to determine the
qualitative and quantitative composition of a mixture of fatty acid
methyl esters obtained in accordance with the method specified in
Annex X B.
The method is not applicable to polymerized fatty acids.
2. REAGENTS
2.1.Carriergas
Inert gas (nitrogen, helium, argon, hydrogen, etc.), thoroughly dried and
with an oxygen content of less than 10 mg/kg.
Note 1.Hydrogen, which is used as a carrier gas only with capillary
columns, can double the speed of analysis but is hazardous.
Safety devices are available.
2.2.Auxiliarygases
2.2.1. Hydrogen(purity ≥99,9 %), feree from organic impurities.
2.2.2. Air or oxygen, free from organic impurities.
2.3.Reference standard
A mixture of methyl esters of pure fatty acids, or the methyl esters of a
fat of known composition, preferably similar to that of the fatty matter
to be analyzed.
Care shall be taken to prevent the oxidation of polyunsaturated fatty
acids.
3. APPARATUS
The instructions given relate to the usual equipment used for gas chro-
matography, employing packed and/or capillary columns and a flame-
ionization detector. Any apparatus giving the efficiency and resolution
specified in4.1.2 is suitable.
3.1.Gaschromatograph
The gas chromatograph shall comprise the following elements.
3.1.1. Injection system
Use anin jectionsystem either:
(a) with packed columns, having the least deadspace possible (in this
case the injection system shall be capable of being heated to a
temperature 20 to 50 ºC higher thanthat of the column ); or
(b) with capillary columns, in which case the injection system shall be
specially designed for use with such columns. It may be of the split
type or it may be of the splitless oncolumnin jector type.
Note 2.Inthe absen ce of fatty acids with less than16 carbonatoms, a
moving needle injector may be used.
3.1.2. Oven
The ovenshall be capable of heatin g the columnto a temperature of at
least 260 ºC and of maintaining the desired temperature to within 1 ºC
with a packed column and within 0,1 ºC with a capillary column. The
last requirement is particularly important when a fused silica tube is
used.
The use of temperature-programmed heating is recommended in all
cases, and in particular for fatty acids with less than 16 carbon atoms.1991R2568 — EN — 21.07.1992 — 004.001 — 41
▼B
3.1.3. Packed column
3.1.3.1. Column, constructed of a material inert to the substances to be analyzed
(i.e. glass or stainless steel) having the following dimensions:
(a) length: 1 to 3 m. A relatively short column should be used when
long-chain fatty acids (above C20) are present. When analyzing
acids with 4 or 6 carbonatoms, it is recommen ded that a column
2 m inlen gth is used;
(b) internal diameter: 2 to 4 mm.
Note 3.If polyunsaturated components with more than three double
bonds are present, they may be decomposed in a stainless steel
column.
Note 4.A system with packed twincolumn s may be used.
3.1.3.2. Packing, comprising the following elements:
(a)support: acid-washed and silanized diatomaceous earth, or other
suitable inert support with a narrow range of grain size (25 µm
range between the limits 125 to 200 µm) the average grain size
being related to the internal diameter and length of the column;
(b)stationary phase: polyester type of polar liquid (e.g. diethylene
glycol polysuccinate, butanediol polysuccinate, ethyleneglycol poly-
adipate, etc.) cyanosilicones or any other liquid permitting the
chromatographic separationrequired (see clause 4). The station ary
phase should amount to 5 to 20 % (m/m) of the packing. A non-
polar stationary phase can be used for certain separations.
3.1.3.3. Conditioning of the column
With the column disconnected, if possible, from the detector, gradually
heat the oven to 185 ºC and pass a current of inert gas through the
freshly prepared columnat a rate of 20 to 60 ml/minfor at least 16
hours at this temperature, and for a further 2 hours at 195 ºC.
3.1.4. Capillary column
3.1.4.1. Tube, made of a material inert to the substances to be analysed (usually
glass or fused silica). The internal diameter shall be between 0,2 and
0,8 mm. The internal surface shall undergo an appropriate treatment
(e.g. surface preparation, inactivation) before receiving the stationary
phase coating. A length of 25 mm is sufficient in most cases.
3.1.4.2. Stationary phase, usually of the type polyglycol (poly(ethylene glycol)
20000), polyester (butanediol polysuccinate) or polar polysiloxane
(cyanosilicones). Bonded (cross-linked) columns are suitable.
Note 5.There is a risk of polar polysiloxanes giving rise to difficulties
in the identification and separation of linolenic acid and C20acids.
The coatings shall be thin, i.e, 0,1 to 0,2 µm.
3.1.4.3. Assembly and conditioning of the column
Observe the normal precautions for assembling capillary columns, i.e.
arrangement of the column in the oven (support), choice and assembly
of joints (leak tightness), positioning of the ends of the column in the
injector and the detector (reduction of dead-spaces). Place the columnunder a flow of carrier gas (e.g. 0,3 bar (30 kPa) for a column of length
25 mm and internal diameter 0,3 mm).
Condition the column by temperature programming of the oven at 3 ºC/
minfrom ambien t temperature to a temperature 10 ºC below the
decomposure limit of the stationary phase. Maintain the oven at this
temperature for one hour until stabilization of the baseline. Return it
to 180 ºC to work under isothermal conditions.
Note 6.Suitably pre-conditioned columns are available commercially.
3.1.5. Detector, preferably capable of being heated to a temperature above
that of the column.
3.2.Syringe
The syringe shall have a maximum capacity of 10 µl, and be graduated
in0,1 µl division s.1991R2568 — EN — 21.07.1992 — 004.001 — 42
▼B
3.3.Recorder
If the recorder curve is to be used to calculate the compositionof the
mixture analysed, an electronic recorder of high precision, compatible
with the apparatus used, is required. The recorder shall have the
following characteristics:
(a) rate of response, below 1,5 s, preferably 1 s (the rate of response is
the time takenfor the recordin g pento pass from 0 to 90 %
following the sudden introduction of a 100 % signal);
(b) width of the paper, 20 cm minimum;
(c) paper speed, adjustable to values between 0,4 and 2,5 cm/min.
3.4.Integrator
Rapid and accurate calculation can be performed with the help of an
electronic integrator. This shall give a linear response with adequate
sensitivity, and the correction for deviation of the base-line shall be
satisfactory.
4. PROCEDURE
The operations described in 4.1 to 4.3 relate to the use of a flame-ioni-
zationdetector.
As an alternative a gas chromatograph employing a catharometer
detector (working on the principle of thermal conductivity changes)
may be used. The operating conditions are then modified as described
inclause 6.
4.1.Testconditions
4.1.1. Selection of optimum operating conditions4.1.1.1. Packed column
In the selection of the test conditions, the following variables should be
taken into account:
(a) the length and diameter of the column;
(b) the nature and amount of the stationary phase;
(c) the temperature of the column;
(d) the carrier gas flow;
(e) the resolutionrequired;
(f) the size of the test portion, selected in such a way that the assembly
of the detector and electrometer gives a linear response;
(g) the durationof an alysis.
In general, the values given in Table 1 and Table 2 will lead to the
desired results, i.e. at least 2 000 theoretical Iplates per metre of column
length for methyl stearate and its elution within about 15 minutes.
Where the apparatus allows it, the injector should be at a temperature of
about 200 ºC and the detector at a temperature equal to or higher than
that of the column.
As a rule, the ratio of the flow-rate of the hydrogensupplied to the
flame-ionization detector to that of the carrier gas varies from 1:2 to
1:1 depending on the diameter of the column. The flow of oxygen is
about 5 to 10 times that of the hydrogen.
Table 1
Internal diameter of column
mmCarrier gas flow
ml/min
21 5 t o 2 5
32 0 t o 4 0
44 0 t o 6 01991R2568 — EN — 21.07.1992 — 004.001 — 43
▼B
Table 2
Concentration of stationary phase
% (m/m)Columntemperature
ºC
5 175
10 180
15 185
20 185
4.1.1.2. Capillary column
The properties of efficiency and permeability of capillary columns
mean that the separation between constituents and the duration of the
analysis are largely dependent on the flow-rate of the carrier-gas in the
column. It will therefore be necessary to optimize the operating condi-
tions by acting on this parameter (or more simply on the headloss of the
column), according to whether one wishes to improve the separations or
to make a rapid analysis.
4.1.2. Determination of the number of theoretical plates (efficiency) and reso-
lution(See Figure 1)
Carry out the analysis of a mixture of methyl stearate and methyl oleate
in about equivalent proportions (for example, methyl esters from cocoa
butter).
Choose the temperature of the columnan d the carrier gas flow so that
the maximum of the methyl stearate peak is recorded about 15 minutes
after the solvent peak. Use a sufficient quantity of the mixture of
methyl esters that the methyl stearate peak occupies about three-quar-
ters of the full scale.
Calculate the number of theoretical plates, n (efficiency), using the
formula:
n¼16dr1
ω1/C20/C21
2
and the resolution, R, using the formula:
R¼2∆
ω1þω2
where:
dr1is the retention distance, in millimetres, from the start of the
chromatogram to the maximum of the peak for methyl stea-
rate;
ω1andω2are the widths, inmillimetres, of the peaks for methyl stea-
rate and methyl oleate respectively, measured between the
points of intersection of the tangents at the points of inflec-
tionof the curve with the base-lin e;
∆is the distance, in millimetres, between the peak maxima for
methyl stearate and methyl oleate;
and the resolution index, lr, using the formula
a
b
where:
a = the height of the smallest peak, measured from the base line;
b = the height of the lowest point of the valley between the two adja-
cent peaks, measured from the base line.1991R2568 — EN — 21.07.1992 — 004.001 — 44
▼M2
▼B
Figure1
Chromatogram for determining the number of theoretical plates (efficiency) and
resolution
The operating conditions to be selected are those which will afford at
least 2 000 theoretical plates per metre of columnlen gth for methyl
stearate and a resolution of at least 1,25.
4.2.Testportion
Using the syringe (3.2) take 0,1 to 2 µl of the solution of methyl esters
prepared according to Annex X B and inject them into the column.
In the case of esters not in solution, prepare a solution of approximately
100 mg/ml in heptane of chromatographic quality, and inject 0,1 to 1 ml
of this solution.
If the analysis is for constituents present only in trace amounts, the size
of the test portionmay be in creased (up to 10-fold).
4.3.Analysis
Generally, the operating conditions shall be those defined in 4.1.1.
Nevertheless, it is possible to work with a lower columntemperature
whenthe determin ationof fatty acids with fewer than12 carbonatoms
is required, or at a higher temperature when determining fatty acids
with more than20 carbonatoms. Onoccasion , it is possible to employ
temperature programming in both these cases. For example, if the
sample contains the methyl esters of fatty acids with fewer than 12
carbonatoms, in ject the sample at 100 ºC (or at 50 to 60 ºC if butyric
acid is present) and immediately raise the temperature at a rate of 4 to
8 ºC/minto the optimum. Incertaincases, the two procedures canbe
combined.
After the programmed heating, continue the elution at a constant
temperature until all the components have been eluted. If the instrument
does not have programmed heating, use it at two fixed temperatures
between100 an d 195 ºC.1991R2568 — EN — 21.07.1992 — 004.001 — 45
▼B
If necessary, it is recommended that an analysis be carried out on two
fixed phases with different polarities to verify the absence of masked
peaks, for example in the case of the simultaneous presence of C18:3and C20:0,o rC18:3and C18:2conjugated.
4.4.Preparationofthereference chromatogramandreference graphs
Analyze the reference standard mixture (2.3) using the same operating
conditions as those employed for the sample, and measure the retention
times or retention distances for the constituent fatty acids. Construct on
semi-logarithmic paper, for any degree of unsaturation, the graphs
showing the logarithm of retention time or distance as a function of
the number of carbon atoms. In isothermal conditions, the graphs for
straight-chainacids of the same degree of un saturationshould be
straight lines. These lines should be approximately parallel.
It is necessary to avoid conditions such that ‘masked peaks’ exist, i.e.
where the resolution is insufficient to separate two constituents.
5. EXPRESSION OF RESULTS
5.1.Qualitativeanalysis
Identify the methyl ester peaks for the sample from the graphs prepared
in 4.4, if necessary by interpolation.
5.2.Quantitativeanalysis
5.2.1. Determination of the composition
Apart from exceptional cases, use the internal normalization method,
i.e. assume that the whole of the components of the sample are repre-
sented on the chromatogram, so that the total of the areas under the
peaks represents 100 % of the constituents (total elution).
If the equipment includes an integrator, use the figures obtained there-
from. If not, determine the area under each peak by multiplying the
height of the peak by its width at mid-height, and where necessary
take into account the various attenuations used during the recording.
5.2.2. Method of calculation
5.2.2.1. General case
Calculate the content of a given component i,expressed as a percentage
by mass of methyl esters, by determining the percentage represented by
the area of the corresponding peak relative to the sum of the areas of all
the peaks, using the following formula:
Ai
ΣA/C2100
where:
Aiis the area under the peak corresponding to component i;
ΣA is the sum of the areas under all the peaks.
Give the result to one decimal place.
Note 7:Inthis gen eral case, the result of the calculationbased onrela-
tive areas is considered to represent a percentage by mass. For
the cases inwhich this assumptionis n ot allowed, see 5.2.2.2.
5.2.2.2. Use of correctionfactors
Incertaincases, for example inthe presen ce of fatty acids with fewer
than eight carbon atoms or of acids with secondary groups, when using
thermal conductivity detectors or where the highest degree of accuracy
is particularly required, correctionfactors should be used to con vert the
percentages of peak areas into mass percentages of the components.
Determine the correction factors with the help of a chromatogram
derived from the analysis of a reference mixture of methyl esters of
known composition, carried out under operating conditions identical
with those used for the sample.
For this reference mixture, the percentage by mass of component iis
givenby the formula:1991R2568 — EN — 21.07.1992 — 004.001 — 46
▼B
mi
Σm/C2100
where:
miis the mass of component i in the reference mixture;
Σm is the total of the masses of the various components of the refer-
ence mixture.
From the chromatogram of the reference mixture (4.4) calculate the
percentage (area/area) for component ias follows:
Ai
ΣA/C2100
where:
Aiis the area under the peak corresponding to component i;
ΣA is the sum of the areas under all the peaks.
The correctionfactor is thencalculated as:
Ki¼mi/C2ΣA
Ai/C2Σm
Commonly, the correction factors are expressed relative to KC16, so that
the relative factors become:
K0
i¼Ki
KC16
For the sample, the content of each component i, expressed as a percen-
tage by mass of methyl esters, is:
K0
i/C2Ai
ΣK0
i/C2Ai/C0/C1 /C2100
Give the results to one decimal place.
5.2.2.3. Use of an internal standard
In certain analyses (for example where not all of the fatty acids are
quantified, such as when acids with four and six carbons are present
alongside acids with 16 and 18 carbons, or when it is necessary to
determine the absolute amount of a fatty acid in a sample) it is neces-
sary to use an Internal Standard. Fatty acids with five, 15 or 17 carbons
are frequently used. The correction factor (if any) for the Internal Stan-
dard should be determined.
The percentage by mass of component i,expressed as methyl esters, is
thengivenby the formula:
ms/C2K0
i/C2Ai
m/C2K0
s/C2As/C2100
where:
Aiis the area under the peak corresponding to component i;
Asis the area under the peak corresponding to the Internal Standard;
K′iis the correction factor for component i(relative to KC1);
K′sis the correction factor for the Internal Standard (relative to KC16);
m is the mass, inmilligrams, of the test portion ;
msis the mass, in milligrams, of the Internal Standard.
Give the results to one decimal place.
6. SPECIAL CASE — DETERMINATION OF TRANS-ISOMERS
It is possible to determine the content of trans-isomers in fatty acids
with a number of carbon atoms between 10 and 24 by separating the
methyl esters using gas chromatography capillary columns having a
specific polarity.1991R2568 — EN — 21.07.1992 — 004.001 — 47
▼M2
▼M2
6.1. A capillary column made of silica having an internal diameter of
between 0,25 mm and 0,32 mm and a length of 50 m, coated with
cyanopropisilicon, the thickness of the coating being between 0,1 and
0,3 µm (type SP 2380, C.P. sil 88, silor 10 and similar types).
6.2. The methyl esters are prepared using the procedure set out in Annex X
B. As a precaution, fatty substances having a free acidity over 3 %
must be neutralized in accordance with 6.1 of Annex VII.
6.3. The operating conditions for gas chromatography are overall as follows:
— columntemperature set between150 ºC an d 230 ºC (for example
165 ºC for 15 minutes then increasing by 5 ºC a minute to 200 ºC);
— injector temperature: 250 ºC if the splitting system is used or the
initial temperature of the column if the on-column system is used;
— detector temperature: 260 ºC;— flow rate of the carrier gas (helium and hydrogen): 1,2 ml a minute.
The quantity injected must be such that in the conditions of sensitivity
employed the height of the peak corresponding to the methyl ester of
the arachidic acid is equal to or greater than20 % of the bottom of
the scale.
6.4. Identification of the various methyl esters is effected on the basis of the
retention times which are compared with those for the reference
mixtures (as indicated at point 2.3).
The esters of trans fatty acids are eluted before the corresponding cis-
isomers. Anexample of a chromatogram is giveninfigure 2.
Figure2:
Gas chromatogram of the trans-isomers of fatty acid using capillary column.
6.5. The efficiency of the column determined in accordance with point 4.1.2
must be such as to allow separationof certaincritical couples, for
example the couple formed by the massif of the transisoleic acids and
the oleic acid peak, trans C18:1/cis C18:1, with a resolution index
greater than2.
6.6. The percentage of the various trans fatty acids is calculated on the basis
of the relationship between the surface of the relevant peak and the sum
of the surfaces of all the peaks present.
The percentages of:
— the trans octadecenoic acids (T 18: 1) indicated in Annex I to this
Regulationas the sum of the tran soleic isomers;
— the cis-trans and trans-cis octadecadienoic acids [(CT/TC) 18: 2]
indicated in Annex I to this Regulation as the sum of the translino-
leic isomers;1991R2568 — EN — 21.07.1992 — 004.001 — 48
▼M2
— the trans-cis-trans, cis-cis-trans, cis-trans-cis, trans-cis-cis, octadeca-
trienoic acids [(TCT + CCT + CTC + TCC) 18: 3], indicated in
Annex I of this Regulation as the sum of the translinolenic isomers
are taken into account.
Note 8:Taking into account the particular characteristics of this
method, please give the results with 2 decimals.
►M27.◄SPECIAL CASE — USE OF A CATHAROMETER DETECTOR
(WORKING ON THE PRINCIPLE OF THERMAL CONDUCTIVITY
CHANGES)
A gas chromatograph employing a detector working on the principle of
thermal conductivity changes (a catharometer) may also be used for the
determination of the qualitative and quantitative composition of a
mixture of fatty acid methyl esters. If it is used, the conditions specified
inclause 3 an d clause 4 should be modified as showninTable 3.
For quantitative analysis, use the correction factors defined in 5.2.2.2.
Table 3
Variable Value/condition
ColumnLen gth: 2 to 4 m
Internal diameter: 4 mm
Support Grainsize between160 an d
200 µm
Concentration of stationary phase 15 to 25 % (m/m)
Carrier gas Helium or, failing this, hydrogen,
with as low an oxygen content as
possible
Auxiliary gases None
Injector temperature From 40 to 60 ºC above that of
the column
Columntemperature 180 to 200 ºC
Flow of carrier gas Usually between60 an d 80 ml/
min
Size of test portion injected Usually between 0,5 and 2 µl
►M28.◄TEST REPORT
The test report shall specify the methods used for the preparationof the
methyl esters and for the gas chromatographic analysis, and the results
obtained. It shall also mention all operating details not specified in this
International Standard, or regarded as optional, together with details of
any incidents which may have influenced the results.
The test report shall include all information necessary for the complete
identification of the sample.1991R2568 — EN — 21.07.1992 — 004.001 — 49
▼B
▼B
ANNEX X B
PREPARATION OF METHYL ESTERS OF FATTY ACIDS IN ACCOR-
DANCE WITH TITLES I AND II OF ANNEX VI TO REGULATION
(EEC)No72/77ORTHEMETHODDESCRIBEDBELOW
FOREWORD
The choice of process will be dictated by the acid compositionan d acidity
of the fatty substance under examination and the gas chromatography
analysis to be carried out.
More specifically:
— only sealed phial processes or processes using dimethyl sulphate can be
used for fatty substances containing fatty acids with fewer than 12
carbonatoms,
— only the methanol-hydrochloric acid or methyl sulphate processes can
be used for fatty substances with acidity of over 3 %,
— only processes using sodium methylate or dimethyl sulphate can be
used for gas chromatography measurements of trans-Isomers,
— the methanol-hexane-sulphuric acid process must be used for the
preparation of the methyl esters of small quantities of fatty substances
from separationby thinlayer chromatography.
The presence of unsaponifiables can be discounted, provided it does not
exceed 3 %, otherwise the methyl esters will have to be prepared from the
fatty acids.
1. SCOPE AND FIELD OF APPLICATION
A descriptionis givenof five processes for the preparationof methyl esters
from fatty substances:
(a) with sodium methylate;
(b) with sodium methylate ina sealed phial;
(c) with methanol-hydrochloric acid in a sealed phial;
(d) with dimethyl sulphate;
(e) with methanol-hexane-sulphuric acid.
ProcessA
2. PRINCIPLE
The fatty substance which is being subjected to analysis is heated under
reflux with methyl alcohol and sodium methylate. The methyl esters
obtained are extracted with ethyl ether.
3. APPARATUS
3.1. 100 ml flask with a reflux condenser with a soda lime tube fitted to the top,
with ground glass joints.
3.2. 50 ml measuring glasses.
3.3. 5 ml measuring pipette marked off in 0,1 ml.
3.4. 250 ml separating funnels.3.5. 200 ml flask.
4. REAGENTS
4.1. Anhydrous methanol.4.2. Solutionof approximately 1 % sodium methylate inmethan ol; this is
prepared by dissolving 0,34 g of metallic sodium in 100 ml of anhydrous
methanol.
4.3. Ethyl ether.
4.4. 10 % sodium chloride solution.4.5. 40 to 60 ºC petroleum ether.1991R2568 — EN — 21.07.1992 — 004.001 — 50
▼B
5. PROCEDURE
5.1. Place inthe 100 ml flask 2 g of the fatty substan ce which has previously
been dried out on sodium sulphate and filtered. Add 35 ml of methanol, fit
the condenser and boil under reflux a few minutes.
5.2. Stop the heating process, remove the condenser and add rapidly 3,5 ml of
sodium methylate solution; refit the condenser and boil under reflux for at
least 3 hours. Methylationis complete whenall the fatty substan ce has
liquified and the reagent mixture is perfectly clear at room temperature.
5.3. Cool and pour the reagent mixture into a 250 ml separating funnel, add 35
to 40 ml of ethyl ether, 100 ml of water and 5 to 6 ml of 10 % sodium
chloride solution. Shake and allow the layers to separate. Transfer the
aqueous phase to a second separating funnel and extract once again with
25 ml of ethyl ether.
Add 50 ml of 40 to 60 ºC petroleum ether to the combined ether extracts.
Water will be separated and can be eliminated.
Wash the ether phase three times with 10 to 15 ml of water, dry onsodium
sulphate and filter through paper, collecting the filtrate in the 200 ml flask.
Evaporate the solvent to 20 ml, completing the process over a water bath in
a current of pure nitrogen.
Process B
2. PRINCIPLE
The fatty substance which is being subjected to analysis is treated with
sodium methylate in a methanol solution, in a sealed phial, at 85 to 90 ºC.
3. APPARATUS
3.1. Strong glass phial with a capacity of approximately 5 ml (height 40 to
45 mm, diameter 14 to 16 mm).
3.2. 1 ml measuring pipette marked off in 0,1 ml.
4. REAGENTS
4.1. Solutionof approximately 1,5 % sodium methylate inmethan ol. This is
prepared by dissolving 0,50 g of metallic sodium in 100 ml of anhydrous
methanol.
5. PROCEDURE
5.1. Place inthe glass phial 2 g of the fatty substan ce, which has previously
beendried out onsodium sulphate an d filtered. Add 0,3 g (approximately
0,4 ml) of sodium methylate solutionan d heat seal the phial.
5.2. Immerse the phial for 2 hours at 85 ºC to 90 ºC, shaking from time to time.
The esterification process is complete when the contents of the phial are
clear after sedimentation of the glycerine and the residue of the reagents.
5.3. Cool at room temperature. Openthe phial whenthe methyl esters are to be
used. These do not require any further treatment before being placed in the
gas chromatography apparatus.
ProcessC
2. PRINCIPLE
The fatty substance which is being subjected to analysis is treated with
methanol-hydrochloric acid, in a sealed phial, at 100 ºC.
3. APPARATUS
3.1. Strong glass phial with a capacity of approximately 5 ml (height 40 to
45 mm, diameter 14 to 16 mm).
3.2. 1 and 2 ml calibrated pipettes.
4. REAGENTS
4.1. Solutionof hydrochloric acid in2 % methan ol. This is prepared from
gaseous hydrochloric acid and anhydrous methanol (Note 1).
4.2. Hexane for gas chromatography.1991R2568 — EN — 21.07.1992 — 004.001 — 51
▼B
5. PROCEDURE
5.1. Place inthe glass phial 0,2 g of the fatty substan ce, which has previously
been dried out on sodium sulphate and filtered, and 2 ml of hydrochloric
acid-methanol solution. Heat seal the phial.
5.2. Immerse the phial at 100 ºC for 40 minutes.5.3. Cool the phial under running water, open, add 2 ml of distilled water and
1 ml of hexane. Centrifuge and remove the hexane phase which is ready for
use.
ProcessD
2. PRINCIPLE
The fatty substance which is being subjected to analysis is saponified with
a methyl alcohol solutionof potassium hydroxide, an d thentreated with
dimethyl sulphate. Whenhydrochloric acid is added, the methyl esters
which have formed are automatically separated. Very pure methyl esters
are obtained by subsequent treatment with alumina.
3. APPARATUS
3.1. Strong test tube with a capacity of approximately 20 ml, with 10/19 ground
glass stopper and safety clips.
3.2. Reflux condensers, with 10/19 ground glass attachment.3.3. Glass filters with sintered disc, G 2 size, 20 mm diameter.
3.4. Glass test tubes with a capacity of approximately 10 ml and a conical base.
3.5. 1 ml and 5 ml syringes.
4. REAGENTS
4.1. Potassium hydroxide, 10 % solutioninmethyl alcohol for gas chromato-
graphy.
4.2. Greenbromocresol in dicator: 0,05 % solutioninmethyl alcohol.
4.3. Dimethyl sulphate (p = 1,335 at 15 ºC).4.4. Concentrated hydrochloric acid (p = 1,19) diluted in equal parts with
methyl alcohol for gas chromatography.
4.5. Brockmann aluminium oxide for adsorption chromatography.
5. PROCEDURE
5.1. Place inthe 20 ml test-tube 2,2 ml of the fatty substan ce, which has
previously beendried out onsodium sulphate an d filtered. Add 5 ml of
the potassium hydroxide solution and a few quartz granules to control
boiling. Attach the reflux condenser and heat over a low flame for five
minutes, shaking. Saponification will be complete when the solution is
clear. Finally, cool with running water and remove the condenser.
5.2. Add two drops of the indicator and, using a syringe, 1 ml of dimethyl
sulphate, slowly. Seal the test tube hermetically and shake for two to three
minutes, immersing the bottom of the test tube in a boiling water bath at
frequent intervals. The reaction is complete when the indicator changes
from blue to yellow. Finally, cool the test tube under running water, then
open and add 5 ml of the hydrochloric acid methanol solution.
5.3. After shaking for a few seconds, lay the test tube at an angle and then tap it
lightly. This will help the methyl esters to rise to the surface inthe form of
anoily mass (Note A).
Remove the methyl esters with a syringe, place in a test tube with a conical
base, add a volume of alumina equal to approximately ¼ of the volume of
the methyl esters, shake and filter with filter paper.
Note A.If the methyl esters do not separate spontaneously, add 5 ml of
water to the test tube and shake.1991R2568 — EN — 21.07.1992 — 004.001 — 52
▼B
Process E
2. PRINCIPLE
The fatty substance which is being subjected to analysis is heated under
reflux with methanol-hexane-sulphuric acid. The methyl esters obtained
are extracted with petroleum ether.
3. APPARATUS
3.1. Test tube of a capacity of approximately 20 ml, fitted with anair reflux
condenser approximately 1 m in length, with ground glass joints.
3.2. 5 ml calibrated pipette.3.3. 50 ml separating funnel.
3.4. 10 ml and 25 ml measuring glasses.
3.5. 15 ml test tube with conical base.
4. REAGENTS
4.1. Methylation reagent: anhydrous methanol-hexane-concentrated sulphuric
acid (p = 1,84) inthe ratio 75:25:1 (V/V/V).
4.2. 40 to 60 ºC petroleum ether.
4.3. Anhydrous sodium sulphate.
5. PROCEDURE
5.1. Place the matter takenfrom the plate inthe 20 ml test tube an d add 5 ml of
methylationreagen t.
5.2. Fit the reflux condenser and heat for 30 minutes in a boiling water bath
(Note 2).
5.3. Transfer quantitatively the mixture into a 50 ml separating funnel, with the
aid of 10 ml distilled water and 10 ml petroleum ether. Shake vigorously,
and allow the phases to separate, remove the aqueous phase and wash the
ether layer twice with 20 ml distilled water. Add to the separating funnel a
small quantity of anhydrous sodium sulphate, shake, allow to settle for a
few minutes and filter, collecting the filtrate in a 15 ml test tube with a
conical base.
Evaporate the solvent over a water bath in a current of nitrogen.
Note 1.Small amounts of gaseous hydrochloric acid can easily be prepared
inthe laboratory by simple displacemen t from the commercial
solution (p = 1,18) by dripping concentrated sulphuric acid (p
= 1,84). The liberated gas is easily dried by bubbling through
conc. sulphuric acid. Since hydrochloric acid is very rapidly
absorbed by methanol, it is advisable to take the usual precautions
in dissolving it, e.g. introduce the gas through a small inverted
funnel with the rim just touching the surface of the liquid. Large
quantities of methanolic hydrochloric acid solution can be prepared
inadvan ce, as it keeps perfectly inglass-stoppered bottles stored in
the dark.
Note 2.To control the boiling point insert a glass rod into the test tube and
limit the temperature of the water bath to 90 ºC.1991R2568 — EN — 21.07.1992 — 004.001 — 53
▼B
ANNEX XI
DETERMINATION OF VOLATILE HALOGENATED SOLVENTS
CONTENTOFOLIVEOIL
1. METHOD
Analysis by gas chromatography using the head space technique.
2. EQUIPMENT
2.1. Gas chromatography apparatus fitted with anelectroncapture detector
(ECD).
2.2. Head space apparatus.
2.3. Gas chromatography column, of glass, 2 m long and 2 mm in diameter,
stationary phase. OV101 10 % or equivalent, impregnating a calcined
diatomaceous earth, acid washed and silanised and of a particle size of 80
to 100 mesh.
2.4. Carrier and auxiliary gas: nitrogen for gas chromatography, suitable for
detectionby electroncapture.
2.5. Glass flasks, 10 to 15 ml, with teflon coating and aluminium stopper with
fitment for entry of syringe.
2.6. Hermetically sealing clamps.
2.7. Gas syringe 0,5 to 2 ml.
3. REAGENTS
Standard: halogenated solvents of a degree of purity suitable for gas chro-
matography.
4. PROCEDURE
4.1. Exactly weigh around 3 g of oil in a glass flask (not to be reused); herme-
tically seal it. Place it in a thermostat at 70 ºC for one hour. Using a
syringe carefully remove 0,2 to 0,5 ml of the head space. Inject this into
the columnof the gas chromatography apparatus regulated as follows:
— injector temperature: 150 ºC,
— columntemperature: 70 to 80 ºC,
— detector temperature: 200 to 250 ºC.
Other temperatures may also be used provided the results remainequiva-
lent.
4.2. Reference solutions: prepare standard solutions using refined olive oil with
no trace of solvents with concentrations ranging from 0,05 to 1 ppm (mg/
kg) and corresponding to the presumed content of the sample. The haloge-
nated solvents may be diluted using pentane.
4.3. Quantitative assessment: correlate the surfaces or the elevations of the
peaks of the sample and of the standard solution of the concentrationpresumed closest. If the deviationis greater than10 % the an alysis must
be repeated in comparison with another standard solution until the devia-
tion is within 10 %. The content is determined on the basis of the average
of the elementary injections.
4.4. Expressionof results: inppm (mg/kg). The detectionlimit for the method
is 0,01 mg/kg.1991R2568 — EN — 21.07.1992 — 004.001 — 54
▼B
ANNEX XII
ORGANOLEPTICASSESSMENTOFVIRGINOLIVEOIL
1. SCOPE
The purpose of this method is to determine the criteria needed to assess
the flavour characteristics of virginolive oil an d to develop the metho-
dology required to do so.
2. FIELD OF APPLICATION
The method described is only applicable to the organoleptic assessment
and classification of virgin olive oil that can be used for direct consump-
tion. It confines itself to grading the virgin oil on a numerical scale
related to the perception of its flavour stimuli, according to the judgement
of a group of selected tasters working as a panel.
3. GENERAL BASIC VOCABULARY FOR SENSORY ANALYSIS
See the Chapter entitled ‘Sensory analysis: general basic vocabulary’.
4. SPECIFIC VOCABULARY FOR OLIVE OIL
4.1. Pleasant sensations, produced by the attributes and characteristics of
quality inolive oils:
Fruity:flavour which is reminiscent of both the odour and taste of sound,
fresh fruit picked as its optimum stage of ripeness.
Ripely fruity: flavour of olive oil obtained from ripe fruit, generally
having a somewhat flat odour and a sweet taste.
Green fruity: flavour of oil obtained from fruit that is still unripe.
4.2. Sensations that may be pleasant to varying degrees, depending on the
intensity with which they are perceived, and which cannot be considered
defects although they do affect the fruity wholeness of the oil:
Apple:flavour of olive oil which is reminiscent of this fruit.
Sweet:pleasant taste, not exactly sugary, but found in oil in which the
bitter, stringent and pungent attributes do not predominate.
Grass:characteristic flavour of certain oils reminiscent of recently mown
grass.
Green leaves (bitter): flavour of oil obtained from excessively green
olives or olives that have beencrushed with leaves an d twigs.
Bitter:characteristic taste of oils obtained from green olives or olives
turning colour. It can be more or less pleasant depending on its intensity.
Harsh:characteristic sensation of certain oils which when tasted produce
a mouthfeel reaction of astringency.
Pungent: biting taste sensation characteristic of oils produced at the start
of the crop year, primarily from olives that are still unripe. It is caused by
the action of phenolic substances on the tips of the trigeminal nerve that
are spread over the entire mouth cavity.
Almond: this flavour may appear intwo forms: that typical of the fresh
almond or that peculiar to dried, sound almonds which can be confused
with incipient rancidity. A distinctive taste is perceived as an aftertaste
when the oil remains in contact with the tongue and the palate. Asso-
ciated with sweet oils which have a flat odour.
Flat or smooth: flavour of olive oil whose organoleptic characteristics are
very weak owning to the loss of their aromatic components.
Hay:characteristic flavour of certain oils reminiscent of more or less
dried grass.
4.3. Sensations that are always unpleasant, even when their intensity is barely
perceptible, and which have to be considered organoleptic defects:
Esparto: characteristic flavour of oil obtained from olives pressed in new
esparto mats. The flavour may differ depending on whether the mats are
made of greenesparto or dried esparto.1991R2568 — EN — 21.07.1992 — 004.001 — 55
▼M3
▼M3
Earthy:characteristic flavour of oil obtained from olives which have been
collected with earth or mud on them and not washed. This flavour may
sometimes be accompanied by a musty-humid flavour.
Old:characteristic flavour of oil that has beenkept too lon g instorage
containers. May also appear in oils which have been packed for an exces-
sively long period.
Grubby: characteristic flavour of oil obtained from olives which have
beenheavily attacked by the grubs of the olive fly (Dacus oleae).
Metallic: flavour that is reminiscent of metal. Characteristic of oils which
have been in prolonged contact, under unsuitable conditions, with food-
stuffs or metallic surfaces during crushing, mixing, pressing or storage.
Mustiness-humidity: characteristic flavour of oils obtained from fruit in
which large numbers of fungi and yeasts have developed, as a result of
its being stored in piles, in humid conditions, for several days.
Rancid: characteristic flavour commonto all oils an d fats that have
undergone a process of auro-oxidation caused by prolonged contact with
the air. This flavour is unpleasant and cannot be corrected.
‘Atrojado’ (fusty): characteristic flavour of oil obtained from olives stored
in piles which have undergone an advance stage of fermentation.
Brine:flavour of oil extracted from olives which have beenpreserved in
saline solutions.
Pomace: characteristic flavour that is reminiscent of the flavour of olive
pomace.
Soapy:flavour producing an olfactory-gustatory sensation reminiscent of
that produced by greensoap.
Vegetable water: characteristic flavour acquired by the oil as a result of
poor decantation and prolonged contact with vegetable water.
Winey-vinegary: characteristic flavour of certain oils reminiscent of wine
or vinegar. Due mainly to the formation of acetic acid, ethyl acetate and
ethanol in larger amounts than is usual in the aroma of olive oil.
Cucumber: flavour produced whenanoil is hermetically packed for too
long, particularly in tin containers, and which is attributed to the forma-
tion of 2,6 nonadienal.
Heated or burnt: characteristic flavour of oils caused by excessive and/or
prolonged heating during processing, particularly when the paste is ther-
mally mixed, if this is done under unsuitable conditions.
Muddy sediment: characteristic flavour of oil recovered from the decanted
sediment in vats and underground tanks.
Pressing mat: characteristic flavour of oil obtained from olives that have
beenpressed indirty pressin g mats inwhich fermen ted residues have
beenleft.
Greasy:odour of olive oil extracted ina plan t where residues of petro-
leum, grease or mineral oil have not been properly removed from the
machinery.
Rough:characteristic perceptionincertainoils which, whentasted,
produce a thick, pasty mouthfeel sensation.
5. GLASS FOR OIL TASTING
See the Chapter entitled ‘Glass for oil tasting’.
6. TEST ROOM
See the Chapter entitled ‘Guide for the installation of a test room’.
7. APPARATUS
The following apparatus, which is required by the taster to perform his
task properly, shall be supplied ineach booth an d shall be withineasy
reach:
— glasses (standardized) containing the samples marked with a legend
consisting of two randomly-picked figures or of two figures and
letters. The marks shall be made with an indelible, odourless pencil,
— watch-glasses with identical marks, to cover the glasses,1991R2568 — EN — 21.07.1992 — 004.001 — 56
▼B
▼B
— grading sheet (see Figure 2) containing the instructions for its use,
— pencil or pen,
— small trays of sliced apple,
— glass of water at ambient temperature.
8. METHODOLOGY
This sectionstipulates the prior kn owledge required to perform the
sensory analysis of virgin olive oils and attempts to standardize the
conduct and procedure of the tasters participating in such tests who must
be aware of both the general and specific recommendations for olive oil
tasting.
8.1.Dutiesofthepanelorganizer orsupervisor (or panel)
The panel organizer shall be a suitably trained, knowledgeably person
who is anexpert onthe kin ds of oils which he will come across inthe
course of his work. He is the key figure in the panel and is responsible
for its organization and running. He shall summon the tasters sufficiently
in advance and shall clarify any doubts they may have as regards the
performance of the tests, but shall refrain from suggesting any opinion
to them onthe sample.
He shall be responsible for inventorying the apparatus and for ensuring
that it is properly cleaned, for preparing and coding the samples and
presenting them to the taster in accordance with the appropriate experi-
mental design, as well as for assembling and statistically processing the
data obtained, so that the best results are obtained with the minimum ofeffort.
The work of the panel supervisor calls for sensory skill, meticulousness
in the preparation of the tests and their rigorous arrangement, as well as
for skill and patience in the planning and execution of the tests. It is the
duty of the panel supervisor to stimulate the morale of the panel members
by encouraging interests, curiosity and a competitive spirit among them.
He shall ensure that his opinion is not known and shall prevent possible
leaders from asserting their criteria over the other tasters. He shall also be
responsible for training, selecting and monitoring the tasters in order to
ascertainwhether they are keepin g up to anadequate level of aptitude.
8.2.Testconditions
8.2.1. Sample size
Each glass shall contain 15 ml of oil.
8.2.2. Test temperature
The oil samples to be tested shall be kept inthe glasses at 28 ºC ± 2 ºC.
This temperature has beenchosenbecause it is the best for easily obser-
ving organoleptic differences, at normal temperature, when oils are used
as a condiment. Another factor that tends to weigh in favour of this value
is that at higher or lower temperatures either the aromatic components
scarcely volatilize or volatile components are produced that are peculiar
to heated oils.
8.2.3. Test times
The morning is the best time for testing oils. It has been proved that there
are optimum perception periods as regards taste and smell during the day.
Meals are preceded by a period inwhich olfactory-gustatory sen sitivity
increases, whereas afterwards this perception decreases.
However, this criterion should not be taken to the extreme where hunger
may distract the tasters, thus decreasing their discriminatory capacity and,
in particular, their preference and acceptance criteria.
9. TASTERS
The people acting as tasters in the organoleptic tests carried out on edible
olive oils shall be trained and selected in accordance with their skills in
distinuishing between similar samples; it should be borne in mind that
their accuracy will improve with training (see appropriate section).
Eight to 12 tasters are required for the test, although it is wise to keep
some extra tasters inreserve to cover possible absen ces.1991R2568 — EN — 21.07.1992 — 004.001 — 57
▼B
9.1.General recommendationsforcandidatesandtasters
These recommendations apply to the conduct of the candidates and tasters
during their work.
When called by the panel supervisor to participate in an organoleptic test,
the taster should be able to attend at the time set beforehand and shall
observe the following:
9.1.1. He shall not smoke at least 30 minutes before the time set for the test.9.1.2. He shall not use any perfume, cosmetic or soap whose small could linger
until the time of the test. He shall use an unperfumed or slightly
perfumed soap to wash his hands which he shall then rinse and dry as
often as necessary to eliminate any smell.
9.1.3. He shall fast at least one hour before the tasting is carried out.9.1.4. Should he feel physically unwell, and in particular if his sense of smell or
taste is affected, or if he is under any psychological effect that prevents
him from concentrating on his work, the taster shall inform the panel
supervisor accordingly with a view to being withdrawn from the test or
to the appropriate decisions being taken, bearing in mind the possible
deviationinthe meanvalues for the rest of the pan el.
9.1.5. Whenhe has complied with the above, the taster shall take up his place
in the booth allotted to him in as orderly and quiet a manner as possible.
9.1.6. Whenseated, he shall check that he has the correct apparatus an d that it
is properly arranged, and shall ensure that the legend on the glass matchs
the legend on the watch-glass.
9.1.7. He shall carefully read the instructions given on the grading sheet and
shall not begin to examine the sample until absolutely sure about the
task he has to perform. If any doubts should arise, he shall discuss the
difficulties encountered privately with the panel supervisor.
9.1.8. The taster shall pick up the glass, keeping it covered with the watch-
glass, and shall bend it gently; he shall then rotate the glass fully in this
position so as to wet the inside as much as possible. Once this stage is
completed, he shall remove the watch-glass and smell the sample taking
even, slow deep breaths until he has formed a criterion on the oil under
assessment. Smelling shall not exceed 30 seconds. If no conclusion has
been reached during this time, he shall take a short rest before trying
again. When the olfactory test has been performed, the taster shall then
judge the flavour (overall olfactory-gustatory-tactile sensation). To do
so, he shall take a small sip of approximately 3 ml of oil. It is very
important to distribute the oil throughout the whole of the mouth cavity,
from the front part of the mouth and tongue along the sides to the back
part and to the palate support, since it is a known fact that the perception
of the four primary tastes, sweet, salty, acid and bitter varies in intensity
depending on the area of the tongue and palate.
It should be stressed that it is essential for a sufficient amount of the oil
to be spread very slowly over the back of the tongue towards the throat
while the taster concentrates on the order in which the bitter and pungent
stimuli appear; if this is not done, both of these stimuli may escape notice
in some oils or else the bitter stimulus may be obscured by the pungent
stimulus.
Taking short, successive breaths, drawing in air through the mouth,
enables the taster not only to spread the sample extensively over the
whole of the mouth but also to perceive the volatile aromatic components
via the back of the nose.
Tactile sensation shall also be taken into consideration. Consequently,
fluidity, stickiness and sharpness or sting shall be noted down when
detected, and if so required for the test, their intensity shall be quantified.
9.1.9. When organoleptically assessing a virgin olive oil, only one sample shall
be evaluated ineach sessionto avoid the con trast effect that could be
produced by immediately tasting other samples.
As successive tastings produce fatigue or loss of sensitivity, it is impor-
tant to use a product that can eliminate the remains of the oil from the
preceding tasting from the mouth.
The use of a small slice of apple (about 15 g) is recommended which,
after being chewed, can be spat out into the spittoon. Then rinse out the
mouth with a little water at ambient temperature. At least 15 minutes
shall lapse between the end of one tasting and the start of the next.1991R2568 — EN — 21.07.1992 — 004.001 — 58
▼B
9.2.Screeningofcandidates.
This stage shall be completed by the panel organizer who shall personally
interview the candidates to familiarize himself with their personality and
surrounding environment. The physio-psychological conditions that have
to be met are not very rigorous since, theoretically, any normal person
should be able to participate. Factors such as sex, age, specific habits
(smoking), etc. have been superseded nowadays by others such as health,
personal interest and having time available for the work.
During the interview, the panel organizer shall explain the characteristics
of his task to the candidate and approximately how much time it will take
up. He shall then obtain information from the candidate allowing him to
assess his interest and motivation and how much real time he has avail-
able. The following questionnaire could help as a reference.
(1)
The organiser shall use this information to screen the candidates and shall
reject those who show little interest in this kind of work, are not readily
available or who are incapable of expressing themselves clearly.
9.3.Determination of the ‘mean threshold’ of the group for ‘character-
isticattributes’
Carefully choose four oils, each one of which is considered representative
of one of the following attributes: ‘atrojado’ (fusty), winey, rancid and
bitter, and has as great and clear an intensity as possible.
Take analiquot part of each oil an d prepare samples, each of whose
concentrations differs by a ratio of 2, comprising successive dilutations
with the appropriate support until no difference can be detected between
the glass containing the support only and the last two or three dilutions.
The last pair shall be two glasses of the support.1991R2568 — EN — 21.07.1992 — 004.001 — 59
(1) Describe what could be gained from the organoleptic assessment of any foodstuff, or, if
you wish, of olive oil.
▼B
Complete the series with glasses containing higher concentrations, until a
total of eight is reached.
Prepare sufficient amounts of the samples prepared at different concentra-
tions so that complete series of each attribute can be given to each
candidate.
To establish the ‘meanthreshold’ of the can didates for each attribute,
give each of them one glass containing 15 ml of any one of the prepared
concentrations, and another glass containing 15 ml of the support solely.
After performing the test, the candidate shall indicate whether they are
the same or different.
Repeat the same test for the remaining concentrations of the attribute
under consideration.
Note down the number of correct answers obtained for each concentration
by all the tasters and give this figure as a percentage of the number of
tests performed.
Then plot as abscissae, in ascending order, the concentrations tested and
as ordinates the % of correct identifications for each concentration.
Figure 1 is a practical example of these instructions. The detection
threshold is determined by extrapolating the ordinate point representing
75 % correct appraisals from the curve onto the abscissae.
This ‘threshold’ concentration, which may be different for each initial oil
because it depends on the intensity of the attribute present, should be
similar for the different groups of candidates to various panels; it is not
linked to any habit or tendentious preference. Consequently, it is a point
of reference common to any normal human group and may be used to
homogenize the various panels by their olfactory-gustatory sensitivity
alone.
On the basis of the threshold concentration obtained for the group,
proceed as follows:
Prepare a series of increasing and decreasing concentrations in such a
way that the ‘threshold concentration’ holds the 10th place in this scale.
Naturally, the 11th and 12th concentrations will be more diluted, as a
result of which it will be more difficult to detect the presence of the oil
possessing the selected attribute.
Taking the C10concentration as the basis, remaining samples can be
prepared in accordance with the following formula:
C10×an, where ‘a’ is a constant, the dilution factor, which is equal to 1,5,
and ‘n’ is the exponent which varies between 9 and − 2.
Example: assuming that the threshold obtained for rancid oil is 0,32; C10= 0,32, onthe basis of which, sin ce ‘a’ = 1,5, the series of samples would
have the following concentrations:
Example 1 2 3456789 1 0 1 1 1 2
Concentration 12,30 8,20 5,47 3,65 2,43 1,62 1,08 0,72 0,48 0,32 0,21 0,14
If the above procedure is repeated for the three remaining attributes on
the basis of their respective thresholds which are also calculated as indi-
cated above, scales with similar aromatic intensities for each stimulus
will be obtained for all the laboratories, even though the defects of the
initial oils may be perceptible at different intensities.
9.4.Selection oftastersbytheintensityratingmethod
Inthe selectionprocedure, there should be two to three times more can di-
dates thanthose required for the pan el so that the people with the best
sensitivity or powers of discrimination can be picked out. It is always
advisable to use the same product as the one that is to be subsequently
analysed (consequently, olive oil will always be used).1991R2568 — EN — 21.07.1992 — 004.001 — 60
▼B
Figure1
When selecting the method, it should not be overlooked that, apart from
being effective, the procedure adopted should be as economical as
possible as regards the quantity of oil, the number of samples to be sent
and the time spent on selection. The effectivencess of a selection proce-
dure lies inthe choice of the optimum levels of the followin g three
dependent variables: (a) ‘cost’ determined by the number of tests; (b)
‘proportion’ of potentially suitable candidates who by chance have been
unfortunately eliminated during screening; and (c) ‘proportion’ of candi-
dates who by chance have got through the selection process although
unsuitable material.
Four points of the selection procedure chosen, the intensity rating test,
which is described in the ASTM (American Society for Testing and
Materials), STP (Special Technical Publication) No 440, page 53, havebeenmodified by:
1. decreasing the number of samples in the series;
2. broadening the range of stimuli with a view to increasing the number
of olfactory-gustatory notes on which selection is based, so as to adapt
them to the most commondefects perceived inolive oil;
3. varying the concentration ratios in the series; and
4. statistically processing the results.
Apparatus required— 1500 ml bottles or glass flasks,
— dark-coloured tasting glasses,
— graduated 10, 15, 1 000 and 1 500 ml test tubes.
Products required
— Merck paraffin(referen ce 7 160, DAB 8, USP XX) or oily support
without taste or odour (recently refined olive oil or another similar
oil),
— oils: ‘atrojado’ (fusty), winey, rancid and bitter.1991R2568 — EN — 21.07.1992 — 004.001 — 61
▼B
9.4.1. Procedure
After preparing the dilutions, go on the selection stage beginning with 25
candidates, in accordance with the methodology described hereafter for
each stimulus:
1. Prepare series of 12 tasting glasses marked with a code (one series per
candidate). Pour 15 ml of each of the various concentrations prepared
according to the formula C10xan, into each respective tasting glass.
2. Whenthe tastin g glasses have beenfilled up, they should be left
covered with a watch-glass inthe tastin g room at a temperature of 20
to 22 ºC for at least anhour before startin g the tests so as to homoge-
nize their temperature with the ambient temperature.
3. The organizer shall then arrange the 12 tasting glasses of each series
in a row in descending order of concentration.
The next step is to ask each candidate to do the test on his own, in
accordance with the following instructions:
9.4.2. Instructions for candidates
The 12 tasting glasses lined up in front of the candidates contain dilutions
of any one of the ‘atrojado’ (fusty), winey, rancid or bitter stimuli. The
distinguishing factor between the contents of the tasting glasses is the
intensity of the odour. The glass with the most intense odour is on the
far left-hand side and the rest of the glasses are placed in descending
order of intensity towards the right. The last tasting glass on the right
may have such a weak odour that it will perhaps be impossible to detect.
Proceed as follows: become familiar with the odour of each of the tasting
glasses in the series. To do so, begin at the right hand side (No 12) and
try to retain the intensity of all the odours, without becoming overtired.
When you feel that you have got used to the scale of concentrations of
the odours, leave the room.
Meanwhile, the organizer shall remove one of the tasting glasses from the
series and shall place it on a level with the last one on the right-hand
side, moving all the others together so as to fill in the space left. Then
returnto the room an d carry onwith the test.
The test involves the following:
The tasting glass withdrawn from the series has to be put back in its
exact place. To do so, smell it and compare it with the others as often
as wished, bearing in mind that if it is to be replaced correctly it must
smell stronger than the sample on its immediate right and weaker than
that onits left. This test will be repeated with three other glasses.
Each candidate shall be issued a form, in addition to the instructions just
described, so as to make the test and the collection of the replies easier.
9.4.3. Obtaining the results
The panel organizer shall record the data for each of the candidates in the
following manner to facilitate their arrangement:
1991R2568 — EN — 21.07.1992 — 004.001 — 62
▼B
9.4.4. Statistical grading procedure
Inthis particular selectioncase, the tastin g glasses that have to be
replaced intheir exact positionshall be the same for all the can didates.
According to the statistical calculations done for this purpose, they shall
correspond to the following positions in the order of the series as regards
each attribute:
‘Atrojado’
— Fusty (Fy)Winey (W) Rancid (Rd) Bitter (Bt)
Glass No Glass No Glass No Glass No
(10, 5, 7, 2) (11, 3, 8, 6) (7, 4, 10, 2) (6, 3, 11, 9)
The number corresponding to the position of the glasses in the order of
the series may not vary since the statistical calulations for this test have
been done with an eye to the probability of the glasses being randomly
put back into their exact position.
In order to make it extremely difficult for any information to be passed
on from one candidate to another, the panel organizer shall ensure that:
1. there is no possible means of contact between the candidates. Different
legends shall be used for each candidate;
2. there is no way in which the candidates can find out the position of
the glasses which have beenwithdrawn ;
3. even thouch all the candidates shall be presented with the same glasses
indicated earlier on, the order in which they are handed over to each
candidate shall vary.
Each candidate shall then be given a grading, depending on his perfor-
mance in the following manner:
Let ei
1,ei
2,…ei
12be the 12 glasses with the 12 corresponding concentra-
tions of attribute ‘i’ (i may be any one of the four attributes: ‘atrojado’ —
fusty, winey, rancid and bitter) arranged in descending order of intensity.
Let ei
kbe one of the glasses picked and K ′the positionit is allocated by
the candidate when replaced in the series. Therefore, the values of K and
K′are whole numbers between 1 and 12 inclusive, corresponding to the
real place number of the glass chosen and that allocated by the candidate
respectively.
Let T (maximum permitted deviation) be a value set beforehand, which
inour case is equal to 3, so that if K ′−K > T, the candidate is automati-
cally rejected(1).
If, onthe con trary, K ′−K≤T, theoretically the candidate is accepted and
may go onwith the test sin ce he or she is able to put the stimulus back
into its exact position or at least very near it.
In this case, the grading awarded a candidate who has assessed a set
stimulus (concentration), for instance in the ‘atrojado’ — fusty series
(Fy), shall be equal to the square of the difference between the exact
number of the glass in the order of the series and the position in which
the candidate has replaced it. That is to say
Ph(Fy)=( K′−K)2.
Since this operation will be conducted by each candidate on four stimuli
(concentrations) of each attribute, the partial grading for the attribute
(e.g.Fy) would be:
ZFy=PFy
h+PFy
j+PFy
l+PFy
m
Some examples are givenbelow to facilitate comprehen sionof this opera-
tion.
Example 1:
Let us assume that the answers given by candidate A for the four stimuli
withdrawnfrom the series for attribute (i) are as follows:1991R2568 — EN — 21.07.1992 — 004.001 — 63
(1) The panel organizer should press the candidate to proceed reasonably, that is to say
without losing any sensitivity through olfactory fatigue.
▼B
Exact positionof the
glass inthe series
(K)Positioninwhich it
was replaced by the
candidate
(K′)Deviationfrom the exact posi-
tion
(K′−K)
7 7 7−7=0
4 5 4−5=−1
10 6 10 − 6 = 4 (1)
2 4 2−4=−2
(1) This candidate is rejected because he has obtained T > 3 in the test.
Example 2:
Let us assume that a candidate rearranges the glasses for an attribute as
follows:
Exact positionof the
glass inthe series
(K)Positioninwhich it
was replaced by the
candidate
(K′)Deviationfrom the exact posi-
tion
(K′−K)
7 7 7−7=0
4 4 4−4=0
10 7 10 − 7 = 3
2 3 2−3=−1
This candidate is not rejected. He has obtained a grading of:
Zi=O2+O2+32+ (−1)2=1 0
The candidate's final grading, sealing his acceptance or rejection or selec-
tion as a taster, depending on his responses to the four attributes under
consideration, would be as follows:
PhFy+PjFy+PlFy+PmFy=ZFy
PhW+PjW+PlW+PmW=ZW
PhRd+PjRd+PlRd+PmRd=ZRd
PhBt+PjBt+PlBt+PmBt=ZBt
Z final = ZFy…ZBt
Where:
Fy = ‘Atrojado’ — fusty
W = WineyRd = RancidBt = Bitter
It is now a question of determining up to what maximum value for Z the
candidate can be considered to have good levels of perception, olfactory
retention and intellectual organization to give the correct answer for the
four stimuli considered. Obviously, Z always has a non negative value
and Z = 0 means that the candidate has recognized and correctly quanti-
fied the whole of the 16 intensities presented (four for each attribute).
Values of Z other than zero indicate that the candidate has recognized
the scale areas from which the selected intensities have been picked, but
withinthese areas has beenun able to locate the exact positionbecause
his ability to discriminate the scale of intensity presented to him for one
or more of the stimuli is not satisfactory.
Therefore, a critical value (Z
c) will have to be determined such that
should the candidate randomly replace all the glasses inside the areas he
had recognized beforehand, the probability of a final grading Z, less than
Zc, is a sufficiently small quantity ( α)which canbe set beforehan d. In
other words, it must be ensured that the probability, using this procedure,
of selecting a taster for the panel who does not show sufficient discrimi-
natory power for the intensities of the stimuli used in the selection
process is less than α.1991R2568 — EN — 21.07.1992 — 004.001 — 64
▼B
Whenthe value for αis set (inour case at 0,05), Zcis obtained from the
probability distributionof the variable Z, which inturndepen ds onthe
probability distributions of the P variables (K ′).
Following the relevant statistical calculations, the value for Zccomes to
34.
When the Z grading for all the candidates has been obtained, any candi-
dates whose grading lies above 34 shall be eliminated.
See the gradings for candidates A and B for an example:
Attribute Candidate A Candidate B
‘Atrojado’ — Fusty (Fy) ZFy=1 0 ZFy=1 2
Winey (W) ZW=1 0 ZW=1 1
Rancid (Rd) ZRd=1 0 ZRd=1 5
Bitter (Bt) ZBt=4 ZBt=0
Σ=3 4 Σ=3 8
Given that the two candidates considered have respective Z values of 34
and 38, candidate A will be retained whereas candidate B will be
rejected. When all the candidates with a grading above 34 have been
eliminated, the remainder shall be classified according to their Z values
until the 12 best candidates have been chosen.
9.5.Training
The chief aims of the training stage are:
(a) to familiarize the tasters with the multiple olfactory-gustatory-tactile
variants found in virgin olive oils;
(b) to familiarize the tasters with the specific sensory methodology;
(c) to heighten individual skill in recognizing, identifying and quanti-
fying the sensory attributes; and
(d) to improve sensitivity and memory as regards the various attributes
considered, so that the end result is precise and consistent assess-
ments.
The training stage normally entails a number of sessions, depending on
the possibilities open to the panel and the study, during which, after indi-
vidually analysing the oils, the tasters discuss the difficulties they have
encountered with the panel organizer and comment on the marks given
so as to unify criteria and opinions.
The standard reached in training after a set number of sessions is assessed
in terms of the percentage increase in the exact replies — should discri-
minatory trials be used — or by analysing the variance in the average
individual marks of the panel when tests using a scale are implemented.
The practical utility of this training period has been discussed at great
length but at present it is considered very effective and even essential if
exact, accurate, sensory data are to be obtained.
9.6.Performance checks
Panels of veteran tasters normally carry out tastings on a regular and
continuous basis, involving sensory tests which require a great effort on
their part. Decisions of great technological and commercial importance
depend in a large majority of cases on their judgement. For this reason,
after their selection and training, the tasters' performance should be
checked to ensure that their results are precise.
After the panels have been set up and have gone through the routine
trials, it would obviously be necessary to regularly check their perfor-
mance at suitable intervals.1991R2568 — EN — 21.07.1992 — 004.001 — 65
▼B
10. PROCEDURE FOR THE ORGANOLEPTIC ASSESSMENT OF
VIRGIN OLIVE OIL
When the conditions indicated in the above standards are met, the neces-
sary facilities are available and the panel has been selected, each taster
shall smell and taste(1) the sample of oil up for analysis contained in
the tasting glass. He shall analyse the olfactory, gustatory, tactile and
kinaesthetic perceptions with the aid of the sheet shown under Figure 2
in which he shall record the ‘notes’ present and their degree of intensity.
His next step shall be to grade the quality of the oil.
10.1.UseofthesheetinFigure2 (flavour description and quality grading).
Some of the most characteristic sensory perceptions more frequently
found in olive oils which describe their flavour are listed on the left-
hand side of the page. Should the taster encounter any other stimuli that
do not correspond to the descriptors listed, he shall note them down under
‘others’, using the descriptor (s) which define them as accurately as
possible.
Perceptible stimuli shall be assessed in proportion to their intensity which
shall be in dicated by a cross (+) inthe appropriate box, inaccordan ce
with the following criteria:
1: barely perceptible,
2: slight,
3: average,
4: great,5: extreme.
On the right-hand side of the sheet, a scale from 1 to 9 points is shown (9
for exceptional quality, 1 for the worst) which the tasters shall use to give
a single, overall grading for the characteristics of the oil being examined.
This grading shall be consistent with the good points and defects of the
oil already noted down on the left-hand side of the sheet.
The first column (defects) of the grading table is divided up into five
sections. Consequently, the classification of the oils shall be based
primarily on the total absence or presence of defective flavours, as well
as on how serious or intense such flavours are. However, since the
grading scale goes up to 9 points, certain nuances or aspects should be
taken into account that help reach a conclusive decision on the total
quality grading and that are described in the second column headed ‘char-
acteristics’.
10.2.Finalgrading
The panel supervisor shall collect the forms filled in by each taster and
shall check that the sensory attributes and the intensities with which
such attributes were perceived and recorded in the profile sheet agree
with the assessment of the oil entered in the grading sheet. If there is a
noticeable difference the superviser shall ask the taster to check his
grading sheet.
If necessary, the taster should repeat the test.
Finally, the panel supervisor should make up a table with the gradings for
the whole group and calculate the arithmetic mean and the degree of
error (of the mean).
Only in the case of revision analyses the group shall repeat the tests with
a view to obtaining an assessment in triplicate of the sample; the final
grade, with a decimal figure, will be the meanof the three gradin gs
given.
If the mean intensity rating for bitterness and/or pungency is greater than
2,5, the oil should be marked accordingly and it should be recorded that
it is bitter and/or pungent.
Expression of results: on the basis of the average grading, the panel
supervisor shall determine the category in which the sample is classified,
in accordance with the limits laid down in Annex I. ►
M3For this
purpose the panel supervisor shall, until 31 October 1993, apply a toler-
ance of + 0,5 if the average grading is not less than 5 points. In the1991R2568 — EN — 21.07.1992 — 004.001 — 66
(1) He may refrain when he finds any extremely or intensely unpleasant attribute in the
odour, recording this on the grading sheet as an exceptional occurrence.
▼B
analysis report he shall indicate only the category in which the sample is
classified. Where analysis in accordance with the first subparagraph of
Article 2 (2) of this Regulationis performed the an alyst shall apply the
same procedure for determining the category. ◄
Note:The samples should be kept sealed inthe refrigerator un til they are
analysed and should be returned to the refrigerator after each
analysis until the test has been done in triplicate.
Figure2
Virginoliveoil
1991R2568 — EN — 21.07.1992 — 004.001 — 67
▼M3
▼B
SENSORYANALYSIS:GENERALBASICVOCABULARY
1. SCOPE
The purpose of this standard is to assemble the general terms used in
sensory analysis and to give their definitions.
2. VOCABULARY
2.1.General terminology
Sensory analysis (noun):
examination of the organoleptic attributes of a product by the sense
organs.
Perception (noun):
sensory awareness of external objects or events.Organoleptic (adjective) (attribute):
describes an attribute of a product, perceptible by the sense organs.Expert(noun):
(with regard to the examination of organoleptic attributes)taster who is specialized in the sensory analysis of a specific product and
has a basic understanding of the preparation of the product and market
preferences.
Taster(noun):
perspicacious, sensitive, trained person selected to evaluate the organo-
leptic attributes of a food with the sense organs.
Panel:group of assessors who have been specially selected and trained and who
assemble to perform the sensory analysis of the product under controlled
conditions.
Sensation (noun):
subjective phenomenon resulting from the stimulation of a sensory
system. This phenomenon can be subjectively discriminated or objec-
tively defined by the sense organ involved, depending on the nature or
kind of stimulus, and its intensity.
Sensitivity (noun):
ability to perceive quantitatively and qualitatively a stimulus of little
intensity or small differences between stimuli by means of the sense
organs.
Tasting(noun):
operation which involves perceiving, analysing and judging the organo-
leptic attributes of a product, particularly the olfactory, gustatory, tactile
and kinaesthetic attributes of a food product.
Acceptance (noun):
the act of an individual or population of favourably accepting a product.Harmony (noun):
attribute of a product which gives rise to an overall pleasant sensation.
This sensation is produced by the perception of the product components
as olfactory, gustatory, tactile and kinaesthetic stimuli because they are
present in suitable concentration ratios.
Acceptability (noun):
state of a product favourably received by anin dividual or populationin
terms of its organoleptic attributes.
Discrimination (noun):
the act of qualitative and/or quantitative differentiation between two or
more stimuli.
Compensation (noun):1991R2568 — EN — 21.07.1992 — 004.001 — 68
▼B
result of the interaction of a combination of stimuli in such a way that
each one is perceived with less intensity than if it acted alone.
Aspect(noun):
combination of organoleptic attributes perceived visually: size, shape,
colour, conformation, turbidness, cleanness, fluidness, foam and efferves-
cence. This term is to be preferred to the term appearence.
Attribute (noun):
a perceptible characteristic.
2.2.Physiologicalterms
Stimulus (noun):
physical or chemical agent which specifically produces the response of
the external or internal sensory receptors.
Taste(noun):
(sense of taste)
sense whose receptors are located in the mouth, particularly on the
tongue, and which are activated by various compounds in solution.
Gustatory (adjective):
describes the attribute of a product which canstimulate the gustatory
apparatus by-awakening the sensations pertaining to one or more of the
four primary tastes: sweet, salty, acid and bitter.
Receptor (noun):
specific structure of a sense organ that can be excited and is capable of
receiving a stimulus and converting it into a nervous discharge.
Note:Receptors are classified interms of the type of en ergy associated
with the stimulus (light, heat, sound, etc.).
Olfaction (noun):
function of the olfactory apparatus to perceive and discriminate between
the molecules that reach it, in gas form from an external environment,
directly or indirectly via the nose.
Intensity (noun):
magnitude of the energy of an attribute that can be measured in terms of
a quantitative scale of values above the threshold.
Adaptation (noun):
temporary modification of sensitivity in perceiving sensory stimuli due to
continous, repeated exposure to a given stimulus or one similar to it.
Inhibition (noun):
lack of response by a sense organ or a part thereof, despite being
subjected to the action of a suitable stimulus whose intensity is above
the threshold.
Response (noun):
action whereby the sensory cells respond to the action of one or more
stimuli related to a given sense organ.
Body(noun):
tactile sensation perceived in the mouth which gives a degree of density,
viscosity, consistency or compactness to a product.
Fragrance (noun):
fresh, pleasant, delicious odour.
To smell (verb):
(active sense applied to smell)
describes the act of perceiving an odour.
Objective (adjective):
(a) describes that which gives a true, verifiable representation of the
object by minimizing the human factors (for instance, preference,
habit, inclination);1991R2568 — EN — 21.07.1992 — 004.001 — 69
▼B
(b) describes the technique which, either by means of sensory or instru-
mental methods, minimizes self-induced errors.
Note:Use of the term ‘instrumental’ as a synonym is not advised.
Subjective (adjective):
describes that which produces a perception that is influenced not only by
the stimulus but also by our way of thinking and feeling.
Kinaesthesis:
sensations resulting from pressure on the sample produced by a move-
ment in the oral cavity or with the fingers (for example: pressing cheese
with fingers)
Threshold (noun)
Absolute threshold:
minimum value of a sensory stimulus which gives rise to:
— the appearance of a sensation (stimulus threshold or detection
threshold), or
— the identification of the sensation (recognition threshold).
Difference threshold:
minimum value of a sensory stimulus which gives rise to a perceptible
difference in the intensity of the sensation.
Terminal threshold:maximum value of a stimulus above which an increase in intensity is not
perceived.
Preference threshold:
minimum quantitative value of a stimulus or critical supra-threshold value
of that stimulus at which anattractionor rejectionrespon se appears in
relationto a n eutral stimulus, for example, inthe choice betweena
sugared solutionan d water.
Note:A distinction should be drawn between an absolute preference
threshold and a differential preference threshold.
Sub-threshold (adjective):
below the absolute threshold.
Supra-threshold (adjective):
above the absolute threshold.Sensory fatigue:
specific form of sen sory adaptationinwhich a decrease insen sitivity
occurs.
Compensation (noun):
result of the interaction of a combination of stimuli in such a way that
each one is perceived with less intensity than if it acted alone.
Synergic (adjective):
joint effect or action of given substances in which the intensity of the
organoleptic attributes resulting from the combination is in excess of the
sum of the intensities of each attribute taken separately.
Contrast effect:increase in response to differences between two simultaneous or consecu-
tive stimuli.
Opposite of the convergence effect.
Convergence effect:
decrease in response to differences between two simultaneous or consecu-
tive stimuli; opposite of contrast effect.
2.3.Terminologyrelated toorganoleptic attributes
Acid(adjective):1991R2568 — EN — 21.07.1992 — 004.001 — 70
▼B
(a) describes the primary taste produced by dilute aqueous solutions of
most acid substances (for example, citric acid, lactic acid, tartaric
acid);
(b) describes the attribute of pure substances or mixtures which produces
this taste.
The corresponding noun is acidity.Sour(adjective):
describes the olfactory-gustatory sensation in which acids generally
produced by fermentation are predominant, as well as the foodstuffs that
produce this sensation.
Some factors that contribute to this sensation are related to the fermenta-
tion, for example the lactic or acetic fermentation, of a food product.
Bitter(adjective):
(a) describes the primary taste produced by dilute aqueous solutions of
various substances such as quinine, caffeine and given alkaloids.
(b) describes the attribute of pure substances or mixtures which produces
this taste.
The corresponding noun is bitterness.
Salty(adjective):
(a) characteristic sensation perceived by the sense of taste, the most
typical example of which is produced by a sodium chloride solution;
(b) describes the attribute of pure substances or mixtures which produces
this taste.
The corresponding noun is saltiness.Sweet(adjective):
(a) describes the primary taste produced by aqueous solutions of various
substances such as sucrose;
(b) describes the attribute of pure substances or mixtures which produces
this taste.
The corresponding noun is sweetness.
Astringent (adjective):
(a) describes the complex sensation produced in the mouth by a dilute
aqueous solution of products such as some tannins (for example,
kaki tannins and sloe tannins).
(b) describes the attribute of pure substances or mixtures which produces
this sensation.
(The corresponding noun is astringency).Flavour(noun):
flavour means the combination of olfactory-gustatory-tactile and kinaes-
thetic sensations which enable an assessor to identify and establish a
multi-level, favourable or unfavourable criterion.
Taste(noun):
(a) sensations perceived when the gustatory papillae are stimulated by
some soluble substances.
(b) attribute of the specific sensation produced by such substances.
Primary taste (noun):
any one of the distinctive tastes of which there are held to be four: sweet,
salty, acid, bitter.
Odour(noun):
(a) combination of sensations perceived by the olfactory organ on
sniffing given volatile substances;
(b) attribute of the specific sensation produced by any one of the above
substances.
Aroma(noun):
(a) pleasant sensations perceived indirectly by the olfactory organ when
tasting a food.1991R2568 — EN — 21.07.1992 — 004.001 — 71
▼B
(b) in perfumery and non-specialized language, this term is also applied
to the same sensations perceived directly through the nose.
After-taste; residual taste (noun):
combination of sensations perceived after the stimulus has disappeared
from the mouth and which differs from the sensations perceived before-
hand.
Aromatic (adjective):
(a) describes the attribute of pure substances or mixtures which when
tasted produce the sensations known as aroma;
(b) describes the products which when examined directly via the nose
produce sensations of fragrance and freshness.
Texture(noun):
characteristics of the solid or rheological state of a product, the combina-
tion of which can stimulate the mechanical receptors during tasting,
particularly those located inthe mouth.
Note:This term refers solely to the objective attributes, not to the sensa-
tions produced which are designated by general terms such as
consistency, fibrousness, greasiness, etc.
Mouthwash:
action whereby a food present in the mouth comes into contact with all
the sensitive areas of the mouth so that the buccal sensations it produces
canbe perceived.
Note:This vocabulary may be enlarged by consulting ISO standards
5492, Parts I to V and other publications such as that by J. L.
Magnen entitled Les cahiers techniques du Centre National de
Coordination des Etudes et Recherches sur la Nutrition et l'Ali-
mentation, etc.
GLASSFOROILTASTING
1. SCOPE
The purpose of this standard is to describe the characteristics of the glass
intended for use in the organoleptic analysis of edible oils (odour, taste,
flavour).
In addition, it describes the adapted heating unit needed to reach and
maintain the right temperature for this analysis.
2. DESCRIPTION OF THE GLASS
The drawing in Figure 1 attempts to establish the optimum characteristics
desirable ina piece of apparatus of this kin d, which canbe specified as
follows:
(a) maximum steadiness, to prevent the glass from tilting and the oil
from being spilled;
(b) a base which easily fits the indentations of the heating unit so that the
bottom of the glass is evenly heated;
(c) a shape that is broadest at the base so that the volatile components of
the oil are readily released but narrowed at the mouth so that the
same components are easily concentrated, thus ensuring that they are
better perceived and identified by the nose;
(d) made of dark-coloured glass to prevent the taster from perceiving the
colour of the oil, thus eliminating any prejudices and impeding the
possible formation of biases or tendencies.
2.1.Dimensions
The glass is sketched in Figure 1, and has the following dimensions:
— total capacity … ................................ 130ml±10ml,
— total height … .................................. 60mm±1mm,
— diameter of mouth … ........................... 50mm±1mm,
— diameter of glass at its widest …............... 70 mm ± 1 mm,— base diameter …............................... 35 mm ± 1 mm,— thickness of glass on sides … ................... 1,5mm±0,2mm,1991R2568 — EN — 21.07.1992 — 004.001 — 72
▼B
— thickness of glass base …...................... 5 mm ± 1 mm.
Each glass shall be equipped with a watch-glass, the diameter of which
shall be 10 mm larger thanthe mouth of the glass. This watch-glass shall
be used as a cover to prevent the loss of aroma and the entry of dust.
2.2.Manufacturingcharacteristics
The glass shall be made of resistant glass; it shall be dark-coloured so
that the colour of its contents cannot be discerned, and shall be free
from scratches or bubbles.
The rim shall be even, smooth and flanged.
The glass shall be annealed so that it stands the temperature changes it
has to undergo in the tests.
2.3.Instructionsforuse
The glasses shall be cleaned using unperfumed soap or detergent and
shall then be rinsed repeatedly until the cleaning agent has been totally
eliminated. The final rinse shall be with distilled water, after which the
glasses shall be left to drainan d thendried ina desiccationstove.
Neither concentrated acids nor chromic acid mixtures shall be used.
The glasses shall be kept inthe stove un til required for use or shall be
kept ina cupboard inwhich they shall be protected from con tamin ation
from any extraneous odours.
Before use, each glass shall be smelled to ensure that no extraneous
odour is present. When the test is being prepared care shall be taken to
record the legend of each glass and the oil it contains. The test organizershall be the only person to know this legend/oil relation.
3. DEVICE FOR HEATING SAMPLES
The samples shall be organoleptically examined at a set temperature
which, inthe case of oils, shall be 28 ± 2º C. For this purpose, a heatin g
device (see Figure 2) shall be installed in each booth within the taster's
reach. It comprises analumin ium block immersed ina thermostatically-
controlled water bath so as to keep a constant temperature. This block
has a series of indentations into which fit the bottoms of the glasses.
The temperature difference between the heating device and the oil
contained in the glasses inserted in the indentations of the various blocks
shall not be more than ± 2º C.
Figure 1—Tastingglass
Dimensions (in mm)1991R2568 — EN — 21.07.1992 — 004.001 — 73
▼B
Figure2—Deviceforheatingsamples(dimensionsinmillimetres)
GUIDEFORTHEINSTALLATIONOFATESTROOM
1. INTRODUCTION
The test room is designed to provide the panel participating in the
sensory tests with a suitable, comfortable, standardized environment
which facilitates work and helps to improve the repeatability and reprodu-
cibility of the results.
2. SCOPE
The purpose of this standard is to specify the basic conditions that have
to be met when installing a test room.1991R2568 — EN — 21.07.1992 — 004.001 — 74
▼B
3. GENERAL SPECIFICATIONS FOR INSTALLATION
The premises, however large they are (see 3.1), shall meet the following
specifications:
They shall be pleasant and suitably lighted (see 3.2) but neutral in style.
For this purpose, a soothing, plain, light colour is recommended for the
walls so that a relaxed atmosphere is created(1).
The premises shall be such that they are easily cleaned and shall be sepa-
rated from any source of noise; consequently, they shall preferably be
soundproofed. They shall also be kept free from extraneous odours for
which purpose, if possible, they shall be fitted with aneffective ven tila-
tion device. If the fluctuations in ambient temperature so warrant, the test
room shall be equipped with air conditioning to keep the atmosphere
close to 20 to 22º C.
3.1.Dimensions
The dimensions of the premises often depend upon the possibilities of the
laboratories or companies. Generally, they should be sufficiently spacious
to permit the installation of 10 booths and an area for preparing the
samples.
However, it is obvious that the larger the area set aside for the installa-
tions, the better, since auxiliary areas can then be provided, for instance,
for cleaning apparatus, arranging culinary preparations and assembling
openpan els.
3.2.Lighting
General lighting, whether from sunlight or lamps (for instance, strip
lighting) shall be uniform, controllable and diffuse.
3.3.Temperatureandhygrometricconditions
The premises shall be kept constantly at a pleasant temperature and under
agreeable hygrometric conditions. Except in special circumstances, a
temperature of 20º to 22º C and hygrometric conditions of 60 to 70 %
relative humidity are recommended.
4. DESCRIPTION OF BOOTHS4.1.General characteristics
The sensory analysis booths shall be placed alongside each other in the
premises. They shall be identical and shall be separated by partitions
which shall be sufficiently high and wide as to isolate the tasters when
seated.
The booths may be made of any appropriate material which is easily
cleaned and looked after (for instance, wood, vitrified plywood, laminated
panelling, etc.). If paint is used it must be completely odour-free when
dry.
The seats provided inthe booths shall be comfortable an d shall have an
adjustable height device.
Each booth shall also be provided with individual lighting, the direction
and intensity of which may be adjusted.
It is highly recommended that the booths be equipped with a button
connected to an outside light which enables the taster to make known to
the attendant outside that he has finished the test, requires further
samples, is missing a piece of apparatus, has noticed some irregularity,
or wishes information, etc. without distracting the other tasters.
4.2.Dimensions
The booths shall be sufficiently large and comfortable. In general, they
shall have the following dimensions:
— width:
0,75 m (without sink)0,85 (with sink);1991R2568 — EN — 21.07.1992 — 004.001 — 75
(1) The colour scheme of the room and its lighting can affect the results of the sensory
analysis.
▼B
— length:
0,50 m (table)
0,20 m excess for partition;
— height of partitions:
0,60 m minimum from table;
— height of table:
0,75 m.
4.3.Arrangement
The table surface shall be such that it is easily cleaned.
Part of this surface shall be used for a sink provided with running
drinking water. However, if this is not practicable, this space may be
used for a tray, spittoonor similar piece of equipmen t.
When the samples have to be kept during the test at a constant tempera-
ture that is above or below ambient temperature, it is advisable to have a
suitable device for this purpose (bain-marie, hot plate, etc.).
A shelf may also be set up at a height of approximately 1,10 metres from
the floor for placing various accessories (glasses, small apparatus, etc.).
If the arrangement of the booths in the test room so permits, it is worth-
while installing a device to facilitate the presentations of the samples.
This may be in the form of a sliding hatch (Figure 1), a revolving vertical
device (Figure 2) suitable for glasses or cups (tall containers), or a hori-
zontally-opening hatch when the containers in which the samples are kept
are small (Figure 3). It is simply a question of ensuring that the opening
is large enough for the trays and glasses containing the samples to pass
through.
See Figure 4 for an example of a test room and additional premises.
5. ADDITIONAL PREMISES
If there is sufficient space, it is advisable to provide separate premises for
preparing the samples (culinary or otherwise), arranging glasses or appa-
ratus and holding discussions prior to or after the tests. If available, such
premises shall be kept clean; in no way shall any smells, noise or conver-
sations from these premises disturb the work of the assessors in the test
room.
Notes:Ideal conditions are described. However, if it were not possible to
have such an installation solely for sensory analyses, the tests
could be performed in premises that meet the minimum condi-
tions described (lighting, temperature, noise, odours) by setting
up mobile booths made up of folding elements in such a way
that, at the very least, they isolate the tasters from each other.1991R2568 — EN — 21.07.1992 — 004.001 — 76
▼B
ARRANGEMENT OF THE BOOTH
Figure1
1991R2568 — EN — 21.07.1992 — 004.001 — 77
▼B
REVOLVING DEVICE FOR PRESENTING SAMPLES
Figure2
1991R2568 — EN — 21.07.1992 — 004.001 — 78
▼B
HATCH FOR PRESENTING SAMPLES
Figure3
1991R2568 — EN — 21.07.1992 — 004.001 — 79
▼B
SENSORY ANALYSIS LABORATORY (Example)
Figure4— Example of a test room
1991R2568 — EN — 21.07.1992 — 004.001 — 80
▼B
ANNEX XIII
PROOF THATREFININGHASTAKENPLACE
1. NEUTRALIZATION AND DECOLORIZATION OF OLIVE OIL IN
THE LABORATORY
1.1.Neutralizationoftheoil
1.1.1. Apparatus
— beaker, 300 ml, tall,
— laboratory centrifuge with 100 ml tubes,— beaker, 250 ml,— round-bottomed flasks, 100 ml,
— separating funnel, 1 litre.
1.1.2. Reagents
— aqueous solutionof 12 % sodium hydroxide,
— ethyl alcohol solution of 1 % phenolphtalein,— pure hexane, AR,
— pure propan-2-ol of AR.
1.1.3. Procedure
(a)Oils witha free fatty acid content, expressed as oleic acid, of less
than 30 %
Place 50 g of crude oil ina tall 300 ml beaker an d heat to 65º C ina
water bath. Add a quantity of 12 % solution of sodium hydroxide
corresponding to the free acid of the oil, with an excess of 5 %, stir-
ring gently all the time. Continue to stir for five minutes, keeping the
temperature at 65º C.
Transfer the mixture into 100 ml centrifuge tubes and separate the
soapy paste by centrifugation. Pour the decanted oil into a 250 ml
beaker and wash with 50 to 60 ml of boiling distilled water,
removing the water by means of a siphon. Repeat the washings until
all traces of residual soap are removed (disappearance of the pink
colouring in the phenolphtalein).
Centrifuge the oil to eliminate any small quantities of residual water.
(b)Oils witha free fatty acid content expressed as oleic acid exceeding
30 %.
In a 1 litre separating funnel place 50 g of crude oil, 200 ml of
hexane, 100 ml of propan-2-ol and a quantity of 12 % solution of
sodium hydroxide corresponding to the free acid of the oil, with an
excess of 0,3 %.
Stir vigorously for one minute. Add 100 ml of distilled water, stir
again and allow to stand.
After separation of the layers, allow the lower layer containing soaps
to drain off. Between the two layers (oily on top and aqueous under-
neath) an intermediary layer often forms made up of mucilages and
insoluble substances which must also be eliminated.
1.2.Decolorization ofneutralizedoil
1.2.1. Apparatus
— round-bottomed flask, 250 ml, with three ground glass necks for the
insertion of:
(a) a thermometer graduated in degrees and allowing readings to be
takenat 90º C;
(b) a mechanical stirrer operating at 250 to 300 revolutions per
minute, equipped to operate in a vacuum;
(c) a vacuum pump connection,
— vacuum pump, with a manometer, capable of giving residual pressure
of 15 to 30 millibars.1991R2568 — EN — 21.07.1992 — 004.001 — 81
▼B
1.2.2. Procedure
Weigh about 100 g of neutralized oil in the three-necked flask. Insert the
thermometer and the stirrer, connect the vacuum pump and heat to 90º C,
stirring all the time. Maintain that temperature, continuing to stir, until
the oil to be analysed is entirely free from water (about 30 minutes).
Thenbreak the vacuum an d add 2 to 3 g of activated earth.
Re-establish the vacuum until a residual pressure of 15 to 30 millibars is
obtained and, maintaining a temperature of 90º C, stir for 30 minutes at
about 250 revolutions per minute.
Filter while still hot ina thermostatic oven(50 to 60º C).1991R2568 — EN — 21.07.1992 — 004.001 — 82
▼B
ANNEX XIV
ADDITIONAL NOTES 2, 3 AND 4 TO CHAPTER15 OF THE
COMBINEDNOMENCLATURE
1.‘Note 2 A: For the purposes of CN codes 1509 and 1510, “olive oil” means
oils derived solely from the treatment of olives, excluding re-
esterified olive oil and mixtures of olive oil with other oils.
The presence of re-esterified olive oil or other oils is ascertained
using the methods set out in Annexes V, VII, IX, X and XII. The
analytical characteristics of the sterol and acidic composition of
all olive oils under CN codes 1509 and 1510 are set out in the
table below.
Table I— Acidic compositionas percen tage
of totalTable II— Sterol compositionas percen tage of
total
Myristic acid M 0,1 Chloresterol M 0,5
Linolenic acid M 0,9 Brassicasterol M 0,2
Arachidic M 0,7 Campesterol M 4,0
Eicosanoic acid M 0,5 Stigmaserol < campesterol
Behenic acid M 0,3 Betasitosterol (1) m 93,0
Lignoceric acid M 0,5 ∆7-stigmasterol M 0,5
M = Maximum.
m = minimum.
(1) Delta-5-23 Stigma sterol + Cholesterol + Betasitosterol + Sitostanol + Delta-5-Avenasterol +
Delta-5-24-Stigmastadienol.
Note 2 B: “Virgin olive oil” means oils derived solely from olives using
mechanical or other physical means under conditions, and particu-
larly thermal conditions, that do not lead to deterioration of the oil,
and which have undergone no treatment other than washing,
decantation, centrifugation or filtration, but excluding oils
extracted from olives using solvents (1510) and defined in
Sections I and II below.
I. For the purposes of subheading 1509 1010, “virgin lampante
olive oil”, whatever its acidity, means olive oil with:
(a) an aliphatic alcohols content not exceeding 400 mg/kg;
(b) an erythrodiol and uvaol content, not exceeding 4,5 %;
(c) a content in saturated fatty acids at the 2-position in the
triglycerides not exceeding 1,3 % and/or
(d) one of the following characteristics:
— (d1) a peroxide number exceeding 20 meq 02/kg;
— (d2) a content in volatile halogenated solvents
exceeding 0,2 mg/kg overall or exceeding 0,1 mg/
kg for any one solvent;
— (d3) a K270(100) extinction coefficient higher than
0,250 and, after treatment of the oil with activated
alumina, not higher than 0,11. Some oils having a
free fatty acid content, expressed as oleic acid, of
more than3,3 g per 100 g after passage through
activated alumina, in accordance with the method
set out in Annex XV, may have a K270extinction1991R2568 — EN — 21.07.1992 — 004.001 — 83
▼B
coefficient higher than 0,11. If so, after neutraliza-
tionan d decolourizationinthe laboratory, they
must have the following characteristics:
—aK270extinction coefficient not higher than
1,20;
— an extinction coefficient variation ( ∆K)(1)i n
the 270 nm region, higher than 0,01 but not
higher than0,16;
— (d4) organoleptic characteristics which include detect-
able defects exceeding the limits of acceptability
and a Panel Test score lower than 3,5.
II. For the purposes of subheading 15091090, “virgin oil” means
olive oil having the following characteristics:
(a) an acid content, expressed as oleic acid, not exceeding 3,3 g
per 100 g;
(b) a peroxide number not exceeding 20 meq 02/kg;
(c) an aliphatic alcohols content not exceeding 300 mg/kg;
(d) a content in volatile halogenated solvents not exceeding
0,2 mg/kg overall and not exceeding 0,1 mg/kg for each
solvent;
(e) a K 270 extinction coefficient not higher than 0,250 and,
after treatment of the oil with activated alumina, not higher
than0,10(2);
(f) an extinction coefficient variation ( ∆K), inthe 270 n m
region, not higher than 0,010;
(g) organoleptic characteristics which may include detectable
defects withinthe limits of acceptability a Pan el Test score
higher than3,5;
(h) an erythrodiol and uvaol content not exceeding 4,5 %;
(i) a content in saturated fatty acids at the 2-position in the
triglycerides not exceeding 1,3 %.
Note 2 C: Subheading 15099000 covers olive oil obtained by the treatment
of olive oils falling within subheading 1509 1010 or 15091090,
whether or not blended with virgin olive oil, having the following
characteristics:
(a) an acid content, expressed as oleic acid, not exceeding 3,3 g
per 100 g;
(b) an aliphatic alcohols content not exceeding 350 mg/kg;
(c) A K270extinction coefficient (100) higher than 0,250 and not
higher than 1,20 and, after treatment of the sample with acti-
vated alumina, higher than 0,10;
(d) an extinction coefficient variation ( ∆K), in the 270 nm region,
higher than 0,010 and not higher than 0,160;
(e) an erythrodiol and uvaol content not exceeding 4,5 %;
(f) a content in saturated fatty acids at the 2-position not
exceeding 1,5 %.
Note 2 D: For the purposes of subheading 1510 0010, “crude oils” means
oils, particularly olive residue oils, with the following characteris-
tics:
(a) an acid content, expressed as oleic acid, greater than 2 g per
100 g;
(b) an erythrodiol and uvaol content exceeding 12 %;
(c) a content in saturated fatty acids at the 2-position in the trigly-
cerides not exceeding 1,8 %.
Note 2 E: Oils under subheading 15100090 includes oils obtained by the
treatment of oils falling within subheading 1510 0010, whether1991R2568 — EN — 21.07.1992 — 004.001 — 84
(1)∆K=Km− 0,5 (Km−4+Km+4)
Kmmeans the extinction coefficient at the wavelength of the peak of the absorption
curve in the 270 nm region.K
m−4and Km+4means the extinction coefficients at wavelengths 4 nm lower and higher
thanthe Km wavelen gth.
(2) If the K270exceeds 0,25, a new test is to be carried out following passage over alumina.
K270may not exceed 0,10.
▼B
or not blended with virgin olive oil, not having the characteristics
of the oils referred to at points I and II provided they have a
content in saturated fatty acids at the 2-position in the triglycerides
not exceeding 2 %.’
2.‘Note 3: Subheadings 1522 0031 and 1522 0039 do not cover:
(a) residues resulting from the treatment of fatty substances
containing oil having an iodine index, determined in accordance
with the method laid down in Annex XVI, lower than 70 or
higher than100;
(b) residues resulting from the treatment of fatty substances
containing oil having an iodine index not lower than 70 or
higher than 100, of which the peak area representing the reten-
tion volume of -sitosterol, determined in accordance with the
Annex to the Regulation mentioned in additional Note 4 below,
is less than93 % of the total sterol peak areas.’
3.‘Note 4: The analytical methods for the determination of the characteristics
of the products referred to above are those laid downintheAnnexes to Regulation (EEC) No 2568/91.’1991R2568 — EN — 21.07.1992 — 004.001 — 85
▼B
ANNEX XV
1. OIL CONTENT OF OLIVE RESIDUE
1.1.Apparatus
— suitable extractionapparatus fitted with a 200 to 250 ml roun d-
bottomed flask,
— electrically heated bath (e.g., sand bath, water bath) or hotplate,— analytical balance,— ovenregulated to a maximum of 80º C,— electrically heated ovenfitted with a thermostatic device regulated to
103 ± 2º C and one that can be swept with a stream of air or operated
at reduced pressure,
— mechanical mill, easy to clean, and one that allows the olive residues to
be ground without a rise in their temperature or any appreciable altera-
tion in their content of moisture, volatile matter or substances
extractable with hexane,
— extractionthimble an d cottonwool or filter paper from which
substances extractable with hexane have already been removed,
— dessicator,— sieve with 1 mm diameter apertures,— small particles of previously dried pumice stone.
1.2.Reagent
Normal hexane, technical grade, which must leave a residue of less than
0,002 g per 100 ml, oncomplete evaporation .
2. PROCEDURE2.1.Preparationofthetestsample
If necessary, use the mechanical mill, which has previously been properly
cleaned, to grind the laboratory sample in order to reduce it to particles that
canpass completely through the sieve.
Use about one twentieth of the sample to complete the process of cleaning
the mill, discard the ground material, grind the remainder and collect, mix
carefully and analyze without delay.
2.2.Testportion
As soon as the grinding operation has been completed, weigh out about 10 g
of the sample to the nearest 0,01 g for testing.
2.3.Preparationoftheextractionthimble
Place the test portioninthe thimble an d plug with cottonwool. If a filter
paper is used, envelope the test portion in it.
2.4.Peliminary drying
If the olive residues are very moist (i.e., moisture and volatile matter
content more than 10 %), carry out preliminary drying by placing the
loaded thimble (or filter paper) inthe ovenheated for anappropriate time
at not more than 80º C in order to reduce the moisture and volatile matter
content to less than 10 %.
2.5.Preparationoftheround-bottomedflask
Weigh to the nearest 1 mg the flask containing one or two particles of
pumice stone, previously dried in the stove at 103 ± 2º C and then cooled
in a dessicator for not less than one hour.
2.6.Initialextraction
Into the extraction apparatus insert the thimble (or filter paper) containing
the test portion. Pour into the flask the requisite quantity of hexane. Fit the
flask to the extractionapparatus an d place the whole onthe electrically
heated bath. Adjust the rate of heating in such a way that the reflux rate
is not less than three drops per second (moderate, not violent boiling). After
four hours extraction, allow to cool. Remove the thimble from the extrac-1991R2568 — EN — 21.07.1992 — 004.001 — 86
▼B
tionapparatus an d place it ina stream of air inorder to drive off most of
the impregnating solvent.
2.7.Secondextraction
Tip the contents of the thimble into the micro-grinder and grind as finely as
possible. Return the ground mixture to the thimble without loss and place it
back inthe extractionapparatus.
Continue the extraction for a further two hours using the same round-
bottomed flask containing the initial extract.
The resultant solution in the extraction flask must be clear. If not, filter it
through a filter paper and wash the original flask and the filter paper
several times with hexane. Collect the filtrate and the washing solvent in
a second round-bottomed flask which has been dried and tared to the
nearest 1 mg.
2.8.Removalofsolventandweighingofextract
Remove the greater part of the solvent by distillation on an electrically
heated bath. Remove the last traces of solvent by heating the flask in the
oven at 103 ± 2º C for 20 minutes. Assist the elimination process either
by blowing in air, or preferably an inert gas, at intervals or by using
reduced pressure.
Leave the flask in a dessicator to cool for at least one hour and weigh to
the nearest 1 mg.
Heat again for 10 minutes under the same conditions, cool in a dessicator
and reweigh.
The difference between the two weighings shall not exceed 10 mg. If it
does, heat again for periods of 10 minutes followed by cooling and
weighing until the weight difference is 10 mg or less. Note the last weight
of the flask.
Carry out duplicate determinations on the test sample.
3. EXPRESSION OF RESULTS3.1.Methodofcalculationandformula
(a) The extract expressed as a percentage by mass of the product as
received is equal to:
S=m
1×100
m0
where: S is the percentage by mass of extract of the product as
received,
m0= is the mass, ingrams, of the test portion ,
m1= is the mass, ingrams, of the extract after dryin g.
Take as the result the arithmetic mean of the duplicate determinations,
providing the repeatability conditions are satisfied.
Express the result to the first decimal place.
(b) The extract is expressed ona dry matter basis by usin g the formula:
S×100
100 − U= oil percentage of extract on a dry basis
where:
S = is the percentage of extract by means of the product as received
(see (a)),
U = is its moisture and volatile matter content.
3.2.Repeatability
The difference between the duplicate determinations carried out simulta-
neously or in rapid sucession by the same analyst shall not exceed 0,2 g
of hexane extract per 100 g of sample.1991R2568 — EN — 21.07.1992 — 004.001 — 87
▼B
If this condition is not satisfied, repeat the analysis on two other test
portions. If, in this case too, the difference exceeds 0,2 g, take as the result
the arithmetic mean of the four determinations.1991R2568 — EN — 21.07.1992 — 004.001 — 88
▼B
ANNEX XVI
DETERMINATIONOFIODINEVALUE
1. SCOPE
This International Standard specifies a method for the determination of the
iodine value of animal and vegetable fats and oils, referred to hereafter as
fats.
2. DEFINITION
For the purposes of this International Standard, the following definition
applies:
2.1.iodine value. The mass of iodine absorbed by the sample under the oper-
ating conditions specified in this International Standard.
The iodine value is expressed as grams of iodine per 100 g of sample.
3. PRINCIPLE
Dissolution of a test portion in solvent and addition of Wijs reagent. After a
specified time, addition of potassium iodide solution and water, and titra-
tion of the liberated iodine with sodium thiosulfate solution.
4. REAGENTS
All reagents shall be of recognized analytical grade:
4.1.water,complying with the requirements of ISO 3696, Grade 3.
4.2.potassium iodide, 100 g/l solution, not containing iodate or free iodine.
4.3.starch,solution.
Mix 5 g of soluble starch in30 ml of water, add this mixture to 1 000 ml of
boiling water, boil for three minutes and allow to cool.
4.4.sodiumthiosulfate, standardvolumetric solutionc(Na2S2O3.5H2O)=0,1mol/
l, standardized not more than seven days before use.
4.5.solvent,prepared by mixing equal volumes of cyclohexane and acetic acid.
4.6.Wijs reagent, containing iodine monochloride in acetic acid. Commercially
available Wijs reagent shall be used.
5. APPARATUS
Usual laboratory apparatus and, in particular, the following:
5.1.glass weighing scoops, suitable for the test portion and for inserting into
the flasks (6.2).
5.2.conical flasks, of 500 ml capacity, fitted with ground glass stoppers and
completely dry.
6. PREPARATION OF THE TEST SAMPLE
The homogenized sample is dried over sodium sulphate and filtered.
7. PROCEDURE
7.1. Test portion
The mass of the test portion varies according to its expected iodine value as
showninTable 1.1991R2568 — EN — 21.07.1992 — 004.001 — 89
▼B
Table 1
Expected iodine valueMass of test portion
(g)
less than5 3,00
5 to 20 1,00
21 to 50 0,40
51 to 100 0,20
101 to 150 0,13
151 to 200 0,10
Weigh the test portion to the nearest 0,1 mg in a glass weighing scoop
(5.1).
7.2. Determination
Place the test portionina 500 ml flask (6.2). Add 20 ml of the solven t
(4.5) to dissolve the fat. Add exactly 25 ml of the Wijs reagent (4.6), insert
the stopper, swirl the contents and place the flask in the dark. Do not use a
mouth pipette for the Wijs reagent.
Similarily, prepare a blank with the solvent and the reagent but omitting
the test portion.
For samples having an iodine valve below 150, leave the flasks in the dark
for one hour; for those with an iodine value above 150 and for polymerized
products or products oxidized to a considerable extent, leave for two hours.
At the end of the time, add 20 ml of the potassium iodide solution (4.2)
and 150 ml of water (4.1) to each of the flasks.
Titrate with the standard volumetric sodium thiosulfate solution (4.4) until
the yellow colour due to iodine has almost disappeared. Add a few drops of
the starch solution (4.3) and continue the titration until the blue colour just
disappears after very vigorous shaking.
Note:Potentiometric determination of the end point is permissible.
7.3. Number of determinations
Carry out two determinations on the same test sample.
8. EXPRESSION OF RESULTS
The iodine value is given by the expression
12;69 c V 1/C0V2 ðÞ
m
where:
c = is the numerical value of the exact concentration, in moles per litre,
of the standard volumetric sodium thiosulfate solution (4.4) used;
V1= is the numerical value of the volume, in millilitres, of the standard
volumetric sodium thiosulfate solution(4.4) used for the blan k test;
V2= is the numerical value of the volume, in millilitres, of the standard
volumetric sodium thiosulfate solution(4.4) used for the determin a-
tion;
m = is the numerical value of the mass, in grams, of the test portion
(7.1).
Take as the result the arithmetic mean of the two determinations, provided
that the requirement for repeatability (9.2) is satisfied.1991R2568 — EN — 21.07.1992 — 004.001 — 90