1991R2568 — EN — 01.11.2003 — 020.001 — 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
►M5CommissionRegulation(EEC) No 3288/92 of 12 November 1992 L 327 28 13.11.1992
►M6CommissionRegulation(EEC) No 183/93 of 29 Jan uary 1993 L 22 58 30.1.1993
►M7amended by Commission Regulation (EEC) No 826/93 of 6 April
1993L 87 6 7.4.1993
►M8CommissionRegulation(EEC) No 620/93 of 17 March 1993 L 66 29 18.3.1993
►M9CommissionRegulation(EC) No 177/94 of 28 Jan uary 1994 L 24 33 29.1.1994
►M10CommissionRegulation(EC) No 2632/94 of 28 October 1994 L 280 43 29.10.1994
►M11CommissionRegulation(EC) No 656/95 of 28 March 1995 L 69 1 29.3.1995
►M12CommissionRegulation(EC) No 2527/95 of 27 October 1995 L 258 49 28.10.1995
►M13CommissionRegulation(EC) No 2472/97 of 11 December 1997 L 341 25 12.12.1997
►M14CommissionRegulation(EC) No 282/98 of 3 February 1998 L 28 5 4.2.1998
►M15CommissionRegulation(EC) No 2248/98 of 19 October 1998 L 282 55 20.10.1998
►M16CommissionRegulation(EC) No 379/1999 of 19 February 1999 L 46 15 20.2.1999
►M17CommissionRegulation(EC) No 455/2001 of 6 March 2001 L 65 9 7.3.2001
►M18CommissionRegulation(EC) No 2042/2001 of 18 October 2001 L 276 8 19.10.2001
►M19CommissionRegulation(EC) No 796/2002 of 6 May 2002 L 128 8 15.5.2002
►M20CommissionRegulation(EC) No 1989/2003 of 6 November 2003 L 295 57 13.11.2003
Corrected by:
►C1Corrigendum, OJ L 347, 28.11.1992, p. 69 (2568/91)
►C2Corrigendum, OJ L 176, 20.7.1993, p. 26 (183/93)
►C3Corrigendum, OJ L 96, 28.3.1998, p. 47 (2472/97)
▼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 in
oils 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 and
the organoleptic characteristics of virgin oil should be defined, in order
to guarantee the purity and quality of the products concerned, withoutprejudice 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 the
Member 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 theadditional 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 disposed
of 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,1991R2568 — EN — 01.11.2003 — 020.001 — 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.
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HAS ADOPTED THIS REGULATION:
Article 1
1. Oils, the characteristics of which comply with those set out in
points 1 and 2 of Annex I to this Regulation, shall be deemed to be
virgin olive oils within the meaning of point 1(a) and (b) of the Annexto RegulationNo 136/66/EEC.
2. Oil, the characteristics of which comply with those set out in
point 3 of Annex I to this Regulation, shall be deemed to be lampante
olive oil within the meaning of point 1(c) of the Annex to RegulationNo 136/66/EEC.
3. Oil, the characteristics of which comply with those set out in
point 4 of Annex I to this Regulation, shall be deemed to be refined
olive 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 5 of Annex I to this Regulation, shall be deemed to be olive oil
composed of refined olive oils and virgin olive oils within the meaningof point 3 of the Annex to Regulation No 136/66/EEC.
5. Oil, the characteristics of which comply with those set out in
point 6 of Annex I to this Regulation, shall be deemed to be crude
olive-pomace oil within the meaning of point 4 of the Annex to Regu-
lationNo 136/66/EEC.
6. Oil, the characteristics of which comply with those set out in
point 7 of Annex I to this Regulation, shall be deemed to be refined
olive-pomace 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 8 of Annex I to this Regulation, shall be deemed to be olive-
pomace 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 determination of the wax content, the method given 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 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,1991R2568 — EN — 01.11.2003 — 020.001 — 3
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— for the determination of stigmastadienes, the method set out in
Annex XVII,
— for determining the content of triglycerides with ECN42, the
method set out in Annex XVIII,
— for determination of the aliphatic alcohol content, the method given
in Annex XIX.
2. Verification by national authorities or their representatives of the
organoleptic characteristics of virgin oils shall be effected by tastingpanels approved by the Member States.
The organoleptic characteristics of an oil as referred to in the first
subparagraph shall be deemed consonant with the category declared if
a panel approved by the Member State confirms the grading.
Should the panel not confirm the category declared as regards the orga-
noleptic characteristics, at the interested party's request the nationalauthorities or their representatives shall have two counter-assessments
carried out by other approved panels, at least one by a panel approved
by the producer Member State concerned. The characteristicsconcerned shall be deemed consonant with the characteristics declaredif at least two of the counter-assessments confirm the declared grade. Ifthat is not the case, the interested party shall be responsible for the cost
of the counter-assessments.
3. When the national authorities or their representatives verify the
characteristics of the oil as provided for inparagraph 1, samples shall
be taken in accordance with international standards EN ISO 661 on thepreparation of test samples and EN ISO 5555 on sampling. However,notwithstanding point 6.8 of standard EN ISO 5555, in the case ofbatches of such oils in immediate packaging not exceeding 100 litres,the sample shall be taken in accordance with Annex Ia to this Regula-
tion.
Without prejudice to standard EN ISO 5555 and Chapter 6 of standard
EN ISO 661, the samples takenshall be put ina dark place away from
strong heat as quickly as possible and sent to the laboratory for analysisno later than:
— the tenth working day after they are taken, during the period from
October to May, and
— the fifth working day after they are taken, during the period from
June to September.
4.►
M20For the purposes of the verificationprovided for inpara-
graph 3, the analyses referred to in Annexes II, III, IX, X and XII and,where applicable, any counter-analyses required under national law,
shall be carried out before the minimum durability date. Where
sampling is done more than four months before the minimum durabilitydate, the analyses shall be carried out no later than the fourth monthafter the month in which the sample was taken. No time limit shallapply to the other analyses provided for in that Regulation ◄
Unless the sample was taken less than one month before the minimum
durability date, if the results of the analyses do not match the charac-teristics of the category of olive oil or olive-residue oil declared, theparty concerned shall be notified no later than one month before theend of the period laid down in the first subparagraph.
5. For the purpose of determining the characteristics of olive oils by
the methods provided for inparagraph 1, the an alysis results shall bedirectly compared with the limits laid downinthis Regulation .1991R2568 — EN — 01.11.2003 — 020.001 — 4
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Article 2a
The national authorities or their representatives may verify whether a
sample is consistent with the category declared:
(a) either by carrying out, in any order, the analyses provided for in
Annex I;
(b) or by following the order set out in Annex Ib on the decision tree
until one of the decisions appearing in the decision tree is reached.
Article►M193◄
Where it is found that the organoleptic characteristics of an oil do not
correspond to its description, the Member State concerned shall,without prejudice to any other penalties, apply administrative financialpenalities, to be determined in the light of the seriousness of the irre-gularity detected.
In assessing the irregularity, attention shall be paid in particular to
natural changes in the characteristics of an oil kept under normal
conditions.
At the beginning of each half-year, the Member States shall inform the
Commission of the number and type of irregularities detected and thepenalties applied during the previous half-year.
Article 4
1. The Member States may approve assessment panels so that
national authorities or their representatives can assess and verify orga-noleptic characteristics.
The terms of approval shall be set by Member States and ensure that:
— the requirements of Annex XII.4 are met,
— the panel head is given training recognised for this purpose by the
Member State,
— continued approval depends on performance in annual checks
arranged by the Member State.
Member States shall notify to the Commission a list of approved panels
and the action taken under this paragraph.
2. Where Member States encounter difficulties in setting up tasting
panels in their territory, they may call on a tasting panel approved in
another Member State.
3. Each Member State draw up a list of tasting panels set up by
professional or inter-branch organizations in accordance with the condi-tions laid down in paragraph 1 and shall ensure that those conditionsare complied with.
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.1991R2568 — EN — 01.11.2003 — 020.001 — 5
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Article 7
The Community provisions concerning the presence of contaminants
shall apply.
As regards halogenated solvents, the limits for all categories of olive
oils are as follows:
— maximum content of each halogenated solvent detected: 0,1 mg/kg,
— maximum total content of halogenated solvents detected: 0,2 mg/
kg.
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 ofRegulationNo 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 .
However, the method set out in Annex XII shall apply from ►
M11
November 1992 ◄,except in sofarasoperations relating to the inter-
vention system are concerned .
That method shall not apply to virgin olive oil prepared for the market
prior to 1 November 1992.
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 — 01.11.2003 — 020.001 — 6
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ANNEXES
Summary
Annex I: Characteristics of olive oil
Annex Ia: Sampling of batches of olive oil or olive-residue oil in
immediate packaging not exceeding 100 litres
Annexe Ib: Decision treeAnnex II: Determination of the free fatty acids
Annex III: Determination of the peroxide value
Annex IV: ►
M6Determination of wax content by capillary column gas-
liquid 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 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 XIX: Method for determining aliphatic alcohol content
Annex XV: Oil content of olive residue
Annex XVI: Determination of iodine value
Annex XVII: Determination of stigmastadienes in vegetable oils
Annex XVIII: Method for determining the content of triglycerides with
ECN421991R2568 — EN — 01.11.2003 — 020.001 — 7
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ANNEX I
CHARACTERISTICSOFOLIVEOILTYPES
CategoryAcidity
(%)
(*)Peroxide value
mEq O2/kg (*)Waxes mg/kg
(**)Saturated
acids in2-
positionof the
triglyceride
(%)Stigmasta-
dienes mg/
kg (1)Difference
between
HPLC ECN42
and theoretical
ECN42K232(*) K270(*) Delta-K (*)Organoleptic
assessment
Medianof
defects (Md)
(*)Organoleptic
assessment
Medianof
fruity (Mf)
(*)
1. Extra virginolive oil ≤0,8 ≤20 ≤250 ≤1,5 ≤0,15 ≤0,2 ≤2,50 ≤0,22 ≤0,01 Md = 0 Mf > 0
2. Virginolive oil ≤2,0 ≤20 ≤250 ≤1,5 ≤0,15 ≤0,2 ≤2,60 ≤0,25 ≤0,01 Md ≤2,5 Mf > 0
3. Lampante olive oil > 2,0 — ≤300(3) ≤1,5 ≤0,50 ≤0,3 — — — Md > 2,5 (2)—
4. Refined olive oil ≤0,3 ≤5 ≤350 ≤1,8 — ≤0,3 — ≤1,10 ≤0,16 — —
5. Blended olive oil
composed of refined
olive oils & virginolive
oils ≤1,0 ≤15 ≤350 ≤1,8 — ≤0,3 — ≤0,90 ≤0,15 — —
6. Crude olive-pomace oil — — > 350(4) ≤2,2 — ≤0,6 — — — — —
7. Refined olive-pomace oil ≤0,3 ≤5 > 350 ≤2,2 — ≤0,5 — ≤2,00 ≤0,20 — —
8. Olive-pomace oil ≤1,0 ≤15 > 350 ≤2,2 — ≤0,5 — ≤1,70 ≤0,18 — —
(1) Sum of isomers that could (or could not) be separated by capillary column.
(2) Or if the medianof defects is less thanor equal to 2,5 an d the medianof fruity is 0.
(3) Oils with a wax content of between 300 mg/kg and 350 mg/kg are considered to be lampante olive oil if the total aliphatic alcohol content is less than or e qual to 350 mg/kg or if the erythrodiol and uvaol content is less than or
equal to 3,5 %.
(4) Oils with a wax content of between 300 mg/kg and 350 mg/kg are considered to be crude olive-pomace oil if the total aliphatic alcohol content is above 35 0 mg/kg and if the erythrodiol and uvaol content is greater than 3,5 %.▼M201991R2568 — EN — 01.11.2003 — 020.001 — 8
CategoryFatty acids content (1)
Sum of
transoleic
isomers
(%)Sum of
translino-
leic and
translino-
lenic
isomers
(%)Sterols composition
Total
sterols
(mg/kg)Erythro-
diol and
uvaol (%)
(**)Myristic
(%)Linolenic
(%)Arachidic
(%)Eicose-
noic
(%)Behenic
(%)Ligno-
ceric
(%)Choles-
terol
(%)Brassi-
casterol
(%)Campes-
terol
(%)Stigmas-
terol
(%)Beta-
sitosterol
(%) (2)Delta-7-
Stigma-
sterol
(%)
1. Extra virgin
olive oil ≤0,05 ≤1,0≤0,6≤0,4≤0,2≤0,2≤0,05≤0,05 ≤0,5≤0,1≤4,0 < Camp. ≥93,0 ≤0,5≥1 000 ≤4,5
2. Virginolive oil ≤0,05 ≤1,0≤0,6≤0,4≤0,2≤0,2≤0,05≤0,05 ≤0,5≤0,1≤4,0 < Camp. ≥93,0 ≤0,5≥1 000 ≤4,5
3. Lampante olive
oil ≤0,05 ≤1,0≤0,6≤0,4≤0,2≤0,2≤0,10≤0,10 ≤0,5≤0,1≤4,0 — ≥93,0 ≤0,5≥1 000≤4,5 (3)
4. Refined olive oil ≤0,05 ≤1,0≤0,6≤0,4≤0,2≤0,2≤0,20≤0,30 ≤0,5≤0,1≤4,0 < Camp. ≥93,0 ≤0,5≥1 000 ≤4,5
5. Blended olive oil
composed of
refined olive oils
& virginolive
oils ≤0,05 ≤1,0≤0,6≤0,4≤0,2≤0,2≤0,20≤0,30 ≤0,5≤0,1≤4,0 < Camp. ≥93,0 ≤0,5≥1 000 ≤4,5
6. Crude olive-
pomace oil ≤0,05 ≤1,0≤0,6≤0,4≤0,3≤0,2≤0,20≤0,10 ≤0,5≤0,2≤4,0 — ≥93,0 ≤0,5≥2 500 > 4,5(4)
7. Refined olive-
pomace oil ≤0,05 ≤1,0≤0,6≤0,4≤0,3≤0,2≤0,40≤0,35 ≤0,5≤0,2≤4,0 < Camp. ≥93,0 ≤0,5≥1 800 > 4,5
8. Olive-pomace oil ≤0,05 ≤1,0≤0,6≤0,4≤0,3≤0,2≤0,40≤0,35 ≤0,5≤0,2≤4,0 < Camp. ≥93,0 ≤0,5≥1 600 > 4,5
(1) Other fatty acids present (%): palmitic: 7,5 to 20,0; palmitoleic: 0,3 to 3,5; heptadecanoic: ≤0,3; heptadecenoic: ≤0,3; stearic: 0,5 to 5,0; oleic: 55,0 to 83,0; linoleic: 3,5 to 21,0.
(2) Sum of: Delta-5-23-Stigmastadienol + Chlerosterol + Beta-Sitosterol + Sitostanol + Delta-5-Avenasterol + Delta-5-24-Stigmastadienol.
(3) Oils with a wax content of between 300 mg/kg and 350 mg/kg are considered to be lampante olive oil if the total aliphatic alcohol content is less than or e qual to 350 mg/kg or if the erythrodiol and uvaol content is less than or
equal to 3,5 %.
(4) Oils with a wax content of between 300 mg/kg and 350 mg/kg are considered to be crude olive-pomace oil if the total aliphatic alcohol content is above 35 0 mg/kg and if the erythrodiol and uvaol content is greater than 3,5 %.
Notes:
(a) The results of the analyses must be expressed to the same number of decimal places as used for each characteristic. The last digit must be increased b y one unit if the following digit is greater than 4.
(b) If just a single characteristic does not match the values stated, the category of an oil can be changed or the oil declared impure for the purposes of t his Regulation.
(c) If a characteristic is marked with an asterisk (*), referring to the quality of the oil, this means the following:
— for lampante olive oil, it is possible for both the relevant limits to be different from the stated values at the same time,
— for virgin olive oils, if at least one of these limits is different from the stated values, the category of the oil will be changed, although they will st ill be classified inon e of the categories of virginolive oil.
(d) If a characteristic is marked with two asterisks (**) this means that for all types of olive-pomace oil, it is possible for both the relevant limits t o be different from the stated values at the same time.▼M201991R2568 — EN — 01.11.2003 — 020.001 — 9
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ANNEX Ia
Sampling of olive oil or olive-pomace oil delivered in immediate packaging
notexceeding100litres
This method of sampling applies to deliveries of olive oil or olive-pomace oil
not exceeding 125000 litres, put up in immediate packaging not exceeding 100
litres.
If the delivery exceeds 125000 litres, it is to be subdivided into batches of
125000 litres or under. If the delivery is less than 125000 litres it shall consti-
tute one batch. The method shall then be applied to each batch.
The minimum number of primary samples to be taken is determined by the size
of the batch inaccordan ce with the table set out inpoin t 1.
The size of the primary sample is determined on the basis of the capacity of the
immediate packaging, in accordance with the table set out in point 2.1.
Delivery, primary sample and laboratory sample shall mean the definitions
giveninstan dard EN ISO 5555.
‘Batch’ shall mean a set of sales units which are produced, manufactured and
packed in circumstances such that the oil contained in each sales unit is consid-
ered to be homogenous in terms of all analytical characteristics.
1. NUMBER OF PRIMARY SAMPLES TO BE TAKEN
The minimum number of primary samples to be taken will be determined
by the size of the batch in accordance with the following table:
Size of batch (litres) less than Minimum number of primary samples
7 500 2
25000 3
75000 4
125000 5
The immediate packs selected to form a primary sample must be adjacent to
each other inthe batch.
In cases of doubt, Member States shall increase the number of primary
samples to be taken.
2. CONTENT OF PRIMARY SAMPLES
2.1Primarysamplesmustcomprisethefollowing:
Where the immediate packaging has a
capacity of:The primary sample shall comprise the
oil from:
(a) 5 litres or more (a) 3 immediate packs
(b) 3 litres or more but less than5
litres(b) 3 immediate packs
(c) 2 litres or more but less than3
litres(c) 3 immediate packs
(d) 1 litre or more but less than2
litres(d) 6 immediate packs
(e) 0,75 litres or more but less than
1 litre(e) 6 immediate packs
(f) less than0,75 litres (f) three times the oil from the
minimum number of packs with
a total capacity of more than1,5
litres1991R2568 — EN — 01.11.2003 — 020.001 — 10
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2.2The primary samples are to be kept in the immediate packaging up to
the time of analysis. The oil in the primary samples shall then, as
applicable, be subdivided into three laboratory samples in order to
carryout:
(a) the analyses referred to in Annexes II, III, IX and X,
(b) the analysis referred to in Annex XII,
(c) the other analyses.
2.3The packs constituting a primary sample shall be subdivided in accor-
dancewiththecontrol proceduresprovidedforinnationallaw.
3. ANALYSES AND RESULTS
(a) Each of the primary samples referred to inpoin t 1 shall be subdivided
into laboratory samples, in accordance with point 2.5 of standard EN
ISO 5555, and analysed as follows:
— determination of free fatty acids, as referred to in the first indent of
Article 2(1),
— determination of the peroxide value, as referred to in the second
indent of Article 2(1),
— spectrophotometric analysis, as referred to in the eighth indent of
Article 2(1),
— determination of the fatty acid composition, as referred to in the
ninth indent of Article 2(1).
(b) Where one of the results of the analyses referred to in (a) for at least
one of the primary samples taken from the same batch does not comply
with the characteristics of the category of oil declared, the whole of the
batch concerned is to be declared not to comply.
Where the results of the analyses referred to in (a) for each of the
primary samples taken from the same batch are not all uniform, given
the repeatability characteristics of the methods concerned, the entire
batch is to be declared non-uniform and each primary sample is to be
subject to the other analysis required. Otherwise, one primary sample
from that batch is to be subject to the other analysis required.
(c) Where one of the results of the analyses referred to in the second para-
graph of point (b) does not comply with the characteristics of the
category of oil declared, the whole of the batch concerned is to be
declared not to comply.
Where all the results of the analyses referred to in the second paragraph
of point (b) comply with the characteristics of the category of oil
declared, the whole batch is to be declared to comply.1991R2568 — EN — 01.11.2003 — 020.001 — 11
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ANNEX Ib
DECISION TREE FOR VERIFYING WHETHER AN OLIVE OIL
SAMPLEISCONSISTENTWITHTHECATEGORYDECLARED
The analysis to verify whether an olive oil or olive-pomace oil is consistent with
the category declared may be undertaken:
(a) either by carrying out in any random order the analyses envisaged for the
purpose of verifying compliance with the characteristics specified in Annex
I; or
(b) by carryin g out inthe order showninthe decisiontree the an alyses speci-
fied therein until one of the decisions appearing in the decision tree is
reached.
The analyses relating to contaminants required for verifying compliance with
European Community standards are to be carried out separately.
The decisiontree applies to all categories of olive oil an d olive-pomace oil. It
consists of tables numbered 1 to 11 which must be approached on the basis of
the declared category of oil concerned in the order set out in the general table.
Key to general tables to 11:
— the double line (=) indicates the route to be followed in case of compliance
(positive answer) with the criteria specified in the preceding box. The dotted
line (…) indicates the alternative route to be followed in case of non-compli-
ance,
— the headin gs inthe boxes intables 1 to 11 refer to the an alyses provided for
inthis Regulationonthe basis of the table of equivalen ce set out in
Appendix 1 to this Annex,
— the letters inbrackets appearin g inthe n egative decisioncircles intables 1 to
11 cross-refer to indicative information given in Appendix 2 to this Annex.
The letters in themselves do not entail the obligation to pursue the analyses
or imply the veracity of the assumptions stated.1991R2568 — EN — 01.11.2003 — 020.001 — 12
▼M20
General table1991R2568 — EN — 01.11.2003 — 020.001 — 13

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Table11991R2568 — EN — 01.11.2003 — 020.001 — 14

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Table21991R2568 — EN — 01.11.2003 — 020.001 — 15

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Table31991R2568 — EN — 01.11.2003 — 020.001 — 16

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Table41991R2568 — EN — 01.11.2003 — 020.001 — 17

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Table51991R2568 — EN — 01.11.2003 — 020.001 — 18

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Table61991R2568 — EN — 01.11.2003 — 020.001 — 19

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Table71991R2568 — EN — 01.11.2003 — 020.001 — 20

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Table81991R2568 — EN — 01.11.2003 — 020.001 — 21

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Table91991R2568 — EN — 01.11.2003 — 020.001 — 22

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Table101991R2568 — EN — 01.11.2003 — 020.001 — 23

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Table111991R2568 — EN — 01.11.2003 — 020.001 — 24

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Appendix 1
Table of equivalence between the annexes to this Regulation and the analyses specified in the
decisiontree
— Acidity Annex II Determination of the free fatty acids
— Peroxide value Annex III Determination of peroxide value
— UV spectrometry Annex IX Spectrophotometric analysis
— Organoleptic assessment Annex XII Organoleptic assessment of virgin
olive oil
— 3,5-Stigmastadienes Annex XVII Method of determining stigmasta-
dienes in vegetable oils
— Trans isomers of fatty acids Annex Xa and Analysis by gas chromatography of
methyl esters of fatty acids
Annex Xb Preparation of methyl esters of fatty
acids
— Fatty acids content Annex Xa and Analysis by gas chromatography of
methyl esters of fatty acids
Annex Xb Preparation of methyl esters of fatty
acids
—∆ECN42 Annex XVIII Determination of the composition of
triglycerides with ECN42 (difference
betweenthe HPLC data an d theore-
tical content)
— Sterols compositionan d total
sterolsAnnex V Determination of the composition
and content of sterols by capillary-
columngas chromatography
— Erythrodiol and Uvaol Annex VI Determination of erythrodiol and
uvaol
— Waxes Annex IV Determination of wax content by
capillary-columngas chromatography
— Aliphatic alcohols Annex XIX Determination of aliphatic alcohols
content by capillary-column gas
chromatography
— Saturated fatty acids in 2-position Annex VII Determination of saturated fatty acids
inposition2 of the triglyceride1991R2568 — EN — 01.11.2003 — 020.001 — 25
▼M20
Appendix 2
Table1
(a) See virginor lampan te olive oil (Quality criteria Table 2, or Quality and
purity criteria Table 4)
(b) See lampante olive oil (Quality and purity criteria Table 4)
Table2
(a) See lampante olive oil (Quality and purity criteria Table 4)
(b) See extra virginolive oil (Quality criteria Table 1)
Table3
(a) Presence of refined oil (olive or others)
(b) Presence of olive-pomace oil
Table4
(a) See extra virginolive oil an d virginolive oil (Quality criteria Table 1and
Table 2)
(b) Presence of refined oil (olive or others)
(c) Presence of olive-pomace oil
(d) Presence of esterified oils
Table7
(a) Presence of olive-pomace oil
(b) Presence of esterified oils
Table8
(a) Presence of refined oil (olive or others)
(b) See lampante olive oil (Quality and purity criteria Table 4)
(c) Presence of esterified oils
Table11
(a) Presence of esterified oils1991R2568 — EN — 01.11.2003 — 020.001 — 26
▼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.)1991R2568 — EN — 01.11.2003 — 020.001 — 27
▼B
1.4.2. Taking the sample
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 0,02
4 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 ►M6of two calculations ◄
carried out.1991R2568 — EN — 01.11.2003 — 020.001 — 28
▼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 — 01.11.2003 — 020.001 — 29
▼B
Expected peroxide value
(meq)Weight of test portion
(g)
0 to 12 5,0 to 2,0
12 to 20 2,0 to 1,2
20 to 30 1,2 to 0,8
30 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 — 01.11.2003 — 020.001 — 30
▼M6
ANNEX IV
DETERMINATION OF WAX CONTENT BY CAPILLARY COLUMN
GAS-LIQUIDCHROMATOGRAPHY
1. SCOPE
This method describes a procedure for the determination of the wax
content of certain fats and oils, under the test conditions.
It may be used in particular to distinguish between olive oil obtained by
pressing and that obtained by extraction (olive-pomace oil).
2. PRINCIPLE
Addition of a suitable internal standard to the fat or oil, then fractiona-
tionby chromatography ona hydrated silica gel column . Recovery of
the fraction eluted first under the test conditions (whose polarity is
less thanthat of the triglycerides), thendirect an alysis by capillary
columngas-liquid chromatography.
3. APPARATUS
3.1. 25-ml Erlenmeyer flask.
3.2. Glass column for chromatography, 15 mm internal diameter and 30-40
cm long.
3.3. Suitable gas-liquid chromatograph with a capillary column, equipped
with a system for direct introduction into the column comprising the
following:
3.3.1 Thermostat-controlled oven for the columns, capable of maintaining the
desired temperature to within1 ºC.
3.3.2. Cold injector for direct introduction into the column.3.3.3. Flame-ionization detector and converter-amplifier.
3.3.4. Recorder-integrator capable of working with the converter-amplifier
(3.3.3.), rate of response below 1 second, with variable paper speed.
3.3.5. Capillary column, glass or fused silico, 10 to 15 m long 0,25 to 0,32
mm internal diameter, internally covered with SE-52 or SE-54 liquid,
or equivalents, to a uniform thickness of 0.10 to 0,30 µm.
3.4. Microsyringe with facilities for on-column injection capacity 10 µl,
equipped with a casehardened needle.
4. REAGENTS
4.1. Silica gel, 70/230 mesh, article 7754 Merck.
Place the gel inthe ovenat 500 ºC for four hours. Allow to cool, then
add 2 % water. Shake well the homogenize slurry. Keep in darkness for
at least 12 hours prior to use.
4.2. n-hexane, for chromatography.
4.3. Ethyl ether, for chromatography.
4.4. n-heptane, for chromatography.4.5. Standard solution of lauryl arachidate, at 0,1 % (m/v) in hexane
(internal standard).
4.6. Carrier gas: hydrogen, pure, for gas-liquid chromatography.
4.7. Auxiliary gases:
— hydrogen, pure, for gas-liquid chromatography,
— air, pure, for gas-liquid chromatography.
5. PROCEDURE
5.1. Separationof the wax friction .
5.1.1. Preparationof the chromatographic column .
Suspend 15 g of silica gel hydrated at 2 % in anhydrous n-hexane and
introduce into the column.1991R2568 — EN — 01.11.2003 — 020.001 — 31
▼M6
Allow to settle spontaneously. Complete settling with the aid of an
electric shaker to make the chromatographic band more homogeneous.
Percolate 30-ml n-hexane to remove any impurities.
5.1.2. Columnchromatography
Weigh exactly 500 mg of the sample into a 25-ml flask, and add a
suitable amount of internal standard, depending on the assumed wax
content, e.g. add 0,1 mg lauryl arachidate in the case of olive oil, and
0,25 to 0,5 mg inthe case of olive-pomace oil.
Transfer the prepared sample to the chromatographic column, prepared
according to 5.1., with the aid of two 2-ml portions of n-hexane.
Allow the solvent to flow to 1 mm above the upper level of the absor-
bent. Then start chromatographic elution; collect 140 ml of the n-
hexane/ethyl ether mixture, at 99:1, at a flow of about 15 drops every
10 seconds (2,1 ml/minute).
Dry the resultant fraction in a rotary evaporator until almost all the
solvent is eliminated. Remove the last 2 or 3 ml of solvent with the
help of a weak current of nitrogen, then add 10 ml n-heptane.
5.2. Gas-liquid chromatographic analysis5.2.1. Preliminary procedure, conditioning of column.
5.2.1.1. Fit the column to the gas-liquid chromatograph, connecting the inlet
port to the on-column system and the outlet port to the detector.
Check the gas-liquid chromatography apparatus (operationof gas loops,
detector and recorder efficiency, etc.).
5.2.1.2. If the columns is being used for the first time, it is advisable to condi-
tionit. Runa light flow of gas through the column , thenswitch onthe
gas-liquid chromatography apparatus. Gradually heat to a temperature
at least 20 ºC above the operating temperature (note). Maintain this
temperature for at least two hours, thenregulate the apparatus to the
operating conditions (regulate gas flow, light flame, connect to elec-
tronic recorder, regulate oven temperature for column, regulate
detector, etc.). Record the signal at a sensitivity at least twice as high
as that required to perform the analysis. The base-line should be linear,
with no peaks of any kind, and must have any deviation.
A negative rectilinear deviation indicates that the column connections
are not correct; a positive deviation indicates that the column has not
been properly conditioned.
Note:Keep the conditioning temperature at all times at least 20 ºC
below the maximum temperature specified for the eluent
employed.
5.2.2. Choice of operating conditions.
5.2.2.1. The operating conditions are generally as follows:
— column temperature: 80 ºC at first, rising by 30 ºC/minute to 120
ºC, then programmed to increase by 5 ºC/minute up to 340 ºC,
— detector temperature: 350 ºC,
— linear speed of carrier gas: hydrogen, 20 to 35 cm/sec,
— instrument sensitivity: 4 to 16 times the minimum attenuation,
— Recorder sensitivity: 1 to 2 mV, from bottom of scale,
— paper speed: 30 cm/hour,
— amount injected: 0,5-1 µl solution.
These conditions may be modified to suit the characteristics of the
columnan d the gas-liquid chromatographic apparatus (inorder to
obtain chromatgrams meeting the following conditions: retention time
of C32 internal standard must be 25 ± 2 minutes and the most represen-
tative peak of the waxes must lie between60 an d 100 % from the
bottom of the scale).
5.2.2.2. Determine the peak integration parameters in such a way as to obtain a
correct evaluationof the peak areas con sidered.
5.2.3. Performance of the analysis
5.2.3.1. Take up 1 µl of the solutionwith the aid of the 10 µl micry-syringe;
draw back the piston until the needle is empty. Introduce the needle in
the injection system and inject quickly after one to two seconds. After
about five seconds, gently extract the needle.1991R2568 — EN — 01.11.2003 — 020.001 — 32
▼M6
5.2.3.2. Perform the recording until the waxes are completely eluted.
The base-line must always satisfy the required conditions (5.2.1.2).
5.2.4. Peak identification
Identify the peaks from the retention times, by comparing them with
mixtures of waxes with known retention times, analysed under the
same conditions.
Figure 1 gives a wax chromatogram of a virginolive oil.
5.2.5. Quantitative analysis5.2.5.1. Determine the areas of the peaks corresponding to the internal standard
and the aliphatic esters from C40 to C46 with the aid of the integrator.
5.2.5.2. Determine the wax content of each of the esters, in mg/kg of fat,
according to the formula:
ester (mg/kg) =A
x·ms·►M91 000◄
As·m
where:
Ax= area of the peak of each ester;
As= area of the lauryl arachidate peak;
ms= mass of the lauryl arachidate added, inmilligram;
m = mass of the sample taken for determination, in grams.
6. EXPRESSION OF THE RESULTS
Give the different wax contents, and the sum of those contents, in mg/
kg of fat.1991R2568 — EN — 01.11.2003 — 020.001 — 33
▼M6
APPENDIX
Determination of linear gas speed
Inject 1 to 3 µl methane (propane) into the gas-liquid chromatographic appa-
ratus, after adjusting it to the normal operating conditions. Measure the time
the gas takes to run through the column, from the moment it is injected until
the peak emerges (tM).
The linear speed in cm/sec. is given by the formula L/tM where L is the length
of the column, in cm, and tM is the time measured in seconds.1991R2568 — EN — 01.11.2003 — 020.001 — 34
▼M6
1991R2568 — EN — 01.11.2003 — 020.001 — 35
▼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. Potassium hydroxide, ►
C1approximately 2 mol/L ethanolic
solution. ◄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-stop-
pered 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 — 01.11.2003 — 020.001 — 36
▼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, ►
M62%◄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 solution in the case of an olive oil and 1 500 µl
for►M6 ◄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.
►M6Oils◄containing appreciable quantities of cholesterol may
show a peak having a retention time identical to cholestanol. If this
occurs the sterol fractionwill have to be an alyzed induplicate with
and without internal standard ►M6or betulinol will have to be used
instead of cholestanol ◄.
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 waterbath 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 — 01.11.2003 — 020.001 — 37
▼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 — 01.11.2003 — 020.001 — 38
▼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 — 01.11.2003 — 020.001 — 39
▼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 ►M6 ◄;
As= area of the ►C1β-cholestanol ◄peak
►M6 ◄;
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 — 01.11.2003 — 020.001 — 40
▼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
Peak IdentificationRelative 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-
ol0,73 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-
ol0,95 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-
ol1,08 1,08
15 ∆-7-stigmastenol [24R,S]-24-Ethyl- ∆-7,24-cholestadien-3 β-
ol1,12 1,12
16 ∆-7-avenasterol [24Z]-24-ethyliden- ∆-7-cholesten-3 β-ol 1,16 1,161991R2568 — EN — 01.11.2003 — 020.001 — 41
▼B
Figure1
Gas chromatogram of the sterol fraction of an unrefined olive oil
1991R2568 — EN — 01.11.2003 — 020.001 — 42
▼B
Figure2
Gas chromatogram of the sterol fraction of a refined olive oil
1991R2568 — EN — 01.11.2003 — 020.001 — 43
▼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 — 01.11.2003 — 020.001 — 44
▼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 ►M6 ◄;
A2= peak area for uvaol ►M6 ◄;
ΣAsterols= total peak area for sterols ►M6 ◄.
The result is expressed to one decimal place.1991R2568 — EN — 01.11.2003 — 020.001 — 45
▼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 — 01.11.2003 — 020.001 — 46
▼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 — 01.11.2003 — 020.001 — 47
▼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 — 01.11.2003 — 020.001 — 48
▼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 — 01.11.2003 — 020.001 — 49
▼M20
▼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 an upper part 270 mm in length and a
diameter of 35 mm and a lower part 270 mm in length and a diameter of
approximately 10mm.
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.
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.1991R2568 — EN — 01.11.2003 — 020.001 — 50
▼M6
▼B
▼M6
▼B
▼B
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.
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 — 01.11.2003 — 020.001 — 51
▼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 — 01.11.2003 — 020.001 — 52
▼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 — 01.11.2003 — 020.001 — 53
▼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 — 01.11.2003 — 020.001 — 54
▼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 — 01.11.2003 — 020.001 — 55
▼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 — 01.11.2003 — 020.001 — 56
▼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 — 01.11.2003 — 020.001 — 57
▼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 — 01.11.2003 — 020.001 — 58
▼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 calculation5.2.2.1. 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 — 01.11.2003 — 020.001 — 59
▼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.1991R2568 — EN — 01.11.2003 — 020.001 — 60
▼M2
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.
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 than2.1991R2568 — EN — 01.11.2003 — 020.001 — 61
▼M2
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;
— 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 — 01.11.2003 — 020.001 — 62
▼B
▼M19
ANNEX X B
PREPARATION OF THE FATTY ACID METHYL ESTERS FROM
OLIVEOILANDOLIVE-POMACEOIL
The following two methods are recommended for preparing the fatty acid
methyl esters from olive oils and olive-pomace oils:
Method A: Trans-esterification with cold methanolic solution of potassium
hydroxide
Method B: Methylation by heating with sodium methylate in methanol
followed by esterificationinacid medium.
Each method will be applied according to the analytical parameter to be deter-
mined and the oil category as indicated below:
(a) determination of difference between actual and theoretical content of trigly-
cerides with ECN42 ( ∆ECN42):
— method A will be applied to samples of all the oil categories after puri-
fication of the oil by passing it through a silica gel column;
(b) determination of the fatty acid composition:
— method A will be applied directly to samples of the following oil cate-
gories:
— virginolive oils with anacidity of less than3,3 %,
— refined olive oil,
— olive oil (blend of virgin olive oils and refined olive oil),
— refined olive-pomace oil,— olive-pomace oil (blend of virgin olive oils and refined olive-pomace
oil);
— method B will be applied directly to samples of the following oil cate-
gories:
— virginolive oil with anacidity of more than3,3 %,
— crude olive-pomace oil;
(c) determination of trans-isomers of fatty acids:
— method A will be applied directly to samples of the following oil cate-
gories:
— virginolive oils with anacidity of less than3,3 %,
— refined olive oil,
— olive oil (blend of virgin olive oils and refined olive oil),
— refined olive-pomace oil,
— olive-pomace oil (blend of virgin olive oils and refined olive-pomace
oil);
— method A will be applied to the following categories of oils after purifi-
cation of the oil by passing it through a silica gel column:
— virginolive oil with anacidity of more than3,3 %,
— crude olive-pomace oil.
PURIFICATION OF OIL SAMPLES
When necessary, the samples will be purified by passing the oil through a silica
gel column, eluting with hexane/diethyl ether (87:13, v/v) as described in
IUPAC method 2.507.
Alternatively, solid-phase extraction on silica gel phase cartridges can be used.
A silica gel cartridge (1 g, 6 ml) is placed ina vacuum elutionapparatus an d
washed with 6 ml of hexane. The vacuum is released to prevent the column
from becoming dry and then a solution of the oil (0,12 g approximately) in
0,5 ml of hexane is loaded into the column and vacuum is applied. The solution
is pulled down and then eluted with 10 ml of hexane/diethyl ether (87:13 v/v)
under vacuum. The combined eluates are homogenised and divided in two
similar volumes. Analiquot is evaporated to dryn ess ina rotary evaporator
under reduced pressure at room temperature. The pomace is dissolved in 1 ml
of heptane and the solution is ready for fatty acid analysis by GC. The second
aliquot is evaporated and the pomace is dissolved in 1 ml of acetone for trigly-
ceride analysis by HPLC, if necessary.1991R2568 — EN — 01.11.2003 — 020.001 — 63
▼M19
METHODS FOR PREPARING THE FATTY ACID METHYL ESTERS
1. Method A: Trans-esterification with cold methanolic solution of
potassium hydroxide
1.1.Purpose
This rapid method is applicable to olive oils and olive-pomace oils with
a free fatty acid content of less than 3,3 %. Free fatty acids are not
esterified by potassium hydroxide. Fatty acid ethyl esters are trans-
esterified at a lower rate than glyceridic esters and may be only
partially methylated.
1.2.Principle
Methyl esters are formed by trans-esterification with methanolic potas-
sium hydroxide as an intermediate stage before saponification takes
place (title 5 inISO-5509:2000, title 5 inIUPAC method 2.301).
1.3.Reagents
Methanol containing not more than 0,5 % (m/m) water.Heptane, chromatographic quality.Potassium hydroxide, approximately 2 N methanolic solution: dissolve
11,2 g of potassium hydroxide in100 ml of methan ol.
1.4.Apparatus
Screw-top test tubes (5 ml volume) with cap fitted with a PTFE joint.Graduated or automatic pipettes, 2 ml and 0,2 ml
1.5.Procedure
Ina 5 ml screw-top test tube weigh approximately 0,1 g of the oil
sample. Add 2 ml of heptane, and shake. Add 0,2 ml of 2 N methanolic
potassium hydroxide solution, put on the cap fitted with a PTFE joint,
tighten the cap, and shake vigorously for 30 seconds. Leave to stratify
until the upper solution becomes clear. Decant the upper layer
containing the methyl esters. The heptane solution is suitable for injec-
tionin to the gas chromatograph. It is advisable to keep the solutionin
the refrigerator until gas chromatographic analysis. Storage of the solu-
tion for more than 12 hours is not recommended.
2. Method B: Methylation by heating with sodium methylate in methanol
followed by esterification in acid medium
2.1.Purpose
This method is applicable to olive oils and olive-pomace oils with a
free fatty acid content of more than 3,3 %.
2.2.Principle
Neutralisation of the free fatty acids and alkaline methanolysis of the
glycerides, followed by esterificationof the fatty acids inacid medium
(title 4.2. inIUPAC method 2.301).
2.3.Reagents
— heptane, chromatographic quality,
— methanol containing not more than 0,05 % (m/m) water,
— sodium methylate, 0,2 N methanolic solution: dissolve 5 g of
sodium in1 000 ml of methan ol (this may be prepared from
commercial solutions),
— phenolphthalein, 0,2 % methanolic solution,— sulphuric acid, 1 N in methanolic solution: add 3 ml of 96 %
sulphuric acid to 100 ml of methanol,
— saturated solutionof sodium chloride inwater.
2.4.Apparatus
— 50 ml flat-bottomed volumetric flask with long, narrow, ground
neck,1991R2568 — EN — 01.11.2003 — 020.001 — 64
▼M19
— reflux condenser: air condenser (1 m long) with ground joint appro-
priate to the neck of the flask,
— boiling chips,
— glass funnel.
2.5.Procedure
Transfer about 0,25 g of the oil sample into a 50 ml ground-necked
volumetric flask. With the aid of a funnel, add 10 ml of 0,2 N sodium
methylate in methanol and the boiling chips. Fit a reflux condenser,
shake, and bring to the boil. The solution should become clear, which
usually occurs inabout 10 min utes. The reactionis complete after 15
minutes. Remove the flask from the source of heat, wait until the reflux
stops, remove the condenser, and add two drops of phenolphthalein
solution. Add a few ml of 1 N sulphuric acid in methanol solution until
the solutionbecomes colourless an d thenadd 1 ml inexcess. Fit the
condenser and boil again for 20 minutes. Withdraw from the source of
heat and cool the flask under running water. Remove the condenser,
add 20 ml of saturated sodium chloride solution, and shake. Add 5 ml
of heptane, plug the flask, and shake vigorously for 15 seconds.
Leave to settle until the two phases have separated. Add saturated
sodium chloride solutionagainun til the aqueous layer reaches the
lower end of the flask neck. The upper layer containing the methyl
esters fills the flask neck. This solution is ready to be injected in the
GC.
Caution: Methylation by method B must be done under a hood.
2.6.AlternativestomethylationMethodB
2.6.1.Method C
2.6.1.1. Principle
The fatty matter undergoing analysis is treated with methanol-hydro-
chloric acid, ina sealed vial, at 100 ºC.
2.6.1.2. Apparatus
— Strong glass vial of a capacity of about 5 ml (height 40 to 45 mm,
diameter 14 to 16 mm).
— 1 and 2 ml graduated pipettes.
2.6.1.3. Reagents
Solutionof hydrochloric acid in2 % methan ol. This is prepared from
gaseous hydrochloric acid and anhydrous methanol (Note 1).
Hexane, chromatographic quality.
Note 1:Commercial solutions of hydrogen chloride in methanol can be
used. 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. Since hydrochloric acid is very rapidly absorbed
by methanol, it is advisable to take the usual precautions when
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. Alternatively, this reagent can be
prepared by dissolutionof acetyl chloride inan hydrous
methanol.
2.6.1.4. Procedure
— Place inthe glass vial 0,2 g of the fatty matter, which has
previously been dried out on sodium sulphate and filtered, and 2 ml
of hydrochloric acid-methanol solution. Heat seal the vial.
— Immerse the vial at 100 ºC for 40 minutes.
— Cool the vial 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.1991R2568 — EN — 01.11.2003 — 020.001 — 65
▼M19
2.6.2.Method D
2.6.2.1. Principle
The fatty matter undergoing analysis is heated under reflux with
methanol-hexane-sulphuric acid. The methyl esters obtained are
extracted with petroleum ether.
2.6.2.2. Apparatus
— Test tube of a capacity of about 20 ml, fitted with anair reflux
condenser approximately 1 m in length, with ground glass joints.
— 5 ml graduated pipette.— 50 ml separating funnel.
— 10 ml and 25 ml measuring beakers.
— 15 ml test tube with conical base.
2.6.2.3. Reagents
— Methylation reagent: anhydrous methanol-hexane-concentrated
sulphuric acid (p = 1,84) inthe ratio 75:25:1 (V/V/V).
— 40 to 60 ºC petroleum ether.
— Anhydrous sodium sulphate.
2.6.2.4. Procedure
Place 0,1 g of oil inthe 20 ml test tube an d add 5 ml of methylation
reagent.
Fit the reflux condenser and heat for 30 minutes in a boiling water bath
(Note 2).
Transfer quantitatively the mixture into a 50 ml separating funnel, with
the aid of 10 ml distilled water and 10 ml petroleum ether. Shake vigor-
ously, and allow the phases to separate, remove the aqueous phase and
wash the ether layer twice with 20 ml distilled water. Add to the separ-
ating 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 2:To control boiling, insert a glass rod into the test tube and limit
the temperature of the water bath to 90 ºC.
3. Precision parameters
The statistical evaluationof the precisionof methods A an d B was
published by the International Olive Oil Council in its method COI/
T.20/CO. No 24.
RECOMMENDATIONS FOR GAS CHROMATOGRAPHIC ANALYSIS OF
THE FATTY ACID ESTERS FROM OLIVE OIL AND OLIVE-POMACE OIL
1. Procedure
The gas chromatographic analysis of solutions of fatty esters in heptane
is to be carried out according to standard ISO-5508 using a capillary
column (50 m length x 0,25 or 0,32 mm i.d.) impregnated with cyano-
propylsilicone phase as indicated for the determination of fatty acid
trans-isomers (COI/T.20/Doc. no. 17).
Figure 1 gives the typical gas chromatographic profile of anolive-
pomace oil containing methyl and ethyl esters of fatty acids, and
trans-isomers of methyl esters.
2. Calculations
2.1. For the calculationof the fatty acid compositionan d ∆ECN42, all the
following fatty acids will be taken into account:
Myristic (C14:0).
Palmitic (C16:0). Sum of the areas of the peaks corresponding to the
methyl and ethyl esters.
Palmitoleic (C16:1). Sum of the areas of the peaks corresponding to the
ω9andω7isomers of the methyl ester.
Margaric (C17:0).Margaroleic (C17:1).1991R2568 — EN — 01.11.2003 — 020.001 — 66
▼M19
Stearic (C18:0).
Oleic (C18:1). Sum of the areas of the peaks corresponding to the ω9
andω7isomers of the methyl ester, ethyl ester, and trans-isomers of the
methyl ester.
Linoleic (C18:2). Sum of the areas of the peaks corresponding to the
methyl and ethyl esters, and the trans-isomers of the methyl ester.
Arachidic (C20:0).Linolenic (C18:3). Sum of the areas of the methyl ester and the trans-
isomers of the methyl ester.
Eicosenoic (C20:1).Behenic (C22:0).
Lignoceric (C24:0).
Squalene will not be taken into account for the calculation of the total
area.
2.2. For the calculation of the percentage of trans-C18:1 the peak corre-
sponding to the methyl esters of this fatty acid is to be used. For the
sum [trans-C18:2 + trans-C18:3], all the peaks corresponding to the
trans-isomers of these two fatty acids are to be added together. For the
calculation of the total area, all the peaks mentioned in 2.1. are to be
taken into account (see COI/T.20/Doc. No. 17).
The calculationof the percen tage of each fatty acid will be carried out
according to the formula:
% X = (Area X x 100)/(total area)
Figure 1: Gas chromatographic profile obtained by the cold methylation
method from olive-pomace oil. The chromatographic peaks correspond to the
methyl and ethyl esters except where otherwise indicated.1991R2568 — EN — 01.11.2003 — 020.001 — 67
▼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 — 01.11.2003 — 020.001 — 68
▼M19
ANNEX XII
ORGANOLEPTICASSESSMENTOFVIRGINOLIVEOILS
1. PURPOSE AND SCOPE
This Annex sets the criteria required for organoleptic assessment of the
virgin oils defined at 1 in the Annex to Regulation No 136/66/EEC and
describes the method of grading them with reference to characteristics.
This method can be used only for grading virgin oils on the basis of fruiti-
ness and intensity of defects by a group of selected trained tasters operating
as a panel in line with section 4.
2. GENERAL
For the general basic vocabulary, the tasting room, the general metho-
dology and the tasting glass compliance with the stipulations of the
International Olive Oil Council is recommended.
3. SPECIFIC VOCABULARY
3.1.Positive attributes
Fruity: range of smells (dependent on variety) characteristic of oil from
healthy fresh fruit, greenor white, perceived directly or retron asally.
Bitter: characteristic taste of oil from green olives or olives turning colour.
Pungent: tingling sensation characteristic of oil made at the beginning of
the season mainly from olives that are still green.
3.2.Negativeattributes
‘Atrojado’ (fusty ): characteristic flavour of oil from piled olives in
advanced anaerobic fermentation.
Mustiness/humidity : characteristic flavour of oil from olives inwhich large
numbers of fungi and yeasts had developed as a result of storage for several
days in humid conditions.
Muddy sediment : characteristic flavour of oil that has remained in contact
with sediment in vats and tanks.
Winey/vinegary : characteristic flavour of certain oils reminiscent of wine or
vinegar, due basically to formation of acetic acid, ethyl acetate and ethanol
by fermentation of the olives.
Metallic: flavour reminiscent of metal, characteristic of oil that has been in
prolonged contact with metal surfaces during crushing, mixing, pressing or
storage.
Rancid: flavour of oil that has become oxidised.
Heated or burnt : characteristic flavour caused by excessive and/or
prolonged heating during production, particularly by thermo-mixing of the
paste in unsuitable conditions.
Hay/wood : characteristic flavour of certainoils from dry olives.
Rough: thick and pasty mouthfeel produced by some oils.
Greasy: flavour reminiscent of diesel, grease or mineral oil.
Vegetable water : flavour acquired by oil through prolonged contact with
the vegetable water.
Brine: flavour of oil from olives preserved insalt solution .
Esparto: characteristic flavour of oil from olives pressed inn ew esparto
mats. It canvary accordin g to whether the mats are of greenor dried
esparto.
Earthy: flavour of oil from olives collected with earth or mud onthem an d
not washed.
Grubby: flavour of oil from olives heavily attacked by grubs of the olive
fly (Bactrocera oleae).
Cucumber : characteristic flavour of oil kept too long in hermetically sealed
containers, notably in tins, attributed to formation of 2,6-nonadienal.1991R2568 — EN — 01.11.2003 — 020.001 — 69
▼M19
4. PANEL
The panel is appointed by the Member State and consists of a panel head
and from eight to twelve tasters. However, for the 2001/02 marketing year,
the panel may consist of fewer than eight tasters.
The panel head must be a soundly trained expert in the various types of oil.
He or she is responsible for the panel and its organisation and operation,
including preparation, coding and presentation of the samples to the tasters
and collection and processing of the data.
He or she selects the testers, sees to their training and checks that their
performance remains of adequate standard.
The testers must be selected and trained on account of their skill in distin-
guishing between similar samples. The International Olive Oil Council's
manual on the selection, training and monitoring of qualified virgin oil
tasters must be followed.
Panels must undertake to participate in national, Community and interna-
tional organoleptic assessments organised for the purposes of periodic
monitoring and harmonisation of perception criteria. They must also
provide the Member State concerned with full information each year on
the composition of the panel and the number of assessments made in their
capacity as anapproved pan el.
5. PROCEDURE FOR ORGANOLEPTIC ASSESSMENT AND GRADING
5.1.Useofprofilesheetbytaster
The profile sheet to be used by the taster is reproduced as Appendix A.Tasters must each smell and then taste (
1) the oil submitted for examination
contained in the tasting glass, analysing their olfactory, gustatory, tactile
and kinaesthetic perceptions and mark on the sheet the intensity of their
perception of each negative and positive attribute.
If negative attributes not listed on the profile sheet are perceived these must
be noted under ‘Other’ using those of the terms defined in 3.2 above that
best describe them.
5.2.Processingofdatabypanelhead
The panel head collects the profile sheets completed by the tasters and
scrutinises the intensities assigned. In the event of an anomaly he or she
will ask tasters to re-examine their sheet and if necessary repeat the test.
The panel head may feed each tester's data into a computer programme for
calculating the median (Appendix B). Input of each sample shall be made
with the help of a grid of 10 vertical columns for the 10 sensory attributes
and one line for each panel member.
If a negative attribute is mentioned under ‘Other’ by 50 % of the panel the
head must calculate the median for this attribute and grade accordingly.
In cases of assessment in connection with monitoring of conformity to stan-
dards and of counter-assessment the panel head shall arrange for the
assessment to be repeated twice at intervals of at least one day. The attri-
bute medians shall be calculated using the data from the profile sheets of
the three assessments.
5.3.Gradingofoils
The oil is graded as follows in line with the median of the defects and the
medianfor ‘fruity’. By this is un derstood the medianof the n egative attri-
bute perceived with greatest intensity. The value of the robust variation
coefficient for this negative attribute must be no greater than 20 %.
(a)extra virgin olive oil : the medianof the defects is 0 an d the medianfor
‘fruity’ is above 0;
(b)virgin olive oil : the medianof the defects is above 0 but n ot above 2,5
and the median for ‘fruity’ is above 0;
(c)ordinary virgin olive oil : the medianof the defects is above 2,5 but n ot
above 6,0; or the median of the defects is not above 2,5 and the median
for ‘fruity’ is 0;1991R2568 — EN — 01.11.2003 — 020.001 — 70
(1) But may refrain from tasting if they note some extremely intense negative attributes;
they will note this exceptional circumstance on the profile sheet.
▼M19
(d)lampante virgin olive oil : the medianof the defects is above 6,0.
From 1 November 2003 categories c) and d) are replaced by:
(c)lampante olive oil : the medianof the defects is above 2,5; or the
median of the defects is not above 2,5 and the median for ‘fruity’ is 0.
5.4.Specialcase
If the medianof a positive attribute other than‘fruity’ is above 5,0 the
panel head must note this on the analysis certificate.1991R2568 — EN — 01.11.2003 — 020.001 — 71
▼M19
APPENDIX A
1991R2568 — EN — 01.11.2003 — 020.001 — 72
▼M19
APPENDIX B
METHOD OF CALCULATING MEDIAN AND CONFIDENCE INTERVALS
Median
The medianis the real n umber Xm characterised by the fact that the probability
(P) that the values of the distribution (X) are below that number (Xm) is not
more than 0,5 and that simultaneously the probability (P) that the values of the
distribution (X) are not above Xm is not less than 0,5. Another definition
considers the median to be the 50th percentile of a distribution of numbers
ranked in order of increase. In other terms the median represents the central
value of an ordered series of uneven numbers or the average of the two central
values of anordered series of evenn umbers.
Robuststandarddeviation
To obtaina reliable estimate of the variability that arises aroun d the median
recourse is required to the Stuart and Kendall method of estimating the robust
standard deviation. The formula for the asymptotic standard deviation S involves
N and IQR. N is the number of observations and IQR the interquartile range, i.e.
the robust estimate of the variability of the data under consideration (the inter-
quartile range covers exactly 50 % of the cases of any probability distribution).
The interquartile range is given by calculating the size of the deviation between
the 75th and the 25th percentiles.
IQR = 75th percentile — 25th percentile
The percentile is the value Xpc characterised by the fact that the probability (P)
that the valves of the distribution are below Xpc is not more than a determined
hundredth and that simultaneously the probability (P) that the valves of the
distribution are not above Xpc is not less than the said hundredth. The
hundredth indicates the distribution fraction used. In the case of the median
this is 50/100.
In other words the percentile is the distribution value corresponding to a deter-mined area plotted from the distribution or density curve. For example, the 25th
percentile represents the distribution value corresponding to an area equal to
0,25 or 25/100.
Robustvariationcoefficient %
The RVC represents a pure number, i.e. without dimension, that indicates the
percentage of variability of the series of numbers analysed against the Av value
of the median. For that reason it is very useful for verifying the reliability of the
panel members.
Confidenceintervalsat95%onthemedian
The confidence intervals (C.I.) at 95 % (value of the error of first kind equal to
0,05 or 5 %) represent the range in which the value of the median would be able1991R2568 — EN — 01.11.2003 — 020.001 — 73
▼M19
to vary should it be possible to repeat the experiment an infinite number of
times. In practice this interval indicates the range of variability of the test under
the operating conditions selected should it be possible to repeat the test several
times. The interval helps evaluate, as in the case of the RVC, the reliability of
the test.
Upper C.I. = Av + (c.S)
Lower C.I = Av - (c.S)
where c in the case of a confidence interval of 0,95 is equal to 1,96.
Grading is effected by comparing the median values with the reference ranges
set insection5.3. The software package permits a visualised gradin g ona table
of the statistics or ona graph.1991R2568 — EN — 01.11.2003 — 020.001 — 74
▼M20
▼M19
▼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 — 01.11.2003 — 020.001 — 75
▼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.
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 — 01.11.2003 — 020.001 — 76
▼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.
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 0,201991R2568 — EN — 01.11.2003 — 020.001 — 77
▼B
Expected iodine valueMass of test portion
(g)
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 — 01.11.2003 — 020.001 — 78
▼M11
ANNEX XVII
METHOD FORTHE DETER MINATION OF STIGMASTADIENES IN
VEGETABLEOILS
1. PURPOSE
Determination of stigmastadienes in vegetable oils containing low
concentrations of these hydrocarbons, particularly in virgin olive oil and
crude olive-residue oil.
2. SCOPE
The standard may be applied to all vegetable oils although measurements
are reliable only where the content of these hydrocarbons lies between
0,01 and 4,0 mg/kg. The method is particularly suited to detecting the
presence of refined vegetable oils (olive, olive residue, sunflower, palm,
etc.) in virgin olive oil since refined oils contained stigmastadienes andvirginoils do n ot.
3. PRINCIPLE
Isolation of unsaponifiable matter. Separation of steroidal hydrocarbon
fraction by column chromatography on silica gel and analysis by capillary
gas chromatography.
4. APPARATUS
4.1. 250 ml flasks suitable for use with a reflux condenser.
4.2. Separating funnels of 500 ml capacity.
4.3. 100 ml round-bottom flasks.
4.4. Rotary evaporator.
4.5. Glass chromatography column (1,5 to 2,0 cm internal diameter by 50 cm
length) with Teflon tap and a plug of glass wool fibre or sintered glass
disc at the bottom. To prepare silica gel column, pour hexane into the
chromatography columnto a depth of approximately 5 cm an d thenfill
with a slurry of silica gel inhexan e (15 g in40 ml) with the help of
hexane portions. Allow to settle and finish settling by applying slight
vibration. Add anhydrous sodium sulphate to a height of approximately
0,5 cm, finally elute the excess hexane.
4.6. Gas chromatograph with flame ionization detector, split or cold on-
column injector and oven programmable to within ± 1 ºC.
4.7. Fused silica capillary columnfor gas chromatography (0,25 or 0,32 mm
internal diameter by 25 m length) coated with 5 %-phenylmethylsilicone
phase, 0,25 mm film thickness.
Note 1:
Other columns of similar or lower polarity can be used.
4.8. Integrator-recorder with possibility of valley-valley integration mode.
4.9. 5 to 10 ml microsyringe for gas chromatography with cemented needle.
4.10. Electrical heating mantle or hot place.
5. REAGENTS
All reagents should be of analytical grade unless otherwise specified. The
water used should be distilled water, or water of at least equivalent
purity.
5.1. Hexane or mixture of alkanes of bp interval 65 to 70 ºC, distilled with
rectifying column.
Note 2:
The solvent must be distilled to remove impurities.
5.2. 96 v/v ethanol.
5.3. Anhydrous sodium sulphate.5.4. Alcoholic potassium hydroxide solutionat 10 %. Add 10 ml of water to
50 g potassium hydroxide, stir, and then dissolve the mixture in ethanol
to 500 ml.1991R2568 — EN — 01.11.2003 — 020.001 — 79
▼M11
Note 3:
Alcoholic potash turns brown on standing. It should be prepared freshly
each day and kept in well stoppered dark glass bottles.
5.5. Silica gel 60 for columnchromatography, 70 to 230 mesh, (Merck, refer-
ence 7734 or similar).
Note 4:
Usually, silica gel can be used directly from the container without any
treatment. However, some batches of silica gel may show low activity
resulting in bad chromatographic separations. Under this circumstance,
the silica gel should be treated inthe followin g way: Activate the silica
gel by heating for a minimum of four hours at 550 ºC. After heating,
place the silica gel in a desiccator while the gel is cooling and then
transfer the silica gel to a stoppered flask. Add 2 % of water and shake
until no lumps can be seen and the powder flows freely.
If batches of silica gel result in chromatograms with interfering peaks, the
silica gel should be treated as above. Analtern ative could be the use of
extra pure silica gel 60 (Merck, reference 7754).
5.6. Stock solution(200 ppm) of cholesta-3,5-dien e (Sigma, 99 % purity) in
hexane (10 mg in 50 ml).
5.7. Standard solution of cholesta-3,5-diene hexane at concentration of
20 ppm, obtained by dilution of above solution.
Note 5:
The solutions 5.6 and 5.7 are stable for a period of at least four months if
kept at less than4 ºC.
5.8. Solution of n-nonacosane in hexane at concentration of approximately
100 ppm.
5.9. Carrier gas for chromatography: helium or hydrogenof 99,9990 % purity.
5.10. Auxiliary gases for flame ionization detector: hydrogen of 99,9990 %
purity and purified air.
6. PROCEDURE6.1.Preparationofunsaponifiablematter
6.1.1. Weigh 20 ± 0,1 g of oil into a 250-ml flask (4.1), add 1 ml of the stan-
dard solutionof cholesta-3,5-dien e (20 µg) and 75 ml of alcoholic potash
at 10 %, fit reflux condenser, and heat to slight boiling for 30 minutes,
Remove the flask containing the sample from the heat and allow the solu-
tionto cool slightly (do n ot allow to cool completely as the sample will
set). Add 100 ml of water and transfer the solution to a separating funnel
(4.2) with the aid of 100 ml of hexane. Shake the mixture vigorously for
30 seconds and allow the separate.
Note 6:
If anemulsionis produced which does n ot rapidly disappear, add small
quantities of ethanol.
6.1.2. Transfer the aqueous phase beneath to a second separating funnel and
extract again with 100 ml of hexane. Once more run off the lower phase
and wash the hexane extracts (combined in another separating funnel)
three times with 100 ml each time of a mixture of ethanol-water (1: 1)
until neutral pH is reached.
6.1.3. Pass the hexane solution through anhydrous sodium sulphate (50 g), wash
with 20 ml hexane and evaporate in a rotary evaporator at 30 ºC under
reduced pressure until dryness.
6.2.Separationofsteroidalhydrocarbonfraction
6.2.1. Take the residue to the fractioning column with the aid of two 1-ml
portions of hexane, run the sample onto the column by allowing the solu-
tionlevel to drop to the top of the sodium sulphate an d start the
chromatographic elutionwith hexan e at a flow rate of 1 ml/minapproxi-
mately. Discard the first 25 to 30 ml of eluate and then collect the
following 40 ml fraction. After collection, transfer this fraction to a 100-
ml round bottomed flask (4.3).
Note 7:
The first fraction contains saturated hydrocarbons (Figure 1 a) and the
second fraction the steroidal ones. Further elution provides squalene and1991R2568 — EN — 01.11.2003 — 020.001 — 80
▼M11
related compounds. To achieve a good separation between saturated and
steroidal hydrocarbons, the optimization of fraction volumes is required.
For this, the volume of the first fractionshould be adjusted so that when
the second fraction is analysed the peaks representing the saturated
hydrocarbons are low (see Figure 1 c); if they do not appear but the
intensity of the standard peak is low, the volume should be reduced.
Anyway, a complete separation between the components of the first and
second fractions is unnecessary; as there is no overlapping of peaks
during GC analysis if GC conditions are ajusted as indicated in 6.3.1.
The optimization of the volume of the second fraction if generally not
needed as a good separation exists with the further components. Never-
theless, the presence of a large peak at approximately 1,5 minutes lower
retention time than the standard is due to squalene, and it is indicative of
a bad separation.
6.2.2. Evaporate the second fraction in a rotary evaporator at 30 ºC under
reduced pressure until dryness, and immediately dissolve the residue in
0,2 ml of hexane. Keep the solution in the refrigerator until analysis.
Note 8:
Residues 6.1.3 and 6.2.2 should not be kept dry and at room temperature.
As soon as they are obtained, the solvent should be added and the solu-
tions should be kept in the refrigerator.
6.3.Gaschromatography
6.3.1. Working conditions for split injection:
— injector temperature: 300 ºC,
— detector temperature: 320 ºC,— integrator-recorder: the parameters for integration should be fixed so
as to give a correct assessment of the areas. Valley-valley integration
mode is recommended,
— sensitivity: about 16 times the minimum attenuation,
— amount of solution injected: 1 µl,
— oven programming temperatures: initial 235 ºC for six minutes and
then rising at 2 ºC/minute up to 285 ºC,
— injector with 1: 15 flow divider,— carrier: helium or hydrogenat about 120 kPa pressure.
These conditions may be adjusted in accordance with the characteristics
of the chromatograph and the column to give chromatograms meeting
the following requirements: internal standard peak within approximately
five minutes of the time given in 6.3.2; the internal standard peak should
be at least 80 % of the full scale.
The gas chromatographic system must be checked injecting a mixture of
the stock solution of cholestadiene (5.6) and n-nonacosane solution (5.8).
The cholesta-3,5-diene peak must appear before the n-nonacosane (Figure
1c); if it does not occur two actions can be undertaken: reduce the oven
temperature and/or use a less polar column.
6.3.2. Peak identification
The internal standard peak appears at approximately 19 minutes and the
3,5-stigmastadiene at a relative retention time of approximately 1,29 (see
Figure 1b). The 3,5-stigmastadiene occurs with small quantities of an
isomer, and usually, both elute together as a single chromatographic
peak. Nevertheless, if the columnis too polar or shows a high resolvin g
power, the isomer canappear as a small peak before an d close to that of
stigmasta-3,5-diene (Figure 2). In order to ensure that the stigmastadienes
are eluted as one peak, it is advisable to replace the column by one which
is either less polar or has a wider internal diameter.
Note 9:
Stigmastadienes for reference can be obtained from the analysis of a
refined vegetable oil by using less amount of sample (1 to 2 g). Stigmas-
tadienes originate a prominent and easily identifiable peak.
6.3.3. Quantitative analysis
The stigmastadienes content is determined according to the formula:
mg/kg of stigmastadienes =
As/C2Mc
Ac/C2Mo1991R2568 — EN — 01.11.2003 — 020.001 — 81
▼M11
where:As= area of stigmastadienes peak (if the peak is resolved
into two isomers, sum of areas of the two peaks),
Ac= area of internal standard (cholestadiene),
Mc= mass of standard added, in micrograms,
Mo= mass of oil taken, in grams.
Detectionlimit: about 0,01 mg/kg.1991R2568 — EN — 01.11.2003 — 020.001 — 82
▼M11
Figure1
Gas chromatograms obtained from olive oil samples analysed on a fused
silica capillary column (0,25 mm internal diameter by 25 m) coated with
5 %-phenylmethylsilicone, 0,25 µm film thickness.
(a) First fraction(30 ml) from a virginoil, spiked with stan dard.
(b) Second fraction (40 ml) from an olive oil containing 0,10 mg/kg of
stigmastadienes.1991R2568 — EN — 01.11.2003 — 020.001 — 83
▼M11
(c) Second fraction (40 ml) containing a small proportion of the first
fraction.
Figure2
Gas chromatogram obtained from a refined olive oil sample analysed on
DB-5 column showing the isomer of 3,5-stigmastadiene.1991R2568 — EN — 01.11.2003 — 020.001 — 84
▼M13
ANNEX XVIII
DETERMINATION OF TRIACYLGLYCEROLS WITH ECN 42
(DIFFERENCE BETWEEN HPLC DATA AND THEORETICAL
CONTENT)
1.Scope
Determination of the composition of triacylglycerols (TAGs) in olive
oils, in terms of their equivalent carbon number by differences between
the analytical results obtained by high performance liquid chromato-
graphy (HPLC) and the theoretical content, calculated starting from
the fatty acid composition.
2.Fieldapplication
The standard is applicable to olive oils. The method is applicable to the
detection of the presence of small amounts of seed oils (rich in linoleic
acid) inevery class of olive oils.
3.Principle
The content of triacylglycerols with ECN42 determined by HPLC
analysis and the theoretical content of triacylglycerols with ECN42
(calculated onthe basis of GLC determin ationof fatty acid composi-
tion) correspond within a certain limit for pure oils. A difference
larger thanthe values stated inthe Regulationfor each type of oil
points out that the oil contains seed oils.
4.Method
The method for calculation of theoretical content of triacylglycerols
with ECN42 and of the difference between the HPLC data and this
one essentially is made by the coordination of analytical data obtained
by means of other methods: it is possible to distinguish three phases:
determination of fatty acid composition by capillary gas chromato-
graphy, calculationof theoretical compositionof triacylglycerols with
ECN42, HPLC determination of ECN42 triacylglycerols
4.1.Apparatus
4.1.1. Round bottom flasks, 250 and 500 ml.4.1.2. Beakers 100 ml.4.1.3. Glass chromatographic column, 21 mm internal diameter, 450 mm
length, with cock and normalized cone (female) at the top.
4.1.4. Separator funnels, 250 ml, with normalized cone (male) at the bottom,
suitable to be connected with the top of the column.
4.1.5. Glass rod, 600 mm length.4.1.6. Glass funnel, 80 mm diameter.4.1.7. Volumetric flasks, 50 ml.4.1.8. Volumetic flasks, 20 ml.4.1.9. Rotative evaporator.4.1.10. High performance liquid chromatography, allowing thermostatic control
of columntemperature.
4.1.11. Injection units for 10 µl delivery.
4.1.12. Detector: differential refractometer. The full scale sensitivity should be
at least 10
−4units of refractive index.
4.1.13. Column: stainless steel tube 250 mm length and 4,5 mm internal
diameter packed with 5 µm diameter particles of slica with 22 to 23 %
carbon in the form of octadecylsilane (note 2).
4.1.14. Recorder and/or integrator.
4.2.Reagents
The reagents should be of analytical purity. Elution solvents should be
de-gassed, and may be recycled several times without effect on the
separations.1991R2568 — EN — 01.11.2003 — 020.001 — 85
▼M13
4.2.1. Petroleum ether 40 to 60 ºC chromatographic grade.
4.2.2. Ethil ether, peroxides free, freshly distilled.4.2.3. Glass chromatographic elutionsolven t: mixture petroleum ether/ethil
ether 87/13 (v/v).
4.2.4. Silicagel, 70-230 mesh, type Merck 7734, with water content standar-
dized at 5 % (w/w).
4.2.5. Glass wool.4.2.6. Acetone.4.2.7. Acetonitrile.4.2.8. HPLC elution solvent: acetonitrile + acetone (proportions to be adjusted
to obtainthe desired separation ; beginwith 50:50 mixture).
4.2.9. Solubilization solvent: acetone.4.2.10. Reference triglycerides commercial triglycerides tripalmitin, triolein,
etc.) may be used and the retention times thence plotted in accordance
with the equivalent carbon number, or alternatively reference chromato-
grams obtained from soya oil, mixture 30:70 soya oil/olive oil and pure
olive oil (see notes 3 and 4 and figure 1, 2, 3, 4).
4.3.Samplepreparation
As a number of interfering substances can rise false positive results, the
sample must always be purified according to IUPAC method 2.507,
used for determination of polar substances in oxidised oils.
4.3.1. Chromatographic columnpreparation
Fill the column(4.1.3) with about 30 ml of elutionsolven t (4.2.3), then
introduce inside the column some glass wool (4.2.5) pushing it to the
bottom of the columnby mean s of the glass rod (4.1.5).
Ina 100 ml beaker, suspen d 25 g of silicagel (4.2.4) in80 ml of elution
mixture (4.2.3), then transfer it inside the column, by means of a glass
funnel (4.1.6).
To ensure the complete transfer of silicagel inside the column, wash the
beaker with the elution mixture and transfer the washing portions inside
the column, too.
Open the cock and let solvent elute from the column until its level is
about 1 cm over the silicagel.
4.3.2. Columnchromatography
Weigh with the accuracy of 0,001 g, 2,5 ± 0,1 g of oil, previously
filtered, homogenized and anhydrified, if necessary, in a 50 ml volu-metric flask (4.1.7). Solve it inabout 20 ml of elutionsolven t (4.2.3),
if necessary, slightly heat it to make the dissolution easily. Cool at
room temperature and adjust the volume with elution solvent.
By means of a volumetric pipette, introduce 20 ml of solution inside the
column prepared according to 4.3.1, open the cock and let solvent elute
to the silicagel layer level.
Then elute with 150 ml of elution solvent (4.2.3), adjusting the solvent
rate at about 2 ml/min(150 ml will take about 60 to 70 min utes to pass
through the column).
The eluated is recovered ina 250 ml roun d bottom flask (4.1.1)
previously tared in an oven and exactly weighted. Eliminate solvent at
reduce pressure (Rotavapor) and weigh the residue that will be used to
prepare the solution for HPLC analysis and for methyl ester prepara-
tion.
The sample recovery from the columnmust be 90 % at least for extra
virgin, virgin, ordinary refined and olive oil categories, and a minimum
of 80 % for lampante and residue olive oils.1991R2568 — EN — 01.11.2003 — 020.001 — 86
▼M13
4.4.HPLCanalysis
4.4.1. Preparationof the samples for chromatographic an alysis
A 5 % solution of the sample 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 (4.2.9).
4.4.2. Procedure
Set up the chromatographic system. Pump elutionsolven t (4.2.8) at a
rate of 1,5 ml/min to purge the entire system. Wait until a stable base
line is obtained. Inject 10 µl of the sample prepared as in4.3.
4.4.3. Calculationan d expressionof results
Use the area normalization method, i.e. assume that the sum of the
areas of the peaks corresponding to TAGs from ECN42 up to ECN52
is equal to 100 %. Calculate the relative percentage of each triglyceride
using the formula:
% triglyceride = area of peak × 100/ sum of peak areas.The results are to be givento withinat least two decimal places.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
bounds, it can be calculated more precisely by taking into account the
originof the double bon d. If n
o,nland nlnare the numbers of double
bonds attributed to oleic, linoleic and linolenic acids respectively, the
equivalent carbon number can be calculated by means of the relation
of the formula:
ECN = CN − dono−dlnl−dlnnln,
where the coefficient do, dland dlncanbe calculated by mean s of the
reference triglycerides. Under the conditions specified in this method,
the relation obtained will be close in:
ECN = CN − (2,60 no) − (2,35 nl) − (2,17 nln)
Note 2:Examples: Lichrosorb (Merck) RP 18 Art 50333
Lichrosphere or equivalent (Merck) 100 CH18 Art
50377.
Note 3:With several reference triglycerides, it is also possible to calcu-
late the resolutionwith respect to triolein :
α= RT' / RT triolein
by use of the reduced retention time RT' = RT − RT solvent.
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 trilinoein from the peaks of the triglycerides with an adja-
cent RT. The elution is carried out up to ECN52 peak.
Note 5:A correct measure of the areas of all peaks of interest for the
present determination is ensured if the second peak corresponding to
ECN50 is 50 % of full scale of the recorder.
4.5.Calculationoftriacylglycerols composition
4.5.1. Determination of fatty acid composition
Fatty acid compositionis carried out by mean s of the EEC gas chroma-
tographic method reported in Annex X A of Regulation (EEC) No
2568/91, by means of a capillary column. The methyl esters preparation
is carried out according to Annex X B (sodium methylate alcohol solu-
tion).1991R2568 — EN — 01.11.2003 — 020.001 — 87
▼M13
4.5.2. Fatty acids for calculation
Glycerides are grouped by their equivalent carbon number (ECN),
taking into account the following equivalencies between ECN and fatty
acids. Only fatty acids with 16 and 18 carbon atoms were taken in
consideration, because only these are important for olive oil.
Fatty acid (FA) AbbreviationMolecular
weight
(MW)ECN
Palmatic acid P 256,4 16
Palmatoleic acid Po 254,4 14Stearic acid S 284,5 18Oleic acid O 282,5 16Linoleic acid L 280,4 14Linolenic acid Ln 278,4 12
4.5.3. Conversion of area % into moles for all fatty acids
moles P ¼
area % P
MW Pmoles S ¼area % S
MW Smoles Po ¼area % Po
MW Po/C148/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C149/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C150(1)
moles O ¼
area % O
MW Omoles L ¼area % L
MW LmolesLn ¼area % Ln
MW Ln
4.5.4. Normalizationof fatty acids to 100 %
moles % P 1 ;2;3 ðÞ ¼moles P /C3100
moles P þSþPoþOþLþLn ðÞ/C148/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C149/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C150(2)moles % S 1 ;2;3 ðÞ ¼
moles S /C3100
moles P þSþPoþOþLþLn ðÞ
moles % Po 1 ;2;3 ðÞ ¼moles Po /C3100
moles P þSþPoþOþLþLn ðÞ
moles % O 1 ;2;3 ðÞ ¼moles O /C3100
moles P þSþPoþOþLþLn ðÞ
moles % L 1 ;2;3 ðÞ ¼moles L /C3100
moles P þSþPoþOþLþLn ðÞ
moles % Ln1 ;2;3 ðÞ ¼moles Ln /C3100
moles P þSþPoþOþLþLn ðÞ
The result gives the percentage of each fatty acid in moles % in the
overall (1,2,3-) positionof the TAGs.
Then the sum of the saturated fatty acids P and S (SFA) and the unsa-
turated fatty acids Po, O, L and Ln (UFA) are calculated:
moles % SFA = moles % P + moles % S/C148/C147/C147/C147/C147/C147/C149/C147/C147/C147/C147/C147/C150(3)moles UFA = 100 − moles % SFA
4.5.5. Calculationof the fatty acid compositionin2- an d 1,3-
positions of TAGs
The fatty acids are distributed to three pools as follows: two identical
for 1- and 3- positions and one for 2- position, with different coeffi-
cients for the saturated (P and S) and unsaturated acids (Po, O, L and
Ln).
4.5.5.1. Saturated fatty acids in2- position[P(2) an d S(2)]
moles % P(2) = moles % P (1,2,3) * 0,06/C148/C147/C147/C147/C147/C147/C149/C147/C147/C147/C147/C147/C150(4)moles % S(2) = moles % S (1,2,3) * 0,061991R2568 — EN — 01.11.2003 — 020.001 — 88
▼M13
4.5.5.2. Unsaturated fatty acids in 2- position [Po(2), O(2), L(2) and Ln(2)]:
moles % Po 2 ðÞ ¼moles % Po 1 ;2;3 ðÞ
moles % UFA/C3100/C0moles % P 2 ðÞ /C0moles % S 2 ðÞ ½/C138/C148/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C149/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C150(5)moles % O 2 ðÞ¼
moles % O 1 ;2;3 ðÞ
moles % UFA/C3100/C0moles % P 2 ðÞ/C0mol % S 2 ðÞ ½/C138
moles % L 2 ðÞ ¼moles % L 1 ;2;3 ðÞ
moles % UFA/C3100/C0moles % P 2 ðÞ /C0moles % S 2 ðÞ ½/C138
moles % Ln2 ðÞ ¼moles % Ln1 ;2;3 ðÞ
moles % UFA/C3100/C0moles % P 2 ðÞ /C0moles % S 2 ðÞ ½/C138
4.5.5.3. Fatty acids in 1,3-positions [P(1,3), S(1,3), Po(1,3) O(1,3), L(1,3) and
Ln(1,3)]:
moles % P 1 ;3ðÞ ¼moles % P 1 ;2;3 ðÞ /C0 moles % P 2 ðÞ
2þmoles % P 1 ;2;3 ðÞ/C148/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C149/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C150(6)moles % S 1 ;3ðÞ ¼
moles % S 1 ;2;3 ðÞ /C0 moles % S 2 ðÞ
2þmoles % S 1 ;2;3 ðÞ
moles % Po 1 ;3ðÞ ¼moles % Po 1 ;2;3 ðÞ /C0 moles % Po 2 ðÞ
2þmoles % Po 1 ;2;3 ðÞ
moles % O 1 ;3ðÞ ¼moles % O 1 ;2;3 ðÞ /C0 moles % O 2 ðÞ
2þmoles % O 1 ;2;3 ðÞ
moles % L 1 ;3ðÞ ¼moles % L 1 ;2;3 ðÞ /C0 moles % L 2 ðÞ
2þmoles % L 1 ;2;3 ðÞ
moles % Ln1 ;3ðÞ ¼moles % Ln1 ;2;3 ðÞ /C0moles % Ln2 ðÞ
2þmoles % Ln1 ;2;3 ðÞ
4.5.6. Calculationof triacylglycerols
4.5.6.1. TAGs with one fatty acid (AAA, here LLL, PoPoPo)
moles % AAA ¼moles % A 1 ;3ðÞ /C3 moles % A 2 ðÞ/C3moles% A 1 ;3ðÞ
10000(7)
4.5.6.2. TAGs with two fatty acids (AAB, here PoPoL, PoLL)
moles % AAB ¼moles % A 1 ;3ðÞ /C3 moles % A 2 ðÞ/C3moles % B 1 ;3ðÞ /C3 2
10000/C148/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C149/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C150(8)
moles % ABA ¼
moles % A 1 ;3ðÞ /C3 moles % B 2 ðÞ/C3moles % A 1 ;3ðÞ
10000
4.5.6.3. TAGs with three different fatty acids (ABC, here OLLn, PLLn, PoOLn,
PPoLn)
moles % ABC ¼moles % A 1 ;3ðÞ /C3 moles % B 2 ðÞ/C3moles % C 1 ;3ðÞ /C3 2
10000/C148/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C149/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C147/C150(9) moles % BCA ¼
moles % B 1 ;3ðÞ /C3 moles % C 2 ðÞ/C3moles % A 1 ;3ðÞ /C3 2
10000
moles % CAB ¼moles % C 1 ;3ðÞ /C3 moles % A 2 ðÞ/C3moles % B 1 ;3ðÞ /C3 2
100001991R2568 — EN — 01.11.2003 — 020.001 — 89
▼M13
4.5.6.4. Triacylglycerides with ECN42
The following triglycerides with ECN42 are calculated according equa-
tion 7, 8 and 9 in order of expected elution in HPLC (normally only
three peaks).
LLLPoLL and the positional isomer LPoLOLLn and the positional isomers OLnL and LnOLPoPoL and the positional isomer PoLPoPoOLn and the positional isomers OPoLn and OLnPoPLLn and the positional isomers LLnP and LnPLPoPoPo
SLnLn and the positional isomer LnSLn
PPoLn and the positional isomers PLnPo and PoPLnThe triacylglycerides with ECN42 are given by the sum of the nine tria-
cylglycerols including their positional isomers. The results to be given
with at least two decimal places.
5.Evaluationoftheresults
The calculated theoretical content and the content determined by the
HPLC analysis are compared. If the difference between HLPC data
minus theoretical data is greater than the values states for the appro-
priate oil category in the Regulation, the sample contains seed oil.
Note:Results are givento withinon e decimal figure.
6.Example (The numbers refer to the sections in the text of the method)
4.5.1. Calculationof moles % fatty acids from GLC data (area %)
The following data are obtained for the fatty acid composition by GLC:
FA
MWP
256,4S
284,5Po
254,4O
282,5L
280,4Ln
278,4
area % 10,0 3,0 1,0 75,0 10,0 1,0
4.5.3. Conversion of area % into moles for all fatty acids
moles P ¼10
256 ;4¼0;03900 moles P See formula (1)
moles S ¼3
284 ;5¼0;01054 moles S See formula (1)
moles Po ¼1
254 ;4¼0;00393 moles Po See formula (1)
moles O ¼75
282 ;5¼0;26549 moles O See formula (1)
moles L ¼10
280 ;4¼0;03566 moles L See formula (1)
moles Ln ¼1
278 ;4¼0;003594 moles LnSee formula (1)
Total = 0,35822 moles TAGs
4.5.4. Normalizationof fatty acids to 100 %
moles % P 1 ;2;3 ðÞ ¼0;03900 moles P /C3100
0;35822 moles¼10;888 % See formula (2)1991R2568 — EN — 01.11.2003 — 020.001 — 90
▼M13
moles % S 1 ;2;3 ðÞ ¼0;01054 moles S /C3100
0;35822 moles¼2;944 % See formula (2)
moles % Po 1 ;2;3 ðÞ ¼0;00393 moles Po /C3100
0;35822 moles¼1;097 % See formula (2)
moles % O 1 ;2;3 ðÞ ¼0;26549 moles O /C3100
0;35822 moles¼74;113 % See formula (2)
moles % L 1 ;2;3 ðÞ ¼0;03566 moles L /C3100
0;35822 moles¼9;956 % See formula (2)
moles % Ln1 ;2;3 ðÞ ¼0;00359 moles Ln /C3100
0;35822 moles¼1;003% See formula (2)
Total moles % = 100,0 %
Sum of the saturated and unsaturated fatty acids in the 1,2,3- position of
TAGs
moles % SFA = 10,888 % + 2,944 % = 13,831 % See formula (3)
moles % UFA = 100,000 % — 13,831 % = 86,169 % See formula (3)
4.5.5. Calculationof the fatty acid compositionin2- an d 1,3-
positions of the TAGs
4.5.5.1. Saturated fatty acids in2- position[P(2) an d S(2)]
moles % P(2) = 10,888 % * 0,06 = 0,653 moles % See formula (4)
moles % S(2) = 2,944 % * 0,06 = 0,177 moles % See formula (4)
4.5.5.2. Unsaturated fatty acids in 1,3-position [Po(1,3), O(1,3), L(1,3) and
Ln(1,3)]
moles % Po 2 ðÞ ¼1;097 %
86;169 %/C3100/C0/C00;659/C00;177 ðÞ ¼ 1;263 moles % See formula (5)
moles % O 2 ðÞ¼74;113 %
86;169 %/C3100/C0/C00;659/C00;177 ðÞ ¼85;295 moles % See formula (5)
moles % L 2 ðÞ ¼9;956 %
86;169 %/C3100/C0/C00;659/C00;177 ðÞ ¼ 11;458 moles % See formula (5)
moles % Ln2 ðÞ ¼1;003 %
86;169 %/C3100/C0/C00;659/C00;177 ðÞ ¼ 1;154 moles % See formula (5)
4.5.5.3. Fatty acids in 1,3-positions [P(1,3), S(1,3), Po(1,3), O(1,3), L(1,3) and
Ln(1,3)]
moles % P 1 ;3ðÞ ¼10;888/C00;659
210;888¼16;005 moles % See formula (6)
moles % S 1 ;3ðÞ ¼2;944/C00;177
22;944¼4;327 moles % See formula (6)
moles % Po 1 ;3ðÞ ¼1;097/C01;263
21;097¼1;015 moles % See formula (6)
moles % O 1 ;3ðÞ ¼74;113/C085;295
274;113¼68;522 moles % See formula (6)
moles % L 1 ;3ðÞ ¼9;956/C011;458
29;956¼9;205 moles % See formula (6)
moles % Ln1 ;3ðÞ ¼1;003/C01;154
21;003¼0;927 moles % See formula (6)
4.5.6. Calculationof triacylglycerols
From the calculated fatty acid composition in sn-2- and sn-1,3- posi-
tions (see above):1991R2568 — EN — 01.11.2003 — 020.001 — 91
▼M13
FA in1,3-pos. 2-pos.
P 16,005 % 0,653 %
S 4,327 % 0,177 %Po 1,015 % 1,263 %
O 68,522 % 85,295 %
L 9,205 % 11,458 %Ln0,927 % 1,154 %Sum 100,0 % 100,0 %
the following triacylglycerols are calculated:
LLL
PoPoPo
PoLL with 1 positional isomer
SLnLn with 1 positional isomerPoPoL with 1 positional isomerPPoLnwith 2 position al isomers
OLLnwith 2 position al isomers
PLLnwith 2 position al isomersPoOLnwith 2 position al isomers.
4.5.6.1. TAGs with one fatty acid (LLL, PoPoPo) See formula (7)
mol % LLL ¼
9;205 % /C311;458 % /C39;205 %
10000= 0,09708 mol LLL
mol % PoPoPo ¼1;015 % /C31;263 % /C31;015 %
10000= 0,00013 mol PoPoPo
4.5.6.2. TAGs with two fatty acids (PoLL, SLnLn, PoPoL) See formula (8)
mol % PoLL þLLPo ¼1;015 % /C311;458 % /C39;205 % /C32
10000= 0,02141
mol % LPoL ¼9;205 % /C31;263 % /C39;205 %
10000= 0,01070
0,03211 mol PoLL
mol % SLnLn þLnLnS ¼4;327 % /C31;154 % /C30;927 % /C32
10000= 0,00093
mol % LnSLn ¼0;927 % /C30;177 % /C30;927 %
10000= 0,00002
0,00095 mol SLnLn
mol % PoPoL þLPoPo ¼1;015 % /C31;263 % /C39;205 % /C32
10000= 0,00236
mol % PoLPo ¼1;015 % /C311;458 % /C31;015 %
10000= 0,00118
0,00354 mol PoPoL
4.5.6.3. TAGs with three different fatty acids (PoPLn, OLLn, PLLn, PoOLn)
See formula (9)
mol % PPoLn ¼16;005 % /C31;263 % /C30;927 % /C32
10000= 0,00375
mol % LnPPo ¼0;927 % /C30;653 % /C31;015 % /C32
10000= 0,000121991R2568 — EN — 01.11.2003 — 020.001 — 92
▼M13
mol % PoLnP ¼1;015 % /C31;154 % /C316;005 % /C32
10000 = 0,00375
0,00762 mol PPoLn
mol % OLLn ¼68;522 % /C311;458 % /C30;927 % /C32
10000= 0,14577
mol % LnOL ¼0;927 % /C385;295 % /C39;205 % /C32
10000= 0,14577
mol % LLnO ¼9;205 % /C31;154 % /C368;522 % /C32
10000= 0,14577
0,43671 mol OLLn
mol % PLLn ¼16;005 % /C311;458 % /C30;927 % /C32
10000= 0,03400
mol % LnPL ¼0;927 % /C30;653 % /C39;205 % /C32
10000= 0,00111
mol % LLnP ¼9;205 % /C31;154 % /C316;005 % /C32
10000= 0,03400
0,06911 mol PLLn
mol % PoOLn ¼1;015 % /C385;295 % /C30;927 % /C32
10000= 0,01605
mol % LnPoO ¼0;927 % /C31;263 % /C368;522 % /C32
10000= 0,01605
mol % OLnPo ¼68;522 % /C31;154 % /C31;015 % /C32
10000= 0,01605
0,04815 mol PoOLn
ECN42= 0,69540 mol TAGs1991R2568 — EN — 01.11.2003 — 020.001 — 93
▼M14
Figure1:Graph of log αagainst f (number of double bonds)
Note:La = lauric acid; My = myristic acid; P = palmitic acid; St =
stearic acid; O = oleic acid; L = linoleic acid; Ln = linolenic
acid.1991R2568 — EN — 01.11.2003 — 020.001 — 94
▼M14
Figure 2:Soyabean oil
Figure 3:Soyabean oil / olive oil 30/70
1991R2568 — EN — 01.11.2003 — 020.001 — 95
▼M14
Figure4:Olive oil
1991R2568 — EN — 01.11.2003 — 020.001 — 96
▼M19
ANNEX XIX
DETERMINATION OF ALIPHATIC ALCOHOLS CONTENT BY
CAPILLARYGASCHROMATOGRAPHY
1. OBJECT
The procedure describes a method for the determination of aliphatic
alcohols content in oils and fats.
2. PRINCIPLE OF THE METHOD
The fatty substance, with 1-eicosanol added as internal standard, is
saponified with ethanolic potassium hydroxide and then the unsaponifi-
able matter extracted with ethyl ether. The alcoholic fractionis
separated from the unsaponifiable matter by chromatography on a basic
silica gel plate; the alcohols recovered from the silica gel are trans-
formed into trimethylsilyl ethers and analysed by capillary gas
chromatography.
3. EQUIPMENT
3.1. 250 ml round-bottomed flask fitted with a reflux condenser having
ground-glass joints.
3.2. 500 ml separating funnel.
3.3. 250 ml round-bottomed flasks.
3.4. Chromatographic tank for thin-layer chromatographic analysis, for glass
plates of dimensions 20 x 20 cm.
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
40µm) 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 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. A temperature-adjustable injection unit with a persilanised glass vapor-
ising element.
3.10.3. A flame ionisation 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.
3.13. Analytical balance sensitive to 1 mg (with 0,1 mg display).
4. REAGENTS
4.1. Potassium hydroxide, approximately 2 N ethanolic solution: 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 opaque glass
bottle.
4.2. Ethyl ether, pure for analysis.
4.3. Anhydrous sodium sulphate, analytical purity.1991R2568 — EN — 01.11.2003 — 020.001 — 97
▼M19
4.4. Glass plates coated with silica gel, without fluorescence indicator,
thickness 0,25 mm (commercially available ready for use).
4.5. Potassium hydroxide, approximately 0,2 N ethanolic solution; 13 g of
potassium hydroxide are dissolved in20 ml of distilled water an d
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. Ethyl ether, for chromatography (see 5.2.2).
4.10. Chloroform, for chromatography.
4.11. Reference solution for thin-layer chromatography: cholesterol or
phytosterols, 5 % solutioninchloroform.
4.12. 0,2 % solutionof 2', 7'-dichlorofluoresceininethan ol. Make slightly
basic by adding a few drops of 2 N alcoholic potassium hydroxide solu-
tion.
4.13. Anhydrous pyridine, for chromatography.
4.14. Hexamethyl disilazane.
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) solution of 1-eicosanol in chloroform (internal standard).
4.18. Carrier gas: hydrogenor helium, gas-chromatographic purity.
4.19. Auxiliary gas: nitrogen, gas-chromatographic purity.
5. PROCEDURE
5.1.Preparationoftheunsaponifiables
5.1.1. Using a 500 µl microsyringe place, into a 250 ml round-bottom flask, a
volume of 0,1 % 1-eicosanol solution in chloroform (4.17) containing a
quantity of 1-eicosanol approximately equal to 10 % of the aliphatic
alcohols content in that portion of sample to be taken for analysis. For
example, to 5 g of sample add 250 µl of the 0,1 % 1-eicosanol solution
if olive oil and 1500 µl if olive pomace oil.
Evaporate to dryness in current of nitrogen and then weigh accurately
5 g of the dry filtered sample into the same flask.
5.1.2. Add 50 ml of 2 N potassium hydroxide ethanolic solution, fit the reflux
condenser and heat the apparatus to slight boiling on a steam bath, stir-
ring continuously throughout the heating process until saponification
has taken place (the solution becomes clear). Continue heating for a
further 20 minutes and then add 50 ml of distilled water through the
condenser. The condenser is then disconnected and the flask cooled to
approximately 30 ºC.
5.1.3. The contents of the flask are quantitatively transferred to a separating
funnel of 500 ml capacity by adding distilled water several times, using
a total of around 50 ml distilled water. Add approximately 80 ml of
ethyl 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. Two further extractions are effected on the aqueous
phase, in the same manner, using each time 60 to 70 ml ethyl ether.
Note 1:Emulsions may be eliminated by adding, using as a spray,
small quantities of ethyl alcohol or methyl alcohol.
5.1.4. The ethyl 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.
Discard the washing water, dry with anhydrous sodium sulphate and
filter, into a flask of 250 ml capacity which has been weighed before-
hand, the funnel and filter being washed with small quantities of ethyl
ether which are added to the total.1991R2568 — EN — 01.11.2003 — 020.001 — 98
▼M19
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 an oven at
100 ºC for approximately a quarter of anhour, an d thenweigh after
cooling in a desiccator.
5.2.Separationofalcoholic fractions
5.2.1. Preparationof basic TLC plates: the silica gel plates (4.4) are immersed
completely, in0,2 N potassium hydroxide solution(4.5) for 10 secon ds,
and then left to dry for two hours under an extractor hood and finally
placed inanovenat 100 ºC for on e hour.
Remove from the oven 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 alcoholic
fraction there is no need to treat the unsaponifiables with
alumina. 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. Place a 65/35 by volume hexane/ethyl ether mixture in the plate-devel-
oping chamber to a depth of approximately 1 cm (*).
Close the chamber using an appropriate cover and leave for half an
hour 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 and 0,3 ml of the solution is streaked as a
uniform strip of minimum thickness, using the 100 µl microsyringe, on
a TLC plate at approximately 2 cm from the bottom of the TLC plate.
Aligned with the origin, 2 to 3 µl of the aliphatic alcohols reference
solution(4.11) are spotted for the iden tificationof the aliphatic alcohols
band after development has been completed.
5.2.4. Place the plate inside the development tank as stated 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.
The plate is then removed from the development chamber and the
solvent evaporated under a hot air current or the plate is left for a while
under the extractor hood.
5.2.5. The plate is sprayed lightly and evenly with the solution of 2', 7'-
dichlorofluoresceinwhenthe plate is observed un der ultra violet light.
The aliphatic alcohols band can be identified through being aligned
with the stain obtained from the reference solution: mark the limits of
the band with a black pencil; outlining the band of aliphatic alcohols
and the band immediately above that, which is the terpenic alcohols
band, together.
Note 4:The aliphatic alcohols band and the terpenic alcohols band are
to be grouped together inview of the possible migrationof
some aliphatic alcohols into the triterpenic alcohols band.
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 pomace inthe flask three times with ethyl ether (approxi-
mately 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 to1991R2568 — EN — 01.11.2003 — 020.001 — 99
(*) In these cases in particular, a 95/5 by volume benzene/acetone eluent mixture must be
used to obtain distinct band separation.
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dryness, place in an oven at 105 ºC for approximately 10 minutes and
thenallow to cool ina desiccator an d weigh.
The pomace inside the test tube is composed of the alcoholic fraction.
5.3.Preparationofthetrimethylsilyl 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 aliphatic alcohols, is added
to the test tube containing the alcoholic fraction, avoiding all absorption
of moisture (Note 6).
Note 5:Solutions which are ready for use are available commercially.
Other silanising 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.
Note 6: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.
5.3.2. Stopper the test tube, shake carefully (without overturning) until the
aliphatic alcohols 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.
5.4.Gaschromatographyanalysis
5.4.1. Preliminary operations, column packing
5.4.1.1. Fit the column in the gas chromatograph, attaching the inlet end to the
injector connected to the splitting system and the outlet end to the
detector. Carry out a general check of the gas chromatography assembly
(tightness of gas fittings, efficiency of the detector, efficiency of the
splitting system and of the 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. A little carrier gas is passed
through the capillary columnan d thenthe gas chromatography
assembly is switched on and gradually heated until a temperature not
less than20 ºC above the operatin g temperature (see Note 7) is
attained. That temperature is held for not less than two hours and then
the assembly is brought to the operating conditions (regulation of gas
flow, split flame ignition, connection to the electronic recorder, adjust-
ment 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 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 connections; a positive drift indicates inadequate condi-
tioning of the column.
Note 7:The conditioning temperature shall be at least 20 ºC less than
the maximum temperature contemplated for the liquid phase
employed.
5.4.2. Choice of operating conditions
5.4.2.1. The guideline 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 to 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 — 01.11.2003 — 020.001 — 100
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The above conditions may be modified according to the characteristics
of the columnan d of the gas chromatograph to obtainchromatograms
satisfying the following conditions:
— alcohol C26retention time shall 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 above requirements are checked by repeated injection of the stan-
dard 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. Analytical procedure
5.4.3.1. Using the microsyringe of 10 µl capacity draw in1 µl of hexane
followed by 0,5 µl of air and subsequently 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. Recording is effected until the TMSE of the aliphatic alcohols present
have been eluted completely. The base line shall always correspond to
the requirements of 5.4.1.2.
5.4.4. Peak identification
The identification of individual peaks is effected according to the reten-
tion times and by comparison with the standard TMSE mixture,
analysed under the same conditions.
A chromatogram of the alcoholic fractionof a virginolive oil is shown
inFigure 1.
5.4.5. Quantitative evaluation
5.4.5.1. The peak areas of 1-eicosanol and of the aliphatic alcohols C22,C24,C26and C28are calculated by electronic integration.
5.4.5.2. The contents of each aliphatic alcohol, expressed in mg/1 000 g fatty
substance, are calculated as follows:
where:
Ax = area of the alcohol peak x
As= area of 1-eicosanol
ms= mass of 1-eicosanol in milligrams
m = mass of sample drawn for determination, in grams.
6. EXPRESSION OF THE RESULTS
The contents of the individual aliphatic alcohols in mg/1 000 g of fatty
substance and the sum of the ‘total aliphatic alcohols’ are reported.1991R2568 — EN — 01.11.2003 — 020.001 — 101
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APPENDIX
Determination of the linear velocity of the gas
1t o3 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 tM is the measured time in seconds.
Figure 1 — Chromatogram of the alcoholic fractionof virginoil
1 = Eicosanol
2 = Decosanol3 = Tricosanol
4 = Tetracosanol
5 = Pentacosanol6 = Hexacosanol
7 = Heptacosanol
8 = Octacosanol1991R2568 — EN — 01.11.2003 — 020.001 — 102