1991R2568 — EN — 01.01.2008 — 021.001

This document is meant purely as a documentation tool and the institutions do not assume any liability for its contents

Amended by:





Corrected by:









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COMMISSION REGULATION (EEC) No 2568/91

of 11 July 1991

on the characteristics of olive oil and olive-residue oil and on the relevant methods of analysis







THE COMMISSION OF THE EUROPEAN COMMUNITIES, Having regard to the Treaty establishing the European Economic Community, 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 descriptions and definitions of olive oil and olive-residue oil marketed within each Member State, in intra-Community trade and in trade with third countries; 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, without prejudice to other existing provisions; Whereas the presence of the characteristics of the different types of oil should be determined uniformly throughout the Community; whereas, to that end, Community methods of chemical analysis and organoleptic evaluation should 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 the additional notes to Chapter 15 of the combined nomenclature; Whereas the method of evaluating the organoleptic characteristics of virgin oil includes the setting up of panels of selected and trained tasters; whereas the period necessary for establishing such a structure should therefore be fixed; whereas in view of the difficulties that some Member States will encounter in setting up panels of tasters, the use of panels in other Member States should be authorized; Whereas, in order to ensure that the system of levies applicable to imports of olive residues functions correctly, a single method for the determination of the oil content of these products should be laid down; Whereas, in order not to harm trade, provision should 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 delivered an opinion within the time limit set by its chairman, HAS ADOPTED THIS REGULATION:







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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 Annex to Regulation No 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 Regulation No 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 Regulation No 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 meaning of point 3 of the Annex to Regulation No 136/66/EEC.

5. Oil, the characteristics of which comply with those set out in point 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 Regulation No 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 Regulation No 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.

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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,

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— for determination of the wax content, the method given in Annex IV,

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— 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,

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— for the determination of the percentage of 2-glyceryl monopalmitate, the method set out in Annex VII,

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— 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 evaluation of the organoleptic characteristics of virgin olive oil, the method set out in Annex XII,

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— for the determination of stigmastadienes, the method set out in Annex XVII,

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— for determining the content of triglycerides with ECN42, the method set out in Annex XVIII,

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— for determination of the aliphatic alcohol content, the method given in Annex XIX.

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2. Verification by national authorities or their representatives of the organoleptic characteristics of virgin oils shall be effected by tasting panels 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 organoleptic characteristics, at the interested party's request the national authorities 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 characteristics concerned shall be deemed consonant with the characteristics declared if at least two of the counter-assessments confirm the declared grade. If that is not the case, the interested party shall be responsible for the cost of the counter-assessments.

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3. When the national authorities or their representatives verify the characteristics of the oil as provided for in paragraph 1, samples shall be taken in accordance with international standards EN ISO 661 on the preparation of test samples and EN ISO 5555 on sampling. However, notwithstanding point 6.8 of standard EN ISO 5555, in the case of batches of such oils in immediate packaging not exceeding 100 litres, the sample shall be taken in accordance with Annex Ia to this Regulation.

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Without prejudice to standard EN ISO 5555 and Chapter 6 of standard EN ISO 661, the samples taken shall be put in a dark place away from strong heat as quickly as possible and sent to the laboratory for analysis no 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.

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4. ►M20 For the purposes of the verification provided for in paragraph 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 durability date, the analyses shall be carried out no later than the fourth month after the month in which the sample was taken. No time limit shall apply 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 characteristics of the category of olive oil or olive-residue oil declared, the party concerned shall be notified no later than one month before the end of the period laid down in the first subparagraph.

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5. For the purpose of determining the characteristics of olive oils by the methods provided for in paragraph 1, the analysis results shall be directly compared with the limits laid down in this Regulation.

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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.

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Article ►M19 3 ◄

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 financial penalities, to be determined in the light of the seriousness of the irregularity 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 the penalties applied during the previous half-year.

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Article 4

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1. The Member States may approve assessment panels so that national authorities or their representatives can assess and verify organoleptic 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.

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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 conditions laid down in paragraph 1 and shall ensure that those conditions are complied with.

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Article 6

1. The oil content of oil cake and other residues resulting from the extraction of olive oil (CN codes 2306 90 11 and 2306 90 19) shall be determined 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.

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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.

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Article 8

1. Member States shall notify the Commission of the measures taken to implement this Regulation.

2. Member States shall send the Commission, at the beginning of each half-year, a statement of the analytical data relating to the tests carried out during the previous half-year.

The results shall be considered by the Management Committee for Oils and Fats in accordance with the procedure laid down in Article 39 of Regulation No 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 publication in the Official Journal of the European Communities.

However, the method set out in Annex XII shall apply from ►M1 1 November 1992 ◄ , except in so far as operations relating to the intervention system are concerned.

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That method shall not apply to virgin olive oil prepared for the market prior to 1 November 1992.

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2. This Regulation shall not apply to olive oil and olive-residue oil packaged before the entry into force of this Regulation and marketed up to 31 October 1992.

This Regulation shall be binding in its entirety and directly applicable in all Member States.









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 tree Annex II: ►M21 Determination of free fatty acids, cold method ◄ Annex III: Determination of the peroxide value Annex IV: ►M6 Determination of wax content by capillary column gas-liquid chromatography ◄ Annex V: Determination of the composition and content of sterols by capillary-column gas chromatography Annex VI: Determination of erythrodiol and uvaol Annex VII: ►M21 Determination of the percentage of 2-glyceryl monopalmitate ◄ Annex VIII: Determination of composition of trilinolein Annex IX: Spectrophotometric investigation in the ultraviolet Annex XA: Analysis by gas chromatography of methyl esters of fatty acids Annex XB: Preparation of methyl esters of fatty acids Annex XI: Determination of the volatile halogenated solvents of olive oil Annex XII: Organoleptic assessment of virgin olive oil Annex XIII: ►M6 Neutralization and decolorization of olive oil in the laboratory ◄ Annex XIV: Additional Notes 2, 3 and 4 to Chapter 15 of the combined nomenclature 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 ECN42

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ANNEX I

OLIVE OIL CHARACTERISTICS







Category Acidity (%) (*) Peroxide index mEq O2/kg (*) Waxes mg/kg (**) 2-glyceril monopalmitate (%) Stigmastadiene mg/kg (1) Difference: ECN42 (HPLC) and ECN42 (theoretical calculation) K232 (*) K270 (*) Delta-K (*) Organoleptic evaluation median defect (Md) (*) Organoleptic evaluation fruity median (Mf) (*) 1. Extra virgin olive oil ≤ 0,8 ≤ 20 ≤ 250 ≤ 0,9 if total palmitic acid % ≤ 14 ≤ 1,0 if total palmitic acid % > 14 ≤ 0,10 ≤ 0,2 ≤ 2,50 ≤ 0,22 ≤ 0,01 Md = 0 Mf > 0 2. Virgin olive oil ≤ 2,0 ≤ 20 ≤ 250 ≤ 0,9 if total palmitic acid % ≤ 14 ≤ 1,0 if total palmitic acid % > 14 ≤ 0,10 ≤ 0,2 ≤ 2,60 ≤ 0,25 ≤ 0,01 Md ≤ 2,5 Mf > 0 3. Lampante olive oil > 2,0 — ≤ 300 (3) ≤ 0,9 if total palmitic acid % ≤ 14 ≤ 1,1 if total palmitic acid % > 14 ≤ 0,50 ≤ 0,3 — — — Md > 2,5 (2) — 4. Refined olive oil ≤ 0,3 ≤ 5 ≤ 350 ≤ 0,9 if total palmitic acid % ≤ 14 ≤ 1,1 if total palmitic acid % > 14 — ≤ 0,3 — ≤ 1,10 ≤ 0,16 — — 5. Olive oil composed of refined and virgin olive oils ≤ 1,0 ≤ 15 ≤ 350 ≤ 0,9 if total palmitic acid % ≤ 14 ≤ 1,0 if total palmitic acid % > 14 — ≤ 0,3 — ≤ 0,90 ≤ 0,15 — — 6. Crude olive-residue oil — — > 350 (4) ≤ 1,4 — ≤ 0,6 — — — — — 7. Refined olive-residue oil ≤ 0,3 ≤ 5 > 350 ≤ 1,4 — ≤ 0,5 — ≤ 2,00 ≤ 0,20 — — 8. Olive-residue oil ≤ 1,0 ≤ 15 > 350 ≤ 1,2 — ≤ 0,5 — ≤ 1,70 ≤ 0,18 — —







Category Acid content (1) Total transoleic isomers (%) Total translinoleic + translinolenic isomers (%) Sterols composition Total sterols (mg/kg) Erythrodiol and uvaol (%) (**) Myristic (%) Linolenic (%) Arachidic (%) Eicosenoic (%) Behenic (%) Lignoceric (%) Cholesterol (%) Brassicasterol (%) Campesterol (%) Stigmasterol (%) Betasitosterol (%) (2) Delta-7-stigmastenol (%) 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. 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 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. Olive oil composed of refined and virgin olive 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-residue 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-residue 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-residue 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

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ANNEX Ia

Sampling of olive oil or olive-pomace oil delivered in immediate packaging not exceeding 100 litres

This method of sampling applies to deliveries of olive oil or olive-pomace oil not exceeding 125 000 litres, put up in immediate packaging not exceeding 100 litres.

If the delivery exceeds 125 000 litres, it is to be subdivided into batches of 125 000 litres or under. If the delivery is less than 125 000 litres it shall constitute 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 in accordance with the table set out in point 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 given in standard 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 considered 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 25 000 3 75 000 4 125 000 5

The immediate packs selected to form a primary sample must be adjacent to each other in the batch.

In cases of doubt, Member States shall increase the number of primary samples to be taken.

2. CONTENT OF PRIMARY SAMPLES

2.1 Primary samples must comprise the following:







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 than 5 litres (b) 3 immediate packs (c) 2 litres or more but less than 3 litres (c) 3 immediate packs (d) 1 litre or more but less than 2 litres (d) 6 immediate packs (e) 0,75 litres or more but less than 1 litre (e) 6 immediate packs (f) less than 0,75 litres (f) three times the oil from the minimum number of packs with a total capacity of more than 1,5 litres

2.2 The 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 carry out:

(a) the analyses referred to in Annexes II, III, IX and X,

(b) the analysis referred to in Annex XII,

(c) the other analyses.

2.3 The packs constituting a primary sample shall be subdivided in accordance with the control procedures provided for in national law.

3. ANALYSES AND RESULTS

(a) Each of the primary samples referred to in point 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 paragraph 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.

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ANNEX Ib

DECISION TREE FOR VERIFYING WHETHER AN OLIVE OIL SAMPLE IS CONSISTENT WITH THE CATEGORY DECLARED

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 carrying out in the order shown in the decision tree the analyses specified 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 decision tree applies to all categories of olive oil and 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-compliance,

— the headings in the boxes in tables 1 to 11 refer to the analyses provided for in this Regulation on the basis of the table of equivalence set out in Appendix 1 to this Annex,

— the letters in brackets appearing in the negative decision circles in tables 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.

General table

Table 1

Table 2

Table 3

Table 4

Table 5

Table 6

Table 7

Table 8

Table 9

Table 10

Table 11









Appendix 1

Table of equivalence between the annexes to this Regulation and the analyses specified in the decision tree







▼M21 — Acidity Annex II Determination of free fatty acids, cold method ▼M20 — 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 stigmastadienes 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 — ΔECN42 Annex XVIII Determination of the composition of triglycerides with ECN42 (difference between the HPLC data and theoretical content) — Sterols composition and total sterols Annex V Determination of the composition and content of sterols by capillary-column gas chromatography — Erythrodiol and Uvaol Annex VI Determination of erythrodiol and uvaol ▼M21 — Saturated fatty acids in position 2 Annex VII Determination of the percentage of 2-glyceryl monopalmitate ▼M20 — 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 in position 2 of the triglyceride









Appendix 2

Table 1

(a) See virgin or lampante olive oil (Quality criteria Table 2, or Quality and purity criteria Table 4)

(b) See lampante olive oil (Quality and purity criteria Table 4)

Table 2

(a) See lampante olive oil (Quality and purity criteria Table 4)

(b) See extra virgin olive oil (Quality criteria Table 1)

Table 3

(a) Presence of refined oil (olive or others)

(b) Presence of olive-pomace oil

Table 4

(a) See extra virgin olive oil and virgin olive oil (Quality criteria Table 1 and Table 2)

(b) Presence of refined oil (olive or others)

(c) Presence of olive-pomace oil

(d) Presence of esterified oils

Table 7

(a) Presence of olive-pomace oil

(b) Presence of esterified oils

Table 8

(a) Presence of refined oil (olive or others)

(b) See lampante olive oil (Quality and purity criteria Table 4)

(c) Presence of esterified oils

Table 11

(a) Presence of esterified oils

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ANNEX II

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DETERMINATION OF FREE FATTY ACIDS, COLD METHOD

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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. ►C1 Diethyl ether ◄ ; 95 % ethanol (v/v), mixture of equal parts by volume. Note: ►C1 Diethyl ether ◄ is highly inflammable and may form explosive peroxides. Special care should be taken in its use. Neutralize precisely at the moment of use with the potassium hydroxide solution (1.2.2), with the addition of 0,3 ml of the phenolpthalein solution (1.2.3) per 100 ml of mixture. Note: If it is not possible to use ►C1 diethyl 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 solution should be colourless or straw coloured. Note: A stable colourless solution of 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 in the distillate the required quantity of potassium hydroxide. Leave for several days and decant the clear supernatant liquid from the precipitate of potassium carbonate. The solution may also be prepared without distillation as 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 solution is ready for use.

1.2.3. Phenolphthalein, 10 g/l solution in 95 to 96 % ethanol (v/v) or alkaline blue, (in the case of strongly coloured fats) 20 g/l solution in 95 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 in 0,05 ml.

1.4. Procedure

1.4.1. Preparation of the specimen for testing (Carry out the test on the filtered sample. Where moisture and impurities together are less than 1 %, use the specimen without further treatment; where they exceed 1 %, it should be filtered.)

1.4.2. Taking the sample Take a sample depending on the presumed acid number in accordance with the following table:



Expected acid value Mass 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 in the conical flask (1.3.2).

1.4.3. Determination Dissolve the sample (1.4.2) in 50 to 150 ml of the previously neutralized mixture of diethyl ►C1 ether ◄ 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 an aqueous solution of 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: expressed as percentage of oleic acid

Acidity as a percentage by weight is equal to:

where:

V = the volume of titrated potassium hydroxide solution used, in millilitres;

c = the exact concentration in moles per litre of the titrated solution of potassium hydroxide used;

M = the molar weight in grams per mole of the acid used to express the result (= 282);

m = the weight in grams of the sample.

▼C1

Take as the result, the arithmetic mean ►M6 of two calculations ◄ carried out.

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ANNEX III

DETERMINATION OF PEROXIDE VALUE

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 in terms of milliequivalents of active oxygen per 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 standardized 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, carbon dioxide).

5.3. 25- or 50-ml burette, graduated in 0,1 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, ►C1 dry 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 ►C1 Mol/L accurately ◄ standardized 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 taken and 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 in diffuse daylight or in artificial 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:







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 °C.

Add about 75 ml of distilled water. Titrate the liberated iodine with the sodium thiosulphate solution ►C1 (6.4) (0,002 Mol/L solution for expected values less than 12, and 0,01 Mol/L solution ◄ for expected values above 12) shaking vigorously, using starch solution (6.5) as indicator.

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 ►C1 0,01 mol/L sodium ◄ thiosulphate solution (6.4), replace the impure reagents.

9. EXPRESSION OF RESULTS

The peroxide value (PV), expressed in milliequivalents of active oxygen per kilogram, is given by the formula:

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 = ►C1 the exact molarity ◄ of the sodium thiosulphate solution (6.4) used;

m = the weight in g, of the test portion.

Take as the result the arithmetic mean of the two determinations carried out.

▼M21









ANNEX IV

DETERMINATION OF WAX CONTENT BY CAPILLARY COLUMN GAS CHROMATOGRAPHY

1. SUBJECT

This method describes a process for determining the wax content of olive oils. Waxes are separated according to the number of their carbon atoms. The method may be used in particular to distinguish between olive oil obtained by pressing and that obtained by extraction (olive-residue oil).

2. PRINCIPLE

Addition of a suitable internal standard to the fat or oil, then fractionation by chromatography on a hydrated silica gel column. Recovery under the test conditions of the fraction eluted first (the polarity of which is less than that of the triglycerides), then direct analysis by capillary column gas chromatography.

3. EQUIPMENT

3.1. 25 ml Erlenmeyer flask.

3.2. Glass column for gas chromatography, internal diameter 15,0 mm, length 30 to 40 cm, fitted with a stopcock.

3.3. Suitable gas chromatograph with a capillary column, equipped with a system for direct introduction into the column comprising the following: 3.3.1. Thermostatic chamber for the columns, equipped with a temperature programmer. 3.3.2. Cold injector for direct introduction into the column. 3.3.3. Flame ionisation detector and converter-amplifier. 3.3.4. Recorder-integrator capable of working with the converter-amplifier (3.3.3), rate of response no slower than 1 second, with variable paper speed. (It is also possible to use computerised systems that allow the acquisition of gas chromatography data via a PC.) 3.3.5. Glass or fused silica capillary column 8 to 12 m long and with an internal diameter of 0,25 to 0,32 mm, with liquid phase, with a uniform film thickness between 0,10 and 0,30 μm. (There are liquid phases suitable for the purpose of type SE-52 or SE-54 available on the market.)

3.4. 10 μl microsyringe for on-column injection, equipped with a hardened needle.

3.5. Electrovibrator.

3.6. Rotary evaporator.

3.7. Muffle furnace.

3.8. Analytical balance with guaranteed precision of ± 0,1 mg.

3.9. Normal laboratory glassware.

4. REAGENTS

4.1. Silica gel with a granule size of between 60 and 200 μm. Place the gel in the furnace at 500 °C for at least four hours. After cooling, add 2 % water in relation to the quantity of sampled silica gel. Shake well to homogenise the 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). (It is also possible to use palmityl palmitate or myristyl stearate.) 4.5.1. Sudan 1 (1-phenyl-azo-2-naphthol).

4.6. Carrier gas: hydrogen or helium, gas-chromatographic purity.

4.7. Auxiliary gases: — pure hydrogen for gas chromatography, — pure air for gas chromatography.

5. PROCEDURE

5.1. Preparation of the chromatographic column.

Suspend 15 g of silica gel (4.1) in the n-hexane (4.2) and introduce it into the column (3.2). Allow to settle spontaneously. Complete settling with the aid of an electrovibrator (3.5) to make the chromatographic layer more homogeneous. Percolate 30 ml of n-hexane to remove any impurities. Using the balance (3.8) weigh exactly 500 mg of the sample into the 25 ml Erlenmeyer flask (3.1), add the appropriate quantity of the internal standard (4.5) according to the presumed wax content. For example, add 0,1 mg of lauryl arachidate for olive oil, and 0,25 to 0,5 mg for olive-residue oil. Transfer the prepared sample to the chromotography column using two 2 ml portions of n-hexane (4.2).

Allow the solvent to flow away until it reaches 1 mm above the upper level of the absorbant then percolate a further 70 ml of n-hexane in order to eliminate the n-alkanes naturally present. Then start the chromatographic elution by collecting 180 ml of the mixture of n-hexane/ethyl ether (ratio 99:1), keeping a rate of flow of approximately 15 drops every 10 seconds. Elution of the sample must be carried out at a room temperature of 22 ± 4 °C.

NB:

— The n-hexane/ethyl ether mixture (99:1) must be prepared every day.

— For a visual check on the correct elution of the waxes 100 μl of 1 % Sudan in the elution mixture can be added to the sample in solution. Since the colourant has an intermediate retention, between waxes and triglycerides, when the coloration has reached the bottom of the column the elution should be suspended because all the waxes will have been eluted.

Dry the fraction thus obtained in a rotary evaporator (3.6.) until virtually all the solvent has been eliminated. Eliminate the final 2 ml of solvent with the aid of a weak current of nitrogen; then add 2-4 ml n-heptane.

5.2. Analysis by gas chromatography

5.2.1. Preparatory work

Fit the column to the gas chromatograph (3.3) by connecting the inlet port to the on-column system and the outlet port to the detector. Perform a general check on the GC apparatus (operation of gas circuits, detector and recorder efficiency, etc.).

If the column is being used for the first time it should be conditioned first. Pass a little gas through the column, then turn on the GC apparatus. Heat gradually until 350 °C is reached after about four hours. Maintain that temperature for at least two hours then regulate the apparatus to operating conditions (set gas flow, light flame, connect to the electronic recorder (3.3.4), set temperature of column chamber, detector, etc.) and record the signal at a sensitivity at least twice as high as that required for the analysis. The baseline must be linear, with no peaks of any kind, and must not show any deviation.

A negative straight-line drift indicates that the column connections are not tight; a positive drift that the column has not been sufficiently conditioned.

5.2.2. Choice of operating conditions

The operating conditions are generally as follows:

— column temperature: —



20 °C/minute 5 °C/minute 20 °C/minute Initially 80 °C (1′) → 240 °C → 325 °C (6′) → 340 °C (10′)

— detector temperature: 350 °C;

— quantity of substance injected: 1 μl of the n-heptane solution (2-4 ml);

— carrier gas: helium or hydrogen at the correct linear velocity for the gas selected (see Appendix);

— instrument sensitivity: suitable for the following conditions:

The conditions may be modified according to the characteristics of the column and the GC apparatus to obtain separation of all the waxes and a satisfactory peak resolution (see figure); the internal standard C32 retention time must be 18 ± 3 minutes. The most representative wax peak must be at least 60 % of the full scale.

The peak integration parameters must be established so as to obtain a correct evaluation of the areas of the peaks in question.

NB: Given the high final temperature, a positive drift of no more than 10 % of the full scale is permitted.

5.3. Performance of the analysis

Sample 1 μl of the solution using the 10 μl microsyringe; withdraw the syringe plunger so that the needle is empty. Place the needle in the injector and after 1-2 seconds inject quickly; remove the needle slowly after about five seconds.

Record until the waxes are completely eluted.

The base line must always satisfy the required conditions.

5.4. Identification of peaks

Identification of the different peaks should be based on retention time by comparison with wax mixtures of known retention times analysed under the same conditions.

The figure is a chromatogram of the waxes of a virgin olive oil.

5.5. Evaluation of quantity

Calculate the areas of the peaks of the internal standard and the aliphatic esters of C40 to C46 using the integrator.

Calculate the wax content of each of the esters in mg/kg fat using the formula:

where:

Ax = area of each ester’s peak, in square millimetres;

As = area of the internal standard’s peak, in square millimetres;

ms = mass of added internal standard, in milligrams;

m = mass of sample for analysis, in grams.

6. EXPRESSION OF RESULTS

Indicate the total of the contents of the various C40 to C46 waxes in mg/kg fat (ppm).

NB: The components to be quantified refer to the peaks with carbon pair numbers between esters C40 and C46, using the example of the olive oil wax chromatogram shown in the figure below. If ester C46 appears twice, it is recommended that to identify it the fraction of the waxes of an olive-residue oil should be analysed where the C46 peak is easy to identify because it is in the clear majority.

The results should be expressed to one decimal place.

Figure

Chromatogram of the waxes of an olive oil ( 5 )

Key:

I.S. = Lauryl arachidate

1. = Diterpenic esters

2 + 2′ = C40 esters

3 + 3′ = C42 esters

4 + 4′ = Esters C44

5. = C46 esters

6. = Sterol esters and triterpenic alcohol









Appendix

Determination of the linear velocity of the gas

Inject 1-3 μl methane (or propane) into the GC apparatus after it has been regulated to normal operating conditions. Measure the time it takes for the gas to flow through the column from the time it is injected to the time the peak appears (tM).

The linear velocity in cm/s is given by the formula L/tM, where L is the length of the column in cm and tM the time measured in seconds.

▼B









ANNEX V

DETERMINATION OF THE COMPOSITION AND CONTENT OF STEROLS BY CAPILLARY-COLUMN GAS CHROMATOGRAPHY

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 unsaponifiables are then extracted with ►C1 diethyl ether ◄ .

The sterol fraction is separated from the unsaponifiable extract by chromatography on a basic silica gel plate. The sterols recovered from the silica gel are transformed into trimethyl-silyl ethers and are analysed by capillary-column gas 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 an accuracy of ± 1 °C; 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 variable 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, ►C1 approximately 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-stoppered dark glass bottles.

4.2. ►C1 Diethyl 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).

4.5. Potassium hydroxide, ►C1 0,2 mol/L ◄ ethanolic solution. Dissolve 13 g of potassium hydroxide in 20 ml of distilled water and 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. ►C1 Diethyl 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, ►M6 2 % ◄ solution in chloroform.

4.12. 2,7-dichlorofluorescein, 0,2 % ethanolic solution. Make slightly basic by adding a few drops of ►C1 2 mol/L ◄ alcoholic potassium hydroxide 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. ►C1 β-cholestanol ◄ , 0,2 % solution (m/V) in chloroform (internal standard).

4.18. Carrier gas: hydrogen or 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 ►C1 introduce, 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 ►C1 dryness in a current ◄ of nitrogen and then weigh accurately 5 g of the dry filtered sample into the same flask. ►M6 Oils ◄ containing appreciable quantities of cholesterol may show a peak having a retention time identical to cholestanol. If this occurs the sterol fraction will have to be analyzed in duplicate with and without internal standard ►M6 or betulinol will have to be used instead of cholestanol ◄ . 5.1.2. Add 50 ml of ►C1 2 mol/L ◄ ethanolic potassium hydroxide solution, fit the reflux condenser and heat to gentle boiling on a water bath with continuous vigorous stirring until saponification takes place (the solution becomes clear). Continue heating for a further 20 minutes, then add 50 ml of ►C1 distilled water to the ◄ top of the condenser, detach the condenser and cool the flask to approximately 30 °C. 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 ►C1 diethyl ether ◄ , shake vigorously for approximately 30 seconds and allow to settle (Note 1). Separate off the lower aqueous phase collecting it in a second separating 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. When the wash water has been removed, dry with anhydrous sodium sulphate and ►C1 filter through anhydrous ◄ sodium sulphate into a previously weighed 250 ml flask, washing the funnel and filter with small quantities of ►C1 diethyl 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 °C for approximately a quarter of an hour, and then weigh after cooling in a desiccator.

5.2. Separation of the sterol fraction. 5.2.1. Preparation of the basic plates. Immerse the silica gel plates (4.4) completely in the ►C1 0,2 mol/L ◄ ethanolic potassium hydroxide solution (4.5) for 10 seconds, then allow to dry in a fume cupboard for two hours and finally place ►C1 in an oven at ◄ 100 °C for one hour. Remove from the stove and keep in a calcium chloride desiccator until required for use (plates treated in this way must be used within 15 days). Note 2. When basic silica gel plates are used to separate the sterol fraction 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 ►C1 mixture into the ◄ plate-developing chamber to a depth of approximately 1 cm. As an alternative 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 an hour 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 approximately 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 chromatographic 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 developing. 5.2.4. Place the plate in the developing chamber prepared as specified in 5.2.2. The ambient temperature should be maintained between 15 and 20 °C. 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 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 ►C1 in 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 °C for approximately 10 minutes and then allow to cool in a desiccator and weigh. The residue contained in the test tube consists of the sterol fraction.

5.3. Preparation of the trimethylsilyl ethers. 5.3.1. Add the silylation reagent, consisting of a 9:3:1 (v/v/v) mixture of pyridine/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-trimethylsilyl, 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 solution is ready for gas chromatographic analysis. 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 deterioration of the reagent. 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 °C above the operating 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 connections; a positive drift indicates inadequate conditioning of the column. Note 6. The conditioning temperature must always be at least 20 °C less than the maximum temperature specified for the stationary phase used. 5.4.2. Choice of operating conditions. 5.4.2.1. The guideline operating conditions are as follows: — column temperature: 260 ± 5 °C, — evaporator temperature: 280 °C, — detector temperature: 290 °C, — 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-chromatograph characteristics so as to obtain chromatograms 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 separated 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. 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 completely 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 comparison with mixtures of sterol TMSE analysed under the same conditions. The sterols are eluted in the following order: cholesterol, brassicasterol, 24-methylene cholesterol, campesterol, campestanol, stigmasterol, Δ 7-campesterol, Δ 5,23-stigmastadienol, ►C1 chlerosterol ◄ , β-sistosterol, sitostanol, Δ 5-avenasterol, Δ 5,24-stigmastadienol ►C1 , Δ 7-stigmasterol, ◄ Δ 7-avenasterol. The retention times for sitosterol for SE-52 and SE-54 columns are shown in Table 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 ►C1 β-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 ►C1 β-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: where: Ax = peak area for sterol x ►M6 ————— ◄ ; As = area of the ►C1 β-cholestanol ◄ peak ►M6 ————— ◄ ; ms = mass of ►C1 β-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. where: Ax = peak area for x; A = total peak area for sterols.









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 column from the time of injection to 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 tM is the measured time in seconds.







Table I Relative retention times for sterols Peak Identification Relative retention time SE 54 column SE 52 column 1 cholesterol Δ-5-cholesten-3β-ol 0,67 0,63 2 cholestanol 5α-cholestan-3β-ol 0,68 0,64 3 brassicasterol [24S]-24-methyl-Δ-5,22-cholestadien-3β-ol 0,73 0,71 4 24-methylene-cholesterol 24-methylene-Δ-5,24-cholesten-3β-ol 0,82 0,80 5 campesterol [24R]-24-methyl-Δ-5-cholesten-3β-ol 0,83 0,81 6 campestanol [24R]-24-methyl-cholestan-3β-ol 0,85 0,82 7 stigmasterol [24R]-24-ethyl-Δ-5,22-cholestadien-3β-ol 0,88 0,87 8 Δ-7-campesterol [24R]-24-methyl-Δ-7-cholesten-3β-ol 0,93 0,92 9 Δ-5,23-stigmastadienol [24R,S]-24-ethyl-Δ-5,23-cholestadien-3β-ol 0,95 0,95 10 chlerosterol [24S]-24-ethyl-Δ-5,25-cholastadien-3β-ol 0,96 0,96 11 β-sitosterol [24R]-24-ethyl-Δ-5-cholestan-3β-ol 1,00 1,00 12 sitostanol 24-ethyl-cholestan-3β-ol 1,02 1,02 13 Δ-5-avenasterol [24Z]-24-ethylidene-5-cholesten-3β-ol 1,03 1,03 14 Δ-5,24-stigmastadienol [24R,S]-24-ethyl-Δ-5,24-cholestadien-3β-ol 1,08 1,08 15 Δ-7-stigmastenol [24R,S]-24-Ethyl-Δ-7,24-cholestadien-3β-ol 1,12 1,12 16 Δ-7-avenasterol [24Z]-24-ethyliden-Δ-7-cholesten-3β-ol 1,16 1,16

Figure 1

Gas chromatogram of the sterol fraction of an unrefined olive oil

Figure 2

Gas chromatogram of the sterol fraction of a refined olive oil









ANNEX VI

DETERMINATION OF ERYTHRODIOL AND UVAOL

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 concentrations 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 ►C1 hydroxide in ethanolic 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. PROCEDURE

5.1. Preparation of the unsaponifiables.

As described at paragraph 5.1.2 of Annex V.

5.2. Separation of erythrodiol and the sterols.

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 solution approximately 1,5 cm from the lower edge in a streak which is as thin and uniform as possible. At one end of the plate place a few microlitres of the solutions of cholesterol and erythrodiol to serve as a reference.

5.2.4. Place the plate in the developing chamber prepared as specified in 5.2.1. The ambient temperature should be about 20 °C. 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-dichlorofluoroscein 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 fluorescence.

5.2.6. Using a metal spatula scrape off the silica gel in the marked areas. Place the material from the plate in a 50 ml flask. Add 15 ml of hot chloroform, 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. Preparation of the trimethylsily esters

As described in paragraph 5.3 of Annex V.

5.4. Gas chromatographic analysis

As described in paragraph 5.4 of the above method. The operating conditions 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

where:

A1 = peak area for erythrodiol ►M6 ————— ◄ ;

A2 = peak area for uvaol ►M6 ————— ◄ ;

Asterols = total peak area for sterols ►M6 ————— ◄ .

The result is expressed to one decimal place.

▼M21









ANNEX VII

DETERMINATION OF THE PERCENTAGE OF 2-GLYCERYL MONOPALMITATE

1. PURPOSE AND SCOPE

This method describes the analysis procedure for determining the percentage of palmitic acid in position 2 of the triglycerides by evaluating 2-glyceryl monopalmitate.

This method can be applied to liquid vegetable oils at ambient temperature (20 °C).

2. PRINCIPLE

After preparation the oil sample is subjected to the action of pancreatic lipase: partial and specific hydrolysis in positions 1 and 3 of the triglyceride molecule causes monoglycerides to appear in position 2. The percentage of 2-glyceryl monopalmitate in the monoglyceride fraction is determined after silylation by capillary-column gas chromatography.

3. APPARATUS AND MATERIALS

3.1. 25 ml Erlenmeyer flask

3.2. 100, 250 and 300 ml beakers

3.3. Glass chromatograph column, internal diameter 21-23 mm, length 400 mm, fitted with a sintered glass disc and a stopcock

3.4. 10, 50, 100 and 200 ml measuring cylinders

3.5. 100 and 250 ml flasks

3.6. Rotary evaporator

3.7. 10 ml conical-bottomed centrifuge tubes with groundglass stopper

3.8. Centrifuge for 10 and 100 ml tubes

3.9. Thermostat permitting a stable temperature of 40 ± 0,5 °C

3.10. 1 and 2 ml graduated pipettes

3.11. 1 ml hypodermic syringe

3.12. 100 μl microsyringe

3.13. 1 000 ml funnel

3.14. Capillary gas chromatograph with an on-column cold injector for direct injection of the sample into the column and a furnace able to maintain the selected temperature to approximately 1 °C

3.15. On-column cold injector for direct injection of the sample into the column

3.16. Flame ionisation detector and electrometer

3.17. Recorder-integrator adapted to the electrometer with a response rate no greater than 1 sec and a variable paper roll rate

3.18. Capillary column made of glass or fused silica 8-12 metres long, 0,25-0,32 mm internal diameter, covered with methylpolysiloxane or phenyl methylpolysiloxane 5 %, 0,10-0,30 μm thick, useable at 370 °C

3.19. 10 μl microsyringe fitted with a hardened needle, at least 7,5 cm long for direct on-column injection.

4. REAGENTS

4.1. Silica gel with a grain size of between 0,063 and 0,200 mm (70/280 mesh) prepared as follows: Place the silica gel in a porcelain capsule, dry in an incubator at 160 °C for four hours, then leave to cool at room temperature in a desiccator. Add water equivalent to 5 % of the mass of the silica gel as follows: Weigh 152 g silica gel into an Erlenmeyer flask then add 8 g of distilled water, stopper and shake gently to distribute the water evenly. Leave to stand for at least 12 hours before use.

4.2. n-hexane (for chromatography)

4.3. Isopropanol

4.4. Isopropanol, 1/1 (v/v) aqueous solution

4.5. Pancreatic lipase. It must have an activity of between 2,0 and 10 lipase units per mg. (Pancreatic lipases with an activity of between 2 and 10 units per mg enzyme are commercially available.)

4.6. Buffer solution of trishydroxymethylaminomethane: 1 M aqueous solution adjusted to pH 8 (potentiometric control) by conc. HCl (1/1 v/v)

4.7. Enzyme-quality sodium cholate, 0,1 % aqueous solution (this solution must be used within two weeks of its preparation)

4.8. Calcium chloride, 22 % aqueous solution

4.9. Diethyl ether for chromatography

4.10. Developer solvent: mixture of n-hexane/diethyl ether (87:13 v:v)

4.11. Sodium hydroxide, 12 % by weight solution

4.12. Phenolphthalein, 1 % solution in ethanol

4.13. Carrier gas: hydrogen or helium, for gas chromatography

4.14. Auxiliary gases: hydrogen, 99 % minimum purity, free from moisture and organic substances, and air, for gas chromatography, of the same purity

4.15. Silanisation reagent: mixture of pyridine/hexamethyldisilazane, trimethylchlorosilane 9/3/1 (v/v/v). (Ready-to-use solutions are commercially available. Other silylation reagents may be used, particularly bis-trimethylsilyl trifluoracetamide + 1 % trimethylchlorosilane, diluted with an identical volume of anhydrous pyridine.)

4.16. Reference samples: pure monoglycerides or monoglyceride mixtures with a known percentage composition similar to that of the sample.

5. METHOD

5.1. Sample preparation

5.1.1. Oils with a free acidity of less than 3 % do not need to be neutralised before chromatography on a silica gel column. Oils with a free acidity of more than 3 % must be neutralised as per point 5.1.1.1. 5.1.1.1. Pour 50 g of oil and 200 ml n-hexane into the 1 000 ml funnel (3.13). Add 100 ml of isopropanol and a quantity of 12 % sodium hydroxide solution (4.11) equivalent to the free acidity of the oil plus 5 %. Shake vigorously for one minute. Add 100 ml of distilled water, shake again and leave to stand. After decanting, remove the lower layer containing the soaps. Remove any intermediate layers (mucilage and insoluble substances). Wash the hexane solution of the neutralised oil with successive portions of 50-60 ml of the 1/1 (v/v) isopropanol/water solution (4.4) until the pink colouration of the phenolphthalein disappears. Remove most of the hexane by vacuum distillation (use a rotary evaporator, for example) and transfer the oil into a 100 ml flask (3.5). Dry the oil in vacuum until the solvent is completely removed. After that procedure is completed, the acidity of the oil should be less than 0,5 %.

5.1.2. Put 1,0 g of the oil prepared as above into a 25 ml Erlenmeyer flask (3.1) and dissolve in 10 ml of developer mixture (4.10). Leave the solution to stand for at least 15 minutes before silica gel column chromatography. If the solution is cloudy centrifuge it to ensure optimum conditions for chromatography. (Ready-to-use 500 mg silica gel SPE cartridges can be used).

5.1.3. Preparation of the chromatography column Pour about 30 ml of the developer solvent (4.10) into the column (3.3), insert a piece of cotton into the bottom part of the column using a glass rod; press to eliminate the air. In a beaker prepare a suspension of 25 g of silica gel (4.1) in about 80 ml of developer solvent and pour it into the column using a funnel. Check that all the silica gel is in the column; wash with developer solvent (4.10), open the stopcock and allow the liquid to reach a level about 2 mm above the level of the silica gel.

5.1.4. Column chromatography Weigh accurately 1,0 g of sample prepared as in point 5.1 into a 25 ml Erlenmeyer flask (3.1). Dissolve the sample in 10 ml of developer solvent (4.10). Pour the solution into the chromatography column prepared as in point 5.1.3. Avoid disturbing the surface of the column. Open the stopcock and pour the sample solution until it reaches the level of the silica gel. Develop with 150 ml of the developer solvent. Adjust the flow rate to 2 ml/min (so that 150 ml enters the column in about 60-70 minutes). Recover the eluate in a previously weighed 250 ml flask. Evaporate the solvent under vacuum and remove the final traces of the solvent under a nitrogen current. Weigh the flask and calculate the recovered extract. (If ready-to-use silica gel SPE cartridges are used use the following method: Put 1 ml of solution (5.1.2) into the prepared cartridges with 3 ml of n-hexane. After percolating the solution develop with 4 ml of n-hexane/diethyl ether 9/1 (v/v). Recover the eluate in a 10 ml tube and evaporate to dry in a nitrogen current. Expose the dry residue to pancreatic lipase (5.2). (It is essential to check the fatty acid composition before and after crossing the SPE cartridge.)

5.2. Hydrolysis by pancreatic lipase

5.2.1. Weigh into the centrifuge tube 0.1 g of the oil prepared as in point 5.1. Add 2 ml of buffer solution (4.6), 0,5 ml of the sodium cholate solution (4.7) and 0,2 ml of the calcium chloride solution, stirring well after each addition. Close the tube with the groundglass stopper and place in the thermostat at 40 + 0,5 °C.

5.2.2. Add 20 mg of lipase, shake carefully (avoid wetting the stopper) and place the tube in the thermostat for exactly two minutes. Then remove it, shake vigorously for exactly 1 minute and leave to cool.

5.2.3. Add 1 ml of diethyl ether, stopper and shake vigorously, then centrifuge and transfer the ether solution into a clean, dry tube using a microsyringe.

5.3. Preparation of the silanised derivatives and gas chromatography

5.3.1. With a microsyringe insert 100 μl of solution (5.2.3) into a 10 ml conical-bottomed tube.

5.3.2. Remove the solvent under a slight nitrogen current, add 200 μl of silanisation reagent (4.15), stopper the tube and leave to stand for 20 minutes.

5.3.3. After 20 minutes, add 1 to 5 ml of n-hexane (depending on the chromatography conditions): the resulting solution is ready for gas chromatography.

5.4. Gas chromatography

Operating conditions:

— Injector temperature (on-column injector) lower than solvent boiling point (68 °C);

— Detector temperature: 350 °C;

— Column temperature: programming of furnace temperature: 60 °C for 1 minute, increasing by 15 °C per minute up to 180 °C, then by 5 °C per minute up to 340 °C, then 340 °C for 13 minutes;

— Carrier gas: hydrogen or helium, set at a linear velocity sufficient to obtain the resolution reflected in Figure 1. The retention time of the C54 triglyceride must be 40 ± 5 minutes (see Figure 2). (The operating conditions indicated above are indicative. Operators will have to optimise them to obtain the desired resolution. The peak corresponding to 2-glyceryl monopalmitate must have a minimum height equal to 10 % of the recorder scale.)

— Quantity of substance injected: 0,5-1 μl of the n-hexane solution (5 ml) (5.3.3).

5.4.1. Identification of the peaks

The individual monoglycerides are identified from their retention times and by comparison with those obtained for standard monoglyceride mixtures under the same conditions.

5.4.2. Quantitative evaluation

The area of each peak is calculated using an electronic integrator.

6. EXPRESSION OF RESULTS

The percentage of glyceryl monopalmitate is calculated from the ratio between the area of the corresponding peak and the areas of the peaks of all the monoglycerides (see Figure 2) using the formula:

where:

Ax = area of the peak corresponding to glyceryl monopalmitate

ΣA = sum of the areas of all the monoglyceride peaks

The result must be to one decimal place.

7. ANALYSIS REPORT

The analysis report must specify:

— reference to this method,

— all the information needed for a full identification of the sample,

— the analysis result,

— any deviation from the method, whether as the result of a decision by the parties concerned or for another reason,

— details to identify the laboratory, the date of the analysis and the signatures of those responsible for the analysis.

Figure 1

Chromatogram of the products of the silanisation reaction obtained by the action of lipase on a refined olive oil with 20 % esterified oil added (100 %)

Key: ‘acides gras libres’ = free fatty acids; ‘Huile d’olive raffinée + 20 % huile estérifiée’ = refined olive oil + 20 % esterified oil; ‘1-2 monopalmitoléine’ = 1-2 monopalmitolein; ‘1-2 mono C18 insat.’ = unsaturated 1-2 mono C18

Figure 2

Chromatogram of:

(A) unesterified olive oil, after lipase; after silanisation; under these conditions (8-12 m capillary column) the wax fraction is eluted at the same time as the diglyceride fraction or slightly afterwards. After lipase, the triglyceride content should not exceed 15 % Key: 1 = Free fatty acids 2 = Monoglycerides 3 = Diglycerides 4 = Triglycerides * = 2-monopalmitine ** = Triglyceride C54

Chromatogram of:

(B) unesterified oil after lipase; after silanisation; under these conditions (8-12 m capillary column) the wax fraction is eluted at the same time as the diglyceride fraction or slightly afterwards. After lipase, the triglyceride content should not exceed 15 %. Key: 1 = Free fatty acids 2 = Monoglycerides 3 = Diglycerides 4 = Triglycerides * = 2-monopalmitine ** = C54 triglyceride

8. NOTES

Lipases with satisfactory activity are commercially available. They can also be prepared in the laboratory in the following manner:

Cool to 0 °C 5 kg of fresh pig’s pancreas. Remove the surrounding solid fat and the connective tissue and grind to a liquid paste in a blender. Stir the paste with 2,5 litres of anhydrous acetone for 4-6 hours, then centrifuge. Extract the residue three more times with the same volume of anhydrous acetone, then twice with an acetone/diethyl ether mixture (1/1 v/v) and twice with diethyl ether.

Vacuum-dry the residue for 48 hours to obtain a stable powder which can be stored for a long time in a refrigerator away from moisture.

Prepare an olive oil emulsion as follows:

In a mixer stir for 10 minutes a mixture of 165 ml of a 100 g/l gum arabic solution, 15 g of crushed ice and 20 ml of a previously neutralised olive oil.

Pour 10 ml of the emulsion into a 50 ml beaker, then 0,3 ml of a 0,2 g/ml sodium cholate solution and then 20 ml of distilled water.

Put the beaker in a thermostat set at 37 °C; introduce the electrodes of the pH meter and the screw agitator.

Using a burette, add a 0,1 N sodium hydroxide solution drop by drop until a pH of 8,3 is obtained.

Add an aliquot of the lipase powder suspension in water (0,1 g/ml of lipase). As soon as the pH meter reads 8,3, start the chronometer and add the sodium hydroxide solution drop by drop at a rate which maintains the pH at 8,3. Note every minute the volume of solution consumed.

Record the data on an x/y graph with the time on the x-axis and millilitres of 0,1 N alkaline solution consumed to keep a constant pH on the y-axis. A linear graph should be obtained.

Lipase activity, expressed in lipase units per mg, is given by the following formula:

where:

A is activity in lipase units/mg

V is the number of millilitres of 0,1 N sodium hydroxide solution per minute (calculated on the basis of the graph)

N is the titre of the sodium hydroxide solution

m is the mass in mg of the test lipase.

A lipase unit is defined as the quantity of enzyme which releases 10 micro-equivalents of acid per minute.

▼M20 —————

▼B









ANNEX IX

SPECTROPHOTOMETRIC INVESTIGATION IN THE ULTRAVIOLET

FOREWORD

Spectrophotometric examination in the ultraviolet can provide information on the quality of a fat, its state of preservation and changes brought about in it by technological processes.

The absorption at the wavelengths specified in the method is due to the presence of conjugated diene and triene systems. These absorptions are expressed as specific extinctions E1 % 1 cm (the extinction of 1 % solution of the fat in the specified solvent, in a thickness of 1 cm) conventionally indicated by K (also referred to as ‘extinction coefficient’).

1. SCOPE

The method describes the procedure for performing a spectrophotometric examination ►C1 of 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 spectrophotometer 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.

▼M6

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.

▼B

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. ▼M6 ————— ▼B

4.2. Basic alumina for column chromatography prepared and checked as described in Appendix 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 °C, hard fats are to be homogenized and filtered at a temperature of not more than 10 °C above the melting point.

5.2. Weigh accurately approximately 0,25 g of the sample so prepared into a 25 ml graduated flask, make up to the mark with the solvent specified and homogenize. The resulting solution must be perfectly clear. If opalescence or turbidity is present filter quickly through paper.

5.3. Fill a cuvette with the solution obtained and measure the extinctions at an appropriate wavelength between 232 and 276 nm, using the solvent used as a reference. 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 °C. 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: where: Kλ = specific extinction at wavelength λ; 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: where Km is the specific extinction at wavelength m, the wavelength for maximum absorption around 270 nm.









APPENDIX I

Preparation of the alumina and testing its activity

A.1.1. Preperation of the alumina

Place alumina which has been previously desiccated in a furnace at 380 to 400 °C for three hours into a hermetically sealed container, add distilled water in the ratio of 5 ml per 100 g of alumina, 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 column the mixture has a specific extinction of more than 0,11 at 268 nm the alumina is acceptable, if not the level of dehydration must be increased.









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 spectrophotometry and dissolve in 0,05 N potassium hydroxide solution in a 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.









ANNEX X A

ANALYSIS BY GAS CHROMATOGRAPHY OF METHYL ESTERS OF FATTY ACIDS

1. SCOPE

This method gives general guidance for the application of gas chromatography, 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. Carrier gas

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. Auxiliary gases

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 chromatography, employing packed and/or capillary columns and a flame-ionization detector. Any apparatus giving the efficiency and resolution specified in 4.1.2 is suitable.

3.1. Gas chromatograph

The gas chromatograph shall comprise the following elements.

3.1.1. Injection system

Use an injection system 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 °C higher than that 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 on column injector type.

Note 2.

In the absence of fatty acids with less than 16 carbon atoms, a moving needle injector may be used.

3.1.2. Oven

The oven shall be capable of heating the column to a temperature of at least 260 °C and of maintaining the desired temperature to within 1 °C with a packed column and within 0,1 °C 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.

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 carbon atoms, it is recommended that a column 2 m in length 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 twin columns 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 polyadipate, etc.) cyanosilicones or any other liquid permitting the chromatographic separation required (see clause 4). The stationary 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 °C and pass a current of inert gas through the freshly prepared column at a rate of 20 to 60 ml/min for at least 16 hours at this temperature, and for a further 2 hours at 195 °C.

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) 20 000), 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 C20 acids. 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 column under 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 °C/min from ambient temperature to a temperature 10 °C 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 °C 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 in 0,1 μl divisions.

3.3. Recorder

If the recorder curve is to be used to calculate the composition of 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 taken for the recording pen to 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-ionization detector.

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 in clause 6.

4.1. Test conditions

4.1.1. Selection of optimum operating conditions 4.1.1.1. 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 resolution required; (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 duration of analysis. 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 °C 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 hydrogen supplied 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 mm Carrier gas flow ml/min 2 15 to 25 3 20 to 40 4 40 to 60



Table 2 Concentration of stationary phase % (m/m) Column temperature °C 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 conditions 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 resolution (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 column and 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-quarters of the full scale. Calculate the number of theoretical plates, n (efficiency), using the formula:

and the resolution, R, using the formula:

where:

dr1 is the retention distance, in millimetres, from the start of the chromatogram to the maximum of the peak for methyl stearate;

ω1 and ω2 are the widths, in millimetres, of the peaks for methyl stearate and methyl oleate respectively, measured between the points of intersection of the tangents at the points of inflection of the curve with the base-line;

Δ is the distance, in millimetres, between the peak maxima for methyl stearate and methyl oleate;

▼M2

and the resolution index, lr, using the formula

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 adjacent peaks, measured from the base line.

▼B

Figure 1

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 column length for methyl stearate and a resolution of at least 1,25.

4.2. Test portion

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 portion may be increased (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 column temperature when the determination of fatty acids with fewer than 12 carbon atoms is required, or at a higher temperature when determining fatty acids with more than 20 carbon atoms. On occasion, 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 carbon atoms, inject the sample at 100 °C (or at 50 to 60 °C if butyric acid is present) and immediately raise the temperature at a rate of 4 to 8 °C/min to the optimum. In certain cases, the two procedures can be 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 between 100 and 195 °C.

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 C18:3 and C20:0, or C18:3 and C18:2 conjugated.

4.4. Preparation of the reference chromatogram and reference 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-chain acids of the same degree of unsaturation should 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. Qualitative analysis

Identify the methyl ester peaks for the sample from the graphs prepared in 4.4, if necessary by interpolation.

5.2. Quantitative analysis

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 represented 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 therefrom. If not, determine the area under each peak by multiplying the height of the peak by its width at mid-height, and where necessary take into account the various attenuations used during the recording.

5.2.2. Method of calculation

5.2.2.1. General case Calculate the content of a given component i, expressed as a percentage by mass of methyl esters, by determining the percentage represented by the area of the corresponding peak relative to the sum of the areas of all the peaks, using the following formula: where: Ai is the area under the peak corresponding to component i; A is the sum of the areas under all the peaks. Giv