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Commission Regulation (EEC) No 2568/91Show full title

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

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  1. Introductory Text

  2. Article 1.(1) Oils, the characteristics of which comply with those set...

  3. Article 2.(1) The characteristics of oils laid down in Annex I...

  4. Article 2a.(1) For the purpose of this Article, ‘ olive oil...

  5. Article 3.. . . . . . . . . ....

  6. Article 3a.. . . . . . . . . ....

  7. Article 3.Where it is found that an oil does not correspond...

  8. Article 4.(1) The Member States may approve assessment panels so that...

  9. Article 5.. . . . . . . . . ....

  10. Article 6.(1) The oil content of oil cake and other residues...

  11. Article 7.The Community provisions concerning the presence of contaminants shall apply....

  12. Article 7a.Natural or legal persons and groups of persons who hold...

  13. Article 8.(1) Member States shall notify the Commission of the measures...

  14. Article 9.Regulation (EEC) No 1058/77 is hereby repealed.

  15. Article 10.(1) This Regulation shall enter into force on the third...

  16. Signature

    1. ANNEXES

      SUMMARY

    2. ANNEX I

      OLIVE OIL CHARACTERISTICS

      1. Quality characteristics

      2. Purity characteristics

      3. Notes:

        1. (a) The results of the analyses must be expressed to the...

        2. (b) If just a single characteristic does not match the values...

        3. (c) For lampante olive oil, both quality characteristics marked with an...

        4. (d) If a characteristic is marked with two asterisks (**), this...

      4. Appendix

        1. Decision trees

    3. ANNEX Ia

      SAMPLING OF OLIVE OIL OR OLIVE-POMACE OIL DELIVERED IN IMMEDIATE PACKAGING

      1. This method of sampling is applied to batches of olive...

      2. ‘Batch’ shall mean a set of sales units which are...

      3. ‘ Increment ’ shall mean the quantity of oil contained...

      4. 1. CONTENT OF PRIMARY SAMPLE

        1. 1.1. Immediate packaging not exceeding 5 litres

        2. 1.2. Immediate packaging exceeding 5 litres

      5. 2. ANALYSES AND RESULTS

        1. 2.1. Each primary sample must be subdivided into laboratory samples, in...

        2. 2.2. Where all the results of the analyses comply with the...

      6. 3. VERIFICATION OF THE CATEGORY OF BATCH

        1. 3.1. In order to verify the batch category, the competent authority...

        2. 3.2. When one of the results of the analyses referred to...

    4. ANNEX Ib

      FLOW-CHART FOR VERIFYING WHETHER AN OLIVE OIL SAMPLE IS CONSISTENT WITH THE CATEGORY DECLARED

      1. General table

      2. Table 1 — Extra Virgin Olive Oil — Quality criteria...

      3. Table 2 — Virgin Olive Oil — Quality criteria

      4. Table 3 — Extra Virgin Olive Oil and Virgin Olive...

      5. Table 4 — Lampante Olive Oil — Purity criteria

      6. Table 5 — Refined Olive Oil — Quality criteria

      7. Table 6 — Olive Oil (composed of refined olive oil...

      8. Table 7 — Refined Olive Oil and olive oil composed...

      9. Table 8 — Crude Olive-Pomace Oil — Purity criteria

      10. Table 9 — Refined Olive-Pomace Oil — Quality criteria

      11. Table 10 — Olive Pomace Oil — Quality criteria

      12. Table 11 — Refined Olive-Pomace Oil and Olive-Pomace Oil —...

    5. ANNEX II

      DETERMINATION OF FREE FATTY ACIDS, COLD METHOD

      1. 1. SCOPE AND FIELD OF APPLICATION

      2. 2. PRINCIPLE

      3. 3. REAGENTS

        1. 3.1 Diethyl ether; 95 % ethanol (v/v), mixture of equal parts...

          1. Note 1: Diethyl ether is highly inflammable and may form explosive peroxides....

          2. Note 2: If it is not possible to use diethyl ether, a...

        2. 3.2 Potassium hydroxide or sodium hydroxide, titrated ethanolic or aqueous solution,...

          1. Note 3: A stable colourless solution of potassium hydroxide (or sodium hydroxide)...

        3. 3.3 Phenolphthalein, 10 g/l solution in 95 to 96 % ethanol...

      4. 4. APPARATUS

      5. 5. PROCEDURE

        1. 5.1 Preparation of the test sample

        2. 5.2 Test portion

        3. 5.3 Determination

          1. Note 4: If the quantity of 0,1 mol/l potassium hydroxide (or sodium...

          2. Note 5: If the solution becomes cloudy during titration, add enough of...

      6. 6. EXPRESSION OF RESULTS

    6. ANNEX III

      DETERMINATION OF PEROXIDE VALUE

      1. 1. Scope

      2. 2. Definition

      3. 3. Principle

      4. 4. Apparatus

        2. 4.1. 3 ml glass scoop.

        3. 4.2. Flasks, with ground necks and stoppers, of about 250 ml...

        4. 4.3. Burette of 5-ml, 10-ml or 25-ml capacity, graduated in at...

        5. 4.4. Analytical balance.

      5. 5. Reagents

        1. 5.1. Chloroform, analytical reagent quality, freed from oxygen by bubbling a...

        2. 5.2. Glacial acetic acid, analytical reagent quality, freed from oxygen by...

        3. 5.3. Potassium iodide, saturated aqueous solution, recently prepared, free from iodine...

        4. 5.4. Sodium thiosulphate, 0,01 mol/l (equivalent to 0,01 N) accurately standardised...

        5. 5.5. Starch solution, 10 g/l aqueous dispersion, recently prepared from natural...

      6. 6. Sample

      7. 7. Procedure

      8. 8. Expression of results

    7. ANNEX IV

      DETERMINATION OF WAX CONTENT BY CAPILLARY COLUMN GAS CHROMATOGRAPHY

      1. 1. SUBJECT

      2. 2. PRINCIPLE

      3. 3. EQUIPMENT

        1. 3.1. 25 ml Erlenmeyer flask.

        2. 3.2. Glass column for gas chromatography, internal diameter 15,0 mm, length...

        3. 3.3. Suitable gas chromatograph with a capillary column, equipped with a...

          1. 3.3.1. Thermostatic chamber for the columns, equipped with a temperature programmer....

          2. 3.3.2. Cold injector for direct introduction into the column.

          3. 3.3.3. Flame ionisation detector and converter-amplifier.

          4. 3.3.4. Recorder-integrator capable of working with the converter-amplifier (3.3.3), rate of...

          5. 3.3.5. Glass or fused silica capillary column 8 to 12 m...

        4. 3.4. 10 μl microsyringe for on-column injection, equipped with a hardened...

        5. 3.5. Electrovibrator.

        6. 3.6. Rotary evaporator.

        7. 3.7. Muffle furnace.

        8. 3.8. Analytical balance with guaranteed precision of ± 0,1 mg.

        9. 3.9. Normal laboratory glassware.

      4. 4. REAGENTS

        1. 4.1. Silica gel with a granule size of between 60 and...

        2. 4.2. n-hexane, for chromatography.

        3. 4.3. Ethyl ether, for chromatography.

        4. 4.4. n-heptane, for chromatography.

        5. 4.5. Standard solution of lauryl arachidate, at 0,1 % (m/v) in...

          1. 4.5.1. Sudan 1 (1-phenyl-azo-2-naphthol).

        6. 4.6. Carrier gas: hydrogen or helium, gas-chromatographic purity.

        7. 4.7. Auxiliary gases:

      5. 5. PROCEDURE

        1. 5.1. Preparation of the chromatographic column.

          1. NB:

        2. 5.2. Analysis by gas chromatography

          1. 5.2.1. Preparatory work

          2. 5.2.2. Choice of operating conditions

        3. 5.3. Performance of the analysis

        4. 5.4. Identification of peaks

        5. 5.5. Evaluation of quantity

      6. 6. EXPRESSION OF RESULTS

        2. Figure Chromatogram of the waxes of an olive oil

      7. Appendix

        1. Determination of the linear velocity of the gas

    8. ANNEX V

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

      1. 1. SCOPE

      2. 2. PRINCIPLE

      3. 3. APPARATUS

      4. 4. REAGENTS

        1. 4.1. . . . . . . . . . ....

        2. 4.2. Potassium hydroxide ethanolic solution, approximately 2 N.

        3. 4.3. . . . . . . . . . ....

        4. 4.4. Potassium hydroxide ethanolic solution, approximately 0,2 N.

        5. 4.5. . . . . . . . . . ....

        6. 4.6. . . . . . . . . . ....

        7. 4.7. . . . . . . . . . ....

        8. 4.8. . . . . . . . . . ....

        9. 4.9. . . . . . . . . . ....

        10. 4.10. . . . . . . . . . ....

        11. 4.11. . . . . . . . . . ....

        12. 4.12. . . . . . . . . . ....

        13. 4.13. . . . . . . . . . ....

        14. 4.14. . . . . . . . . . ....

        15. 4.15. . . . . . . . . . ....

        16. 4.16. . . . . . . . . . ....

        17. 4.17. . . . . . . . . . ....

        18. 4.18. Sample solutions of sterol trimethylsilyl ethers.

        19. 4.19. . . . . . . . . . ....

        20. 4.20. . . . . . . . . . ....

        21. 4.21. . . . . . . . . . ....

        22. 4.22. . . . . . . . . . ....

        23. 4.23. . . . . . . . . . ....

        24. 4.24. . . . . . . . . . ....

        25. 4.25. . . . . . . . . . ....

      5. 5. PROCEDURE

        1. 5.1. . . . . . . . . . ....

          1. 5.1.1. . . . . . . . . . ....

            1. Note 1: . . . . .

          2. 5.1.2. . . . . . . . . . ....

          3. 5.1.3. . . . . . . . . . ....

          4. 5.1.4. . . . . . . . . . ....

          5. 5.1.5. . . . . . . . . . ....

        2. 5.2. Separation of the sterol and triterpene dialcohols fraction (erythrodiol +...

          1. 5.2.1. . . . . . . . . . ....

            1. Note 3: . . . . .

          2. 5.2.2. . . . . . . . . . ....

            1. Note 4: . . . . .

          3. 5.2.3. . . . . . . . . . ....

          4. 5.2.4. . . . . . . . . . ....

          5. 5.2.5. . . . . . . . . . ....

          6. 5.2.6. . . . . . . . . . ....

        3. 5.3. . . . . . . . . . ....

          1. 5.3.1. . . . . . . . . . ....

            1. Note 6: . . . . .

          2. 5.3.2. . . . . . . . . . ....

            1. Note 7: . . . . .

        4. 5.4. . . . . . . . . . ....

          1. 5.4.1. . . . . . . . . . ....

            1. 5.4.1.1. Fit the column (point 3.11) in the gas chromatograph, by...

            2. 5.4.1.2. If the column is being used for the first time,...

              1. Note 8: . . . . .

          2. 5.4.2. . . . . . . . . . ....

            1. 5.4.2.1. The operating conditions are as follows:

          3. 5.4.3. Analytical procedure

            1. 5.4.3.1. By using the 10 μl microsyringe, take 1 μl of...

            2. 5.4.3.2. . . . . . . . . . ....

          4. 5.4.4. Peak identification

          5. 5.4.5. . . . . . . . . . ....

            1. 5.4.5.1. . . . . . . . . . ....

            2. 5.4.5.2. Calculate the concentration of each individual sterol, in mg/kg of...

      6. 6. EXPRESSION OF THE RESULTS

        1. 6.1. Report individual sterol concentrations as mg/kg of fatty material and...

        2. 6.2. Calculate the percentage of each individual sterol from the ratio...

        3. 6.3. . . . . . . . . . ....

        4. 6.4. Calculate the percentage of erythrodiol and uvaol:

      7. Appendix

        1. Determination of the linear speed of the gas

    9. ANNEX VI

      DETERMINATION OF ERYTHRODIOL AND UVAOL

      1. INTRODUCTION

      2. 1. SCOPE

      3. 2. PRINCIPLE OF THE METHOD

      4. 3. APPARATUS

        1. 3.1. . . . . . . . . . ....

      5. 4. REAGENTS

        1. 4.1. . . . . . . . . . ....

        2. 4.2. . . . . . . . . . ....

      6. 5. PROCEDURE

        1. 5.1. Preparation of the unsaponifiables.

        2. 5.2. Separation of erythrodiol and the sterols.

          1. 5.2.1. . . . . . . . . . ....

          2. 5.2.2. . . . . . . . . . ....

          3. 5.2.3. . . . . . . . . . ....

          4. 5.2.4. . . . . . . . . . ....

          5. 5.2.5. . . . . . . . . . ....

          6. 5.2.6. . . . . . . . . . ....

        3. 5.3. Preparation of the trimethylsily esters

        4. 5.4. Gas chromatographic analysis

      7. 6. EXPRESSION OF THE RESULTS

    10. ANNEX VII

      DETERMINATION OF THE PERCENTAGE OF 2-GLYCERYL MONOPALMITATE

      1. 1. PURPOSE AND SCOPE

      2. 2. PRINCIPLE

      3. 3. APPARATUS AND MATERIALS

        1. 3.1. 25 ml Erlenmeyer flask

        2. 3.2. 100, 250 and 300 ml beakers

        3. 3.3. Glass chromatograph column, internal diameter 21-23 mm, length 400 mm,...

        4. 3.4. 10, 50, 100 and 200 ml measuring cylinders

        5. 3.5. 100 and 250 ml flasks

        6. 3.6. Rotary evaporator

        7. 3.7. 10 ml conical-bottomed centrifuge tubes with groundglass stopper

        8. 3.8. Centrifuge for 10 and 100 ml tubes

        9. 3.9. Thermostat permitting a stable temperature of 40 ± 0,5 °C...

        10. 3.10. 1 and 2 ml graduated pipettes

        11. 3.11. 1 ml hypodermic syringe

        12. 3.12. 100 μl microsyringe

        13. 3.13. 1 000 ml funnel

        14. 3.14. Capillary gas chromatograph with an on-column cold injector for direct...

        15. 3.15. On-column cold injector for direct injection of the sample into...

        16. 3.16. Flame ionisation detector and electrometer

        17. 3.17. Recorder-integrator adapted to the electrometer with a response rate no...

        18. 3.18. Capillary column made of glass or fused silica 8-12 metres...

        19. 3.19. 10 μl microsyringe fitted with a hardened needle, at least...

      4. 4. REAGENTS

        1. 4.1. Silica gel with a grain size of between 0,063 and...

        2. 4.2. n-hexane (chromatography grade). Hexane may be replaced by iso-octane (2,2,4-...

        3. 4.3. Isopropanol

        4. 4.4. Isopropanol, 1/1 (v/v) aqueous solution

        5. 4.5. Pancreatic lipase. It must have an activity of between 2,0...

        6. 4.6. Buffer solution of trishydroxymethylaminomethane: 1 M aqueous solution adjusted to...

        7. 4.7. Enzyme-quality sodium cholate, 0,1 % aqueous solution (this solution must...

        8. 4.8. Calcium chloride, 22 % aqueous solution

        9. 4.9. Diethyl ether for chromatography

        10. 4.10. Developer solvent: mixture of n-hexane/diethyl ether (87:13 v:v)

        11. 4.11. Sodium hydroxide, 12 % by weight solution

        12. 4.12. Phenolphthalein, 1 % solution in ethanol

        13. 4.13. Carrier gas: hydrogen or helium, for gas chromatography

        14. 4.14. Auxiliary gases: hydrogen, 99 % minimum purity, free from moisture...

        15. 4.15. Silanisation reagent: mixture of pyridine/hexamethyldisilazane, trimethylchlorosilane 9/3/1 (v/v/v). (Ready-to-use solutions...

        16. 4.16. Reference samples: pure monoglycerides or monoglyceride mixtures with a known...

      5. 5. METHOD

        1. 5.1. Sample preparation

          1. 5.1.1. Oils with a free acidity of less than 3 %...

            1. 5.1.1.1. Pour 50 g of oil and 200 ml n-hexane into...

          2. 5.1.2. Put 1,0 g of the oil prepared as above into...

          3. 5.1.3. Preparation of the chromatography column

          4. 5.1.4. Column chromatography

        2. 5.2. Hydrolysis by pancreatic lipase

          1. 5.2.1. Weigh into the centrifuge tube 0.1 g of the oil...

          2. 5.2.2. Add 20 mg of lipase, shake carefully (avoid wetting the...

          3. 5.2.3. Add 1 ml of diethyl ether, stopper and shake vigorously,...

        3. 5.3. Preparation of the silanised derivatives and gas chromatography

          1. 5.3.1. With a microsyringe insert 100 μl of solution (5.2.3) into...

          2. 5.3.2. Remove the solvent under a slight nitrogen current, add 200...

          3. 5.3.3. After 20 minutes, add 1 to 5 ml of n-hexane...

        4. 5.4. Gas chromatography

          1. 5.4.1. Identification of the peaks

          2. 5.4.2. Quantitative evaluation

      6. 6. EXPRESSION OF RESULTS

      7. 7. ANALYSIS REPORT

        1. Figure 2

          1. ( A ) unesterified olive oil, after lipase; after silanisation; under these conditions...

          2. ( B ) unesterified oil after lipase; after silanisation; under these conditions (8-12...

      8. 8. NOTES

        1. Note 1. PREPARATION OF THE LIPASE

        2. Note 2. MONITORING LIPASE ACTIVITY

    11. ANNEX VIII

      DETERMINATION OF TRILINOLEIN CONTENT

      1. 1. SCOPE

      2. 2. FIELD OF APPLICATION

      3. 3. PRINCIPLE

      4. 4. APPARATUS

        1. 4.1. . . . . . . . . . ....

        2. 4.2. . . . . . . . . . ....

        3. 4.3. . . . . . . . . . ....

        4. 4.4. . . . . . . . . . ....

        5. 4.5. . . . . . . . . . ....

      5. 5. REAGENTS

        1. 5.1. . . . . . . . . . ....

        2. 5.2. . . . . . . . . . ....

        3. 5.3. . . . . . . . . . ....

        4. 5.4. . . . . . . . . . ....

        5. 5.5. . . . . . . . . . ....

        6. 5.6. . . . . . . . . . ....

      6. 6. PREPARATION OF SAMPLES

      7. 7. PROCEDURE

        1. 7.1. . . . . . . . . . ....

      8. 8. CALCULATION AND EXPRESSION OF RESULTS

        1. Note 1.

        2. Note 2. Examples:

        3. Note 3.

        4. Note 4.

        5. Note 5:

      9. . . . . . . . . . ....

      11. . . . . . . . . . ....

    12. ANNEX IX

      SPECTROPHOTOMETRIC INVESTIGATION IN THE ULTRAVIOLET

      1. FOREWORD

      2. 1. SCOPE

      3. 2. PRINCIPLE OF THE METHOD

      4. 3. EQUIPMENT

        1. 3.1. A spectrophotometer suitable for measurements at ultraviolet wavelengths (220 nm...

          1. 3.1.1. Wavelength scale: This may be checked using a reference material...

          2. 3.1.2. Absorbance scale: This may be checked using commercially available sealed...

        2. 3.2. Rectangular quartz cuvettes, with covers, suitable for measurements at the...

        3. 3.3. One- mark volumetric flasks, capacity 25 ml, class A.

        4. 3.4. Analytical balance, capable of being read to the nearest 0,0001...

      5. 4. REAGENTS

      6. 5. PROCEDURE

        1. 5.1. The sample must be perfectly homogeneous and without suspended impurities....

        2. 5.2. Weigh accurately approximately 0,25 g (to the nearest 1 mg)...

        3. 5.3. If necessary, correct the baseline (220-290 nm) with solvent in...

        4. 5.4. After measuring the absorbance at 268 or 270 nm, measure...

      7. 6. EXPRESSION OF THE RESULTS

        1. 6.1. Record the specific extinctions (extinction coefficients) at the various wavelengths...

        2. 6.2. Variation of the specific extinction (ΔΚ)

    13. ANNEX X

      DETERMINATION OF FATTY ACID METHYL ESTERS BY GAS CHROMATOGRAPHY

      1. 1. SCOPE

      2. 2. PRINCIPLE

      3. PART A PREPARATION OF THE FATTY ACID METHYL ESTERS FROM OLIVE OIL...

        1. 1. SCOPE

        2. 2. FIELD OF APPLICATION

        3. 3. METHODOLOGY

          1. 3.1. Trans-esterification with methanolic solution of potassium hydroxide at room temperature...

            1. 3.1.1. Principle

            2. 3.1.2. Reagents

              1. 3.1.2.1. Methanol containing not more than 0,5 % (m/m) water.

              2. 3.1.2.2. Hexane, chromatographic quality.

              3. 3.1.2.3. Heptane, chromatographic quality.

              4. 3.1.2.4. Diethyl ether, stabilised for analysis.

              5. 3.1.2.5. Acetone, chromatographic quality.

              6. 3.1.2.6. Elution solvent for purifying the oil by column/SPE chromatography, mixture...

              7. 3.1.2.7. Potassium hydroxide, approximately 2M methanolic solution: dissolve 11,2 g of...

              8. 3.1.2.8. Silica gel cartridges, 1 g (6 ml), for solid phase...

            3. 3.1.3. Apparatus

              1. 3.1.3.1. Screw-top test tubes (5 ml volume) with cap fitted with...

              2. 3.1.3.2. Graduated or automatic pipettes, 2 ml and 0,2 ml.

            4. 3.1.4. Purification of oil samples

            5. 3.1.5. Procedure

      4. PART B ANALYSIS OF FATTY ACID METHYL ESTERS BY GAS CHROMATOGRAPHY

        1. 1. SCOPE

        2. 2. REAGENTS

          1. 2.1. Carrier gas

            1. Note 1: Hydrogen can double the speed of analysis but is hazardous....

          2. 2.2. Auxiliary gases

            1. 2.2.1. Hydrogen (purity ≥ 99,9 %), free from organic impurities.

            2. 2.2.2. Air or oxygen, free from organic impurities.

            3. 2.2.3. Nitrogen (purity > 99 %).

          3. 2.3. Reference standard

        3. 3. APPARATUS

          1. 3.1. Gas chromatograph

            1. 3.1.1. Injection system

            2. 3.1.2. Oven

            3. 3.1.3. Capillary column

              1. 3.1.3.1. Tube, made of a material inert to the substances to...

              2. 3.1.3.2. Stationary phase, polar polysiloxane (cyanopropylsilicone) bonded (cross-linked) columns are suitable....

                1. Note 2: There is a risk that polar polysiloxanes may give rise...

              3. 3.1.3.3. Assembly and conditioning of the column

                1. Note 3: Suitably pre-conditioned columns are available commercially.

            4. 3.1.4. Flame ionisation detector and converter-amplifier

          2. 3.2. Syringe

          3. 3.3. Data acquisition system

        4. 4. PROCEDURE

          1. 4.1. Test conditions

            1. 4.1.1. Selection of optimum operating conditions for capillary columns

            2. 4.1.2. Determination of the resolution (see Appendix A)

        5. 5. EXPRESSION OF RESULTS

          1. 5.1. Qualitative analysis

          2. 5.2. Quantitative analysis

            1. 5.2.1. Determination of the composition

            2. 5.2.2. Method of calculation

              1. 5.2.2.1. General case

                1. Note 4: For fats and oils, the mass fraction of the fatty...

              2. 5.2.2.2. Use of correction factors

                1. Note 5: These correction factors are not identical to the theoretical FID...

                2. Note 6: The calculated value corresponds to the percentage of mass of...

              3. 5.2.2.3. Use of an internal standard

        6. 6. TEST REPORT

        7. 7. PRECISION

          1. 7.1. Results of interlaboratory test

          2. 7.2. Repeatability

          3. 7.3. Reproducibility

      5. Appendix A

        1. Figure 1

        2. ω 0,5 width at half height of the triangle (ABC)...

      6. Appendix B

        1. Figure 1 Gas chromatographic profile obtained by the cold methylation...

        2. The chromatographic peaks correspond to the methyl and ethyl esters...

    14. ANNEX XI

      DETERMINATION OF VOLATILE HALOGENATED SOLVENTS CONTENT OF OLIVE OIL

      1. 1. METHOD

      2. 2. EQUIPMENT

        1. 2.1. Gas chromatography apparatus fitted with an electron capture detector (ECD)....

        2. 2.2. Head space apparatus.

        3. 2.3. Gas chromatography column, of glass, 2 m long and 2...

        4. 2.4. Carrier and auxiliary gas: nitrogen for gas chromatography, suitable for...

        5. 2.5. Glass flasks, 10 to 15 ml, with teflon coating and...

        6. 2.6. Hermetically sealing clamps.

        7. 2.7. Gas syringe 0,5 to 2 ml.

      3. 3. REAGENTS

      4. 4. PROCEDURE

        1. 4.1. Exactly weigh around 3 g of oil in a glass...

        2. 4.2. Reference solutions: prepare standard solutions using refined olive oil with...

        3. 4.3. Quantitative assessment: correlate the surfaces or the elevations of the...

        4. 4.4. Expression of results: in ppm (mg/kg). The detection limit for...

    15. ANNEX XII

      THE INTERNATIONAL OLIVE COUNCIL’S METHOD FOR THE ORGANOLEPTIC ASSESSMENT OF VIRGIN OLIVE OIL

      1. 1. PURPOSE AND SCOPE

      2. 2. GENERAL BASIC VOCABULARY FOR SENSORY ANALYSIS

      3. 3. SPECIFIC VOCABULARY

        1. 3.1. Negative attributes

          1. 3.1.1. Other negative attributes

        2. 3.2. Positive attributes

        3. 3.3. Optional terminology for labelling purposes

      4. 4. GLASS FOR OIL TASTING

      5. 5. TEST ROOM

      6. 6. ACCESSORIES

      7. 7. PANEL LEADER AND TASTERS

        1. 7.1. Panel leader

          1. 7.1.1. Deputy panel leader

        2. 7.2. Tasters

      8. 8. TEST CONDITIONS

        1. 8.1. Presentation of the sample

        2. 8.2. Test and sample temperature

        3. 8.3. Test times

        4. 8.4. Tasters: general rules of conduct

      9. 9. PROCEDURE FOR THE ORGANOLEPTIC ASSESSMENT AND CLASSIFICATION OF VIRGIN OLIVE...

        1. 9.1. Tasting technique

          1. 9.1.1. The tasters shall pick up the glass, keeping it covered...

          2. 9.1.2. When organoleptically assessing a virgin olive oil, it is recommended...

        2. 9.2. Use of the profile sheet by tasters

        3. 9.3. Use of the data by the panel leaders

        4. 9.4. Classification of the oil

          1. Note 1: When the median of the bitter and/or pungent attribute is...

        5. 9.5 Criteria for the acceptance and rejection of duplicates

      10. Appendix

        1. Method for calculating the median and the confidence intervals

          1. Median

          2. Robust standard deviation

          3. Robust coefficient of variation (%)

          4. Confidence intervals of the median at 95%

          5. References

            1. (1) Wilkinson, L. 1990. Systat: The system for statistics. Evanston, IL.SYSTAT...

            2. (2) Cicchitelli, G. 1984. Probabilità e Statistica. Maggioli Editore, Rimini.

            3. (3) Massart, D.L.; Vandeginste, B.G.M.; Deming, Y.; Michotte, L. 1988. Chemometrics....

            4. (4) Kendall, M.G.; Stuart, A. 1967. The advanced theory of statistics....

            5. (5) McGill, R.; Tukey, J.W.; Larsen, W.A. 1978. Variation of Box...

            6. (6) IOC/T.28/Doc. No 1 September 2007, Guidelines for the accreditation of...

            7. (7) IOC/T.20/Doc. No 14.

            8. (8) IOC/T.20/Doc. No 15.

            9. (9) ISO/IEC 17025:05.

    16. ANNEX XIII

      NEUTRALIZATION AND DECOLORIZATION OF OLIVE OIL IN THE LABORATORY

      1. 1. NEUTRALIZATION AND DECOLORIZATION OF OLIVE OIL IN THE LABORATORY

        1. 1.1. Neutralization of the oil

          1. 1.1.1. Apparatus

          2. 1.1.2. Reagents

          3. 1.1.3. Procedure

            1. (a) Oils with a free fatty acid content, expressed as oleic...

            2. (b) Oils with a free fatty acid content expressed as oleic...

        2. 1.2. Decolorization of neutralized oil

          1. 1.2.1. Apparatus

          2. 1.2.2. Procedure

    17. ANNEX XIV

      ADDITIONAL NOTES 2, 3 AND 4 TO CHAPTER 15 OF THE COMBINED NOMENCLATURE

      1. 2. A. . . . . . . . . ....

        1. A. . . . . . . . . . ....

        2. B. . . . . . . . . . ....

          1. I. . . . . . . . . . ....

          2. II. . . . . . . . . . ....

        3. C. . . . . . . . . . ....

        4. D. . . . . . . . . . ....

        5. E. . . . . . . . . . ....

      2. 3. . . . . . . . . . ....

      3. 4. . . . . . . . . . ....

    18. ANNEX XV

      1. 1. OIL CONTENT OF OLIVE RESIDUE

        1. 1.1. Apparatus

        2. 1.2. Reagent

      2. 2. PROCEDURE

        1. 2.1. Preparation of the test sample

        2. 2.2. Test portion

        3. 2.3. Preparation of the extraction thimble

        4. 2.4. Peliminary drying

        5. 2.5. Preparation of the round-bottomed flask

        6. 2.6. Initial extraction

        7. 2.7. Second extraction

        8. 2.8. Removal of solvent and weighing of extract

      3. 3. EXPRESSION OF RESULTS

        1. 3.1. Method of calculation and formula

        2. 3.2. Repeatability

    19. ANNEX XVI

      DETERMINATION OF IODINE VALUE

      1. 1. SCOPE

      2. 2. DEFINITION

        1. 2.1. iodine value. The mass of iodine absorbed by the sample...

      3. 3. PRINCIPLE

      4. 4. REAGENTS

        1. 4.1. water, complying with the requirements of ISO 3696, Grade 3....

        2. 4.2. potassium iodide, 100 g/l solution, not containing iodate or free...

        3. 4.3. starch, solution.

        4. 4.4. sodium thiosulfate, standard volumetric solution c (Na2S2O3.5H2O) = 0,1 mol/l,...

        5. 4.5. solvent, prepared by mixing equal volumes of cyclohexane and acetic...

        6. 4.6. Wijs reagent, containing iodine monochloride in acetic acid. Commercially available...

      5. 5. APPARATUS

        1. 5.1. glass weighing scoops, suitable for the test portion and for...

        2. 5.2. conical flasks, of 500 ml capacity, fitted with ground glass...

      6. 6. PREPARATION OF THE TEST SAMPLE

      7. 7. PROCEDURE

        1. 7.1. Test portion

        2. 7.2. Determination

          1. Note:

        3. 7.3. Number of determinations

      8. 8. EXPRESSION OF RESULTS

    20. ANNEX XVII

      METHOD FOR THE DETERMINATION OF STIGMASTADIENES IN VEGETABLE OILS

      1. 1. PURPOSE

      2. 2. SCOPE

      3. 3. PRINCIPLE

      4. 4. APPARATUS

        1. 4.1. 250 ml flasks suitable for use with a reflux condenser....

        2. 4.2. Separating funnels of 500 ml capacity.

        3. 4.3. 100 ml round-bottom flasks.

        4. 4.4. Rotary evaporator.

        5. 4.5. Glass chromatography column (1,5 to 2,0 cm internal diameter by...

        6. 4.6. Gas chromatograph with flame ionization detector, split or cold on-column...

        7. 4.7. Fused silica capillary column for gas chromatography (0,25 or 0,32...

          1. Note 1:

        8. 4.8. Integrator-recorder with possibility of valley-valley integration mode.

        9. 4.9. 5 to 10 ml microsyringe for gas chromatography with cemented...

        10. 4.10. Electrical heating mantle or hot place.

      5. 5. REAGENTS

        1. 5.1. Hexane or mixture of alkanes of b.p. interval 65 to 70 °C,...

        2. 5.2. 96 v/v ethanol.

        3. 5.3. Anhydrous sodium sulphate.

        4. 5.4. Alcoholic potassium hydroxide solution at 10 %. Add 10 ml...

          1. Note 3:

        5. 5.5. Silica gel 60 for column chromatography, 70 to 230 mesh,...

          1. Note 4:

        6. 5.6. Stock solution (200 ppm) of cholesta-3,5-diene (Sigma, 99 % purity)...

        7. 5.7. Standard solution of cholesta-3,5-diene hexane at concentration of 20 ppm,...

          1. Note 5:

        8. 5.8. Solution of n-nonacosane in hexane at concentration of approximately 100...

        9. 5.9. Carrier gas for chromatography: helium or hydrogen of 99,9990 %...

        10. 5.10. Auxiliary gases for flame ionization detector: hydrogen of 99,9990 %...

      6. 6. PROCEDURE

        1. 6.1. Preparation of unsaponifiable matter

          1. 6.1.1. Weigh 20 ± 0,1 g of oil into a 250-ml...

            1. Note 6:

          2. 6.1.2. Transfer the aqueous phase beneath to a second separating funnel...

          3. 6.1.3. Pass the hexane solution through anhydrous sodium sulphate (50 g),...

        2. 6.2. Separation of steroidal hydrocarbon fraction

          1. 6.2.1. Take the residue to the fractioning column with the aid...

            1. Note 7:

          2. 6.2.2. Evaporate the second fraction in a rotary evaporator at 30...

            1. Note 8:

        3. 6.3. Gas chromatography

          1. 6.3.1. Working conditions for split injection:

          2. 6.3.2. Peak identification

            1. Note 9:

          3. 6.3.3. Quantitative analysis

      7. Figure 1

      8. Gas chromatogram obtained from a refined olive oil sample analysed...

    21. ANNEX XVIII

      DETERMINATION OF THE DIFFERENCE BETWEEN ACTUAL AND THEORETICAL CONTENT OF TRIACYLGLYCEROLS WITH ECN 42

      1. 1. SCOPE

      2. 2. FIELD OF APPLICATION

      3. 3. PRINCIPLE

      4. 4. METHOD

        1. 4.1. Apparatus

          1. 4.1.1. Round-bottomed flasks, 250 and 500 ml.

          2. 4.1.2. Beakers 100 ml.

          3. 4.1.3. Glass chromatographic column, 21 mm internal diameter, 450 mm length,...

          4. 4.1.4. Separating funnels, 250 ml, with normalised cone (male) at the...

          5. 4.1.5. Glass rod, 600 mm length.

          6. 4.1.6. Glass funnel, 80 mm diameter.

          7. 4.1.7. Volumetric flasks, 50 ml.

          8. 4.1.8. Volumetric flasks, 20 ml.

          9. 4.1.9. Rotary evaporator.

          10. 4.1.10. High performance liquid chromatograph, allowing thermostatic control of column temperature....

          11. 4.1.11. Injection units for 10 μl delivery.

          12. 4.1.12. Detector: differential refractometer. The full scale sensitivity should be at...

          13. 4.1.13. Column: stainless steel tube 250 mm length x 4,5 mm...

          14. 4.1.14. Data processing software.

          15. 4.1.15. Vials, of about 2 ml volumes, with Teflon-layered septa and...

        2. 4.2. Reagents

          1. 4.2.1. Petroleum ether 40-60 °C chromatographic grade or hexane. Hexane may be...

          2. 4.2.2. Ethyl ether, peroxide-free, freshly distilled.

          3. 4.2.3. Elution solvent for purifying the oil by column chromatography mixture...

          4. 4.2.4. Silica gel, 70-230 mesh, type Merck 7734, with water content...

          5. 4.2.5. Glass wool.

          6. 4.2.6. Acetone for HPLC.

          7. 4.2.7. Acetonitrile or propionitrile for HPLC.

          8. 4.2.8. HPLC elution solvent: acetonitrile + acetone (proportions to be adjusted...

          9. 4.2.9. Solubilisation solvent: acetone.

          10. 4.2.10. Reference triglycerides: commercial triglycerides (tripalmitin, triolein, etc.) may be used...

          11. 4.2.11. Solid phase extraction column with silica phase 1 g, 6...

          12. 4.2.12. Heptane, chromatographic quality. Heptane may be replaced by iso-octane (2,2,4-trimethyl...

        3. 4.3. Sample preparation

          1. 4.3.1. Chromatographic column preparation

          2. 4.3.2. Column chromatography

          3. 4.3.3. SPE purification

        4. 4.4. HPLC analysis

          1. 4.4.1. Preparation of the samples for chromatographic analysis

          2. 4.4.2. Procedure

          3. 4.4.3. Calculation and expression of results

        5. 4.5. Calculation of triacylglycerols composition (moles %) from fatty acid composition...

          1. 4.5.1. Determination of fatty acid composition

          2. 4.5.2. Fatty acids for calculation

          3. 4.5.3. Conversion of area % into moles for all fatty acids...

          4. 4.5.4. Normalisation of fatty acid moles to 100 % (2)

          5. 4.5.5. Calculation of the fatty acid composition in 2- and 1,...

            1. 4.5.5.1. Saturated fatty acids in 2-position [P(2) and S(2)] (4):

            2. 4.5.5.2. Unsaturated fatty acids in 2-position [Po(2), O(2), L(2) and Ln(2)]...

            3. 4.5.5.3. Fatty acids in 1,3-positions [P(1,3), S(1,3), Po(1,3), O(1,3), L(1,3) and...

          6. 4.5.6. Calculation of triacylglycerols

            1. 4.5.6.1. TAGs with one fatty acid (AAA, here LLL, PoPoPo) (7)...

            2. 4.5.6.2. TAGs with two fatty acids (AAB, here PoPoL, PoLL) (8)...

            3. 4.5.6.3. TAGs with three different fatty acids (ABC, here OLLn, PLLn,...

            4. 4.5.6.4. Triacylglycerols with ECN42

      5. 5. EVALUATION OF THE RESULTS

      6. 6. EXAMPLE (THE NUMBERS REFER TO THE SECTIONS IN THE TEXT...

        1. — 4.5.1. Calculation of moles % fatty acids from GLC data (normalised...

        2. — 4.5.3 Conversion of area % into moles for all fatty acids...

        3. — 4.5.4 Normalisation of fatty acid moles to 100 % (see formula...

        4. — 4.5.5 Calculation of the fatty acid composition in 2- and 1,3-positions...

          1. — 4.5.5.1 Saturated fatty acids in 2-position [P(2) and S(2)] (see formula...

          2. — 4.5.5.2 Unsaturated fatty acids in 2-position [Po(1,3), O(1,3), L(1,3) and Ln(1,3)]...

          3. — 4.5.5.3 Fatty acids in 1,3-positions [P(1,3), S(1,3), Po(1,3), O(1,3), L(1,3) and...

        5. — 4.5.6. Calculation of triacylglycerols

          1. — 4.5.6.1. TAGs with one fatty acid (LLL, PoPoPo) (see formula (7))...

          2. — 4.5.6.2 TAGs with two fatty acids (PoLL, SLnLn, PoPoL) (see formula...

          3. — 4.5.6.3 TAGs with three different fatty acids (PoPLn, OLLn, PLLn, PoOLn)...

      11. (b)

      12. (b)

    22. ANNEX XIX

      1. DETERMINATION OF THE STEROL COMPOSITION AND CONTENT AND ALCOHOLIC COMPOUNDS BY...

        1. 1. SCOPE

        2. 2. PRINCIPLE

      2. PART 1 PREPARATION OF THE UNSAPONIFIABLE MATTER

        1. 1. SCOPE

        2. 2. PRINCIPLE

        3. 3. APPARATUS

        4. 4. REAGENTS

        5. 5. PROCEDURE

          1. Note 1: Animal or vegetable oils and fats containing appreciable quantities of...

          2. Note 2: Any emulsion can be destroyed by adding small quantities of...

      3. PART 2 SEPARATION OF THE ALCOHOLIC COMPOUNDS FRACTIONS

        1. 1. SCOPE

        2. 2. PRINCIPLE

        3. 3. APPARATUS

        4. 4. REAGENTS

        5. 5. REFERENCE METHOD: SEPARATION OF THE ALCOHOLIC COMPOUNDS BY BASIC THIN-LAYER...

          1. Note 3: The developing mixture should be replaced for every test, in...

          2. Note 4: Higher temperature could worsen the separation.

        6. 6. SEPARATION OF THE ALCOHOLIC FRACTION BY HPLC

          1. Note 5: Carefully control the pressure of the HPLC pump, the ethyl...

      4. PART 3 GAS CHROMATOGRAPHIC ANALYSIS OF THE ALCOHOLIC COMPOUNDS FRACTIONS

        1. 1. SCOPE

        2. 2. PRINCIPLE

        3. 3. APPARATUS

        4. 4. REAGENTS

        5. 5. PREPARATION OF THE TRIMETHYLSILYL ETHERS

          1. Note 6: Ready for use solutions are available commercially. Other silylation reagents,...

          2. Note 7: The slight opalescence, which may form, is normal and does...

        6. 6. GAS CHROMATOGRAPHIC ANALYSIS

          1. 6.1. Preliminary operations, capillary column conditioning

            1. Note 8: The conditioning temperature must always be at least 20 °C less...

          2. 6.2. Operating conditions

            1. 6.2.1. Aliphatic alcohols

            2. 6.2.2. Sterol and triterpenic dialcohols

          3. 6.3. Analytical procedure

          4. 6.4. Peak identification

          5. 6.5. Quantitative evaluation

        7. 7. EXPRESSION OF THE RESULTS

      5. Appendix

        1. Figure 1 — TLC of the unsaponifiable fraction from olive...

        2. Figure 2 — GC-FID chromatographic profile of the sterol and...

        3. Figure 3 — GC-FID chromatographic profile of the sterol and...

        4. Figure 4 — GC-FID chromatographic profile of aliphatic alcohols and...

        5. Figure 5 — GC-FID chromatographic profile of aliphatic alcohols and...

        6. Figure 6 — HPLC Chromatogram of an olive oil unsaponifiable...

    23. ANNEX XX

      Method for the determination of the content of waxes, fatty acid methyl esters and fatty acid ethyl esters by capillary gas chromatography

      1. 1. PURPOSE

      2. 2. PRINCIPLE

      3. 3. APPARATUS

        1. 3.1. Erlenmeyer flask, 25 ml .

        2. 3.2. Glass column for liquid chromatography, internal diameter 15 mm, length...

        3. 3.3. Gas chromatograph suitable for use with a capillary column, equipped...

          1. 3.3.1. Thermostat-controlled oven with temperature programming .

          2. 3.3.2. Cold injector for direct on-column injection

          3. 3.3.3. Flame ionisation detector and converter-amplifier .

          4. 3.3.4. Recorder-integrator (Note 1) for use with the converter-amplifier (point 3.3.3),...

            1. Note 1: Computerised systems may also be used where the gas chromatography...

          5. 3.3.5. Capillary column, fused silica (for analysis of the waxes and...

            1. Note 2: Suitable commercial liquid phases are available for this purpose such...

        4. 3.4. Microsyringe , 10 μl, with hardened needle, for direct on-column...

        5. 3.5. Electric shaker .

        6. 3.6. Rotary evaporator .

        7. 3.7. Muffle oven .

        8. 3.8. Analytical balance for weighing to an accuracy of ± 0,1...

        9. 3.9. Usual laboratory glassware.

      4. 4. REAGENTS

        1. 4.1. Silica gel , 60-200 μm mesh. Place the silica gel...

        2. 4.2. n-hexane, chromatography grade or residue grade. Hexane may be replaced...

        3. 4.3. Ethyl ether, chromatography grade

        4. 4.4. n-heptane , chromatography grade, or iso-octane

        5. 4.5. Standard solution of lauryl arachidate ( Note 3 ), at...

          1. Note 3: Palmityl palmitate, myristyl stearate or arachidyl laureate may also be...

        6. 4.6. Standard solution of methyl heptadecanoate, at 0,02 % (m/V) in...

        7. 4.7. Sudan 1 (1-phenylazo-2-naphthol) .

        8. 4.8. Carrier gas: hydrogen or helium, pure, gas chromatography grade ....

          1. WARNING

        9. 4.9. Auxiliary gases :

          1. WARNING

      5. 5. PROCEDURE

        1. 5.1. Preparation of the chromatography column

          1. Note 4: The n-hexane/ethyl ether (99:1) mixture should be freshly prepared every...

          2. Note 5: 100 μl of Sudan I dye at 1 % in...

        2. 5.2. Gas chromatography analysis

          1. 5.2.1. Preliminary procedure

          2. 5.2.2. Choice of operating conditions for waxes and methyl and ethyl...

            1. Note 6: Due to the high final temperature, positive drift is allowed...

        3. 5.3. Performance of the analysis

        4. 5.4. Peak identification

        5. 5.5. Quantitative analysis of the waxes

          1. 5.5.1. Quantitative analysis of the methyl and ethyl esters

      6. 6. EXPRESSION OF RESULTS

        1. Note 7: The components for quantification refer to the peaks with even...

      7. Appendix A

        1. Determination of linear gas speed

    24. ANNEX XXa

      METHOD FOR THE DETECTION OF EXTRANEOUS OILS IN OLIVE OILS

      1. 1. SCOPE

      2. 2. PRINCIPLE

      3. 3. MATERIAL AND REAGENTS

        1. 3.1. Oil purification

          1. 3.1.1. . . . . . . . . . ....

          2. 3.1.2. . . . . . . . . . ....

          3. 3.1.3. . . . . . . . . . ....

          4. 3.1.4. . . . . . . . . . ....

          5. 3.1.5. . . . . . . . . . ....

          6. 3.1.6. . . . . . . . . . ....

          7. 3.1.7. . . . . . . . . . ....

        2. 3.2. HPLC analysis of triacylglycerols

          1. 3.2.1. . . . . . . . . . ....

          2. 3.2.2. . . . . . . . . . ....

          3. 3.2.3. . . . . . . . . . ....

        3. 3.3. Preparation of fatty acid methyl esters

          1. 3.3.1. . . . . . . . . . ....

          2. 3.3.2. . . . . . . . . . ....

          3. 3.3.3. . . . . . . . . . ....

          4. 3.3.4. . . . . . . . . . ....

        4. 3.4. GC analysis of FAMEs

          1. 3.4.1 . . . . . . . . . ....

          2. 3.4.2 . . . . . . . . . ....

          3. 3.4.3 . . . . . . . . . ....

          4. 3.4.4 . . . . . . . . . ....

          5. 3.4.5. . . . . . . . . . ....

      4. 4. APPARATUS

        1. 4.1. . . . . . . . . . ....

        2. 4.2. . . . . . . . . . ....

        3. 4.3. HPLC equipment composed of:

        4. 4.4 Capillary gas chromatography equipment described in Annex X A, provided...

        5. 4.5. . . . . . . . . . ....

      5. 5. ANALYTICAL PROCEDURE

        1. 5.1. Oil purification

        2. 5.2. HPLC analysis of triacylglycerols

        3. 5.3. Preparation of fatty acid methyl esters

        4. 5.4. GC analysis of fatty acid methyl esters

      6. 6. INTEGRATION OF CHROMATOGRAPHIC PEAKS

        1. 6.1. HPLC chromatogram

        2. 6.2. GC chromatogram

      7. 7. DETECTION OF EXTRANEOUS OILS IN OLIVE OILS

    25. ANNEX XXI

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