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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 the oils laid down in Annex...

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 Article 9.Regulation (EEC) No 1058/77 is hereby repealed.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

      1. 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) If a characteristic is marked with an asterisk (*), referring...

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

    3. ANNEX Ia

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

      1. This method of sampling applies to deliveries of olive oil...

      2. If the delivery exceeds 125 000 litres, it is to...

      3. The minimum number of primary samples to be taken is...

      4. The size of the primary sample is determined on the...

      5. Delivery, primary sample and laboratory sample shall mean the definitions...

      6. ‘ Batch ’ shall mean a set of sales units...

      7. 1. NUMBER OF PRIMARY SAMPLES TO BE TAKEN

      8. 2. CONTENT OF PRIMARY SAMPLES

        1. 2.1 Primary samples must comprise the following:

        2. 2.2 The primary samples are to be kept in the immediate...

        3. 2.3 The packs constituting a primary sample shall be subdivided in...

      9. 3. ANALYSES AND RESULTS

    4. ANNEX Ib

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

      1. The analysis to verify whether an olive oil or olive-pomace...

      2. either by carrying out in any random order the analyses...

      3. The analyses relating to contaminants required for verifying compliance with...

      4. The decision tree applies to all categories of olive oil...

      5. Key to general tables to 11:

      6. the double line (=) indicates the route to be followed...

      7. Appendix 1

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

      8. Appendix 2

        1. Table 1

        2. Table 2

        3. Table 3

        4. Table 4

        5. Table 7

        6. Table 8

        7. Table 11

    5. ANNEX II

      DETERMINATION OF FREE FATTY ACIDS, COLD METHOD

      1. 1. DETERMINATION OF ACIDITY

        1. 1.1. Principle

        2. 1.2. Reagents

          1. 1.2.1. Diethyl ether; 95 % ethanol (v/v), mixture of equal parts...

            1. Note:

            2. Note:

          2. 1.2.2. Potassium hydroxide, titrated ethanolic solution, c(KOH) about 0,1 mol/l or,...

            1. Note:

          3. 1.2.3. Phenolphthalein, 10 g/l solution in 95 to 96 % ethanol...

        3. 1.3. Apparatus

          1. 1.3.1. analytical balance;

          2. 1.3.2. 250 ml conical flask;

          3. 1.3.3. 10 ml burette, graduated in 0,05 ml.

        4. 1.4. Procedure

          1. 1.4.1. Preparation of the specimen for testing

          2. 1.4.2. Taking the sample

          3. 1.4.3. Determination

            1. Note 1.

            2. Note 2.

            3. Note 3.

        5. 1.5. Acidity: expressed as percentage of oleic acid

    6. ANNEX III

      DETERMINATION OF PEROXIDE VALUE

      1. 1. SCOPE

      2. 2. FIELD OF APPLICATION

      3. 3. DEFINITION

      4. 4. PRINCIPLE

      5. 5. APPARATUS

        1. Note:

        2. 5.1. 3 ml glass scoop.

        3. 5.2. Flasks, with ground necks and stoppers, of about 250 ml...

        4. 5.3. 25- or 50-ml burette, graduated in 0,1 ml.

      6. 6. REAGENTS

        1. 6.1. Chloroform, analytical reagent quality, freed from oxygen by bubbling a...

        2. 6.2. Glacial acetic acid, analytical reagent quality, freed from oxygen by...

        3. 6.3. Potassium iodide, saturated aqueous solution, recently prepared, free from iodine...

        4. 6.4. Sodium thiosulphate, 0,01 or 0,002 Mol/L accurately standardized aqueous solution,...

        5. 6.5. Starch solution, 10 g/l aqueous dispersion, recently prepared from natural...

      7. 7. SAMPLE

      8. 8. PROCEDURE

      9. 9. 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 BY CAPILLARY-COLUMN GAS CHROMATOGRAPHY

      1. 1. SCOPE

      2. 2. PRINCIPLE OF THE METHOD

      3. 3. APPARATUS

        1. 3.1. 250 ml flask fitted with a reflux condenser having ground-glass...

        2. 3.2. 500 ml separating funnels.

        3. 3.3. 250 ml flasks.

        4. 3.4. Complete apparatus for analysis by thin-layer chromatography using 20 ×...

        5. 3.5. Ultraviolet lamp having a wavelength of 366 or 254 nm....

        6. 3.6. 100 μl and 500 μl microsyringes.

        7. 3.7. A cylindrical filter funnel with a G3 porous septum (porosity...

        8. 3.8. 50 ml vacuum conical flask with a 12/21 ground-glass female...

        9. 3.9. A 10 ml test tube with a tapering bottom and...

        10. 3.10. Gas chromatograph suitable for use with a capillary column, provided...

          1. 3.10.1. a thermostatic chamber for columns capable of maintaining the desired...

          2. 3.10.2. a temperature-adjustable vaporization unit with a persilanized glass vapourizing element;...

          3. 3.10.3. a flame ionization detector and converter-amplifier;

          4. 3.10.4. an integrator-recorder suitable for use with the converter-amplifier (3.10.3) having...

        11. 3.11. A glass or fused-silica capillary column of length 20 to...

        12. 3.12. A 10 μl gas chromatography microsyringe with a hardened needle....

      4. 4. REAGENTS

        1. 4.1. Potassium hydroxide, approximately 2 mol/L ethanolic solution. Dissolve 130 g...

        2. 4.2. Diethyl ether, analytical purity.

        3. 4.3. Anhydrous sodium sulphate, analytical purity.

        4. 4.4. Glass plates coated with silica gel, without fluorescence indicator, thickness...

        5. 4.5. Potassium hydroxide, 0,2 mol/L ethanolic solution. Dissolve 13 g of...

        6. 4.6. Benzene, for chromatography. (See 5.2.2)

        7. 4.7. Acetone, for chromatography. (See 5.2.2)

        8. 4.8. Hexane, for chromatography. (See 5.2.2)

        9. 4.9. Diethyl ether, for chromatography. (See 5.2.2)

        10. 4.10. Chloroform, analytical purity. (See 5.2.2)

        11. 4.11. Reference solution for thin-layer chromatography: cholesterol or phytosterols, 2 %...

        12. 4.12. 2,7-dichlorofluorescein, 0,2 % ethanolic solution. Make slightly basic by adding...

        13. 4.13. Anhydrous pyridine, for chromatography.

        14. 4.14. Hexamethyl disilazane.

        15. 4.15. Trimethylchlorosilane.

        16. 4.16. Reference solutions of sterol trimethylsilyl ethers. To be prepared at...

        17. 4.17. β-cholestanol, 0,2 % solution (m/V) in chloroform (internal standard).

        18. 4.18. Carrier gas: hydrogen or helium, gas-chromatographic purity.

        19. 4.19. Auxiliary gases:

      5. 5. PROCEDURE

        1. 5.1. Preparation of the unsaponifiables.

          1. 5.1.1. Using the 500 μl microsyringe introduce, into a 250 ml...

          2. 5.1.2. Add 50 ml of 2 mol/L ethanolic potassium hydroxide solution,...

          3. 5.1.3. Transfer the contents of the flask quantitatively into a 500...

            1. Note 1.

          4. 5.1.4. Pool the ether extracts into a single separating funnel and...

          5. 5.1.5. Distil the ether down to a few ml, then bring...

        2. 5.2. Separation of the sterol fraction.

          1. 5.2.1. Preparation of the basic plates. Immerse the silica gel plates...

            1. Note 2.

          2. 5.2.2. Place a 95:5 (v/v) benzene/acetone mixture into the plate-developing chamber...

            1. Note 3.

          3. 5.2.3. Prepare an approximately 5 % solution of the unsaponifiables (5.1.5)...

          4. 5.2.4. Place the plate in the developing chamber prepared as specified...

          5. 5.2.5. Spray the plate lightly and uniformly with the 2,7-dichlorofluoroscein solution....

          6. 5.2.6. Using a metal spatula scrape off the silica gel in...

        3. 5.3. Preparation of the trimethylsilyl ethers.

          1. 5.3.1. Add the silylation reagent, consisting of a 9:3:1 (v/v/v) mixture...

            1. Note 4.

          2. 5.3.2. Stopper the test tube, shake carefully (without overturning) until the...

            1. Note 5.

        4. 5.4. Gas chromatographic analysis.

          1. 5.4.1. Preliminary operations, column packing.

            1. 5.4.1.1. Fit the column in the gas chromatograph, attaching the inlet...

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

              1. Note 6.

          2. 5.4.2. Choice of operating conditions.

            1. 5.4.2.1. The guideline operating conditions are as follows:

          3. 5.4.3. Analytical procedure.

            1. 5.4.3.1. Using the 10 μl microsyringe take 1 μl of hexane,...

            2. 5.4.3.2. Continue recording until the TMSE of the sterols present are...

          4. 5.4.4. Peak identification.

          5. 5.4.5. Quantitative evaluation.

            1. 5.4.5.1. Calculate the areas of the β-cholestanol and the sterol peaks...

            2. 5.4.5.2. Calculate the concentration of each individual sterol in mg/100 g...

      6. 6. EXPRESSION OF THE RESULTS

        1. 6.1 Record individual sterol concentrations as mg/100 g of fatty material...

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

      7. APPENDIX

        1. Determination of the linear velocity 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. The apparatus described in Annex V (determination of the content...

      5. 4. REAGENTS

        1. 4.1. The reagents described in Annex V (determination of the content...

        2. 4.2. Reference solution of erythrodiol, 0,5 % solution in chloroform.

      6. 5. PROCEDURE

        1. 5.1. Preparation of the unsaponifiables.

        2. 5.2. Separation of erythrodiol and the sterols.

          1. 5.2.1. See paragraph 5.2.1 of Annex V.

          2. 5.2.2. See paragraph 5.2.2 of Annex V.

          3. 5.2.3. Prepare a 5 % solution of the unsaponifiables in chloroform....

          4. 5.2.4. Place the plate in the developing chamber prepared as specified...

          5. 5.2.5. Spray the plate lightly and uniformly with the alcoholic 2,7-dichlorofluoroscein...

          6. 5.2.6. Using a metal spatula scrape off the silica gel in...

        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 (for chromatography)

        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 for measuring extinction in the ultraviolet between 220...

          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 a reference material...

        2. 3.2. Rectangular quartz cells, with covers, having an optical length of...

        3. 3.3. 25 ml graduated flasks.

        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 in question must be perfectly homogeneous and without...

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

        3. 5.3. Fill a quartz cell with the solution obtained and 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)

      8. Appendix

    13. ANNEX X A

      ANALYSIS BY GAS CHROMATOGRAPHY OF METHYL ESTERS OF FATTY ACIDS

      1. 1. SCOPE

      2. 2. REAGENTS

        1. 2.1. Carrier gas

          1. Note 1.

        2. 2.2. Auxiliary gases

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

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

        3. 2.3. Reference standard

      3. 3. APPARATUS

        1. 3.1. Gas chromatograph

          1. 3.1.1. Injection system

            1. Note 2.

          2. 3.1.2. Oven

          3. 3.1.3. Packed column

            1. 3.1.3.1. Column, constructed of a material inert to the substances to...

              1. Note 3.

              2. Note 4.

            2. 3.1.3.2. Packing, comprising the following elements:

            3. 3.1.3.3. Conditioning of the column

          4. 3.1.4. Capillary column

            1. 3.1.4.1. Tube, made of a material inert to the substances to...

            2. 3.1.4.2. Stationary phase, usually of the type polyglycol (poly(ethylene glycol) 20...

              1. Note 5.

            3. 3.1.4.3. Assembly and conditioning of the column

              1. Note 6.

          5. 3.1.5. Detector, preferably capable of being heated to a temperature above...

        2. 3.2. Syringe

        3. 3.3. Recorder

        4. 3.4. Integrator

      4. 4. PROCEDURE

        1. 4.1. Test conditions

          1. 4.1.1. Selection of optimum operating conditions

            1. 4.1.1.1. Packed column

            2. 4.1.1.2. Capillary column

          2. 4.1.2. Determination of the number of theoretical plates (efficiency) and resolution...

        2. 4.2. Test portion

        3. 4.3. Analysis

        4. 4.4. Preparation of the reference chromatogram and reference graphs

      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 7:

            2. 5.2.2.2. Use of correction factors

            3. 5.2.2.3. Use of an internal standard

      6. 6. SPECIAL CASE — DETERMINATION OF TRANS-ISOMERS

        1. 6.1. A capillary column made of silica having an internal diameter...

        2. 6.2. The methyl esters are prepared using procedure B set out...

        3. 6.3. The operating conditions for gas chromatography are overall as follows:...

        4. 6.4. Identification of the various methyl esters is effected on the...

        5. 6.5. The efficiency of the column determined in accordance with point...

        6. 6.6. The percentage of the various trans fatty acids is calculated...

          1. Note 8:

      7. 7. SPECIAL CASE — USE OF A CATHAROMETER DETECTOR (WORKING ON...

      8. 8. TEST REPORT

    14. ANNEX X B

      PREPARATION OF THE FATTY ACID METHYL ESTERS FROM OLIVE OIL AND OLIVE-POMACE OIL

      1. The following two methods are recommended for preparing the fatty...

      2. Method A Trans-esterification with cold methanolic solution of potassium hydroxide...

      3. Each method will be applied according to the analytical parameter...

      4. determination of difference between actual and theoretical content of triglycerides...

      5. PURIFICATION OF OIL SAMPLES

      6. METHODS FOR PREPARING THE FATTY ACID METHYL ESTERS

        1. 1. Method A: Trans-esterification with cold methanolic solution of potassium hydroxide...

          1. 1.1. Purpose

          2. 1.2. Principle

          3. 1.3. Reagents

          4. 1.4. Apparatus

          5. 1.5. Procedure

        2. 2. Method B: Methylation by heating with sodium methylate in methanol...

          1. 2.1. Purpose

          2. 2.2. Principle

          3. 2.3. Reagents

          4. 2.4. Apparatus

          5. 2.5. Procedure

          6. 2.6. Alternatives to methylation Method B

            1. 2.6.1. Method C

              1. 2.6.1.1. Principle

              2. 2.6.1.2. Apparatus

              3. 2.6.1.3. Reagents

                1. Note 1:

              4. 2.6.1.4. Procedure

            2. 2.6.2. Method D

              1. 2.6.2.1. Principle

              2. 2.6.2.2. Apparatus

              3. 2.6.2.3. Reagents

              4. 2.6.2.4. Procedure

                1. Note 2:

        3. 3. Precision parameters

      7. RECOMMENDATIONS FOR GAS CHROMATOGRAPHIC ANALYSIS OF THE FATTY ACID ESTERS...

        1. 1. Procedure

        2. 2. Calculations

          1. 2.1. For the calculation of the fatty acid composition and ΔECN42,...

          2. 2.2. For the calculation of the percentage of trans-C18:1 the peak...

    15. 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...

    16. ANNEX XII

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

      1. 1. PURPOSE AND SCOPE

      2. 2. GENERAL

      3. 3. SPECIFIC VOCABULARY

        1. 3.1. Positive attributes

        2. 3.2. Negative attributes

        3. 3.3. Optional terminology for labelling purposes

      4. 4. PANEL

      5. 5. PROCEDURE FOR ORGANOLEPTIC ASSESSMENT AND GRADING

        1. 5.1. Use of profile sheet by taster

        2. 5.2. Processing of data by panel head

        3. 5.3. Grading of oils

        4. 5.4. Special case

      6. Appendix A

        1. Profile sheet for virgin olive oil

      7. Appendix B

        1. METHOD FOR CALCULATING THE MEDIAN AND CONFIDENCE INTERVALS

          1. Median

          2. Robust standard deviation

          3. Robust variation coefficient %

          4. Confidence intervals at 95 % on the median

    17. 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

    18. 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. . . . . . . . . . ....

    19. 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

    20. 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

    21. 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 bp interval 65 to...

          1. Note 2:

        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...

    22. 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.

          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...

        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)

    23. ANNEX XIX

      DETERMINATION OF ALIPHATIC ALCOHOLS CONTENT BY CAPILLARY GAS CHROMATOGRAPHY

      1. 1. OBJECT

      2. 2. PRINCIPLE OF THE METHOD

      3. 3. EQUIPMENT

        1. 3.1. 250 ml round-bottomed flask fitted with a reflux condenser having...

        2. 3.2. 500 ml separating funnel.

        3. 3.3. 250 ml round-bottomed flasks.

        4. 3.4. Chromatographic tank for thin-layer chromatographic analysis, for glass plates of...

        5. 3.5. Ultraviolet lamp having a wavelength of 366 or 254 nm....

        6. 3.6. 100 μl and 500 μl microsyringes.

        7. 3.7. A cylindrical filter funnel with a G3 porous septum (porosity...

        8. 3.8. 50 ml vacuum conical flask with a 12/21 ground-glass female...

        9. 3.9. A 10 ml test tube with a tapering bottom and...

        10. 3.10. Gas chromatograph for use with a capillary column, and provided...

        11. 3.11. Glass or fused silica capillary column, of length 20 to...

        12. 3.12. Microsyringe for gas chromatography, of 10 μl capacity with hardened...

        13. 3.13. Analytical balance sensitive to 1 mg (with 0,1 mg display)....

      4. 4. REAGENTS

        1. 4.1. Potassium hydroxide, approximately 2 N ethanolic solution: 130 g potassium...

        2. 4.2. Ethyl ether, pure for analysis.

        3. 4.3. Anhydrous sodium sulphate, analytical purity.

        4. 4.4. Glass plates coated with silica gel, without fluorescence indicator, thickness...

        5. 4.5. Potassium hydroxide, approximately 0,2 N ethanolic solution; 13 g of...

        6. 4.6. Benzene, for chromatography (see 5.2.2).

        7. 4.7. Acetone, for chromatography (See 5.2.2).

        8. 4.8. Hexane, for chromatography (see 5.2.2).

        9. 4.9. Ethyl ether, for chromatography (see 5.2.2).

        10. 4.10. Chloroform, for chromatography.

        11. 4.11. Reference solution for thin-layer chromatography: cholesterol or phytosterols, 5 %...

        12. 4.12. 0,2 % solution of 2', 7'-dichlorofluorescein in ethanol. Make slightly...

        13. 4.13. Anhydrous pyridine, for chromatography.

        14. 4.14. Hexamethyl disilazane.

        15. 4.15. Trimethylchlorosilane.

        16. 4.16. Standard solutions of trimethylsilyl ethers of aliphatic alcohols from C...

        17. 4.17. A 0,1 % (m/v) solution of 1-eicosanol in chloroform (internal...

        18. 4.18. Carrier gas: hydrogen or helium, gas-chromatographic purity.

        19. 4.19. Auxiliary gas: nitrogen, gas-chromatographic purity.

      5. 5. PROCEDURE

        1. 5.1. Preparation of the unsaponifiables

          1. 5.1.1. Using a 500 μl microsyringe place, into a 250 ml...

          2. 5.1.2. Add 50 ml of 2 N potassium hydroxide ethanolic solution,...

          3. 5.1.3. The contents of the flask are quantitatively transferred to a...

            1. Note 1:

          4. 5.1.4. The ethyl ether extracts are combined in a separating funnel...

          5. 5.1.5. Distil the ether down to a few ml, then bring...

        2. 5.2. Separation of alcoholic fractions

          1. 5.2.1. Preparation of basic TLC plates: the silica gel plates (4.4)...

            1. Note 2:

          2. 5.2.2. Place a 65/35 by volume hexane/ethyl ether mixture in the...

            1. Note 3:

          3. 5.2.3. An approximately 5 % solution of unsaponifiable matter (5.1.5) in...

          4. 5.2.4. Place the plate inside the development tank as stated in...

          5. 5.2.5. The plate is sprayed lightly and evenly with the solution...

            1. Note 4:

          6. 5.2.6. Using a metal spatula scrape off the silica gel in...

        3. 5.3. Preparation of the trimethylsilyl ethers

          1. 5.3.1. The reagent for silylation, consisting of a mixture of 9:3:1...

            1. Note 5:

            2. Note 6:

          2. 5.3.2. Stopper the test tube, shake carefully (without overturning) until the...

        4. 5.4. Gas chromatography analysis

          1. 5.4.1. Preliminary operations, column packing

            1. 5.4.1.1. Fit the column in the gas chromatograph, attaching the inlet...

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

              1. Note 7:

          2. 5.4.2. Choice of operating conditions

            1. 5.4.2.1. The guideline operating conditions are as follows:

            2. 5.4.2.2. The above requirements are checked by repeated injection of the...

            3. 5.4.2.3. The parameters for the integration of peaks shall be set...

          3. 5.4.3. Analytical procedure

            1. 5.4.3.1. Using the microsyringe of 10 μl capacity draw in 1...

            2. 5.4.3.2. Recording is effected until the TMSE of the aliphatic alcohols...

          4. 5.4.4. Peak identification

          5. 5.4.5. Quantitative evaluation

            1. 5.4.5.1. The peak areas of 1-eicosanol and of the aliphatic alcohols...

            2. 5.4.5.2. The contents of each aliphatic alcohol, expressed in mg/

      6. 6. EXPRESSION OF THE RESULTS

      7. APPENDIX

        Determination of the linear velocity of the gas

        1. 1 to 3 μl of methane or propane are injected...

        2. The linear velocity in cm/s is given by L/tM, where...

    24. 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 (the purity must...

        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

    25. ANNEX XXI

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