Quality characteristics Purity characteristics Notes: (a)The results of the analyses must be expressed to the...(b)If just a single characteristic does not match the values...(c)For lampante olive oil, both quality characteristics marked with an...(d)If a characteristic is marked with two asterisks (**), this...Decision trees 1.CONTENT OF PRIMARY SAMPLE 1.1.Immediate packaging not exceeding 5 litres 1.2.Immediate packaging exceeding 5 litres 2.ANALYSES AND RESULTS 2.1.Each primary sample must be subdivided into laboratory samples, in...2.2.Where all the results of the analyses comply with the...3.VERIFICATION OF THE CATEGORY OF BATCH 3.1.In order to verify the batch category, the competent authority...3.2.When one of the results of the analyses referred to...General table Table 1 — Extra Virgin Olive Oil — Quality criteria...Table 2 — Virgin Olive Oil — Quality criteria Table 3 — Extra Virgin Olive Oil and Virgin Olive...Table 4 — Lampante Olive Oil — Purity criteria Table 5 — Refined Olive Oil — Quality criteria Table 6 — Olive Oil (composed of refined olive oil...Table 7 — Refined Olive Oil and olive oil composed...Table 8 — Crude Olive-Pomace Oil — Purity criteria Table 9 — Refined Olive-Pomace Oil — Quality criteria Table 10 — Olive Pomace Oil — Quality criteria Table 11 — Refined Olive-Pomace Oil and Olive-Pomace Oil —...1.SCOPE AND FIELD OF APPLICATION 2.PRINCIPLE 3.REAGENTS 3.1Diethyl ether; 95 % ethanol (v/v), mixture of equal parts...Note 1:Diethyl ether is highly inflammable and may form explosive peroxides....Note 2:If it is not possible to use diethyl ether, a...3.2Potassium hydroxide or sodium hydroxide, titrated ethanolic or aqueous solution,...Note 3:A stable colourless solution of potassium hydroxide (or sodium hydroxide)...3.3Phenolphthalein, 10 g/l solution in 95 to 96 % ethanol...4.APPARATUS 5.PROCEDURE 5.1Preparation of the test sample 5.2Test portion 5.3Determination Note 4:If the quantity of 0,1 mol/l potassium hydroxide (or sodium...Note 5:If the solution becomes cloudy during titration, add enough of...6.EXPRESSION OF RESULTS 1.Scope 2.Definition 3.Principle 4.Apparatus 4.1.3 ml glass scoop. 4.2.Flasks, with ground necks and stoppers, of about 250 ml...4.3.Burette of 5-ml, 10-ml or 25-ml capacity, graduated in at...4.4.Analytical balance. 5.Reagents 5.1.Chloroform, analytical reagent quality, freed from oxygen by bubbling a...5.2.Glacial acetic acid, analytical reagent quality, freed from oxygen by...5.3.Potassium iodide, saturated aqueous solution, recently prepared, free from iodine...5.4.Sodium thiosulphate, 0,01 mol/l (equivalent to 0,01 N) accurately standardised...5.5.Starch solution, 10 g/l aqueous dispersion, recently prepared from natural...6.Sample 7.Procedure 8.Expression of results 1.SUBJECT 2.PRINCIPLE 3.EQUIPMENT 3.1.25 ml Erlenmeyer flask. 3.2.Glass column for gas chromatography, internal diameter 15,0 mm, length...3.3.Suitable gas chromatograph with a capillary column, equipped with a...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...3.3.5.Glass or fused silica capillary column 8 to 12 m...3.4.10 μl microsyringe for on-column injection, equipped with a hardened...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...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...4.5.1.Sudan 1 (1-phenyl-azo-2-naphthol). 4.6.Carrier gas: hydrogen or helium, gas-chromatographic purity. 4.7.Auxiliary gases: 5.PROCEDURE 5.1.Preparation of the chromatographic column. NB: 5.2.Analysis by gas chromatography 5.2.1.Preparatory work 5.2.2.Choice of operating conditions 5.3.Performance of the analysis 5.4.Identification of peaks 5.5.Evaluation of quantity 6.EXPRESSION OF RESULTS FigureChromatogram of the waxes of an olive oil Determination of the linear velocity of the gas 1.SCOPE 2.PRINCIPLE 3.APPARATUS 4.REAGENTS 4.1.. . . . . . . . . ....4.2.Potassium hydroxide ethanolic solution, approximately 2 N. 4.3.. . . . . . . . . ....4.4.Potassium hydroxide ethanolic solution, approximately 0,2 N. 4.5.. . . . . . . . . ....4.6.. . . . . . . . . ....4.7.. . . . . . . . . ....4.8.. . . . . . . . . ....4.9.. . . . . . . . . ....4.10.. . . . . . . . . ....4.11.. . . . . . . . . ....4.12.. . . . . . . . . ....4.13.. . . . . . . . . ....4.14.. . . . . . . . . ....4.15.. . . . . . . . . ....4.16.. . . . . . . . . ....4.17.. . . . . . . . . ....4.18.Sample solutions of sterol trimethylsilyl ethers. 4.19.. . . . . . . . . ....4.20.. . . . . . . . . ....4.21.. . . . . . . . . ....4.22.. . . . . . . . . ....4.23.. . . . . . . . . ....4.24.. . . . . . . . . ....4.25.. . . . . . . . . ....5.PROCEDURE 5.1.. . . . . . . . . ....5.1.1.. . . . . . . . . ....Note 1:. . . . . 5.1.2.. . . . . . . . . ....5.1.3.. . . . . . . . . ....5.1.4.. . . . . . . . . ....5.1.5.. . . . . . . . . ....5.2.Separation of the sterol and triterpene dialcohols fraction (erythrodiol +...5.2.1.. . . . . . . . . ....Note 3:. . . . . 5.2.2.. . . . . . . . . ....Note 4:. . . . . 5.2.3.. . . . . . . . . ....5.2.4.. . . . . . . . . ....5.2.5.. . . . . . . . . ....5.2.6.. . . . . . . . . ....5.3.. . . . . . . . . ....5.3.1.. . . . . . . . . ....Note 6:. . . . . 5.3.2.. . . . . . . . . ....Note 7:. . . . . 5.4.. . . . . . . . . ....5.4.1.. . . . . . . . . ....5.4.1.1.Fit the column (point 3.11) in the gas chromatograph, by...5.4.1.2.If the column is being used for the first time,...Note 8:. . . . . 5.4.2.. . . . . . . . . ....5.4.2.1.The operating conditions are as follows: 5.4.3.Analytical procedure 5.4.3.1.By using the 10 μl microsyringe, take 1 μl of...5.4.3.2.. . . . . . . . . ....5.4.4.Peak identification 5.4.5.. . . . . . . . . ....5.4.5.1.. . . . . . . . . ....5.4.5.2.Calculate the concentration of each individual sterol, in mg/kg of...6.EXPRESSION OF THE RESULTS 6.1.Report individual sterol concentrations as mg/kg of fatty material and...6.2.Calculate the percentage of each individual sterol from the ratio...6.3.. . . . . . . . . ....6.4.Calculate the percentage of erythrodiol and uvaol: Determination of the linear speed of the gas INTRODUCTION 1.SCOPE 2.PRINCIPLE OF THE METHOD 3.APPARATUS 3.1.. . . . . . . . . ....4.REAGENTS 4.1.. . . . . . . . . ....4.2.. . . . . . . . . ....5.PROCEDURE 5.1.Preparation of the unsaponifiables. 5.2.Separation of erythrodiol and the sterols. 5.2.1.. . . . . . . . . ....5.2.2.. . . . . . . . . ....5.2.3.. . . . . . . . . ....5.2.4.. . . . . . . . . ....5.2.5.. . . . . . . . . ....5.2.6.. . . . . . . . . ....5.3.Preparation of the trimethylsily esters 5.4.Gas chromatographic analysis 6.EXPRESSION OF THE RESULTS 1.PURPOSE AND SCOPE 2.PRINCIPLE 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,...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...3.15.On-column cold injector for direct injection of the sample into...3.16.Flame ionisation detector and electrometer 3.17.Recorder-integrator adapted to the electrometer with a response rate no...3.18.Capillary column made of glass or fused silica 8-12 metres...3.19.10 μl microsyringe fitted with a hardened needle, at least...4.REAGENTS 4.1.Silica gel with a grain size of between 0,063 and...4.2.n-hexane (chromatography grade). Hexane may be replaced by iso-octane (2,2,4-...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...4.6.Buffer solution of trishydroxymethylaminomethane: 1 M aqueous solution adjusted to...4.7.Enzyme-quality sodium cholate, 0,1 % aqueous solution (this solution must...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...4.15.Silanisation reagent: mixture of pyridine/hexamethyldisilazane, trimethylchlorosilane 9/3/1 (v/v/v). (Ready-to-use solutions...4.16.Reference samples: pure monoglycerides or monoglyceride mixtures with a known...5.METHOD 5.1.Sample preparation 5.1.1.Oils with a free acidity of less than 3 %...5.1.1.1.Pour 50 g of oil and 200 ml n-hexane into...5.1.2.Put 1,0 g of the oil prepared as above into...5.1.3.Preparation of the chromatography column 5.1.4.Column chromatography 5.2.Hydrolysis by pancreatic lipase 5.2.1.Weigh into the centrifuge tube 0.1 g of the oil...5.2.2.Add 20 mg of lipase, shake carefully (avoid wetting the...5.2.3.Add 1 ml of diethyl ether, stopper and shake vigorously,...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...5.3.2.Remove the solvent under a slight nitrogen current, add 200...5.3.3.After 20 minutes, add 1 to 5 ml of n-hexane...5.4.Gas chromatography 5.4.1.Identification of the peaks 5.4.2.Quantitative evaluation 6.EXPRESSION OF RESULTS 7.ANALYSIS REPORT Figure 2 ( A )unesterified olive oil, after lipase; after silanisation; under these conditions...( B )unesterified oil after lipase; after silanisation; under these conditions (8-12...8.NOTES Note 1.PREPARATION OF THE LIPASE Note 2.MONITORING LIPASE ACTIVITY 1.SCOPE 2.FIELD OF APPLICATION 3.PRINCIPLE 4.APPARATUS 4.1.. . . . . . . . . ....4.2.. . . . . . . . . ....4.3.. . . . . . . . . ....4.4.. . . . . . . . . ....4.5.. . . . . . . . . ....5.REAGENTS 5.1.. . . . . . . . . ....5.2.. . . . . . . . . ....5.3.. . . . . . . . . ....5.4.. . . . . . . . . ....5.5.. . . . . . . . . ....5.6.. . . . . . . . . ....6.PREPARATION OF SAMPLES 7.PROCEDURE 7.1.. . . . . . . . . ....8.CALCULATION AND EXPRESSION OF RESULTS Note 1. Note 2. Examples: Note 3. Note 4. Note 5: FOREWORD 1.SCOPE 2.PRINCIPLE OF THE METHOD 3.EQUIPMENT 3.1.A spectrophotometer suitable for measurements at ultraviolet wavelengths (220 nm...3.1.1.Wavelength scale: This may be checked using a reference material...3.1.2.Absorbance scale: This may be checked using commercially available sealed...3.2.Rectangular quartz cuvettes, with covers, suitable for measurements at the...3.3.One- mark volumetric flasks, capacity 25 ml, class A. 3.4.Analytical balance, capable of being read to the nearest 0,0001...4.REAGENTS 5.PROCEDURE 5.1.The sample must be perfectly homogeneous and without suspended impurities....5.2.Weigh accurately approximately 0,25 g (to the nearest 1 mg)...5.3.If necessary, correct the baseline (220-290 nm) with solvent in...5.4.After measuring the absorbance at 268 or 270 nm, measure...6.EXPRESSION OF THE RESULTS 6.1.Record the specific extinctions (extinction coefficients) at the various wavelengths...6.2.Variation of the specific extinction (ΔΚ) 1.SCOPE 2.PRINCIPLE PART APREPARATION OF THE FATTY ACID METHYL ESTERS FROM OLIVE OIL...1.SCOPE 2.FIELD OF APPLICATION 3.METHODOLOGY 3.1.Trans-esterification with methanolic solution of potassium hydroxide at room temperature...3.1.1.Principle 3.1.2.Reagents 3.1.2.1.Methanol containing not more than 0,5 % (m/m) water. 3.1.2.2.Hexane, chromatographic quality. 3.1.2.3.Heptane, chromatographic quality. 3.1.2.4.Diethyl ether, stabilised for analysis. 3.1.2.5.Acetone, chromatographic quality. 3.1.2.6.Elution solvent for purifying the oil by column/SPE chromatography, mixture...3.1.2.7.Potassium hydroxide, approximately 2M methanolic solution: dissolve 11,2 g of...3.1.2.8.Silica gel cartridges, 1 g (6 ml), for solid phase...3.1.3.Apparatus 3.1.3.1.Screw-top test tubes (5 ml volume) with cap fitted with...3.1.3.2.Graduated or automatic pipettes, 2 ml and 0,2 ml. 3.1.4.Purification of oil samples 3.1.5.Procedure PART BANALYSIS OF FATTY ACID METHYL ESTERS BY GAS CHROMATOGRAPHY 1.SCOPE 2.REAGENTS 2.1.Carrier gas Note 1:Hydrogen can double the speed of analysis but is hazardous....2.2.Auxiliary gases 2.2.1.Hydrogen (purity ≥ 99,9 %), free from organic impurities. 2.2.2.Air or oxygen, free from organic impurities. 2.2.3.Nitrogen (purity > 99 %). 2.3.Reference standard 3.APPARATUS 3.1.Gas chromatograph 3.1.1.Injection system 3.1.2.Oven 3.1.3.Capillary column 3.1.3.1.Tube, made of a material inert to the substances to...3.1.3.2.Stationary phase, polar polysiloxane (cyanopropylsilicone) bonded (cross-linked) columns are suitable....Note 2:There is a risk that polar polysiloxanes may give rise...3.1.3.3.Assembly and conditioning of the column Note 3:Suitably pre-conditioned columns are available commercially. 3.1.4.Flame ionisation detector and converter-amplifier 3.2.Syringe 3.3.Data acquisition system 4.PROCEDURE 4.1.Test conditions 4.1.1.Selection of optimum operating conditions for capillary columns 4.1.2.Determination of the resolution (see Appendix A) 5.EXPRESSION OF RESULTS 5.1.Qualitative analysis 5.2.Quantitative analysis 5.2.1.Determination of the composition 5.2.2.Method of calculation 5.2.2.1.General case Note 4:For fats and oils, the mass fraction of the fatty...5.2.2.2.Use of correction factors Note 5:These correction factors are not identical to the theoretical FID...Note 6:The calculated value corresponds to the percentage of mass of...5.2.2.3.Use of an internal standard 6.TEST REPORT 7.PRECISION 7.1.Results of interlaboratory test 7.2.Repeatability 7.3.Reproducibility 1.METHOD 2.EQUIPMENT 2.1.Gas chromatography apparatus fitted with an electron capture detector (ECD)....2.2.Head space apparatus. 2.3.Gas chromatography column, of glass, 2 m long and 2...2.4.Carrier and auxiliary gas: nitrogen for gas chromatography, suitable for...2.5.Glass flasks, 10 to 15 ml, with teflon coating and...2.6.Hermetically sealing clamps. 2.7.Gas syringe 0,5 to 2 ml. 3.REAGENTS 4.PROCEDURE 4.1.Exactly weigh around 3 g of oil in a glass...4.2.Reference solutions: prepare standard solutions using refined olive oil with...4.3.Quantitative assessment: correlate the surfaces or the elevations of the...4.4.Expression of results: in ppm (mg/kg). The detection limit for...1.PURPOSE AND SCOPE 2.GENERAL BASIC VOCABULARY FOR SENSORY ANALYSIS 3.SPECIFIC VOCABULARY 3.1.Negative attributes 3.1.1.Other negative attributes 3.2.Positive attributes 3.3.Optional terminology for labelling purposes 4.GLASS FOR OIL TASTING 5.TEST ROOM 6.ACCESSORIES 7.PANEL LEADER AND TASTERS 7.1.Panel leader 7.1.1.Deputy panel leader 7.2.Tasters 8.TEST CONDITIONS 8.1.Presentation of the sample 8.2.Test and sample temperature 8.3.Test times 8.4.Tasters: general rules of conduct 9.PROCEDURE FOR THE ORGANOLEPTIC ASSESSMENT AND CLASSIFICATION OF VIRGIN OLIVE...9.1.Tasting technique 9.1.1.The tasters shall pick up the glass, keeping it covered...9.1.2.When organoleptically assessing a virgin olive oil, it is recommended...9.2.Use of the profile sheet by tasters 9.3.Use of the data by the panel leaders 9.4.Classification of the oil Note 1:When the median of the bitter and/or pungent attribute is...9.5Criteria for the acceptance and rejection of duplicates Method for calculating the median and the confidence intervals Median Robust standard deviation Robust coefficient of variation (%) Confidence intervals of the median at 95% References (1)Wilkinson, L. 1990. Systat: The system for statistics. Evanston, IL.SYSTAT...(2)Cicchitelli, G. 1984. Probabilità e Statistica. Maggioli Editore, Rimini. (3)Massart, D.L.; Vandeginste, B.G.M.; Deming, Y.; Michotte, L. 1988. Chemometrics....(4)Kendall, M.G.; Stuart, A. 1967. The advanced theory of statistics....(5)McGill, R.; Tukey, J.W.; Larsen, W.A. 1978. Variation of Box...(6)IOC/T.28/Doc. No 1 September 2007, Guidelines for the accreditation of...(7)IOC/T.20/Doc. No 14. (8)IOC/T.20/Doc. No 15. (9)ISO/IEC 17025:05. 1.NEUTRALIZATION AND DECOLORIZATION OF OLIVE OIL IN THE LABORATORY 1.1.Neutralization of the oil 1.1.1.Apparatus 1.1.2.Reagents 1.1.3.Procedure (a)Oils with a free fatty acid content, expressed as oleic...(b)Oils with a free fatty acid content expressed as oleic...1.2.Decolorization of neutralized oil 1.2.1.Apparatus 1.2.2.Procedure 2.A. . . . . . . . . ....A.. . . . . . . . . ....B.. . . . . . . . . ....I.. . . . . . . . . ....II.. . . . . . . . . ....C.. . . . . . . . . ....D.. . . . . . . . . ....E.. . . . . . . . . ....3.. . . . . . . . . ....4.. . . . . . . . . ....1.OIL CONTENT OF OLIVE RESIDUE 1.1.Apparatus 1.2.Reagent 2.PROCEDURE 2.1.Preparation of the test sample 2.2.Test portion 2.3.Preparation of the extraction thimble 2.4.Peliminary drying 2.5.Preparation of the round-bottomed flask 2.6.Initial extraction 2.7.Second extraction 2.8.Removal of solvent and weighing of extract 3.EXPRESSION OF RESULTS 3.1.Method of calculation and formula 3.2.Repeatability 1.SCOPE 2.DEFINITION 2.1.iodine value. The mass of iodine absorbed by the sample...3.PRINCIPLE 4.REAGENTS 4.1.water, complying with the requirements of ISO 3696, Grade 3....4.2.potassium iodide, 100 g/l solution, not containing iodate or free...4.3.starch, solution. 4.4.sodium thiosulfate, standard volumetric solution c (Na2S2O3.5H2O) = 0,1 mol/l,...4.5.solvent, prepared by mixing equal volumes of cyclohexane and acetic...4.6.Wijs reagent, containing iodine monochloride in acetic acid. Commercially available...5.APPARATUS 5.1.glass weighing scoops, suitable for the test portion and for...5.2.conical flasks, of 500 ml capacity, fitted with ground glass...6.PREPARATION OF THE TEST SAMPLE 7.PROCEDURE 7.1.Test portion 7.2.Determination Note: 7.3.Number of determinations 8.EXPRESSION OF RESULTS 1.PURPOSE 2.SCOPE 3.PRINCIPLE 4.APPARATUS 4.1.250 ml flasks suitable for use with a reflux condenser....4.2.Separating funnels of 500 ml capacity. 4.3.100 ml round-bottom flasks. 4.4.Rotary evaporator. 4.5.Glass chromatography column (1,5 to 2,0 cm internal diameter by...4.6.Gas chromatograph with flame ionization detector, split or cold on-column...4.7.Fused silica capillary column for gas chromatography (0,25 or 0,32...Note 1: 4.8.Integrator-recorder with possibility of valley-valley integration mode. 4.9.5 to 10 ml microsyringe for gas chromatography with cemented...4.10.Electrical heating mantle or hot place. 5.REAGENTS 5.1.Hexane or mixture of alkanes of b.p. interval 65 to 70 °C,...5.2.96 v/v ethanol. 5.3.Anhydrous sodium sulphate. 5.4.Alcoholic potassium hydroxide solution at 10 %. Add 10 ml...Note 3: 5.5.Silica gel 60 for column chromatography, 70 to 230 mesh,...Note 4: 5.6.Stock solution (200 ppm) of cholesta-3,5-diene (Sigma, 99 % purity)...5.7.Standard solution of cholesta-3,5-diene hexane at concentration of 20 ppm,...Note 5: 5.8.Solution of n-nonacosane in hexane at concentration of approximately 100...5.9.Carrier gas for chromatography: helium or hydrogen of 99,9990 %...5.10.Auxiliary gases for flame ionization detector: hydrogen of 99,9990 %...6.PROCEDURE 6.1.Preparation of unsaponifiable matter 6.1.1.Weigh 20 ± 0,1 g of oil into a 250-ml...Note 6: 6.1.2.Transfer the aqueous phase beneath to a second separating funnel...6.1.3.Pass the hexane solution through anhydrous sodium sulphate (50 g),...6.2.Separation of steroidal hydrocarbon fraction 6.2.1.Take the residue to the fractioning column with the aid...Note 7: 6.2.2.Evaporate the second fraction in a rotary evaporator at 30...Note 8: 6.3.Gas chromatography 6.3.1.Working conditions for split injection: 6.3.2.Peak identification Note 9: 6.3.3.Quantitative analysis Figure 1 1.SCOPE 2.FIELD OF APPLICATION 3.PRINCIPLE 4.METHOD 4.1.Apparatus 4.1.1.Round-bottomed flasks, 250 and 500 ml. 4.1.2.Beakers 100 ml. 4.1.3.Glass chromatographic column, 21 mm internal diameter, 450 mm length,...4.1.4.Separating funnels, 250 ml, with normalised cone (male) at the...4.1.5.Glass rod, 600 mm length. 4.1.6.Glass funnel, 80 mm diameter. 4.1.7.Volumetric flasks, 50 ml. 4.1.8.Volumetric flasks, 20 ml. 4.1.9.Rotary evaporator. 4.1.10.High performance liquid chromatograph, allowing thermostatic control of column temperature....4.1.11.Injection units for 10 μl delivery. 4.1.12.Detector: differential refractometer. The full scale sensitivity should be at...4.1.13.Column: stainless steel tube 250 mm length x 4,5 mm...4.1.14.Data processing software. 4.1.15.Vials, of about 2 ml volumes, with Teflon-layered septa and...4.2.Reagents 4.2.1.Petroleum ether 40-60 °C chromatographic grade or hexane. Hexane may be...4.2.2.Ethyl ether, peroxide-free, freshly distilled. 4.2.3.Elution solvent for purifying the oil by column chromatography mixture...4.2.4.Silica gel, 70-230 mesh, type Merck 7734, with water content...4.2.5.Glass wool. 4.2.6.Acetone for HPLC. 4.2.7.Acetonitrile or propionitrile for HPLC. 4.2.8.HPLC elution solvent: acetonitrile + acetone (proportions to be adjusted...4.2.9.Solubilisation solvent: acetone. 4.2.10.Reference triglycerides: commercial triglycerides (tripalmitin, triolein, etc.) may be used...4.2.11.Solid phase extraction column with silica phase 1 g, 6...4.2.12.Heptane, chromatographic quality. Heptane may be replaced by iso-octane (2,2,4-trimethyl...4.3.Sample preparation 4.3.1.Chromatographic column preparation 4.3.2.Column chromatography 4.3.3.SPE purification 4.4.HPLC analysis 4.4.1.Preparation of the samples for chromatographic analysis 4.4.2.Procedure 4.4.3.Calculation and expression of results 4.5.Calculation of triacylglycerols composition (moles %) from fatty acid composition...4.5.1.Determination of fatty acid composition 4.5.2.Fatty acids for calculation 4.5.3.Conversion of area % into moles for all fatty acids...4.5.4.Normalisation of fatty acid moles to 100 % (2) 4.5.5.Calculation of the fatty acid composition in 2- and 1,...4.5.5.1.Saturated fatty acids in 2-position [P(2) and S(2)] (4): 4.5.5.2.Unsaturated fatty acids in 2-position [Po(2), O(2), L(2) and Ln(2)]...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...4.5.6.Calculation of triacylglycerols 4.5.6.1.TAGs with one fatty acid (AAA, here LLL, PoPoPo) (7)...4.5.6.2.TAGs with two fatty acids (AAB, here PoPoL, PoLL) (8)...4.5.6.3.TAGs with three different fatty acids (ABC, here OLLn, PLLn,...4.5.6.4.Triacylglycerols with ECN42 5.EVALUATION OF THE RESULTS 6.EXAMPLE (THE NUMBERS REFER TO THE SECTIONS IN THE TEXT...— 4.5.1.Calculation of moles % fatty acids from GLC data (normalised...— 4.5.3Conversion of area % into moles for all fatty acids...— 4.5.4Normalisation of fatty acid moles to 100 % (see formula...— 4.5.5Calculation of the fatty acid composition in 2- and 1,3-positions...— 4.5.5.1Saturated fatty acids in 2-position [P(2) and S(2)] (see formula...— 4.5.5.2Unsaturated fatty acids in 2-position [Po(1,3), O(1,3), L(1,3) and Ln(1,3)]...— 4.5.5.3Fatty acids in 1,3-positions [P(1,3), S(1,3), Po(1,3), O(1,3), L(1,3) and...— 4.5.6.Calculation of triacylglycerols — 4.5.6.1.TAGs with one fatty acid (LLL, PoPoPo) (see formula (7))...— 4.5.6.2TAGs with two fatty acids (PoLL, SLnLn, PoPoL) (see formula...— 4.5.6.3TAGs with three different fatty acids (PoPLn, OLLn, PLLn, PoOLn)...DETERMINATION OF THE STEROL COMPOSITION AND CONTENT AND ALCOHOLIC COMPOUNDS BY...1.SCOPE 2.PRINCIPLE PART 1
PREPARATION OF THE UNSAPONIFIABLE MATTER
1.SCOPE 2.PRINCIPLE 3.APPARATUS 4.REAGENTS 5.PROCEDURE Note 1:Animal or vegetable oils and fats containing appreciable quantities of...Note 2:Any emulsion can be destroyed by adding small quantities of...PART 2
SEPARATION OF THE ALCOHOLIC COMPOUNDS FRACTIONS
1.SCOPE 2.PRINCIPLE 3.APPARATUS 4.REAGENTS 5.REFERENCE METHOD: SEPARATION OF THE ALCOHOLIC COMPOUNDS BY BASIC THIN-LAYER...Note 3:The developing mixture should be replaced for every test, in...Note 4:Higher temperature could worsen the separation. 6.SEPARATION OF THE ALCOHOLIC FRACTION BY HPLC Note 5:Carefully control the pressure of the HPLC pump, the ethyl...PART 3
GAS CHROMATOGRAPHIC ANALYSIS OF THE ALCOHOLIC COMPOUNDS FRACTIONS
1.SCOPE 2.PRINCIPLE 3.APPARATUS 4.REAGENTS 5.PREPARATION OF THE TRIMETHYLSILYL ETHERS Note 6:Ready for use solutions are available commercially. Other silylation reagents,...Note 7:The slight opalescence, which may form, is normal and does...6.GAS CHROMATOGRAPHIC ANALYSIS 6.1.Preliminary operations, capillary column conditioning Note 8:The conditioning temperature must always be at least 20 °C less...6.2.Operating conditions 6.2.1.Aliphatic alcohols 6.2.2.Sterol and triterpenic dialcohols 6.3.Analytical procedure 6.4.Peak identification 6.5.Quantitative evaluation 7.EXPRESSION OF THE RESULTS 1.PURPOSE 2.PRINCIPLE 3.APPARATUS 3.1.Erlenmeyer flask, 25 ml . 3.2.Glass column for liquid chromatography, internal diameter 15 mm, length...3.3.Gas chromatograph suitable for use with a capillary column, equipped...3.3.1.Thermostat-controlled oven with temperature programming . 3.3.2.Cold injector for direct on-column injection 3.3.3.Flame ionisation detector and converter-amplifier . 3.3.4.Recorder-integrator (Note 1) for use with the converter-amplifier (point 3.3.3),...Note 1:Computerised systems may also be used where the gas chromatography...3.3.5.Capillary column, fused silica (for analysis of the waxes and...Note 2:Suitable commercial liquid phases are available for this purpose such...3.4.Microsyringe , 10 μl, with hardened needle, for direct on-column...3.5.Electric shaker . 3.6.Rotary evaporator . 3.7.Muffle oven . 3.8.Analytical balance for weighing to an accuracy of ± 0,1...3.9.Usual laboratory glassware. 4.REAGENTS 4.1.Silica gel , 60-200 μm mesh. Place the silica gel...4.2.n-hexane, chromatography grade or residue grade. Hexane may be replaced...4.3.Ethyl ether, chromatography grade 4.4.n-heptane , chromatography grade, or iso-octane 4.5.Standard solution of lauryl arachidate ( Note 3 ), at...Note 3:Palmityl palmitate, myristyl stearate or arachidyl laureate may also be...4.6.Standard solution of methyl heptadecanoate, at 0,02 % (m/V) in...4.7.Sudan 1 (1-phenylazo-2-naphthol) . 4.8.Carrier gas: hydrogen or helium, pure, gas chromatography grade ....WARNING 4.9.Auxiliary gases : WARNING 5.PROCEDURE 5.1.Preparation of the chromatography column Note 4:The n-hexane/ethyl ether (99:1) mixture should be freshly prepared every...Note 5:100 μl of Sudan I dye at 1 % in...5.2.Gas chromatography analysis 5.2.1.Preliminary procedure 5.2.2.Choice of operating conditions for waxes and methyl and ethyl...Note 6:Due to the high final temperature, positive drift is allowed...5.3.Performance of the analysis 5.4.Peak identification 5.5.Quantitative analysis of the waxes 5.5.1.Quantitative analysis of the methyl and ethyl esters 6.EXPRESSION OF RESULTS Note 7:The components for quantification refer to the peaks with even...Determination of linear gas speed 1.SCOPE 2.PRINCIPLE 3.MATERIAL AND REAGENTS 3.1.Oil purification 3.1.1.. . . . . . . . . ....3.1.2.. . . . . . . . . ....3.1.3.. . . . . . . . . ....3.1.4.. . . . . . . . . ....3.1.5.. . . . . . . . . ....3.1.6.. . . . . . . . . ....3.1.7.. . . . . . . . . ....3.2.HPLC analysis of triacylglycerols 3.2.1.. . . . . . . . . ....3.2.2.. . . . . . . . . ....3.2.3.. . . . . . . . . ....3.3.Preparation of fatty acid methyl esters 3.3.1.. . . . . . . . . ....3.3.2.. . . . . . . . . ....3.3.3.. . . . . . . . . ....3.3.4.. . . . . . . . . ....3.4.GC analysis of FAMEs 3.4.1. . . . . . . . . ....3.4.2. . . . . . . . . ....3.4.3. . . . . . . . . ....3.4.4. . . . . . . . . ....3.4.5.. . . . . . . . . ....4.APPARATUS 4.1.. . . . . . . . . ....4.2.. . . . . . . . . ....4.3.HPLC equipment composed of: 4.4Capillary gas chromatography equipment described in Annex X A, provided...4.5.. . . . . . . . . ....5.ANALYTICAL PROCEDURE 5.1.Oil purification 5.2.HPLC analysis of triacylglycerols 5.3.Preparation of fatty acid methyl esters 5.4.GC analysis of fatty acid methyl esters 6.INTEGRATION OF CHROMATOGRAPHIC PEAKS 6.1.HPLC chromatogram 6.2.GC chromatogram 7.DETECTION OF EXTRANEOUS OILS IN OLIVE OILS
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 fats1, as last amended by Regulation (EEC) No 3577/902, 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 1058/773, as last amended by Regulation (EEC) No 1858/884;
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: