[F1ANNEX X B U.K. PREPARATION OF THE FATTY ACID METHYL ESTERS FROM OLIVE OIL AND OLIVE-POMACE OIL
Textual Amendments
F1 Substituted by Commission Regulation (EC) No 796/2002 of 6 May 2002 amending Regulation (EEC) No 2568/91 on the characteristics of olive oil and olive-pomace oil and on the relevant methods of analysis and the additional notes in the Annex to Council Regulation (EEC) No 2658/87 on the tariff and statistical nomenclature and on the Common Customs Tariff.
The following two methods are recommended for preparing the fatty acid methyl esters from olive oils and olive-pomace oils:
:
Trans-esterification with cold methanolic solution of potassium hydroxide
:
Methylation by heating with sodium methylate in methanol followed by esterification in acid medium.
Each method will be applied according to the analytical parameter to be determined and the oil category as indicated below:
determination of difference between actual and theoretical content of triglycerides with ECN42 (ΔECN42):
method A will be applied to samples of all the oil categories after purification of the oil by passing it through a silica gel column;
determination of the fatty acid composition:
method A will be applied directly to samples of the following oil categories:
virgin olive oils with an acidity of less than 3,3 %,
refined olive oil,
olive oil (blend of virgin olive oils and refined olive oil),
refined olive-pomace oil,
olive-pomace oil (blend of virgin olive oils and refined olive-pomace oil);
method B will be applied directly to samples of the following oil categories:
virgin olive oil with an acidity of more than 3,3 %,
crude olive-pomace oil;
determination of trans-isomers of fatty acids:
method A will be applied directly to samples of the following oil categories:
virgin olive oils with an acidity of less than 3,3 %,
refined olive oil,
olive oil (blend of virgin olive oils and refined olive oil),
refined olive-pomace oil,
olive-pomace oil (blend of virgin olive oils and refined olive-pomace oil);
method A will be applied to the following categories of oils after purification of the oil by passing it through a silica gel column:
virgin olive oil with an acidity of more than 3,3 %,
crude olive-pomace oil.
PURIFICATION OF OIL SAMPLES U.K.
When necessary, the samples will be purified by passing the oil through a silica gel column, eluting with hexane/diethyl ether (87:13, v/v) as described in IUPAC method 2.507.
Alternatively, solid-phase extraction on silica gel phase cartridges can be used. A silica gel cartridge (1 g, 6 ml) is placed in a vacuum elution apparatus and washed with 6 ml of hexane. The vacuum is released to prevent the column from becoming dry and then a solution of the oil (0,12 g approximately) in 0,5 ml of hexane is loaded into the column and vacuum is applied. The solution is pulled down and then eluted with 10 ml of hexane/diethyl ether (87:13 v/v) under vacuum. The combined eluates are homogenised and divided in two similar volumes. An aliquot is evaporated to dryness in a rotary evaporator under reduced pressure at room temperature. The pomace is dissolved in 1 ml of heptane and the solution is ready for fatty acid analysis by GC. The second aliquot is evaporated and the pomace is dissolved in 1 ml of acetone for triglyceride analysis by HPLC, if necessary.
METHODS FOR PREPARING THE FATTY ACID METHYL ESTERS U.K.
1. Method A: Trans-esterification with cold methanolic solution of potassium hydroxide U.K.
1.1. Purpose U.K.
This rapid method is applicable to olive oils and olive-pomace oils with a free fatty acid content of less than 3,3 %. Free fatty acids are not esterified by potassium hydroxide. Fatty acid ethyl esters are trans-esterified at a lower rate than glyceridic esters and may be only partially methylated.
1.2. Principle U.K.
Methyl esters are formed by trans-esterification with methanolic potassium hydroxide as an intermediate stage before saponification takes place (title 5 in ISO-5509:2000, title 5 in IUPAC method 2.301).
1.3. Reagents U.K.
Methanol containing not more than 0,5 % (m/m) water.
Heptane, chromatographic quality.
Potassium hydroxide, approximately 2 N methanolic solution: dissolve 11,2 g of potassium hydroxide in 100 ml of methanol.
1.4. Apparatus U.K.
Screw-top test tubes (5 ml volume) with cap fitted with a PTFE joint.
Graduated or automatic pipettes, 2 ml and 0,2 ml
1.5. Procedure U.K.
In a 5 ml screw-top test tube weigh approximately 0,1 g of the oil sample. Add 2 ml of heptane, and shake. Add 0,2 ml of 2 N methanolic potassium hydroxide solution, put on the cap fitted with a PTFE joint, tighten the cap, and shake vigorously for 30 seconds. Leave to stratify until the upper solution becomes clear. Decant the upper layer containing the methyl esters. The heptane solution is suitable for injection into the gas chromatograph. It is advisable to keep the solution in the refrigerator until gas chromatographic analysis. Storage of the solution for more than 12 hours is not recommended.
2. Method B: Methylation by heating with sodium methylate in methanol followed by esterification in acid medium U.K.
2.1. Purpose U.K.
This method is applicable to olive oils and olive-pomace oils with a free fatty acid content of more than 3,3 %.
2.2. Principle U.K.
Neutralisation of the free fatty acids and alkaline methanolysis of the glycerides, followed by esterification of the fatty acids in acid medium (title 4.2. in IUPAC method 2.301).
2.3. Reagents U.K.
heptane, chromatographic quality,
methanol containing not more than 0,05 % (m/m) water,
sodium methylate, 0,2 N methanolic solution: dissolve 5 g of sodium in 1 000 ml of methanol (this may be prepared from commercial solutions),
phenolphthalein, 0,2 % methanolic solution,
sulphuric acid, 1 N in methanolic solution: add 3 ml of 96 % sulphuric acid to 100 ml of methanol,
saturated solution of sodium chloride in water.
2.4. Apparatus U.K.
50 ml flat-bottomed volumetric flask with long, narrow, ground neck,
reflux condenser: air condenser (1 m long) with ground joint appropriate to the neck of the flask,
boiling chips,
glass funnel.
2.5. Procedure U.K.
Transfer about 0,25 g of the oil sample into a 50 ml ground-necked volumetric flask. With the aid of a funnel, add 10 ml of 0,2 N sodium methylate in methanol and the boiling chips. Fit a reflux condenser, shake, and bring to the boil. The solution should become clear, which usually occurs in about 10 minutes. The reaction is complete after 15 minutes. Remove the flask from the source of heat, wait until the reflux stops, remove the condenser, and add two drops of phenolphthalein solution. Add a few ml of 1 N sulphuric acid in methanol solution until the solution becomes colourless and then add 1 ml in excess. Fit the condenser and boil again for 20 minutes. Withdraw from the source of heat and cool the flask under running water. Remove the condenser, add 20 ml of saturated sodium chloride solution, and shake. Add 5 ml of heptane, plug the flask, and shake vigorously for 15 seconds.
Leave to settle until the two phases have separated. Add saturated sodium chloride solution again until the aqueous layer reaches the lower end of the flask neck. The upper layer containing the methyl esters fills the flask neck. This solution is ready to be injected in the GC.
Caution : Methylation by method B must be done under a hood.
2.6. Alternatives to methylation Method B U.K.
2.6.1. Method C U.K.
2.6.1.1. Principle U.K.
The fatty matter undergoing analysis is treated with methanol-hydrochloric acid, in a sealed vial, at 100 °C.
2.6.1.2. Apparatus U.K.
Strong glass vial of a capacity of about 5 ml (height 40 to 45 mm, diameter 14 to 16 mm).
1 and 2 ml graduated pipettes.
2.6.1.3. Reagents U.K.
Solution of hydrochloric acid in 2 % methanol. This is prepared from gaseous hydrochloric acid and anhydrous methanol (Note 1).
Hexane, chromatographic quality.
Note 1: U.K.
Commercial solutions of hydrogen chloride in methanol can be used. Small amounts of gaseous hydrochloric acid can easily be prepared in the laboratory by simple displacement from the commercial solution (p = 1,18) by dripping concentrated sulphuric acid. Since hydrochloric acid is very rapidly absorbed by methanol, it is advisable to take the usual precautions when dissolving it, e.g. introduce the gas through a small inverted funnel with the rim just touching the surface of the liquid. Large quantities of methanolic hydrochloric acid solution can be prepared in advance, as it keeps perfectly in glass-stoppered bottles stored in the dark. Alternatively, this reagent can be prepared by dissolution of acetyl chloride in anhydrous methanol. U.K.
2.6.1.4. Procedure U.K.
Place in the glass vial 0,2 g of the fatty matter, which has previously been dried out on sodium sulphate and filtered, and 2 ml of hydrochloric acid-methanol solution. Heat seal the vial.
Immerse the vial at 100 °C for 40 minutes.
Cool the vial under running water, open, add 2 ml of distilled water and 1 ml of hexane.
Centrifuge and remove the hexane phase, which is ready for use.
2.6.2. Method D U.K.
2.6.2.1. Principle U.K.
The fatty matter undergoing analysis is heated under reflux with methanol-hexane-sulphuric acid. The methyl esters obtained are extracted with petroleum ether.
2.6.2.2. Apparatus U.K.
Test tube of a capacity of about 20 ml, fitted with an air reflux condenser approximately 1 m in length, with ground glass joints.
5 ml graduated pipette.
50 ml separating funnel.
10 ml and 25 ml measuring beakers.
15 ml test tube with conical base.
2.6.2.3. Reagents U.K.
Methylation reagent: anhydrous methanol-hexane-concentrated sulphuric acid (p = 1,84) in the ratio 75:25:1 (V/V/V).
40 to 60 °C petroleum ether.
Anhydrous sodium sulphate.
2.6.2.4. Procedure U.K.
Place 0,1 g of oil in the 20 ml test tube and add 5 ml of methylation reagent.
Fit the reflux condenser and heat for 30 minutes in a boiling water bath (Note 2).
Transfer quantitatively the mixture into a 50 ml separating funnel, with the aid of 10 ml distilled water and 10 ml petroleum ether. Shake vigorously, and allow the phases to separate, remove the aqueous phase and wash the ether layer twice with 20 ml distilled water. Add to the separating funnel a small quantity of anhydrous sodium sulphate, shake, allow to settle for a few minutes and filter, collecting the filtrate in a 15 ml test tube with a conical base.
Evaporate the solvent over a water bath in a current of nitrogen.
Note 2: U.K.
To control boiling, insert a glass rod into the test tube and limit the temperature of the water bath to 90 °C. U.K.
3. Precision parameters U.K.
The statistical evaluation of the precision of methods A and B was published by the International Olive Oil Council in its method COI/T.20/CO. No 24.
RECOMMENDATIONS FOR GAS CHROMATOGRAPHIC ANALYSIS OF THE FATTY ACID ESTERS FROM OLIVE OIL AND OLIVE-POMACE OIL U.K.
1. Procedure U.K.
The gas chromatographic analysis of solutions of fatty esters in heptane is to be carried out according to standard ISO-5508 using a capillary column (50 m length x 0,25 or 0,32 mm i.d.) impregnated with cyanopropylsilicone phase as indicated for the determination of fatty acid trans-isomers (COI/T.20/Doc. no. 17).
Figure 1 gives the typical gas chromatographic profile of an olive-pomace oil containing methyl and ethyl esters of fatty acids, and trans-isomers of methyl esters.
2. Calculations U.K.
2.1. For the calculation of the fatty acid composition and ΔECN42, all the following fatty acids will be taken into account: U.K.
Myristic (C14:0).
Palmitic (C16:0). Sum of the areas of the peaks corresponding to the methyl and ethyl esters.
Palmitoleic (C16:1). Sum of the areas of the peaks corresponding to the ω9 and ω7 isomers of the methyl ester.
Margaric (C17:0).
Margaroleic (C17:1).
Stearic (C18:0).
Oleic (C18:1). Sum of the areas of the peaks corresponding to the ω9 and ω7 isomers of the methyl ester, ethyl ester, and trans-isomers of the methyl ester.
Linoleic (C18:2). Sum of the areas of the peaks corresponding to the methyl and ethyl esters, and the trans-isomers of the methyl ester.
Arachidic (C20:0).
Linolenic (C18:3). Sum of the areas of the methyl ester and the trans-isomers of the methyl ester.
Eicosenoic (C20:1).
Behenic (C22:0).
Lignoceric (C24:0).
Squalene will not be taken into account for the calculation of the total area.
2.2. For the calculation of the percentage of trans-C18:1 the peak corresponding to the methyl esters of this fatty acid is to be used. For the sum [trans-C18:2 + trans-C18:3], all the peaks corresponding to the trans-isomers of these two fatty acids are to be added together. For the calculation of the total area, all the peaks mentioned in 2.1. are to be taken into account (see COI/T.20/Doc. No. 17). U.K.
The calculation of the percentage of each fatty acid will be carried out according to the formula:
