COMMISSION REGULATION (EEC) N° 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 N° 136/66/EEC of

22 September 1966 on the establishment of a common organization of the market in oils and fats (1), as last amended by Regulation (EEC) N° 3577/90 (2), and in particular Article 35a thereof,

Whereas the Annex to Regulation N° 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) N° 1058/77 (3), as last amended by Regulation (EEC) N° 1858/88 (4);

Whereas the Management Committee for Oils and Fats has not delivered an opinion within the time limit set by its chairman,

HAS ADOPTED THIS REGULATION:

Article 1

1. Oils, the characteristics of which comply with those set out in points 1, 2 and 3 of Annex I to this Regulation, shall be deemed to be virgin olive oil within the meaning of point 1 (a), (b) and (c) of the Annex to Regulation No 136/66/EEC.

2. Oil, the characteristics of which comply with those set out in point 4 of Annex I to this Regulation, shall be deemed to be lampante virgin olive oil within the meaning of point 1 (d) of the Annex to Regulation N° 136/66/EEC.

3. Oil, the characteristics of which comply with those set out in point 5 of Annex I to this Regulation, shall be deemed to be refined olive oil within the meaning of point 2 of the Annex to Regulation N° 136/66/EEC.

4. Oil, the characteristics of which comply with those set out in point 6 of Annex I to this Regulation, shall be deemed to be pure olive oil within the meaning of point 3 of the Annex to Regulation N° 136/66/EEC.

5. Oil, the characteristics of which comply with those set out in point 7 of Annex I to this Regulation, shall be deemed to be olive-residue oil within the meaning of point 4 of the Annex to Regulation N° 136/66/EEC.

6. Oil, the characteristics of which comply with those set out in point 8 of Annex I to this Regulation, shall be deemed to be refined olive-residue oil within the meaning of point 5 of the Annex to Regulation N° 136/66/EEC.

7. Oil, the characteristics of which comply with those set out in point 9 of Annex I to this Regulation, shall be deemed to be olive-residue oil within the meaning of point 6 of the Annex to Regulation N° 136/66/EEC.

Article 2

1. The characteristics of the oils laid down in Annex I shall be determined in accordance with the methods of analysis set out below:

- for the determination of the free fatty acids, expressed as the percentage of oleic acid, the method set out in Annex II,

- for the determination of the peroxide index, the method set out in Annex III,

- for the determination of aliphatic alcohols, the method set out in Annex IV,

- for the determination of the sterol content, the method set out in Annex V,

- for the determination of erythrodiol and uvaol, the method set out in Annex VI,

- for the determination of the saturated fatty acids in position 2 of the triglyceride, the method set out in Annex VII,

- for the determination of the trilinolein content, the method set out in Annex VIII,

- for spectrophotometric analysis, the method set out in Annex IX,

- for the determination of the fatty acid composition, the method set out in Annex X A and X B,

- for the determination of the volatile halogenated solvents, the method set out in Annex XI,

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

- for proof that refining has taken place, the method set out in Annex XIII.

2. Evaluation of the organoleptic characteristics shall be carried out by an analyst and, as appropriate, with the assistance of a specialist, according to the procedure described in the tasting notes referred to in Annex XII. Where analysis shows different characteristics to those resulting from the description of the product, the sample must be examined by a panel of tasters in accordance with the provisions of Annex XII.

Any second analysis shall be carried out by the panel according to the said provisions.

In order to ascertain the organoleptic characteristics in connection with operations relating to the intervention system, the panel of tasters will carry out this evaluation in accordance with the provisions of Annex XII.

Article 3

Until 31 October 1992, the introduction of the analysis methods provided for in Article 2 shall not impede the use by the Member States of other tested and scientifically valid methods, provided that products recognized as complying with the rules in force governing Community methods shall be allowed to move freely. Before using other methods, the Member States concerned shall notify them to the Commission.

Where one of the other methods produces a result different from that produced by the common method, the result obtained by the latter method shall be determinant.

Article 4

1. For the purpose of assessing organoleptic characteristics, the Member States shall set up panels of trained and selected tasters in accordance with the rules laid down by the method set out in Annex XII.

2. Where a Member State encounters difficulties in setting up a panel in its territory, it may use the services of a panel operating in another Member State.

Article 5

The additional notes 2, 3 and 4 to Chapter 15 of the combined nomenclature are replaced by those contained in Annex XIV.

Article 6

1. The oil content of oil cake and other residues resulting from the extraction of olive oil (CN codes 2306 90 11 and 2306 90 19) shall be determined using the method set out in Annex XV.

2. The oil content referred to in paragraph 1 shall be expressed as a percentage of the weight of oil to the weight of dry matter.

Article 7

The Community provisions concerning the presence of undesirable substances, other than those referred to in Annex XI, shall apply.

Article 8

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

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

The results shall be considered by the Management Committee for Oils and Fats in accordance with the procedure laid down in Article 39 of Regulation N° 136/66/EEC.

Article 9

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

Article 10

1. This Regulation shall enter into force on the third day following its publication in the Official Journal of the European Communities.

However, the method set out in Annex XII shall apply from

1 January 1992, except in so far as operations relating to the intervention system are concerned.

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

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

Done at Brussels, 11 July 1991.

For the Commission

RAY MAC SHARRY

Member of the Commission

(1) OJ No 172, 30. 9. 1966, p. 3025/66.

(2) OJ N° L 353, 17. 12. 1990, p. 23.

(3) OJ N° L 128, 24. 5. 1977, p. 6.

(4) OJ N° L 166, 1. 7. 1988, p. 10.

ANNEXES

Summary

Page

Annex I:

Characteristics of olive oil .

4

Annex II:

Determination of the free fatty acids .

6

Annex III:

Determination of the peroxide value .

8

Annex IV:

Determination of aliphatic alcohols content by capillary gas chromatography .

10

Annex V:

Determination of the composition and content of sterols by capillary-column gas chromatography .

15

Annex VI:

Determination of erythrodiol and uvaol .

23

Annex VII:

Determination of the saturated fatty acids in position 2 of the triglyceride .

25

Annex VIII:

Determination of composition of trilinolein .

29

Annex IX:

Spectrophotometric investigation in the ultraviolet .

33

Annex XA:

Analysis by gas chromatography of methyl esters of fatty acids .

36

Annex XB:

Preparation of methyl esters of fatty acids .

44

Annex XI:

Determination of the volatile halogenated solvents of olive oil .

48

Annex XII:

Organoleptic assessment of virgin olive oil .

49

Annex XIII:

Proof that refining has taken place .

75

Annex XIV:

Additional Notes 2, 3 and 4 to Chapter 15 of the combined nomenclature .

77

Annex XV:

Oil content of olive residue .

80

Annex XVI:

Determination of iodine value .

82

ANNEX I

CHARACTERISTICS OF OLIVE OIL

>TABLE>

>TABLE>

ANNEX II

DETERMINATION OF THE FREE FATTY ACIDS

1.

DETERMINATION OF ACIDITY

The determination of free fatty acids in olive oils. The content of free fatty acids is expressed as acidity calculated conventionally.

1.1.

Principle

A sample is dissolved in a mixture of solvents and the free fatty acids present titrated using an ethanolic solution of potassium hydroxide.

1.2.

Reagents

All the reagents should be of recognized analytical quality and the water used either distilled or of equivalent purity.

1.2.1.

Diethyl oxide; 95 % ethanol (v/v), mixture of equal parts by volume.

Note: Diethyl oxide is highly inflammable and may form explosive peroxides. Special care should be taken in its use.

Neutralize precisely at the moment of use with the potassium hydroxide solution (1.2.2), with the addition of 0,3 ml of the phenolpthalein solution (1.2.3) per 100 ml of mixture.

Note: If it is not possible to use diethyl oxide, a mixture of solvents containing ethanol and toluene may be used. If necessary, ethanol may be replaced by propanol-2.

1.2.2.

Potassium hydroxide, titrated ethanolic solution, c(KOH) about 0,1 mol/l or, if necessary, c(KOH) about 0,5 mol/l.

The exact concentration of the ethanolic solution of potassium hydroxide must be known and checked immediately prior to use. Use a solution prepared at least five days before use and decanted into a brown glass bottle with a rubber stopper. The solution should be colourless or straw coloured.

Note: A stable colourless solution of potassium hydroxide may be prepared as follows. Bring to the boil 1 000 ml of ethanol with 8 g of potassium hydroxide and 0,5 g of aluminium shavings and continue boiling under reflux for one hour. Distill immediately. Dissolve in the distillate the required quantity of potassium hydroxide. Leave for several days and decant the clear supernatant liquid from the precipitate of potassium carbonate.

The solution may also be prepared without distillation as follows: to 1 000 ml of ethanol add 4 ml of aluminium butylate and leave the mixture for several days. Decant the supernatant liquid and dissolve the required quantity of potassium hydroxide. The solution is ready for use.

1.2.3.

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

1.3.

Apparatus

Usual laboratory equipment including:

1.3.1.

analytical balance;

1.3.2.

250 ml conical flask;

1.3.3.

10 ml burette, graduated in 0,05 ml.

1.4.

Procedure

1.4.1.

Preparation of the specimen for testing

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

1.4.2.

Taking the sample

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

>TABLE>

Weigh the sample in the conical flask (1.3.2).

1.4.3.

Determination

Dissolve the sample (1.4.2) in 50 to 150 ml of the previously neutralized mixture of diethyl oxide and ethanol (1.2.1).

Titrate while stirring with the 0,1 mol/l solution of potassium hydroxide (1.2.2) (see Note 2) until the indicator changes (the pink colour of the phenolphtalein persists for at least 10 seconds).

Note 1. The titrated ethanolic solution of potassium hydroxide (1.2.2) may be replaced by an aqueous solution of potassium or sodium hydroxide provided that the volume of water introduced does not induce phase separation.

Note 2. If the quantity of 0,1 mol/l potassium hydroxide solution required exceeds 10 ml, use the 0,5 mol/l solution.

Note 3. If the solution becomes cloudy during titration, add enough of the solvents (1.2.1) to give a clear solution.

1.5.

Acidity: expressed as percentage of oleic acid

Acidity as a percentage by weight is equal to:

V × c × 1 000 × 100 = V × c × M

V × c ×

M

1 000

×

100

m

=

V × c × M

10 × m

where:

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

c

=

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

M

=

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

m

=

the weight in grams of the sample.

The arithmetic mean of the two calculations should be taken as the result.

ANNEX III

DETERMINATION OF PEROXIDE VALUE

1.

SCOPE

This Standard describes a method for the determination of the peroxide value of oils and fats.

2.

FIELD OF APPLICATION

This Standard is applicable to animal and vegetable oils and fats.

3.

DEFINITION

The peroxide value is the quantity of those substances in the sample, expressed in terms of milliequivalents of active oxygen per kilogram, which oxidize potassium iodide under the operating conditions described.

4.

PRINCIPLE

Treatment of the test portion, in solution in acetic acid and chloroform, by a solution of potassium iodide. Titration of the liberated iodine with standardized sodium thiosulphate solution.

5.

APPARATUS

All the equipment used shall be free from reducing or oxidizing substances.

Note: Do not grease ground surfaces.

5.1.

3 ml glass scoop.

5.2.

Flasks, with ground necks and stoppers, of about 250 ml capacity, dried beforehand and filled with a pure, dry inert gas (nitrogen or, preferably, carbon dioxide).

5.3.

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

6.

REAGENTS

6.1.

Chloroform, analytical reagent quality, freed from oxygen by bubbling a current of pure, dry inert gas through it.

6.2.

Glacial acetic acid, analytical reagent quality, freed from oxygen by bubbling a current of pure, dry gas through it.

6.3.

Potassium iodide, saturated aqueous solution, recently prepared, free from iodine and iodates.

6.4.

Sodium thiosulphate, 0,01 or 0,002 N accurately standardized aqueous solution, standardized just before use.

6.5.

Starch solution, 10 g/l aqueous dispersion, recently prepared from natural soluble starch.

7.

SAMPLE

Take care that the sample is taken and stored away from the light, kept cold and contained in completely filled glass containers, hermetically sealed with ground-glass or cork stoppers.

8.

PROCEDURE

The test shall be carried out in diffuse daylight or in artificial light. Weigh in a glass scoop (5.1) or, failing this, in a flask (5.2), to the nearest 0,001 g, a mass of the sample in accordance with the following table, according to the expected peroxide value:

>TABLE>

Unstopper a flask (5.2) and introduce the glass scoop containing the test portion. Add 10 ml of chloroform (6.1). Dissolve the test portion rapidly by stirring. Add 15 ml of acetic acid (6.2), then 1 ml of potassium iodide solution (6.3). Insert the stopper quickly, shake for one minute, and leave for exactly five minutes away from the light at a temperature from 15 to 25 oC.

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

Carry out two determinations on the same test sample.

Carry out simultaneously a blank test. If the result of the blank exceeds 0,05 ml of 0,01 N sodium thiosulphate solution (6.4), replace the impure reagents.

9.

EXPRESSION OF RESULTS

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

P.V. = V × T × 1 000

PV =

V × T × 1 000

m

where:

V = the number of ml of the standardized sodium thiosulphate solution (6.4) used for the test, corrected to take into account the blank test;

T

=

the exact normality of the sodium thiosulphate solution (6.4) used;

m

=

the weight in g, of the test portion.

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

ANNEX IV

DETERMINATION OF ALIPHATIC ALCOHOLS CONTENT BY CAPILLARY GAS CHROMATOGRAPHY

1.

OBJECT

The procedure describes a method for the determination of eliphatic alcohols content in oils and fats.

2.

PRINCIPLE OF THE METHOD

The fatty substance, with 1-eicosanol added as internal standard, is saponified with methanolic potassium hydroxide and then the unsaponifiable matter extracted with ethyl ether.

The alcoholic fraction is separated from the unsaponifiable matter by chromatography on a layer of potassium hydroxide impregnated silica gel; the alcohols recovered from the silica gel are transformed into trimethylsilyl ethers and analyzed by capillary gas chromatography.

3.

APPARATUS

3.1.

250 ml round-bottom flask with reflux condenser and ground glass joints.

3.2.

Separating funnels of 500 ml capacity.

3.3.

Flasks of 250 ml capacity.

3.4.

Chromatographic tank for thin-layer chromatographic analysis, for glass plates of dimensions

20 × 20 cm.

3.5.

UV light of wavelenght 366 or 254 nm, for examination of TLC plates.

3.6.

Microsyringe to deliver 100 ml and 500 ml.

3.7.

Sintered glass filtering funnel with porous frit G 3 (porosity 15 to 40 m) of approximate diameter 2 cm and approximate height 5 cm suitable for filtering under vacuum and 12/21 ground male joint.

3.8.

Vacuum flask of 50 ml capacity with 12/21 ground glass female joint for use with filter funnel (3.7).

3.9.

Test tube of 10 ml capacity with conical bottom and plug.

3.10.

Gas chromatograph for use with a capillary column, and provided with a splitting system composed of:

3.10.1.

thermostatic chamber for columns (column oven) to hold the temperature desired with a precision of

p1 oC;

3.10.2.

thermostatic vaporization assembly (injection port) with silanizet coated glass;

3.10.3.

flame ionization detector, and converter amplifier;

3.10.4.

recorder-integrator for operation with the converter-amplifier (3.10.3), with response time not exceeding one second and with variable paper-speed.

3.11.

Glass or fused silica capillary column, of length 20 to 30 m, internal diameter 0,25 to 0,32 mm, with SE-52 or SE-54 liquid phase or equivalent, with a film thickness between 0,10 and 0,30 mm.

3.12.

Microsyringe for gas chromatography, of 10 ml capacity with hardened needle.

4.

REAGENTS

4.1.

Potassium hydroxide, approximately 2 N ethanolic solution: 130 g potassium hydroxide (minimum concentration 85 %) is dissolved, with cooling, in 200 ml distilled water and then made up to one litre with ethanol. The solution should be stored in a well-stoppered dark coloured glass bottle.

4.2.

Ethyl ether, pure for analysis.

4.3.

Anhydrous sodium sulphate, pure for analysis.

4.4.

Silica gel TLC glass plates, without florescence indicator, of thickness 0,25 mm (may be obtained commercially prepared).

4.5.

Potassium hydroxide, approximately 0,2 N ethanolic solution; 13 g of potassium hydroxide are dissolved in 20 ml of distilled water and made up to one litre with ethanol.

4.6.

Benzene, for chromatography. (see 5.2.2).

4.7.

Acetone, for chromatography. (see 5.2.2).

4.8.

Hexane, for chromatography. (see 5.2.2).

4.9.

Ethyl ether, for chromatography. (see 5.2.2).

4.10.

Chloroform, for chromatography.

4.11.

Reference solution for thin layer chromatography: mixture of 5 % C20 to C28 alcohols in chloroform.

4.12.

0,2 % solution of 2,7-dichlorofluorescein in ethanol. This is made slightly basic by adding a few drops of 2 N potassium hydroxide solution.

4.13.

Anhydrous pyridine, for chromatography.

4.14.

Hexamethyldisilazane.

4.15.

Trimethylchlorosilane.

4.16.

Standard solutions of trimethylsilyl ethers of aliphatic alcohols from C20 to C28. They may be prepared from mixtures of pure alcohols at the time they are required for use.

4.17.

A 0,1 % (m/v) solution of 1-eicosanol in CHCl3 (internal standard).

4.18.

Carrier gases: hydrogen and helium, pure for gas chromatography.

4.19.

Auxiliary gases:

- hydrogen, pure for gas chromatography,

- air, pure for gas chromatography.

5.

PROCEDURE

5.1.

Preparation of unsaponifiable matter.

5.1.1.

Using a 500 ml microsyringe place, into a 250 ml round-bottom flask, a volume of 0,1 % 1-eicosanol (1-eneicosanol may also be used) solution (4.17) containing a quantity of 1-eicosanol approximately equal to 10 % of the aliphatic alcohol content in that portion of sample to be taken for analysis. For example, to 5 g of sample add 250 ml of the 0,1 % 1-eicosanol solution if olive oil or seed oil and 1 500 ml if olive residue oil. Evaporate the internal standard solution to dryness under N2.

Quantitatively weigh into the flask approximately 5 g of dry, filtered sample.

5.1.2.

50 ml of 2 N potassium hydroxide ethanolic solution are added, reflux condenser is fitted and the apparatus heated to slight boiling on steam bath and stirred continuously throughout the heating process until saponification has taken place (the solution becomes clear). Heating is continued for a further 20 minutes and then 50 ml of distilled water are added through the condenser, the condenser is then diconnected and the flask is cooled to approximately 30 oC.

5.1.3.

The contents of the flask are quantitatively transferred to a separating funnel of 500 ml capacity with the aid of 2 × 25 ml distilled water. Approximately 80 ml of ethyl ether are added, the whole shaken vigorously for 30 seconds and then left to stratify (Note 1).

The aqueous phase beneath is transferred to a second separating funnel. Two further extractions are effected on the aqueous phase, in the same manner, using each time 60 to 70 ml ethyl ether.

Note 1. Emulsions may be eliminated by adding, using as a spray, small quantities of ethyl alcohol or methyl alcohol.

5.1.4.

The ethyl ether extracts are combined in a separating funnel and washed with distilled water (50 ml at a time) until the washing water gives a neutral reaction.

Discard the aqueous phase, dry with anhydrous sodium sulphate and filter, into a flask of 250 ml capacity which has been weighed beforehand, the funnel and filter being washed with small quantities of ethyl ether which are added to the total.

5.1.5.

The ether is evaporated with gentle geating to a few ml and then dried under a slight vacuum or under a current of nitrogen; drying is finished in an oven at 100 oC for approximately 15 minutes and the residue weighed after cooling in a dessicator.

5.2.

Separation of alcoholic fractions.

5.2.1.

Preparation of basic TLC plates (4.4) are immersed completely, in 0,2 N potassium hydroxide solution (4.5) for 10 seconds, and then left to dry under a hood for two hours and finally, they are placed in an oven at 100 oC for one hour.

The plates are taken from the oven and stored in a calcium chloride dessicator until required for use. The plates thus treated must be used within two weeks.

Note 2. When basic silica gel plates are used for separation of the alcoholic fraction, there is no need for the unsaponifiable substances to be treated with A1203. It follows that all acid compounds (fatty acids and others) are retained at the origin thereby obtaining both aliphatic alcohol and terpenic alcohol bands which are both separated distinctly from the sterol band.

5.2.2.

A solution of 95:5 by volume of benzene and acetone is introduced into the development chamber to an approximate depth of 1 cm. Alternatively, a 65:35 by volume mixture of hexane and ethyl ether may be used. The tank is closed and left for not less than half an hour to allow equilibration between vapour and liquid. Strips of filter paper dipping into the eluent may be affixed to the inside surfaces of the tank to reduce the development time by approximately one-third and obtain more uniform, regular elution of the components.

Note 3. The developing solution must be replaced for each analysis in order to obtain reproducible developing conditions.

5.2.3.

An approximately 5 % solution of unsaponifiable matter (5.1.5) in chloroform is prepared and 0,3 ml of the solution is streaked as a uniform strip of minimum thickness, by means of the microsyringe of 100 ml capacity, on a TLC plate at approximately 2 cm from the bottom of the TLC plate. Aligned with the origin, 2 to 3 ml of the aliphatic alcohol reference solution (4.11) are spotted for the identification of the aliphatic alcohol band after development has been completed.

5.2.4.

The plate is placed inside the development tank as stated in 5.2.2. The temperature shall be kept between 15 and 20 oC. The tank is closed immediately, and the sample is left to elute until the front of the solvent has reached a distance 1 cm from the top of the plate. The plate is then removed from the development tank and the solvent evaporated under a hot air current or the plate is left for a while under the hood.

5.2.5.

The plate is sprayed lightly and evenly with the solution of 2,7-dichlorofluorescein when the plate is observed under ultra violet light, the aliphatic alcohol band is identified by comparison to the aliphatic alcohols in the band immediately above that, which is the triterpenic alcohol band, are outlined together.

Note 4. The requirement for the grouping of the aliphatic alcohol band and the triterpenic alcohol band together is determined by the possible migration of some aliphatic alcohols into the triterpenic alcohol band.

5.2.6.

The silica gel enclosed in the area defined is scraped with a metal spatula. The material removed is broken into fine fragments, and introduced into a filter funnel (3.7), 10 ml of hot chloroform are added, and the contents mixed thoroughly with the metal spatula and filtered under vacuum, the filtrate being collected in the flask (3.8) which is connected to the filter funnel.

The residue inside the funnel is washed 3 × 10 ml with ethyl ether collecting the filtrate in the same flask fitted to the funnel. The filtrate is evaporated to a volume of approximately 4 to 5 ml and the residual solution is poured into a test tube of 10 ml capacity (3.9) which has been weighed beforehand; the test tube is dried by light heating under a gentle nitrogen current. Redissolve the residue with a few drops of acetone, dry again, then place in an oven at 105 oC for 10 minutes, remove and cool in the dessicator and weigh.

The residue inside the test tube is composed of the alcoholic fraction.

5.3.

Preparation of trimethylsily ethers.

5.3.1.

The reagent for silylation, consisting of a mixture of 9:3:1 by volume (Note 5) of pyridinehexamethyldisilazane-trimethylchlorosilane in the proportion of 50 ml for each milligram of alcohols, is added to the test tube containing the alcoholic fraction, avoiding all absorption of moisture (Note 6).

Note 5. Solutions ready for use are commercially available; silanizing reagents such as N, 0-bis (trimethylsilyl) trifluoroacetamide + 1 % trimethylchlorosilane for mixing with the same volume of anhydrous pyridine.

5.3.2.

The test tube is stoppered and shaken carefully without overturning until the alcohols have been solubilized. It is then left for at least 15 minutes at room temperature and subsequently centrifuged for some minutes; the clear solution is ready for gas chromatographic analysis.

Note 6. Any formation of slight opalescence is normal and causes no interference. The formation of a white flocculate or the appearance of a pink colouring are signs of the presence of moisture or the deterioration of the reagent. In this case the test shall be repeated.

5.4.

Gas chromatography analysis.

5.4.1.

Preliminary operations and conditioning of the capillary column.

5.4.1.1.

The capillary column is fitted inside the gas chromatograph by connecting the beginning of the column to the evaporator which is connected to the splitting system and the end of the column to the detector.

General checking of the gas chromatography assembly is performed (tightness of gas fittings, efficiency of the detector, efficiency of the splitting system and of the recording system, etc.).

5.4.1.2.

Capillary columns being used for the first time should be conditioned. A little carrier gas is caused to flow through the capillary column and then the gas chromatography assembly is switched on and gradual heating accomplished until a temperature not less than 20 oC above the operating temperature (see Note 7) is attained. That temperature is held for not less than two hours and then the assembly is brought to the operating conditions (regulation of gas flow, split flame ignition, connection to the electronic recorder, adjustment of the temperature of the capillary column oven, the detector and the injector, etc.) and the signal is adjusted to a sensitivity not less than twice the highest level contemplated for the execution of the analysis. The base line tracing shall be linear, devoid of spikes of whatever nature and shall show no signs of drift.

Negative rectilinear drift is indicative of imperfect tightness of column connections whereas a positive drift is indicative of insufficient conditioning of the column.

Note 7. The temperature of conditioning shall be at least 20 oC less than the maximum temperature contemplated for the liquid phase employed.

5.4.2.

Selection of operating conditions.

5.4.2.1.

General operating conditions are as follows:

- column temperature: the initial isotherm is set at 180 oC for eight minutes and then programmed at 5 oC/minute to 260 oC and a further 15 minutes at 260 oC,

- temperature of evaporator: 280 oC,

- temperature of detector: 290 oC,

- linear velocity of carrier gas: helium 20 to 35 cm/s, hydrogen 30 to 50 cm/s,

- splitting ratio: 1:50 at 1:100,

- sensitivity of instrument: 4 to 16 times the minimum attenuation,

- sensitivity of recording: 1 to 2 mV fs,

- paper speed: 30 to 60 cm/h,

- quantity of substance injected: 0,5 to 1 ml of TMSE solution.

The above conditions may be modified according to the characteristics of the column and of the gas chromatograph in order that chromatograms satisfying the following conditions be obtained:

- alcohol retention time C26 shall be 18 p5 minutes,

- the alcohol C22 peak shall be 80 p20 % of the full scale value for olive oil and 40 p20 % of the full scale value for seed oil.

5.4.2.2.

The aforesaid requirements are checked by repeated injection of the standard TMSE mixture of alcohols and the operating conditions are adjusted to yield the best possible results.

5.4.2.3.

The parameters for the integration of peaks shall be set so that a correct appraisal of the areas of the peaks considered is obtained.

5.4.3.

Execution of analysis.

5.4.3.1.

Using the microsyringe of 10 ml capacity draw in 1 ml of hexane followed by 0,5 ml of air and subsequently 0,5 to 1 ml of the sample solution; the plunger of the microsyringe is raised to empty the needle.

The needle is introduced through the septum of the injection assembly and after one to two seconds the solution is injected rapidly and the needle extracted slowly after approximately five seconds.

5.4.3.2.

Recording is effected until the TMSE of the alcohols present have been eluted completely. The base line shall always correspond to the requirements of 5.4.1.2.

5.4.4.

Identification of peaks.

The identification of individual peaks is effected according to the retention times and by comparison with the standard TMSE mixture, analyzed under the same conditions.

A chromatogram of the alcoholic fraction of a virgin olive oil is shown in Figure 1.

5.4.5.

Quantitative appraisal.

5.4.5.1.

The peak areas of 1-eicosanol and of the aliphatic alcohols C22 to C28 are calculated by electronic integration.

5.4.5.2.

The contents of each alcohol, expressed in mg/100 g fatty substance are calculated as follows:

alcohol x = Ax . ms . 100

alcohol x =

Ax 7 ms 7 100

As 7 m

where:

Ax = area of the alcohol peak ×, in square millimetres;

As

=

area of 1-eicosanol in square millimetres;

ms

=

mass of 1-eicosanol in milligrams;

m

=

mass of sample drawn for determination, in grams.

6.

EXPRESSION OF RESULTS

The contents of the individual aliphatic alcohols in mg/100 g of fatty substance and the sum of the 'total aliphatic alcohols` are reported.

APPENDIX

Determination of the linear velocity of the gas

1 to 3 ìl of methane or propane are injected into the gas chromatograph set at normal operating conditions and the time taken for the methane or propane to flow through the column from the instant of injection to the instant the peak elutes (tM) is measured using a stop clock.

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

>START OF GRAPHIC>

Figure 1 Chromatogram of the alcoholic fraction of a virgin olive oil.

1 = Eicosanol (SI)

2 = Decosanol,

3 = Tricosanol,

4 = Tetracosanol,

5 = Pentacosanol,

6 = Hexacosanol,

7 = Heptacosanol,

8 = Octacosanol.

>END OF GRAPHIC>

ANNEX V

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

1.

SCOPE

The method describes a procedure for determining the individual and total sterols content of fatty substances.

2.

PRINCIPLE OF THE METHOD

The fatty substance, with added a-cholestanol as an internal standard, is saponified with potassium hydroxide in ethanolic solution and the unsaponifiables are then extracted with ethyl ether.

The sterol fraction is separated from the unsaponifiable extract by chromatography on a basic silica gel plate. The sterols recovered from the silica gel are transformed into trimethyl-silyl ethers and are analysed by capillary-column gas chromatography.

3.

APPARATUS

3.1.

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

3.2.

500 ml separating funnels.

3.3.

250 ml flasks.

3.4.

Complete apparatus for analysis by thin-layer chromatography using 20 × 20 cm glass plates.

3.5.

Ultraviolet lamp having a wavelength of 366 or 254 nm.

3.6.

100 ml and 500 ml microsyringes.

3.7.

A cylindrical filter funnel with a G3 porous septum (porosity 15 to 40 mm) of diameter approximately 2 cm and a depth of some 5 cm, with an attachment suitable for filtration under vacuum and a 12/21 male ground glass joint.

3.8.

50 ml vacuum conical flask with a 12/21 ground-glass female joint which can be fitted to the filter funnel (3.7).

3.9.

A 10 ml test tube with a tapering bottom and a sealing stopper.

3.10.

Gas chromatograph suitable for use with a capillary column, provided with a splitting system consisting of:

3.10.1.

a thermostatic chamber for columns capable of maintaining the desired temperature with an accuracy of p1 oC;

3.10.2.

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

3.10.3.

a flame ionization detector and converter-amplifier;

3.10.4.

an integrator-recorder suitable for use with the converter-amplifier (3.10.3) having a response time of not more than one second and a variable paper speed.

3.11.

A glass or fused-silica capillary column of length 20 to 30 m, internal diameter 0,25 to 0,32 mm, entirely coated with SE-52 or SE-54 liquid or equivalent in a uniform thickness between 0,10 and 0,30 mm.

3.12.

A 10 ml gas chromatography microsyringe with a hardened needle.

4.

REAGENTS

4.1.

Potassium hydroxide, approximately 2 N ethanolic solution. Dissolve 130 g of potassium hydroxide (minimum title 85 %) with cooling in 200 ml of distilled water and then make up to one litre with ethanol. Keep the solution in well-stoppered dark glass bottles.

4.2.

Ethyl ether, analytical purity.

4.3.

Anhydrous sodium sulphate, analytical purity.

4.4.

Glass plates coated with silica gel, without fluorescence indicator, thickness 0,25 mm (commercially available ready for use).

4.5.

Potassium hydroxide, 0,2 N ethanolic solution. Dissolve 13 g of potassium hydroxide in 20 ml of distilled water and make up to one litre with ethanol.

4.6.

Benzene, for chromatography. (See 5.2.2)

4.7.

Acetone, for chromatography. (See 5.2.2)

4.8.

Hexane, for chromatography. (See 5.2.2)

4.9.

Ethyl ether, for chromatography. (See 5.2.2)

4.10.

Chloroform, analytical purity. (See 5.2.2)

4.11.

Reference solution for thin-layer chromatography: cholesterol or phytosterols, 5 % solution in chloroform.

4.12.

2,7-dichlorofluorescein, 0,2 % ethanolic solution. Make slightly basic by adding a few drops of 2 N alcoholic potassium hydroxide solution.

4.13.

Anhydrous pyridine, for chromatography.

4.14.

Hexamethyl disilazane.

4.15.

Trimethylchlorosilane.

4.16.

Reference solutions of sterol trimethylsilyl ethers. To be prepared at the time of use from pure sterols or mixtures of sterols obtained from oils containing them.

4.17.

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

4.18.

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

4.19.

Auxiliary gases:

- hydrogen, gas-chromatographic purity,

- air, gas-chromatographic purity.

5.

PROCEDURE

5.1.

Preparation of the unsaponifiables.

5.1.1.

Using the 500 ml microsyringe introduce a volume of 0,2 % a-cholestanol solution in chloroform (4.17) containing an amount of cholestanol corresponding to approximately 10 % of the sterol content of the sample aliquot taken for the determination into the 250 ml flask. For example, for 5 g of sample add 500 ml of the 0,2 % a-cholestanol solution in the case of an olive oil and 1 500 ml for seed oils or olive-pomaca oil.

Evaporate to dryness in current of nitrogen and then weigh accurately 5 g of the dry filtered sample into the same flask.

Animal or vegetable oils and fats containing appreciable quantities of cholesterol may show a peak having a retention time identical to cholestanol. If this occurs the sterol fraction will have to be analyzed in duplicate with and without internal standard.

5.1.2.

Add 50 ml of 2 N ethanolic potassium hydroxide solution, fit the reflux condenser and heat to gentle boiling on a water bath with continuous vigorous stirring until saponification takes place (the solution becomes clear). Continue heating for a further 20 minutes, then add 50 ml of distilled water from the top of the condenser, detach the condenser and cool the flask to approximately 30 oC.

5.1.3.

Transfer the contents of the flask quantitatively into a 500 ml separating funnel using several rinses of distilled water, amounting in all to about 50 ml. Add approximately 80 ml of ethyl ether, shake vigorously for approximately 30 seconds and allow to settle (Note 1).

Separate off the lower aqueous phase collecting it in a second separating funnel. Perform two further extractions on the aqueous phase in the same way using 60 to 70 ml of ethyl ether on each occasion.

Note 1. Any emulsion can be destroyed by adding small quantities of ethyl or methyl alcohol by means of a spray.

5.1.4.

Pool the ether extracts into a single separating funnel and wash with distilled water (50 ml at a time) until the wash water gives a neutral reaction.

When the wash water has been removed, dry with anhydrous sodium sulphate and filter on anhydrous sodium sulphate into a previously weighed 250 ml flask, washing the funnel and filter with small quantities of ethyl ether.

5.1.5.

Distil the ether down to a few ml, then bring to dryness under a slight vacuum or in a current of nitrogen, completing drying in a stove at 100 oC for approximately a quarter of an hour, and then weigh after cooling in a desiccator.

5.2.

Separation of the sterol fraction.

5.2.1.

Preparation of the basic plates. Immerse the silica gel plates (4.4) completely in the 0,2 N ethanolic potassium hydroxide solution (4.5) for 10 seconds, then allow to dry in a fume cupboard for two hours and finally place in a stove at 100 oC for one hour.

Remove from the stove and keep in a calcium chloride desiccator until required for use (plates treated in this way must be used within 15 days).

Note 2. When basic silica gel plates are used to separate the sterol fraction there is no need to treat the unsaponifiables with alumina. In this way all compounds of an acid nature (fatty acids and others) are retained on the spotting line and the sterols band is clearly separated from the aliphatic and triterpene alcohols band.

5.2.2.

Place a 95:5 (v/v) benzene/acetone mixture in the plate-developing chamber to a depth of approximately 1 cm. As an alternative a 65:35 (v/v) hexane/ethyl ether mixture may be used. Close the chamber with the appropriate cover and leave thus for approximately half an hour so that liquid-vapour equilibrium is established. Strips of filter paper dipping into the eluent may be placed on the internal surfaces of the chamber. This reduces developing time by approximately one-third and brings about more uniform and regular elution of the components.

Note 3. The developing mixture should be replaced for every test in order to achieve perfectly reproducible elution conditions.

5.2.3.

Prepare an approximately 5 % solution of the unsaponifiables (5.1.5) in chloroform and, using the 100 ml microsyringe, streak a chromatographic plate (5.2.1) with 0,3 ml approximately 2 cm from one end in a streak which is as thin and as uniform as possible. In line with the streak place 2 to 3 ml of the sterol reference solution (4.11) at one end of the plate so that the sterol band can be identified after developing.

5.2.4.

Place the plate in the developing chamber prepared as specified in 5.2.2. The ambient temperature should be maintained between 15 and 20 oC. Immediately close the chamber with the cover and allow to elute until the solvent front reaches approximately 1 cm from the upper edge of the plate. Remove the plate from the developing chamber and evaporate the solvent in a flow of hot air or by leaving the plate for a short while under a hood.

5.2.5.

Spray the plate lightly and uniformly with the 2,7-dichlorofluoroscein solution. When the plate is observed under ultraviolet light the sterol band can be identified through being aligned with the stain obtained from the reference solution. Mark the limits of the band along the edges of the fluorescence with a black pencil.

5.2.6.

Using a metal spatula scrape off the silica gel in the marked area. Place the finely comminuted material removed into the filter funnel (3.7). Add 10 ml of hot chloroform, mix carefully with the metal spatula and filter under vacuum, collecting the filtrate in the conical flask (3.8) attached to the filter funnel.

Wash the residue in the flask three times with ethyl ether (approximately 10 ml each time) collecting the filtrate in the same flask attached to the funnel. Evaporate the filtrate to a volume of 4 to 5 ml, transfer the residual solution to the previously weighed 10 ml test tube (3.9), evaporate to dryness by mild heating in a gentle flow of nitrogen, make up again using a few drops of acetone, evaporate again to dryness, place in a stove at 105 oC for approximately 10 minutes and then allow to cool in a desiccator and weigh.

The residue contained in the test tube consists of the sterol fraction.

5.3.

Preparation of the trimethylsilyl ethers.

5.3.1.

Add the silylation reagent, consisting of a 9:3:1 (v/v/v) mixture of pyridine/hexamethyl disilazane/trimethyl chlorosilane (Note 4) in the ratio of 50 ml for every milligram of sterols to the test tube containing the sterol fraction, avoiding any uptake of moisture (Note 5).

Note 4. Solutions which are ready for use are available commercially. Other silanizing reagents such as, for example, bis-trimethylsilyl, trifluor acetamide + 1 % trimethyl chlorosilane, which has to be diluted with an equal volume of anhydrous pyridine, are also available.

5.3.2.

Stopper the test tube, shake carefully (without overturning) until the sterols are completely dissolved. Stand for at least 15 minutes at ambient temperature and then centrifuge for a few minutes. The clear solution is ready for gas chromatographic analysis.

Note 5. The slight opalescence which may form is normal and does not cause any interference. The formation of a white floc or the appearance of a pink colour are indicative of the presence of moisture or deterioration of the reagent. If these occur the test must be repeated.

5.4.

Gas chromatographic analysis.

5.4.1.

Preliminary operations, column packing.

5.4.1.1.

Fit the column in the gas chromatograph, attaching the inlet end to the evaporator connected to the splitting system and the outlet end to the detector.

Carry out general checks on the gas chromatograph unit (leaks from the gas circuits, detector efficiency, efficiency of the splitting system and recording system, etc.).

5.4.1.2.

If the column is being used for the first time it is recommended that it should be subjected to conditioning. Pass a gentle flow of gas through the column and then switch on the gas chromatography unit and begin gradual heating up to a temperature of at least 20 oC above the operating temperature (Note 6). Hold this temperature for at least two hours, then place the entire unit in operating mode (adjustment of gas flows and splitting, ignition of the flame, connection with the electronic recorder, adjustment of the column chamber, detector and injector temperature, etc.) and then record the signal with a sensitivity at least two times greater than that intended for the analysis. The course of the base line must be linear, without peaks of any kind, and must not drift.

A negative straight-line drift indicates leakage from the column connections; a positive drift indicates inadequate conditioning of the column.

Note 6. The conditioning temperature must always be at least 20 oC less than the maximum temperature specified for the stationary phase used.

5.4.2.

Choice of operating conditions.

5.4.2.1.

The guideline operating conditions are as follows:

- column temperature: 260 p5 oC,

- evaporator temperature: 280 oC,

- detector temperature: 290 oC,

- linear velocity of the carrier gas: helium 20 to 35 cm/s, hydrogen 30 to 50 cm/s,

- splitting ratio: from 1:50 to 1:100,

- instrument sensitivity: from 4 to 16 times the minimum attenuation,

- recording sensitivity: 1 to 2 mV f.s.,

- paper speed: 30 to 60 cm/hour,

- amount of substance injected: 0,5 to 1 ml of TMSE solution.

These conditions may be varied in the light of column and gas-chromatograph characteristics so as to obtain chromatograms which meet the following requirements:

- the retention time for b-sitosterol should be 20 p5 minutes,

- the campesterol peak should be: for olive oil (mean content 3 %) 15 p5 % of full scale; for soya oil (mean content 20 %) 80 p10 % of full scale,

- all the sterols present must be separated. In addition to being separated the peaks must also be completely resolved, i.e. the peak trace should return to the base line before leaving for the next peak. Incomplete resolution is however tolerated provided that the peak at TRR 1,02 can be quantified using the perpendicular.

5.4.3.

Analytical procedure.

5.4.3.1.

Using the 10 ml microsyringe take 1 ml of hexane, draw in 0,5 ml of air and then 0,5 to 1 ml of the sample solution. Raise the plunger of the syringe further so the needle is emptied. Push the needle through the membrane of the injection unit and after one to two seconds inject rapidly, then slowly remove the needle after some five seconds.

5.4.3.2.

Continue recording until the TMSE of the sterols present are completely eluted

The base line must continue to meet the requirements (5.4.1.2).

5.4.4.

Peak identification.

Identify individual peaks on the basis of retention times and by comparison with mixtures of sterol TMSE analysed under the same conditions.

The sterols are eluted in the following order: cholesterol, brassicasterol, 24-methylene cholesterol, campesterol, campestanol, stigmasterol, D7-campesterol, D5,23-stigmastadienol, clerosterol, b-sistosterol, sitostanol, D5-avenasterol, D5,24-stigmastadienol, D7-sigmastenol, D7-avenasterol.

The retention times for sitosterol for SE-52 and SE-54 columns are shown in Table 1.

Figures 1 and 2 illustrate typical chromatograms for some oils.

5.4.5.

Quantitative evaluation.

5.4.5.1.

Calculate the areas of the a-cholestanol and the sterol peaks using the integrator. Ignore peaks for any compounds which are not included among those listed in Table 1. The response coefficient for a-cholestanol is to be equal to 1.

5.4.5.2.

Calculate the concentration of each individual sterol in mg/100 g of fatty material as follows:

sterol x = Ax . ms . 100

sterol x =

Ax 7 ms 7 100

As 7 m

where:

Ax = peak area for sterol ×, in square millimetres;

As

=

area of the á-cholestanol peak, in square millimetres;

ms

=

mass of á-cholestanol added, im milligrams;

m

=

mass of the sample used for determination, in grams.

6.

EXPRESSION OF THE RESULTS

6.1

Record individual sterol concentrations as mg/100 g of fatty material and their sum as 'total sterols`.

6.2

Calculate the percentage of each individual sterol from the ratio of the relevant peak area to the total peak area for sterols.

% of sterol x = ÓA . 100

% of sterol x =

Ax

ÓA

. 100

where:

Ax = peak area for x;

ÓA

=

total peak area for sterols.

APPENDIX

Determination of the linear velocity of the gas

With the gas chromatograph set to normal operating conditions inject 1 to 3 ml of methane (or propane) and measure the time taken by the gas to pass through the column from the time of injection to the time at which the peak appears (tM).

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

>TABLE>

>START OF GRAPHIC>

Figure 1

Gas chromatogram of the sterol fraction of an unrefined olive oil

>END OF GRAPHIC>

>START OF GRAPHIC>

Figure 2

Gas chromatogram of the sterol fraction of a refined olive oil

>END OF GRAPHIC>

ANNEX VI

DETERMINATION OF ERYTHRODIOL AND UVAOL

INTRODUCTION

Erythrodiol (commonly understood as the glycols erythrodiol and uvaol together) is a constituent of the unsaponifiable fraction, characteristic of some types of fatty substances. It is found at considerably higher concentrations in solvent-extracted olive oil than in other oils, such as pressed olive oil and grape pip oil, which also contain it, and so its presence may demonstrate the presence of solvent-extract olive oil.

1.

SCOPE

The method describes a procedure for detecting erythrodiol in fatty substances.

2.

PRINCIPLE OF THE METHOD

The fatty substance is saponified with potassium hydroxide in methanolic ethanolic solution. The unsaponifiable fraction is then extracted with ethyl ether and purified by passage over a column of alumina.

The unsaponifiables are subjected to thin-layer chromatography on a silica gel plate until the bands corresponding to the sterol and erythrodiol fractions are separated. The sterols and the erythrodiol recovered from the plate are transformed into trimethylsilyl ethers and the mixture is analysed by gas chromatography.

The result is expressed as the percentage of erythrodiol in the mixture of erythrodiol and sterols.

3.

APPARATUS

3.1.

The apparatus described in Annex V (determination of the content of sterols).

4.

REAGENTS

4.1.

The reagents described in Annex V (determination of the content of sterols).

4.2.

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

5.

PROCEDURE

5.1.

Preparation of the unsaponifiables.

As described at paragraph 5.1.2 of Annex V.

5.2.

Separation of erythrodiol and the sterols.

5.2.1.

See paragraph 5.2.1 of Annex V.

5.2.2.

See paragraph 5.2.2 of Annex V.

5.2.3.

Prepare a 5 % solution of the unsaponifiables in chloroform.

Using the 0,1 ml microsyringe, streak a chromatographic plate with 0,3 ml of solution approximately 1,5 cm from the lower edge in a streak which is as thin and uniform as possible.

At one end of the plate place a few microlitres of the solutions of cholesterol and erythrodiol to serve as a reference.

5.2.4.

Place the plate in the developing chamber prepared as specified in 5.2.1. The ambient temperature should be about 20 oC. Immediately close the chamber with the cover and allow to elute until the solvent front reaches approximately 1 cm from the upper edge of the plate. Remove the plate from the developing chamber and evaporate the solvent in a flow of hot air.

5.2.5.

Spray the plate lightly and uniformly with the alcoholic 2,7-dichlorofluoroscein solution. When the plate is observed under ultralviolet light the sterol and erythrodiol bands can be identified through being aligned with the references. Mark with a spot just outside the edges of the fluorescence.

5.2.6.

Using a metal spatula scrape off the silica gel in the marked areas. Place the material from the plate in a 50 ml flask. Add 15 ml of hot chloroform, shake well and filter through a funnel with a sintered glass disc so that the silica gel is transferred to the filter. Wash three times with hot chloroform (10 ml each time) collecting the filtrate in a 100 ml flask. Evaporate the filtrate to a volume of 4 to 5 ml, transfer to a calibrated 10 ml conical-bottomed centrifuge tube, dry by gently heating in a current of nitrogen and weigh.

5.3.

Preparation of the trimethylsily esters

As described in paragraph 5.3 of Annex V.

5.4.

Gas chromatographic analysis

As described in paragraph 5.4 of the above method. The operating conditions of the gas chromatograph in analysis must be such as to perform the sterol analysis and separate the TMSE from the erythrodiol and uvaol.

Once the sample has been injected, continue recording until the sterols present, the erythrodiol and the uvaol have been eluted. Then identify the peaks (the retention times for erythrodiol and uvaol relative to b-sitosterol are about 1,45 and 1,55 respectively) and calculate the areas as for the sterols.

6.

EXPRESSION OF THE RESULTS

Erythrodiol % = A1 + A2 + Ó Asterols × 100

Erythrodiol % =

A1 + A2

A1 + A2 + Ó Asterols

× 100

where:

A1

= peak area for erythrodiol in square millimetres;

A2

= peak area for uvaol in square millimetres;

Ó Asterols

= total peak area for sterols in square millimetres.

The result is expressed to one decimal place.

ANNEX VII

DETERMINATION OF FATTY ACIDS IN THE 2-POSITION IN THE TRIGLYCERIDES OF OILS AND FATS

1.

SCOPE

This Standard describes a method for the determination of the composition of that fraction of the fatty acids of an oil or fat which is esterified at the 2-position (or internal position) of the glycerol.

2.

FIELD OF APPLICATION

This Standard is applicable to oils and fats having a melting point below 45 oC, owing to the peculiarities of the action of pancreatic lipase.

It is not applicable unreservedly to oils and fats containing substantial amounts of: fatty acids with 12 or fewer carbon atoms (coconut and palmkernel oils, butterfat), or highly unsaturated fatty acids (with more than four double bounds) containing 20 or more carbon atoms (fish and marine animals oils), or fatty acids containing oxygenated groups, other than the acid group.

3.

PRINCIPLE

Possible neutralization of acid oils and fats in a solvent. Purification by passing onto an alumina column. Partial hydrolysis of triglycerides by pancreatic lipase during a determined time. Separation of the formed monoglycerides by thin-layer chromatography and methanolysis of these monoglycerides. Analysis of these methyl esters by gas-liquid chromatography.

4.

APPARATUS

4.1.

100 ml round-bottomed flask.

4.2.

25 ml round bottomed flask, with ground joint.

4.3.

1 m-long air condenser, to fit the flask 4.2.

4.4.

250 ml conical flask.

4.5.

50 ml beaker.

4.6.

500 ml separating funnel.

4.7.

Chromatographic glass column, 13 mm internal diameter, 400 mm in length, fitted with a fritted glass disc and a tap.

4.8.

10 ml centrifuge tube, with ground glass stopper.

4.9.

5 ml burette, graduated in 0,05 ml.

4.10.

1 ml hypodermic syringe, fitted with a thin needle.

4.11.

Microsyringe, to deliver drops of 3 to 4 ml.

4.12.

Spreader for thin-layer chromatography.

4.13.

Glass plates for thin-layer chromatography, 20 × 20 cm.

4.14.

Glass developing tank for thin-layer chromatography, with ground-glass lid, suitable for the 20 × 20 plates.

4.15.

Spray for thin-layer chromatography.

4.16.

Oven regulated at 103 p2 oC.

4.17.

Thermostat regulatable between 30 and 45 oC to within 0,5 oC.

4.18.

Rotary evaporator.

4.19.

Vibrating electric shaker, allowing vigorous agitation of the centrifuge tube.

4.20.

Ultraviolet lamp for the examination of the thin-layer plates.

For the control of the lipase activity:

4.21.

pH meter.

4.22.

Spiral stirer.

4.23.

5 ml burette.

4.24.

Stop-watch.

For the possible preparation of the lipase:

4.25.

Laboratory stirrer, suitable for the dispersion and mixture of heterogeneous materials.

5.

REAGENTS

5.1.

n-hexane, or, failing this, light petroleum (bp 30 to 50 oC), chromatographic quality.

5.2.

2-propanol, or ethanol, 95 % (v/v), analytical reagent quality.

5.3.

2-propanol, or ethanol, 1/1 aqueous solution.

5.4.

Diethyl ether, free from peroxides.

5.5.

Acetone.

5.6.

Formic acid, at least 98 % (m/m).

5.7.

Developing solvent: mixture of n-hexane (5.1), diethyl ether (5.4) and formic acid (5.6) in proportions 70/30/1 (v/v/v).

5.8.

Activated alumina for chromatography, neutral, grade Brockmann I.

5.9.

Silica powder, with binder, of suitable quality for thin-layer chromatography.

5.10.

Pancreatic lipase of suitable quality (Notes 1 and 2).

5.11.

Sodium hydroxide, 120 g/l aqueous solution.

5.12.

Hydrochloric acid, aqueous solution 6 N.

5.13.

Calcium chloride (CaCl2), 220 g/l aqueous solution.

5.14.

Sodium cholate (enzymatic quality), 1 g/l aqueous solution.

5.15.

Buffer solution: 1 M aqueous solution of tris-hydroxymethylaminomethane bring to pH 8 by addition of hydrochloric acid (5.12) (check by potentiometer).

5.16.

Phenolphthalein, 10 g/l solution in 95 % (v/v) ethanol.

5.17.

2m,7m-dichlorofluorescein, 2 g/l solution in 95 % (v/v) ethanol, rendered slightly alkaline by the addition of one drop of 1 N sodium hydroxide solution per 100 ml.

For the control of the lipase activity:

5.18.

Neutralized oil.

5.19.

Sodium hydroxide, 0,1 N aqueous solution.

5.20.

Sodium cholate (enzymatic quality), 200 g/l aqueous solution.

5.21.

Gum arabic, 100 g/l aqueous solution.

6.

PREPARATION OF THE SAMPLE

If the sample has an acidity below 3 %, determined according to Annex II, purify directly over alumina according to 6.2.

If the sample has an acidity over 3 %, determined according to Annex II, neutralize by alkali in the presence of a solvent according to 6.1, then pass over alumina according to 6.2.

6.1.

Neutralization by alkali in the presence of solvent

Into a separating funnel (4.6) introduce about 10 g of the crude oil and add 100 ml of hexane (5.1), 50 ml of 2-propanol (5.2), a few drops of phenolphthalein solution (5.16), and an amount of the sodium hydroxide solution (5.11) corresponding to the free acidity of the oil plus 0,3 % excess. Shake vigorously for one minute, add 50 ml of distilled water, shake again and leave to settle.

After separation, remove the bottom soap layer. Also remove any intermediate layers (mucilage, insoluble matter). Wash the hexane solution of the neutralized oil with successive 25 to 30 ml portions of the 2-propanol solution (5.3) until the pink colour of the phenolphthalein disappears.

Remove most of the hexane by distillation under vacuum in the rotary evaporator (4.18), dry the oil at 30 to 40 oC under vaccum with the help of a current of pure nitrogen until the hexane has been removed completely.

6.2.

Purification through alumina

Prepare a suspension of 15 g of activated alumina (5.8) in 50 ml hexane (5.1) and pour it, while stirring, onto the chromatographic column (4.7). Let the alumina settle evenly, and allow the solvent level to fall to within 1 to 2 mm above the absorbent. Carefully pour onto the column a solution of 5 g of oil in 25 ml of hexane (5.1); collect the whole of the effluent from the column in a round-bottomed flask (4.1).

7.

Preparation of the chromatographic plates

Thoroughly clean the glass plates (4.13) with ethanol, light petroleum and acetone to eliminate any trace of fatty matter.

In a conical flask (4.4) place 30 g of silica powder (5.9). Add 60 ml of distilled water. Stopper and shake vigorously for one minute. Transfer the slurry immediately to the spreader (4.12) and coat the clean plates with a layer 0,25 mm thick.

Dry the plates in the air for 15 minutes and then for an hour in the oven (4.16) at 103 p2 oC. Cool the plates in a desiccator to room temperature before use.

Prepared plates are available in commerce.

8.

PROCEDURE

8.1.

Hydrolysis with pancreatic lipase.

Into the centrifuge tube (4.8) weigh about 0,1 g of the prepared sample, if the sample is a liquid oil proceed directly as below.

Add 20 mg of lipase (5.10) and 2 ml of the buffer solution (5.15). Shake well, but carefully, and then add 0,5 ml of the sodium cholate solution (5.14) and 0,2 ml of the calcium chloride solution (5.13). Close the tube with the ground stopper, shake cautiously (avoid wetting the stopper) and put the tube immediately in the thermostat (4.17) maintained at 40 p0,5 oC and shake by hand for exactly one minute.

Remove the tube from the thermostat, and agitate vigorously by means of the electric shaker (4.19) for exactly two minutes.

Cool immediately in running water; add 1 ml of hydrochloric acid (5.12) and 1 ml of diethyl ether (5.4). Stopper and mix vigorously by means of the electric shaker. Allow to stand and remove the organic layer by means of the syringe (4.10), if necessary after centrifuging.

8.2.

Separation of the monoglycerides by thin-layer chromatography

Apply the extract to the chromatographic plate with the microsyringe (4.11), about 1,5 cm from the bottom edge, in a thin, uniform line, as narrow as possible. Set the plate in the well-saturated developing tank (4.14) and develop with the developing solvent (5.7) at about 20 oC, up to about 1 cm from the top edge of the plate.

Dry the plate in the air at the temperature of the tank, and spray it with the 2m,7m-dichlorofluorescein solution (5.17). Identify the monoglyceride band (Rf about 0,035) under ultraviolet light (4.20).

8.3.

Analysis of the monoglycerides by gas-liquid chromatography

Remove the band obtained in 8.2 with the help of a spatula (avoid removing components remaining on the base line) and transfer in the methylation flask (4.2).

Treat the collected silica directly by the methods described in Annex X B alternative so as to convert the monoglycerides into methyl esters, and then examine the esters by gas chromatography as described in Annex X A.

9.

EXPRESSION OF RESULTS

Calculate the fatty acid composition in the 2-position to one decimal place (Note 3).

10.

NOTES

Note 1: Checking the activity of the lipase

Prepare an oil emulsion by shaking a mixture of 165 ml of the solution of gum arabic (5.21), 15 g of crushed ice and 20 ml of a neutralized oil (5.18) in a suitable agitator.

In a beaker (4.5) place 10 ml of this emulsion, followed successively by 0,3 ml of the sodium cholate solution (5.20) and and 20 ml of distilled water.

Put the beaker into a thermostat maintained at 37 p0,5 oC (Note 4); insert the electrodes of a pH meter (4.21) and a spiral stirrer (4.22).

By means of a burette (4.23) add dropwise the sodium hydroxide solution (5.19) until the pH reaches 8,5.

Add sufficient of an aqueous suspension of the lipase (see below). As sson as the pH meter indicates a pH of 8,3, start the stop-watch (4.24) and drip in the sodium hydroxide solution (5.19) at such a rate as to maintain the pH at 8,3. Read off the volume of alkali solution consumed every minute.

Record the observations in the form of a graph, using the time readings as abscissae and the ml of alkali solution required to maintain the pH constant as ordinates. A linear graph should be obtained.

The lipase suspension mentioned above is a 1 per thousand (m/m) suspension in water. For each test sufficient of this suspension should be used so that about 1 ml of the alkali solution is consumed in four to five minutes. Usually about 1 to 5 mg of the powder is required.

The lipase unit is defined as the amount of enzyme which will liberate 10 m-equivalents of acid per minute. Then the activity A of the powder used, measured in lipase units per mg, is given by the formula:

Note 1: A = V × 10

A =

V × 10

m

where V is the number of the sodium hydroxide solution (5.19) consumed per minute, calculated from the graph, m is the mass in mg, of the test portion of the powder.

Note 2: Preparation of the lipase

Lipases having satisfactory lipase activity are available commercially. But it is also possible to prepare them in the laboratory as follows:

Chill 5 kg of fresh pig pancreas to 0 oC; remove the surrounding solid fat and connective tissue and triturate in a blender so as to obtain a pasty fluid. Stir this paste with the stirrer (4.25) for four to six hours with 2,5 l of anhydrous acetone and centrifuge. Extract the residue thrice more with the same volume of acetone, then twice with 1/1 (V/V) mixture of acetone and diethyl ether, and twice with diethyl ether.

Dry the residue in vacuo for 48 hours to obtain a stable powder, which should be stored in a refrigerator.

Note 3: In every case it is advisable to determine the composition of the total fatty acids of the same sample, since the comparison with that of the acids in the 2-position will help in the interpretation of the figures obtained.

Note 4: The temperature of hydrolysis is set at 37 oC, as a liquid oil is being used. However, it is set at 40 oC for the test sample, so as to allow the examination of fats with melting points up to 45 oC.

ANNEX VIII

DETERMINATION OF COMPOSITION OF TRILINOLEIN

1.

SCOPE

Determination of composition of triglyceride in olive oils in terms of thehir equivalent carbon number by high-performance liquid chromatography.

The present Standard describes a method of separation and quantitative determination of the triglyceride composition of vegetable oils in terms of their molecular weight and degree of unsaturation as a function of their equivalent carbon number (see Note 1).

2.

FIELD OF APPLICATION

This standard is applicable to all vegetable oils containing triglycerides of long-chain fatty acids. The method is especially appliable to the detection of the presence of small quantities of semi-drying oils (rich in linoleic acid) in vegetable oils containing oleic acid as the predominant unsaturated fatty acid, such as olive oil.

3.

PRINCIPLE

Separation of triglycerides according to their equivalent carbon number by high-performance liquid chromatography (reversed phase polarity) and interpretation of the chromatograms.

4.

APPARATUS

4.1.

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

4.2.

Injection unit for 10 ml delivery.

4.3.

Detector: differential refractometer. The full-scale sensitivity should be at least 10-% unit of refractive index.

4.4.

Column: stainless steel tube 250 mm in length and of internal diameter 4,5 mm, packed with 5 mm diameter particles of silica with 22 to 23 % carbon in the form of octadecylsilane (Note 2).

4.5.

Recorder and/or integrator.

5.

REAGENTS

The reagents should be of analytical purity. Elution solvents should be de-gassed, and may be recycled several times without effect on the separations.

5.1.

Chloroform.

5.2.

Acetone.

5.3.

Acetonitrile.

5.4.

Elution solvent: acetonitrile + acetone (proportions to be adjusted to obtain the desired separation; begin with 50:50 mixture).

5.5.

Solubilization solvent: acetone or 1:1 acetone-chloroform mixture.

5.6.

Reference triglycerides: either commercial triglycerides (tripalmitin, triolein, etc.) may be used and the retention times thence plotted in accordance with the equivalent carbon number, or alternatively a reference chromatogram obtained from soya oil (see Notes 3 and 4 and Figures 1 and 2).

6.

PREPARATION OF SAMPLES

A 5 % solution of the samples to be analysed is prepared by weighing 0,5 ± 0,001 g of the sample into a 10 ml graduated flask and making up to 10 ml with the solubilization solvent (5.5).

7.

PROCEDURE

7.1.

Set up the chromatographic system. Pump elution solvent (5.4) at a rate of 1,5 ml/mm to purge the entire system. Wait until a stable base line is obtained.

Inject 10 ml of the sample prepared as in 6.

8.

CALCULATION AND EXPRESSION OF RESULTS

Use the internal standardization method, i.e. assume that the sum of the areas of the peaks corresponding to the various triglycerides is equal to 100 %. Calculate the relative percentage of each triglyceride using the formula:

% triglyceride = sum of peak areas × 100

% triglyceride =

area of peak

sum of peak areas

× 100

The result to be given to one decimal place.

Note 1. The elution order can be determined by calculating the equivalent carbon numbers, often defined by the relation ECN = CN 2n, where CN is the carbon number and n is the number of double bonds; it can be calculated much more precisely by taking into account the origin of the double bond. If no, n1 and n1n are the numbers of double bonds attributable to oleic, linoleic and linolenic acids respectively, the equivalent carbon number can be calculated by means of a relation of the formula:

ECN = CN do no d1 n1 d1n n1n

where the coefficients do, d1 and d1n can be calculated by means of the reference triglycerides. Under the conditions specified in this method the relation obtained will be close to:

ECN = CN [2,60 no] [2,35 n1] [2,17 n1n]

Note 2. Examples: Lichrosorb (Merck) RP18 Art 50333;

Lichrosphere or equivalent (Merck) 100 CH18 Art 50377.

Note 3. With several reference triglycerides it is also possible to calculate the resolution with respect to triolein,

á = RTm/RTmolein

by use of the reduced retention time RTm = RT RTsolvent.

The graph of log á against f (number of double bonds) enables the retention values to be determined for all the triglycerides of fatty acids contained in the reference triglycerides - see Figure 2.

Note 4. The efficiency of the column should permit clear separation of the peak of trilinolein from the peaks of the triglycerides with an adjacent RT.

>START OF GRAPHIC>

Figure 1

Chromatogram of a sample of soya oil

>END OF GRAPHIC>

P = palmitic acid St = linolenic acid

P

= palmitic acid

St

= stearic acid

O = oleic acid

L

= linoleic acid

ln

= linolenic acid

>START OF GRAPHIC>

Figure 2

Graph of log á against f (number of double bonds)

>END OF GRAPHIC>

ANNEX IX

SPECTROPHOTOMETRIC INVESTIGATION IN THE ULTRAVIOLET

FOREWORD

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

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

1.

SCOPE

The method describes the procedure for performing a spectrophotometric examination of fats in the ultraviolet.

2.

PRINCIPLE OF THE METHOD

The fat in question is dissolved in the required solvent and the extinction of the solution is then determined at the specified wavelengths with reference to pure solvent. Specific extinctions are calculated from the spectrophotometer readings.

3.

EQUIPMENT

3.1.

A spectrophotometer for measuring extinction in the ultraviolet between 220 and 360 nm, with the possibility of reading individual nanometric units.

3.2.

Rectangular quartz cuvettes, with covers, having an optical length of 1 cm. When filled with water

or other suitable solvent the cuvettes should not show differences between them of more than 0,01 extinction units.

3.3.

25 ml graduated flasks.

3.4.

Chromatography column having a length of 450 mm and a diameter of 35 mm with a discharge tube of diameter approximately 10 mm.

4.

REAGENTS

4.1.

Spectrophotometrically pure iso-octane (2,2,4-trimethylpentane). With reference to distilled water this should have a transmittance of not less than 60 % at 220 nm and not less than 95 % at 250 nm, or

- spectrophotometrically pure cyclohexane: with reference to distilled water this should have a transmittance of not less than 40 % at 220 nm and not less than 95 % at 250 nm, or

- another suitable solvent capable of completely dissolving the fat (e.g. ethyl alcohol for castor oil).

4.2.

Basic alumina for column chromatography prepared and checked as described in Appendix I.

4.3.

n-hexane, for chromatography.

5.

PROCEDURE

5.1.

The sample in question must be perfectly homogeneous and without suspected impurities. Oils which are liquid at ambient temperature are to be filtered through paper at a temperature of approximately 30 oC, hard fats are to be homogenized and filtered at a temperature of not more than 10 oC above the melting point.

5.2.

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

5.3.

Fill a cuvette with the solution obtained and measure the extinctions at an appropriate wavelength between 232 and 276 nm, using the solvent used as a reference.

The extinction values recorded must lie within the range 0,1 to 0,8. If not the measurements must be repeated using more concentrated or more dilute solutions as appropriate.

5.4.

When a determination of specific extinction is required after passage over alumina, proceed as follows. Place 30 g of basic alumina in suspension in hexane in the chromatography column. After the adsorbent has settled remove the excess hexane down to approximately 1 cm above the top of the alumina.

Dissolve 10 g of the fat, homogenized and filtered as described in 5.1, in 100 ml of hexane and pour the solution into the column. Collect the eluate and evaporate off all the solvent under vacuum at a temperature below 25 oC.

Proceed immediately as specified in 5.2 using the fat so obtained.

6.

EXPRESSION OF THE RESULTS

6.1.

Record the specific extinctions (extinction coefficients) at the various wavelengths calculated as follows:

Kl = c 7 s

Kl =

Eë

c 7 s

where:

Kë = specific extinction at wavelength l;

Eë

=

extinction measured at wavelength ë;

c

=

concentration of the solution in g/100 ml;

s

=

thickness of the cuvette in cm.

The results are to be expressed to two decimal places.

6.2.

Spectrophotometric analysis of olive oil in accordance with the official method in the EEC regulations specifies determination of the specific extinction in iso-octane solution at wavelengths of 232 and 270 nm and the determination K, which is given by:

DK = Km Km 4 + Km + 4

DK = Km

Km 4 + Km + 4

2

where Km is the specific extinction at wavelength m, the wavelength for maximum absorption around 270 nm.

APPENDIX I Preparation of the alumina and testing its activity

A.1.1.

Preperation of the alumina

Place alumina which has been previously desiccated in a furnace at 380 to 400 oC for three hours into a hermetically sealed container, add distilled water in the ratio of 5 ml per 100 g of alumina, immediately close the container, shake repeatedly, and then allow to rest for at least 12 hours before use.

A.1.2.

Checking the activity of the alumina

Prepare a chromatographic column with 30 g of alumina. Working as described in paragraph 5.4 pass a mixture consisting of:

- 95 % virgin olive oil having a specific extinction of less than 0,18 at 268 nm,

- 5 % ground-nut oil treated with earth in the refining process, having a specific extinction of not less than 4 at 268 nm

through the column.

If after passage through the column the mixture has a specific extinction of more than 0,11 at 268 nm the alumina is acceptable, if not the level of dehydration must be increased.

APPENDIX II Calibration of the spectrophotometer

A.2.

The equipment must be checked at intervals (at least every six months) for both wavelength response and the accuracy of the response.

A.2.1.

The wavelength may be checked using a mercury vapour lamp or by means of suitable filters.

A.2.2.

In order to check the response of the photocell and the photomultiplier proceed as follows: weigh 0,2000 g of pure potassium chromate for spectrophotometry and dissolve in 0,05 N potassium hydroxide solution in a 1 000 ml graduated flask and make up to the mark. Take precisely 25 ml of the solution obtained, transfer to a 500 ml graduated flask and dilute up to the mark using the same potassium hydroxide solution.

Measure the extinction of the solution so obtained at 275 nm, using the potassium hydroxide solution as a reference. The extinction measured using a 1 cm cuvette should be 0,200 p0,005.

ANNEX X A

ANALYSIS BY GAS CHROMATOGRAPHY OF METHYL ESTERS OF FATTY ACIDS

1.

SCOPE

This method gives general guidance for the application of gas chromatography, using packed or capillary columns, to determine the qualitative and quantitative composition of a mixture of fatty acid methyl esters obtained in accordance with the method specified in Annex X B.

The method is not applicable to polymerized fatty acids.

2.

REAGENTS

2.1.

Carrier gas

Inert gas (nitrogen, helium, argon, hydrogen, etc.), thoroughly dried and with an oxygen content of less than 10 mg/kg.

Note 1. Hydrogen, which is used as a carrier gas only with capillary columns, can double the speed of analysis but is hazardous. Safety devices are available.

2.2.

Auxiliary gases

2.2.1.

Hydrogen (purity 799,9 %), feree from organic impurities.

2.2.2.

Air or oxygen, free from organic impurities.

2.3.

Reference standard

A mixture of methyl esters of pure fatty acids, or the methyl esters of a fat of known composition, preferably similar to that of the fatty matter to be analyzed.

Care shall be taken to prevent the oxidation of polyunsaturated fatty acids.

3.

APPARATUS

The instructions given relate to the usual equipment used for gas chromatography, employing packed and/or capillary columns and a flame-ionization detector. Any apparatus giving the efficiency and resolution specified in 4.1.2 is suitable.

3.1.

Gas chromatograph

The gas chromatograph shall comprise the following elements.

3.1.1.

Injection system

Use an injection system either:

(a) with packed columns, having the least deadspace possible (in this case the injection system shall be capable of being heated to a temperature 20 to 50 oC higher than that of the column); or

(b) with capillary columns, in which case the injection system shall be specially designed for use with such columns. It may be of the split type or it may be of the splitless on column injector type.

Note 2. In the absence of fatty acids with less than 16 carbon atoms, a moving needle injector may be used.

3.1.2.

Oven

The oven shall be capable of heating the column to a temperature of at least 260 oC and of maintaining the desired temperature to within 1 oC with a packed column and within 0,1 oC with a capillary column. The last requirement is particularly important when a fused silica tube is used.

The use of temperature-programmed heating is recommended in all cases, and in particular for fatty acids with less than 16 carbon atoms.

3.1.3.

Packed column

3.1.3.1.

Column, constructed of a material inert to the substances to be analyzed (i.e. glass or stainless steel) having the following dimensions:

(a) length: 1 to 3 m. A relatively short column should be used when long-chain fatty acids (above C20) are present. When analyzing acids with 4 or 6 carbon atoms, it is recommended that a column 2 m in length is used;

(b) internal diameter: 2 to 4 mm.

Note 3. If polyunsaturated components with more than three double bonds are present, they may be decomposed in a stainless steel column.

Note 4. A system with packed twin columns may be used.

3.1.3.2.

Packing, comprising the following elements:

(a) support: acid-washed and silanized diatomaceous earth, or other suitable inert support with a narrow range of grain size (25 mm range between the limits 125 to 200 mm) the average grain size being related to the internal diameter and length of the column;

(b) stationary phase: polyester type of polar liquid (e.g. diethylene glycol polysuccinate, butanediol polysuccinate, ethyleneglycol polyadipate, etc.) cyanosilicones or any other liquid permitting the chromatographic separation required (see clause 4). The stationary phase should amount to 5 to 20 % (m/m) of the packing. A non-polar stationary phase can be used for certain separations.

3.1.3.3.

Conditioning of the column

With the column disconnected, if possible, from the detector, gradually heat the oven to 185 oC and pass a current of inert gas through the freshly prepared column at a rate of 20 to 60 ml/min for at least 16 hours at this temperature, and for a further 2 hours at 195 oC.

3.1.4.

Capillary column

3.1.4.1.

Tube, made of a material inert to the substances to be analysed (usually glass or fused silica). The internal diameter shall be between 0,2 and 0,8 mm. The internal surface shall undergo an appropriate treatment (e.g. surface preparation, inactivation) before receiving the stationary phase coating. A length of 25 mm is sufficient in most cases.

3.1.4.2.

Stationary phase, usually of the type polyglycol (poly(ethylene glycol) 20 000), polyester (butanediol polysuccinate) or polar polysiloxane (cyanosilicones). Bonded (cross-linked) columns are suitable.

Note 5. There is a risk of polar polysiloxanes giving rise to difficulties in the identification and separation of linolenic acid and C20 acids.

The coatings shall be thin, i.e, 0,1 to 0,2 mm.

3.1.4.3.

Assembly and conditioning of the column

Observe the normal precautions for assembling capillary columns, i.e. arrangement of the column in the oven (support), choice and assembly of joints (leak tightness), positioning of the ends of the column in the injector and the detector (reduction of dead-spaces). Place the column under a flow of carrier gas (e.g. 0,3 bar (30 kPa) for a column of length 25 mm and internal diameter 0,3 mm).

Condition the column by temperature programming of the oven at 3 oC/min from ambient temperature to a temperature 10 oC below the decomposure limit of the stationary phase. Maintain the oven at this temperature for one hour until stabilization of the baseline. Return it to 180 oC to work under isothermal conditions.

Note 6. Suitably pre-conditioned columns are available commercially.

3.1.5.

Detector, preferably capable of being heated to a temperature above that of the column.

3.2.

Syringe

The syringe shall have a maximum capacity of 10 ml, and be graduated in 0,1 ml divisions.

3.3.

Recorder

If the recorder curve is to be used to calculate the composition of the mixture analysed, an electronic recorder of high precision, compatible with the apparatus used, is required. The recorder shall have the following characteristics:

(a) rate of response, below 1,5 s, preferably 1 s (the rate of response is the time taken for the recording pen to pass from 0 to 90 % following the sudden introduction of a 100 % signal);

(b) width of the paper, 20 cm minimum;

(c) paper speed, adjustable to values between 0,4 and 2,5 cm/min.

3.4.

Integrator

Rapid and accurate calculation can be performed with the help of an electronic integrator. This shall give a linear response with adequate sensitivity, and the correction for deviation of the base-line shall be satisfactory.

4.

PROCEDURE

The operations described in 4.1 to 4.3 relate to the use of a flame-ionization detector.

As an alternative a gas chromatograph employing a catharometer detector (working on the principle of thermal conductivity changes) may be used. The operating conditions are then modified as described in clause 6.

4.1.

Test conditions

4.1.1.

Selection of optimum operating conditions

4.1.1.1.

Packed column

In the selection of the test conditions, the following variables should be taken into account:

(a) the length and diameter of the column;

(b)

the nature and amount of the stationary phase;

(c)

the temperature of the column;

(d)

the carrier gas flow;

(e)

the resolution required;

(f)

the size of the test portion, selected in such a way that the assembly of the detector and electrometer gives a linear response;

(g)

the duration of analysis.

In general, the values given in Table 1 and Table 2 will lead to the desired results, i.e. at least 2 000 theoretical plates per metre of column length for methyl stearate and its elution within about 15 minutes.

Where the apparatus allows it, the injector should be at a temperature of about 200 oC and the detector at a temperature equal to or higher than that of the column.

As a rule, the ratio of the flow-rate of the hydrogen supplied to the flame-ionization detector to that of the carrier gas varies from 1:2 to 1:1 depending on the diameter of the column. The flow of oxygen is about 5 to 10 times that of the hydrogen.

>TABLE>

>TABLE>

4.1.1.2.

Capillary column

The properties of efficiency and permeability of capillary columns mean that the separation between constituents and the duration of the analysis are largely dependent on the flow-rate of the carrier-gas in the column. It will therefore be necessary to optimize the operating conditions by acting on this parameter (or more simply on the headloss of the column), according to whether one wishes to improve the separations or to make a rapid analysis.

4.1.2.

Determination of the number of theoretical plates (efficiency) and resolution (See Figure 1)

Carry out the analysis of a mixture of methyl stearate and methyl oleate in about equivalent proportions (for example, methyl esters from cocoa butter).

Choose the temperature of the column and the carrier gas flow so that the maximum of the methyl stearate peak is recorded about 15 minutes after the solvent peak. Use a sufficient quantity of the mixture of methyl esters that the methyl stearate peak occupies about three-quarters of the full scale.

Calculate the number of theoretical plates, n (efficiency), using the formula:

n = 16 [ ù1 ]²

n = 16 [

dr1

ù1

]²

and the resolution, R, using the formula:

R = ù1 + ù2

R =

2D

ù1 + ù2

where:

dr1

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

ù1 and ù2

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

D

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

>START OF GRAPHIC>

Figure 1

Chromatogram for determining the number of theoretical plates (efficiency) and resolution

>END OF GRAPHIC>

The operating conditions to be selected are those which will afford at least 2 000 theoretical plates per metre of column length for methyl stearate and a resolution of at least 1,25.

4.2.

Test portion

Using the syringe (3.2) take 0,1 to 2 ml of the solution of methyl esters prepared according to Annex X B and inject them into the column.

In the case of esters not in solution, prepare a solution of approximately 100 mg/ml in heptane of chromatographic quality, and inject 0,1 to 1 ml of this solution.

If the analysis is for constituents present only in trace amounts, the size of the test portion may be increased (up to 10-fold).

4.3.

Analysis

Generally, the operating conditions shall be those defined in 4.1.1.

Nevertheless, it is possible to work with a lower column temperature when the determination of fatty acids with fewer than 12 carbon atoms is required, or at a higher temperature when determining fatty acids with more than 20 carbon atoms. On occasion, it is possible to employ temperature programming in both these cases. For example, if the sample contains the methyl esters of fatty acids with fewer than 12 carbon atoms, inject the sample at 100 oC (or at 50 to 60 oC if butyric acid is present) and immediately raise the temperature at a rate of 4 to 8 oC/min to the optimum. In certain cases, the two procedures can be combined.

After the programmed heating, continue the elution at a constant temperature until all the components have been eluted. If the instrument does not have programmed heating, use it at two fixed temperatures between 100 and 195 oC.

If necessary, it is recommended that an analysis be carried out on two fixed phases with different polarities to verify the absence of masked peaks, for example in the case of the simultaneous presence of C18:3 and C20:0, or C18:3 and C18:2 conjugated.

4.4.

Preparation of the reference chromatogram and reference graphs

Analyze the reference standard mixture (2.3) using the same operating conditions as those employed for the sample, and measure the retention times or retention distances for the constituent fatty acids. Construct on semi-logarithmic paper, for any degree of unsaturation, the graphs showing the logarithm of retention time or distance as a function of the number of carbon atoms. In isothermal conditions, the graphs for straight-chain acids of the same degree of unsaturation should be straight lines. These lines should be approximately parallel.

It is necessary to avoid conditions such that 'masked peaks` exist, i.e. where the resolution is insufficient to separate two constituents.

5.

EXPRESSION OF RESULTS

5.1.

Qualitative analysis

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

5.2.

Quantitative analysis

5.2.1.

Determination of the composition

Apart from exceptional cases, use the internal normalization method, i.e. assume that the whole of the components of the sample are represented on the chromatogram, so that the total of the areas under the peaks represents 100 % of the constituents (total elution).

If the equipment includes an integrator, use the figures obtained therefrom. If not, determine the area under each peak by multiplying the height of the peak by its width at mid-height, and where necessary take into account the various attenuations used during the recording.

5.2.2.

Method of calculation

5.2.2.1.

General case

Calculate the content of a given component i, expressed as a percentage by mass of methyl esters, by determining the percentage represented by the area of the corresponding peak relative to the sum of the areas of all the peaks, using the following formula:

ÓA × 100

Ai

ÓA

× 100

where:

Ai is the area under the peak corresponding to component i;

ÓA

is the sum of the areas under all the peaks.

Give the result to one decimal place.

Note 7: In this general case, the result of the calculation based on relative areas is considered to represent a percentage by mass. For the cases in which this assumption is not allowed, see 5.2.2.2.

5.2.2.2.

Use of correction factors

In certain cases, for example in the presence of fatty acids with fewer than eight carbon atoms or of acids with secondary groups, when using thermal conductivity detectors or where the highest degree of accuracy is particularly required, correction factors should be used to convert the percentages of peak areas into mass percentages of the components.

Determine the correction factors with the help of a chromatogram derived from the analysis of a reference mixture of methyl esters of known composition, carried out under operating conditions identical with those used for the sample.

For this reference mixture, the percentage by mass of component i is given by the formula:

Óm × 100

mi

Óm

× 100

where:

mi is the mass of component i in the reference mixture;

Óm

is the total of the masses of the various components of the reference mixture.

From the chromatogram of the reference mixture (4.4) calculate the percentage (area/area) for component i as follows:

Ai

ÓA

× 100

where:

Ai

is the area under the peak corresponding to component i;

ÓA

is the sum of the areas under all the peaks.

The correction factor is then calculated as:

Ki = mi × ÓA

Ki=

mi× ÓA

Ai× Óm

Commonly, the correction factors are expressed relative to KC16, so that the relative factors become:

Kmi = KC16

Kmi=

Ki

KC16

For the sample, the content of each component i, expressed as a percentage by mass of methyl esters, is:

Ó (Kmi × Ai) × 100

Kmi× Ai

Ó (Kmi× Ai)

× 100

Give the results to one decimal place.

5.2.2.3.

Use of an internal standard

In certain analyses (for example where not all of the fatty acids are quantified, such as when acids with four and six carbons are present alongside acids with 16 and 18 carbons, or when it is necessary to determine the absolute amount of a fatty acid in a sample) it is necessary to use an Internal Standard. Fatty acids with five, 15 or 17 carbons are frequently used. The correction factor (if any) for the Internal Standard should be determined.

The percentage by mass of component i, expressed as methyl esters, is then given by the formula:

ms × Kmi × Ai × 100

ms× Kmi× Ai

m × Kms× As

× 100

where:

Ai is the area under the peak corresponding to component i;

As

is the area under the peak corresponding to the Internal Standard;

Kmi

is the correction factor for component i (relative to KC1);

Kms

is the correction factor for the Internal Standard (relative to KC16);

m

is the mass, in milligrams, of the test portion;

ms

is the mass, in milligrams, of the Internal Standard.

Give the results to one decimal place.

6.

SPECIAL CASE - USE OF A CATHAROMETER DETECTOR (WORKING ON THE PRINCIPLE OF THERMAL CONDUCTIVITY CHANGES)

A gas chromatograph employing a detector working on the principle of thermal conductivity changes (a catharometer) may also be used for the determination of the qualitative and quantitative composition of a mixture of fatty acid methyl esters. If it is used, the conditions specified in clause 3 and clause 4 should be modified as shown in Table 3.

For quantitative analysis, use the correction factors defined in 5.2.2.2.

>TABLE>

7.

TEST REPORT

The test report shall specify the methods used for the preparation of the methyl esters and for the gas chromatographic analysis, and the results obtained. It shall also mention all operating details not specified in this International Standard, or regarded as optional, together with details of any incidents which may have influenced the results.

The test report shall include all information necessary for the complete identification of the sample.

ANNEX X B

PREPARATION OF METHYL ESTERS OF FATTY ACIDS IN ACCORDANCE WITH TITLES I AND II OF ANNEX VI TO REGULATION (EEC) N° 72/77 OR THE METHOD DESCRIBED BELOW

FOREWORD

The choice of process will be dictated by the acid composition and acidity of the fatty substance under examination and the gas chromatography analysis to be carried out.

More specifically:

- only sealed phial processes or processes using dimethyl sulphate can be used for fatty substances containing fatty acids with fewer than 12 carbon atoms,

- only the methanol-hydrochloric acid or methyl sulphate processes can be used for fatty substances with acidity of over 3 %,

- only processes using sodium methylate or dimethyl sulphate can be used for gas chromatography measurements of trans-Isomers,

- the methanol-hexane-sulphuric acid process must be used for the preparation of the methyl esters of small quantities of fatty substances from separation by thin layer chromatography.

The presence of unsaponifiables can be discounted, provided it does not exceed 3 %, otherwise the methyl esters will have to be prepared from the fatty acids.

1.

SCOPE AND FIELD OF APPLICATION

A description is given of five processes for the preparation of methyl esters from fatty substances:

(a) with sodium methylate;

(b)

with sodium methylate in a sealed phial;

(c)

with methanol-hydrochloric acid in a sealed phial;

(d)

with dimethyl sulphate;

(e)

with methanol-hexane-sulphuric acid.

Process A

2.

PRINCIPLE

The fatty substance which is being subjected to analysis is heated under reflux with methyl alcohol and sodium methylate. The methyl esters obtained are extracted with ethyl ether.

3.

APPARATUS

3.1.

100 ml flask with a reflux condenser with a soda lime tube fitted to the top, with ground glass joints.

3.2.

50 ml measuring glasses.

3.3.

5 ml measuring pipette marked off in 0,1 ml.

3.4.

250 ml separating funnels.

3.5.

200 ml flask.

4.

REAGENTS

4.1.

Anhydrous methanol.

4.2.

Solution of approximately 1 % sodium methylate in methanol; this is prepared by dissolving 0,34 g of metallic sodium in 100 ml of anhydrous methanol.

4.3.

Ethyl ether.

4.4.

10 % sodium chloride solution.

4.5.

40 to 60 oC petroleum ether.

5.

PROCEDURE

5.1.

Place in the 100 ml flask 2 g of the fatty substance which has previously been dried