1.

In the list of Annexes, the title of Annex IV is changed to: Determination of wax content by capillary column gas-liquid chromatography.

2.

In the summary, the title of Annex XIII ‘Proof that refining has taken place’ is replaced by ‘Neutralization and decolorization of olive oil in the laboratory.’

3.

The first table in Annex I is replaced by the following table:

4.

The following footnote is added to the second table in Annex I:

‘For the purpose or establishing purity, where K270 exceeds the limit for the category concerned, it shall be determined again after passage over alumina.’

5.

In Annex II, point 1.5in fine, the words ‘of the two calculations’ are replaced by ‘of two calculations’.

6.

In Annex IV, point 5.1.1, the words ‘or seed oil’ are deleted.

7.

In Annex IV, point 5.2.2, the first two sentences are replaced by the following:

‘A solution of 65:35 by volume of hexane and ethyl ether is introduced into the development chamber to an approximate depth of 1 cm.

.’

.

8.

In Annex IV, ►C1  point 5.2.5.2 and point 6., the number‘100’ is replaced by ‘1 000’and the words ‘in square millimetres’ are deleted.

9.

In the Appendix to Annex IV, figure 1is replaced by the following:

▼C1

1 = Eicosanol

2 = Decosanol

3 = Tricosanol

4 = Tetracosanol

5 = Pentacosanol

6 = Hexacosanol

7 = Heptacosanol

8 = Octacosanol

10.

Annex IV is replaced by the following text and diagram:

‘ANNEX IV

DETERMINATION OF WAX CONTENT BY CAPILLARY COLUMN GAS-LIQUID CHROMATOGRAPHY

1.   SCOPE

This method describes a procedure for the determination of the wax content of certain fats and oils, under the test conditions.

It may be used in particular to distinguish between olive oil obtained by pressing and that obtained by extraction (olive-pomace oil).

2.   PRINCIPLE

Addition of a suitable internal standard to the fat or oil, then fractionation by chromatography on a hydrated silica gel column. Recovery of the fraction eluted first under the test conditions (whose polarity is less than that of the triglycerides), then direct analysis by capillary column gas-liquid chromatography.

3.   APPARATUS

4.   REAGENTS

5.   PROCEDURE

5.1.   Separation of the wax friction.

5.1.1.   Preparation of the chromatographic column.

Suspend 15 g of silica gel hydrated at 2 % in anhydrous n-hexane and introduce into the column.

Allow to settle spontaneously. Complete settling with the aid of an electric shaker to make the chromatographic band more homogeneous. Percolate 30-ml n-hexane to remove any impurities.

5.1.2.   Column chromatography

Weigh exactly 500 mg of the sample into a 25-ml flask, and add a suitable amount of internal standard, depending on the assumed wax content, e.g. add 0,1 mg lauryl arachidate in the case of olive oil, and 0,25 to 0,5 mg in the case of olive-pomace oil.

Transfer the prepared sample to the chromatographic column, prepared according to 5.1., with the aid of two 2-ml portions of n-hexane.

Allow the solvent to flow to 1 mm above the upper level of the absorbent. Then start chromatographic elution; collect 140 ml of the n-hexane/ethyl ether mixture, at 99:1, at a flow of about 15 drops every 10 seconds (2,1 ml/minute).

Dry the resultant fraction in a rotary evaporator until almost all the solvent is eliminated. Remove the last 2 or 3 ml of solvent with the help of a weak current of nitrogen, then add 10 ml n-heptane.

5.2.   Gas-liquid chromatographic analysis

5.2.1.   Preliminary procedure, conditioning of column.

5.2.2.   Choice of operating conditions.

5.2.3.   Performance of the analysis

5.2.4.   Peak identification

Identify the peaks from the retention times, by comparing them with mixtures of waxes with known retention times, analysed under the same conditions.

Figure 1 gives a wax chromatogram of a virgin olive oil.

5.2.5.   Quantitative analysis

6.   EXPRESSION OF THE RESULTS

Give the different wax contents, and the sum of those contents, in mg/kg of fat.

APPENDIX

Determination of linear gas speed

Inject 1 to 3 μl methane (propane) into the gas-liquid chromatographic apparatus, after adjusting it to the normal operating conditions. Measure the time the gas takes to run through the column, from the moment it is injected until the peak emerges (tM).

The linear speed in cm/sec. is given by the formula L/tM where L is the length of the column, in cm, and tM is the time measured in seconds.’

11.

In Annex V, point 4.11., ‘5 %’is replaced by ‘2 %’.

12.

In Annex V, point 5.1.1., first subparagraph, ‘seed oils or’ is deleted.

13.

In Annex V, point 5.1.1., third subparagraph, ‘animal or vegetable oils and fats’ is replaced by ‘oils’.

14.

In Annex V, point 5.1.1.in fine, the following is added:

‘or betulinol will have to be used instead of cholestanol’

.

15.

In Annex V, point 5.4.5.2., ‘in square millimetres’ is deleted.

16.

In Annex VI, point 6., ‘in square millimetres’ is deleted.

17.

In Annex IX, point 3.4. is replaced by the following:

18.

In Annex IX, point 4.1., the second indentis deleted.

19.

In Annex XIII, the title ‘Proof that refining has taken place’ is replaced by the following ‘Neutralization and decolorization of olive oil in the laboratory’.

20.

Annex XIV is replaced by the following:

‘ANNEX XIV

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