Commission Regulation (EEC) No 2568/91Show full title

[F1 5. PROCEDURE U.K.

5.1. Preparation of the chromatography column U.K.

Suspend 15 g of silica gel (point 4.1) in n-hexane (point 4.2) and introduce into the column (point 3.2). Allow to settle spontaneously. Complete settling with the aid of an electric shaker to make the chromatographic bed more homogeneous. Percolate 30 ml of n-hexane to remove any impurities. Weigh exactly about 500 mg of the sample into the 25-ml flask (point 3.1), using the analytical balance (point 3.8), and add a suitable amount of internal standard (point 4.5), depending on the assumed wax content, e.g. add 0,1 mg of lauryl arachidate in the case of olive oil, 0,25-0,50 mg in the case of olive-pomace oil and 0,05 mg of methyl heptadecanoate for olive oils (point 4.6).

Transfer the prepared sample to the chromatography column with the aid of two 2-ml portions of n-hexane (point 4.2).

Allow the solvent to flow to 1 mm above the upper level of the absorbent. Percolate a further of n-hexane/ethyl ether (99:1) and collect 220 ml at a flow of about 15 drops every 10 seconds. ( This fraction contains the methyl and ethyl esters and waxes ). ( Note 4 ) ( Note 5 ).

Note 4: The n-hexane/ethyl ether (99:1) mixture should be freshly prepared every day U.K.

Note 5: 100 μl of Sudan I dye at 1 % in the elution mixture can be added to the sample solution to check visually that the waxes are eluted properly. U.K.

The retention time of the dye lies in between that of the waxes and triacylglycerols. Hence, when the dye reaches the bottom of the chromatography column, elution has to be suspended because all the waxes have been eluted.

Evaporate the resultant fractions in a rotary evaporator until the solvent is almost removed. Remove the last 2 ml under a weak current of nitrogen. Collect the fraction containing the methyl and ethyl esters is diluted with 2-4 ml of n-heptane or iso-octane.

5.2. Gas chromatography analysis U.K.

5.2.1. Preliminary procedure U.K.

Fit the column to the gas chromatograph (point 3.3), connecting the inlet port to the on-column system and the outlet port to the detector. Check the gas chromatography apparatus (operation of gas loops, efficiency of detector and recorder system, etc.).

If the column is being used for the first time, it is advisable to condition it. Run a light flow of gas through the column, then switch on the gas chromatography apparatus. Gradually heat until a temperature of 350 °C is reached after approximately 4 h.

Maintain this temperature for at least 2 h, then regulate the apparatus to the operating conditions (regulate gas flow, light flame, connect to electronic recorder (point 3.3.4), regulate oven temperature for column, regulate detector, etc.). Record the signal at a sensitivity at least twice as high as that required for the analysis. The base line should be linear, with no peaks of any kind, and must not have any drift.

Negative straight-line drift indicates that the column connections are not correct while positive drift indicates that the column has not been properly conditioned.

5.2.2. Choice of operating conditions for waxes and methyl and ethyl esters (Note 6). U.K.

The operating conditions are generally as follows:

— Column temperature

:

20 °C/min 5 °C/min

80 °C at first (1′) 140 °C 335 °C (20)

— Detector temperature

:

350 °C.

— Amount injected

:

1 μl of n-heptane solution (2-4 ml).

— Carrier gas

:

helium or hydrogen at the optimal linear speed for the gas chosen (see Appendix A).

— Instrument sensitivity

:

suitable for fulfilling the above conditions.

Note 6: Due to the high final temperature, positive drift is allowed but may not exceed more than 10 % of the full-scale value. U.K.

These conditions may be modified to suit the characteristics of the column and the gas chromatograph in order to separate all the waxes and fatty acid methyl and ethyl esters and to obtain satisfactory peak separation (see Figures 2, 3 and 4) and a retention time of 18 ± 3 minutes for the lauryl arachidate internal standard. The most representative peak of the waxes must be over 60 % of the full-scale value while the methyl heptadecanoate internal standard for the methyl and ethyl esters must reach the full-scale value.

The peak integration parameters should be determined in such a way as to obtain a correct evaluation of the peak areas considered.

5.3. Performance of the analysis U.K.

Take up 10 μl of the solution with the aid of the 10 μl micro-syringe, drawing back the plunger until the needle is empty. Introduce the needle into the injection system and inject quickly after 1–2 s. After about 5 s, gently extract the needle.

Perform the recording until the waxes or stigmastadienes are completely eluted, depending on the fraction being analysed.

The base line must always meet the required conditions.

5.4. Peak identification U.K.

Identify the peaks from the retention times by comparing them with mixtures of waxes with known retention times, analysed under the same conditions. The alkyl esters are identified from mixtures of methyl and ethyl esters of the chief fatty acids in olive oils (palmitic and oleic).

Figure 1 provides a chromatogram of the waxes in a virgin olive oil. Figures 2 and 3 show the chromatograms of two retail extra virgin olive oils, one with methyl and ethyl esters and the other without them. Figure 4 gives the chromatograms for a top-quality extra virgin olive oil and the same oil spiked with 20 % deodorised oil.

5.5. Quantitative analysis of the waxes U.K.

Determine the area of the peaks corresponding to the lauryl arachidate internal standard and the aliphatic esters from C ₄₀ to C ₄₆ with the aid of the integrator.

Determine the total waxes content by adding each individual wax, in mg/kg of fat, as follows:

where:

A _x

=

area corresponding to the peak for the individual ester, in computer counts

A _s

=

area corresponding to the peak for the lauryl arachidate internal standard, in computer counts

m _s

=

mass of the lauryl arachidate internal standard added, in milligrams;

m

=

mass of the sample taken for determination, in grams.

5.5.1. Quantitative analysis of the methyl and ethyl esters U.K.

With the aid of the integrator, determine the areas of the peaks corresponding to the methyl heptadecanoate internal standard, the methyl esters of the C ₁₆ and C ₁₈ fatty acids and the ethyl esters of the C ₁₆ and C ₁₈ fatty acids.

Determine the content of each alkyl ester, in mg/kg of fat, as follows:

where:

A _x

=

area corresponding to the peak for the individual C ₁₆ and C ₁₈ ester, in computer counts

A _s

=

area corresponding to the peak for the methyl heptadecanoate internal standard, in computer counts

m _s

=

mass of the methyl heptadecanoate internal standard added, in milligrams;

m

=

mass of the sample taken for determination, in grams.]

Textual Amendments

F1 Inserted by Commission Regulation (EU) No 61/2011 of 24 January 2011 amending Regulation (EEC) No 2568/91 on the characteristics of olive oil and olive-residue oil and on the relevant methods of analysis.