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Commission Regulation (EEC) No 2568/91 of 11 July 1991 on the characteristics of olive oil and olive-residue oil and on the relevant methods of analysis
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Suspend 15 g of silica gel (4.1) in the n-hexane (4.2) and introduce it into the column (3.2). Allow to settle spontaneously. Complete settling with the aid of an electrovibrator (3.5) to make the chromatographic layer more homogeneous. Percolate 30 ml of n-hexane to remove any impurities. Using the balance (3.8) weigh exactly 500 mg of the sample into the 25 ml Erlenmeyer flask (3.1), add the appropriate quantity of the internal standard (4.5) according to the presumed wax content. For example, add 0,1 mg of lauryl arachidate for olive oil, and 0,25 to 0,5 mg for olive-residue oil. Transfer the prepared sample to the chromotography column using two 2 ml portions of n-hexane (4.2).
Allow the solvent to flow away until it reaches 1 mm above the upper level of the absorbant then percolate a further 70 ml of n-hexane in order to eliminate the n-alkanes naturally present. Then start the chromatographic elution by collecting 180 ml of the mixture of n-hexane/ethyl ether (ratio 99:1), keeping a rate of flow of approximately 15 drops every 10 seconds. Elution of the sample must be carried out at a room temperature of 22 ± 4 °C.
The n-hexane/ethyl ether mixture (99:1) must be prepared every day.
For a visual check on the correct elution of the waxes 100 μl of 1 % Sudan in the elution mixture can be added to the sample in solution. Since the colourant has an intermediate retention, between waxes and triglycerides, when the coloration has reached the bottom of the column the elution should be suspended because all the waxes will have been eluted.
Dry the fraction thus obtained in a rotary evaporator (3.6.) until virtually all the solvent has been eliminated. Eliminate the final 2 ml of solvent with the aid of a weak current of nitrogen; then add 2-4 ml n-heptane.
Fit the column to the gas chromatograph (3.3) by connecting the inlet port to the on-column system and the outlet port to the detector. Perform a general check on the GC apparatus (operation of gas circuits, detector and recorder efficiency, etc.).
If the column is being used for the first time it should be conditioned first. Pass a little gas through the column, then turn on the GC apparatus. Heat gradually until 350 °C is reached after about four hours. Maintain that temperature for at least two hours then regulate the apparatus to operating conditions (set gas flow, light flame, connect to the electronic recorder (3.3.4), set temperature of column chamber, detector, etc.) and record the signal at a sensitivity at least twice as high as that required for the analysis. The baseline must be linear, with no peaks of any kind, and must not show any deviation.
A negative straight-line drift indicates that the column connections are not tight; a positive drift that the column has not been sufficiently conditioned.
The operating conditions are generally as follows:
column temperature:
20 °C/minute | 5 °C/minute | 20 °C/minute | ||||
Initially 80 °C (1′) | → | 240 °C | → | 325 °C (6′) | → | 340 °C (10′) |
detector temperature: 350 °C;
quantity of substance injected: 1 μl of the n-heptane solution (2-4 ml);
carrier gas: helium or hydrogen at the correct linear velocity for the gas selected (see Appendix);
instrument sensitivity: suitable for the following conditions:
The conditions may be modified according to the characteristics of the column and the GC apparatus to obtain separation of all the waxes and a satisfactory peak resolution (see figure); the internal standard C 32 retention time must be 18 ± 3 minutes. The most representative wax peak must be at least 60 % of the full scale.
The peak integration parameters must be established so as to obtain a correct evaluation of the areas of the peaks in question.
NB : Given the high final temperature, a positive drift of no more than 10 % of the full scale is permitted. U.K.
Sample 1 μl of the solution using the 10 μl microsyringe; withdraw the syringe plunger so that the needle is empty. Place the needle in the injector and after 1-2 seconds inject quickly; remove the needle slowly after about five seconds.
Record until the waxes are completely eluted.
The base line must always satisfy the required conditions.
Identification of the different peaks should be based on retention time by comparison with wax mixtures of known retention times analysed under the same conditions.
The figure is a chromatogram of the waxes of a virgin olive oil.
Calculate the areas of the peaks of the internal standard and the aliphatic esters of C 40 to C 46 using the integrator.
Calculate the wax content of each of the esters in mg/kg fat using the formula:
where:
=
area of each ester’s peak, in square millimetres;
=
area of the internal standard’s peak, in square millimetres;
=
mass of added internal standard, in milligrams;
=
mass of sample for analysis, in grams.]
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