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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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Point in time view as at 01/03/2014.
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Then draw off the soap solution as completely as possible into a second separating funnel. Perform two further extractions on the water-alcohol phase in the same way using 60 to 70 ml of ethyl ether (point 4.10).
Note 2 : Any emulsion can be destroyed by adding small quantities of ethanol (point 4.11). U.K.
When the wash water has been removed, filter on anhydrous sodium sulphate (point 4.5) into a previously weighed 250 ml flask, washing the funnel and filter with small quantities of ethyl ether (point 4.10).
Remove from the oven and keep in a calcium chloride desiccator (point 3.13) until required for use (plates treated in this way must be used within 15 days).
Note 5 : Higher temperature could worsen the separation. U.K.
Wash the residue in the flask three times with ethyl ether (point 4.3) (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 (point 3.9), evaporate to dryness by mild heating, in a gentle flow of nitrogen, make up again using a few drops of acetone (point 4.8), evaporate again to dryness,
The residue contained in the test tube must consist of the sterol and triterpene dialchols fractions.
Pyridine can be replaced by the same amount of acetonitrile.
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.).
A negative straight-line drift indicates leakage from the column connections; a positive drift indicates inadequate conditioning of the column.
Column temperature: 260 ± 5 °C;
Injector temperature: 280-300 °C;
Detector temperature: 280-300 °C;
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 full scale;
Amount of substance injected: 0,5 to 1 μl of TMSE solution.
These conditions may be changed according to the characteristics of the column and gas chromatograph, so as to obtain chromatograms, which meet the following requirements:
The retention time for the ß-sitosterol peak should be at 20 ± 5 min;
The campesterol peak should be: for olive oil (mean content 3 %) 20 ± 5 % of full scale; for soybean oil (average content 20 %) 80 ± 10 % of full scale;
All the present sterols must be separated. In addition to being separated the peaks, they 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 RRT 1,02 (Sitostanol) can be quantified using the perpendicular.
An automatic injector can be used as well.
Identify individual peaks on the basis of retention times and by comparison with the mixture of sterol and triterpene dialcohols TMSE, analysed under the same conditions (see Appendix).
The sterols and triterpene dialcohols are eluted in the following order: cholesterol, brassicasterol, ergosterol, 24-methylen-cholesterol, campesterol, campestanol, stigmasterol, Δ7-campesterol, Δ5,23-stigmastadienol, clerosterol, ß-sistosterol, sitostanol, Δ5-avenasterol, Δ5,24-stigmastadienol, Δ7-stigmastenol, Δ7-avenasterol, erythrodiol and uvaol.
The retention times for ß-sitosterol, for SE-52 and SE-54 columns, are shown in Table 1.
Figures 1 and 2 show typical chromatograms for some oils.
where:
=
peak area for sterol x, in computing system counts;
=
area of the α-cholestanol peak, in computing system counts;
=
mass of added α-cholestanol, in milligrams;
=
mass of the sample used for determination, in grams.]
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