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

[F1 6. GAS CHROMATOGRAPHIC ANALYSIS U.K.

6.1. Preliminary operations, capillary column conditioning U.K.

Fit the column (3.3) in the gas chromatograph, by attaching the inlet end to the split injector 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.).

If the column is being used for the first time, it is recommended that it should be subjected to conditioning: passing a gentle flow of gas through the column itself, then switch on the gas chromatography unit and begin a gradual heating, up to a temperature of at least 20 °C above the operating temperature (Note 8). 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 computing system, adjustment of the column, detector and injector temperature, etc.) and then record the signal with a sensitivity at least two times greater than that one intended for the analysis. The course of the base line must be linear, without peaks of any kind, and must not show drift. A negative straight-line drift indicates leakage from the column connections; a positive drift indicates inadequate conditioning of the column.

Note 8: The conditioning temperature must always be at least 20 °C less than the maximum temperature specified for the stationary phase used. U.K.

6.2. Operating conditions U.K.

Optimize the temperature programme and the carrier gas flow so that chromatograms similar to Figures 3 to 6 are obtained.

The following parameters were tested and found useful:

6.2.1. Aliphatic alcohols U.K.

Oven Program	180 °C (8 min.) → 260 °C (at 5 °C/min) → 260 °C (15 min)
Injector Temperature	280 °C
Detector Temperature	290 °C
Linear Velocity of Carrier gas	Helium (20 to 30 cm/s); Hydrogen (30 to 50 cm/s)
Split Ratio	1:50 to 1:100
Volume Injected	0,5 to 1 μL of TMSE solution

6.2.2. Sterol and triterpenic dialcohols U.K.

Oven Program	260 ± 5 °C Isothermal
Injector Temperature	280 – 300 °C
Detector Temperature	280 – 300 °C
Linear Velocity of Carrier gas	Helium (20 to 30 cm/s); Hydrogen (30 to 50 cm/s)
Split Ratio	1:50 to 1:100
Volume 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:

• Alcohol C26 retention time shall be 18 ± 5 minutes.

• Alcohol C22 peak shall be 80 ± 20 % of the full-scale value for olive oil and 40 ± 20 % of the full-scale value for olive-pomace oil.

• The retention time for the β-sitosterol peak should be at 20 ± 5 min.

• The campesterol peak should be: for olive oil (mean content 3 %) 20 ± 5 % of full scale.

• All the present sterols 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 RRT 1,02 (Sitostanol) can be quantified using the perpendicular.

6.3. Analytical procedure U.K.

By using the 10 μl microsyringe (3.4), take 1 μl of hexane, draw in 0,5 μl of air and then 0,5 to 1 μl of the sample solution. Raise the plunger of the syringe further, so the needle is emptied. Push the needle through the membrane of the injector and after one to two seconds, inject rapidly, and then slowly remove the needle after around five seconds. An automatic injector can be used as well.

Carry out the recording until the TMSE of the corresponding alcoholic compounds present are completely eluted. The base line must continue to meet the requirements of the corresponding operating conditions (6.2.1 or 6.2.2).

6.4. Peak identification U.K.

Identify individual peaks on the basis of retention times and by comparison with the mixture of the aliphatic and triterpenic alcohols or the sterol and triterpene dialcohols TMSE, analysed under the same conditions. A chromatogram of the aliphatic and triterpenic alcohols fraction is showed in Figure 3 and the corresponding chromatograms for sterols and triterpenic dialcohols are showed in Figure 2.

The aliphatic alcohols are eluted in the following order: C20-ol (I.S.), C22-ol, C23-ol, C24-ol, C25-ol, C26-ol, C27-ol and C28-ol.

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, β-sistosterol, sitostanol, Δ5-avenasterol, Δ5,24-stigmastadienol, Δ7-stigmastenol, Δ7-avenasterol, erythrodiol and uvaol.

6.5. Quantitative evaluation U.K.

The peak areas of 1-eicosanol and of the aliphatic alcohols C22, C24, C26, C28 are calculated by a data acquisition system. The response factor for 1-eicosanol should be considered equal to 1.

Calculate the areas of the α-cholestanol and the sterol and triterpene dialcohols peaks by using the computing system. Ignore peaks for any compound, which are not included (ergosterol must not be calculated) among those listed in Table 1. The response factor for α-cholestanol should be considered equal to 1.

Calculate the concentration of each individual alcoholic compound, in mg/kg of fatty material, as follows:

where:

A _x

=

Peak area for alcoholic compound x, in computing system counts.

A _s

=

Area of the 1-eicosanol/α-cholestanol peak, in computing system counts.

m _s

=

Mass of added 1-eicosanol/α-cholestanol, in milligrams.

m

=

Mass of the sample used for determination, in grams.]

Textual Amendments

F1 Substituted by Commission Implementing Regulation (EU) 2019/1604 of 27 September 2019 amending Regulation (EEC) No 2568/91 on the characteristics of olive oil and olive-residue oil and on the relevant methods of analysis.