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[F1ANNEX X U.K. DETERMINATION OF FATTY ACID METHYL ESTERS BY GAS CHROMATOGRAPHY

Textual Amendments

PART B U.K. ANALYSIS OF FATTY ACID METHYL ESTERS BY GAS CHROMATOGRAPHY

4. PROCEDURE U.K.

The operations described in 4.1 to 4.3 are for the use of a flame-ionisation detector.

4.1. Test conditions U.K.
4.1.1. Selection of optimum operating conditions for capillary columns U.K.

Owing to the efficiency and permeability of capillary columns, the separation of the constituents and the duration of the analysis are largely dependent on the flow-rate of the carrier gas in the column. It will therefore be necessary to optimise the operating conditions by adjusting this parameter (or simply column head loss) depending on whether the aim is to improve separation or speed up analysis.

The following conditions have proved to be suitable for the separation of FAMEs (C 4 to C 26 ). Examples of chromatograms are shown in Appendix B:

Injector temperature: 250 °C
Detector temperature: 250 °C
Oven temperature: 165 °C (8 min) to 210 °C at 2 °C/min
Carrier gas hydrogen: column head pressure, 179 kPa
Total flow: 154,0 ml/min;
Split ratio: 1:100
Injection volume: 1 μl
4.1.2. Determination of the resolution (see Appendix A) U.K.

Calculate the resolution, R, of two neighbouring peaks I and II, using the formula:

R = 2 × (( d dr(II) – d r(I) )/(ω (I) + ω (II) )) or R = 2 × (( t r(II) – t r(I) )/(ω (I) + ω (II) )) (USP) (United States Pharmacopeia),

or

R = 1,18 × (( t r(II) – t r(I) )/(ω 0,5(I) + ω 0,5(II) )) (EP, BP, JP, DAB), (JP (Japanese Pharmacopeia), EP (Pharmacopée Européenne), BP (British Pharmacopeia))

where:

  • d r(I) is the retention distance of peak I;

  • d r(II) is the retention distance of peak II;

  • t r(I) is the retention time of peak I;

  • t r(II) is the retention time of peak II;

  • ω (I) is the width of the base of peak I;

  • ω (II) is the width of the base of peak II;

  • ω 0,5 is the peak width of the specified compound, at mid-height of the peak;

If ω (I) ≈ ω (II) , calculate R using the following formulas:

R = ( d r(II) – d r(I) )/ω = ( d r(II) – d r(I) )/4σ

where:

σ is the standard deviation (see Appendix A, Figure 1).

If the distance dr between the two peaks d r(II) - d r(I) is equal to 4σ, the resolution factor R = 1.

If two peaks are not separated completely, the tangents to the inflection points of the two peaks intersect at point C. In order to completely separate the two peaks, the distance between the two peaks must be equal to:

d r(II) - d r(I) = 6 σ from where R = 1,5 (see Appendix A, Figure 3).]