Textual Amendments
This part gives general guidance for the application of capillary column gas chromatography to determine the qualitative and quantitative composition of a mixture of fatty acid methyl esters obtained in accordance with the method specified in Part A.
The part is not applicable to polymerised fatty acids.
Inert gas (helium or hydrogen), thoroughly dried and with an oxygen content of less than 10 mg/kg.
Mixture of methyl esters of pure fatty acids, or the methyl esters of a fat of known composition, preferably similar to that of the fatty matter to be analysed. Cis and trans isomers of octadecenoic, octadecadienoic and octadecatrienoic methyl esters are useful for the identification of trans isomers of unsaturated acids.
Care should be taken to prevent the oxidation of polyunsaturated fatty acids.
The instructions given are for the usual equipment used for gas chromatography, employing capillary columns and a flame-ionisation detector.
The gas chromatograph shall comprise the following elements.
Use an injection system with capillary columns, in which case the injection system should be specially designed for use with such columns. It may be of the split type or the splitless on-column injector type.
The oven shall be capable of heating the capillary column to a temperature of at least 260 °C and of maintaining the desired temperature to within 0,1 °C. The last requirement is particularly important when a fused silica tube is used.
The use of temperature-programmed heating is recommended in all cases, and in particular for fatty acids with less than 16 carbon atoms.
The coatings shall be thin, i.e. 0,1 to 0,2 μm.
Observe the normal precautions for assembling capillary columns, i.e. arrangement of the column in the oven (support), choice and assembly of joints (leak tightness), positioning of the ends of the column in the injector and the detector (reduction of dead-spaces). Place the column under a flow of carrier gas (e.g. 0,3 bar (30 kPa) for a column of length 25 m and internal diameter 0,3 mm).
Condition the column by temperature programming of the oven at 3 °C/min from ambient temperature to a temperature 10 °C below the decomposition limit of the stationary phase. Maintain the oven at this temperature for one hour until stabilisation of the baseline. Return it to 180 °C to work under isothermal conditions.
The syringe shall have a maximum capacity of 10 μl, graduated in 0,1 μl divisions.
Data acquisition system connected online with the detectors and employed with a software program suitable for peak integration and normalisation.
The operations described in 4.1 to 4.3 are for the use of a flame-ionisation detector.
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 |
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).
Identify the methyl ester peaks of the sample from the chromatogram in Appendix B, figure 1, if necessary by interpolation, or by comparison with those of the methyl esters reference mixtures (as indicated at point 2.3).
Calculate the mass fraction w i of the individual fatty acid methyl esters, expressed as a percentage by mass of methyl esters, as follows:
Calculate the content of a given component i, expressed as a percentage by mass of methyl esters, by determining the percentage represented by the area of the corresponding peak relative to the sum of the areas of all the peaks, using the following formula:
w i = (A i /ΣA) × 100
where:
A i is the area under the peak of the individual fatty acid methyl ester i ;
ΣA is the sum of the areas under all the peaks of all the individual fatty acid methyl esters.
The results are expressed to two decimal places.
In certain cases, for example in the presence of fatty acids with less than eight carbon atoms or of acids with secondary groups, the areas shall be corrected with specific correction factors (Fci). These factors shall be determined for each single instrument. For this purpose suitable reference materials with certified fatty acid composition in the corresponding range shall be used.
For this reference mixture, the mass percentage of the FAME i is given by the formula:
w i = (m i /Σ m) × 100
where
m i is the mass of the FAME i in the reference mixture;
Σm is the total of the masses of the various components as FAMEs of the reference mixture.
From the chromatogram of the reference mixture, calculate the percentage by area for the FAME i as follows:
w i = (A i /ΣA) × 100
where:
A i is the area of the FAME i in the reference mixture;
ΣA is the sum of all the areas of all the FAMEs of the reference mixture.
The correction factor F c is then
F c = (m i × ΣA)/(A i /Σm)
For the sample, the percentage by mass of each FAME i is:
w i = (F i × A i )/Σ (F i × A i )
The results are expressed to two decimal places.
In certain analyses (for example where not all of the fatty acids are quantified, such as when acids with four and six carbons are present alongside acids with 16 and 18 carbons, or when it is necessary to determine the absolute amount of a fatty acid in a sample) it is necessary to use an Internal Standard. Fatty acids with 5, 15 or 17 carbons are frequently used. The correction factor (if any) for the Internal Standard should be determined.
The percentage by mass of component i, expressed as methyl esters, is then given by the formula:
w i = (m IS × F i × A i )/(m × F IS × A IS )
where:
A i is the area the FAME i ;
A IS is the area of the internal standard;
F i is the correction factor of the fatty acid i , expressed as FAME;
F IS is the correction factor of the internal standard;
m is the mass of the test portion, in milligrams
m IS is the mass of the internal standard, in milligrams.
The results are expressed to two decimal places.
The test report shall specify the methods used for the preparation of the methyl esters and for the gas chromatographic analysis. It shall also mention all operating details not specified in this Standard Method, or regarded as optional, together with details of any incidents which may have influenced the results.
The test report shall include all the information necessary for complete identification of the sample.
Details of an interlaboratory test on the precision of the method are set out in Annex C to standard IOC/T.20/Doc. No 33. The values derived from this interlaboratory test may not be applicable to concentration ranges and matrices other than those given.
The absolute difference between two independent single test results, obtained using the same method on identical test material in the same laboratory by the same operator using the same equipment within a short interval of time, will in not more than 5 % of cases be greater than r given in tables 1 to 14 in Annex C to standard IOC/T.20/Doc. No 33.
The absolute difference between two single test results, obtained using the same method on identical test material in different laboratories with different operators using different equipment, will in not more than 5 % of cases be greater than R given in tables 1 to 14 in Annex C to standard IOC/T.20/Doc. No 33.]