GB/T 22507-2008Animal and vegetable fats and oils -- Determination of the content of trans fatty acid isomers of vegetable fats and oils -- Gas chromatographic method (English PDF)

5.1 Carrier gas

preferentially helium or hydrogen, or otherwise nitrogen, of gas chromatographic quality, dried and with oxygen removed by suitable filters.

WARNING - Hydrogen, which is used only with capillary columns, can double the speed of the analysis (in comparison with helium) but is hazardous. Safety devices are available and it is essential that a suitable device be incorporated into the apparatus.

10.1 General

In conjunction with the analysis of the test sample (or a series of test samples), analyze a blank sample (n-heptane) and a reference sample of CRM (5.2).

10.2 GC conditions

10.2.1 Set up the gas chromatograph with one of the recommended

combinations of temperature and column as described in Table 1.

Measure the average carrier gas linear velocity as indicated in Table 1. with a split ratio of approximately 1:100.

See Annex B for typical chromatograms obtained with the conditions given in

Table 1.

The two C18:1 cis isomers should be clearly separated (see Annex B).

NOTE 2: Hydrogenated marine oils can give rise to a much wider range of trans isomers, making identification and quantification more difficult.

The C20:1 cis natural isomer should be positioned exactly amidst the last eluting trans isomer C18:3 (tcc) and the C18:3 ccc peak (linolenic acid) in (high temperature) refined oils.

If the separation is sufficient for this type of analysis, in (high temperature) refined oils there should be a small peak for the C18:1 trans isomer, two approximately equally sized C18:2 trans isomers, and four (sometimes five)

C18:3 trans isomers.

For partially hydrogenated oils and fats, the separation of the C18:1 13trans and the C18:1 9cis isomers should be visible on the chromatogram. This is required for an accurate peak-split between cis and trans isomers (see Annex

B).

The 18:1 13trans isomer always elutes with the 18:1 14trans isomer. Therefore, the peak for these two isomers should be identified as 18:1 (13+14) trans.

10.4 Peak identification

For (high temperature) refined oils and fats, the trans isomers are limited in number, as only geometrical isomers with the double bond(s) at the same natural position are formed. These specific isomers are for the C18 type of fatty acids: C18:1 9trans, C18:2 9c12t and C18:2 9t12c and for C18:3 the tct, cct, ctc and tcc 9.12.15 isomers (in some samples the C18:2 9t12t and C18:3 ttc isomers are found as well in very small amounts).

For partially hydrogenated oils and fats, the trans double-bond-containing isomers are identified using the equivalent chain length (ECL). For accurate peak identification with this system, the ECL values have to be determined after suitable calibration with available cis and trans fatty acid isomer standards

1 (see also Annex C).

The first sample in an analysis batch is always a blank (n-heptane). No peaks should be detected in the blank run. Repeat this test after every ten samples.

Per analysis batch (i.e. methylation performed in one batch) at least one reference sample (5.2) is analyzed to check the performance of the methylation and GC analysis. The methylated fatty acids of the reference material are injected after each set of ten samples.

Fatty acid isomer standards are available from many chemicals suppliers (e.g. Nu-Check Prep Inc., US; Sigma,

US; Larodan, Sweden).

relative to this value. For example, the corrected response factor for C10:0 becomes 1.10. See Annex D for a list of FID factors.

11.4 Calculation of the relative mass fraction

Calculate the relative mass fraction of each component by the equation (3):

Where, wx is the relative mass fraction of component x, in percent by peak area;

Ax is the area of the peak corresponding to component x, in area units;

At is the sum of the corrected areas of all peaks, excluding the solvent peak, in area units;

f x is the correction factor for component x.

11.5 Calculation of the content of trans fatty acid isomers

11.5.1 (High temperature) refined oils and fats

Calculate the trans fatty acid isomers content of (high temperature) refined oils and fats as the sum of the relative mass fractions of the C18:1 trans, C18:2 trans and C18:3 trans fatty acid methyl esters, relative to all fatty acid methyl esters. The maximum possible peaks which may be formed are: C18:1 trans (1 peak), C 18:2 trans (2 peaks) and C18:3 trans (4 peaks). See also Figures A.4 and A.5.

Report the result to the nearest 0.01 % (mass fraction).

11.5.2 Partially hydrogenated oils and fats

Calculate the content of trans fatty acid isomers of partially hydrogenated oils and fats as the sum of the relative mass fractions of all trans double-bond- containing fatty acid methyl esters, relative to all fatty acid methyl esters.

Report the result to the nearest 0.1 % (mass fraction).

12.1 Interlaboratory test

Details of an interlaboratory test on the precision of the method are summarized

Annex E (Informative)

Results of interlaboratory test

The precision of the method was established in 1995 by an interlaboratory test carried out in accordance with ISO 5725-1

[7] , ISO 5725-2

[8] and MEN 6303

[9]

In this test 60 laboratories participated. The results of 38 laboratories were accepted. Six samples have been investigated (blind) in duplicates of sunflower seed oil, soyabean oil and rapeseed oil (see Table E.1). A report of the test has been published (see reference

[10]

).

NOTE: Some partially hydrogenated oils may have trans fatty acid levels in excess of the range obtained from the interlaboratory trial.

Table E.1 Results of interlaboratory test

Parameter

Sample

Sunflower seed oil

Soyabean oil

Rapeseed oil

Number of laboratories retained after eliminating 38 38 37

Number of observations retained after eliminating 144 147 143

Mean trans fatty acid isomers content, % (by mass) 0.34 0.78 1.09

Repeatability standard deviation (sr), % (by mass) 0.029 0.045 0.047

Repeatability relative standard deviation, % 8.64 5.85 4.34

Repeatability limit (r) r = 2.8 sr, % (by mass) 0.08 0.13 0.13

Reproducibility standard deviation (sR), % (by mass) 0.077 0.109 0.143

Reproducibility relative standard deviation, % 22.64 14.07 13.14

Reproducibility limit (R) R = 2.8 sR, % (by mass) 0.21 0.31 0.40

In 1999. Unilever Research Vlaardingen in cooperation with the Institute for

Interlaboratory Studies organized an international interlaboratory test for the determination of trans fatty acid isomers in various types of edible oils. In this interlaboratory study 62 laboratories in 29 different countries participated; 57 laboratories sent in results. In accordance with ISO 5725-2 the uniform level method was used to calculate the limits of repeatability and reproducibility. Six samples were investigated in duplicates of sunflower seed oil, rapeseed oil and olive oil (see Tables E.2 to E.4).