🍎 Fatty acid composition of oils and fats — methyl esters (FAME), gas chromatography GC-FID

Food chemistry PN-EN ISO 12966-1

Overview

Part 1 of the standard ISO 12966 gives an overview of the chromatographic determination of fatty acids, free and bound, in animal and vegetable oils and fats after their conversion into methyl esters (FAME). The quantitative and qualitative determination of the composition of the fatty acids by the method of gas chromatography is widely used in the analysis of lipids — for characterising fats and oils and fatty foods after the extraction of the fat from the matrix. The series ISO 12966 (parts 1–4) replaced ISO 5508, ISO 5509 and ISO 15304; it concerns crude, refined, partly and fully hydrogenated fats and fatty acids, but it is not suitable for milk fat and ruminant fat, for products enriched with conjugated linoleic acid (CLA) or for polymerised and oxidised fats. The preparation of the esters is described by parts 2 (chemical methods) and 3 (trimethylsulfonium hydroxide, TMSH), and the conditions of the capillary analysis by part 4.

The blocking of the carboxyl groups in the methyl esters makes the formation of hydrogen bonds impossible, which lowers the boiling and melting points with respect to the acids and makes GC analysis possible. The standard sets out the methods of esterification: the rapid transmethylation with KOH in methanol (it does not esterify free acids; also for fats with short-chain acids, a risk of the formation of soaps), the general transmethylation/methylation with NaOCH3 (not recommended for lauric oils), the methylation with BF3 (a 14 % methanolic solution, bought in, highly toxic; for fish oils), methanolic H2SO4 or HCl (for samples with a higher content of free acids; 3 h in a sealed ampoule) and TMSH (very rapid, C4–C26, the esterification of free acids about 80 %, small amounts of trans isomers).

For the separation, WCOT capillaries of an internal diameter of 0.1–0.53 mm and a film thickness of 0.1–0.3 µm are used, of a length of 10–100 m depending on the resolution required; doubling the length of the column increases the resolution only by a factor of 1.41. For complex mixtures of geometric and positional isomers of polyunsaturated acids, columns of 100 m with a biscyanopropyl phase (SP-2560) or a highly substituted cyanopropyl phase (CP-Sil 88) are recommended. The carrier gas with FID should be hydrogen (with a mass detector — helium). In the instructions of the University of Gdańsk the FAME analysis of lard is carried out on an RTX-5 column 30 m × 0.25 mm × 0.25 µm with a temperature programme of 120–220 °C.

Method principle

The methyl esters prepared according to ISO 12966-2 or ISO 12966-3 (for example the transesterification of a solution of the fat in isooctane with a methanolic solution of KOH with a subsequent neutralisation with sodium hydrogen sulfate) dissolved in a solvent are injected into the injector, where they evaporate; the separation takes place on a capillary column of a chosen polarity and length, and the substances leave the column at characteristic retention times, compared with the retention times of a standard FAME mixture. Detection: a flame ionisation detector (FID). The percentage proportions of the fatty acids are calculated from the peak areas by the method of internal normalisation (the peak area as a percentage of the sum) or, more accurately, with an internal standard or with response correction factors. With a mass detector the differences of fragmentation of the individual FAME have to be taken into account.

Applications

• Characterisation and identification of vegetable oils and animal fats (the profile of the fatty acids, saturated acids, MUFA, PUFA)
• Determination of trans and conjugated isomers in hydrogenated and frying fats
• The declaration of the nutritional value of food after the extraction of the fat from the matrix
• Detection of the adulteration of oils (for example olive oil) and control of conformity with the specification
• Calculation of the iodine value from the composition of the fatty acids (ISO 3961, Annex B)

Key parameters

Standard

Standard number

PN-EN ISO 12966-1:2015-01

Title (PL)

Oleje i tłuszcze roślinne oraz zwierzęce — Chromatografia gazowa estrów metylowych kwasów tłuszczowych — Część 1: Przewodnik do nowoczesnej chromatografii gazowej estrów metylowych kwasów tłuszczowych

Title (EN)

Animal and vegetable fats and oils — Gas chromatography of fatty acid methyl esters — Part 1: Guidelines on modern gas chromatography of fatty acid methyl esters

Step-by-step procedure

1. Choice of the method of esterification

   Choose the method according to Table 1 of the standard: KOH/methanol (rapid, without the esterification of free acids), NaOCH3 (not for lauric oils), BF3 (fish oils), methanolic H2SO4/HCl (a high content of free acids, 3 h in an ampoule) or TMSH (very rapid, split only).

2. Preparation of the FAME (the example of the University of Gdańsk, KOH)

   Into a graduated test tube introduce 1 ml of a solution of the fat in isooctane (10 mg/ml), add 3 ml of isooctane and shake; add 400 µl of the methanolic solution of KOH and shake vigorously for about 30 s — after the turbidity the glycerol separates out and the mixture becomes clear.

   ⏱ Time: 30 s

3. Neutralisation

   Add about 1 g of sodium hydrogen sulfate, shake; after the salt has settled, collect with a pipette the upper layer with the methyl esters into a 1.5 ml vial.

4. GC conditions

   Install a column suitable for the purpose (a polar one of 100 m for isomers; in the exercises an RTX-5 of 30 m), set the injector and the detector (for example 300 °C), the temperature programme (for example 120→220 °C, 3 °C/min), the carrier gas (hydrogen with FID; at the University of Gdańsk argon 1.5 ml/min).

5. Analysis of the standard

   Inject 1 µl of the standard FAME mixture and establish the retention times of the individual esters; with 100 m polar columns take into account the order of elution from ISO 12966-4.

6. Analysis of the sample

   Inject 1 µl of the solution of the FAME of the sample under the same conditions; identify the peaks by comparing the retention times.

7. Calculations

   By the method of internal normalisation calculate the percentage proportion of every acid from the peak areas (a percentage of the sum of the areas) or apply an internal standard / correction factors; the result may be converted into mg/g of product.

8. Test report

   Give the method of esterification (the part of the standard), the column and the chromatographic conditions, the manner of calculation and the composition of the fatty acids.

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• Boron trifluoride in methanol is highly toxic — work only in closed vials and under a fume hood
• Methanolic solutions of KOH and of acids are corrosive; methanol is toxic — goggles, gloves, a fume hood
• Hydrogen as the carrier gas — check the tightness of the installation, a leak detector; isooctane is flammable
• A hot injector and detector (300 °C) — do not touch them during the work

Frequently asked questions