🍎 Preparation of fatty acid methyl esters (FAME) from oils and fats — four methods: rapid transmethylation with KOH in methanol (5.2), general alkaline–acidic procedure (5.3), BF3 (5.4) and acid-catalysed transmethylation (5.5), PN-EN ISO 12966-2

Food chemistry PN-EN ISO 12966-2

Overview

WHAT THE STANDARD COVERS. We have a preview of ISO 12966-2:2017 (second edition) — from the table of contents to the beginning of 5.2.5 (page 3 of 15); PN-EN ISO 12966-2:2017-05 introduces the text unchanged (IDT). Scope (Clause 1): methods for preparing fatty acid methyl esters from animal and vegetable fats and oils, fatty acids and soaps — four methylation methods: (a) a "rapid" transmethylation under alkaline conditions, (b) a "general" transmethylation/methylation under successively alkaline and acidic conditions, (c) BF3 transmethylation, (d) an alternative acid-catalysed transmethylation of glycerides. The esters are used for gas chromatography (GLC), thin-layer chromatography (TLC) and infrared spectrometry (IR). Milk and milk products (and fat derived from them) are EXCLUDED from the scope — according to the foreword, this is the only change from the 2011 edition.

WHY ESTERS AND WHICH METHOD TO CHOOSE (introduction and Clause 4). Triacylglycerols are not volatile enough and free fatty acids chromatograph poorly, so methyl esters are formed before GC; incomplete conversion, changes in the structure of the acids (positional and geometric isomers) and non-FAME artefacts change the composition result. Transmethylation is reversible — it requires a large excess of methanol, and water hinders its completion; alkaline procedures do not form FAME from free acids (soaps are formed). The rapid method (5.2) is suitable for routine analysis of edible oils and fats with acids from butyric acid (C4:0) upwards and for determining butyric and caproic acid with an internal standard, but it does not esterify free acids; the standard suggests it only at a free fatty acid content ≤ 0.5 % (m/m), and the general method (5.3) above 0.5 %; partially hydrolysed fat can be converted into FAME by procedure 5.5. The general method works for all oils and fats, including distillates and acid oils, but is not recommended for lauric oils — short-chain esters are lost during heating under reflux. BF3 — because of its toxicity only "as a last resort", not for milk fats; high BF3 concentrations (50 %) produce methoxy artefacts from unsaturated acids, the reagent has a limited shelf life (store in a refrigerator), and each new batch is checked before use. Keto, epoxy, hydroxy and hydroperoxy groups, cyclopropyl and cyclopropenyl rings and acetylenic acids may decompose during methylation. With BF3, isooctane gives the best recovery of the esters, but only about 75 % of methyl caproate is recovered. Short-chain esters are lost through volatility (heating under reflux), solubility in water (aqueous extraction) and during evaporation of the solvent; Annex A gives a simple TLC test of the completeness of derivatization.

THE RAPID METHOD — WHAT IS IN THE PREVIEW (5.2). Principle: the esters are formed by transmethylation with a methanolic potassium hydroxide solution; free acids are not esterified. Reagents: methanol with not more than 0.5 % water, water of grade 3 according to ISO 3696, anhydrous sodium hydrogen sulfate, isooctane (2,2,4-trimethylpentane) of chromatographic purity, methanolic KOH about 2 mol/l (13.1 g of KOH with a content of 85 g/100 g per 100 ml of anhydrous methanol, dissolved with slight heating), internal standard — valeric acid methyl ester (methyl pentanoate, 250 mg in a 50 ml flask of isooctane; reference solution 10 ml in 100 ml) — only for determining butyric and caproic acid, and NaCl solution (40 g in 100 ml of water). Each new batch of reagent is checked by preparing the ester of pure oleic acid — additional peaks disqualify the reagent. Apparatus: 10 ml tubes with a screw cap with a PTFE seal, pipettes according to ISO 8655-2, 3 ml vials, class A volumetric flasks according to ISO 1042. Note to 5.2.1: a similar procedure is applied according to COI/T.20/Doc. No. 24:2001 to olive oil (virgin with an acidity below 3.3 %, refined, blended) and to olive-pomace oil. The procedure starts with a test portion of about 0.1 g in a 10 ml tube — then the preview breaks off.

STATUS (state of the cards on 29 September 2026). The card of PN-EN ISO 12966-2:2017-05 (Polish version) has no "Withdrawn" header — the standard is CURRENT: publication date of the Polish version 14-11-2022, 24 pages, price group M, Food, Agriculture and Forestry Sector, KT 92 for Oilseeds, Vegetable and Animal Fats and their By-products, ICS 67.200.10, "Introduces: EN ISO 12966-2:2017 [IDT], ISO 12966-2:2017 [IDT]", "Replaces: PN-EN ISO 12966-2:2011 - English version". The card of the English version of the same edition: published 17-05-2017, 30 pages, price group P, the same "Scope" field. The card of PN-EN ISO 12966-2:2011 (English version): "Withdrawn and replaced by PN-EN ISO 12966-2:2017-05", published 31-03-2011, "Withdrawal date: 17-05-2017", 17 pages, "Replaces: PN-EN ISO 5509:2001 - Polish version". ISO 12966-2:2017 was reviewed and confirmed in 2022 (page of the standard at ISO).

HOW MANY LABORATORIES AND IN WHAT FORM (copy of the accreditation-scope database, data loaded up to 17 September 2026, read on 29 September 2026). All notations of number 12966-2: 19 entries at 11 laboratories. "PN-EN ISO 12966-2:2017-05" — 10 laboratories, "PN-EN ISO 12966-2" without a year — AB 444. Many laboratories narrow the standard to one or two methods (the notes stand next to the standard number both in the PCA documents and in the service's database): only the rapid method 5.2 — AB 539 ("excluding points 5.3; 5.4; 5.5") and AB 1179 ("point 5.2"); only the general method 5.3 — AB 819 ("point 5.3"); 5.2 and 5.3 — AB 565 ("p. 5.2, p. 5.3") and AB 1679 ("excluding points 5.4 and 5.5"); only BF3 (5.4) — AB 562 ("excluding points 5.2, 5.3 and 5.5", three entries); the whole standard — AB 343, AB 444, AB 515, AB 552 and AB 775. The "Laboratories" tab searches for notations BEGINNING WITH "PN-EN ISO 12966-2" and will show all 11 laboratories — checked with the tab query on the labcoda.pl production server on 29 September 2026: 11. Current scopes on the PCA website (read on 29 September 2026, issues from 03.11.2025 to 18.09.2026): all entries stand in the valid issues, every pair of range numbers from the copy occurs in the current document and no entry is suspended (the suspension footnotes in the scope of AB 1179 concern other entries).

SUBJECT AND QUANTITIES. Erucic acid — AB 343 (vegetable oils and fats, infant formulae, mustard), AB 539 (oils), AB 552 (oils, foods for particular nutritional uses, mustard), AB 562 (oils and fats, infant formulae), AB 565 (edible fats) and AB 775 (rapeseed) — ranges from (0.1 – 1.5) % (AB 343, infant formulae) to (10.0 – 55.0) % of fat (AB 552, mustard); AB 565 gives its range only in g/kg — (1.0 – 50.0) g/kg. Fatty acid composition (from C4:0 or C6:0 upwards) — AB 444, AB 515, AB 819, AB 1179 and AB 1679 (also liquid biofuels: vegetable oil), AB 562 — omega-3 acids and linoleic acid. Technique everywhere: gas chromatography with flame ionisation detection (GC-FID). The AB 562 entry with omega-3 acids (BF3 method) also includes milk and milk products in its subject — the 2017 edition excludes them from the scope of the standard, and the introduction excludes milk fats from the BF3 method.

ALONGSIDE THIS STANDARD. In the same entries: PN-EN ISO 12966-4:2015-07 (determination by capillary gas chromatography) — 9 laboratories (AB 343, AB 444, AB 515, AB 552, AB 562, AB 775, AB 819, AB 1179, AB 1679) and PN-EN ISO 12966-1:2015-01 (with or without corrigendum AC:2015-06) — 8 (AB 515, AB 539, AB 552, AB 562, AB 775, AB 819, AB 1179, AB 1679); also PN-ISO 1444:2000 — AB 343 (for oils, infant formulae and mustard), procedures IB-05 issue 6 of 12.05.2021 and IB-17 issue 4 of 10.03.2021 — AB 1179. PN-EN ISO 12966-1 has a separate entry in the Knowledge Base. The card of PN-EN ISO 12966-4:2015-07 (Polish version, state on 29 September 2026): "Withdrawn and replaced by PN-EN ISO 12966-4:2026-08 - English version", publication of the Polish version 03-10-2022, "Withdrawal date: 31-08-2026", 28 pages, KT 92, in the "Additional information" field: "Current in conformity assessment: 30-09-2026 - See the Communication of the President of PKN"; the card of PN-EN ISO 12966-4:2026-08 (English version): published 31-08-2026, 52 pages, "Introduces: EN ISO 12966-4:2026 [IDT], ISO 12966-4:2026 [IDT]", "Replaces" both versions of the 2015-07 edition. Eight of these laboratories have the 2015-07 edition in their scope, AB 444 — a notation without a year. The "Scope" field of the 2015-07 card: a method for determining FAME obtained by transesterification or esterification of fats, oils and fatty acids by capillary GLC, separation of the methyl esters from C8 to C24 — of saturated and of cis- and trans-monounsaturated and polyunsaturated fatty acids; not for fat from ruminant milk or products enriched with conjugated linoleic acid (CLA), milk and milk products outside the scope, not for di-, tri-, polymerised and oxidised fatty acids. The "Scope" field of the 2026-08 card: separation of the esters from C4 to C24 (likewise: saturated and cis- and trans-monounsaturated and polyunsaturated fatty acids), also fats extracted from food products; not for milk and milk products or products enriched with CLA, not for di-, tri-, polymerised, hydroxylated and oxidised fatty acids; Annex E — FAME composition as area % in liquid vegetable oils. Butyric acid (C4:0) is determined in these entries by AB 444, AB 819 and AB 1179, caproic acid (C6:0) as the first acid of the list by AB 515 — all with Part 4 in the 2015-07 edition (AB 444 without a year); in Part 2 short chains are covered by the rapid method with an internal standard (5.2); from Part 2, AB 1179 cites only 5.2, AB 819 only the general method 5.3, and AB 444 and AB 515 the whole standard. The card of PN-ISO 1444:2000 (Polish version): no "Withdrawn" header, "Meat and meat products — Determination of free fat content", extraction of the dried residue with n-hexane or light petroleum, published 14-06-2000, 7 pages, KT 93 for Meat, Eggs and their Products, introduces ISO 1444:1996 [IDT]. Erucic acid without this standard is determined by 8 laboratories, exclusively according to in-house procedures (AB 311, AB 313, AB 377, AB 381, AB 438, AB 509, AB 591, AB 1275); all these rows stand in the current PCA scopes without suspension (read on 29 September 2026).

WHERE A SEEMINGLY CORRECT RESULT IS EASY TO GET. The rapid method does not esterify free fatty acids: the composition result then describes the acids bound in glycerides, and the free acids in the solution may give additional peaks and hinder identification of the esters (Clause 4). The standard allows the assumption that free acids have the same distribution as the triglycerides, but usually only in crude oils — in refined and fractionated oils it may be different. Second, short chains (C4–C6) are lost on heating, in water and on evaporation of the solvent — and with BF3 only about 75 % of methyl caproate is recovered. Third, BF3 from an aged batch or at a high concentration produces artefacts — additional peaks may be taken for fatty acids.

WHAT WE DO NOT GIVE. The preview ends at the beginning of the procedure of the rapid method (5.2.5), so we do not give the further steps of the rapid method, the reagents and procedures of the general (5.3), BF3 (5.4) or acid-catalysed (5.5) methods, or the TLC test of Annex A. This part does not give the chromatographic separation conditions — they are in PN-EN ISO 12966-4.

Method principle

The fatty acids bound in acylglycerols are converted into methyl esters by transmethylation in methanol with an alkaline catalyst (KOH — the rapid method, without esterification of free acids), in two steps, alkaline and acidic (the general method, also for free acids), with BF3 (methylation of free acids and transmethylation of esters) or with an acid catalyst (partially hydrolysed fats). The esters are extracted with isooctane and subjected to gas chromatography according to another part of the standard. The choice of method depends on the free acid content (≤ 0.5 % — rapid, > 0.5 % — general), the presence of short chains and of labile groups; BF3 — only as a last resort.

Applications

• Erucic acid in oils, fats, rapeseed, mustard and infant formulae — 6 laboratories
• Fatty acid composition of foods, oils and biofuels from vegetable oil (from C4:0) — 6 laboratories
• Preparation of FAME for GC, TLC and IR; without milk and milk products (excluded from the scope of the 2017 edition)

Key parameters

Standard

Standard number

PN-EN ISO 12966-2:2017-05 (wersja polska z 14-11-2022 i wersja angielska; EN ISO 12966-2:2017, ISO 12966-2:2017 IDT)

Title (PL)

Oleje i tłuszcze roślinne oraz zwierzęce — Chromatografia gazowa estrów metylowych kwasów tłuszczowych — Część 2: Przygotowanie estrów metylowych kwasów tłuszczowych

Title (EN)

Animal and vegetable fats and oils — Gas chromatography of fatty acid methyl esters — Part 2: Preparation of methyl esters of fatty acids

Step-by-step procedure

1. Choice of method

   Free fatty acid content ≤ 0.5 % — rapid method (5.2); > 0.5 % — general (5.3); partially hydrolysed fat — 5.5; BF3 (5.4) only as a last resort; milk and milk products outside the scope.

2. Checking the reagents

   Each new batch of reagent or solvent is checked on the ester of pure oleic acid — unexpected peaks disqualify the reagent.

3. Laboratory sample

   According to ISO 661; solid fat is heated to just above its melting point (5.1).

4. Rapid method — the beginning

   About 0.1 g of sample in a 10 ml screw-cap tube; for determination with the internal standard — an exact test portion; the further steps are outside the preview — we DO NOT GIVE them.

5. Chromatography

   Esters in isooctane to GC according to another part of the standard (PN-EN ISO 12966-4); we DO NOT GIVE that part.

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• The methanolic potassium hydroxide solution is toxic and corrosive — protect the eyes and protect against chemical burns (warning in Clause 5)
• Isooctane is flammable and toxic if swallowed and inhaled, explosive limits 1.1–6.0 % by volume; BF3 — toxic, only as a last resort; work in an efficient fume hood

Frequently asked questions