⛽ Determination of free and total glycerol and mono-, di- and triglycerides in fatty acid methyl esters (FAME) — 80–125 mg of sample with four internal standards (1,2,4-butanetriol, glycerol mono-, di- and trinonadecanoate) is silylated with MSTFA in pyridine, diluted with n-heptane and analysed by GC-FID on a 15 m column programmed to 370 °C; limit of quantification 0.001 % (m/m) for free glycerol and 0.10 % (m/m) for glycerides; total glycerol by calculation, PN-EN 14105

Petrochemistry PN-EN 14105

Overview

WHAT THE STANDARD COVERS. Scope from the PKN catalogue card of PN-EN 14105:2025-04 (English version) — its first two paragraphs repeat Clause 1 of the text described below; we read the text of the standard in the sample of SIST EN 14105:2024 (the text of EN 14105:2024 without changes) from the title page through the contents, European foreword and Clauses 1–8 up to and including the beginning of 9.7 (column performance check, formula 1). Outside the sample remain Clauses 10–13 (determination of results — integration, glycerol calibration function, free glycerol, glycerides, total glycerol; expression of results; precision; test report) and Annexes A–D (example chromatograms, calculation and example of the calibration function, results of the interlaboratory test).

ACCORDING TO THE TEXT OF EN 14105:2024 (Clauses 1–9.7). The standard was prepared by CEN/TC 307 “Oilseeds, vegetable and animal fats and oils and their by-products — Methods of sampling and analysis” (secretariat AFNOR); it superseded EN 14105:2020, and the only technical change is the specification of the weighing accuracy. Scope (1): determination of free glycerol and residual mono-, di- and triglycerides in FAME, with calculation of total glycerol; under the described conditions the limit of quantification is 0.001 % (m/m) for free glycerol and 0.10 % (m/m) for all glycerides; the method is suitable for FAME from rapeseed, sunflower, soybean and palm oil, animal oils and fats and their mixtures, and not suitable for FAME from coconut and palm kernel oils (or with their addition), because the glyceride peaks overlap. Notes: “% (m/m)” means mass fraction; under the usual GC conditions of this standard squalene may co-elute with glycerol alpha-monostearate — if squalene is suspected, EN 17057 may be used. Warning: the use of the standard may involve hazardous materials and operations; safety measures are established by the user. References (2): EN ISO 5555 and EN ISO 3170 (sampling). Principle (4): glycerol and mono- and diglycerides are converted into more volatile and more stable silyl derivatives in the presence of pyridine and MSTFA (N-methyl-N-trimethylsilyltrifluoroacetamide); after silylation the sample is analysed by gas chromatography on a short thin-film capillary column, with an on-column or equivalent injector and a flame ionization detector; glycerol is determined after calibration against the internal standard 1,2,4-butanetriol, and mono-, di- and triglycerides — directly against an internal standard for each group: glycerol mononondecanoate (Mono C19), dinonadecanoate (Di C38), trinonadecanoate (Tri C57). Reagents (5): MSTFA; pyridine with at most 0.1 % water, stored over a molecular sieve (or pyridine for silylation); tetrahydrofuran; n-heptane; glycerol; 1,2,4-butanetriol; Mono C19, Di C38, Tri C57. Apparatus (6): gas chromatograph with on-column or equivalent injector, programmable oven and FID; capillary column programmable to 400 °C (“high-temperature” type), recommended: phase 100 % dimethylpolysiloxane or 5 % diphenyl-, length 15 m, diameter 0.32 mm, film 0.1 µm; class A volumetric flasks of 50, 20 and 10 ml; 10 ml vials with screw caps and PTFE-coated septa; precision pipettes or syringes of 1 ml, 100 µl and 500 µl; 10 ml cylinder; balance with a weighing accuracy of ± 1 mg or better and a reading of ± 0.1 mg or better; carrier gas — hydrogen or helium; auxiliary gases. Solutions (7): 1,2,4-butanetriol 1 mg/ml (40–60 mg in 50 ml of pyridine); glycerol 0.5 mg/ml (40–60 mg in 10 ml of pyridine, then 1 ml to 10 ml); reference glycerides 2.5 mg/ml (40–60 mg of each in one 20 ml flask of tetrahydrofuran) — the solution shall be completely clear at ambient temperature, a precipitate after storage at 4 °C shall dissolve spontaneously without heating, stability at 4 °C — almost 3 months; commercial mixture of monoglycerides (monopalmitin, monostearin, monoolein in equal masses), approx. 100 mg in 10 ml of pyridine — for locating the peaks; four calibration solutions (Table 1): 10, 40, 70 and 100 µl of glycerol solution with 80 µl of butanetriol solution. Sampling (8): according to EN ISO 5555 or EN ISO 3170. Conditions (9.1): adjusted to the column and carrier gas; recommended analysis time approx. 30–35 min so that the triglycerides elute; example (Table 2): 50 °C for 1 min, 15 °C/min to 180 °C, 7 °C/min to 230 °C, 10 °C/min to 370 °C and 15 min, detector 380 °C, hydrogen 80 kPa or constant flow approx. 2 ml/min, injection 1 µl. Calibration (9.2): to each calibration solution 150 µl of MSTFA, close tightly, shake, 15 min at room temperature, add 8 ml of n-heptane; analysis of only the first part of the programme, up to 230 °C; samples after derivatization are stable for a few hours, and silylated standard solutions — for only one day. Monoglyceride mixture (9.3): 200 µl of solution and 150 µl of MSTFA, without contact with moisture, then as in 9.2. Samples (9.4): 80–125 mg of homogenized sample to the nearest 0.1 mg into a 10 ml vial; 80 µl of butanetriol solution, 200 µl of reference glyceride solution, 200 µl of pyridine and 200 µl of MSTFA, without contact with moisture; close, shake, 15 min, 8 ml of n-heptane, injection 1 µl; warning: polyethylene Pasteur pipettes used to transfer the sample into autosampler vials may give contaminant peaks overlapping the monoglycerides — this is checked with a blank using these pipettes. Identification (9.5): by the retention times of the calibration solutions; the elution zones of methyl esters and monoglycerides overlap, therefore injection of the silylated commercial mixture is recommended for identifying the monoglycerides; example chromatograms of FAME from rapeseed and palm oil (also with ester dimers) are in Annex A. Calibration (9.6): only for glycerol the dependence of the mass ratio on the area ratio is examined and the calibration function determined; for glycerides a linear detector response in the range considered is assumed. Column check (9.7): with each analysis the relative response factor RF for Di C38 against Tri C57 is calculated — RF = (A Di C38 / M Di C38) / (A Tri C57 / M Tri C57), where A are the peak areas and M the masses of the standards in mg.

STATUS (catalogue cards as of 03.10.2026). The card of PN-EN 14105:2025-04 (English version, published 02-04-2025, 29 pages, price group N) has no “Withdrawn” header: Food, Agriculture and Forestry Sector, KT 92 Oilseeds, Vegetable and Animal Fats and their By-products, ICS 67.200.10, “Introduces: EN 14105:2024 [IDT]”, “Supersedes: PN-EN 14105:2021-05 — English version”. The card of PN-EN 14105:2021-05 (English version) — “Withdrawn” 02-04-2025, “Replaced by: PN-EN 14105:2025-04”; it itself superseded PN-EN 14105:2012 in the Polish version. Between the 2020 and 2024 editions the text of the standard lists one technical change — the specification of the weighing accuracy; for the result this means the requirement of a balance with an accuracy of ± 1 mg and a reading of ± 0.1 mg (6.11). The PKN search (query “PN-EN 14105”, 03.10.2026) also shows the editions 2004 and 2012 (Polish versions) and 2011 (English version).

HOW MANY LABORATORIES AND IN WHAT FORM (copy of the accreditation scope database, load up to 17.09.2026, read 03.10.2026). The number 14105 appears in 9 records at 6 laboratories: AB 009, AB 387, AB 391, AB 484, AB 1668, AB 1679. In the current scopes on the PCA website (read 03.10.2026, issues from 19.09.2025 to 30.07.2026) AB 391, AB 484, AB 1668 and AB 1679 give “PN-EN 14105:2025-04”, and AB 009 and AB 387 — the designation alone without year (AB 387 in a flexible scope, for “Liquid fuels E”). At AB 009 (issue no. 28 of 03.06.2026) the whole item with PN-EN 14105 is set in bold italics, and the footnote says: “Accreditation suspended at the request of the body in the part of the scope marked in bold italics from 03.06.2026 to 30.11.2026” (our translation) — on the day of reading the accreditation of this item is suspended. Without suspensions the standard is thus held by 5 laboratories. The “Laboratories” tab (“PN-EN 14105”) shows all 6, including AB 009; checked with the tab query on the production server labcoda.pl on 03.10.2026.

WHAT THE LABORATORIES TEST (PCA items). Fatty acid methyl esters (FAME) as a biocomponent and liquid biofuel, at AB 387 — liquid fuels in a flexible scope; technique: GC-FID. Ranges (% m/m): AB 391 — free glycerol 0.005–0.050, monoglycerides 0.25–1.25, di- and triglycerides 0.10–0.50 each; AB 484 (two items) — free glycerol 0.001–0.050, monoglycerides 0.05–1.25 and 0.10–1.25, diglycerides 0.05–0.50 and 0.10–0.50, triglycerides 0.05–0.50 and 0.10–0.40; AB 1668 — monoglycerides 0.10–1.00, di- and triglycerides 0.10–0.50, free glycerol 0.005–0.050; AB 1679 — monoglycerides 0.25–0.70, diglycerides 0.07–0.20, triglycerides 0.05–0.20, free glycerol 0.002–0.200; AB 009 (suspended item) — free glycerol 0.002–0.050, monoglycerides 0.25–1.25, diglycerides 0.10–0.50, triglycerides 0.10–0.40; AB 387 gives no ranges. Total glycerol “by calculation” is given by AB 009, AB 1668 and AB 1679. The lower limits for glycerides of 0.05 % (AB 484, AB 1679) and 0.07 % (AB 1679) lie below the limit of quantification of Clause 1 (0.10 % m/m); the lower limits for free glycerol (from 0.001 %) — at it or above.

WHERE A SEEMINGLY CORRECT RESULT IS EASY. At silylation: the standard requires pyridine with at most 0.1 % water and avoidance of contact with moisture (5.2, 9.3, 9.4); silylated standards are stable only one day, samples — a few hours (9.2). At identification: monoglycerides elute in the methyl ester zone, and squalene may elute with glycerol alpha-monostearate — without injecting the silylated monoglyceride mixture a peak is easily misassigned (note 2 to Clause 1, 9.5); polyethylene pipettes give peaks overlapping the monoglycerides (9.4). At the column: too short a programme does not elute the triglycerides — recommended analysis time 30–35 min (9.1); the relative response factor of Di C38 against Tri C57 (RF of 9.7) is calculated with each analysis as a check of column performance — the criterion for assessing it is in the part we did not read. At the raw material: FAME with coconut or palm kernel oil give overlapping glyceride peaks — the method is not suitable for them (1). At calculation: total glycerol is a calculated quantity — its components are free glycerol (measured against the calibration function) and three glyceride groups (measured against three internal standards, assuming linearity, 9.6); an error in any component passes into the sum.

WHAT WE DO NOT GIVE. We did not read Clauses 10–13 and the annexes, so we do not give the formulas for converting glycerides into glycerol, the integration method, the criterion for RF, precision data or test report requirements. We also do not give requirements for glycerol in biodiesel (e.g. from product standards), because we did not read their texts.

Method principle

Glycerol and mono- and diglycerides are converted with MSTFA in pyridine into volatile and stable trimethylsilyl derivatives; after dilution with n-heptane the sample is separated on a short thin-film high-temperature column programmed to 370 °C, with an on-column injector and a flame ionization detector. Glycerol is calculated from the calibration function against 1,2,4-butanetriol, and each glyceride group — directly against its internal standard (Mono C19, Di C38, Tri C57); total glycerol is calculated from the results.

Applications

• Fatty acid methyl esters (FAME) from rapeseed, sunflower, soybean and palm oil, animal oils and fats and their mixtures — free and total glycerol, mono-, di- and triglycerides (Clause 1)
• Not applicable to FAME from coconut and palm kernel oils or with their addition (Clause 1)
• Biocomponents and liquid biofuels (FAME), liquid fuels — in the accreditation scopes of 6 laboratories at PCA, at one the item is suspended until 30.11.2026

Key parameters

Standard

Standard number

PN-EN 14105:2025-04

Title (PL)

Produkty przetwarzania olejów i tłuszczów — Estry metylowe kwasów tłuszczowych (FAME) — Oznaczanie zawartości wolnego i ogólnego glicerolu oraz mono-, di- i triacylogliceroli

Title (EN)

Fat and oil derivatives — Fatty acid methyl esters (FAME) — Determination of free and total glycerol and mono-, di-, triglyceride contents

Step-by-step procedure

1. Solutions

   Butanetriol 1 mg/ml, glycerol 0.5 mg/ml (pyridine), reference glycerides 2.5 mg/ml (THF); four calibration solutions 10–100 µl of glycerol with 80 µl of butanetriol. PN-EN 14105:2025-04 current (PKN catalogue card as of 03.10.2026).

2. Glycerol calibration

   150 µl MSTFA, tightly closed, shake, 15 min, 8 ml n-heptane; analysis up to 230 °C; calibration function from the dependence of the mass ratio on the area ratio.

3. Sample

   80–125 mg of FAME; 80 µl butanetriol, 200 µl reference glycerides, 200 µl pyridine, 200 µl MSTFA, without moisture; 15 min; 8 ml n-heptane; 1 µl into the GC.

4. Identification and check

   Retention times of the standards and of the silylated monoglyceride mixture; RF of Di C38 against Tri C57 with each analysis.

5. Result

   Free glycerol from the calibration function, glycerides against the internal standards, total glycerol by calculation. Formulas (Clause 10) outside the sample read.

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• Use of the standard may involve hazardous materials and operations — safety measures are established by the user (warning in Clause 1)
• Pyridine, tetrahydrofuran and n-heptane are flammable and harmful; MSTFA reacts with moisture — work in a fume hood, in tightly closed vials
• Hydrogen as carrier gas — tightness of the installation and safeguards according to the chromatograph instructions

Frequently asked questions