🍎 Unsaponifiable matter content of animal and vegetable fats and oils (not waxes) — about 5 g of sample saponified for 1 h under reflux with 50 ml of about 1 mol/l ethanolic KOH, three extractions with 50 ml of hexane each (or light petroleum 40–60 °C), washing of the extract with 10 % ethanol until no colour with phenolphthalein, evaporation, drying of the residue at 103 °C for 15 min periods until the mass difference is below 1.5 mg, blank ≤ 1.5 mg, optional correction for free fatty acids (0.28·V·c); result in % by mass as the mean of two determinations; results systematically lower than with the ISO 3596 method; PN-EN ISO 18609:2002

Food chemistry PN-EN ISO 18609

Overview

WHAT THE STANDARD COVERS. We read the text of the standard in the iTeh sample of ISO 18609:2000 (first edition of 15.08.2000, English text) — PN-EN ISO 18609:2002 adopts EN ISO 18609:2001 and ISO 18609:2000 as identical (PKN search). The sample covers the foreword, clauses 1–12 and the beginning of Annex A (participants, samples and summary of results); Tables A.1–A.4 with results of individual laboratories and statistics are not in the sample.

ACCORDING TO THE TEXT OF ISO 18609:2000. The standard was prepared by ISO/TC 34 "Agricultural food products", Subcommittee SC 11 "Animal and vegetable fats and oils"; the first edition replaces ISO 3596-2:1988 and its Amendment 1:1999 as a minor revision. Scope (1): determination of the unsaponifiable matter content of animal and vegetable fats and oils using three hexane extractions; applicable to all fats and oils but not to waxes; caution: compared with the method in ISO 3596, this method gives systematically low results. Reference (2): ISO 661 (preparation of test sample). Definition (3.1): unsaponifiable matter — all substances present in the product which, after saponification with potassium hydroxide and extraction with hexane, are not volatile under the specified conditions; note: it includes natural lipids such as sterols, higher hydrocarbons and alcohols, aliphatic and terpenic alcohols, as well as foreign organic matter extracted by the solvent and not volatile at 103 °C (e.g. mineral oils). Principle (4): the fat is saponified by boiling under reflux with ethanolic potassium hydroxide; unsaponifiable matter is extracted from the soap solution with hexane or, failing this, light petroleum; the solvent is evaporated and the residue weighed after drying. Reagents (5): n-hexane or light petroleum distilling at 40–60 °C, bromine number below 1, both free from residue; 10 % (V/V) ethanol; phenolphthalein 10 g/l in 95 % ethanol; ethanolic KOH about 1 mol/l — 60 g KOH in 50 ml water, made up to 1000 ml with 95 % ethanol, colourless or straw-yellow. Apparatus (6): 250 ml round-bottomed flasks with ground neck, reflux condenser, 250 ml separating funnels with PTFE stopcocks and stoppers, boiling water bath, oven (103 ± 2) °C or vacuum drying apparatus, e.g. rotary evaporator. Sampling (7) is not part of the method — ISO 5555 recommended; test sample according to ISO 661 (8). Test portion (9.1): about 5 g to 0.01 g. Saponification (9.2): 50 ml KOH solution and anti-bumping granules, gentle boiling for 1 h under reflux; after stopping heating, 50 ml water through the condenser. Extraction (9.3): after cooling, the solution is transferred to a separating funnel; the flask and granules are rinsed with hexane (50 ml in total) into the funnel; shaken for 1 min, periodically releasing pressure; after separation the lower layer is run off into a second funnel; an emulsion is broken with small quantities of ethanol, concentrated KOH or sodium chloride solution; the aqueous ethanolic soap solution is extracted twice more with 50 ml of hexane and the three extracts are combined. Washing (9.4): combined extracts washed three times with 25 ml of 10 % ethanol, drawing off the aqueous ethanolic phase (leaving 2 ml, rotating the funnel and drawing off the rest to the stopcock), and further until the washings no longer turn pink with a drop of phenolphthalein. Evaporation (9.5): the hexane solution is transferred quantitatively through the top of the funnel into a 250 ml flask dried at 103 °C and weighed to 0.1 mg; the solvent is evaporated on a boiling water bath. Drying (9.6.1): 15 min at 103 °C with the flask almost horizontal, cooling in a desiccator, weighing to 0.1 mg; or drying under the maximum vacuum of a water pump on a boiling water bath for about 15 min; drying is repeated in 15 min periods until the loss between weighings is less than 1.5 mg; if constant mass is not obtained after three periods, the residue is probably contaminated and the determination is repeated. Correction (9.6.2): if a correction for free fatty acids is needed, the residue is dissolved in 4 ml of hexane, 20 ml of ethanol neutralized to a faint pink with phenolphthalein is added and titrated with 0.1 mol/l ethanolic KOH to the same colour; the acids are calculated as oleic acid. Two determinations (9.7); blank without test portion — if the residue exceeds 1.5 mg, technique and reagents are checked (9.8). Result (10): content in % by mass = 100·(m1 − m2 − m3)/m0, where m0 — mass of test portion, m1 — mass of residue, m2 — mass of blank residue, m3 — mass of free fatty acids = 0.28·V·c (V — volume of KOH in ml, c — concentration in mol/l); the result is the arithmetic mean of two determinations. Test report (12): identification of the sample, sampling method (if known), method with reference to the standard, details not specified and incidents, result. Annex A: study organized by the Institut des corps gras Centre technique industriel, 14 laboratories from 6 countries (France, Germany, Hungary, Malaysia, Netherlands, United Kingdom); samples: crude sunflower oil, crude palm oil, crude tallow; two laboratories eliminated for sample A (Cochran and Dixon tests) and one for sample C (Dixon test); mean contents 0.15–0.58 % by mass; repeatability limit about 0.06 % by mass with CVr 3.6–10.5 %; reproducibility limit about 0.18 % by mass with CVR 9–36 %.

STATUS (as of 03.10.2026). The PKN search engine (query "PN-EN ISO 18609", 03.10.2026) returns one result: PN-EN ISO 18609:2002 — Polish version, "Introduces: EN ISO 18609:2001 [IDT], ISO 18609:2000 [IDT]", without a withdrawal note.

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 18609 appears in 2 records at 2 laboratories: AB 285 and AB 433. In the current PCA documents (read 03.10.2026; AB 285 issue no. 28 of 13.01.2026, AB 433 issue no. 26 of 11.06.2026) the number appears at the same 2 laboratories; in each document the number of page markers equals the number of PDF pages, without duplicates; searching all PCA documents downloaded from the BIP on 03.10.2026 found no laboratories outside the database copy. Form at both: "PN-EN ISO 18609:2002". None of these items is suspended. The "Laboratories" tab (query "PN-EN ISO 18609") shows both laboratories — checked by a tab query on the labcoda.pl production server on 03.10.2026.

WHAT THE LABORATORIES TEST (PCA items). AB 285: vegetable and animal oils and fats — unsaponifiable matter content (0.10–5.00) %, gravimetric method. AB 433: vegetable and animal oils and fats — unsaponifiable matter (1.4–31.6) g/kg, gravimetric method.

WHERE A SEEMINGLY CORRECT RESULT IS EASY TO GET. Choice of method: the standard explicitly warns that results are systematically lower than according to ISO 3596 (1) — results of the two methods are not compared without qualification. Scope: the method does not cover waxes (1). Washing: the extract is washed until no pink colour with phenolphthalein (9.4) — remaining soaps increase the residue mass. Drying: constant mass means a loss below 1.5 mg between weighings after 15 min; no constant mass after three periods means contamination and repetition (9.6.1). Blank: a residue above 1.5 mg indicates the solvent or technique (9.8) — hexane must be free from residue (5.1). Free acids: the correction m3 = 0.28·V·c is optional (9.6.2) — the report should state whether it was applied. Content of the result: unsaponifiable matter also includes foreign substances not volatile at 103 °C, e.g. mineral oils (3.1, note) — the result does not distinguish them from sterols. Units: AB 285 gives %, AB 433 — g/kg (1.4–31.6 g/kg is 0.14–3.16 %).

WHAT WE DO NOT GIVE. We did not read Tables A.1–A.4 with the results of individual laboratories and the full statistics of the interlaboratory study — the sample contains only their summary. We do not summarize ISO 3596, ISO 661 or ISO 5555 in this entry.

Method principle

The fat is saponified by boiling under reflux with ethanolic potassium hydroxide; an aqueous ethanolic soap solution is formed. Components that do not saponify (sterols, higher hydrocarbons and alcohols, and also e.g. mineral oils) are extracted three times with hexane, the extract is washed free of residual soaps, the solvent evaporated, and the residue dried at 103 °C and weighed. The blank and — optionally — free fatty acids determined by titration are subtracted from the mass.

Applications

• All animal and vegetable oils and fats, except waxes (1)
• Sterols, higher hydrocarbons and alcohols and foreign substances not volatile at 103 °C, e.g. mineral oils (3.1, note)
• Vegetable and animal oils and fats — unsaponifiable matter — 2 accreditation scope records of 2 laboratories in PCA

Key parameters

Standard

Standard number

PN-EN ISO 18609:2002

Title (PL)

Oleje i tłuszcze roślinne oraz zwierzęce — Oznaczanie substancji niezmydlających się — Metoda ekstrakcji heksanem

Title (EN)

Animal and vegetable fats and oils — Determination of unsaponifiable matter — Method using hexane extraction

Step-by-step procedure

1. Test portion and saponification

   About 5 g (± 0.01 g), 50 ml ethanolic KOH, 1 h boiling under reflux, 50 ml water (9.1, 9.2). PN-EN ISO 18609:2002 — no withdrawal note (PKN, 03.10.2026).

2. Extraction

   Three times 50 ml hexane, shaking 1 min, combining extracts (9.3).

3. Washing

   10 % ethanol in 25 ml portions until no pink colour with phenolphthalein (9.4).

4. Evaporation and drying

   Flask weighed to 0.1 mg; 103 °C in 15 min periods until loss < 1.5 mg (9.5, 9.6.1).

5. Correction and result

   Optionally free acids (0.28·V·c); % = 100·(m1 − m2 − m3)/m0; blank ≤ 1.5 mg; mean of two determinations (9.6.2–10).

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• When shaking hexane in a separating funnel, pressure is released periodically by inverting the funnel and cautiously opening the stopcock (9.3)

Frequently asked questions