ISO 18395:2005

Overview

ISO 18395:2005 specifies a validated laboratory method for the determination of monoacylglycerols, diacylglycerols, triacylglycerols and free glycerol in animal and vegetable fats and oils using high-performance size-exclusion chromatography (HPSEC/GPC). The method is intended primarily for products (e.g. emulsifiers) where mono- and diacylglycerols are major constituents (>10 %) and triacylglycerols are <20 %. It is not applicable to dairy fats or oils with a wide range of fatty acid chain lengths and has restricted applicability to C8/C10 (caprylic/capric) based mixtures (only monoacylglycerol and glycerol determinable).

Key topics and technical requirements

• Principle: dissolve sample in tetrahydrofuran (THF) and separate acylglycerols by molecular size using gel permeation chromatography (GPC) with THF mobile phase and a refractive index (RI) detector.
• Reagents: THF stabilized with BHT; standards for glycerol and representative mono-, di- and triacylglycerols with fatty-acid distributions similar to the sample.
• Apparatus essentials:
  • GPC column combination with effective molecular-mass range up to 4 000 Da (examples: Plgel columns, 300 mm × 7.5 mm, 5 µm, 100 Å).
  • Column oven (35 °C or 40 °C), RI detector at matching temperature, HPLC pump, 20 µl injection loop.
  • Typical flow rate: 0.8–1.0 ml/min.
• Sample preparation and calibration:
  • Test portion prepared per ISO 661 and dissolved in THF (ultrasonic bath).
  • Use at least two calibration solutions bracketing expected concentrations; in many cases response factors are ≈1 so results by area% are acceptable.
  • Determine glycerol response factor when glycerol >3 %.
• Calculation and reporting: contents expressed as mass fraction (%) to one decimal place; baseline integration is horizontal. Notes on possible interference: free fatty acids often co-elute with monoacylglycerols - subtract calculated fatty-acid contribution if acid value >1.

Applications and users

ISO 18395:2005 is used by:

• Analytical and quality-control laboratories in the fats, oils and emulsifier industries
• Food ingredient manufacturers and formulators assessing acylglycerol composition
• Regulatory and specification testing bodies verifying product labels and purity
• Research labs studying acylglycerol-based formulations

Practical uses include product specification, batch release testing, formulation control and troubleshooting of emulsifier quality.

Related standards

• ISO 661 - Preparation of test sample (referenced for sample prep)
• ISO 5555 - Recommended sampling methods (sampling guidance)
• ISO 5725-2 - Precision (used for interlaboratory precision evaluation reported in Annex B)

Keywords: ISO 18395:2005, HPSEC, GPC, monoacylglycerols, diacylglycerols, triacylglycerols, glycerol, fats and oils, emulsifiers, refractive index detector.