Overview
EN ISO 29841:2014 (identical to ISO 29841:2009) defines a standardized laboratory procedure for determining chlorophyll degradation products-pheophytin a, pheophytin a′ and pyropheophytin a-in vegetable fats and oils. The method separates pigments from triglycerides by miniaturized silica gel column cleanup and quantifies the degradation products using reverse-phase HPLC (RP18-HPLC) with photometric detection at 410 nm or, for higher sensitivity (e.g., some seed oils), fluorescence detection (excitation 430 nm, emission 670 nm). The procedure is intended for quality control, research and regulatory testing of vegetable oils.
Key Topics and Requirements
- Scope: Applicable only to vegetable fats and oils; sampling is addressed by ISO 5555 and sample preparation by ISO 661.
- Principle: Silica gel mini-column separation → eluate concentrated → RP18-HPLC analysis; pigments monitored photometrically or by fluorescence.
- Reagents & solvents: Analytical-grade acetone, methanol, diethyl ether, petroleum ether, n‑heptane (or n‑hexane), and defined solvent mixtures for elution and mobile phase.
- Apparatus: RP18 HPLC column (250 mm × 4.0–4.6 mm, 5 µm), pump, 20 µl injector or autosampler, photometric detector (410 nm) or fluorescence detector (430/670 nm).
- Procedure highlights: Clean-up on silica cartridge, elution of pheophytin fraction with acetone, solvent evaporation at ≤20 °C, immediate HPLC injection to avoid pigment degradation.
- Quantification: Calculate pyropheophytin a content as the percentage of pyropheophytin peak area relative to the sum of pheophytin a, a′ and pyropheophytin a; results reported to one decimal place.
- Precision: Method precision established by interlaboratory studies (see Annex B); repeatability and reproducibility values are provided for olive oil matrices.
Applications and Who Uses It
- Olive oil quality control: Assessing thermal or storage-induced chlorophyll degradation (freshness and authenticity indicators).
- Edible oil producers: Routine QC during refining, storage and product release.
- Analytical and regulatory laboratories: Conformance testing, comparative studies and enforcement of quality standards.
- Research institutions: Studying pigment stability, refining effects and processing conditions on oil quality.
- Seed oil testing: Method supports higher-sensitivity detection (fluorescence) for oils like rapeseed.
Keywords: EN ISO 29841:2014, ISO 29841:2009, vegetable fats and oils, pheophytin, pyropheophytin, chlorophyll degradation, HPLC, silica gel column, fluorescence detection, oil quality control.
Related Standards
- ISO 661 - Preparation of test sample (fats and oils)
- ISO 5555 - Recommended sampling for oils
- ISO 5725-1 / ISO 5725-2 - Precision of measurement methods (interlaboratory study procedures)
For laboratories and producers, EN ISO 29841:2014 provides a validated HPLC-based protocol to quantify chlorophyll degradation markers that are important indicators of oil processing, storage and quality.