Overview
ISO 5558:1982 specifies a thin-layer chromatographic (TLC) method for the detection and identification of selected antioxidants in animal and vegetable fats and oils. The method targets nordihydroguaiaretic acid (NDGA), propylgallate, octylgallate, dodecylgallate, tert‑butylhydroquinone (TBHQ), butylhydroxyanisole (BHA), alpha‑tocopherol, and butylhydroxytoluene (BHT). It combines liquid‑liquid extraction (n‑hexane / acetonitrile) with silica‑gel TLC, chemical visualization and Rf comparison against standards.
Key topics and technical requirements
- Scope: qualitative detection and identification of eight common antioxidants in fats and oils.
- Sample extraction:
- Dissolve a 7.5–10 g test portion in n‑hexane.
- Extract antioxidants with acetonitrile (acetonitrile saturated with n‑hexane) in multiple washes.
- Back‑wash extracts with n‑hexane saturated with acetonitrile, combine, evaporate under reduced pressure (water bath ≤ 40 °C).
- Re‑dissolve residue in methanol (2 mL or 1 mL for low-level samples).
- Chromatography:
- Use 200 × 200 mm silica‑gel plates, 0.4 mm layer; activate plates (60 °C, 1 h).
- Apply sample (10 µL and 20 µL) plus standard spots; develop in solvent vapor‑saturated tank.
- Developing solvent: 2 volumes n‑hexane : 2 volumes benzene : 1 volume glacial acetic acid (benzene caution noted).
- Detection and visualization:
- Indicator: 2,6‑dichloro‑p‑benzoquinone‑4‑chloroimide in 96% ethanol.
- Spray plate, heat at 103 ± 2 °C for 10–15 min, compare Rf values with standards.
- Performance notes:
- Detection limits: ~10 mg/kg for most antioxidants; BHT ~20 mg/kg.
- BHT is not completely isolated by this method; alpha‑tocopherol spots may appear diffuse.
- Safety: benzene is toxic and flammable - follow appropriate laboratory safety and ventilation practices.
Applications and users
- Routine screening and identification of added or residual antioxidants in edible oils and animal fats.
- Useful for:
- Food quality control and assurance laboratories.
- Regulatory and compliance testing (food safety agencies).
- Oil processors and ingredient manufacturers monitoring antioxidant additives.
- Analytical chemists validating presence/identity before quantitative analysis (e.g., GC/ HPLC).
- Practical value: cost‑effective TLC screening to confirm presence/identity prior to more resource‑intensive tests.
Related standards
- ISO 5558:1982 complements other ISO methods for fats and oils and chromatographic analysis produced by ISO TC 34 (Agricultural food products). Laboratories commonly use TLC screening alongside quantitative ISO or national methods (GC, HPLC) for antioxidant determination.
Keywords: ISO 5558:1982, antioxidants detection, thin‑layer chromatography, TLC, animal and vegetable fats and oils, extraction with acetonitrile, n‑hexane, BHT, BHA, TBHQ, propylgallate, NDGA.
