Overview
ISO 9648:1988 specifies a standardized spectrophotometric method for the determination of tannin content in sorghum grain. The method uses extraction with dimethylformamide (DMF), chemical complexation with ferric ammonium citrate and ammonia, and absorbance measurement at 525 nm against a tannic acid calibration curve. ISO 9648 is intended to allow reliable, comparable results across laboratories and is referenced to ISO procedures for sampling and moisture determination.
Key topics and technical requirements
- Principle: Extraction of sorghum tannins with 75% (w/v) dimethylformamide, centrifugation, reaction of supernatant with ferric ammonium citrate and ammonia, spectrometric reading at 525 nm; tannin content determined from a tannic acid calibration curve.
- Reagents:
- Tannic acid standard (calibration; origin affects curve - a reference product example is Merck No. 773).
- Dimethylformamide 75% (W/V) - WARNING: DMF is harmful by inhalation/skin contact.
- Ferric ammonium citrate solution (3.5 g/L) - iron content must be 17–20% (m/m).
- Ammonia solution (8.0 g/L NH3).
- Apparatus & sample prep:
- Crush sample to pass a 0.5 mm sieve; determine moisture per ISO 712 before expressing results on dry matter.
- Centrifuge capable of 3 000 g; centrifuge tubes (~50 mL) and 10 mm spectrometer cells.
- Typical extraction: add DMF to weighed test portion, stir ~60 min, centrifuge 10 min at 3 000 g, then process aliquots for color development.
- Calibration & measurement:
- Prepare calibration series (example: 0–0.5 mg/mL tannic acid in DMF).
- Plot absorbance vs concentration; the curve should not be forced through the origin.
- The analytical result is the difference between two absorbance measurements as specified in the method.
- Precision: Precision was evaluated by an inter‑laboratory study per ISO 5725; consult the standard for numerical repeatability and reproducibility criteria.
Applications and users
ISO 9648 is used by:
- Grain quality and feed laboratories assessing tannin levels in sorghum varieties.
- Plant breeders, cereal chemists and food scientists monitoring polyphenol levels.
- Quality control teams in milling, feed formulation and processing where tannin-related antinutritional effects are relevant.
- Research laboratories linking tannin content to metabolizable energy and animal nutrition (the method shows a negative correlation with metabolizable energy in feeding trials).
Related standards
- ISO 712 - Cereals and cereal products: determination of moisture (routine reference method).
- ISO 950 - Cereals: sampling of grain.
- ISO 5725 - Precision of measurement methods (repeatability and reproducibility).
Keywords: ISO 9648, sorghum tannin determination, tannin content sorghum, spectrophotometric method 525 nm, dimethylformamide extraction, ferric ammonium citrate, tannic acid calibration.