SOP: Determination of Iron
GIFSQ — Global Institute for Food Safety & Quality
Principle
Iron in the food is converted to the ferric form with oxidizing agents such as potassium persulphate or hydrogen peroxide, and treated with potassium thiocyanate to form the red ferric thiocyanate complex, which is measured colorimetrically at 480 nm.
Apparatus required
- Stoppered measuring cylinders
- Colorimeter or spectrophotometer (480 nm)
Reagents
a. Concentrated H₂SO₄ (iron-free)
b. Saturated potassium persulphate (K₂S₂O₈) solution: shake 7 to 8 g of reagent-grade iron-free potassium persulphate with 100 ml of water in a glass-stoppered bottle; the undissolved excess settles and compensates for loss by decomposition; shake briefly before use; keep in the refrigerator
c. 3 N potassium thiocyanate (KSCN) solution: dissolve 146 g of reagent-grade potassium thiocyanate in water and dilute to 500 ml; filter if turbid; add 20 ml of pure acetone to improve the keeping quality
d. Standard iron solution: dissolve 0.702 g of reagent-grade crystalline ferrous ammonium sulphate [FeSO₄·(NH₄)₂SO₄·6H₂O] in 100 ml of water; add 5 ml of concentrated H₂SO₄, warm slightly and add concentrated potassium permanganate solution drop by drop until one drop produces a permanent colour; transfer to a 1-litre volumetric flask, rinse with water and make up to volume; this solution contains 0.1 mg of ferric iron per ml and is stable indefinitely
Sample preparation
Destroy the organic matter of the sample by dry ashing and prepare the ash solution as described in SOP: Destruction of Organic Matter; use the ash solution for colour development.
Procedure — colour development
Into three separate stoppered measuring cylinders, pipette the solutions as given below:
| Solution | Blank (ml) | Standard (ml) | Sample (ml) |
|---|---|---|---|
| Standard iron solution (1 ml = 0.1 mg of Fe) | 0.0 | 1.0 | 0.0 |
| Sample ash solution | 0.0 | 0.0 | 5.0 |
| Water | 5.0 | 4.0 | 0.0 |
| Conc. H₂SO₄ | 0.5 | 0.5 | 0.5 |
| Potassium persulphate | 1.0 | 1.0 | 1.0 |
| Potassium thiocyanate | 2.0 | 2.0 | 2.0 |
In each case, make up the volume to 15 ml with water
↓
Measure the colour at 480 nm, setting the blank at 100% transmission
↓
To construct a calibration curve if desired: pipette 0.0, 0.5, 1.0, 1.5, 2.0 and 2.5 ml of standard iron solution into stoppered measuring cylinders; add 0.5 ml of concentrated H₂SO₄, 1.0 ml of potassium persulphate and 2.0 ml of potassium thiocyanate and make up to 15.0 ml; measure the colour as before; plot absorbance against concentration and read the iron in the sample aliquot directly from the curve.
Calculation
Iron, mg/100 g = (OD of sample ÷ OD of standard) × 0.1 × (Total volume of ash solution ÷ ml of ash solution taken) × 100 ÷ Wt of sample taken for ashing
Where,
- OD of sample and OD of standard = optical densities measured at 480 nm
- 0.1 = mg of iron in the 1.0 ml of standard iron solution used
- ml of ash solution taken = 5.0 ml (the aliquot used for colour development)
- Total volume of ash solution = volume to which the ash solution was made up (ml)
- Wt of sample taken for ashing = weight of sample ashed (g)
References
- Wong, S.Y., J. Biol. Chem., 77, 409 (1928).