Quantitative estimation of iron (Wong's thiocyanate method)
Iron in the ash solution is kept ferric by potassium persulphate and forms red ferric thiocyanate; the colour is read at about 480 nm against an iron standard.
Aim and principle
Aim: To estimate the iron content of a food (bajra) from its ash solution by Wong's thiocyanate method. Principle: Ashing releases iron from the food, and dilute acid dissolves it. Potassium persulphate oxidises any ferrous iron (Fe²⁺) to ferric iron (Fe³⁺) and keeps it ferric. In acid solution Fe³⁺ combines with thiocyanate from potassium thiocyanate to form the blood-red ferric thiocyanate complex; the acid also stops Fe³⁺ from forming insoluble ferric hydroxide. The depth of the red colour is proportional to the iron present (Beer-Lambert law) and is read in a colorimeter at about 480 nm (blue-green filter) against standards made from ferrous ammonium sulphate. Alternative: in the α,α′-dipyridyl or o-phenanthroline method, iron is reduced to Fe²⁺ with hydroxylamine and forms a red complex read at about 510-520 nm; this colour is more stable than the thiocyanate colour.
Requirements
| Requirement | What it does |
|---|---|
| Ash solution of the food (10 g in 100 mL) | Source of iron |
| Dilute sulphuric acid | Keeps the medium acid, so Fe³⁺ stays in solution and the colour forms |
| Potassium persulphate (saturated solution) | Oxidises Fe²⁺ to Fe³⁺ and steadies the red colour |
| Potassium thiocyanate (concentrated, e.g. 3 N) | Forms red ferric thiocyanate with Fe³⁺ |
| Standard iron: ferrous ammonium sulphate, stock 100 µg Fe/mL (0.702 g per litre with a little sulphuric acid); working 10 µg Fe/mL | Makes the standard curve |
| Iron-free distilled water | Avoids iron contamination, since the method measures micrograms |
| Apparatus | Colorimeter (blue-green filter, about 480 nm), cuvettes, acid-washed test tubes and pipettes, volumetric flasks |
Procedure
Typical volumes; every tube ends at 10 mL. Follow the exact volumes in your lab manual.
- 1Set up tubes: blank (water), standards with 1, 2, 3, 4 and 5 mL working standard (10-50 µg Fe), and test with 5 mL ash solution, in duplicate.
- 2Make every tube up to 6.5 mL with iron-free water.
- 3Add 1 mL dilute sulphuric acid and 1 mL saturated potassium persulphate; mix.
- 4Add 1.5 mL potassium thiocyanate and mix (volume now 10 mL).the red colour appears at once
- 5Set zero with the blank and read absorbance at about 480 nm within 10-15 minutes.the colour fades on standing
- 6Plot absorbance against µg iron and read the test value from the line.
Observation: standard curve readings (example)
| Tube | Working standard (mL) | Iron (µg) | Absorbance at 480 nm |
|---|---|---|---|
| Blank | 0 | 0 | 0.000 |
| S1 | 1.0 | 10 | 0.110 |
| S2 | 2.0 | 20 | 0.220 |
| S3 | 3.0 | 30 | 0.330 |
| S4 | 4.0 | 40 | 0.440 |
| S5 | 5.0 | 50 | 0.550 |
| Test (5 mL ash solution) | - | read from graph | 0.385 |
Worked example: iron in bajra
10 g bajra was ashed and made up to 100 mL. 5 mL of the ash solution, taken as the test, gave absorbance 0.385; the 40 µg iron standard gave 0.440 (curve as in the table). Find iron in mg per 100 g.
- Slope of the standard curve = 0.440 ÷ 40 = 0.011 absorbance per µg iron.
- Iron in the test tube = 0.385 ÷ 0.011 = 35 µg. (By proportion: 0.385 ÷ 0.440 × 40 = 35 µg.)
- This is in 5 mL of ash solution, so 100 mL holds 35 × 100 ÷ 5 = 700 µg = 0.70 mg, from 10 g bajra.
- Per 100 g = 0.70 × 100 ÷ 10 = 7.0 mg.
- In one line: Fe (mg/100 g) = µg from graph × (100 ÷ 5) × (100 ÷ 10) ÷ 1000 = 35 × 20 × 10 ÷ 1000 = 7.0.
Bajra contains about 7.0 mg iron per 100 g.
Precautions and result
- Wear gloves and goggles; handle concentrated acid in a fume hood. Never pipette acids or thiocyanate by mouth.
- Use acid-washed glassware and iron-free distilled water; tap water, rust or dust can add iron.
- Add persulphate before thiocyanate, so all the iron is ferric; Fe²⁺ gives no red colour.
- Read every tube within about 15 minutes of adding thiocyanate, as the red colour fades.
- Keep the test reading inside the standard range; if it is above the top standard, take less ash solution.
- Result: The iron content of the given food (bajra) is ___ mg per 100 g.
Iron-deficiency anaemia is the most common nutritional deficiency in India, especially in adolescent girls, women and young children. Food iron values from estimations like this fill food composition tables used to plan iron-rich diets, fortified foods and programmes such as Anaemia Mukt Bharat. Ashing gives total iron; how much is absorbed depends on its form (haem or non-haem), on enhancers like vitamin C, and on inhibitors like phytate, tannins in tea, and calcium.
Short answer: “What is the role of potassium persulphate in the thiocyanate method for iron?” It oxidises ferrous iron to ferric iron and keeps it ferric, because only Fe³⁺ forms the red complex with thiocyanate. It also steadies the colour.
Short answer: “Why must glassware be acid-washed for iron estimation?” Iron is measured in micrograms, so traces of iron on glass, in tap water or from rust would give falsely high values.
Structured question: “Describe the colorimetric estimation of iron in a food by the thiocyanate method.” Principle: persulphate keeps iron ferric; Fe³⁺ + thiocyanate in acid gives red ferric thiocyanate; Beer-Lambert law. Reagents, standard curve with ferrous ammonium sulphate (10-50 µg), procedure, reading at about 480 nm within 15 minutes, calculation per 100 g (e.g. 35 µg × 20 × 10 ÷ 1000 = 7.0 mg) and precautions.
"Persulphate prepares, thiocyanate tints": persulphate makes the iron ferric, thiocyanate turns it blood red.