Quantitative estimation of phosphorus (Fiske-Subbarow method)
Phosphate forms phosphomolybdic acid with acid molybdate; ANSA reduces it to molybdenum blue, read at about 660 nm against a phosphate standard.
Aim and principle
Aim: To estimate the phosphorus content of a food (ragi) from its ash solution by the Fiske-Subbarow method. Principle: Ashing turns organic phosphorus (phytate, phospholipids, phosphoproteins) into inorganic phosphate, so the method gives total phosphorus. In strong acid, phosphate combines with ammonium molybdate to form phosphomolybdic acid. The reducing agent ANSA (1-amino-2-naphthol-4-sulphonic acid, dissolved in sodium bisulphite and sodium sulphite) reduces part of the molybdenum in this complex, giving an intense blue called molybdenum blue. The high acidity keeps free molybdate from being reduced, so the blank stays almost colourless. The depth of blue is proportional to the phosphate (Beer-Lambert law) and is read in a colorimeter with a red filter (about 660 nm) against standards made from potassium dihydrogen phosphate.
Requirements
| Requirement | What it does |
|---|---|
| Ash solution of the food (10 g in 100 mL), diluted 1 in 10 | Source of phosphate, brought into the range of the standards |
| Ammonium molybdate (about 2.5%) in sulphuric acid | Forms phosphomolybdic acid with phosphate; the acid keeps free molybdate from turning blue |
| ANSA reagent (1-amino-2-naphthol-4-sulphonic acid in sodium bisulphite and sodium sulphite) | Reducing agent that turns phosphomolybdic acid into molybdenum blue; keep in a brown bottle |
| Standard phosphate: KH₂PO₄ stock 100 µg P/mL (0.439 g per litre), working 10 µg P/mL | Makes the standard curve |
| 10% trichloroacetic acid (only for serum) | Removes proteins before testing blood |
| Apparatus | Colorimeter or spectrophotometer (red filter, about 660 nm), cuvettes, test tubes, graduated pipettes, 50 mL volumetric flask |
Procedure
Typical volumes; every tube ends at 10 mL. Follow the exact volumes in your lab manual.
- 1Dilute 5 mL of the ash solution to 50 mL with distilled water (1 in 10).
- 2Set up tubes: blank (water only), standards with 1, 2, 3, 4 and 5 mL working standard (10-50 µg P), and test with 1 mL diluted ash solution, in duplicate.
- 3Make every tube up to 8 mL with distilled water.
- 4Add 1 mL ammonium molybdate reagent to each tube and mix.
- 5Add 0.4 mL ANSA reagent, mix, and make up to 10 mL with water.
- 6Stand for 10 minutes for the blue colour to develop.same time for every tube
- 7Set zero with the blank and read the absorbance of standards and test at about 660 nm.
- 8Plot absorbance (y-axis) against µg phosphorus (x-axis) and read the test value from the line.
Observation: standard curve readings (example)
| Tube | Working standard (mL) | Phosphorus (µg) | Absorbance at 660 nm |
|---|---|---|---|
| Blank | 0 | 0 | 0.00 |
| S1 | 1.0 | 10 | 0.12 |
| S2 | 2.0 | 20 | 0.24 |
| S3 | 3.0 | 30 | 0.36 |
| S4 | 4.0 | 40 | 0.48 |
| S5 | 5.0 | 50 | 0.60 |
| Test (1 mL of the 1 in 10 dilution) | - | read from graph | 0.34 |
Worked example: phosphorus in ragi
10 g ragi was ashed and made up to 100 mL. 5 mL of this was diluted to 50 mL, and 1 mL of the dilution was taken as the test. Test absorbance = 0.34; the 30 µg standard read 0.36 (curve as in the table). Find phosphorus in mg per 100 g.
- Slope of the standard curve = 0.36 ÷ 30 = 0.012 absorbance per µg phosphorus.
- Phosphorus in the test tube = 0.34 ÷ 0.012 = 28.33 µg. (By proportion: 0.34 ÷ 0.36 × 30 = 28.33 µg.)
- This is in 1 mL of the dilution, so the 50 mL dilution holds 28.33 × 50 = 1416.7 µg, all from 5 mL of ash solution.
- 100 mL ash solution holds 1416.7 × 100 ÷ 5 = 28,333 µg = 28.33 mg, from 10 g ragi.
- Per 100 g = 28.33 × 100 ÷ 10 = 283.3 mg.
- In one line: P (mg/100 g) = µg from graph × (50 ÷ 1) × (100 ÷ 5) × (100 ÷ 10) ÷ 1000 = 28.33 × 50 × 20 × 10 ÷ 1000 = 283.3.
Ragi contains about 283 mg phosphorus per 100 g.
Precautions and result
- Wear gloves and goggles. The molybdate reagent is made in strong sulphuric acid: prepare it in a fume hood, adding acid to water, never water to acid. Never pipette by mouth.
- Rinse glassware well with distilled water; many detergents contain phosphate and give falsely high readings.
- Keep ANSA in a brown bottle and discard it when it darkens; old reagent gives a weak colour.
- Add the reagents in the same order to every tube, and read all tubes after the same standing time.
- Keep the test reading inside the standard range; if it is above the top standard, dilute further and multiply by the extra factor.
- Result: The phosphorus content of the given food (ragi) is ___ mg per 100 g.
Phosphorus works with calcium in bones and teeth and is part of ATP, DNA and phospholipids. Because the calcium : phosphorus ratio of a diet affects bone health, both are usually estimated from the same ash solution. In cereals, millets and pulses much of the phosphorus is held as phytate, which also binds iron, zinc and calcium; ashing gives total phosphorus, not the absorbable part. The same molybdenum-blue reaction measures serum phosphate, which is low in rickets.
Short answer: “What is molybdenum blue? Name the reducing agent that forms it.” It is the intense blue complex formed when phosphomolybdic acid (phosphate + ammonium molybdate in acid) is partly reduced. In the Fiske-Subbarow method the reducing agent is ANSA, 1-amino-2-naphthol-4-sulphonic acid.
Short answer: “Why is the food ashed before phosphorus is estimated?” Ashing turns organic phosphorus (phytate, phospholipids) into inorganic phosphate, the only form that reacts with molybdate. So the method measures total phosphorus.
Structured question: “Describe the estimation of phosphorus in a food by the Fiske-Subbarow method.” Principle: phosphate + acid molybdate → phosphomolybdic acid; ANSA reduces it to molybdenum blue; Beer-Lambert law. Reagents, standard curve with KH₂PO₄ (10-50 µg), procedure (molybdate, ANSA, 10 minutes, about 660 nm), calculation with the dilution factor, and result per 100 g.
"Molybdate makes it, ANSA blues it": phosphate + molybdate → phosphomolybdic acid → (ANSA) → molybdenum blue.