Iodine clock: estimating hydrogen peroxide concentration by serial dilution
Serially dilute a 2.0% hydrogen peroxide stock by half, four times, then time how long each dilution takes to turn a starch–iodide–thiosulfate mixture blue-black. Use the same timing method on an unknown 'patient sample' to estimate its hydrogen peroxide concentration by interpolation.
WHAT YOU'LL LEARN
- ✓Why a 'clock' reaction stays invisible until a fixed reagent runs out, then changes almost instantly
- ✓Building a five-point calibration series by halving a stock solution with a fixed transfer-and-top-up method
- ✓Why clock time is roughly inversely proportional to concentration over this range
- ✓Repeating an unknown measurement three times and averaging, to guard against a single anomalous reading
- ✓Estimating an unknown concentration by interpolating a mean time against your own calibration curve, rather than reading off a formula
ON YOUR BENCH
- □Solution R1 — dilute sulfuric acid, 0.05 mol/dm³
- □Solution R2 — starch solution, 0.4%
- □Solution R3 — potassium iodide solution, 0.5 mol/dm³
- □Solution R4 — sodium thiosulfate solution, 0.01 mol/dm³
- □Solution H — 2.0% hydrogen peroxide stock, for serial dilution
- □Solution U — unknown 'patient sample'
- □Solution W — distilled water, for dilution
- □10 cm³ syringes ×2 and 1 cm³ syringes ×5
- □Beakers, 50–100 cm³ ×5
- □Large test-tubes, 40–50 cm³ ×8, and a rack to hold them
- □Glass rod
- □Containers labelled 'For washing' and 'For waste'
- □Paper towels, glass marker pen
- □Stop-clock or timer showing seconds
- □Eye protection
The protocol, step by step
The same guide is printed and waiting at your bench.
- 01
Set up the serial dilution
Label five beakers 2.0%, 1.0%, 0.5%, 0.25% and 0.125%. Starting from stock H (2.0%), transfer 10 cm³ of each beaker's contents into the next using a 10 cm³ syringe, then top up with 10 cm³ of distilled water W using the second syringe. Each step exactly halves the concentration.
- 02
Mix the reagent cocktail
For each concentration, add a measured volume of R1 (acid), R2 (starch), R3 (iodide) and R4 (thiosulfate) to a test-tube using the 1 cm³ syringes, then add the H₂O₂ dilution last.
- 03
Start the clock and watch for the end-point
Start the stop-clock the instant the H₂O₂ dilution is added. Iodide is slowly oxidised to iodine, but thiosulfate reduces it straight back as fast as it forms — so nothing visible happens until the fixed, small amount of thiosulfate is used up. Record the time, to the nearest whole second, the moment the tube turns blue-black.
- 04
Stop at 180 seconds if nothing happens
If no colour change has appeared after 180 seconds, stop timing and record 'more than 180' rather than waiting indefinitely — this is expected for the most dilute sample.
- 05
Repeat for all five concentrations
Work through 2.0%, 1.0%, 0.5%, 0.25% and 0.125% and record all five times in a table.
- 06
Time the unknown sample U three times
Run the identical method on sample U three times (U1, U2, U3), using a fresh tube each time, and record all three times.
- 07
Calculate and interpolate
Calculate the mean of the three U timings. Plot your five-point calibration series (concentration vs. time) and read off the concentration of H₂O₂ in sample U that corresponds to that mean time by interpolation.
What you should see
- →A clear inverse trend: the 2.0% dilution turns blue-black fastest, and each successive halving takes roughly twice as long.
- →The most dilute sample (0.125%) often exceeds the 180 second cutoff — recording 'more than 180' there is a normal, expected result, not an error.
- →Sample U's mean time should fall between two of your five calibration points, so its concentration is read off by interpolation rather than extrapolation.
- →Repeating U three times and checking the results are close together is what justifies using the mean rather than a single reading.
| 2.0% | 1.0% | 0.5% | 0.25% | 0.125% | |
|---|---|---|---|---|---|
| Time /s | 20 | 40 | 80 | 160 | more than 180 |
⚠ BEFORE YOU START
- •Wear eye protection throughout — R1 is an irritant and solution H is an irritant at higher concentrations.
- •Dispose of all reacted mixtures into the container labelled 'For waste', not down the sink.
- •Wipe up any spills immediately with paper towels and rinse the affected area using the 'For washing' container.
Try it, then run it for real.
Practise the whole thing on the virtual bench, then book real lab time and do it with your own hands.