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Iron by spectrophotometry: a calibration curve and an unknown

Make a series of iron standards with pipettes and volumetric flasks, develop the colour and read the absorbance at 510 nm, fit the calibration line by least squares, then measure the unknown and reagent blanks and report the iron concentration with its confidence interval and detection limit.

Difficulty ●●○ ≈ 30 min

Aim Determine the iron concentration of an unknown water sample by external calibration, with its 95 % confidence interval, detection limit and quantitation limit.
Iron(II)–phenanthroline complex · 510 nm · 1.00 cm cell

Iron by spectrophotometry: a calibration curve

① Make standards: pipette 10.00 mg/L iron stock into 50 mL flasks

Every pipette and flask carries a small deterministic error (uniform within its class A tolerance), so the true concentrations differ slightly from the nominal ones — but you only know the nominal values. Absorbance = ε·b·c + a reagent blank of 0.010 with ε = 1.11 × 10⁴ L mol⁻¹ cm⁻¹ (Fortune & Mellon 1938), plus photometer noise σ = 0.0015 + 0.3 % × A and 0.2 % stray light. The unknown’s concentration is set by the sample number, between 1.2 and 3.8 mg/L. Flask colours are computed from two absorption bands (a teaching fit matching ε at 510 nm) for a 2 cm path.

Procedure

  1. Choose the volumes: with class A pipettes transfer 10.00 mg/L iron stock into 50 mL flasks to make a set of standards from 0 to 5 mg/L (at least 5 points, including a blank).
  2. To each flask add hydroxylamine hydrochloride (reducing Fe³⁺ to Fe²⁺), 1,10-phenanthroline and sodium acetate buffer; make up to the mark, mix, and let the colour develop for 10 minutes.
  3. Measure the absorbance of every standard at 510 nm in a 1.00 cm cell, fit the calibration line and check the residual plot.
  4. Measure at least 5 reagent blanks and work out the blank’s standard deviation and the detection limit.
  5. Develop the unknown the same way, read it 3–5 times, and find its concentration with a 95 % confidence interval from the calibration line; finally reveal the true value to check.

What to notice

  • The standards darken from nearly colourless to orange-red — absorbance proportional to concentration (Beer’s law).
  • Even with every pipette inside its class A tolerance, the standards do not lie exactly on a line; the residuals should scatter randomly.
  • The unknown’s confidence interval is narrowest in the middle of the calibration range; more readings of the unknown narrow it, but only until the calibration line itself becomes the limit.
  • With only 2–3 standards t is large (ν = n − 2) and the interval becomes surprisingly wide.

In a real lab

  • 1,10-Phenanthroline is toxic and hydroxylamine hydrochloride is an irritant: wear gloves and goggles, and collect the waste as heavy-metal and organic waste.
  • Rinse the cuvette with the solution to be measured, wipe fingerprints and drops off the optical faces, and zero the instrument on the reagent blank before each series.
  • Colour development takes time and depends on pH (stable between pH 3 and 9); develop every solution under the same conditions and measure them at about the same time.
  • If the sample has matrix interferences (other coloured ions, strong complexing agents), run a spike-recovery test or switch to standard addition.

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