Plan quantitative analysis using UV/visible spectroscopy

A reliable UV/visible analysis is more than one substitution into A = εcl. It controls background absorption, chooses a sensitive wavelength, obtains a valid concentration relationship and handles sample preparation correctly.

GCE A-Level H3 Chemistry 9813-2027

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Learning objectives

Plan quantitative analysis using UV/visible spectroscopy

Plan from sample to reported result

Decide whether the concentration will come from a known molar absorption coefficient or from a calibration graph made with standards. A calibration is especially useful when matrix and instrument effects need to be matched.

Blank, standards and calibration

A reagent blank contains the solvent and all reagents except the analyte. It corrects for absorption by the cuvette, solvent and reagents so the measured signal is attributed to the analyte as far as possible.

Calibration standards contain known analyte concentrations spanning the expected sample range. Their blank-corrected absorbances are plotted against concentration and a best-fit line is obtained within the linear region.

A suitable analytical wavelength is commonly at or near λmax, where sensitivity is high, provided interferences do not absorb strongly there.

A complete quantitative method

Prepare a blank and several standards accurately, using the same solvent, reagents and treatment as the sample. Set the spectrophotometer to the chosen wavelength, zero with the blank, and measure standards and sample in matched cuvettes.

Plot absorbance against concentration and check that the standards are linear. Read or calculate the concentration of the measured sample only if its absorbance lies within that calibrated range.

If the sample is too concentrated, dilute it accurately, remeasure, then multiply the measured concentration by the dilution factor to recover the original concentration. Report units and suitable significant figures.

Worked example: calibration plus dilution

A calibration line is A = 2500c, with c in mol dm⁻³. A sample made by diluting 10.0 cm³ to 100.0 cm³ gives A = 0.500.

The diluted concentration is 0.500/2500 = 2.00 × 10⁻⁴ mol dm⁻³. The dilution factor is 100.0/10.0 = 10.0, so the original concentration is 2.00 × 10⁻³ mol dm⁻³.

Try this

Find the original concentration from A = 2500c, A = 0.500 and a 10.0 cm³ to 100.0 cm³ dilution.

Check your answer

The measured solution is 2.00 × 10⁻⁴ mol dm⁻³; multiplying by the dilution factor 10.0 gives 2.00 × 10⁻³ mol dm⁻³.

Practise interpolation

Blank-corrected standards: 2.0, 4.0 and 6.0 μmol dm⁻³ give absorbances 0.10, 0.20 and 0.30. An unknown gives A = 0.25.

Before calculating, check two things: whether the standards lie on a straight line through the origin, and whether the unknown's absorbance lies inside the range the standards cover.

Try this

Check the calibration data, find its gradient, decide whether interpolation is justified, then find the concentration corresponding to A = 0.25.

Check your answer

Each 2.0 μmol dm⁻³ step adds 0.10, and the line passes through the origin, so it is linear with gradient 0.050 per μmol dm⁻³. A = 0.25 lies between the 4.0 and 6.0 μmol dm⁻³ standards, so interpolation is justified: c = 0.25/0.050 = 5.0 μmol dm⁻³.

Decide what to do with an out-of-range sample

The calibration standards give reliable, linear absorbances up to A = 0.80.

Decide what the evidence supports before choosing a calculation.

Try this

An unknown has A = 1.40, but the highest reliable standard has A = 0.80. What should you do?

Check your answer

Do not extrapolate beyond the highest standard. Dilute the unknown accurately (recording the volumes), remeasure so its absorbance falls within the calibration range, calculate the diluted concentration, then multiply by the dilution factor.

Common mistake: treating the blank as pure water

The blank must match everything except the analyte. If coloured reagent is present in the samples but absent from the blank, its absorbance is incorrectly assigned to the analyte.

Another common mistake is to forget the dilution factor or apply it in the wrong direction. A diluted solution has a lower concentration, so the original value must be larger.

Try this

Correct: ‘The blank is always distilled water, whatever reagents are used.’

Check your answer

The blank contains the same solvent and reagents as the standards and sample but no analyte, so background absorption is corrected.

Check your understanding

A strong plan names the analyte or chromophore, wavelength choice, matched blank, standards, cuvette control, calibration range, sample measurement, dilution treatment and final units.

Next, move to infrared spectroscopy and describe how molecular bonds stretch before interpreting an IR spectrum.

Try this

Give the full sequence for determining an unknown concentration by calibration.

Check your answer

Prepare blank and standards, choose the wavelength, blank the instrument, measure standards, verify a linear calibration, measure the sample within range, calculate or interpolate c, then reverse any dilution.