Spectrophotometry (Beer–Lambert law)

Principle

• A spectrophotometer measures how much light of a selected wavelength a solution absorbs.
• Beer–Lambert law: A = εbc, where A is absorbance, ε the molar absorptivity, b the light path (usually 1 cm) and c the concentration.
• Absorbance is directly proportional to concentration; transmittance (T) is not. A = –log T = 2 – log %T.
• The law holds only for dilute solutions and monochromatic light; at high concentration the curve bends (loss of linearity).

Reagents and equipment

• Light source: tungsten or tungsten-halogen lamp for visible range; deuterium lamp for UV.
• Monochromator (filter, prism or diffraction grating) selects the wavelength; slits control bandwidth.
• Cuvette: glass or plastic for visible work; quartz (silica) needed below about 340 nm.
• Detector: photodiode, photodiode array or photomultiplier tube; converts light to an electrical signal.

Procedure

• Select the wavelength of maximum absorbance of the coloured product.
• Zero the instrument with a reagent blank (corrects for reagent colour).
• Read standards and tests under identical conditions.
• Calculation: concentration of test = (A test / A standard) × concentration of standard.

Quality control and pitfalls

• Check wavelength accuracy with didymium or holmium oxide filters; photometric accuracy with neutral density filters or certified solutions.
• Stray light causes falsely low absorbance, most obvious at high absorbance values.
• Scratched, dirty or fingerprinted cuvettes, air bubbles, turbidity, lipaemia, haemolysis and icterus give erroneous readings.
• Samples above the linear range must be diluted and re-read, multiplying by the dilution factor.

Clinical use

• Basis of most colorimetric and kinetic assays in clinical chemistry (glucose, proteins, enzymes at 340 nm using NADH/NADPH).