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Study Notes: Introduction to the Spectrophotometer

The spectrophotometer is an analytical machine that measures the transmission of a beam of light through a liquid sample (% Transmission) or the amount of light that does not pass through a liquid (extinction). The spectrophotometer uses specially designed sample containers (called cuvettes) to hold the liquid sample in the beam of light.

The wavelength of the beam of light may be varied for different applications, which may include measuring the:

  • turbidity (‘murkiness’) of a liquid
  • appearance or disappearance of a coloured compound during a chemical reaction
  • intensity of colour that may be related to concentration of a solute
  • wavelength of the colour in a liquid.

The spectrophotometer that we will use has the following features:

  • a single sample (cuvette) holder
  • is used for only two applications
    • measuring the turbidity of a bacterial suspension to determine the concentration of bacterial cells (used to check the calibration of the spectrophotometer)
    • measuring apple pulp browning enzyme levels
  • is set to a wavelength of 686 nm or 590 nm (nm = nanometre = 10-9 metre) respectively for each of the applications
  • reports in % Transmission (%T)
  • is of a simple manual design and is not automated.

The spectrophotometer is set-up according to an in-house SOP, calibrated by zeroing against air (i.e. no sample cuvette in the cuvette holder) and setting 100%T against a ‘blank’ solution.

Some simple precautions in the use of the spectrophotometer include:

  • allowing the lamps and electronics to warm up
  • using the correct wavelength
  • wiping fingerprints and spilt sample off the outside of the cuvette before measuring
  • carrying out the set-up procedure in the correct order
  • performing calibration checks after set up
  • closing the door to the cuvette compartment before reading the result
  • cleaning up any spills inside the cuvette compartment
  • ensuring that %T or transmission is used as appropriate.
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