## 6. Measuring solute concentrations in real samples using colorimetry

The concentration of solute is a key measurement in a very wide range of studies. Most techniques involve the use of optical instruments to measure the absorption of the solution. Beer's Law states that the absorbance of light at a particular wavelength is proportional to the amount of the absorbing substance in the solution for a given pathlength through the solution. You can find out more about Beer's Law at this webpage from Sheffield Hallam University:

http://teaching.shu.ac.uk/hwb/chemistry/tutorials/molspec/beers1.htm

In some cases, the solution has a natural colour, that is stronger the more concentrated the solution. An example would be coloured salt solutions such as copper sulphate (CuSO4). However, most analyses require that a chemical reaction is set up to produce a coloured endproduct whose concentration is proportional to the concentration of the reactant (the solute being tested).

Precision instruments are used to measure the absorption of light. A colorimeter is an instrument that uses light at a single wavelength, whilst a spectrophotometer allows the user to select the wavelength used.

Samples to be measured are placed in cuvettes that are optically identical and have the same pathlength (the distance the light beam passes through the solution). The instrument is zeroed against a sample with all of the reagents but none of the reactant, so that it fulfils the requirements for a blank that is optically equal to real samples with a zero concentration.

The zero sample (control) can be represented in this diagram, where a light beam passes through a sample in a cuvette, and produces an output from the detector:

A sample with low concentration absorbs more light than the control:

A sample with high concentration absorbs even more light:

The method involves making measurements of a series of standards. These allow the relationship between absorbance and reactant concentration to be established. The data obtained from this calibration are used in a linear regression to obtain an equation relating absorbance and concentration. This can then be used to calculate reactant concentration in real samples from their optical absorbance using the same chemical reaction.