How to use a spectrometer?

PRINCIPLE


The measurement is based on the use of Beer-Lambert’s law: \large A = \log \frac {I_{0}}{I} = \varepsilon \cdot l \cdot C

It includes two steps:

  1. Finding ε: this is the calibration stage. In practice, the length L is fixed by the cuvettes in which the absorbance is measured. The absorbance measurement of several solutions with different concentrations gives a range of values Ai and Ci that allow to draw the graph, which is generally linear. Its slope is ε x L.
  2. Counter-calibration: the absorbance measurement gives a value Ax which leads to the concentration Cx after graphical analysis based on the calibration curve.
Both steps use measurements of A that are detailed in the following protocol:
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