Calibration Curve

In most cases we are trying to measure the absorbance of an unknown solution

to determine the concentration of a molecule in the solution. The best way to

accomplish this is to measure the absorbance spectra of solutions where we

know the concentrations. These measurements then allow us to construct a

graph for how concentration and absorbance are related.

When we create this graph, it is important to use the wavelength of maximum

absorbance because that will give us the biggest change in signal as the

concentration changes, and it is also insensitive to small wavelength changes.

In constructing a calibration curve for the red form of orange carotenoid protein,

we would keep the measuring wavelength the same (525 nm) and measure how

the absorbance changes at this wavelength as we change the concentration. This

would lead to a graph like Figure 8.7

Here the data points represent the absorbances at each concentration, and the

dotted line represents a line of best fit with the equation of the line displayed on

the graph.

This calibration curve graph and the equation for the line now allow us to

determine the concentration of an unknown sample by measuring just its

66

Figure 8.7: Example calibration curve for the red form of orange carotenoid protein. The line of

best fit for the data is with . The raw data is available as a

supplementary resource.