Spectrophotometry analysis

The general approach for spectrophotometry analysis is as follows:

1. Choose wavelength

According to the Beer-Lambert Law, for each wavelength, absorbance is proportional to concentration. The wavelength of maximum absorbance is selected for a given sample and use in the absorbance measurements.

2. Planning the calibration graph

To be able to determine the concentration of the analytes in the sample, a calibration curve is needed. The calibration curve is prepared by measuring several concentrations, which create a linear graph. These measurements include concentration levels that are both lower and higher than the expected concentration of the analyte in the real sample.

On the x-axis is the concentration of standard solutions in M - moles per liter. On the y-axis is the absorbance in A - the absorbance unit. A linear line on the graph consists of 5 experimental points.  An equation for the line shows that the absorbance can be calculated by multiplying the concentration by 2.

Figure 1. Calibration curve.

3. Determine the concentration of an unknown sample

Once the calibration curve has been set up, the absorbance of any unknown solution can be measured at the same wavelength and its concentration read from the graph or calculated from the slope.

It's important to prepare a reference blank each time a measurement is performed using the spectrophotometer. The reference blank is a solution that contains no analyte of interest.

The blank calibrates the spectrophotometer and sets the absorption of the solvent to zero. This ensures the result relates only to the analyte and not to any other compound in the solvent.


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