Worksheet 4.2: An absorption photometer for instruction in school (5/6)

Measurement of the absorbance

Generally, the absorption coefficient can be determined by single measurements of intensity spectra and their processing as it is described in detail in the section about the Analysis of photometer data. In a similar way, the decadic extinction coefficient and the absorbance can be calculated. The spectra of dark current has to be subtracted from every reference and sample spectrum.

Photometer read file The photometer's software enables the automatic processing of measurements and calculation in order to obtain spectra of the absorbance. The procedure is started by a click on the symbol on the left (A stands for absorbance). The dialogue leads to the following data windows.

First step: without a cuvette in the holder an intensity spectrum is measured and checked for possible overload. With "Set Automatically ...", the integration time can be optimised.

Second step: The dark current spectrum is determined. For that, the angled plate is positioned in the cuvette holder in a way that it prevents light beams from entering the polychromator. Otherwise the power supply of the lamp can be cut off.

Third step: The cuvette is placed in the holder and filled with the reference liquid. The lamp is switched on and the reference spectrum is measured.

Fourth and last step: The reference liquid is replaced by the sample. From now on, dark currency corrected spectra of the absorbance are measured. The given example shows the absorbance spectrum of the chlorophyll sample, of which the intensity spectrum has been shown previously.