Absorption photometry is the main physical measurement principle of our analytical systems. It consists of passing a certain intensity of light, of a specific wavelength, through a liquid and then measuring the intensity of light that is not absorbed by this liquid.

Biosystems analysers use LEDs (light emitting diodes) of different wavelengths as light sources, except the BioSystems A15/Y15 analysers that use a halogen lamp, with the suitable interferential filters to get the desired light wavelength, usually between 340 and 750 nm. All analysers have transparent cuvettes where liquids are dispensed and the optical measurements can be made. The light intensity is measured using high precision silicon photodiodes.

The analysers measure first the intensity of light that reaches the photodetector when we have water in the cuvette (Io) and then the intensity that reaches it when we have the problem liquid that we want to study (I). The difference between these two intensities is due to the absorption of light caused by the problem liquid. This liquid is normally a chemical reaction in which certain substances called chromophores appear. They are molecules that absorb light of a certain wavelength, which cause us to observe a drop in the light intensity that reaches the detector. To express the degree of light absorption we can use the physical magnitude called absorbance (A) which is defined as:

                                     A = log (Io / I)

It is very convenient to use this magnitude because the absorbance is directly proportional to the concentration of the chromophore in the reaction (C). In other words, the more concentrated the chromophore, the more light is absorbed. And the absorbance is also proportional to the distance that the light has to pass through the reaction (L). This distance is called the optical path length of the cuvette. These two facts are included in the Beer-Lambert law, which states that the measured absorbance is proportional to the concentration of the chromophore and the optical path length. The proportionality constant is called molar absorptivity or molar absorption coefficient (α) of the chromophore, which depends on the wavelength and is specific to each chromophore. This law is expressed, therefore, as:

                                          A = α C L

In the clinical laboratory, absorbance values are standardized to an optical path length of 1 cm.

The Beer-Lambert law enables the concentration of the chromophore to be calculated by measuring its absorbance. But what really matters is not the concentration of the chromophore in the chemical reaction, but the concentration of the analyte in the sample. The analyser is able to relate the measured absorbance of the reaction to the concentration of the analyte, through the appropriate analysis mode and calibration defined and programmed in the instrument software. The complete analysis process is explained in more detail in the article How does a clinical biochemistry analyser work?.

In the case of turbidimetry reagents, when the reagent reacts with the sample, aggregates of particles appear that scatter light in different directions. Therefore, we also observe a decrease in light intensity that we can quantify with an absorbance, even if in this case it is due to the dispersion of light and not to its absorption.  And in a completely analogous way, we can also define the appropriate analysis mode and calibration that, based on the measured absorbances, allows us to determine the concentration of the desired analyte in the sample.