• Light source
• Monochromator
• Sample in cuvette
• Detector

• The solution containing the sample must be homogeneous: it must be clear and not milky.
• The sample should absorb light at the measured wavelength.
• The concentration should be low, because at high concentrations Lambert-Beer's law no longer applies.
• Now the light passes through the sample, loses intensity and therefore only has intensity I.

Extinction as a central value

What is extinction?

The absorbance is the decadal logarithm of the ratio of the initial intensity and the intensity after sampling. The concentration can be determined by the loss of intensity.

Extinction should not be confused with absorption. Extinction includes all light-attenuating events. The following light-attenuating events can occur in our sample:

• The absorption of the wavelength by the molecules of the sample in the solution,
• the refraction of light in an inhomogeneous, milky solution on the particles of the sample,
• the reflection on the liquid surface or the cuvette.

Preparation of the sample

In order to specifically measure absorption, the following things must be taken into account:

• The sample must be well dissolved: There should be no more particles floating around that would make the sample milky or inhomogeneous.
• A calibration measurement must be carried out with the cuvette and the solvent. This means that the solvent (usually water) and the cuvette must be placed in the photometer and calibrated. (Press the Calibrate button).

The photometer now measures the extinction again. However, since no dye is included, only the reflection on the water and on the cuvette is measured, which is subtracted from the extinction in the subsequent measurements. If these steps are followed, you can successfully measure the absorbance together with the extinction.

Photometry and Lambert-Beer's law

What does extinction tell us? Lambert-Beer's law applies here. This represents the extinction in connection with our substance, its concentration and the layer thickness of the optical medium. The layer thickness is, so to speak, the width of the cuvette, which is usually standardized to 1 cm. If the layer thickness remains constant, as does the molar, decadal extinction coefficient (absorption of the corresponding wavelength), there is a linear function here. This line increases as the concentration of the sample in the solution increases.

This allows the concentration of the substance being sought to be determined. Note: Lambert-Beer's law only applies at low concentrations of the "dye". Thus there is a natural upper limit for the extinction. As an example, let's assume our solution is completely black, so no more light comes through. If you add more dye, the concentration increases, but no light comes through the solution for measurements anyway.

In reality, there is no material that exactly fulfills Lambert's law. In particular, the radiance of any surface has a directional dependence and this changes as the direction from which the surface is illuminated changes. Even standards that are used to calibrate measuring devices can only be well described by Lambert's law in certain reflection directions and wavelength ranges. At wavelengths outside the visible spectral range and at reflection or illumination directions of more than a few 10° to the vertical, deviations of several 100% from Lambert's law can occur, even with normal ones. [1]

Creating a spectrum with a photometer

As a rule, to determine the concentration of an analyte in the sample, it is sufficient to measure the extinction or absorption of the solution. However, if you want to characterize your analyte more precisely, you may have to record a spectrum.

As a rule, the extinction is measured individually for each wavelength at a specified concentration and layer thickness. However, since one does not want to carry out a calibration for every wavelength, modern spectrometers are used today that take on this task independently. Spectral analysis is an important method for identifying and/or determining the concentration of unknown substances.

UV/VIS spectroscopy

When we talk about spectra, we are no longer talking about photometry, but rather about spectroscopy or, more precisely, UV/VIS spectroscopy. The name comes from the fact that these spectra are recorded from the UV (Ultra Violet) to the visible (Visible) range of light.

UV/VIS spectroscopy is based on measuring the absorbance of visible and ultraviolet light by the sample. The spectral, ie wavelength-dependent, information can be obtained either by selecting and scanning the wavelength of the incident light in front of the sample (see dual-beam spectrometer) or by separating the wavelengths of the transmitted light after the sample (diode array spectrometer). The ratio of the spectral intensity of the transmitted and incident light provides the transmission spectrum. The logarithmic reciprocal of the transmission gives the extinction spectrum.

Basically, extinction provides information about absorption, scattering, diffraction and reflection on and in the sample. Phenomena of radiation absorption are often evaluated in UV/VIS spectroscopy, since the photon energy of visible and ultraviolet light corresponds to the transition energy of the states of outer electrons of many atoms and molecules. By absorbing photons in the visible and ultraviolet spectral range, valence electrons (e.g. those in the p and d orbitals) can be excited, that is, transformed into a state of higher energy. The transmission or extinction spectrum therefore allows the identification and quantitative determination of analytes. 

Here is an overview of spectrophotometers and HowTo's.

[1]  Andreas Höpe, Kai-Olaf Hauer: Three-dimensional appearance characterization of diffuse standard reflection materials. In: Metrologia. Band 47, Nr. 3, April 2010, S. 295–304, doi:10.1088/0026-1394/47/3/021.