The absorption spectrometry refers to a variety of techniques that employ the interaction of electromagnetic radiation with matter. In absorption spectrometry, the intensity of a beam of light measured before and after the interaction with a sample is compared. The words transmission and referral refer to the direction of travel of the light beams measured before and after absorption. Experimental descriptions usually assume that there is a single direction of incidence of light on the sample and that a plane perpendicular to this direction passes through the sample.
In transmission, light is scattered from the sample to a detector on the opposite side of the sample. In remission, light is scattered from the sample to a detector on the same side of the sample.
The emitted radiation can be formed by two kinds of radiation: specular reflection (when the angle of reflection is equal to the frequency angle) and diffuse reflection (in all other angles).
Another descriptor associated with absorption spectrometry is the variety of wavelengths of radiation that is used in the incident light beam. For example, we talk about infrared or microwave spectrometry, which are in turn examples of absorption spectrometry. On the other hand, there are also references to other wavelength ranges, such as-ray spectrometry, which usually denotes emission spectrometry. This article deals mainly with ultraviolet-visible spectrometry.
UV-visible spectrometry refers to techniques where you measure how a sample absorbs much light of a particular wavelength (color). Since color can often correlate with the presence and structure of a particular chemical, and since absorbance is an easy and inexpensive measure to make, absorbance spectrometry is widely used in qualitative, quantitative and structural calculations. For example, DNA absorbs light in the ultraviolet range (which is why sunlight is dangerous), and therefore the amount of DNA in a sample can be determined by measuring the absorbance of ultraviolet light.
The relationship between visible color and absorbance color is complicated; a sample that looks red does not absorb in the red but absorbs in other wavelengths (colors) so that the light that passes through the sample is enriched in red.
The word “color” is used to indicate that absorbance spectrometry deals not only with light in the visible range (photons with a wavelength of approximately 400 to 700 nanometers) but also with wavelengths that are outside the range of human vision (infrared, ultraviolet, x-ray). However, the principles are quite similar for both visible and non-visible light.
Technically, absorption spectrometry is based on the absorption of photons by one or more substances present in a sample (which can be a solid, liquid, or gas), and the subsequent promotion of the electron (or electrons) from one energy level to another in that substance. The sample can be a pure, homogeneous substance or a complex mixture. The wavelength at which the incident photon is absorbed is determined by the difference in available energy levels of the different substances present in the sample. This is the selectivity of absorbance spectrometry, the ability to generate photon (light) sources that are absorbed only by some components in a sample. Typically, X-rays are used to reveal the chemical composition, while wavelengths near ultraviolet and infrared are used to distinguish the configurations of various isomers in detail. In absorption spectroscopy, the absorbed photons are not emitted again (as in the fluorescence ), but the energy that is transferred to the chemical compound in the absorbance of a photon is lost by non-radiant means, such as the transfer of energy by heat to other molecules.
Although the relative intensity of the absorption lines does not vary with the concentration, at any given wavelength the measured absorbance (- log (I / I0)) is proportional to the molar concentration of the absorbing species and the thickness of the sample by which light passes. This is known as Beer-Lambert’s law. The graph of the amount of radiation absorbed concerning the wavelength for a particular compound is known as the absorption spectrum. The normalized absorption spectrum is characteristic for each particular compound, does not change with concentration and is like the chemical “fingerprint” of the compound. At the wavelengths corresponding to the resonant energy levels of the sample, some of the incident photons are absorbed, which causes a drop in the measured transmission intensity and a slope in the spectrum. The absorption spectrum can be measured using a spectrometer. Knowing the shape of the spectrum, the optical path length and the amount of radiation absorbed, the structure and concentration of the compound can be determined.