# Optoelectronic Instrumentation and Measurement Strategies for Optical Chemical (Bio)Sensing
## 3 Spectroscopic Techniques
### Absorbance, Reflectance, and Luminescence  
Radiation absorption in the UV-visible region is one of the most popular methods in conventional analytical chemistry. When a ray of light of a certain wavelength and intensity (\(l_{0}\)) comes into contact with the dissolution of a chemical compound, the compound will absorb some of the radiation from the light (\(I_{\text{a}}\)). The remaining light (\(I\)) will pass through the solution and reach the light detector. It is true that \(I_{0}=I_{\text{a}}+I\). Absorbance indicates the amount of light absorbed by the sample. The absorbance coefficient is defined as:  
\[A_{\lambda}=log_{10}(I_{0}/I) \tag{3}\]  
The Lambert-Beer law defines the relationship between absorbance and analyte concentration in the solution (\(C\), mol L\({}^{-1}\)) as:  
\[A_{\lambda}=log_{10}(I_{0}/I)=\varepsilon\cdot\!I\cdot\!C \tag{4}\]  
where \(\varepsilon\) is the molar absorption coefficient (L mol\({}^{-1}\) cm\({}^{-1}\)) and \(I\) the length of the optical path in the cuvette containing the sample (cm). It is important to note that the molar absorption coefficient is a function of the wavelength; therefore, the Lambert-Beer law is only true at a single wavelength (monochromatic light). The identification and quantification of a substance in a solution is carried out from the analysis of the absorption spectrum, which is a graphical representation indicating the amount of light absorbed (\(\varepsilon\)) at different wavelengths. A detailed study of this spectrum is required because in the solution there may be more than one light-absorbing substance. Figure 6: Optical phenomena due to light–matter interaction. When a chemical sensor is in an optically opaque medium or one with a weak ability to transmit light, the intensity of the reflected light can be used to measure the concentration of that substance. The reflection of light can be specular or diffuse. Specular reflection (SR) occurs on the surface of the medium without any transmission mediation and is governed by Snell's law. In diffuse reflection (DR), the light penetrates into the medium and reappears on the surface after suffering partial absorption and a series of multiple dispersions. The reflectance coefficient for a given wavelength is defined as:  
\[\rho_{\lambda}=\frac{I_{0}}{I_{\text{DR}}} \tag{5}\]  
This coefficient depends on obvious physical parameters and includes information on the quantity of a specific substance; therefore, reflectance can be used as an instrumentation parameter in the design of a sensor for that substance. As with absorbance, the identification and quantification of a substance is achieved through the analysis of the reflected light spectrum (reflectance spectroscopy). The processing of the spectrum to obtain the content information for a substance is complex, and as in the case of absorbance, mathematical and statistical methods are used. Fluorescence and phosphorescence are two phenomena involving photoluminescence molecules, consisting of the emission of light from excited electronic states of the molecule. To explain these phenomena, it is common to use to a diagram of the energy levels of a photoluminescent molecule, as shown in Figure 7, known as the Jablonski diagram [36]. A brief explanation of this diagram can help explain the difference between fluorescence and phosphorescence processes. When a molecule absorbs UV or visible radiation, the electrons pass from the _single_ fundamental state, \(S_{0}\), to a vibrational state of higher energy, \(S_{1}\) or \(S_{2}\). From these states, the molecules can return to their fundamental state, without radiation emission or with emission; this emission is known as fluorescence. The fluorescent process, characterized by the total time between excitation from the fundamental state and return, is very short (ns). Molecules can also return to the ground state via another less usual route (low probability of occurrence), through the process called cross-system crossing.