7.1.2. Infrared (IR)

Infrared spectrophotometry is the most widely used technique for pilot-level applications. This is done with the aid of an infrared spectrophotometer, which can distinguish molecular structures by producing wavelengths with a spectral range of 500 to 4000 nm. That is, the transmittance response for a given wavelength shows the difference in the links caused by photodegradation [38].

This method works by exposing a sample to ultraviolet radiation, which causes changes in the vibrational states of the sample's constituent molecules. Radiation absorption by a sample indicates the type of bonds and functional groups present [25,44]. It is useful to divide the infrared region into three regions called near infrared (NIR), middle infrared (MIR), and far infrared (FIR) from the standpoint of instrumentation and applications [44,45]. The vast majority of traditional analytical applications of infrared spectroscopy are focused on the use of the middle infrared (4000-600 cm<sup>−</sup>1) and near infrared, allowing this method to be converted into a quantitative technique. The Fourier transform technique, which converts a time domain spectrum to a frequency domain spectrum using a mathematical operation, allows for the generation of spectra that are quick, accurate, and have elevated signal/noise (S/N) relationships [46].

There are several measurement techniques to obtain this type of spectra; however, some of the most common are described below:

Transmission: IR radiation is passed into the sample in this measurement process, recording the amount of energy absorbed by the sample. The IR spectrum is obtained using a reference experiment by comparing the radiation recorded after going through the sample. With the proper accessories, this method analyzes gaseous, liquid, and solid samples [47].

Reflection: Infrared radiation is reflected on the sample. The sample's molecular information is extracted by analyzing the reflected radiation and comparing it to the incident radiation. To use this measuring tool, the sample must be reflective or mounted on a reflective surface [44,45].

ATR mode: It is a sampling mode in which an infrared beam is projected onto a crystal with a high refractive index. The beam reflects off the inside of the glass, generating an evanescent wave that enters the sample. This must be in near proximity to the crystal. A portion of the evanescent wave's energy is absorbed, and the reflected radiation (containing chemical information about the sample) is directed to the detector. It is a very versatile method for measuring liquid and solid samples without manually processing them [47].

In order to gain as much detail as possible on the pigment in question, the methods previously described are usually used as a supplement. As in the case of Quijano-Ortega et al. [48], both FTIR and ATR were used jointly to determine carotenoids present in *Cucurbita* spp., where structural conformations typical of carotenoids could be determined, for example the double bonding between carbons, deformation of CH3 groups and even the stable existence of CH2 chains [48].
