*2.2.3 Methodology*

The radiation energy is the main element in the IR spectrophotometer. It acts as a function of wavelength and provides energy to reach a maximum at a wavelength (μm) equal to 2897/T, where T is the absolute temperature (K). This radiation energy is usually providing a short-wavelength limit of the spectrum (∼2 μm) and it decreases as the wavelength gets longer [17]. The radiation energy coming from the source falls on to the samples, causing the excitations followed by molecular vibrations in the sample by which it can give IR spectrum that can be detected by detectors. The detectors can change the radiation energy into an electrical signal that can be amplified and processed to yield a spectrum. The spectrum gives information about various functional present in the sample.

#### *2.2.4 Applications of IR spectroscopy*

a.Identification of compounds: IR spectroscopy assists in finding out various chemical compounds and functional groups in organic molecules, such as aliphatic, aromatic, saturated and unsaturated hydrocarbons, amino acids, ether and hydroxyl groups, halogens, nitrogen, phosphorous, silicon, sulfur-oxy compounds etc.

The aliphatic and aromatic hydrocarbons can be analyzed by C▬H and C▬C stretching and bending vibrations, most of these vibrations are unique for each molecule, and are generally described as skeletal vibrations. The C〓C▬C bond in the ring structure of aromatic compounds is diagnosed by characteristic stretching and bending vibrations [18].

For example, the spectrum of 1-hexene shows characteristic absorptions of a double bond. The C▬H stretch at 3080 cm−1 corresponds to the alkene 〓C▬H bonds. The absorption at 1642 cm−1 results from stretching of the C〓C double bond. The diagram represents the IR Spectra of 1-hexene (**Figure 8**) [18].


**Figure 8.** *IR spectra of 1-hexene.*

#### **Figure 9.**

*Enzymatic reaction of PK. (A) Series of overlaid spectra (solid lines) of infrared absorbance changes upon PEP and ADP addition to PK, observed for 30 min.*

of chondrule in NWA 2086 CV3 meteorite by using IR spectroscopy along with optical microscopy and electron microprobe. This study revealed that the alternations have brought changes in intensity and wavelength positions of olivine peaks with the advancement of alteration and related Fe/Mg substitution inward of the chondrule and also it was identified that there is a good correlations between Fo% composition and positions of 830 and 860 cm−1 IR peaks [22].

#### **2.3 Fourier transform infrared spectroscopy (FTIR)**

#### *2.3.1 Principle*

FTIR works on the principle of IR spectroscopy. Nevertheless, the instrumentation is different from IR spectroscopy.

#### *2.3.2 Instrumentation*

FTIR spectrometer consists of a light source, a sample holder, a monochromator, and a detector which are like that of IR spectrophotometer, but the major difference is an interferometer, which makes this instrument highly advanced than normal IR spectrophotometer. The interferometer specially consists of a compensator plate, a beam splitter, a fixed mirror, and a scanning mirror, which are connected to a detector. The advantages of FTIR over the existing dispersive infrared instrument are spectral quality, data collection speed, reproducibility of data, and ease of maintenance and use. Instrumentation of the FTIR is shown in the following (**Figure 10**) [23].

#### *2.3.3 Applications*

Fourier transform infrared (FTIR) spectroscopy is a powerful analytical tool in identifying chemical constituents and elucidating structures in various forms in real-world samples.

*Spectroscopy and Spectrophotometry: principles and Applications for Colorimetric and Related… DOI: http://dx.doi.org/10.5772/intechopen.101106*

**Figure 10.** *Schematic diagram of FTIR spectrometer.*

**Figure 11.**

*FTIR spectrum of poly-3-hydroxybutyrate (PHB).*


shows peaks at 1724 cm−1 and 1279 cm−1 corresponding to C▬O stretching and the adsorption band respectively in the ester group. The following (**Figure 11**) shows the C–O stretching and the adsorption band of poly-3-hydroxybutyrate (PHB) [25].


### **2.4 Raman spectroscopy**
