**1. Introduction**

Every compound, that is present in the nature, has a property to absorb, transmit, or reflect light (electromagnetic radiation) at a certain wavelength. This property of the compounds, helps to measure quantitatively by using spectrophotometric techniques. Spectrophotometry is a technique which deals with the measurement of the interaction of light with materials. When light falls on a material that can be reflected, transmitted, scattered, or absorbed, and at the same time the material on which light has fallen can emit absorbed light with different frequency. This is due to the gained energy from the light (e.g., electroluminescence) or due to its temperature (incandescence) [1]. Different types of spectroscopy and spectrophotometry is well known and widely used technique to identify and quantify

#### **Figure 1.** *Electromagnetic Spectrum.*

compounds in the field of research as well as in the industrial and chemical laboratories. For example, in chemistry, and pharmacy, UV-visible spectrophotometry is a basic technique to analyze the samples based on the application of the Beer-Lambert-Bouguer Law. In biochemistry and molecular biology, spectrophotometric analysis is essential for determining biomolecule concentration of a solution and is employed ubiquitously for determining the concentration of DNA, RNA, or protein [2]. In clinical laboratories both manual and automated spectrophotometric are extensively used for the determination of blood, urine, and body fluid samples [3].

Several types of spectroscopic and spectrophotometric methods are applied to analyze the samples. Among them, there are two primary methods which are highly utilized; absorption spectrophotometry, which is based on the absorption of radiation at specific wavelength of light to get absorption spectrum, and UV-visible spectrophotometry, which is apprehensive with the reflectance of specific spectra of a given material within UV and visible range of electromagnetic radiation spectrum (**Figure 1**) [4].

#### **2. Principle, instrumentation and applications of various spectrophotometric techniques**

A spectroscopic/spectrophotometric instrument basically consists of four important components: a light/radiation source, a collimator, a monochromator, and a detector. The monochromator comprehends a fixed entrance slit, a dispersing element such as a prism or a diffraction grating, and a moving exit slit (**Figure 2**) [6].

#### **2.1 UV-visible spectrophotometry**

#### *2.1.1 Principle*

Law of absorption is the basic principle of UV-visible spectrophotometry. This law discusses the relation between thickness of the absorbing material and the concentration of the sample solution, which is popularly known as Beer-Lambert law or simply Beer's law. This law states that the amount of light absorbed is proportional to the concentration of the absorbing substance and to the thickness of the absorbing material [7].

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

**Figure 2.** *Basic instrumentation of spectrophotometer [5].*

$$\log\_{10} = \frac{I\_0}{I} = ab\,\text{C} \tag{1}$$

where *I*o = the intensity of the incident light, *I* = the intensity of the transmitted light, *a* = absorption, *b* = the absorbing thickness, *C* = the concentration of the absorbing material.

#### *2.1.2 Instrumentation*

The UV-visible spectrophotometer consists of a light source, sample holders, a monochromator, and a detector [8].

*Light source*: Hydrogen lamp and the deuterium lamps are used as UV light source, whereas for visible source tungsten filament lamp is the most used.

*Sample holders*: In UV and Visible ranges, cuvettes are used as sample holders, which are made from quartz or ordinary glass. Generally, in the UV region quartz or silica cell are used, whereas in the visible region glass cell are used and these cuvettes have a standard path length is usually 1 cm.

*Monochromators*: A monochromator converts polychromatic radiation into monochromatic radiations by which the wavelengths of these radiations translate into very narrow bands.

*Detectors*: Photovoltaic cells, phototubes and photomultiplier are commonly used detectors in the UV and visible range [7]. The following block diagram (**Figure 3**) shows main parts of UV-Visible spectrophotometer [7].

#### *2.1.3 Applications of UV-visible spectrophotometry*

a.Pharmaceutical analysis: UV-visible Spectrophotometry has been widely used technique in the determination of drug concentration in pharmaceutical analysis.

For example, this technique is used in the determination of etravirine in bulk and pharmaceutical formulations. The spectrum of etravirineis shown below. This is acting as an anti-viral drug, and it showed the maximum absorption at 414 nm

**Figure 3.** *Block diagram of UV-visible spectrophotometer.* (visible range) by reacting with NaOH and 1,2-napthaquinone 4-sulphonate. The details are shown in the following (**Figure 4**) [9].


**Figure 4.** *UV-visible spectrum of etravirine.*

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

**Figure 5.** *UV absorption spectrum of DNA.*

**Figure 6.**

*Optical absorbance spectrums of four oil samples with different CI based on ASTM D 1500 after applying ND filter [14].*

#### **2.2 Infrared spectrophotometry**

#### *2.2.1 Principle*

When a molecule absorbs the light of higher wavelength rather than UV and visible, then there is a possibility of vibrational transitions in the molecules. These vibrational transitions of the molecules lead to the formation of an IR spectrum.

**Figure 7.** *Interaction human serum albumin (HAS) with citrate-capped gold nanoparticles (AuNPs) [15].*

These vibrational transitions due to occurrence of electronic transitions when a given substance absorbs light energy.

#### *2.2.2 Instrumentation*

Like UV-visible spectrophotometer, IR spectrometer also consists of a light source, a sample holder, a monochromator, and a detector [7].

*Light source*: Xenon and tungsten lamps are typically used as light sources in the near IR-region [16].

*Sample holders*: In IR region quartz cuvettes usually used as sample holders. *Monochromator*: Gratings are used as monochromators in the IR region.

*Detectors*: Commonly used detectors in the IR spectrophotometry are indium gallium arsenide (InGaAs) semiconductor materials.
