**7.3 UV-spectrophotometric methods**

For the quantification of components present in solution, the UV absorption spectroscopy works on the principle of Beer-Lambert law [17] (**Figure 6**). According to Beer-Lambert law,

$$\mathbf{A} = \log \mathbf{I}\_0 \text{ / } \mathbf{I} = \text{ε.с.} \text{l.}$$

where A = Absorbance. I0 = intensity of incident light.

I = intensity of emergent light.

ɛ = molar absorptivity.

**Figure 6.**

*Flow diagram of working principle of UV spectrophotometer.*


The natural Ultra-Violet (UV) absorption methods and chemical reactions spectrophotometric methods are having importance in pharmacopeia. In these methods quantitative data of reflection or transmission by the analyte as function of wavelength is measured. The method is based on the fact, that functional group of analyte absorbs UV radiation at specific wavelength in a solvent system. The λmax is the term used for maximum absorption of wavelength which is independent of concentration. These methods require less time and less labor consumption. The method also gives best precision. The UV-Visible methods are applied for multicomponent analysis of samples [15].

#### **7.4 Brief introduction of other spectroscopic techniques**

The Near Infrared spectroscopy (NIRS) is one of the spectroscopic techniques that can be applied for multicomponent analysis of all types of samples and having advantage of non-destructive technique. For the purpose of raw material testing, quality control of finished product and to monitor the process, the NIR spectroscopy plays important role in recent years. The great advantage of NIR is there no requirement of sample pre-treatment, the use of fiber optic probes and both the chemical and physical parameters can be obtained in single spectrum [15].

The Nuclear Magnetic Resonance (NMR) spectroscopy is one of the advantageous techniques over UV and IR spectroscopy that it can detect the intermediate products like ions, reaction complexes, solvents of chemical reaction. The NMR spectroscopy provides unique information on the structure of intermediate due to which there is no need to restore various hypotheses to explain the mechanism of the process [18]. The mass spectrometry is one of the outstanding techniques in all type's spectroscopies due to its sensitivity, detection limits and its wide range of application. It is widely applied in biochemical problems like proteome, metabolone, drug discovery and metabolism. This technique can also be applied for pollution control, food control, forensic science and natural product or process monitoring. It can also be applied in atomic physics, reaction physics, reaction kinetics, inorganic chemical analysis, ion-molecule reactions and determination of thermodynamic parameters [19].

#### **7.5 Hyphenated techniques**

Generally, for the qualitative and quantitative analysis of the samples, the separation technique is combined with the identification technique. In the analysis,

**167**

**Author details**

Mahesh Mukund Deshpande

maheshdeshpande83@gmail.com

provided the original work is properly cited.

Sangamner, M.S., India

*Analytical, Bioanalytical, Stability-Indicating Methods: Key Part of Regulatory Submissions*

chromatographic and electrophoresis are used for separation or isolation of the required components. The quantitative determination or structural information of the sample under study is performed by spectrophotometry. The Hyphenated technique is nothing but the combination of both above said techniques that are separation and spectrophotometric technique. The various hyphenated techniques like Liquid chromatography-Mass spectrometry (LC-MS), Gas chromatography-Mass spectrometry, Liquid Chromatography-Nuclear Magnetic resonance (LC-NMR), Liquid chromatography-Fourier transform infra-red spectroscopy (LC-FTIR), Capillary electrophoresis-Mass spectrometry (CE-MS) are used widely for qualitative and quantitative analysis. There may be a combination of more than one separation or detection technique like Liquid chromatography-Photodiode array-Mass spectrometry (LC-PDA-MS), Liquid chromatography-Mass spectrometry-Mass spectrometry (LC-MS-MS), Liquid chromatography-Nuclear magnetic resonance-mass spectrometry (LC-NMR-MS), Liquid chromatography-photodiode array-nuclear magnetic resonance-Mass spectrometry (LC-PDA-NMR-MS) [20].

Department of Pharmaceutical Chemistry, Amrutvahini College of Pharmacy,

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

\*Address all correspondence to: mahesh\_deshpande11@rediffmail.com;

*DOI: http://dx.doi.org/10.5772/intechopen.93566*

*Analytical, Bioanalytical, Stability-Indicating Methods: Key Part of Regulatory Submissions DOI: http://dx.doi.org/10.5772/intechopen.93566*

chromatographic and electrophoresis are used for separation or isolation of the required components. The quantitative determination or structural information of the sample under study is performed by spectrophotometry. The Hyphenated technique is nothing but the combination of both above said techniques that are separation and spectrophotometric technique. The various hyphenated techniques like Liquid chromatography-Mass spectrometry (LC-MS), Gas chromatography-Mass spectrometry, Liquid Chromatography-Nuclear Magnetic resonance (LC-NMR), Liquid chromatography-Fourier transform infra-red spectroscopy (LC-FTIR), Capillary electrophoresis-Mass spectrometry (CE-MS) are used widely for qualitative and quantitative analysis. There may be a combination of more than one separation or detection technique like Liquid chromatography-Photodiode array-Mass spectrometry (LC-PDA-MS), Liquid chromatography-Mass spectrometry-Mass spectrometry (LC-MS-MS), Liquid chromatography-Nuclear magnetic resonance-mass spectrometry (LC-NMR-MS), Liquid chromatography-photodiode array-nuclear magnetic resonance-Mass spectrometry (LC-PDA-NMR-MS) [20].
