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Highlights of Spectroscopic Analysis – A Review

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 ijlpr 2021; doi 10.22376/ijpbs/lpr.2021.11.2.P136-145

International Journal of Life science and Pharma Research

Pharmaceutical analysis for Novel drug formulation

Review Article

Highlights of Spectroscopic Analysis – A Review

Sasikala M*1&2 , Mohan S1 and Swarnakumari S1

1
Karpagam College of Pharmacy, Coimbatore – 641032, Tamil Nadu, India
2
Karpagam Academy of Higher Education, Coimbatore – 641021, Tamil Nadu, India

Abstract: Spectroscopic techniques are broadly employed to study the chemical structure of an analyte
accurately. The electromagnetic radiation used in each method is allowed to interact with the molecule. The
electric and magnetic property of the radiation is interacted with the similar properties of the chemical
substance. Hence, the analyte is identified and characterized for the presence of atoms, bonds, functional
groups, basic nucleus, molecular formula and molecular weight. The study is highly relevant to have a reference
covering most of the important analytical techniques. The investigation implies content of basics for the
interpretation of a spectrum, rules/laws followed, sampling techniques, sample cells, mechanism involved in
analysis, units of measurements, spectral ranges, light sources, nature of samples to be analyzed, kind of
solvents utilized, appearance of spectra and major uses of individual spectroscopic analysis. This also includes
every significant aspect of various spectroscopic methods and represents qualitative & quantitative parameters
which insists for the research and experiments carried by the specific spectral process. Thereby, review
regarding combination of entire spectroscopic principle, instrumentation and applications will be giving fruitful
information to all analytical persons Pharma students.

Keywords: Atoms; Molecules; Spectrum; Interpretation; Applications.

*Corresponding Author Recieved On 17 September 2020

Sasikala M , Karpagam College of Pharmacy, Coimbatore – Revised On 15 February 2021

641032, Tamil Nadu, India Accepted On 23 February 2021
Karpagam Academy of Higher Education, Coimbatore – Published On 06 March 2021
641021, Tamil Nadu, India

Funding This research did not receive any specific grant from any funding agencies in the public, commercial or not for profit sectors.

Citation Sasikala M, Mohan S and Swarnakumari S , Highlights of Spectroscopic Analysis – A Review.(2021).Int. J. Life Sci. Pharma Res.11(2),
P136-145 http://dx.doi.org/10.22376/ijpbs/lpr.2021.11.2.P136-145

This article is under the CC BY- NC-ND Licence (https://creativecommons.org/licenses/by-nc-nd/4.0)

Copyright @ International Journal of Life Science and Pharma Research, available at www.ijlpr.com

Int J Life Sci Pharma Res., Volume11., No 2 (March) 2021, pp P136-145

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1. INTRODUCTION element. Flame emission spectroscopy allows quantitative

measurement of the optical emission from excited atoms to
Spectroscopy is the evaluation of the interaction between examine analyte concentration. These extreme temperature
matter and electromagnetic radiation. Reliably, it emerged atomization sources provide sufficient energy to endorse the
through the study of visible light dispersed accord to its atoms into high energy levels. The atoms crumble back to
wavelength by a prism. It is associated with the absorption, lower levels by emitting radiation4. Mass spectrometry is an
emission or scattering of electromagnetic radiation by analytical weapon beneficial for estimating the mass-to-charge
atoms or molecules. The consequence of such an ratio (m/z) of one or more molecules present in a sample5.
interaction has researchers contemplating analytical Fluorescence spectroscopy is valuable in applications such as
findings1. UV-Vis Spectroscopy is accustomed to conclude detecting and quantifying organic compounds. Industrial
analyte concentration by the absorption of light over the applications include testing surface quality and cleanliness.
range through a liquid sample. Infrared spectroscopy Laser-induced fluorescence spectroscopy permits exciting
is choicely used in industry besides in research. It is a lasers to energies fluorophores in the proposed constituents
conservative and reliable approach for measurement, which are emitted during relaxation within a reach of
quality control and dynamic measurements2. It is applied to nanoseconds6. Electron Spin Resonance spectroscopy
determine functional groups in molecules. Generally, determines the absorption of microwave radiation resemble to
stronger bonds and light atoms will vibrate at a high the energy splitting of an unpaired electron when it is placed in
stretching frequency. Nuclear magnetic resonance a strong magnetic field. X-ray powder diffraction is a
spectroscopy is the widest use to determine the structure constructive tool for the identification of unknown crystalline
of organic molecules in solution and used in advanced materials and determination of solids which is decisive to
medical imaging techniques, such as in magnetic resonance studies in geology, environmental science, material science,
imaging (MRI)3. Atomic absorption spectrometry is an engineering and biology7. From the correlation of entire
analytical technique that quantifies the concentrations of spectroscopic analysis, the aim and objective of this review is
components. It is thus sensitive that it can work through to accomplish content of learning about each method in a
parts per billion of a gram in a test. The technique single article. Therefore, the highlights of every important
comprises wavelengths of light specifically absorbed by an feature of spectroscopy are focused in this study.

2. METHODS/STUDY

Fig 1. Classification of Spectroscopic Analysis

2.1 Comparison and Difference of Principle, Instrumentation and Applications between various spectroscopic
analysis (Figure 1 and table 1)

2.1.1 Electronic Spectroscopy 8-12

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Table 1. Comparison and Difference of Principle, Instrumentation and Applications between Electronic Spectroscopy
Electronic Spectroscopy
S. Absorption Spectroscopy Emission Spectroscopy
Parameters
No. Atomic Absorption Fluorescence Flame Emission
UV/Visible Spectroscopy
Spectroscopy Spectroscopy Spectroscopy
ElectroMagnetic Ultraviolet and Visible Ultraviolet and Visible Ultraviolet and Visible Ultraviolet and Visible
01
Radiation used radiation radiation radiation radiation
Mercury arc/ Xenon No radiation source is
02 Light Source D2/ W lamp Hollow Cathode lamp
arc lamp used
Filters/ prisms/ grating Filters/ prisms/ grating Filters/ prisms/ grating Filters/ prisms/ grating
03 Monochromators
monochromators monochromators monochromators monochromators
Sample cells/ Cylindrical/ rectangular Cylindrical flat Cylindrical/ Cylindrical flat
04
Cuvettes Quartz/ Glass cells bottomed Glass cells rectangular Glass cells bottomed Glass cells
Monophasic Monophasic
Phase of Sample Monophasic diluted Monophasic diluted
05 Colourless/coloured diluted Colourless diluted
analyzed solutions solutions
solutions solutions
Polar solvents like
Polar solvents like Distilled Polar solvents like Polar solvents like
06 Solvents used Distilled water and
water and Methanol Distilled water Distilled water
Methanol
Concentration
07 µg – mg µg – mg µg – mg µg – mg
range of analysis
Physical properties
08 Absorption Absorption Emission Emission
of sample analyzed
Chemical
09 properties of Chromophores Valence electrons Fluorophores Valence electrons
sample analysed
Change occurs at Change occurs at Change occurs at
Change occurs at electronic
10 Principle electronic level of an electronic level of a electronic level of an
level of a molecule
atom molecule atom
Moving of electrons Moving of electrons Moving of electrons
Moving of electrons from
from HOMO (ground from LUMO (excited from LUMO (excited
11 Mechanism HOMO (ground state) to
state) to LUMO state) to HOMO state) to HOMO
LUMO (excited state)
(excited state) (ground state) (ground state)
Photo tubes, Photovoltaic Photo tubes, Photo tubes, Photo tubes,
12 Detectors cells and PhotoMultiplier Photovoltaic cells and Photovoltaic cells and Photovoltaic cells and
tubes PhotoMultiplier tubes PhotoMultiplier tubes PhotoMultiplier tubes
% Fluorescence
Absorbance versus Absorbance versus % Flame intensity
Appearance of intensity versus
13 Wavelength Wavelength versus Wavelength
spectrum Wavelength
(Figure 2) (Figure 3) (Figure 5)
(Figure 4)
14 Qualitative respect λmax (λex) λmax (λex) λmax (λem) λmax (λem)
Concentration versus
Quantitative Concentration versus Concentration versus Concentration versus
15 % Fluorescence
respect Absorbance Absorbance % Flame intensity
intensity
Applications to
16 Chromophoric substances Metallic salts Fluorophoric analytes Metallic salts
analyze
Interpretation
17 Beer – Lambert’s law Beer – Lambert’s law Beer – Lambert’s law Beer – Lambert’s law
Laws
Interpretation
18 Woodward – Fieser rule Bohr’s equation ---- Bohr’s equation
Rules
Radiation Range of
19 200 – 800nm 200 – 400nm 400 – 800nm 400 – 800nm
analysis
Detection of impurities
Laser induced
Structure elucidation of
fluorescence
organic compounds
spectroscopy of
Qualitative analysis Environmental Environmental
human tissues for
Chemical kinetics Clinical Clinical
General cancer diagnosis
20 Detection of functional Pharmaceutical Pharmaceutical
applications Study of Marine
groups Examination of Agricultural Agricultural
Petroleum Pollutants
polynuclear hydrocarbons Petrochemicals Petrochemicals
Accurate
Molecular weight
determination
determination As HPLC
of glucose
detector

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2.1.2 Appearance of Spectrum

Fig 2: UV/Visible spectrum

Fig 3: Atomic Absorption spectrum

Fig 4: Fluorescence spectrum

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Fig 5: Flame Emission spectrum

2.2 Structural Elucidation Spectroscopy (Part I, table 2):

Table 2. Comparison and Difference of Principle, Instrumentation and Applications between Structural
Elucidation Spectroscopy (Part I)13,14
S. Structural Elucidation Spectroscopy
Parameters
No. IR Spectroscopy NMR Spectroscopy Mass Spectrometry
ElectroMagnetic
01 Infra red Radio Waves No radiation is used
Radiation used
Nernst Glower
02 Light Source NMR Probes High energy electron beams
Globar Source
Filters/ prisms/ grating Filters/ prisms/ grating
03 Monochromators Electric and Magnetic field Analyzers
monochromators monochromators
Sample cells/ Circular shaped Alkali
04 Cylindrical Glass cells Septum type Injectors
Cuvettes metal halide windows
Monophasic diluted
Phase of Sample solutions with
05 Solid/ Liquid/ Gas Gas
analyzed Tetramethylsilane as
internal standard
Potassium bromide/
Nujol mull (Solids),
Deprotonated/Deuterated
06 Solvents used Carbon tetrachloride/ Volatile solvents
Solvents
Carbon disulphide
(Solutions)
Concentration range
07 ng – µg µg – mg ng – µg
of analysis
Physical properties of Nuclear Magnetic
08 Molecular Vibrations Ionization/ Fragmentation
sample analysed Resonance
Force Constant/ Bond
Chemical properties Protons (Odd Mass
09 strength and Atomic Positively charged ions
of sample analysed number)
mass
Change occurs at Spin changes occurs at Acceleration of ions based on m/e
10 Principle
vibrational energy level nuclear level ratio
Stretching and bending Arrangement of ions as per m/e ratio
11 Mechanism Flipping of nuclear spin
vibrations by analyzers
Thermopiles/ Ion detectors
Thermoelectric/ Electron Multipliers
12 Detectors Thermocouple PhotoMultiplier tubes Faraday cups
detectors (MCT Ion – to – Photon detectors
detector) Micro channel plates
Appearance of % Transmittance Intensity of signal versus % Relative intensity versus Mass to
13
spectrum versus Wavenumber Chemical shift Charge ratio
Wave numbers in
14 Qualitative respect Chemical shift Mass to Charge ratio
Fingerprint region and

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Functional group
region
Concentration versus
Concentration versus Concentration versus % Relative
15 Quantitative respect % Transmittance
Intensity of signal (Figure 7) intensity (Figure 8)
(Figure 6)
Molecules with
Applications to Structural elucidation of Molecular formula and Molecular
16 asymmetric and Dipole
analyze Organic molecules mass
moment
17 Interpretation Laws Hooke’s law ----- -----
Nitrogen rule
3n – 5 (Linear
Ring rule
molecule) n + 1 rule
Rule of 13
18 Interpretation Rules 3n – 6 (Non – Linear Pascal’s triangle
Stevenson’s rule
molecule) Coupling Constant
Even Electron rule
Isotopic rule
Radiation Range of
19 4000 – 667cm-1 60 – 200MHz Depends on Molecular weight
analysis
● Pharmaceutical analysis
Identification of ● Bioavailability studies
substances ● Drug metabolism studies
Studying progress of ● pharmacokinetics
reactions ● Characterization of potential drugs
Determination of Protein hydration ● Drug degradation product analysis
molecular structure Ionization state ● Screening of drug candidates
20 General applications Detection of impurities Protein folding ● Identifying drug targets
Monitoring the Molecular dynamics ● Biomolecule characterization
structural plasticity of Solution structure ● Proteins and peptides
plant cell walls ● Oligonucleotides
Protein quantitation ● Environmental analysis
Non-invasive blood ● Pesticides on foods, Soil and
glucose monitoring groundwater contamination
● Forensic analysis/clinical

2.2.1 Appearance of Spectrum

Fig 6: FTIR spectrum

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Fig 7: NMR spectrum

Fig 8: Mass spectrum

2.3 Structural Elucidation Spectroscopy (Part II, table 3)

Table 3. Comparison and Difference of Principle, Instrumentation and Applications between Structural
Elucidation Spectroscopy (Part II)15-17
S. Parameters Structural Elucidation Spectroscopy
No. ESR Spectroscopy X – ray Diffraction
Spectroscopy
01 Electro Magnetic Microwaves X – rays
Radiation used
02 Light Source Klystron source Metal atom with high energy
electron beams
03 Monochromators Isolator and Attenuator Crystal type monochromator
04 Sample cells/ Cuvettes Cylindrical Glass cells Metallic pan
05 Phase of Sample analyzed Monophasic diluted solutions with Diphenyl Picryl Solids
Hydrazyl radical as internal standard
06 Solvents used Polar Solvents -----
07 Concentration range of µg – mg µg – mg
analysis
08 Physical properties of Electron spin Resonance Diffraction
sample analysed
09 Chemical properties of Free radicals/ Lone-pair electrons Electrons in crystal planes
sample analysed
10 Principle Spin changes occurs at electron level Diffraction of X – rays by
electrons in a crystal plane
11 Mechanism Flipping of electron spin Analysing diffraction pattern
12 Detectors Square law detector at low energy levels Photographic detectors

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Linear detector at high power levels Coulter – Counter methods

Geiger – Muller counter
Scintillation counter (Semi –
conductive detectors)
13 Appearance of spectrum Intensity of signal versus External/Applied magnetic Intensity of signal versus Angle of
field Diffraction
14 Qualitative respect Applied magnetic field 2θ (Angle)
15 Quantitative respect Concentration versus Intensity of signal (Figure 9) Concentration versus Intensity
of signal (Figure 10)
16 Applications to analyze Structural elucidation of Organic molecules Particle size and crystal structure
determination
Polymer characterization
17 Interpretation Laws ----- Bragg’s law
18 Interpretation Rules 2nI + 1 rule (No. of Hyperfine lines)
Pascal’s triangle
Coupling Constant
19 Radiation Range of analysis 9,000 – 10,000MHz 0 – 100Å
20 General applications Biology (as spin – labels or spin – probes to study Crystal perfections, Crystallite
dynamic organization of lipids in biological membrane) size, % Crystallinity Quantitative
Chemistry (in chemical reactions) analysis, Phase identification,
Physics (Electrochemical systems and in materials Change in lattice, Atomic
exposed to UV light) substitutions

2.3.1 Appearance of Spectrum

Fig 9: ESR spectrum

Fig 10: XRD spectrum

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3. CONCLUSION 4. ACKNOWLEDGEMENT

The study includes information on different electromagnetic I wholeheartedly acknowledge my institution Karpagam
radiations, their range in EMR spectrum, the part of College of Pharmacy, Coimbatore to provide me facilities to
instrument to convert it into monochromatic light, refer and update the sources required for this study.
individual sample cells, actual principle and mechanism of
sample and radiations, various detectors, laws governing 5. AUTHORS CONTRIBUTION STATEMENT
each analysis, rules used for interpretation and general
applications of all basic spectroscopic techniques. Sasikala M, Mohan S and Swarnakumari S contributed
Absolutely, this will guide to get idea regarding the maximum to complete this study and bring it to publication.
fundamentals of each method in concise format. Benefiters
are researchers, scientists, chemists, pharmacy students, 6. CONFLICTS OF INTEREST
pharmacists, clinical chemists and most importantly
competitive exams appearing candidates. Conflict of interest declared none.

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