Muhammad 

Sajid Hamid Akash
Kanwal Rehman

Essentials
of Pharmaceutical
Analysis
Essentials of Pharmaceutical Analysis
Muhammad Sajid Hamid Akash •
Kanwal Rehman

Essentials
of Pharmaceutical Analysis
Muhammad Sajid Hamid Akash Kanwal Rehman
Department of Pharmaceutical Chemistry Department of Pharmacy
Government College University University of Agriculture
Faisalabad, Pakistan Faisalabad, Pakistan

ISBN 978-981-15-1546-0 ISBN 978-981-15-1547-7 (eBook)

https://doi.org/10.1007/978-981-15-1547-7

# Springer Nature Singapore Pte Ltd. 2020

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Contents

1 Introduction to Pharmaceutical Analysis . . . . . . . . . . . . . . . . . . . . . 1

1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.2 Types of Pharmaceutical Analysis . . . . . . . . . . . . . . . . . . . . . 2
1.2.1 Qualitative Analysis . . . . . . . . . . . . . . . . . . . . . . . . 2
1.2.2 Quantitative Analysis . . . . . . . . . . . . . . . . . . . . . . . 2
1.2.3 Classical Methods for Pharmaceutical Analysis . . . . . 2
1.2.4 Instrumental Methods for Pharmaceutical
Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.2.5 Radiochemical Methods for Pharmaceutical
Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
1.2.6 Thermal Methods for Pharmaceutical Analysis . . . . . 4
1.3 Where We Do Pharmaceutical Analysis . . . . . . . . . . . . . . . . . 4
1.4 Socio-Economic Impact of Pharmaceutical Analysis . . . . . . . . 4
1.5 Regulatory Issues for Pharmaceutical Analysis: The Present
Situation and Future Trends . . . . . . . . . . . . . . . . . . . . . . . . . . 5
1.6 Basic Requirements for Pharmaceutical Analysis . . . . . . . . . . . 5
1.7 Terms Used in Analytical Techniques . . . . . . . . . . . . . . . . . . . 8
1.7.1 Analyte . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
1.7.2 Analytical Blank . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
1.7.3 Standard Solution . . . . . . . . . . . . . . . . . . . . . . . . . . 10
1.7.4 Indicators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
1.7.5 Calibration of Analytical Method
for Pharmaceutical Analysis . . . . . . . . . . . . . . . . . . 11
1.7.6 Statistical Analysis . . . . . . . . . . . . . . . . . . . . . . . . . 11
1.7.7 Sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
1.7.8 Accuracy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
1.7.9 Precision . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
1.7.10 Repeatability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
1.7.11 Reproducibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
1.8 Properties of Drug . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
1.8.1 Physical Properties of Drugs . . . . . . . . . . . . . . . . . . 17
1.8.2 Chemical Properties of Drugs . . . . . . . . . . . . . . . . . 17
1.9 Standard Operating Procedures (SOP)/Protocols . . . . . . . . . . . 17

v
vi Contents

1.10 Applications of Pharmaceutical Analysis . . . . . . . . . . . . . . . . . 17

Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
2 Introduction to Spectrophotometric Techniques . . . . . . . . . . . . . . . 19
2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
2.1.1 Photometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
2.1.2 Spectrophotometry . . . . . . . . . . . . . . . . . . . . . . . . . 20
2.2 Spectrum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
2.3 Electromagnetic Radiations . . . . . . . . . . . . . . . . . . . . . . . . . . 20
2.3.1 Frequency (ν) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
2.3.2 Wavelength (λ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
2.3.3 Levels of Electromagnetic Radiations . . . . . . . . . . . . 21
2.4 Principle of Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
2.5 Spectrophotometer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
2.5.1 Components of Spectrophotometer . . . . . . . . . . . . . 22
2.5.2 Single-Beam Spectrophotometer . . . . . . . . . . . . . . . 23
2.5.3 Double-Beam Spectrophotometer . . . . . . . . . . . . . . . 23
2.5.4 Types of Spectrophotometric Techniques . . . . . . . . . 24
2.5.5 Absorption Spectroscopy . . . . . . . . . . . . . . . . . . . . 24
2.5.6 Emission Spectroscopy . . . . . . . . . . . . . . . . . . . . . . 24
2.5.7 Scattering Spectroscopy . . . . . . . . . . . . . . . . . . . . . 25
2.6 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
2.6.1 Quantitative Analysis . . . . . . . . . . . . . . . . . . . . . . . 25
2.6.2 Qualitative Analysis . . . . . . . . . . . . . . . . . . . . . . . . 26
2.6.3 Enzyme Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
2.6.4 Molecular Weight Determination . . . . . . . . . . . . . . . 26
2.6.5 Physicochemical Properties . . . . . . . . . . . . . . . . . . . 27
Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
3 Ultraviolet-Visible (UV-VIS) Spectroscopy . . . . . . . . . . . . . . . . . . . 29
3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
3.2 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
3.3 Theory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
3.4 Electronic Transitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
3.4.1 Types of Electronic Transitions . . . . . . . . . . . . . . . . 32
3.5 Origin of Absorption Spectra . . . . . . . . . . . . . . . . . . . . . . . . . 33
3.5.1 σ-Electrons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
3.5.2 π-Electrons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
3.5.3 n-Electrons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
3.6 Effect of Solvent on Absorption Spectra . . . . . . . . . . . . . . . . . 34
3.7 Effect of Electronic Transitions on Absorption Spectra . . . . . . 34
3.7.1 Effect of π ! π Electronic Transitions
on Absorption Spectra . . . . . . . . . . . . . . . . . . . . . . . 34
3.7.2 Effect of n ! π Electronic Transitions
on Absorption Spectra . . . . . . . . . . . . . . . . . . . . . . . 34
Contents vii

3.8 Components of UV-VIS Spectrophotometer . . . . . . . . . . . . . . 34

3.8.1 Light Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
3.8.2 Monochromator . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
3.8.3 Sample Device/Cuvette . . . . . . . . . . . . . . . . . . . . . . 36
3.8.4 Detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
3.8.5 Recorder . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
3.9 Types of UV-VIS Spectrophotometer . . . . . . . . . . . . . . . . . . . 38
3.9.1 Single-Beam UV-VIS Spectrophotometer . . . . . . . . . 38
3.9.2 Double-Beam UV-VIS Spectrophotometer . . . . . . . . 38
3.10 Absorbance Laws . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
3.10.1 Beer’s Law . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
3.10.2 Lambert’s Law . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
3.10.3 Beer–Lambert Law . . . . . . . . . . . . . . . . . . . . . . . . . 40
3.11 Terms Used in UV-VIS Spectroscopy . . . . . . . . . . . . . . . . . . . 46
3.11.1 Chromophore . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
3.11.2 Auxochrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
3.12 Absorption and Intensity Shifts in UV-VIS Spectroscopy . . . . . 48
3.12.1 Bathochromic Shift (Red Shift) . . . . . . . . . . . . . . . . 49
3.12.2 Hypsochromic Shift (Blue Shift) . . . . . . . . . . . . . . . 50
3.12.3 Hyperchromic Shift . . . . . . . . . . . . . . . . . . . . . . . . 50
3.12.4 Hypochromic Shift . . . . . . . . . . . . . . . . . . . . . . . . . 51
3.13 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
3.13.1 Determination of Molecular Weight . . . . . . . . . . . . . 52
3.13.2 Detection of Impurities . . . . . . . . . . . . . . . . . . . . . . 52
3.13.3 Quantitative Analysis . . . . . . . . . . . . . . . . . . . . . . . 52
3.13.4 Qualitative Analysis of Pharmaceuticals . . . . . . . . . . 53
3.13.5 Detection of Functional Group . . . . . . . . . . . . . . . . 53
3.13.6 Chemical Kinetics . . . . . . . . . . . . . . . . . . . . . . . . . 53
3.13.7 Determination of Unknown Concentration . . . . . . . . 54
3.13.8 Determination of Extent of Conjugation . . . . . . . . . . 54
3.13.9 Structural Elucidation of Organic Compounds . . . . . 55
3.13.10 As HPLC Detector . . . . . . . . . . . . . . . . . . . . . . . . . 55
3.14 Advantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
3.15 Disadvantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
4 Infrared Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
4.2 Regions of IR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
4.3 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
4.4 Modes of Molecular Vibrations . . . . . . . . . . . . . . . . . . . . . . . 59
4.4.1 Stretching Vibration . . . . . . . . . . . . . . . . . . . . . . . . 59
4.4.2 Bending Vibrations . . . . . . . . . . . . . . . . . . . . . . . . . 59
4.5 Components of IR Spectrophotometer . . . . . . . . . . . . . . . . . . 62
4.5.1 Radiation Source . . . . . . . . . . . . . . . . . . . . . . . . . . 62
viii Contents

4.5.2 Sample Cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

4.5.3 Monochromator . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
4.5.4 Detectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
4.5.5 Read-Out Device . . . . . . . . . . . . . . . . . . . . . . . . . . 65
4.6 Sampling Techniques for IR Spectroscopy . . . . . . . . . . . . . . . 65
4.6.1 Solid Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
4.6.2 Liquid Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
4.6.3 Gas Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
4.7 Types of IR Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
4.7.1 Dispersive IR Spectroscopy . . . . . . . . . . . . . . . . . . . 67
4.7.2 FT-IR Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . 67
4.7.3 Near-IR Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . 68
4.8 Regions of IR Spectrum . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
4.9 Interpretations of IR Spectrum . . . . . . . . . . . . . . . . . . . . . . . . 68
4.9.1 IR Spectra of Alkanes . . . . . . . . . . . . . . . . . . . . . . . 69
4.9.2 IR Spectra of Alkenes . . . . . . . . . . . . . . . . . . . . . . . 69
4.9.3 IR Spectra of Alkynes . . . . . . . . . . . . . . . . . . . . . . . 69
4.9.4 IR Spectra of Aromatic Compounds . . . . . . . . . . . . . 74
4.9.5 IR Spectra of Ethers . . . . . . . . . . . . . . . . . . . . . . . . 74
4.10 Applications of IR Spectroscopy . . . . . . . . . . . . . . . . . . . . . . 74
4.10.1 Medical Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . 74
4.10.2 Identification of Unknown Substance . . . . . . . . . . . . 74
4.10.3 Determination of Molecular Structure . . . . . . . . . . . 77
4.10.4 Studying the Progress of Chemical Reaction . . . . . . . 77
4.10.5 Detection of Impurities . . . . . . . . . . . . . . . . . . . . . . 78
4.10.6 Analysis of Isomers . . . . . . . . . . . . . . . . . . . . . . . . 78
4.11 Advantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
4.12 Disadvantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
5 Atomic Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
5.2 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
5.3 Types of Atomic Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . 83
5.4 Advantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
5.5 Disadvantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
5.6 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
6 Atomic Absorption Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
6.2 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
6.3 Components of AAS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
6.3.1 Radiation Source . . . . . . . . . . . . . . . . . . . . . . . . . . 87
6.3.2 Chopper . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
Contents ix

6.3.3 Atomizers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
6.3.4 Nebulization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
6.3.5 Monochromators . . . . . . . . . . . . . . . . . . . . . . . . . . 92
6.3.6 Detectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
6.3.7 Amplifier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
6.3.8 Read-Out Device . . . . . . . . . . . . . . . . . . . . . . . . . . 93
6.4 Working of AAS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
6.5 Types of AAS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
6.5.1 Single-Beam AAS . . . . . . . . . . . . . . . . . . . . . . . . . 94
6.5.2 Double-Beam AAS . . . . . . . . . . . . . . . . . . . . . . . . . 94
6.6 How Can We Obtain the Data of AAS? . . . . . . . . . . . . . . . . . 95
6.7 How Do We Analyze the Data of AAS? . . . . . . . . . . . . . . . . . 96
6.8 Interferences of AAS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
6.8.1 Ionization Interference . . . . . . . . . . . . . . . . . . . . . . 97
6.8.2 Back Ground Absorption of Source Radiation
Interference . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
6.8.3 Transport Interference . . . . . . . . . . . . . . . . . . . . . . . 98
6.8.4 Cation–Cation Interference . . . . . . . . . . . . . . . . . . . 98
6.8.5 Anion–Cation Interference . . . . . . . . . . . . . . . . . . . 98
6.8.6 Oxide Formation Interference . . . . . . . . . . . . . . . . . 98
6.8.7 Spectral Interferences . . . . . . . . . . . . . . . . . . . . . . . 98
6.8.8 Chemical Interferences . . . . . . . . . . . . . . . . . . . . . . 98
6.8.9 Physical Interferences . . . . . . . . . . . . . . . . . . . . . . . 99
6.8.10 Vaporization Interferences . . . . . . . . . . . . . . . . . . . . 99
6.9 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
6.10 Precautionary Measures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
6.11 Advantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
6.12 Disadvantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
7 Atomic Emission Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
7.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
7.2 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
7.3 Types of Emission Spectra Used in AES . . . . . . . . . . . . . . . . . 104
7.3.1 Line Spectra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
7.3.2 Band Spectra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
7.3.3 Continuous Spectra . . . . . . . . . . . . . . . . . . . . . . . . . 105
7.4 Components of AES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
7.4.1 Emission Source . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
7.4.2 Monochromator . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
7.4.3 Detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
7.4.4 Readout Device . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
7.5 Working of AES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
7.6 Comparison Between AAS and AES . . . . . . . . . . . . . . . . . . . 107
7.7 Interferences of AES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
x Contents

7.8 Applications of AAS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108

7.8.1 Analysis of Pharmaceuticals . . . . . . . . . . . . . . . . . . 108
7.8.2 Biomedical Applications . . . . . . . . . . . . . . . . . . . . . 108
7.9 Advantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
7.10 Disadvantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
8 Molecular Emission Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . 111
8.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
8.2 Types of Luminescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
8.2.1 Fluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
8.2.2 Phosphorescence . . . . . . . . . . . . . . . . . . . . . . . . . . 113
8.2.3 Chemiluminescence . . . . . . . . . . . . . . . . . . . . . . . . 113
8.3 Theory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
8.4 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
8.5 Types of Fluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
8.6 Instrumentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
8.6.1 Radiation Source . . . . . . . . . . . . . . . . . . . . . . . . . . 116
8.6.2 Filters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
8.6.3 Detectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
8.6.4 Amplifiers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
8.6.5 Read-out Device . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
8.7 Factors Affecting the Fluorescence Intensity . . . . . . . . . . . . . . 117
8.8 Quenching . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
8.8.1 Reasons of Quenching . . . . . . . . . . . . . . . . . . . . . . 118
8.8.2 Types of Quenching . . . . . . . . . . . . . . . . . . . . . . . . 118
8.9 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
9 Mass Spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
9.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
9.2 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
9.3 Instrumentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
9.3.1 The Inlet System . . . . . . . . . . . . . . . . . . . . . . . . . . 124
9.3.2 The Ion Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
9.3.3 Electrostatic System . . . . . . . . . . . . . . . . . . . . . . . . 128
9.3.4 Ion Separator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
9.3.5 Ion Collector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
9.3.6 Vacuum System . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
9.4 Types of Peaks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
9.4.1 Molecular Peak . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
9.4.2 Fragment Peak . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
9.4.3 Rearrangement Ion Peak . . . . . . . . . . . . . . . . . . . . . 132
9.4.4 Metastable Ion Peak . . . . . . . . . . . . . . . . . . . . . . . . 133
9.4.5 Multicharged Ion Peak . . . . . . . . . . . . . . . . . . . . . . 133
Contents xi

9.4.6 Base Peak . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133

9.4.7 Negative Ion Peak . . . . . . . . . . . . . . . . . . . . . . . . . 133
9.5 Types of Mass Spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . 133
9.5.1 Gas Chromatography-Mass
Spectrometry (GC-MS) . . . . . . . . . . . . . . . . . . . . . . 133
9.5.2 Liquid Chromatography-Mass
Spectrometry (LC-MS) . . . . . . . . . . . . . . . . . . . . . . 134
9.5.3 Chemical Ionization Mass Spectrometry (CI-MS) . . . 134
9.5.4 Field Ionization Mass Spectrometry (FI-MS) . . . . . . 134
9.5.5 Fast Atom Bombardment Mass
Spectrometry (FAB-MS) . . . . . . . . . . . . . . . . . . . . . 134
9.6 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
9.7 Advantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
9.8 Disadvantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
10 Nuclear Magnetic Resonance Spectroscopy . . . . . . . . . . . . . . . . . . . 137
10.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
10.2 Types of Nuclear Shielding . . . . . . . . . . . . . . . . . . . . . . . . . . 139
10.3 Intensities of Resonance Signals . . . . . . . . . . . . . . . . . . . . . . . 139
1
10.3.1 H NMR Signal Intensities . . . . . . . . . . . . . . . . . . . 139
13
10.3.2 C NMR Signal Intensities . . . . . . . . . . . . . . . . . . . 139
10.4 One-Dimensional NMR Spectroscopy . . . . . . . . . . . . . . . . . . 140
10.5 Two-Dimensional NMR Spectroscopy . . . . . . . . . . . . . . . . . . 140
10.6 NMR Spectra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
10.7 Components of NMR Spectroscopy . . . . . . . . . . . . . . . . . . . . 141
10.7.1 The Magnet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
10.7.2 A Radiofrequency Oscillator . . . . . . . . . . . . . . . . . . 141
10.7.3 The Sample Holder . . . . . . . . . . . . . . . . . . . . . . . . . 142
10.7.4 A Radiofrequency Receiver . . . . . . . . . . . . . . . . . . . 142
10.7.5 A Recorder . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
10.8 Solvents Used in NMR Spectra . . . . . . . . . . . . . . . . . . . . . . . 142
10.9 How to Interpret NMR Spectra . . . . . . . . . . . . . . . . . . . . . . . . 142
10.10 Types of NMR Spectra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
10.10.1 Wide-Line Spectra . . . . . . . . . . . . . . . . . . . . . . . . . 144
10.10.2 High-Resolution Spectra . . . . . . . . . . . . . . . . . . . . . 144
10.11 Advantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
10.12 Disadvantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
10.13 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
11 Introduction to Chromatographic Techniques . . . . . . . . . . . . . . . . . 147
11.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
11.2 Principle of Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . 148
11.3 Terms Used in Chromatography . . . . . . . . . . . . . . . . . . . . . . . 148
xii Contents

11.3.1 Mobile Phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148

11.3.2 Stationary Phase . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
11.3.3 Supporting Medium . . . . . . . . . . . . . . . . . . . . . . . . 148
11.3.4 Eluate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
11.3.5 Eluent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
11.3.6 Elution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
11.3.7 Chromatogram . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
11.3.8 Retention Time . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
11.4 Types of Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
11.4.1 Based on the Nature of Mobile Phase . . . . . . . . . . . . 149
11.4.2 Based on the Mechanism of Separation . . . . . . . . . . 150
11.5 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154
Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
12 Thin Layer Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
12.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
12.2 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
12.3 Types of Thin Layer Chromatography . . . . . . . . . . . . . . . . . . 158
12.3.1 Based on Nature of Phases . . . . . . . . . . . . . . . . . . . 158
12.3.2 Based on Purpose of Use . . . . . . . . . . . . . . . . . . . . 158
12.4 Components of Thin Layer Chromatography . . . . . . . . . . . . . . 159
12.4.1 Developing Chamber . . . . . . . . . . . . . . . . . . . . . . . 159
12.4.2 Solid Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
12.4.3 Stationary Phase . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
12.4.4 Mobile Phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
12.4.5 Pipette . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
12.4.6 Forceps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
12.5 Working on Thin Layer Chromatography . . . . . . . . . . . . . . . . 159
12.5.1 Preparation of TLC Plates . . . . . . . . . . . . . . . . . . . . 160
12.6 Advantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
12.7 Disadvantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
12.8 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
13 Column Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
13.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
13.2 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
13.3 Types of Column Chromatography . . . . . . . . . . . . . . . . . . . . . 168
13.4 Components of Column Chromatography . . . . . . . . . . . . . . . . 169
13.4.1 Stationary Phase . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
13.4.2 Column . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170
13.5 Working of Column Chromatography . . . . . . . . . . . . . . . . . . . 171
13.5.1 Isocratic Elution Technique . . . . . . . . . . . . . . . . . . . 172
13.5.2 Gradient Elution Technique . . . . . . . . . . . . . . . . . . . 172
13.6 Detection of Components . . . . . . . . . . . . . . . . . . . . . . . . . . . 172
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13.7 Factors Affecting on Column Chromatography . . . . . . . . . . . . 173

13.8 Advantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
13.9 Disadvantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
13.10 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174
14 High Performance Liquid Chromatography . . . . . . . . . . . . . . . . . . 175
14.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
14.2 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
14.3 Types of HPLC Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . 176
14.3.1 Based on Mode of Chromatography . . . . . . . . . . . . . 176
14.3.2 Based on Principle of Separation . . . . . . . . . . . . . . . 176
14.3.3 Based on Scale of Operation . . . . . . . . . . . . . . . . . . 177
14.3.4 Based on the Types of Analysis . . . . . . . . . . . . . . . . 177
14.4 HPLC System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
14.5 Components of HPLC System . . . . . . . . . . . . . . . . . . . . . . . . 177
14.5.1 Solvent Reservoir . . . . . . . . . . . . . . . . . . . . . . . . . . 177
14.5.2 Pump . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178
14.5.3 Sample Injector . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
14.5.4 Column . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
14.5.5 Detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
14.5.6 Computer System . . . . . . . . . . . . . . . . . . . . . . . . . . 182
14.6 Factors Affecting on HPLC . . . . . . . . . . . . . . . . . . . . . . . . . . 183
14.7 Advantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
14.8 Disadvantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
14.9 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184
15 Gas Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185
15.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185
15.2 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186
15.3 Types of Gas Chromatography . . . . . . . . . . . . . . . . . . . . . . . . 186
15.3.1 Gas–Solid Chromatography . . . . . . . . . . . . . . . . . . . 186
15.3.2 Gas–Liquid Chromatography . . . . . . . . . . . . . . . . . . 186
15.4 Components of Gas Chromatography . . . . . . . . . . . . . . . . . . . 187
15.4.1 Carrier Gas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
15.4.2 Columns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188
15.4.3 Temperature Programmer . . . . . . . . . . . . . . . . . . . . 189
15.4.4 Sample Injector . . . . . . . . . . . . . . . . . . . . . . . . . . . 190
15.4.5 Detectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190
15.4.6 Recorder and Read-Out Device . . . . . . . . . . . . . . . . 191
15.5 Factors Affecting on Gas Chromatography . . . . . . . . . . . . . . . 191
15.6 Advantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192
15.7 Disadvantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192
15.8 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192
Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
xiv Contents

16 Introduction to Thermal Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . 195

16.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195
16.2 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196
16.3 Types of Thermal Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . 196
16.4 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
16.5 Advantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198
16.6 Disadvantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198
Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198
17 Differential Scanning Calorimetry . . . . . . . . . . . . . . . . . . . . . . . . . . 199
17.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
17.2 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
17.3 Types of Differential Scanning Calorimeter . . . . . . . . . . . . . . . 200
17.3.1 Heat Flux DSC . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
17.3.2 Power Compensated DSC . . . . . . . . . . . . . . . . . . . . 200
17.4 Instrumentation and Working . . . . . . . . . . . . . . . . . . . . . . . . . 200
17.4.1 Sample Preparation for DSC Analysis . . . . . . . . . . . 201
17.4.2 Purge Gases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201
17.4.3 Heating Rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
17.5 Calibration of DSC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
17.6 DSC Curves . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
17.6.1 Factors Affecting DSC Curve . . . . . . . . . . . . . . . . . 202
17.7 Advantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203
17.8 Disadvantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203
17.9 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204
Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
18 Differential Thermal Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
18.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
18.2 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208
18.3 Instrumentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208
18.3.1 Furnace Assembly . . . . . . . . . . . . . . . . . . . . . . . . . 208
18.3.2 Sample and Reference Holder with Temperature
Detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
18.3.3 Furnace Temperature Programmer . . . . . . . . . . . . . . 209
18.3.4 Amplifier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210
18.3.5 Read-Out Device . . . . . . . . . . . . . . . . . . . . . . . . . . 210
18.3.6 Insulator for Furnace and Sample Holder . . . . . . . . . 210
18.4 Working of DTA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210
18.5 Characteristics of DTA Curve . . . . . . . . . . . . . . . . . . . . . . . . 211
18.6 Factors Affecting on DTA Curve . . . . . . . . . . . . . . . . . . . . . . 211
18.6.1 Sample Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211
18.6.2 Instrumental Factors . . . . . . . . . . . . . . . . . . . . . . . . 212
18.6.3 Physical Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . 212
18.6.4 Chemical Factors . . . . . . . . . . . . . . . . . . . . . . . . . . 212
Contents xv

18.7 Advantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213

18.8 Disadvantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
18.9 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
19 Thermo Gravimetric Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
19.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
19.2 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216
19.3 Types of Thermogravimetry . . . . . . . . . . . . . . . . . . . . . . . . . . 216
19.3.1 Dynamic TGA . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216
19.3.2 Isothermal TGA . . . . . . . . . . . . . . . . . . . . . . . . . . . 216
19.3.3 Quasistatic TGA . . . . . . . . . . . . . . . . . . . . . . . . . . . 216
19.4 Instrumentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216
19.4.1 Microbalance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216
19.4.2 An Auto Sampler . . . . . . . . . . . . . . . . . . . . . . . . . . 217
19.4.3 Thermocouple . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217
19.4.4 Furnace . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217
19.4.5 Temperature Programmer . . . . . . . . . . . . . . . . . . . . 217
19.4.6 Recording System . . . . . . . . . . . . . . . . . . . . . . . . . . 218
19.5 Working of TGA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218
19.5.1 Sample Preparation . . . . . . . . . . . . . . . . . . . . . . . . . 218
19.5.2 Heating Rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218
19.5.3 Purge Gas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219
19.6 Calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219
19.6.1 Blank Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219
19.6.2 Calibration of Mass Changes . . . . . . . . . . . . . . . . . . 219
19.6.3 Calibration of Temperature . . . . . . . . . . . . . . . . . . . 219
19.7 Factors Affecting the TG Curve . . . . . . . . . . . . . . . . . . . . . . . 220
19.7.1 Instrumental Factors . . . . . . . . . . . . . . . . . . . . . . . . 220
19.7.2 Sample Characteristics . . . . . . . . . . . . . . . . . . . . . . 220
19.7.3 Particle Size of Sample . . . . . . . . . . . . . . . . . . . . . . 221
19.8 Advantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
19.9 Disadvantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
19.10 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222
About the Authors

Dr. Muhammad Sajid Hamid Akash (PAS Gold Medal) is Associate Professor
and Chairman at the Department of Pharmaceutical Chemistry, GC University
Faisalabad, Pakistan. He did his BPharm and MPhil from BZU, Multan, Pakistan,
while PhD from Zhejiang University, China. His research focuses on metabolic
disorders and treatment strategies. He has about 100 articles with a cumulative
impact factor of 250 and 2400 citations with h-index 28. He has authored 1 book
and 26 book chapters. He is working on three HEC-funded research projects and has
completed two projects. Moreover, he has remained a prestigious awardee of PCST
to win “Research Productivity Award” in 2015, 2016, 2017, and 2018. Based on his
outstanding contribution, the Pakistan Academy of Sciences awarded him the most
prestigious award “PAS Gold Medal” in the field of health sciences.

Dr. Kanwal Rehman is an Assistant Professor at the Department of Pharmacy,

University of Agriculture Faisalabad, Pakistan. Dr. Rehman completed her PhD in
Pharmacology and Toxicology at Zhejiang University, Hangzhou, China, in the
course of which she did excellent work on exploring arsenic-induced carcinogenic
and anticancer effects. She has a keen interest in the pathogenesis of metabolic
disorders like diabetes mellitus and obesity, as well as new therapeutic modalities for
their treatment. She has published more than 90 articles in peer-reviewed journals, as
well as 26 book chapters with international publishers like Springer Nature, Elsevier,
Material Research Forum LLC, and CRC Press. She is currently working on three
research projects funded by the Higher Education Commission (HEC) of Pakistan
and previously completed one HEC-funded project. Currently, she is investigating
treatment strategies like the use of natural biogenic compounds against different risk
factors for metabolic disorders including pancreatitis, cardiometabolic disorders, and
hormonal imbalance. Dr. Rehman was selected for the “Research Productivity
Award” by the Pakistan Council for Science and Technology (PCST) in 2016.

xvii
Introduction to Pharmaceutical Analysis
1

Abstract
Pharmaceutical analysis is a broader term which can be defined in many
ways. It is the series of processes that are used for identification, determination,
separation, purification, and structure elucidation of the given compound used
in the formulation of pharmaceutical products. The components, to which the
pharmaceutical analysis is done, are normally active pharmaceutical ingredients,
pharmaceutical excipients, contaminants present in pharmaceutical products,
or drug metabolites. In pharmaceutical analysis, the samples are typically
finished pharmaceutical products, biological samples, impurities, contaminants,
and pharmaceutical raw materials. Pharmaceutical analysis can be done using
various analytical techniques. This chapter discusses in details the fundamentals
of pharmaceutical analysis including its types and its associated important
terminologies.

Keywords
Pharmaceutical analysis · Spectroscopy · Chromatography · Analytical
techniques

1.1 Introduction

Pharmaceutical analysis is a broader term and there are many ways to define
it. It is the process or series of processes that can be used for the identification,
determination, separation, purification, and structure elucidation of the given
compound used in the formulation of pharmaceutical products. The components,
to which the pharmaceutical analysis is done, are normally active pharmaceutical
ingredients (APIs), pharmaceutical excipients (disintegrants, binders, surfactants,
suspending agents, viscosity increasing agents, polymers, adhesives, lubricants,
etc.), contaminants present in pharmaceutical products, or drug metabolites. In
pharmaceutical analysis, the samples are typically finished pharmaceutical

# Springer Nature Singapore Pte Ltd. 2020 1

M. S. H. Akash, K. Rehman, Essentials of Pharmaceutical Analysis,
https://doi.org/10.1007/978-981-15-1547-7_1
2 1 Introduction to Pharmaceutical Analysis

products (tablets, capsules, syrups, creams, lotions, ointments, injections, etc.),

biological samples (blood and/or urine, tissue samples that contain one or more
ingredients), impurities, contaminants, and pharmaceutical raw materials. Pharma-
ceutical analysis can be done using various analytical techniques.

1.2 Types of Pharmaceutical Analysis

Pharmaceutical analysis can be classified into the following major types based on
the purpose (identification and/or determination) of analysis:

1.2.1 Qualitative Analysis

It is the type of pharmaceutical analysis, in which non-quantifiable method of

determination of what constituent or substance is present in an unknown sample
or compound. For example, identification of the specific constituent, element, or
functional group present in the sample. Identification means to confirm the identity
of analyte/s in given sample. Therefore, identification can also be referred to as
qualitative analysis. For example, identification of mefenamic acid in Ponstan™
forte tablet is performed with the help of pharmaceutical analysis to make sure
that Ponstan™ forte tablet contains exactly the same API as claimed in the label.

1.2.2 Quantitative Analysis

It is the type of pharmaceutical analysis which is intended to measure the exact

concentration of the substance of interest in a given sample. In quantitative analysis,
as we intend to measure the exact concentration of substance of interest, therefore,
it is also referred to as determination. For example, Ponstan™ forte tablet contains
500 mg of mefenamic acid/tablet. So, the quantity of mefenamic acid (500 mg) in
individual Ponstan™ forte tablet is determined by the help of pharmaceutical
analysis after compression and before releasing into the packing. Determination of
exact amount of mefenamic acid in Ponstan™ forte tablet is to make sure that
Ponstan™ forte tablet contains correct API that is exactly or close to 500 mg.

1.2.3 Classical Methods for Pharmaceutical Analysis

Following are the most commonly used classical methods that are used in pharma-
ceutical analysis:

1.2.3.1 Gravimetry
It is the type of classical method of pharmaceutical analysis, in which weight of
the sample is determined after the precipitation is occurred.
1.2 Types of Pharmaceutical Analysis 3

1.2.3.2 Titrimetry
It is the type of classical method of pharmaceutical analysis, in which volume of
the sample is determined after the chemical reaction (neutralization, oxidation,
reduction, complex and precipitate formation, etc.) occurred in the sample solution.

1.2.3.3 Volumetry
It is the type of classical method of pharmaceutical analysis, in which the volume of
the gas evolved from the sample is determined when the reaction in the sample
solution is occurred.

1.2.4 Instrumental Methods for Pharmaceutical Analysis

Following are the most commonly used instrumental methods for pharmaceutical
analysis:

1.2.4.1 Optical Methods for Pharmaceutical Analysis

Optical methods are further classified into the following two types:

Absorption of Radiation Methods for Pharmaceutical Analysis

1. UV-VIS spectroscopy.
2. Atomic absorption spectroscopy.
3. IR spectroscopy.

Emission of Radiation Methods for Pharmaceutical Analysis

1. Atomic emission spectroscopy.
2. Molecular fluorescence spectroscopy.
3. Flame spectroscopy.
4. Mass spectrophotometry.
5. NMR spectroscopy.
6. Fluorimetry.
7. Phosphorimetry.

1.2.4.2 Chromatographic Methods for Pharmaceutical Analysis

1. Column chromatography.
2. Thin layer chromatography (TLC).
3. Gas liquid chromatography (GLC).
4. High-performance liquid chromatography (HPLC).
5. Liquid chromatography (LC).
6. Capillary electrophoresis.

1.2.4.3 Electrochemical Methods for Pharmaceutical Analysis

1. Potentiometry.
2. Polarography.
4 1 Introduction to Pharmaceutical Analysis

1.2.5 Radiochemical Methods for Pharmaceutical Analysis

1. Ionization method.
2. Liquid scintillation method.
3. Radiometric methods that include radiometric titration, radiochromatography,
radioimmunoassay, and isotope dilution method.

1.2.6 Thermal Methods for Pharmaceutical Analysis

1. Differential thermal analysis (DTA).

2. Thermogravimetric analysis (TGA).
3. Differential scanning calorimetry (DSC).

1.3 Where We Do Pharmaceutical Analysis

This is the important question that where can we do the pharmaceutical analysis?
Pharmaceutical analysis basically plays a significant role in the development
and validation of the processes for good manufacturing practices of high-quality
pharmaceutical products. Pharmaceutical analysis is usually carried out in the
following fields:

1. Identification and determination of APIs from the bulk drug and/or raw materials.
2. Identification and determination of intermediates of drug during its synthesis.
3. Identification and determination of different stages of drug during the research
and development of pharmaceutical products.
4. Identification and determination of in-process quality control of pharmaceutical
products during manufacturing.
5. Identification and determination of impurities and degradation products in phar-
maceutical products during manufacturing and storage.
6. Identification and determination of API, pharmaceutical excipients, impurities,
contaminations, and drug metabolites during drug stability.
7. Identification and determination of metabolites in biological samples containing
the pharmaceutical products.
8. Identification and determination of various toxins, poisons, and narcotics in
biological fluids.

1.4 Socio-Economic Impact of Pharmaceutical Analysis

High-quality pharmaceutical products exhibit good impact on society by its

own way. Pharmaceutical analysis is considered as an important branch of applied
analytical chemistry. Pharmaceutical analysis makes sure the high-quality of
pharmaceutical products which has direct impact on the economy of the drug therapy
1.6 Basic Requirements for Pharmaceutical Analysis 5

that can also be assessed with respect to the public and financial point of view.
Diseased people recover soon after using the high-quality pharmaceutical products
as compared to those diseased people who use sub-standard and/or low-quality
pharmaceutical products.

1.5 Regulatory Issues for Pharmaceutical Analysis: The Present

Situation and Future Trends

Owing to the globalization of pharmaceutical market and sharpening concurrence

among the pharmaceutical companies, the term pharmaceutical analysis has become
one of the battlefields in struggle which has increased the importance of issues that
are directly related to the drug safety and efficacy. To fulfill the legal requirements,
analytical techniques for pharmaceutical analysis need to be harmonized. The
first step for that purpose was the establishment of European Pharmacopoeia in
1970 which provided the basis for the establishment of national pharmacopoeias
by the other countries. The second step was the formation of International Confer-
ence on Harmonization (ICH) in 1990 with the aim of harmonizing the efforts of
registration agencies, principal pharmacopoeias, and pharmaceutical industries to
improve the quality of pharmaceutical products using various analytical techniques
for pharmaceutical analysis. The guidelines of ICH have been declared as authorita-
tive worldwide with respect to drug-related issues. By following the ICH-guidelines,
pharmaceutical analysis is an important field which increases the safety of drug
therapy, but it also acts as a source of inexhaustible intellectual pleasures at the
same time.

1.6 Basic Requirements for Pharmaceutical Analysis

To perform the pharmaceutical analysis, the following are the basic and/or
fundamental requirements:

1. The first step to perform the pharmaceutical analysis is to define the information
you need. The procedures involved in pharmaceutical analysis are generally
complex and consist of multiple steps; therefore, it is very important to define
the purpose of analysis at this stage to avoid any kind of ambiguity.
2. The second step is the selection of most appropriate methods for pharmaceutical
analysis which can be selected by keeping in view the purpose of pharmaceuti-
cal analysis.
3. Third step is the collection of required number of samples. This is a very
important step. It is crucial to take samples in a representative manner in
order to make a precise picture of purpose of analysis. If we talk about the
identification and determination of mefenamic acid in Ponstan™ forte tablet,
final form of Ponstan™ forte tablet should be taken in a systematic way during
the entire time scale of production to give an average of the total production.
6 1 Introduction to Pharmaceutical Analysis

The collected samples should be stored at recommended temperature. The

sample storage is very crucial because there may be compositional changes
in the constituents during storage. Before sampling, the following points should
be defined:
a. Purpose of sampling?
b. Type of tests intended to be applied to the samples?
c. Type of pharmaceutical products/materials to be sampled?
d. Are sampling facilities adequate?
e. Are responsibilities of the samplers clear?
Generally, there is no specific rule for sampling, but whenever possible,
sample should be obtained from each package or container. In case of single
package, contents should be thoroughly mixed and then sampling should
be done. In case of large number of sampling, each sample should be labeled
properly by mentioning the following information:
a. Name of the sample.
b. Date of sampling.
c. Amount of the sample taken.
d. Name of the sampler, etc.
4. After the collection of the required number of the samples from pharmaceutical
product, the next step is the preparation of sample and standard solution to
perform pharmaceutical analysis. Samples are normally pretreated in some way
and this is called sample preparation. Sample preparation can be very simple
or quite complicated, depending on the nature of the sample. For example, in
case of Ponstan™ forte tablet, the sample is prepared after pulverization of
the tablets, dissolution of the tablets, and filtration of material that has not been
dissolved.
5. Next step is to specify the quantities and concentrations for pharmaceutical
analysis. For example, quantitative pharmaceutical analysis is performed when
analyte is present in a solution. A solution is a homogeneous mixture of two or
more substances in which the minor species in a solution is known as solute. The
analyte (whose concentration is to be determined) is an example of a solute.
The major species in a solution is known as solvent. The amount of analyte
(solute) is normally expressed as the concentration which means the amount
of solute per volume unit of solution.
6. Once the quantity and concentration have been defined, next step is to do the
analysis.
7. Calculate the results and estimate reliability.
8. Convert results to information.
9. Data concerning each analysis should be entered in the laboratory notebook
by any way.
10. Fundamental apparatus required for pharmaceutical analysis: Following are
some important basic equipment that more or less commonly used during the
pharmaceutical analysis of pharmaceutical product depending upon the purpose
and nature of pharmaceutical analysis to be performed:
1.6 Basic Requirements for Pharmaceutical Analysis 7

a. Volumetric flasks: These are the standard equipment that are used to transfer
known volumes of the sample and/or standard solutions with exact concen-
tration and/or volume for pharmaceutical analysis (Fig. 1.1). It contains a
narrow neck. There is a mark on the neck of flask for the measurement. The
liquid sample is filled up-to this mark to obtain the exact volume. It is
available in different sizes to measure the desired volume at a specific
temperature and this temperature is usually printed on the volumetric flask.
b. Graduated cylinders: These are used to deliver and/or transfer the known
volumes of the sample and/or standard solutions during pharmaceutical
analysis (Fig. 1.2).
c. Burettes: It is a long tube made of glass and there is a tap at the bottom side
(Fig. 1.3). There is a graduation scale (usually in milliliter) on the tube that
makes it possible to continuously read the volume of the liquid delivered
from the burette. They are mostly used in titration where a solution is added
gradually into a sample until end point reached where the titration is
terminated. Then the consumption of solution can be read off on the burette
with high accuracy.
d. Pipettes: These are used to deliver and/or transfer the known volumes
(usually in milliliters and in some cases in microliters) of the sample and/or
standard solutions during pharmaceutical analysis. There are three types of
pipettes (Fig. 1.4) that are (1) transfer pipettes, (2) graduated pipettes, and
(3) automatic pipettes.
e. Apparatus for filtration: Depending on the nature and purpose of pharma-
ceutical analysis, various types of filtration apparatus and methods can be
used during pharmaceutical analysis.
f. Balances: Quantitative analysis is primarily based on exact weighing of the
analyte which is under consideration. Therefore, weighing should be done
with the help of an analytical balance in order to get a maximum possible
accuracy. Thus, an analytical balance is considered as a fundamental instru-
ment use in the pharmaceutical laboratory.
g. Equipment for analysis: Type of the equipment depends on the purpose of the
pharmaceutical analysis.

Fig. 1.1 Schematic

representation of volumetric
flasks having different
measuring capacity

500ml 250ml 100ml

8 1 Introduction to Pharmaceutical Analysis

Fig. 1.2 Schematic

representation of graduated
cylinders having different
measuring capacity

1.7 Terms Used in Analytical Techniques

During pharmaceutical analysis, several important terminologies are used. We

have described the most important ones here.

1.7.1 Analyte

In pharmaceutical analysis, the substance that is to be identified and/or quantified is

known as analyte. The sample contains one or more than one analyte. For example, if
we want to determine the exact quantity of mefenamic acid in Ponstan™ forte tablet,
then in this analysis, mefenamic acid will be analyte and rest of the tablet material
that contains different pharmaceutical excipients will be the sample matrix.
1.7 Terms Used in Analytical Techniques 9

Fig. 1.3 Schematic

representation of burette

1.7.2 Analytical Blank

It contains all the reagents or solvents that are used in the pharmaceutical analysis but
do not contain any of analyte in it. It is used to check whether the reagents and/or
indicators do not contribute to the final results required. It is usually used to calibrate
the instruments and/or validate the methods for pharmaceutical analysis. The pri-
mary purpose of using analytical blank is to trace sources of artificially induced
contamination.
10 1 Introduction to Pharmaceutical Analysis

Fig. 1.4 Schematic representation of different types of pipettes

1.7.3 Standard Solution

Standard solution is used in quantitative pharmaceutical analysis. It is defined as

the solution which has known amount and/or concentration of the reagent in
predetermined volume of the solution. The known amount and/or concentration of
the reagent in standard solution is mostly expressed by one of the following ways:

1.7.3.1 Molarity
The number of moles of solute in 1 L of the solution is known as molarity. In
pharmaceutical analysis, it is abbreviated as “M.”

1.7.3.2 Normality
The number of equivalents of solute in 1 L of the solution is known as normality.
It is abbreviated as “N.”
In pharmaceutical analysis, the following two types of the standard solutions
are used:

1.7.3.3 Primary Standard Solution

Such type of standard solution is made of primary standard substances. Primary
standard substance is almost 99.9% pure and is dissolved in the known volume of the
solvent. A primary standard solution is typically used to determine the unknown
concentration of the analyte. This solution is often used to make the secondary
standard solutions. An ideal primary standard substance must have the following
properties:

1. It must have high level of purity.

2. It must have low reactivity and high stability.
3. It must not be hygroscopic in nature.
4. It must be non-toxic.
1.7 Terms Used in Analytical Techniques 11

The most common example of primary standard solutions is NaCl solution in

which NaCl is used as primary standard substance for silver nitrate (AgNO3)
reactions. Zinc is used as primary standard substance for EDTA solutions when it
is dissolved in sulfuric acid and/or hydrochloric acid. Potassium hydrogen phthalate
is used as primary standard substance for perchloric acid.

1.7.3.4 Secondary Standard Solution

The solution has already been standardized with primary standard solution and
then it is used in specific pharmaceutical analysis. Such type of standard solution
is known as secondary standard solution. Secondary standard solutions are mostly
used to calibrate the equipment and validate the analytical methods. For example,
NaOH solution. Once its concentration has been standardized with primary standard
solution, it is then used as secondary standard solution. The other examples of
secondary standard solutions are oxalic acid and copper sulfate solutions. The
secondary standard solutions must have the following features:

1. Its concentration should be stable for longer period of time.

2. It should have the ability to rapidly react with analyte to complete the reaction
by simple chemical equation.
3. It should have the ability to produce the sharp end point.

1.7.4 Indicators

In pharmaceutical analysis, indicators play their pivotal role to indicate the com-
pleteness of the chemical reaction. They are weak organic acids or bases whose
solutions change their color due to change in pH of the solution. In chemical
reaction, indicators either produce or disappear specific colors and it is based
on the presence of the hydrogen ion concentration in solution. For example, phenol-
phthalein produces pink color and methyl orange produces red to yellow color.

1.7.5 Calibration of Analytical Method for Pharmaceutical Analysis

It can be defined as the comparison of the value of particular parameter measured

by the system under strictly pre-defined conditions with pre-set standard values.

1.7.6 Statistical Analysis

Pharmaceutical analysis does not mean just to perform the experiment in laboratory
by dealing and/or handling with chemicals and reagents. Interpretation of the data
using various statistical analysis tools enables the analyst to obtain the results that are
close to the theoretical values. Statistical analysis ensures the analyst that the method
has an appropriate precision and accuracy with minimum errors.
12 1 Introduction to Pharmaceutical Analysis

1.7.6.1 Errors
In pharmaceutical analysis, the difference between the true or standard value with
observed value is known as error. There is a degree of uncertainty that is associated
with every measurement and one can at its best decrease this uncertainty to an
acceptable value. While doing pharmaceutical analysis, there may be uncertainty
that is related with measurements. It is because of minor errors that may happen
in various stages of pharmaceutical analysis. Thus, analytical result is considered
as an estimation of the true value and/or content of analyte in the sample under
consideration. Therefore, it is important to decrease these uncertainties to a mini-
mum level to ensure that the result is as close to the true value as possible. Error
can be calculated by the help of the following formula:

Error ¼ True value  Observed value:

And %error is calculated by the help of the following formula:

True value  Observed value

%error ¼  100:
True value

1.7.6.2 Types of Errors

The possible errors encountered during pharmaceutical analysis are broadly classi-
fied into the following two categories:

Determinate Errors
These errors are determinable and presumably can be either avoided or corrected.
Such type of errors are also known as systemic errors. These types of errors are
known to the pharmaceutical analyst. These have the following types:

Instrumental Errors
These errors are occurred due to the use of the defective and non-calibrated equip-
ment. It is common to all instruments due to their limited accuracy. Calibration of
an instrument in one range may not be valid for entire range. For example, a transfer
pipette is intended to transfer 10.00 mL. But it is taking up slightly greater volume
(10.06 mL). It is because pipette was not calibrated due to which it will give a greater
volume (10.06 mL) than expected one (10.00 mL). It will give rise to a systematic
error and this error can affect all pharmaceutical analysis during which this uncali-
brated pipette has been employed.

Personal Errors
Such type of errors is exclusively caused by the personal mistakes and/or care-
lessness of the analyst. These can be reduced by experience. For example, mistake
of the analyst during reading and/or calculation.
1.7 Terms Used in Analytical Techniques 13

Operative Errors
These errors are physical in nature and occur when an appropriate analytical
technique is not followed. Most common examples are transfer of solutions, effer-
vescence during solution, incomplete drying of samples, mathematical errors, etc.

Reagent Errors
These types of errors are dependent on the quality of the reagent used during
pharmaceutical analysis. For example, some reagents are supplied in impure
form but are pretended to be absolutely pure. When these reagents are used in
pharmaceutical analysis, such type of errors occur.

Constant Errors
Sometimes, the errors remain constant throughout the pharmaceutical analysis
irrespective of the amount and/or volume of the sample used. For example, in case
of titration, the end point appears at 5 mL but for proper visibility of the end point, if
we add 0.1 mL, then it would be 5.1 mL instead of 5 mL. If we use 10 mL, then
it would be 10.1 and if we use 15 mL, it would be 15.1 mL. Here, 0.1 mL is known
as constant error irrespective of the volume used.

Proportional Errors
Proportional increase in the absolute error is known as proportional error. For
example, 5.1 mL instead of 5 mL, 10.2 mL instead of 10 mL, 15.3 mL instead of
15 mL. Here, the proportional error is 0.1 mL for 5 mL, 0.2 mL for 10 mL, and
0.3 mL for 15 mL.

Errors in Method
These are inherent in the method and they cannot be changed. These errors occur
due to the selection of the wrong method. For example, if the synthesis of one
product takes place in 3 h, but we have given only 2 h, then the final product will
be defective.

Absolute Error
It is calculated by measuring the difference between a measured value and true value.

Relative Error
It is an absolute error divided by true value.

Indeterminate Errors
These are accidental or random errors that may or may not be known to the analyst.
Analyst has no control over such type of errors. It follows random distribution.
A mathematical law of probability can be applied to it. Generally, it is easily
estimated by measuring the standard deviation for several replicate measurements.
14 1 Introduction to Pharmaceutical Analysis

Sources of Errors
Following are the possible sources that may lead to the occurrence of different
types of errors:

1. Improper sampling.
2. During sample preparation.
3. Improper and/or careless analysis.
4. Improper function of the equipment.
5. Improper calibration of the equipment.
6. Wrong data and/or improper observation.
7. Wrong calculation.
8. Wrong method selection.
9. Improper handling of materials during transport and/or storage.

1.7.6.3 Limit of Detection

It can be defined as the smallest amount of given analyte that can be detected by
using a particular analytical method with known confidence level, but cannot
be necessarily quantified as an exact amount. It is abbreviated as LOD.

1.7.6.4 Limit of Quantification

It is defined as the minimum concentration and/or amount of the analyte at which the
quantitative pharmaceutical analysis can be performed. It is abbreviated as LOQ.
LOQ is actually a range of the analyte concentration where the detector responds
the change in the concentration of the analyte.

1.7.6.5 Linearity
It refers to the mathematical relationship that can be graphically represented as a
straight line as in two quantities that are directly proportional to each other (Fig. 1.5).

1.7.6.6 Range of Method

During pharmaceutical analysis, analytical method measures the analyte within
from minimum up to maximum concentration. It measures both repeatability and
reproducibility for a measurement system.

1.7.6.7 Selectivity
The selectivity of a given method reflects to its potential to measure the analyte
alone in a given sample consisting of a number of compounds. For examples, the
comparison is between the UV spectroscopy and fluorescence spectroscopy. There
are various compounds which show UV absorption than there are those that
only exhibit strong fluorescence. Therefore, fluorescence is considered as a more
selective method in this case.
1.7 Terms Used in Analytical Techniques 15

Fig. 1.5 Schematic representation of linear relationship between the constant and dependent
variables

1.7.7 Sensitivity

Sensitivity of a particular method shows how responsive it is to a small change in

the amount of a given analyte.

1.7.8 Accuracy

During pharmaceutical analysis, the term precision and accuracy is used to describe
the quality of analytical measurements. The term accuracy tells how much the
analytical results match with the true value. It is generally expressed by the differ-
ence between the observed value and true value which is actually an error. The good
accuracy means there is the lowest possible difference or error. To find the accuracy
of analytical method, true value of the samples must be known, but in case of
unknown sample, an experienced analyst can obtain the true value by analyzing
the unknown samples with well-defined procedures and/or standard operating
procedures. The most suitable way to obtain the true value is to add the known
concentration and/or amount of the analyte in a solution to prepare a standard
solution (reference solution).

1.7.9 Precision

In quantitative pharmaceutical analysis, analysis of a given sample is performed

multiple times to ensure that the obtained results are not affected by the random
errors. Variability among the replicate measurements is known as precision. It means
that different values are how much close to each other. Precision is usually expressed
as standard deviation or relative standard deviation.
16 1 Introduction to Pharmaceutical Analysis

1.7.10 Repeatability

It is the term which is carried out in an assay of the particular sample by a single
operator or sum total of all the operations carried out by the same operator. For
repeatability, conditions that must be followed are same location, measurement
procedure, observer, measuring instrument, and environmental conditions. Repeti-
tion should be done within short interval of time.

1.7.11 Reproducibility

It is referred as degree to which consistent results are produced, if an experiment is

performed multiple times. Reproducibility is confirmed by employing multiple
operators or by changing the location or instrument. It has the following two types:

1.7.11.1 Within-Laboratory Reproducibility

Experiment is repeated by the different analyst but in the same laboratory with same
equipment.

1.7.11.2 Between-Laboratory Reproducibility

Experiment is repeated by employing different operators, equipment, and laboratory.

1.7.11.3 Dilution
Dilution refers to the process in which concentration of solute is reduced in given
sample and it is simply done by adding the more solvent. Serial dilutions are made so
that concentration of analyte falls within given range of an instrument. In order to
obtain the dilution factor, simply divide the final volume of the solution by initial
volume of the solution. For example, mix 0.1 mL of solution with 9.9 mL of solvent.
The final volume of the solution is 10 mL. Divide final volume (10 mL) by initial
volume (0.1 mL), dilution factor 100 is obtained and it is written as 1:100 dilution.

1.7.11.4 Analysis of Variance

It is abbreviated as ANOVA. It has a significant role in the field of pharmaceutical
analysis. ANOVA can be calculated by dividing the total variance present in
experimental data over several factors and/or components. ANOVA is a parametric
test which requires sum of the squares of the data and treatment sum of squares.

1.8 Properties of Drug

Pharmaceutical analysis of drug is totally dependent on the nature and/or properties

of the drug to be analyzed. Drug that is to be analyzed possesses two types of
properties that are as follows:
1.10 Applications of Pharmaceutical Analysis 17

1.8.1 Physical Properties of Drugs

Physical properties are measured or observed without any change in the matter
composition. Physical properties include appearance, odor, color, melting point,
boiling point, solubility, density, polarity, opacity, viscosity, and many others.
These properties play an important role in the design of analytical methods for
pharmaceutical analysis.

1.8.2 Chemical Properties of Drugs

Chemical properties are observed whenever a substance undergoes a chemical

change. Ionization, functional groups, partition coefficient, pH, degradation and
stereochemistry, heat of combustion, and enthalpy change are the important chemi-
cal properties that should be considered during the selection of an appropriate
analytical method for pharmaceutical analysis.

1.9 Standard Operating Procedures (SOP)/Protocols

SOP is a set of step-by-step instructions complied by an organization to help analyst

carry out routine analysis in the laboratory. Pharmaceutical analysis is totally based
on these SOPs and/or protocols. First, the SOPs and/or protocols are developed in
laboratories by experts. SOPs must be repeatable and reproducible. SOPs and/or
protocols must be in accordance with the guidelines of ICH and pharmacopoeias.
SOPs help the analyst to perform pharmaceutical analysis and also able the analyst
to avoid any kind of possible errors.

1.10 Applications of Pharmaceutical Analysis

Pharmaceutical analysis is a broader field and its applications are vast which are as
follows:

1. Identification and determination of APIs in bulk drug and/or raw material and
finished dosage forms of the pharmaceutical products.
2. Identification and determination of impurities in bulk drug and/or raw material
and finished dosage forms of the pharmaceutical products.
3. Identification and determination of contaminants in bulk drug and/or raw material
and finished dosage forms of the pharmaceutical products.
4. Identification and determination of various drug metabolites and/or intermediates
in biological samples.
18 1 Introduction to Pharmaceutical Analysis

Further Reading
Beckett AH, Stenlake JB Text book of practical pharmaceutical chemistry, Vol. I and II.
A&C Black, London
Connors KA (2007) A textbook of pharmaceutical analysis. Wiley, Hoboken
Görög S (2007) The changing face of pharmaceutical analysis. TrAC Trends Anal Chem
26(1):12–17
Griffin JP, O’Grady J (2006) The textbook of pharmaceutical medicine. Wiley, Hoboken
Hansen SH, Pedersen-Bjergaard S, Rasmussen K (2011) Introduction to pharmaceutical chemical
analysis. Wiley, Hoboken
Kar A (2005) Pharmaceutical drug analysis. New Age International, Chennai
LibreTexts™. Pharmaceutical analysis. Accessed 2 May 2019
Ohannesian L, Streeter AJ (2002) Handbook of pharmaceutical analysis. Marcel Dekker, New York
Sudha PC (2012) Pharmaceutical analysis. Pearson Education India, Chennai
Valcárcel M (2012) Principles of analytical chemistry: a textbook. Springer, Berlin
Waters Corporation. Pharmaceutical analysis. Accessed 11 June 2019
Watson DG (2015) Pharmaceutical analysis E-book: a textbook for pharmacy students and
pharmaceutical chemists. Elsevier Health Sciences, Amsterdam
Introduction to Spectrophotometric
Techniques 2

Abstract
Spectrometric techniques are used to measure the interaction of different fre-
quency components of electromagnetic radiations (EMR) with that of matter.
After interaction with matter, these radiations are absorbed by the matter. It is not
possible that we look at the matter instead, we observe the interaction of light with
different degrees of freedom of matter and/or substance. This chapter describes
the basics of spectrophotometry and various types of spectrophotometric
techniques. Spectrophotometry is a key method that is frequently used for
identification and quantification of raw materials and pharmaceutical products.

Keywords
Electromagnetic radiations · Frequency · Electronic energy level ·
Spectrophotometer · Types of spectrophotometric techniques

2.1 Introduction

Spectrometric techniques are used to measure the interaction of different frequency

components of electromagnetic radiations (EMR) with that of matter. Electromag-
netic radiations interact with matter at specific energy levels. After interaction with
matter, these radiations are absorbed then atoms/molecules of analyte present in
sample move from one energy state (usually ground state having low energy level) to
another energy state (usually excited stated having high energy level). It is not
possible that we look at the matter and/or substance but its ghost when the matter
and/or substance interacts with light. Instead, we observe the interaction of light with
different degrees of freedom of matter and/or substance. In spectrophotometry, we
either measure the absorbance or emission of EMR after its interaction with matter.
Interaction of EMR with matter is directly dependent on the energy of radiation.
Spectrophotometry is used to understand how different frequency components
of EMR interact with matter and how we can use this information to understand

# Springer Nature Singapore Pte Ltd. 2020 19

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20 2 Introduction to Spectrophotometric Techniques

the quantity of analyte present in sample. Broadly speaking, spectrophotometry is

basically the set of tools that can be used together in different ways to understand the
chemical properties and/or nature of the analyte present in sample.

2.1.1 Photometry

It is the branch of science that deals with the measurement of light. Photometer is a
tool that is used for the measurement of intensity of light. It is the measurement of the
amount of luminous light (either absorbed or emitted) falling on the surface of
analyte present in sample from a source of radiation.

2.1.2 Spectrophotometry

It is the phenomenon of measurement of the intensity of light at selected

wavelengths. This method depends on the light absorbing and/or emission property
of either the analyte or a derivative of analyte being analyzed. Spectrophotometry is
used in pharmaceutical analysis for the identification of the analyte through the
spectrum that is absorbed and emitted by an analyte present in the sample solution.

2.2 Spectrum

It is defined as the measurement of response as a function of wavelength or more

precisely the frequency.

2.3 Electromagnetic Radiations

Electromagnetic radiations (EMR) contain discrete energy packets. These discrete

energy packets are known as photons. A photon contains an oscillating magnetic
field and an oscillating electric field (Fig. 2.1). Both of these fields fall perpendicular
to one another.

2.3.1 Frequency (n)

Frequency is defined as the number of oscillations produced by an electrical field

radiation per second. Hertz (Hz) is the unit used for frequency (1 Hz ¼ 1 cycle per
second).
2.3 Electromagnetic Radiations 21

Electric Field
d
Wavelength (λ) el Direction of Current
Fi
ic
n et
ag
M

Fig. 2.1 Schematic representation of levels of electromagnetic radiations

2.3.2 Wavelength (l)

It is the measurement of distance that exists between two adjacent parts of a wave
present in the same phase or the distance present between two adjacent troughs or
crest.

2.3.3 Levels of Electromagnetic Radiations

In EMR, three levels exist which are as follows:

2.3.3.1 Electronic Energy Level

Molecules exist in the lowest energy level (E0) at room temperature that is known as
ground state. Whenever, the EMR are absorbed by the molecules, valence electrons
are promoted to next higher energy state abbreviated as E1 that is also known as
excited state. This shifting of electrons from lower energy level (ground state) to
higher energy level (excited state) by absorbing the energy in the form of EMR is
known as electronic transition and the difference is calculated as: DE ¼ E1Eo.

2.3.3.2 Vibrational Energy Level

These are the less energy levels than electronic energy levels. The difference
between these energy levels is comparatively small such as 0.01–10 kcal/mole.
For example, when infrared (IR) radiations are absorbed by the molecules, they
are excited from lower vibrational energy level to higher vibrational energy level and
start vibrating with relatively higher amplitude.

2.3.3.3 Rotational Energy Level

Rotational energy levels are quantized and discrete. The difference between these
levels is relatively smaller as compared to that of vibrational energy levels. Overall,
order of different energy level is represented as follows:
22 2 Introduction to Spectrophotometric Techniques

ΔErotational < ΔEvibrational < ΔEelectronic :

2.4 Principle of Spectroscopy

The principle of spectroscopy is based upon the measurement of spectrum of given

analyte present in sample having either atoms or molecules. Spectrum consists of the
graph of intensity radiations either absorbed or emitted by the sample versus
wavelength (λ) or frequency (ν). Spectrometer is a device which is used to measure
the spectrum of analyte. It is more advanced instrument than colorimeter. It is
because there are filters present in colorimeter which allow wide range of
wavelengths of radiation to pass through, whereas in spectrophotometer, grating
and/or prism is utilized in order to split the light beam into multiple wavelengths.
With the help of selective mechanism, specific wavelength of EMR may interact
with sample solution.

2.5 Spectrophotometer

This is an optical instrument that is used to measure the intensity of EMR relative to
the specific wavelength. It is useful for the measurement of analyte concentration
present in sample solution by measuring the amount of EMR absorbed and/or
emitted by the sample solution. The sample to be analyzed by spectrophotometer
must be in solution form and the solvent that contains analyte must be optically
transparent in specific wavelength region.

2.5.1 Components of Spectrophotometer

Following are the four major components of spectrophotometer:

1. Light source: It provides the polychromatic light to the monochromator which

diffracts the light. The sources of light depend on the type of spectrophotometric
technique. For UV-radiation, the most common sources of light are hydrogen
lamp and the deuterium lamp. For visible radiation, the most common sources are
tungsten filament and carbon arc. For IR-radiation, the most common sources are
Nernst Glower and Global.
2. Monochromator: It splits polychromatic light into individual wavelengths and
separates these individual wavelengths into narrow bands.
3. Cuvette and/or sample holder: It holds the sample and allows the specific
wavelength to pass through itself. Cuvettes are transparent in nature and are
made of ordinary glass or quartz material.
4. Photosensitive detector: Detector must be highly sensitive and long-term stable
that have the ability to detect the low level of the radiant energy.
2.5 Spectrophotometer 23

Fig. 2.2 Schematic representation of single-beam spectrophotometer

5. Read-out device: It has the ability to interpret the signals received from the
detector.

According to the number of beams used, spectrophotometer has the following

two types:

2.5.2 Single-Beam Spectrophotometer

Single beam passes through sample present in cuvette (Fig. 2.2). At first, the
spectrophotometer is standardized by putting a reference solution in the cuvette,
and the resulting absorbance is subtracted from absorbance of sample solution in
order to remove solvent effect. Single-beam spectrophotometer follows Beer–
Lambert law to determine the unknown concentration of the analyte.

2.5.2.1 Advantages
1. Less expensive.
2. High sensitivity due to non-splitting of the light source.

2.5.2.2 Disadvantages
Single-beam spectrophotometer is not suitable due to the lack of the compensation of
the following disturbances:

1. Analysis is time consuming as it has single cell. At first, the reference solution is
loaded to record the reading, then cuvette is washed and sample solution is loaded
to record the recording.
2. Electronic circuit fluctuations.
3. Voltage fluctuations.
4. Mechanical component’s instability.
5. Drift in energy of light sources.

2.5.3 Double-Beam Spectrophotometer

A double-beam spectrophotometer is used for the comparison of the light intensity

between two light paths, one beam passes through a reference and the other from the
test sample (Fig. 2.3).
24 2 Introduction to Spectrophotometric Techniques

Fig. 2.3 Schematic representation of double-beam spectrophotometer

2.5.3.1 Advantages
1. It offers better detection than single-beam spectrophotometer.
2. Instability factors, lamp drift, voltage fluctuations, and stray light do not affect the
measurement of light as these factors influence the measurement of results in
single-beam spectrophotometer.

According to the range and type of EMR used, it has the following types:

1. UV-VIS spectrophotometer.
2. IR spectrophotometer.
3. Atomic absorption spectrophotometer (AAS).
4. Atomic fluorescence spectrophotometer (AFS).
5. X-ray fluorescence spectrophotometer (XFS).
6. Mass spectrophotometer (MS).

2.5.4 Types of Spectrophotometric Techniques

Types of the spectrophotometric technique depend on the quantitative pharmaceuti-

cal analysis of analyte which is measured by the interaction of different frequency
components of EMR with analyte. Mostly, the spectrophotometric techniques can be
classified into the following major types:

2.5.5 Absorption Spectroscopy

In this spectrophotometric technique, we measure the amount of EMR that is being

absorbed by the sample (Fig. 2.4). The examples are UV-VIS spectroscopy, atomic
absorption spectroscopy, infra-red spectroscopy, and nuclear magnetic resonance
spectroscopy.

2.5.6 Emission Spectroscopy

In this spectrophotometric technique, when EMR interacts with sample, the sample
emits the radiations of specific wavelength which are then detected to predict the
2.6 Applications 25

Fig. 2.4 Schematic

representation of absorption
of light in atomic absorption
spectroscopy

amount of the sample. In emission spectrophotometric technique, the sample first

absorbs the EMR and then emits the light of specific wavelength. The examples are
atomic emission spectroscopy, fluorescence emission spectroscopy.

2.5.7 Scattering Spectroscopy

In this technique, the substance scatters the EMR at specific wavelength and polari-
zation angles. Scattering of light gives the information regarding the molecular
structure. The scattering phenomenon is much faster than the absorption and/or
emission phenomenon. One of the most important examples of light scattering
spectroscopy is Raman spectroscopy. It is used for qualitative purpose. It is helpful
for the identification of molecules and chemical bonds.

2.6 Applications

Spectroscopic techniques have wide range of applications in the field of pharmaceu-

tical analysis. The most important are as follows:

2.6.1 Quantitative Analysis

Spectrophotometer is widely used in the quantitative pharmaceutical analysis. In

quantitative pharmaceutical analysis, the unknown concentration of analyte may be
determined with the help of absorption spectrophotometry because most of the
biological organic compounds have the ability to absorb EMR in UV-VIS region.
For example, nucleic acids and proteins absorb at 254 nm and 280 nm, respectively.
26 2 Introduction to Spectrophotometric Techniques

2.6.2 Qualitative Analysis

UV-VIS spectrophotometer is used to identify the compounds both not only in pure
form but also in biological preparations. Identification of compounds can be done by
plotting the absorption spectrum of the analyte because the absorption at specific
wavelength gives some hints to the structure of the compound. For example,
absorption at 220–280 nm indicates that the analyte may be aliphatic, alicyclic
hydrocarbons or their derivatives. Absorption at 250–330 nm indicates that the
analyte may contain more than two conjugated double bonds.

2.6.3 Enzyme Assay

Enzyme assay is basically performed with the help of spectrophotometer. In this

assay, the substrate absorbs light in UV region. For example, lactate dehydrogenase
(LDH) is an enzyme that converts lactate to pyruvate with the help of co-enzyme
NAD+. In this reaction, LDH transfer the electrons from lactate to NAD+ as shown in
the following equation.

Lactate þ NADþ $ Pyruvate þ NADH þ Hþ :

In spectrophotometer, NADH absorbs radiations at the wavelength of 340 nm in

UV range, while NAD+ does not absorb radiation at this wavelength.
Another example of enzyme assay is pyruvate kinase that converts phosphoenol-
pyruvate to pyruvate and produces one molecule of ATP. Then pyruvate is converted
into lactate.

Phosphoenolpyruvate þ ADP $ Pyruvate þ ATP

Pyruvate þ NADH þ Hþ $ Lactate þ NADþ :

In this reaction, NADH is oxidized to NAD+ by converting pyruvate into lactate.

As NAD+ does not absorb radiations at 340 nm in UV range, the absorbance
decreases which indicate the conversion of pyruvate to lactate.

2.6.4 Molecular Weight Determination

Spectrophotometer can help to determine the molecular weight of the analyte.

Spectrophotometer can only determine the molecular weight of the small
compounds.
Further Reading 27

2.6.5 Physicochemical Properties

Spectrophotometer can be used to determine the following physicochemical

properties of the analyte:

1. Heats of formation for molecular addition in compounds and/or complexes.

2. Determination of the empirical formula of the substrates.
3. Dissociation constants of acids and bases.
4. Determination of the rate of reactions of the substrates.
5. Quantitative and qualitative determination of the complexes.
6. Hydration equilibration of carbonyl compounds.

Further Reading
Beckett A, Stenlake J (1997) Practical pharmaceutical chemistry, part II, vol 1. CBS Publications
and Distributors, New Delhi, pp 275–300
Gauglitz G, Dakin JP (2017) Spectroscopic analysis. In: John PD, Robert GWB (eds) Handbook of
optoelectronics, vol 2. CRC Press, Boca Raton, pp 569–600
Gauglitz G, Moore DS, Vo-Dinh T (2014) Handbook of spectroscopy. Wiley, Hoboken
LibreTexts™. Introduction to spectroscopy. Accessed 2 Sept 2019
Pavia DL, Lampman GM, Kriz GS, Vyvyan JA (2014) Introduction to spectroscopy. Cengage
Learning, Boston
Sudha PC (2012) Pharmaceutical analysis. Pearson Education India, Chennai
Waters Corporation. Introduction to spectroscopy. Accessed 10 Aug 2019
Watson DG (2015) Pharmaceutical analysis E-book: a textbook for pharmacy students and phar-
maceutical chemists. Elsevier Health Sciences, Amsterdam
Ultraviolet-Visible (UV-VIS) Spectroscopy
3

Abstract
UV-VIS spectroscopy is considered as the most important spectrophotometric
technique that is most widely used for the analysis of variety of compounds. This
technique works on the basis of the measurement of interaction of electromag-
netic radiations (EMR) with matter at particular wavelength. In this chapter, we
have briefly described the UV-VIS spectroscopy by covering the fundamentals
of UV-VIS spectroscopy, origin of spectra along with the types of electronic
transitions. We have also described the effect of solvents on the absorption
spectra of analyte. For proper working of UV-VIS spectroscopy and to get
accurate results, it is very important to understand the components of UV-VIS
spectroscopy and their individual role in the proper functioning of UV-VIS
spectrophotometer. In UV-VIS spectroscopy, absorption of light is the basic
phenomenon and we have also described the various absorbance laws on which
UV-VIS spectroscopy works. At the end of this chapter, we have also discussed
the various terms that are used in this spectroscopy along with the diverse
applications of this analytical technique.

Keywords
Electronic transitions · Absorption spectra · Absorbance laws · Beer–Lambert
law · Chromophore

3.1 Introduction

UV-VIS spectroscopy is considered as the oldest analytical technique that can be

defined as the spectrophotometric technique which is used to measure the intensity
of light in UV (10–400 nm) and VIS (400–800 nm) regions as a function of
wavelength. The wavelengths of UV and VIS radiations are usually expressed in
nanometers (nm). The analyte absorbs the light of specific wavelength (UV and VIS
only) and the amount of radiation absorbed by the analyte is measured. The spectrum

# Springer Nature Singapore Pte Ltd. 2020 29

M. S. H. Akash, K. Rehman, Essentials of Pharmaceutical Analysis,
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30 3 Ultraviolet-Visible (UV-VIS) Spectroscopy

Fig. 3.1 Schematic representation of wave phenomenon

produced after the absorption of UV-VIS light results from the interaction of EMR in
UV-VIS region with the analyte. It forms the basis to analyze a variety of substances
like organic, inorganic, biochemical, and pharmaceutical compounds. In UV-VIS
spectroscopy, absorption of radiations occurs at electronic energy levels (one of the
three basic energy levels, i.e., electronic, vibrational, and rotational energy levels) of
molecules; therefore, this technique is also known as electronic spectroscopy.
Electromagnetic waves (Fig. 3.1) are described in terms of frequency (v), wave-
length (λ), and distance present between the two peaks. The wavelength can be
defined as the distance between the two adjacent parts of a wave existing in the same
phase, i.e., the distance between the adjacent troughs or crest (Fig. 3.1). Frequency
can be defined as the number of times electrical field radiation oscillates in 1 s. Hertz
(Hz) is the unit used for frequency and 1 Hz ¼ 1 cycle per second.
The arithmetic relationship among the speed of light (c), the wavelength (λ), and
frequency (ν) can be written as:

c ¼ vλ:

According to the laws of quantum mechanics, photon is subjected to energy, so

that the above equation may be considered as:

hc
E ¼ hν ¼ ,
λ
where E represents the radiation energy, v is the frequency, h is the Planck’s
constant, c is speed of light, and λ is the wavelength of light that is going to be
absorbed.

3.2 Principle

UV-VIS spectroscopy works on the basis of the absorption phenomenon of light

and the amount of absorbed light is directly proportional to the amount of analyte
present in a sample solution. As the concentration of analyte is increased, absorption
3.4 Electronic Transitions 31

of light is also increased linearly, whereas the transmission of light is decreased

exponentially. In UV-VIS region, the absorption of radiation depends on the
electronic configuration of the absorbing species like atoms, molecules, ions, or
complexes.
An electronic energy level consists of various vibrational energy levels, whereas a
single vibrational energy level consists of various rotational energy levels. When a
photon interacts with a molecule, it may induce a transition in electronic energy
levels if energy provided by the photon matches with energy difference in these
levels. The amount of radiations absorbed by the analyte is measured and plotted
against the wavelength of EMR in order to obtain the spectrum. Thus, a typical
UV-VIS spectrum is a plot of wavelength or frequency versus the intensity of
absorption.

3.3 Theory

In a molecule, electrons are associated with more than one nucleus and they are
involved in making bonds among the various atoms. These bonding electrons are
susceptible to transitions in various energy levels under the provocation of appropri-
ate radiations. The details of various levels of electronic transitions have been briefly
described in the following sub-sections.

3.4 Electronic Transitions

The absorption of electronic energy levels (UV-VIS radiations) by an organic

molecule is associated with the excitation of valence electrons from ground state
to excited state (Fig. 3.2). After absorption of energy, the electronic transitions are
generally occurred from the excited state that contain the highest molecular orbital
(this phenomenon is known as antibonding). The wavelength of the absorbed
radiation depends upon the difference of energy between the orbital (E2) which is
originally occupied by the electron (ground state) and the orbital to which it is
promoted (E1).

Fig. 3.2 Schematic E1 (Excited state)

representation of electronic hv
energy levels
Absorption of light energy
Light
E2 (Ground state)
32 3 Ultraviolet-Visible (UV-VIS) Spectroscopy

Fig. 3.3 Schematic

representation of various
levels of electronic transitions

3.4.1 Types of Electronic Transitions

Electrons in an organic molecule are involved in bonding as strong (σ bond), weak

(π bond) or present as non-bonding lone pair. Based on the nature of the bond present
in organic molecule, absorption of electronic transitions that are associated with the
absorption of UV-VIS radiations are of following four types (Fig. 3.3).

3.4.1.1 s ! s Electronic Transition

These electrons are held firmly within the molecule. Large amount of energy is
required for the transition of σ (bond) to σ (antibonding) that is corresponding to the
far UV region with the range of 120–200 nm. This type of transition is present in
saturated hydrocarbons such as CH3–CH3 that contain only strongly bound σ
electrons and the excitation of these electrons from σ orbital to σ orbital requires
large amount of energy. Methane (CH4) shows σ ! σ electronic transition at λmax at
122 nm.

3.4.1.2 p ! p Electronic Transition

This type of transition is present in the compounds that contain double and/or triple
bonds or aromatic rings and the excitation of electrons requires less energy as
compared to the σ ! σ electronic transition which indicates that π ! π electronic
transition occurs at larger wavelength (160–190 nm). For example, ethylene shows
absorption at 171 nm, whereas a conjugated unsaturated bond such as 1,3-butadiene
absorbs at a much higher wavelength (217 nm). Acetone (CH3)2C¼O and benzene
(C6H6) show π ! π electronic transition at λmax of 122 nm and 255 nm,
respectively.

3.4.1.3 n ! s Electronic Transition

The excitation of electrons in an unshared pair form on an atom to an antibonding
sigma orbital is called as n ! σ electronic transition. This type of transition is
present in saturated molecules such as halides, ethers, alcohols, etc. The excitation in
n ! π electronic transition involves the excitation of an electron from n orbital
(non-bonding) of the heteroatom to an antibonding sigma orbital (σ) of the mole-
cule. The energy required for the transition of electrons from n orbital (non-bonding)
3.5 Origin of Absorption Spectra 33

to antibonding sigma orbital (σ) is less than that of the σ ! σ electronic transition
which results in the absorption of longer wavelength of the UV-VIS region (between
150 and 250 nm). For example, methyl alcohol shows n ! σ electronic transition at
183 nm, whereas trimethylamine at 227 nm. Trimethylamine does not show n ! σ
electronic transition in aqueous acid because the protonated amine does not contain
non-bonding electrons. Chloromethane (CH3Cl) and benzene (C6H6) show n ! σ
electronic transition at λmax of 173 nm and 277 nm, respectively.

3.4.1.4 n ! p Electronic Transition

In this type of transition, the unshared pair electrons on heteroatoms are excited to
antibonding pi star (π) orbital and in this orbital, the non-bonding electrons are held
loosely; therefore, the transition requires longer wavelengths. This type of transition
occurs in compounds that contain double bonds involving heteroatoms having
unshared pair of electrons such as C¼O, C¼S, N¼O, etc. For example, ketones
and aldehydes exhibit two bands at 180–200 nm (π ! π) and 280 nm (n ! π).
n ! π electronic transition is not strong as the electrons in non-bonding n orbital
are situated at perpendicular to the plant of the π bond and hence the chance of the
jump of an electron from n orbital to pi star orbital (π) becomes low.

3.5 Origin of Absorption Spectra

The absorption of UV-VIS radiations is based on the electrons in molecules that may
be promoted to higher energy levels after the absorption of energy. The electrons
present in molecules can be categorized into the following types:

3.5.1 s-Electrons

These electrons are involved in making single covalent bond and high energy
radiation is required in order to excite them.

3.5.2 p-Electrons

These electrons are involved in making either double or triple bond and relatively
low energy radiation is required in order to excite them.

3.5.3 n-Electrons

These types of electrons are attached with nitrogen, oxygen, and chlorine as a lone
pair of electrons and also known as non-bonding electrons. These electrons require
less energy for excitation as compared to both σ and π electrons.
34 3 Ultraviolet-Visible (UV-VIS) Spectroscopy

3.6 Effect of Solvent on Absorption Spectra

Nature of the solvent (either polar or non-polar) in which the analyte is present
may interfere with the absorption spectra of analyte. Saturated hydrocarbons are
non-polar solvents and if are highly pure, they do not interfere with the solute
molecules neither in ground state nor in excited state. The absorption spectra of
analyte in such type of solvent are similar to that in pure gaseous state. Polar solvents
like water or alcohols may stabilize or destabilize orbitals of molecules both in
ground and excited state. Solvents (both polar and non-polar) play distinguished
role in the absorption spectra determined by UV-VIS spectroscopy. Therefore, the
solvent should not interact with the analyte and should not interfere with the
absorption spectra of analyte.

3.7 Effect of Electronic Transitions on Absorption Spectra

Despite the nature of the solvent, electronic transition state may also affect the
spectrum of the analyte. Among the four types of electronic transitions (n ! π,
n ! σ, π ! π, and σ ! σ), n ! π and π ! π electronic transitions affect the
absorption spectrum of analyte.

3.7.1 Effect of p ! p Electronic Transitions on Absorption Spectra

In π ! π transition, the excited state shows more polarity as compared to that in

the ground state, whereas the dipole–dipole interaction with solvent decreases the
energy of electrons at excited state as compared to that in ground state. Thus, polar
solvents decease the π ! π electronic transition energy and the absorption maxima
of the analyte appears ~10–20 nm (red shift or bathochromic shift) when the polar
solvent is changed from hexane to ethanol.

3.7.2 Effect of n ! p Electronic Transitions on Absorption Spectra

In n ! π electronic transitions, polar solvents with hydrogen bonds contain more

readily polar molecules at ground state as compared to that of their excited state and
hence in polar solvents, the electronic transition energy is increased.

3.8 Components of UV-VIS Spectrophotometer

Following are the main components of UV-VIS spectrophotometer:

3.8 Components of UV-VIS Spectrophotometer 35

3.8.1 Light Sources

It provides a sufficient light in the form of polychromatic light that is appropriate for
marking the measurements. Usually, light source provides polychromatic light over
a wide range of spectrum (Fig. 3.4). In UV-VIS spectrophotometer, two types of
light sources are used. For UV sources, deuterium, hydrogen, tungsten, mercury, and
xenon lamps are used, whereas for VIS sources, tungsten, mercury vapor, and carbon
lamps are used.

3.8.2 Monochromator

It is a device that receives the polychromatic light as input from a lamp and provides
output in the form of monochromatic light (Fig. 3.5). This device is used to disperse
the radiations of polychromatic light according to the wavelength.

3.8.2.1 Components of Monochromator

Monochromator consists of three essential components (Fig. 3.6) that are collec-
tively involved to disperse polychromatic light into monochromatic light. These are
entrance slit, dispersing device, and exit slit.

Fig. 3.4 Schematic

representation of light source

Fig. 3.5 Schematic

representation of
monochromatic light

Fig. 3.6 Schematic

representation of components Entrance slit
of monochromator White light

Dispersion device

Monochromatic light
Exit slit
36 3 Ultraviolet-Visible (UV-VIS) Spectroscopy

Entrance Slit
It sharply defines the incoming polychromatic beam of light and imparts it on
dispersing element.

Dispersing Device
It is a special plate that contains hundreds of parallel grooved lines. These lines are
used to separate the polychromatic light (white light) into visible light spectrum. It
disperses the polychromatic light that is coming from the entrance slit into its
component wavelengths with the help of focusing lens. Dispersing element, such
as prism or grating is usually made up of quartz, fused silica, or glass.

Exit Slit
Exit slit allows the minimal wavelength together with the band of wavelengths on
either side. The position of exit slit is not fixed and it is mostly needed to adjust it by
rotation to vary the minimal wavelength passing through the exit slit.

3.8.2.2 Working of Monochromator

Polychromatic light is collimated and hits the dispersing element at an angle and
splits into its component wavelength by prism or grating after entering monochro-
mator through the entrance slit. By rotating the dispersing element and exit slit,
radiation of required particular wavelength is obtained from monochromator via
exit slit.

3.8.3 Sample Device/Cuvette

Cuvettes are available in various forms for UV-VIS region (Fig. 3.7). They are
specifically designed to hold the sample for spectrophotometric analysis. They vary
with respect to shape, size, path length, and transmission characteristics of required
wavelength. Cuvettes are made up of plastic, glass, or optical grade quartz that does

Fig. 3.7 Schematic representation of sample device or cuvette

3.8 Components of UV-VIS Spectrophotometer 37

not absorb the λ range of interest. Before to do the analysis, cuvette should be
cleaned thoroughly and they should be free from all types of impurities that may alter
the spectroscopic reading.

3.8.4 Detector

It is a device that can be used for the measurement of the amount of light passing
through the sample and convert the light signals (coming from the sample) into the
electrical signals. In order to detect the radiation in UV-VIS spectroscopy, three
kinds of photosensitive devices are used which are as follows:

3.8.4.1 Photovoltaic Cells or Barrier Layer Cell

It contains metallic base plate such as iron or aluminum that acts as an electrode.
Semiconductor, like silicon or germanium, is deposited on its surface. Over it, thin
layer made of gold or silver is present that acts as second collector tube. When light
falls over Se, electrons are generated at Se-Ag surface and these electrons are
collected by the silver. Excess of electrons on silver surface produces the voltage
difference between the silver and base of cell.

3.8.4.2 Phototubes or Photoemissive Tubes

It contains an evacuated glass tube. A light sensitive cathode is present inside
it. Cathode is coated from inner side with light sensitive material like silver oxide
and potassium oxide. When cathode is exposed to radiation, there is the emission of
photoelectrons and they are collected by anode and are returned through the external
circuit. Current is amplified and recorded by this process.

3.8.4.3 Photomultiplier Tubes

It is the most commonly used type of detector. It contains a photoemissive cathode. It
contains numerous dynodes (emit numerous electrons in response to each electron
hitting them) and an anode. A photon strikes to the cathode after entering the tube
and it causes emission of numerous electrons that accelerate to the first dynode. First
dynode is 90 V more positive as compared to that of cathode, several electrons
emitted for each incident electron are further accelerated and more electrons are
produced by this process, eventually electrons are collected at anode. By this way,
each original photon produces 106–107 electrons, resulting current is amplified and
recorded.

3.8.5 Recorder

Spectrometer provides the signals to the recorder and intensity of signals depends
upon light absorption by the analyte at a particular wavelength. These signals are
amplified by amplifier and elaborated by the personal computer. Digital read-out
38 3 Ultraviolet-Visible (UV-VIS) Spectroscopy

devices like light emitting diode or liquid crystal display (LCD) are used for clarity
purpose that removes the ambiguity.

3.9 Types of UV-VIS Spectrophotometer

Design of spectrophotometer is based on the fundamental principle of light absorp-

tion by absorbing species. Based on the number of cuvettes and beams used,
UV-VIS spectrophotometer is classified into the following two types:

3.9.1 Single-Beam UV-VIS Spectrophotometer

In this type of spectrophotometer, single beam and/or cuvette is used for the analysis
(Fig. 3.8). In single-beam UV-VIS spectrophotometer, a single light beam passes
across the cuvette. The spectrophotometer is calibrated by putting the reference
and/or standard solution in the cuvette and then the resulting value of absorbance
is subtracted from sample measurements in order to remove the effects produced by
the solvent and cell. This type of spectrophotometer is appropriate for applications in
the wavelength range of 190–1100 nm. Various types of samples including nucleic
acid, proteins, and a number of organic molecules are often analyzed in this region.
Whereas, the visible region of the electromagnetic spectrum is from 340 to 750 nm,
and this region is appropriate for the analysis of colored samples.

3.9.2 Double-Beam UV-VIS Spectrophotometer

In this type of spectrophotometer, two beams and/or cuvettes are used for the
analysis (Fig. 3.9). The light beam coming from the source of light is split into the
sample and reference beam with the help of mechanical chopper. The role of
reference beam is to monitor the intensity of light energy while the sample beam

Fig. 3.8 Schematic representation of single-beam spectrophotometer

Fig. 3.9 Schematic representation of double-beam spectrophotometer

3.10 Absorbance Laws 39

shows absorption of light from the sample. The value of observed absorbance which
is the ratio of the sample and reference beam is recombined before it enters into the
monochromator. This arrangement compensates the effects of light reference and
sample beam due to drift in lamp intensity, electronic and mechanical fluctuations
which affect the reference and sample beams equally. Although, double-beam
UV-VIS spectrophotometers are bit complicated as compared to that of single
beam, still they have some advantages which are as follows:

1. The sample and reference run simultaneously. There is no need to adjust zero at
each wavelength or replace the blank with sample.
2. The ratio of the powers of reference and sample is constantly obtained.
3. The scanning speed is rapid over a wide range of wavelength and digital read-out
device is used to record the electrical signals.

3.10 Absorbance Laws

There are basically three laws for spectroscopy that describe the absorbance of light
through a material. The details of these laws have been described below.

3.10.1 Beer’s Law

It can be described as the beam intensity of the monochromatic light is decreased

exponentially when the concentration of analyte increases arithmetically (Fig. 3.10).
In quantitative analysis, primarily concerned with solutions, the effect of concentra-
tion of the colored constituent in solution depends upon the light absorption or
transmission. It can be expressed as:

I ¼ Ieklc :

3.10.1.1 Beer Derivation

In 1852, Beer described the following relationship between the concentration of a
solution and absorbance of light:

Fig. 3.10 Schematic representation of % transmittance vs. absorbance

40 3 Ultraviolet-Visible (UV-VIS) Spectroscopy

I0 kn c kn c
log ¼ or A ¼
I T 2:303 2:303
where c is the concentration of analyte in sample solution, kn is a proportionality
constant, and A is the absorbance of light.
The amount of light absorbed can be measured by multiple ways:

T ¼ P=Po

%T ¼ 100T:

And the absorbance (A) can be calculated as:


Po
A ¼ log 10
P


1
A ¼ log 10
T


100
A ¼ log 10
%T
A ¼ 2  log 10 %T:

The last equation (A ¼ 2  log10 %T) allows to calculate the absorbance of light
from % transmittance data.

3.10.2 Lambert’s Law

The rate of decrease in the intensity of incident light with the thickness of the
medium is directly proportional to the intensity of incident light. It can also be stated
that the intensity of emitted light is decreased exponentially as the thickness of
absorbing medium is increased. It is expressed as:

I ¼ Iek2l :

3.10.3 Beer–Lambert Law

Beer–Lambert law (or Lambert–Beer law) describes a linear relationship between

the absorbance of light and the concentration of absorbing species. The Beer–
Lambert law is written as:

I ¼ I o ekcl :
3.10 Absorbance Laws 41

This law is derived by combining the Beer’s law and Lambert’s law that
associates the light absorption with the properties of sample across which the light
travels.

3.10.3.1 Deviations from Beer–Lambert Law

Ideally, Beer’s law is applicable only with truly monochromatic radiation.
According to the Beer’s law, a linear calibration curve for the absorbance of
monochromatic light versus the analyte concentration in a series of standard
solutions must be a straight line with an intercept of 0, but practically, calibration
curve is not found to be linear (Fig. 3.11).
If the incident radiation consists of just two wavelengths λ0 and λ00 , with powers
P0 and P000, considering that A ¼ log (P/P0), then the power of the radiation to come
0

out from (P) the cell for each wavelength would be:
0
P0 ¼ P00 10ε bC
00
P00 ¼ P000 10ε bC

where ε0 and ε00 are the molar absorptivities of each wavelength. Therefore, the
measured absorbance Am may be
  !
P0 þ P00 P00 þ P000
Am ¼  log ¼ log :
P00 þ P000 0 00
P00 10ε bC þ P000 10ε bC

The last equation indicates a non-linear relation between Am and C. The

proportionality between Am and C is restored only if ε0 ¼ ε00 . The same situation

Fig. 3.11 Schematic

Positive deviation
representation of deviations
from Beer’s law Ideal condition

Negative deviation
Absorbance

Concentration
42 3 Ultraviolet-Visible (UV-VIS) Spectroscopy

Fig. 3.12 Schematic

representation of absorbance
versus wavelength (left) and
concentration (right)

occurs when a radiation consists of many wavelengths. The situation is illustrated in

Fig. 3.12.
When a polychromatic radiation beam is used, its preferable position (in terms of
central wavelength) is on the top of a relatively wide absorption peak (position 1 in
Fig. 3.12). In this case, the proportionality between absorbance (A) and concentra-
tion (C) is maintained, because the molar absorptivities are practically the same for
all wavelengths. On the other hand, marked deviations are expected when the band is
positioned in spectral regions such as the sides of absorption peaks (position 2 in
Fig. 3.12), where a wide range of molar absorptivity values is expected. It has been
found that deviations from Beer’s law are insignificant for the measurement of
absorbance at the maximum of narrow peaks, if the effective bandwidth of the
incident beam is less than 1/10 of the width of the absorption peak at half height.

3.10.3.2 Limitations from Beer–Lambert Law

Beer–Lambert law shows a direct correlation between the absorbance (A) of given
analyte to the concentration (c) and path length (b) of the sample. This relationship is
linear but, under certain conditions, this relationship breaks down and gives a
non-linear relationship. The most important deviations from Beer–Lambert law
may be categorized into the following three categories:

Real Deviations
Beer and Lambert laws describe the absorption characteristics of solutions that have
relatively low concentrations (<10 mM) of solute and/or analyte dissolved in
it. When analyte concentration in a given solution is higher (>10 mM), then the
analyte begins to behave differently owing to the interactions with surrounding
solvent molecules or other solute molecules present in solution and hydrogen
bonding also plays a significant role in this regard.
3.10 Absorbance Laws 43

Chemical Deviations
This type of deviation is observed owing to the presence of particular chemical
species in sample which is being analyzed. Various factors are involved in chemical
deviation notably association, dissociation, polymerization, complex formation, and
interaction of analyte with solvent to make a product having different absorption
properties. Followings are the examples of chemical deviations of Beer–Lambert
law:

1. Phenomenon of resonance transformation occurs in phenol red. Its medium

changes from acidic form (yellow) to the basic form (red) (Fig. 3.13). Due to
this resonance, electron distribution of the bonds in phenol molecule changes as
the pH of the solvent in which it is dissolved changes from acidic medium to the
basic medium. UV-Visible spectroscopy is an electron-related phenomenon, and
absorption spectrum of the sample changes with the change in pH of the solvent.
Acidic and basic forms of phenol red along with their UV spectra at different
pH exhibit the chemical deviations of Beer–Lambert law in UV-Visible
spectroscopy.

Fig. 3.13 Effect of pH on

phenol red along with their
UV spectra demonstrates
chemical deviations of Beer–
Lambert law in UV-Visible
spectroscopy
44 3 Ultraviolet-Visible (UV-VIS) Spectroscopy

2. Concentrated solution of benzoic acid in sample has lower pH and contains

higher proportion of unionized form than that of dilute solution. The absorption
wavelength of unionized benzoic acid is 273 nm, whereas for ionized form of
benzoic acid, the absorption wavelength is 268 nm. The increased wavelength of
unionized form of benzoic acid indicates that increasing the concentration
of benzoic acid increases the wavelength (273 nm) with positive deviation from
Beer’s law. Whereas, the lower absorption of wavelength (268 nm) at low
concentration of benzoic acid gives negative deviation from Beer’s law.
3. Methylene blue at the concentration of 105 M exists as monomer with λmax of
660 nm, but at concentration above 104 M exists as dimer with λmax of 600 nm.
4. In unbuffered solution of potassium dichromate, the dissociation of dichromate
ions is observed by lowering the pH.
5. Insufficient time for the completion of reaction (incomplete reaction) also
produces deviation from Beer’s law. For example, determination of iron using
thioglycolic acid before completion of reaction.

Instrumental Deviations
These deviations may occur while measuring the absorbance of analyte. Experimen-
tal measurements are usually made in terms of transmittance (T), which is defined as:

T ¼ I=I o

where Io denotes initial intensity of light and I is the intensity of light when it passes
through the sample. The relationship between T and A can also be represented as
follows:

A ¼  log T ¼  log ðI=I o Þ:

Modern light absorption devices generally display the data either as absorbance,
transmittance, or %-transmittance. Beer’s law may be used in order to measure the
unknown concentration of an analyte by measuring the amount of light being
absorbed by the sample (Fig. 3.14). When the absorptivity coefficient is not
known, the unknown concentration of analyte may be estimated by using a working
curve of absorbance versus concentration derived from standards.

Fig. 3.14 Schematic

representation of absorption
of light by sample
3.10 Absorbance Laws 45

3.10.3.3 Due to Polychromatic Radiation

Beer–Lambert law is strictly followed when a source of monochromatic light exists
but practically, grafting unit (monochromator) or filter is used to create a monochro-
matic beam of light from the source of polychromatic light. Deviations from Beer–
Lambert law are minimal, when the molar absorptivity of analyte remains essentially
constant at selected wavelength on spectrometer. If the molar absorptivity of analyte
changes at selected wavelength, then the absorbance of analyte does not follow
Beer–Lambert law. It is observed (Fig. 3.15) that the deviations in absorbance over
the wavelengths are minimal when the wavelength observed is at the λmax. Due to
this reason absorption measurements are taken at wavelengths.
Figure 3.15 shows a difference in deviations in absorbance when values are
obtained at λmax of absorbance (band A) vs. other wavelengths of absorbance
(band B). Figure 3.15b shows the deviations in Beer–Lambert law due to the
observations made at wavelengths other than λmax.

3.10.3.4 Due to the Presence of Scattered Radiation

Scattered radiation refers to radiation from the device that is outside the nominal
selected wavelength band. Generally scattered radiation wavelength varies from the

a Large change in absorbance

per unit change in wavelength
Minimal change in absorbance
per unit change in wavelength

Band A

Band B
Absorbance

Wavelength
b
Band A
Absorbance

Band B

Concentration

Fig. 3.15 Deviation from Beer–Lambert law due to polychromatic radiation

46 3 Ultraviolet-Visible (UV-VIS) Spectroscopy

selected wavelength band. It has been observed that radiations from a monochroma-
tor are often contaminated with small quantity of scattered radiation. Usually,
scattered radiation is produced due to the reflection and/or scattering of light by
the surface of grating, mirrors, filters, lenses, and windows. When the analyte
absorbs at the wavelength of the scattered radiation, a deviation from Beer–Lambert
law is observed comparable to the deviation due to the polychromatic radiation.

3.10.3.5 Due to Mismatched Cuvettes

Beer–Lambert law cannot be followed when the cuvettes containing the analyte and
the blank solutions have variable path lengths, or unequal optical characteristics. In
such cases when absorbance versus concentration is plotted, the curve will have an
intercept k and the equation will be defined as:

A ¼ εbc þ k:

3.10.3.6 Theoretical Limitations

Beer’s law is applicable only if the concentration of analyte is low. At higher
concentrations, the individual particles of analyte no longer behave independently
with one another and do not follow this law. The resulting interaction between the
particles of analyte may change the value of a or ε. The values of a or ε depend on
sample’s refractive index, whereas the refractive index varies with the analyte’s
concentration.

3.11 Terms Used in UV-VIS Spectroscopy

In UV-VIS spectrophotometry, two main terms, namely chromophore and

auxochrome, are used. The detailed descriptions of these two terms are as follows:

3.11.1 Chromophore

Chromophore is derived from the Greek word “Chromophorus” which mean the
color carrier and it can be defined as “the part of a molecule that is covalently bonded
with unsaturated group responsible for imparting the color after absorbing the light
in UV or VIS region” is called as chromosphere, or it can also be defined as “the
functional group containing multiple bonds capable of absorbing radiations above
200 nm.” It can also be defined as “any structural feature that is present in the
molecule and responsible to absorb EMR and hence impart color to the compound.”
For example, in nitro compounds, the yellow color is produced due to presence of
NO2 group and hence NO2 is considered as chromophore. Typical examples of
chromophore are NO2, N¼O, C¼C, C¼N, CN, C¼O, C¼S, etc.
White light is composed of various colors (radiations). When a specific com-
pound is placed in the path of white light, a specific color is produced in human eye
3.11 Terms Used in UV-VIS Spectroscopy 47

Table 3.1 Various regions Region Complementary color Wavelength (nm)

of visible spectrum and
Violet Yellow-green 400–435
their corresponding colors
Blue Yellow 435–480
Green-blue Orange 480–490
Blue-green Red 490–500
Green Purple 500–560
Yellow-green Violet 560–580
Yellow Blue 580–595
Orange Green-blue 595–650
Red Blue-green 650–800

because it absorbs some specific color from the white light while transmitting the
remaining colors. The precise color of the compound that it reflects depends on the
wavelength it absorbs from the white light. For instance, if a compound absorbs blue
light (435–480 nm) from the white light, it will transmit the yellow light; then
compound will produce the yellow light to the human eye. The transmitted or
reflected color from the compound is known as the complementary color of the
absorbed color. Table 3.1 contains the complementary color of the white light which
can be produced from each region.
All the compounds that contain chromophore in their molecules generally contain
π electrons and many of them also contain non-bonding (n) electrons. Those
functional groups that contain both n and π electrons undergo three types of
electronic transitions, i.e., n ! π, π ! π, and n ! σ, in addition to σ ! σ
electronic transitions.
Saturated hydrocarbons only contain σ electrons and hence σ ! σ electronic
transitions are involved in these compounds which require energy that is only
available in vacuum UV region. Therefore, saturated hydrocarbons do not show
absorption in UV-VIS region (200–800 nm) and thus can be used as solvent for the
spectral analysis throughout this region. Similarly, saturated compounds that possess
heteroatoms, such as halogens, nitrogen, and oxygen, contain non-bonding
(n) electrons in addition to σ electrons. That is why, in addition to σ ! σ electronic
transition, n ! σ electronic transition is involved and majority of such types of
compounds that involve σ ! σ electronic transition do not show absorption in
UV-VIS region and hence are commonly used as solvent for spectral analysis of
analyte.
Following points should also be considered to interpret UV-VIS spectrum:

1. Non-conjugated alkenes have the absorbance at less than 200 nm of wavelength.

Therefore, they are inaccessible to UV spectrophotometer. For example, if double
bonds are conjugated in a compound, λmax is shifted towards longer wavelength.
1,5-hexadiene (H C 2
CH
) has λmax ¼ 178 nm and 2,4-hexadiene
2

CH3
( H 3C ) has λmax ¼ 227 nm.
48 3 Ultraviolet-Visible (UV-VIS) Spectroscopy

2. Non-conjugated compound with carbonyl group gives a weak absorption band in

O

the 200–300 nm region. For example, acetone ( ) which has λmax ¼ 279 nm
H3 C
C
CH3

and that cyclohexane ( ) has λmax ¼ 291 nm.

3. Conjugation of C¼C and carbonyl group shifts the λmax of both groups to a longer
wavelength. For example, ethylene (H2C CH2) has λmax ¼ 171 nm, acetone
O O

( H 3C
C ) has λmax ¼ 279 nm, and crotonaldehyde (H C
CH3 2
C
CH3
) has λmax ¼ 290 nm.

3.11.2 Auxochrome

The wavelength of absorption maxima for specific molecule or compound not only
depends on the nature of the functional group present in the molecule, but also on the
nature of chromophore. There are certain functional groups, such as –SH, –NH2, –
OH, and halogens, which are not chromophore themselves (do not show absorption
at wavelength higher than 200 nm), once they are attached to a chromophore, they
enhance the absorption of chromophore to shift towards a longer wavelength,
accompanied by an increased intensity. Such functional groups are known as
auxochromes. Auxochromes when attached to a particular chromophore, they alter
the ability of a chromophore to absorb light and alter the intensity of light absorption.
Auxochrome in fact is a color enhancing group that contains non-bonding
(n) electrons that do not absorb EMR themselves in near UV region, but when
they attached to a chromophore, alter the wavelength and intensity of absorption of
that chromophore. Therefore, they can be defined as “any group whose presence
bring about the shift of absorption band towards a longer wavelength.” For example,
benzene ( ) shows λmax at 255 nm, but if any group (e.g., –OH) when attached to
benzene, it increases the λmax such as phenol ( ), it has λmax at 270 nm. In phenol,
–OH group is an auxochrome. Similarly, when –NH2 group is attached to benzene
ring, then it increases the λmax towards longer wavelength (aniline ( ) has λmax at
280 nm) as compared to that of –OH group which indicates that –NH2 group is more
powerful than the –OH group.

3.12 Absorption and Intensity Shifts in UV-VIS Spectroscopy

The subsituent group when attached on a basic structure of chromophore and

replaces hydrogen, cause the change of the intensity and position of absorption
band of the chromophore. The subsituent groups do not show the absorption in
UV-radiation themselves. But due to the presence of substituent group, absorption of
the principal chromophore is modified. Subsituents (possibly the auxochromes)
may either increase or decrease the intensity of absorption, and the possibily the
3.12 Absorption and Intensity Shifts in UV-VIS Spectroscopy 49

Hyperchromic effect

Absorbance (A)
Blue shift Red shift

Hypochromic effect

λmax
Wavelength (λ)

Descriptive term Nature of the shift

Bathochromic shift (Red shift) Towards longer wavelength
Hypsochromic shift (Blue shift) Towards shorter wavelength
Hyperchromic effect Towards higher absorbance
Hypochromic effect Towards lower absorbance

Fig. 3.16 Schematic representation of absorption and intensity shifts (upper part of the figure) and
their relevant descriptive terms (lower part of the figure in the form of table)

Fig. 3.17 Effect of hydroxyl

group on absorption maxima
of p-nitrophenol

wavelengths as well (Fig. 3.16). Following are the four types of absorption and
intensity shifts:

3.12.1 Bathochromic Shift (Red Shift)

If λmax of the given compound shifts towards the longer wavelength, this type of shift
is known as bathochromic shift or red shift. Bathochromatic shift is produced due to
the presence of an auxochrome in compound or by changing the pH of the medium
in which compound is present. For example, auxochrome groups such as –OH,
–OCH3 when attached on a compound, increase the absorption of compound
at longer wavelength. p-nitrophenol shows bathohromic shift in alkaline medium
(Fig. 3.17) due to the negatively charged oxygen atom that delocalizes more
effectively as compared to unshared pair of electron.
50 3 Ultraviolet-Visible (UV-VIS) Spectroscopy

3.12.2 Hypsochromic Shift (Blue Shift)

If λmax of the given compound shifts towards the shorter wavelength, this type of
shift is known as hypsochromic shift or blue shift. Hypsochromic shift is produced
due to the presence of functional group that causes the removal of conjugation or
with pH change of the medium in which compound is present. For example, aniline
shows hypsochromic shift in acidic medium, because it loses conjugation
(Fig. 3.18).

3.12.3 Hyperchromic Shift

If absorption intensity (ε) of the given compound increases, this type of shift is called
as hyperchromic shift. It may occur due to introduction of an auxochrome which
ultimately results in the increase in intensity of coumpound. For example, when
methyl group (auxochrome) is introduced in the structure of pyridine, it increases the
intensity of pyridine from 2750 to 3560 (Fig. 3.19).
Hyperchromic effect may also occur due to the change of solvent. For example,
phenol shows bathochromic shift along with hyperchromic effect in the presence of
alkaline media (Fig. 3.20).

Fig. 3.18 Effect of acidic

medium on absorption
maxima of aniline

Fig. 3.19 Effect of

auxochrome on intensity of
pyridine

Fig. 3.20 Effect of medium on bathochromic shift along with hyperchromic effect
3.13 Applications 51

Fig. 3.21 Acidic medium shows hypsochromic shift and hypochromic effect in aniline

Fig. 3.22 Effect of

auxochrome on absorption
intensity of naphthalene

3.12.4 Hypochromic Shift

If absorption intensity (ε) of the given compound decreases, this type of shift is
called as hypochromic shift. For example, aniline shows hypsochromic and hypo-
chromic shift in acidic medium. For example, analine shows hypochromic effect and
hypsochromic shift in acidic medium (Fig. 3.21).
It may occur due to the introduction of an auxochrome which ultimately results in
the decrease in intensity of coumpound. For example, naphthalene shows absorption
intensity (ε) at 19,000, but when methyl group (auxochrome) is introduced in the
structure of naphthalene, it decreases the intensity of 2-methyl naphthalene from
19,000 to 10,250 (Fig. 3.22).

3.13 Applications

UV-VIS spectroscopy is regularly used in different fields of science for the qualita-
tive and/or quantitative analysis of different types of analytes. The analysis is usually
carried out when analyte is in solution form but the analyte presents even in gas or
solid, may also be analyzed using UV-VIS spectroscopy. Followings are the impor-
tant applications of UV-VIS spectroscopy:
52 3 Ultraviolet-Visible (UV-VIS) Spectroscopy

3.13.1 Determination of Molecular Weight

Molecular weights of the organic compounds may be determined with the help of
UV-VIS spectroscopy. This method is based upon the formation of a derivative.

3.13.2 Detection of Impurities

UV-VIS spectrophotometric analysis is considered as the best method for detection

of impurities that may be present in organic molecules. Additional peaks observed in
the spectra of pure compound indicate the presence of impurities in pure material
(Fig. 3.23).

3.13.3 Quantitative Analysis

UV-VIS spectroscopy may be utilized for the quantitative analysis of compounds.

This analysis is based on the Beer–Lambert law. Various drugs either in the form of
raw material or in the form of final dosage form may be analyzed by making a
solution of given drug in a suitable solvent and measuring the absorbance at
particular wavelength. For instance, diazepam tablets may be analyzed by measuring
the absorbance of diazepam in 0.5% sulfuric acid in methanol at 284 nm wavelength.
Quantitative analysis of the drug can be done by using reference standard and/or
multiple standard methods and single compound analysis by using separation
technique after extraction and/or after chromatographic separation.

Fig. 3.23 Schematic representation of UV-VIS spectra of standard PCM and/or with impurity
3.13 Applications 53

3.13.4 Qualitative Analysis of Pharmaceuticals

UV-VIS absorption spectroscopy can be used to characterize those compounds

which absorb UV and/or visible radiation. Identification is done by comparing the
absorption spectra of unknown compound and/or analyte with the spectra of known
compounds. A record of UV-VIS absorption curves can be found in certain reference
books.

3.13.5 Detection of Functional Group

It is possible to detect the presence of certain functional group in compound with the
help of UV–VIS spectroscopy. Absence of a band at particular wavelength indicates
the evidence for absence of particular group in compound (Fig. 3.24).

3.13.6 Chemical Kinetics

UV-Visible spectroscopy can be used to study the chemical kinetics of the rate of
chemical reactions. In order to determine the kinetics of reaction, the change in the
concentration of either a reactant or product with respect to time is measured. As
absorbance is directly proportional to the concentration of analyte, UV-VIS spectro-
photometry can be used to follow the course of a reaction. The method is based upon
the fact that one of the reactants of product exhibiting a suitable absorption in UV
region is not overlapped by absorption spectra of other species that may be present in
sample. This method can be employed to study such rates which must be relatively
slow (half-lives of the order of a minute).

0.5

0.45
0.4
0.40 CH3
Absorbance

Absorbance

Benzene 0.3 CH3

0.35
0.2
0.30
0.1 Toluene
0.25
0
170 180 190 200 265 270 275
Wavelength (nm) Wavelength (nm)

Fig. 3.24 Schematic representation for detection of functional group using UV-VIS spectroscopy
54 3 Ultraviolet-Visible (UV-VIS) Spectroscopy

Fig. 3.25 Schematic

representation for
determination of unknown
concentration of a compound
using UV-VIS spectroscopy

3.13.7 Determination of Unknown Concentration

Unknown concentration of any compound can also be measured using UV-VIS

spectroscopy (Fig. 3.25). It involves the following steps:

1. First of all, prepare the samples.

2. Then prepare series of standard solutions having known concentrations.
3. Set spectrophotometer to the wavelength at which sample shows maximum
absorption of light.
4. Draw the standard plot.
5. Finally, measure the absorption of unknown analyte and determine its concentra-
tion with the help of standard plot.

3.13.8 Determination of Extent of Conjugation

It is helpful to determine the relationship between various groups mainly with

respect to conjugation that usually can occur between the

• Two or more carbon–carbon double or triple bonds, e.g., in aromatic compounds.

• Double bonds between carbon and oxygen.
• Double bonds present in various other organic compounds.

It may reveal the existence of an aromatic ring and total number and locations of
various substituents that are attached to the carbons of conjugated system.
Further Reading 55

3.13.9 Structural Elucidation of Organic Compounds

From the location and combination of peaks, structural elucidation of organic

compounds can also be done using UV-VIS spectroscopy. It indicates the degree
of unsaturation and existence of heteroatoms in organic compounds.

3.13.10 As HPLC Detector

UV-VIS spectrophotometer can also be utilized as a detector in combination with

HPLC. The presence of an analyte gives a response which can be assumed to be
proportional to the concentration.

3.14 Advantages

1. Analysis is quick.
2. Sample analysis is easy.
3. Absorption spectrum provides valuable information regarding the presence of
analyte in sample.

3.15 Disadvantages

1. Lack of sensitivity and selectivity.

2. Limited to UV/VIS absorbing compounds.
3. Need spectrophotometer capable of reading in the UV-VIS region.
4. Samples should be in solution form.
5. Mixture of substances poses difficulty to analyze and requires prior separation.
6. Interferences from sample mixture make the measurement difficult.
7. Samples should be in solution. Mixture of substances poses difficulty to analyze
and requires prior separation.
8. Interference from the sample’s matrix makes the measurement difficult.

Further Reading
Beckett A, Stenlake J (1997) Practical pharmaceutical chemistry, part II, vol 1. CBS Publications
and Distributors, New Delhi, pp 275–300
Fifield FW (2000) Principles and practice of analytical chemistry. Blackwell, Oxford
Förster HUV (2004) Vis spectroscopy. In: Karge HG, Weitkamp J (eds) Characterization I
molecular sieves – science and technology, vol 4. Springer, Berlin, pp 337–426
Gauglitz G (2000) Ultraviolet and visible spectroscopy. In: Ullmann’s encyclopedia of industrial
chemistry. Wiley, Weinheim
Gauglitz G, Dakin JP (2017) Spectroscopic analysis. In: John PD, Robert GWB (eds) Handbook of
optoelectronics, vol 2. CRC Press, Boca Raton, pp 569–600
56 3 Ultraviolet-Visible (UV-VIS) Spectroscopy

Gorog S (2018) Ultraviolet-visible spectrophotometry in pharmaceutical analysis. CRC Press,

Boca Raton
Gurdeep R, Anand K (2016) Instrumental methods of chemical analysis. Himalaya Publishing
House, Mumbai
http://rxpharmaworld.blogspot.com/2016/12/spectrophotometry-uvvisible-spectroscopy.html
Kemp W (2017) Organic spectroscopy. Macmillan International Higher Education, London
LibreTextsTM. UV-VIS spectroscopy. Accessed 12 Aug 2019
Pavia DL, Lampman GM, Kriz GS, Vyvyan JA (2014) Introduction to spectroscopy. Cengage
Learning, Belmont
Perkampus H-H (2013) UV-VIS spectroscopy and its applications. Springer, Berlin
Picollo M, Aceto M, Vitorino T (2018) UV-Vis spectroscopy. Phys Sci Rev 4
Schoonheydt RA (2010) UV-VIS-NIR spectroscopy and microscopy of heterogeneous catalysts.
Chem Soc Rev 39(12):5051–5066
Sudha PC (2012) Pharmaceutical analysis. Pearson Education India, Chennai
Waters Corporation. UV-VIS spectroscopy. Accessed 15 Aug 2019
Watson DG (2015) Pharmaceutical analysis E-book: a textbook for pharmacy students and phar-
maceutical chemists. Elsevier Health Sciences, Amsterdam
Infrared Spectroscopy
4

Abstract
Infrared spectroscopy deals with the interaction of infrared (IR) region of
electromagnetic spectrum with that of analyte present in sample. The absorption
of electromagnetic radiation (EMR) in IR region is based on resonant frequency,
i.e., absorption happens when natural frequency of vibration of bonds and
groups matches with applied frequency. In this chapter, we have comprehen-
sively described the various types of vibration of bonds or groups. There are
multiple components associated with IR spectroscopy that are needed for its
proper functioning. IR spectroscopy is useful for the analysis of sample existing
in any form either solid, liquid, or gas. However, different sampling techniques
are used for the preparation of sample to be analyzed by IR spectroscopy.
A comparison of different types of IR spectroscopy is also given in this chapter.
At the end, various applications of this spectroscopy have been described along
with advantages and disadvantages.

Keywords
Regions of IR · Modes of molecular vibrations · Sampling techniques · Types of
IR spectroscopy · Interpretations of IR spectra

4.1 Introduction

Infrared spectroscopy is used to determine the absorbance of given sample within

the infrared (IR) region of the electromagnetic radiations (EMR). IR radiation exists
between the microwave and visible regions of EMR. Wavelength of IR radiation is
longer than visible but smaller than microwaves. However, frequency of IR radiation
is lower than visible but greater than microwaves. IR is a kind of EMR which has
wavelength region longer than visible light, but shorter than radio wave. Different
regions of EMR have been shown in Fig. 4.1.

# Springer Nature Singapore Pte Ltd. 2020 57

M. S. H. Akash, K. Rehman, Essentials of Pharmaceutical Analysis,
https://doi.org/10.1007/978-981-15-1547-7_4
58 4 Infrared Spectroscopy

Fig. 4.1 Schematic representation of regions of electromagnetic radiations

Table 4.1 Regions of IR IR regions Range

and their ranges
Near IR region 0.8–2.5 μm (12,500–4000 cm
1)
Mid IR region 2.5–15 μm (4000–667 cm
1)
Far IR region 15–200 μm (667–100 cm
1)

4.2 Regions of IR

There are three types of IR regions that have been described in Table 4.1. The unit
commonly used is wavenumbers (cm
1), i.e., 4000–400 cm
1. Wavenumber is
proportional to energy (E) and frequency (v), but reciprocal to wavelength.

4.3 Principle

Molecules consist of atoms that are linked by different chemical bonds. The
movement of atoms and their associated chemical bonds resembles with spring
and balls system. This characteristic movement is known as natural frequency
of vibration. Applied IR frequency is equal to the natural frequency of vibration.
Owing to vibrational motion, there is a change in dipole moment of the molecule.
Covalent bond oscillation results in oscillation of dipole of the molecule producing
an electromagnetic (EM) field. The more dipole moment changes because of the
vibrational motion, the more intense the electromagnetic field is produced. Basically,
IR spectroscopy is the measurement of the reflection, absorption, and emission of
IR spectrum.
4.4 Modes of Molecular Vibrations 59

4.4 Modes of Molecular Vibrations

Any slight change in the shape of molecule via bending of bonds, stretching of
bonds, or internal rotation around a single bond is known as molecular vibration
(Fig. 4.2). When the waves of EMR interact with matter, they change the appearance
of bending and/or stretching vibrations. Based on the types of vibrations that appear
in the form of bands in spectra, molecular vibrations are classified into the following
types.

4.4.1 Stretching Vibration

The vibration along the line of bond is known as stretching vibrations. These
vibrations occur in radial direction and change bond length either by increasing
the bond length or decreasing the bond length. These vibrations also involve the
change in interatomic distance along the axis of the bond between the two atoms.
Such types of vibrations require higher energy, i.e., 4000–1250 cm
1 and appear
at shorter wavelength. Stretching vibrations have the following two types:

4.4.1.1 Symmetrical Stretching Vibration

In this kind of stretching, the interatomic distance between the two bonds increases
or decreases simultaneously (Fig. 4.3).

4.4.1.2 Asymmetrical Stretching Vibration

In this kind of stretching, the length of one bond is increased and the length of
another bond is decreased. The interatomic distance between the two atoms is
alternate and/or opposite to each other (Fig. 4.4).

4.4.2 Bending Vibrations

These types of molecular vibrations are either latitudinal or longitudinal in direction

that results in the change of angle between the two adjacent bonds. Latitudinal

Fig. 4.2 Schematic representation of modes of molecular vibrations

60 4 Infrared Spectroscopy

Fig. 4.3 Schematic

representation of stretching
vibration

Fig. 4.4 Schematic

representation of stretching
vibration

bending vibrations are known as in-plane bending vibrations, whereas the longitu-
dinal bending vibrations are known as out-plane bending vibrations. These
vibrations require less energy so easily happens at higher wavelength.

4.4.2.1 In-Plane Bending Vibrations

In-plane bending vibrations involve a change in bond angle and they occur in
the same plane. These are of the following two types:

Scissoring Vibration
Scissoring vibration happens when two atoms approach to each other. The bond
angles are decreased in this type of vibration (Fig. 4.5).
4.4 Modes of Molecular Vibrations 61

Fig. 4.5 Schematic

representation of scissoring
vibration

Fig. 4.6 Schematic

representation of rocking
vibration

Rocking Vibration
Rocking vibration happens when movement of atoms occurs towards the same
direction without any change in the angle of bond (Fig. 4.6).

4.4.2.2 Out-Plane Bending Vibrations

As the name indicates, out-plane bending vibrations take place away from the plane.
These are of the following two types:

Wagging Vibration
Wagging vibration happens when two atoms move towards the same side of the
plane. They move either up or down of the plane (Fig. 4.7).

Twisting Vibration
Twisting vibration happens when two atoms move to opposite sides of the plane.
One atom moves up from the plane and second moves down from the plane
(Fig. 4.8).
62 4 Infrared Spectroscopy

Fig. 4.7 Schematic

representation of wagging
vibration

Fig. 4.8 Schematic

representation of twisting
vibration

4.5 Components of IR Spectrophotometer

The following are the components of IR spectrophotometer:

4.5.1 Radiation Source

Radiation source is an important component of IR spectrophotometer. It continu-

ously emits IR radiation having sufficient intensity. Radiation source should have the
following properties:

1. Intensity of radiation should be continuous over the λ range and covers a

wide λ range.
4.5 Components of IR Spectrophotometer 63

2. Intensity of radiation should be constant over long periods of time.

3. Source should have the normal operating temperatures, i.e., between 1100 and
1500 K.
4. Source should have maximum intensity between 4000 and 400 cm
1.
5. Sources should be enclosed in an insulator to reduce noise.

The important examples of modern sources for radiations include furnace

ignitors, diesel engine heaters. The following are the important sources of IR
radiation:

4.5.1.1 Mid-IR Sources

The sources for mid-IR radiation have the following types:

1. Nernst glowers: It is made up of zirconium oxide, cerium oxide, and/or thorium

oxide. It contains heated ceramic rods with cylindrical bar. It can be heated
electrically up to 1500  C and emits radiations between 0.4 and 20 μm. As the
temperature increases, resistance decreases proportionally. If the care is not taken,
overheating may cause the burning of overall apparatus.
2. Globar: It contains bar that is made of silicon carbide which can be heated
electrically to emit the continuous IR radiations. It is more sensitive than the
Nernst glower.
3. Heated wire coils: It can also be heated electrically up to ~1100  C. Its shape
is similar to the incandescent light bulb filament. Nichrome wire is commonly
used but sometimes, rhodium is also used. Due to the softness and flexibility
in wire, it may easily fracture and burn out.

4.5.1.2 Near-IR Sources

One of the most important examples of sources for near-IR radiation is quartz
halogen lamp. It contains tungsten wire filament in which iodine vapors are sealed
in a quartz bulb. Upon heating, the tungsten is evaporated and then redeposited
on the filament. This phenomenon of tungsten redeposition on filament increases
the stability. The output range is between 25,000 and 2000 cm
1.

4.5.1.3 Far-IR Sources

The most important example of far-IR sources for radiation is high pressure mercury
discharge lamp which contains inert gas and two electrodes. It is made of quartz bulb
that contains elemental mercury.

4.5.1.4 Laser Sources for IR Radiation

It has the following two types:

1. Gas-phase (tunable CO2) laser.

2. Solid-state laser.
64 4 Infrared Spectroscopy

4.5.2 Sample Cell

It holds the sample and allows the monochromatic light to pass through it for the
measurement of IR spectra. The samples to be analyzed may be in solid, liquid,
or gaseous forms. It is usually made up of alkali halides, e.g., NaCl or KBr. These
halides are soluble in water; therefore, aqueous solvents cannot be used for analysis
because these solvents dissolve the sample cell if it is composed of alkali halides.
Only organic solvents such as carbon disulfide (CS2) and carbon tetrachloride (CCl4)
are used for analytical purpose.

4.5.3 Monochromator

Monochromator is used to split the polychromatic radiation into component

wavelengths either by using the prisms (metal halide or NaCl prism) or grating
or both. Quality of mirrors and slit width determine the resolution of IR spectrum.
Rock salt prism that is made up of metal with polished front surface is usually used
in the range of 650–4000 cm
1.

4.5.4 Detectors

Detector is a device that receives the light signals from the sample cell and converts
it into electrical signals.

4.5.4.1 Types of Detectors

Depending upon the characteristics of the material used in the composition of
the detector and nature of the analyte whose spectrum is to be determined, there
are two types of the detectors which are as follows:

Thermal Detectors
They consist of a tiny active element used for the detection. Insulation of that
element or decreased size of that element increases the detector response. Such
detectors are widely used because of linearity in response but they have some
demerits including slow processing time and less sensitivity.

1. Bolometers: These are sensitive to electrical resistance. The older ones were
made up of platinum wire (resistance change ¼ 0.4% per  C). Modern ones
are made of silicon that is placed in a Wheatstone bridge. Their diameter is
few micrometers and their response time is very fast.
2. Thermocouples: These detectors are made up of two wires from different metals,
welded together at both ends, e.g., bismuth and antimony. One welded joint
known as hot junction is exposed to IR radiation. The other joint known as cold
junction is kept at constant temperature. Hot junction becomes hotter than the
4.6 Sampling Techniques for IR Spectroscopy 65

cold junction. Potential difference between the hot and cold junction is a
function of IR radiation. It has slow response time and cannot be used for FTIR
spectrophotometry.
3. Thermistors: These detectors contain fused mixture of various metal oxides.
Owing to increase in temperature, there is a decrease in resistance in these
detectors which is a function of IR radiation. The approximate resistance change
is about 5% per  C but the response time is very slow.
4. Pyroelectric devices: These are made of ferroelectric materials, dielectric
materials, or semiconductors. A thin crystal of a material is placed between
the two electrodes. Any change in the temperature changes the polarization
of the material which is a function of IR radiation exposed. The electrodes
measure the change in polarization in response to the change in temperature.
They have a fast response time.

Photon-Sensitive Detectors
These are made of materials such as indium antimonide, indium gallium arsenide,
lead selenide. These materials are semiconductor in nature but they are very sensitive
and have very fast response time.

4.5.5 Read-Out Device

It receives the electrical signals from the detector and translates it into easy to
interpret form.

4.6 Sampling Techniques for IR Spectroscopy

In IR spectroscopy, the sample used for analysis is in the form of solid, liquid, or
gas. Inappropriate sample influences the overall results of analysis. Therefore,
keeping in view the nature of the sample, different sampling techniques are used
which are as follows:

4.6.1 Solid Samples

In case of solid samples, following techniques may either be used to prepare the solid
samples for IR spectroscopy:

4.6.1.1 Mulling
In this technique, the solid sample is ground to make it powder (Fig. 4.9). Few
drops of viscous liquid like Nujol are added to make thick slurry of the sample
which is then pressed between salt plates to form a thin film.
66 4 Infrared Spectroscopy

Fig. 4.9 Schematic

representation of solid sample
preparation mulling technique

Fig. 4.10 Schematic

representation of solid sample
preparation pelleting
technique

4.6.1.2 Pelleting
In this technique, 1 mg of the ground sample is mixed with 100 mg of dry powder
of KBr. Mixture is then compressed under very high pressure and small disk with
very smooth surfaces is formed that looks like a glass (Fig. 4.10).

4.6.1.3 Thin Film Formation

Molten sample is deposited on the surface of NaCl or KBr plates and sample is then
allowed to dry to form a thin film on the substrate. This technique is good for
qualitative identification of polymers but it cannot be used for quantitative analysis.
4.7 Types of IR Spectroscopy 67

4.6.2 Liquid Samples

Many liquid samples are analyzed as it is. There is no need to use a specific
technique to prepare the liquid samples if IR spectroscopy is to be carried out
but dilution with an appropriate solvent such as CCl4, CS2, and CH3Cl may be
necessary. If the solvent is used for the dilution of liquid sample, then the solvent
must be transparent in the region of interest. Salt plates are hygroscopic in nature
and water-soluble samples should be avoided to be in contact with these plates.
Therefore, special cells like BaF2 and AgCl are used for water containing samples.

4.6.3 Gas Samples

Like the liquid samples, gas samples do not require any special technique to prepare
sample for IR spectroscopy. For gaseous samples, special gas cells are used.
These cells have longer path lengths generally about 10 cm (commercial ones
are up to 120 cm) to compensate very low concentrations of gas samples. For
quantitative analysis, both temperature and pressure are controlled.

4.7 Types of IR Spectroscopy

The following are the types of IR spectroscopy:

4.7.1 Dispersive IR Spectroscopy

It contains filter and/or grating monochromator. The main components of dispersive

IR spectrometer are radiation source, monochromator, and detector. Dispersive
spectrometers usually contain a double-beam design having two equivalent beams
coming from the same source and pass through the reference and sample chambers
as an independent beam.

4.7.2 FT-IR Spectroscopy

Fourier-transform infrared (FT-IR) spectroscopy is one of the most important

types of IR spectroscopy which is used to obtain the IR spectra of high resolution
over the wide range of spectrum either by the absorption or emission of the
analyte which is either solid, liquid, and/or gaseous in nature. Generally, FT-IR
spectrometer contains a source of radiation, interferometer, sample compartment,
detector, amplifier, analog-to-digital converter, and computer. The source is used
to generate radiations that pass through the sample via interferometer and
reach to the detector. The amplifier amplifies the signals and converts them to digital
68 4 Infrared Spectroscopy

Table 4.2 Difference between dispersive IR spectroscopy and FT-IR spectroscopy

S. No. Dispersive IR spectroscopy FT-IR spectroscopy
1. There are numerous moving parts, Mirror is the only part that moves during
causing mechanical slippage the experiment
2. Calibration against the reference spectra Use of laser provides greater frequency
is needed to measure the frequency accuracy (up to 0.01 cm
1)
3. Stray light gives spurious readings Stray light do not affect the detector
4. Only a small amount of IR beam is A much larger beam is used at all time.
allowed to pass to increase the resolution Data collection is comparatively easy
5. Only radiation of a narrow frequency All frequency of radiation falls on the
range falls on the detector at one time detector simultaneously
6. Scanning speed is slow Scanning speed is high

signals by analog-to-digital converter. Ultimately, the signals are transferred to the

computer in which FT is carried out.
The difference between the dispersive IR and FT-IR spectroscopy is described in
Table 4.2:

4.7.3 Near-IR Spectroscopy

It is used for the quantitative analysis of liquid and solid samples having OH,
NH, CH bonds. It is commonly used for quantitative characterization of polymers,
food, proteins, and agricultural products. Pharmaceutical tablets can be analyzed
nondestructively with the help of this technique. Forensic analysis of unknown
wrapped powders that may be believed to be drugs and/or narcotics, are analyzed
without destroying the wrappers.

4.8 Regions of IR Spectrum

In IR spectra, there are four primary regions which may help to interpret the spectra
of unknown compound. These four regions of IR spectra have been described
in Fig. 4.11.

4.9 Interpretations of IR Spectrum

Bending and stretching vibrations occur with a particular frequency as the charges
and atoms involved are different for different bonds. A schematic representation of
IR spectra has been illustrated in Fig. 4.12. In the following sub-sections, IR spectra
of few compounds have been interpreted accordingly.
4.9 Interpretations of IR Spectrum 69

Fig. 4.11 Schematic representation of different regions of IR

4.9.1 IR Spectra of Alkanes

Consider the IR spectra of octane (Fig. 4.13). The combination of C–H and C–C
bonds (e.g., octane) is as follows: C–C stretching and bending appears between 1360
and 1470 cm
1, CH2–CH2 bond appears between 1450 and 1470 cm
1, CH2–CH3
bond appears between 1360 and 1390 cm
1, sp3 C–H bond between 2800 and
3000 cm
1.

4.9.2 IR Spectra of Alkenes

Consider the spectra of 1-octene (Fig. 4.14). Addition of the vinyl C–H and C¼C
bonds (e.g., 1 octene) is as follows: C¼C stretching is appeared at 1620–1680 cm
1
which becomes weaker as the substitution increases in the structure. Vinyl C–H
stretch happens at 3000–3100 cm
1.

4.9.3 IR Spectra of Alkynes

Consider the spectra of 1-octyne (Fig. 4.15). Addition of CC and vinyl C–H bonds
(e.g., 1 octyne) is as follows: CC stretch appears between 2100 and 2260 cm
1.
Strength of CC depends on asymmetry of bond. This bond is strongest if CC is
present at terminal alkynes and weakest for symmetrical internal alkynes. C–H for
terminal alkynes appears between 3200 and 3300 cm
1.
 70
4

Fig. 4.12 Schematic representation of interpretation of IR spectra

Infrared Spectroscopy
 4.9 Interpretations of IR Spectrum

Fig. 4.13 Schematic IR spectra of alkanes

71
 72
4

Fig. 4.14 Schematic IR spectra of alkenes

Infrared Spectroscopy
 4.9 Interpretations of IR Spectrum

Fig. 4.15 Schematic IR spectra of alkynes

73
74 4 Infrared Spectroscopy

4.9.4 IR Spectra of Aromatic Compounds

Consider the spectra of ethyl benzene (Fig. 4.16). Because of the delocalization
of electrons in the ring, the order of C–C bond is 1.5. The energy of stretching
frequency for such bonds is relatively low as compared to that of the normal C¼C.
They are represented as a pair of sharp bands, i.e., 1500 and 1600 cm
1 (lower
frequency band is stronger). C–H bonds outside the ring appear between 3000 and
3100 cm
1 as that of vinyl C–H.

4.9.5 IR Spectra of Ethers

Consider the spectra of di-isopropyl ether (Fig. 4.17). In di-isopropyl ether,

addition of C–O–C asymmetric bond and vinyl C–H bonds shows a strong band
for antisymmetric C–O–C stretch at 1050–1150 cm
1.

4.10 Applications of IR Spectroscopy

4.10.1 Medical Diagnosis

IR spectroscopy is used for the detection, prevention (early diagnosis), monitoring,

and diagnosis (under investigation) of several life-threatening diseases notably
cardiovascular diseases, cancers, and diabetes mellitus. As biological samples
are of proteins, lipids, carbohydrates, and nucleic acids. All these biochemical
substances have unique fingerprints, so any change in them because of the presence
disease can be detected using IR spectroscopy.

4.10.2 Identification of Unknown Substance

Small difference in the structure and composition of molecule results in a significant

change in the peaks of spectra in specific region. To compare spectrums of unknown
substances, computer search systems are required. IR instruments do not offer
computer search systems to identify the unknown compounds from the stored IR
spectral data. Instead, the position and magnitudes of the peaks in the spectrum of
unknown substance are compared with the profiles of pure compounds stored in
the library of the computer search system. Computer then matches the profiles
similar to that of the analyte and result is displayed (Fig. 4.18). It is very important
that sample and the reference should be analyzed in the same conditions such as
same physical state, temperature, solvent, etc. One of the main disadvantages is that
enantiomers cannot be distinguished because their spectra are identical to each other.
 4.10
Applications of IR Spectroscopy
75

Fig. 4.16 Schematic IR spectra of aromatic compounds

 76
4

Fig. 4.17 Schematic IR spectra of aromatic ethers

Infrared Spectroscopy
4.10 Applications of IR Spectroscopy 77

Fig. 4.18 Schematic representation of comparison of IR spectra of unknown compound with

that of known one

4.10.3 Determination of Molecular Structure

Identification is carried out on the basis of position of the absorption bands present
in the spectrum. For example, C¼O at 1717 cm
1. Therefore, absence of the
band associated with a particular group points out the absence of that particular
group in given compound.

4.10.4 Studying the Progress of Chemical Reaction

IR spectroscopy is used to observe the rate of reaction. It can be done by predicting

the disappearance of particular absorption band in the reactants. This shows the
utilization of that species and an increase in the rate of the absorption bands in
the product shows the formation of particular product. For example, O–H appears
at 3600–3650 cm
1, C¼O appears at 1680–1760 cm
1.
78 4 Infrared Spectroscopy

4.10.5 Detection of Impurities

Presence of impurities in specific compound can be determined by comparing

the sample spectrum with the spectrum of pure reference compound. Detection is
favored when impurity possess a strong band in IR region where the main substance
does not possess a band. For example, the presence of ketone (as impurity) in
alcohols can be determined by IR spectroscopy.

4.10.5.1 Detection of API in Final Dosage Form

IR spectroscopy is used to determine the % content of required ingredient and/or
API in the final dosage form.

4.10.5.2 Biosimilar and Bioequivalence Studies

Biosimilar and bioequivalence studies of the pharmaceuticals of different
competitors can be performed and compared with IR spectroscopy.

4.10.5.3 Determination of Drug–Polymer Interaction

IR spectroscopy notably FT-IR spectroscopy can be used to determine the drug–
polymer interaction in the final dosage form.

4.10.6 Analysis of Isomers

IR spectroscopy can be used to find the following types of isomers of the

compounds.

4.10.6.1 Geometrical Isomerism

Trans-isomer gives a simpler spectrum than the cis-isomer due to the presence
of symmetrical carbon in structure.

4.10.6.2 Conformers (Rotational Isomers)

They can be identified with the help of high-resolution IR spectrometers.

4.10.6.3 Tautomerism
Presence of two or more chemical compounds capable of interconverting, usually
by exchanging a hydrogen atom between the two atoms, can be determined with
the help of IR spectroscopy. For example, thiocarboxylic acid.
4.10 Applications of IR Spectroscopy 79

4.10.6.4 Functional Group Isomerism

Isomerism shown by the compounds having same molecular formula but different
functional groups can also be determined using IR spectroscopy. For example,
dimethyl ether (CH3–O–CH3) and ethanol (CH3–CH2–OH) have same molecular
formula, i.e., C2H6O but have different functional groups. In dimethyl ether, the
functional group is ether (–O–), whereas in ethanol, the functional group is alcohol
(–OH). The characteristic peak of –OH group appears at 3500–3100 cm
1 which
differentiate the ethanol from dimethyl ether.

4.10.6.5 Determination of Symmetrical Shapes of Molecules

With the help of IR spectroscopy, symmetry of the molecules may be determined.
For example, nitrogen dioxide (NO2) if linear in shape then only two bands appear,
but if it is bent, then three bands appear. The actual IR spectrum of NO2 shows three
peaks at 750, 1323, and 1616 cm
1. Likewise, IR spectrum is also used to determine
the structures of XeF2 (linear in shape), XeF4 (square planar), and XeF6 (octahedral).

4.10.6.6 Identification of Functional Groups

IR region can be divided into two regions, group frequency region
(4000–1500 cm
1) and fingerprint region (1500–400 cm
1). Group frequency
region gives the determination of functional group, whereas fingerprint region can
be used to identify the atom in molecule.

4.10.6.7 Study of Inorganic Complexes

During the complex formation, large number of new bonds formation occurs
which can be easily detected by IR spectroscopy.

4.10.6.8 Detection of Moisture in Samples

In the presence of lattice water, spectra will comprise of three characteristic bands
at 3600–3200 cm
1, 1650 cm
1, and 600–300 cm
1.

4.10.6.9 Characterization of Heterogeneous Catalysts

It is useful to determine the nature of molecules attached with catalyst surfaces.
For example, characterization of olefin polymerization catalysis with silica gel.
80 4 Infrared Spectroscopy

4.10.6.10 Forensic Analysis

Polymer degradation can be analyzed in both criminal and civil cases. For example,
it is used to determine the blood alcohol content in suspected drunk driver.

4.10.6.11 Analysis of Multilayered Polymeric Film

Identities of polymer materials in the multilayered film can be determined with
the help of IR spectroscopy.

4.11 Advantages

1. Easy to use.
2. Inexpensive.
3. Analysis takes <10 min.
4. IR spectrophotometry can eliminate the common possible errors that appear
in other spectrophotometric techniques.
5. Same sample cell is utilized for all kind of determinations.
6. All measurements are done based on the defined spectrum without dependence
on λ intensity.

4.12 Disadvantages

1. Sample cell is made up of alkali halides; therefore, it is very sensitive to the

absorption of water.
2. In case of excessive amount of moisture, penetration distance of absorbing light
decreases.

Further Reading
LibreTextsTM. Infrared spectroscopy. Accessed 22 July 2019
Pavia DL, Lampman GM, Kriz GS, Vyvyan JA (2008) Introduction to spectroscopy.
Cengage Learning, Belmont
Smith BC (2018) Infrared spectral interpretation: a systematic approach. CRC Press, Boca Raton
Stuart B (2000) Infrared spectroscopy. In: Kirk-Othmer encyclopedia of chemical technology.
Wiley, Hoboken, pp 1–18
Stuart B (2004) Infrared spectroscopy: fundamentals and applications. Wiley, West Sussex
10:0470011149
Sudha PC (2012) Pharmaceutical analysis. Pearson Education India, Chennai
Waters Corporation. Infrared spectroscopy. Accessed 10 July 2019
Watson DG (2015) Pharmaceutical analysis E-book: a textbook for pharmacy students and
pharmaceutical chemists. Elsevier Health Sciences, Amsterdam
Wilson K, Walker J (2010) Principles and techniques of biochemistry and molecular biology.
Cambridge University Press, Cambridge
Atomic Spectroscopy
5

Abstract
Atomic spectroscopy is a type of spectroscopic technique which is used for both
quantitative and qualitative analysis of an element present in sample through
mass spectrum. Atomic spectroscopic technique has two techniques; atomic
absorption spectroscopy (AAS) and atomic emission spectroscopy (AES) which
involve the energy absorption in the process of excitation or energy emission in
the process of decay, respectively. AAS and AES have been briefly described in
Chaps. 6 and 7, respectively. In this chapter, we have focused on the basic
mechanism of atomic spectroscopy along with its advantages, disadvantages,
and applications.

Keywords
Types of atomic spectroscopy · Atomic absorption spectroscopy · Atomic
emission spectroscopy

5.1 Introduction

In an atom, electrons are arranged according to their energy levels. They are
arranged in sub-shells; the sub-shells are arranged in shells and shells are arranged
around the nucleus. Electrons near the nucleus have lower energy level (also known
as ground state) than the electrons that are much far away that have high energy level
(also known as excited state). However, they experience stronger attraction in the
nucleus than those ones that are further away from the nucleus. Ground state is a
status where the atom’s electrons are in their lowest possible energy level (stable).
Excited state is another status where the atom’s electrons absorb enough energy to be
promoted to a higher level (Fig. 5.1). Therefore, they are not in their lowest energy
level (unstable). Generally, atoms are in their “ground state” but when an atom
receives enough input of energy that their electrons require to be promoted to a
higher energy level, they will then turn to their “excited state.” Since, an atom’s

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82 5 Atomic Spectroscopy

Fig. 5.1 Schematic representation of absorption and excitation of energy by the electron in atomic
spectroscopy

excited state is very unstable, it rapidly “jumps” back down to its ground state. This
“jump” then causes the atom to release the energy it absorbed in the form of photons
of light.

5.2 Principle

Samples containing metals of unknown concentration can be analyzed using both

types of spectroscopic techniques, i.e., AAS and AES. Both techniques possess the
same instrumentation. The only difference is that in AAS, light absorbed by the
excited atoms or ions of the metal whose concentration is going to be measured is
measured, whereas in AES, the light emitted by the excited atoms or ions of the
metal whose concentration is going to be measured is measured. In AAS, when a
light of certain wavelength passes through an atom or ion, it excites that atom or ion
from ground state (having low energy level) to an excited state (having high energy
level). As the number of atoms or ions of the metal of interest present in sample is
increased in the path of light, the amount of light absorbed is also increased and this
wavelength is directly proportional to the concentration of absorbing atoms or ions
of metal of interest. As the wavelength of the light absorbed is specific for each
element, the measurement of this absorbed light gives a quantitative measurement of
the amount of analyte present in the sample. In AES, the sample is subjected to high
energy thermal environment to produce the excited state which can emit the light. As
5.5 Disadvantages 83

the wavelength of light emitted by atoms or ions of metal of interest is specific for
each element present in the sample, so, the measurement of this emission spectrum
which is the collection of emission lines from the excited atoms gives a qualitative
measurement of the analyte present in the sample. As the number of atoms or ions is
increased, the amount of the emission intensity will also be increased.

5.3 Types of Atomic Spectroscopy

Following are the main types of atomic spectroscopy:

1. Atomic absorption spectroscopy: This technique has been briefly described in

Chap. 6.
2. Atomic emission spectroscopy: This technique has been briefly described in
Chap. 7.

5.4 Advantages

1. High accuracy and sensitivity.

2. Accurate precision.
3. Easy to use.
4. Helpful in detecting the trace elements in the presence of higher concentration of
other elements.
5. Can reach previously inaccessible places.

5.5 Disadvantages

1. Used to determine ppm levels of metals, so could not be used for the analysis of
light elements, e.g., carbon, hydrogen, oxygen, nitrogen, phosphorus, sulfur,
halogens, and noble gases.
2. Only liquid samples can be detected, hence solid samples need vaporization prior
to detection.
3. Samples need dilution prior to their determination.
4. Another drawback is that flame atomization produces both atoms and ions but
only atoms are detected through this technique because atoms which undergo
ionization do not undergo atomic absorption.
5. Atomic spectrophotometer is costly.
84 5 Atomic Spectroscopy

5.6 Applications

1. More or less than 68 elements can be determined using this technique with good
precision. Therefore, in pharmaceutical industry, this technique is helpful for the
analysis of active pharmaceutical ingredient (API), raw material, or intermediates
used for drug development.
2. In pharmaceutical industry or drug testing labs, these techniques are used to
determine a metal catalyst that can be present in a drug after purification, e.g.,
detection of trace elements in multivitamin formulations, zinc in insulin, cobalt in
vitamin B12, lithium in anti-depressants, etc.
3. In medical laboratory, both AAS and AES can be used to analyze the tissue
samples for the detection of type and amount of toxic metals. Tissue samples may
be blood, urine, hair, bone marrow, or nails. A typical example is the measure-
ment of electrolyte sodium or potassium in the plasma.
4. These techniques have also found their importance in mining industry as they can
be used for quantitative and qualitative determination of precious metals like
silver, gold, etc.
5. Water analysis can also be done using atomic spectroscopy which may include
drinking water, sewerage water, or marine water, e.g., analysis of leaching of zinc
and lead from tin-lead solder in water.
6. An oldest application of atomic spectroscopy is the analysis of animal products,
animal feeds, and vegetables in food industry. Food samples are analyzed to
detect the mineral and trace elements.
7. As this process relies on atomic absorption or emission, it can reach previously
inaccessible places. For example, miners use this technique to determine whether
the rock contains enough metals or not.

Further Reading
Fernández B, Lobo L, Pereiro R (2019) Atomic absorption spectrometry. Fundamentals, instru-
mentation and capabilities. In: Worsfold P, Poole C, Townshend A, Miró M (eds) Encyclopedia
of analytical science, 3rd edn. Academic, Oxford, pp 137–143
Fifield FW (2000) Principles and practice of analytical chemistry. Blackwell Science, Hoboken
Gauglitz G, Moore DS, Vo-Dinh T (2014) Handbook of spectroscopy. Wiley, Hoboken
Hollas JM (2002) Basic atomic and molecular spectroscopy. Royal Society of Chemistry, London
Kemp W (2017) Organic spectroscopy. Macmillan International Higher Education, London
Labmate Online. Atomic absorption spectroscopy. Accessed 12 Sept 2019
LibreTextsTM. UV-VIS spectroscopy. Accessed 12 Aug 2019
Pavia DL, Lampman GM, Kriz GS, Vyvyan JA (2014) Introduction to spectroscopy. Cengage
Learning, Boston
Schrenk W (2012) Analytical atomic spectroscopy. Springer, Berlin
Sneddon J (2002) Advances in atomic spectroscopy. Elsevier, Amsterdam
Sudha PC (2012) Pharmaceutical analysis. Pearson Education India, Chennai
Waters Corporation. UV-VIS spectroscopy. Accessed 15 Aug 2019
Watson DG (2015) Pharmaceutical analysis E-book: a textbook for pharmacy students and phar-
maceutical chemists. Elsevier Health Sciences, Amsterdam
Atomic Absorption Spectroscopy
6

Abstract
In this chapter, we have discussed the components of AAS. The two types of
AAS and their comparison have been discussed in detail. The comprehensive
method for working of AAS has also been elaborated here. Moreover, we have
also discussed the various types of interferences that may influence the results of
AAS. Applications of AAS along with precautionary measures, advantages, and
disadvantages have also been discussed here.

Keywords
Components of AAS · Hollow cathode lamp · Electrodeless discharge lamp ·
Total consumption burner

6.1 Introduction

Atomic absorption spectroscopy (AAS) is a spectroscopic technique which is widely

used for elemental analysis. The basic principle involved in AAS is the absorption of
energy by the atoms at ground state in the gaseous state and the amount of light
absorbed by the atom at ground state is determined. Once the amount of light
absorbed by the atom at ground state is determined, we can estimate the unknown
concentration of metal accordingly.
In 1952, Australian scientist Alan Walsh was working on the measurement of
small concentrations of metals using atomic emission spectroscopy (this technique
has been discussed in detail in Chap. 7). The idea of AAS came into his mind as he
was gardening at his Melbourne home. On the normal Sunday morning, he had the
idea about looking at the light absorbed by the atoms except than looking at the light
they emit. Alan Walsh did not just discover a process that has the ability to save
lives, but also proven that atoms will only absorb light that has the exact value
requires to promote their electron to a higher level. AAS is a technique in which
radiations are absorbed by non-exited atoms in vaporized form. This technique is

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86 6 Atomic Absorption Spectroscopy

used to determine the amount of several metals (e.g., Cu, Fe, Zn, Mg) in soil, blood,
urine, air, water, food, etc. In electromagnetic spectrum, AAS uses the visible part of
electromagnetic radiation to detect the presence of metals present in sample. The
main energy source used in this technique is hollow cathode lamp and electrodeless
discharge lamp. AAS is especially used for the estimation of the trace metals in
samples independent from the molecular form of the interested metal which is
present in the sample. For example, we can also estimate the total content of metal
present in the water sample either that metal exists as salt form such as chloride and
sulfate.

6.2 Principle

When a sample having metallic species is placed into a flame, the vapor of metallic
species is formed. The phenomenon behind is that the atoms of a specific element in
ground state absorb the radiation of light of their own specific wavelength and
jumped up from low energy state (ground state) to the higher energy state (excited
state). The absorption of light energy that has the right wavelength to be absorbed by
the metal, promotes the electrons of metal from the sample to jump up from a lower
energy level (ground state) to a higher energy level (excited state) as clearly shown in
Fig. 6.1.
In AAS, two processes and/or phenomenon are involved that are as follows:

1. When light energy is absorbed by the metal sample, free atoms are produced from
the sample metal but are not ionized.
2. Once the free atoms are produced, they further absorb radiation from an external
source.

These free atoms once produced in the flame after absorption of radiation cause a
transition of these atoms from the ground state (having low energy level) to an
excited state (having high energy level after absorption of light). The amount of light
absorbed by specific element present in the sample is directly proportional to the
density of atoms present in the flame and here, AAS determines how much light

Fig. 6.1 Schematic representation of excitation of atom after absorption of light

6.3 Components of AAS 87

absorbed by the sample. Once it is determined that how much light energy was
absorbed by the atom of analyte present in sample, the concentration of metal in the
sample can be estimated. The total amount of the light of specific wavelength
absorbed by the sample solution is calculated using the following equation:


Total amount of light absorbed by the metal present in sample ¼ ле2 =mc Nf

where e ¼ charge on the electron, C ¼ speed of light, N ¼ the total number of atoms,
f ¼ strength of oscillator.

6.3 Components of AAS

A schematic diagram of AAS has been shown in Fig. 6.2 which contains the
following components:

6.3.1 Radiation Source

The radiation source is one of the main components of AAS. It is of two types. The
detail description of these radiation sources is as follows:

6.3.1.1 Hollow Cathode Lamp (HCL)

In HCL, the cathode has a hollow cup. The sample which contains metal whose
concentration is to be determined is placed in the cup of HCL. In HCL, anode is
made up of tungsten wire. In a tube, an inert gas is placed between the two
electrodes. The lamp window is made up of quartz, silica, or glass (Fig. 6.3).
When a voltage is applied between the two electrodes, the inert gas becomes
partially ionized at the anode and moves towards cathode. As a result, cathode
vaporizes metal atoms present in it and gives rise to spectrum of that metal. In the
overall phenomenon, the pressure of lamp is important which should be kept from
1 to 5 Torr, because ionization at the low filling pressure remains largely confined to
the interior of the cathode. The noble gas ions, traveling toward the cathode, start to

Fig. 6.2 Schematic representation of components of atomic absorption spectroscopy

88 6 Atomic Absorption Spectroscopy

Fig. 6.3 Schematic representation of hollow cathode lamp

Fig. 6.4 Schematic representation of electrodeless discharge lamp

strike the interior of the cathode thereby sputtering off atoms of the metal composing
it, in sufficient numbers to give rise to a cloud of metallic atoms. The spectral lines
produced in HCL after the absorption of light by the metal. The spectrum lines
emitted by HCL of the metal represent the type of metal that is present in the cathode.
For example, the spectrum of copper is obtained from the emission of copper
cathode lamp which is then absorbed by the copper atoms present in lamp.

6.3.1.2 Electrodeless Discharge Lamp

One of the main drawbacks of using HCL is that it is very difficult to construct it
from those elements that are volatile in nature, for example, germanium and arsenic.
To overcome this drawback, an alternative source of light has been introduced that is
known as electrode discharge lamp (EDL). EDL is an evacuated tube (Fig. 6.4) and
the metal whose concentration is to be determined from the sample is placed in an
evacuated tube. The tube contains argon whose pressure is kept low and sealed off.
When the sealed tube is kept in a microwave discharge cavity, the argon is shifted
towards a plasma state which is responsible for the excitation of metal that is sealed
inside the tube. The emission of radiation from the metal is a spectrum of the metal
present in it.
6.3 Components of AAS 89

Fig. 6.5 Schematic representation of working of chopper

6.3.2 Chopper

Chopper is just like a rotating wheel which is placed between the radiation source
and the flame. The reason to use chopper is that when the steady light comes from the
lamp, chopper converts it in to a pulsating light (Fig. 6.5). As a result, the pulsating
current is produced in photocell. The emitted light from the flame produces a steady
current. The steady current and pulsating current both are present but only pulsating
current is amplified and recorded by read-out device.

6.3.3 Atomizers

Atomizers are the devices that are responsible to carry out the phenomenon of
atomization. Atomization is actually the phenomenon of separation of atoms
and/or molecules present in sample into individual molecules or atom and breaking
molecules into the atoms of metal whose concentration is going to be determined.
Atomization is a process in which the free atoms of the metal of interest whose
concentration is going to be determined are produced by heat. This can be done by
exposing the analyte present in sample, in flame at high temperature. There are two
types of atomizers used in AAS.

6.3.3.1 Flame Atomizers

This is common method in which flame is used. The liquid sample which contains
the metal of unknown concentration or whose concentration is going to be deter-
mined is converted into the gaseous state by using the flame. The flame is also
responsible for the conversion of molecular form of the metal into its atomic form
which can be achieved at vapor state. In flame atomizers, two types of burners are
usually used and their details have been described in the following sub-sections:
90 6 Atomic Absorption Spectroscopy

Total Consumption Burner

In this type of flame atomizer, the sample solution which contains metal of unknown
concentration, oxidizing gases, and the fuel is allowed to pass through separate
passages to meet at the opening of the base of flame as shown in Fig. 6.6. The
mixture of hydrogen or acetylene with oxygen is used in this type of burner and it
gives rise to intense hot flames. The sample is present in the liquid form which is
broken up by the flame and is converted into the droplets which are then evaporated
and ultimately burns by leaving behind the residues which are reduced into atoms of
the metal of unknown concentration present in sample solution.

Regions of the Flame in Burner

Following are the three main regions of the flame in total combustion burner
(Fig. 6.7).
Primary combustion zone: Thermal equilibrium cannot be obtained in this region.
Therefore, it is seldom used for spectroscopic analysis. This region exists at the tip
and/or opening of the flame.
Interzonal combustion region: This region is situated between the primary and
secondary combustion zone. This region is relatively narrow region in the flames of

Fig. 6.6 Schematic

representation of total
combustion burner
6.3 Components of AAS 91

Fig. 6.7 Schematic

representation of regions of
the burner

burner and often rich in free atoms of the metal of unknown concentration. This
region is the hottest part of the flame and is widely used in spectroscopic analysis of
the metals of unknown concentration.
Secondary combustion zone: In this region of flame, the species of metal of
unknown concentration are converted into stable molecular oxides and then dis-
persed into the surroundings of the flame. This part of flame is also seldom used in
spectroscopic analysis of the metals.

Premixed Burner
In this kind of burner, the fuel gas and oxidants cause the aspiration into a large
chamber. This is carried out under the pressure. The fine droplets of the premixed
fuel and oxidants along with larger drops of the sample that contain the metal of
unknown concentration are collected at the outlet of the chamber and then
introduced into the flame. The excess amount is then drained out.

6.3.4 Nebulization

Nebulization is the phenomenon in which the liquid sample is converted into the fine
droplets before entering of the liquid sample into the burner. This process is used for
the formation of small and fine droplets. The conversion of liquid sample into fine
droplets is known as nebulization. This is the common method of nebulization in
92 6 Atomic Absorption Spectroscopy

Fig. 6.8 Schematic representation of nebulization phenomenon in atomic absorption spectroscopy

which a gas is used having a high velocity, called pneumatic nebulization. A

schematic representation of nebulization phenomenon has been described in
Fig. 6.8.

6.3.5 Monochromators

Monochromator is an essential part of AAS. It is used for the separation of all the
thousands of spectral lines into individual ones. If good monochromator is not used,
the efficiency of AAS is reduced because of the limit of detection of individual
spectral lines of metallic species is severely compromised. Monochromator uses the
light of specific wavelength with respect to the metal of unknown concentration. The
sample which contains the metal of unknown concentration absorbs the light of
specific wavelength and excludes all the other wavelengths. That specific light
coming from the monochromator permits the detection of the interested element
and/or metal whose concentration is going to be determined even in the presence of
others elements.

6.3.6 Detectors

These are the devices that are used to detect which wavelength is being absorbed by
the sample having the metal whose concentration is going to be determined.

6.3.7 Amplifier

It amplifies the signals received from the detector and then transfers to the read-out
device.
6.4 Working of AAS 93

6.3.8 Read-Out Device

The most widely used read-out devices in AAS are as follows:

1. Digital voltmeter
2. Simple galvanometer
3. Potentiometer
4. Computer

6.4 Working of AAS

Before the use of spectrometer, it should be calibrated by standard solution of known

concentration of solute which has to be determined in the test solution. Cuvettes are
filled with standard solution or reference solution and placed in sample holder in
spectrometer. The light of specific wavelength is directed towards the sample and/or
reference solution. Before reaching of light ray towards the sample or blank, the light
will pass through a series of prism, diffraction grating, and mirrors. The prism splits
light into different wavelengths, the mirror is used for navigation of light and the
diffraction grating allows the light of the required wavelength to pass through it and
reaches towards the cuvette containing reference or standard solution. It analyzes the
reflected light and compare with predetermined standard solutions. Some part of
light is absorbed by the solution and remaining part is transmitted towards the
detector. The detector measures the intensity of transmitted light and converts it
into electrical signals. These electrical signals send to the read-out device (galva-
nometer). The galvanometer measures electrical signals and displays in digital form.
This digital representation is actually the absorbance and/or optical density of the
analyte present in sample solution. A schematic representation of working of AAS
has been described in Fig. 6.9.

Fig. 6.9 Schematic representation of working of atomic absorption spectroscopy

94 6 Atomic Absorption Spectroscopy

6.5 Types of AAS

Following are the two main types of AAS:

6.5.1 Single-Beam AAS

The light of wavelength between 325 and 1000 nm is used in single-beam AAS. The
light travels from the sample solution and/or reference solution separately
(Fig. 6.10). The light source, e.g., HCL, emits the sharp atomic line of the element
whose determination is going to be determined. The chopper is interposed between
the light source and flame. The light is modulated easily by rotating the chopper
located between the light source and the flame and can also be achieved by pulsing
the power. Modulation helps to differentiate the light coming from the radiation
source lamp for the emission. The modulated light is then led to the flame where the
atoms of the element present in sample are in ground state (low energy level) and
after absorption of light radiation, these atoms are jumped to the excited state (high
energy level). The monochromator, which separates the wavelength of interest, is
then led to the detector followed by read-out device.

6.5.2 Double-Beam AAS

The light of the wavelength between the range of 185 and 1000 nm is used in double-
beam AAS. It has two cells which can be used separately for the sample solution and
the reference one. The light coming from the monochromator is split by an instru-
ment which is known as splitter into two beams. One of the two beams is used for the
reference and other one is used for the sample solution (Fig. 6.11). The chances of
error are eliminated in double-beam AAS which may occur due to fluctuations in
light output and sensitivity of detector.

Fig. 6.10 Schematic representation of single-beam atomic absorption spectrophotometer

Fig. 6.11 Schematic representation of double-beam atomic absorption spectrophotometer

6.6 How Can We Obtain the Data of AAS? 95

Table 6.1 Comparison between single and double beam atomic absorption spectrophotometer
Single-beam AAS Double-beam AAS
It is simple in working Its working is complicated
It is less expensive This equipment is costly
In single-beam AAS, the automation is low While in double-beam AAS, the speed of
automation is high
The efficiency of light is more in single-beam The efficiency of light is less in double-beam
in AAS AAS
Due to single beam in AAS, reference and Due to the presence of double beam, reference
sample are rung separately and more time is and sample are rung simultaneously and less
consumed to analyze the sample time is required for analysis
Single-beam AAS has low stability because it Double-beam AAS has maximum stability
depends upon the intensity of single beam because it depends on the ratio of intensity of
light between the two beams
Due to the presence of single beam, the There are lesser chances of fluctuation in the
chances of fluctuation in the intensity of light intensity of light and readings of data as
and reading of data are more separate light beam is used for the reference
and the sample solution
Due to single beam, the reference and the As double beam is present, the reference and
sample are placed separately the sample are kept at the same time
In single-beam AAS, the light throughput is In double-beam AAS, the light throughput less
high and more resolution is attained and decreased resolution is attained
Single-beam AAS takes time to warm-up and Double-beam AAS does not take much time to
takes time to start analysis warm-up and ready for analysis

Fig. 6.12 Schematic representation of collection of data from atomic absorption

spectrophotometer

6.5.2.1 Difference Between Single- and Double-Beam AAS

The comparison between single-beam and double-beam AAS has been described in
Table 6.1.

6.6 How Can We Obtain the Data of AAS?

Consider the following Fig. 6.12, we can obtain the data by the following way:
Number 1 mentioned in Fig. 6.12 reflects the intensity of light coming through the
radiation source is measured. Number 2 mentioned in Fig. 6.12 indicates that the
96 6 Atomic Absorption Spectroscopy

light can then be absorbed by the atoms of the metal whose concentration is going to
be determined that has been vaporized in the flame. This wavelength can cause the
transition of the electrons that are present in ground state (low energy level) into a
higher energy level (excited state). Number 3 mentioned in Fig. 6.12 indicates that
the higher the concentration of the metal present in the sample, greater will be the
absorbance of light. Number 4 mentioned in Fig. 6.12 indicates that the intensity of
light after passing through the sample which contains the metal of unknown concen-
tration is measured again and compared with the first result that was obtained with
blank and/or reference.

6.7 How Do We Analyze the Data of AAS?

The data can be analyzed by comparing the light intensity absorbed by the sample
which contained the metal of unknown concentration with blank and/or the reference
at the same time. For the construction of calibration curve, it is important to measure
the absorbance of different known concentrations of the metal whose unknown
concentration present in samples is going to be determined. The instrument is
calibrated using several solutions of known concentrations of the metal. The absor-
bance of each known solution is measured. In calibration curve, the absorbance
obtained by the spectrophotometer is plotted against its known concentration
(Fig. 6.13a). Through the calibration curve, we can then determine the unknown
concentration of metal present in the sample. Once the calibration curve is
constructed, it can be used to determine the unknown concentration of an element
in a solution. We can determine the unknown concentration of analyte with the help
of this calibration curve. The sample solution that contains metal of unknown
concentration is introduced into the flame of AAS. The metal present in sample
absorbs the radiation of light having specific wavelength that is measured with the
help of detector and interpreted by read-out device. The unknown concentration of
the element is then estimated from the calibration curve which is obtained by
comparing the absorbance of unknown concentration with that of absorbance of
the known concentration as represented in Fig. 6.13b.

6.8 Interferences of AAS

Interferences can be defined as increasing or reducing the extent of absorption of

light due to any physical or chemical interference. It is achieved with the test element
in aqueous solution. In the sample, the element other than the one of interested
element may absorb the radiation of specific wavelength which is being used for
specific element. Following are the most commonly observed interferences when
AAS is used:
6.8 Interferences of AAS 97

Fig. 6.13 Schematic representation of calibration curve for known concentrations of standard
solution (a) and calculation of concentration of unknown analyte (b) with the help of standard curve

6.8.1 Ionization Interference

The absorption of radiation is low due to the formation of ions rather than atoms. To
overcome this problem, ionization suppressors are usually added to prevent the
formation of ions during the combustion of sample solution in flame.
98 6 Atomic Absorption Spectroscopy

6.8.2 Back Ground Absorption of Source Radiation Interference

Due to incomplete atomization, presence of particles causes the absorption of

background source radiation. This problem can be overcome by increasing the
temperature of flame.

6.8.3 Transport Interference

Rate of nebulization, viscosity, density, vapor pressure, surface tension and rate of
aspiration of the sample are the factors that may also affect the results of AAS.

6.8.4 Cation–Cation Interference

Sometimes, the signal intensity of the interested element present in the sample is
irregularly decreased. These are neither the ionic nor the spectral in nature and their
interactions mechanism is unknown.

6.8.5 Anion–Cation Interference

The intensity of radiation that is emitted by an element is affected due to the presence
of certain anions in the sample solution and thus causes a serious analytical error.

6.8.6 Oxide Formation Interference

If oxygen is present in the flame, then the stable oxides with free metals are produced
that are responsible for this kind of interference. The intensity of emitted radiation is
lowered due to the removal of a large percentage of free metal atoms from the flame.
The oxides of alkaline earth metals are subjected to this kind of interference.

6.8.7 Spectral Interferences

It includes spectral overlap, molecular absorption, and light scattering. Few

examples of the spectral interferences of some metals are presented in Table 6.2.

6.8.8 Chemical Interferences

It may include thermal stability and ionization ability of the molecules present in the
sample.
6.9 Applications 99

Table 6.2 Examples of spectral interferences of interfering elements with that of target elements
Target element Spectral line (nm) Interfering element Spectral line (nm)
Aluminum (Al) 308.215 Vanadium (V) 308.211
Calcium (Ca) 422.673 Ge 422.657
Cadmium (Cd) 228.802 Arsenic (As) 228.812
Cobalt (Co) 252.136 Indium (In) 252.137
Copper (Cu) 324.754 Eu 324.753
Iron (Fe) 271.903 Platinum (Pt) 271.904
Ga 403.298 Mn 403.307
Mercury (Hg) 253.652 Cobalt (Co) 253.649
Mn 403.307 Ga 403.298
Sb 217.023 Lead (Pb) 216.999
Si 250.690 Vanadium (V) 250.690
Zn 213.856 Iron (Fe) 213.859

6.8.9 Physical Interferences

It includes viscosity, surface tension, and density of the sample solution that contains
metal of unknown concentration.

6.8.10 Vaporization Interferences

The rate of vaporization of the salt particle present in the sample solution of metal
whose concentration is going to be determined may alter due to the presence of some
component in the sample solution.

6.9 Applications

1. It is one of the most widely used techniques for qualitative analysis of the
metals.
2. It is also used for quantitative analysis of the metals even they are present in the
trace amounts.
3. It can also be used for simultaneous multicomponent analysis but this can be
done by using multicomponent hallow cathode lamp.
4. Detection of metals in biological fluids: It is used for elemental profiling in
biological fluids such as in urine and blood samples.
5. Determination of metallic elements in food products: Metallic element such as
copper, nickel, and zinc are the most common toxic elements that may be
present. AAS can be used to detect any of these metals from the raw material
that is used in the processing the of food products.
6. Detection of metals in pharmaceutical products: In pharmaceutical products, the
small amount of a catalyst that is used in manufacturing process may be present
100 6 Atomic Absorption Spectroscopy

in the final product. By using AAS, the amount of catalyst present in the final
product can be determined. This is also used for detection of many trace
elements such as zinc in multivitamins, lithium in antidepressants, etc. It can
be used for detection of traces of metals in parenteral preparations.
7. Detection of metals in environment: AAS is widely used to monitor—e.g., it can
be used to find out the levels of various elements in rivers, drinking water, sea
water, air, and petrol especially for the leaching of zinc.
8. Detection of trace elements in cosmetic products: The content of heavy metals
such as lead and cadmium used in cosmetic products can be analyzed
using AAS.
9. It is also used in mining industry for the detection and determination of precious
metals.
10. AAS is a life-saving technique: In Canada, AAS was used to determine the
unsafe levels of lead in children who was lived nearby a lead smelter. In Japan,
from 1932 to 1968, AAS was used to identify the reason why over 3000
residents who lived near the Minamata Bay started showing neurological
problems and pregnant women started giving birth to impaired children.
Scientists started taking samples and performed AAS process; AAS results
showed a very high concentration of mercury in their blood. This was resulted
on stopping Chisso corporation dumped about 27 tons of mercury in the bay.
11. It is also used for the analysis of gas for the purity.

6.10 Precautionary Measures

AAS is used for the analysis of many different elements. It can be potentially
harmful as well

1. Exhaust system: A large amount of heat, fumes, and vapors are produced in AAS
that may be harmful for operator. It is very important to use the exhaust system to
expel the excess amount of heat, fumes, and vapors that are produced during the
working of AAS.
2. Gas cylinders: It must be located outside of the laboratory in a cool and well-
ventilated area. Proper ventilation is practiced for protection of formation of
potentially hazardous toxic fumes.
3. Flammable solvents: The combination of solvent and flame is a harmful situation
to the operator. The analysis through AAS always uses solvent with the highest
flashpoint. It is recommended to use the covered containers and smallest practical
volume.
4. Burners: It is recommended that the burners should be kept close to any door
and/or material made up of wooden and/ plastic material. Before the operation,
the inspection of the entire burner system is necessary.
5. UV radiation: Hazardous UV radiation is emitted from flames, analytical
furnaces, hollow cathode lamps. Do not view the flames directly unless you are
wearing protective goggles. During the working on AAS, the door or flame shield
must be closed.
Further Reading 101

6.11 Advantages

This technique has following advantages:

1. Highly sensitive: It is highly sensitive technique for the detection of metals.

2. High accuracy: It is a method of high accuracy, if appropriate standards are
followed.
3. High selectivity: It is used for the detection of single element in the sample.
4. Wide applicability: It is widely used in pharmaceutical analysis, elemental analy-
sis, and water analysis.
5. Highly specificity: It is highly specific because element of the particular metal can
absorb the specific wavelength of light.
6. This is independent from the nature of flame.
7. It is very easy to operate.
8. The sample preparation is very easy.

6.12 Disadvantages

This technique has the following disadvantages:

1. It is applicable to analysis of metals only.

2. It is time consuming technique.
3. It is more expensive technique.
4. It needs separate lamp for each element to be determined. Each sample is
analyzed separately.
5. In aqueous solution, signal is predominantly affected by the anion.
6. Thermal interference may affect the overall result.
7. It has relative low precision.
8. Analysis cannot be done simultaneously.
9. Only liquid samples can be used. The solid sample for analysis is first converted
into a vapor form and then analyzed.
10. It cannot be used for analysis of lighter metals such as hydrogen, oxygen, sulfur,
halogens, and noble gases.

Further Reading
Fernández B, Lobo L, Pereiro R (2019) Atomic absorption spectrometry. Fundamentals, instru-
mentation and capabilities. In: Worsfold P, Poole C, Townshend A, Miró M (eds) Encyclopedia
of analytical science, 3rd edn. Academic Press, Oxford, pp 137–143
Labmate Online. Atomic absorption spectroscopy. Accessed 12 Sept 2019
LibreTextsTM. Atomic absorption spectroscopy. Accessed 12 Aug 2019
Metcalfe E (1987) Atomic absorption and emission spectroscopy. Wiley, Hoboken
Sudha PC (2012) Pharmaceutical analysis. Pearson Education India, Chennai
102 6 Atomic Absorption Spectroscopy

van Loon JA (2012) Analytical atomic absorption spectroscopy: selected methods. Elsevier,
Amsterdam
Waters Corporation. Atomic absorption spectroscopy. Accessed 15 Aug 2019
Watson DG (2015) Pharmaceutical analysis E-book: a textbook for pharmacy students and phar-
maceutical chemists. Elsevier Health Sciences, Amsterdam
Atomic Emission Spectroscopy
7

Abstract
Atomic emission spectroscopy is the oldest elemental analysis among spectro-
scopic techniques and still it is popular. This technique is used specifically to
determine the quantity of element in the sample. In this chapter, we have
explained the basic mechanism of atomic emission spectroscopy along with its
instrumentation, working, applications, advantages, and some disadvantages.

Keywords
Types of emission spectra · Components of AES · Inductively coupled plasma ·
Interferences of AES

7.1 Introduction

Atomic emission spectroscopy is a type of atomic spectroscopy that is frequently

used in order to measure the number of elements found in various samples. The
atoms of analyte are excited and promoted to relatively higher energy level by
providing the sufficient amount of energy. This energy is obtained from heat energy
provided from the atomization sources. Afterward, the excited atoms are shifted
back to lower energy levels with the emission of light energy (Fig. 7.1). Emitted
energy waves are passed across monochromators/filters before detection by
photomultiplier tubes.

7.2 Principle

In atomic emission, the sample is subjected to high energy thermal environment to

produce the excited state which can emit light in the form of photon. As the
wavelength of light emitted by atoms or ions is specific for each element present
in the sample, so the measurement of this emission spectrum which is the collection

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104 7 Atomic Emission Spectroscopy

Fig. 7.1 Schematic representation of excitation and emission of energy in atomic emission
spectroscopy

Fig. 7.2 Schematic representation of emission spectra in atomic emission spectroscopy

of emission lines from the excited atoms gives a quantitative measurement of the
concentration of analyte in the sample. As the numbers of atoms or ions is increased,
the amount of the emission intensity will also be increased. Every element has a
unique set of energy levels and hence it will have a unique set of wavelength in
which energy will be released, therefore these wavelengths are specific for every
element present in the sample.

7.3 Types of Emission Spectra Used in AES

When a light beam is passed through prism/grating, it is divided into its components
constituting various colors. This array of colors is known as spectrum. When an
analyte is heated at high temperature by electric or thermal method, there is the
emission of light. When the light, coming after passing through the prism, is
analyzed with spectroscope, the spectrum so obtained is known as emission spec-
trum (Fig. 7.2). It has following three types:

7.3.1 Line Spectra

They consist of sharply defined and often widely and irregularly spaced individual
line of the single wavelength. It is also known as atomic spectra and used for the
analysis of the atoms.
7.4 Components of AES 105

7.3.2 Band Spectra

They consist of group of lines. It is also known as molecular spectra.

7.3.3 Continuous Spectra

They are obtained when solids are heated to incandescence. They are characterized
by the absence of any sharp lines as a function of wavelength.

7.4 Components of AES

It has following components including sample containing analyte, source of energy

to excite the atoms or ions in sample, a monochromator, a detector, and a readout
device/computer (Fig. 7.3).

7.4.1 Emission Source

Plasma, arcs, sparks, and flames are the most commonly used emission sources.
Plasma is a homogeneous mixture of gaseous electrons, ions, and atoms at very high
temperature. There are two types of plasma most frequently used as atomic emission
source, namely inductively coupled plasma (ICP) and direct current plasma (DCP).

7.4.1.1 Inductively Coupled Plasma

ICP is comprised of three concentric quartz tubes. Argon gas stream flows through
these tubes at a rate of 5–20 L/min. Outer tube has diameter of approximately 2.5 cm
and topmost of the outer tube is encircled by a radiofrequency powered induction
coil generating power of almost 2 KW with frequency of approximately 27–41 MHz.
The outer coil also generates a very strong magnetic field. A spark is used to ionize
the argon and this ionized argon then interacts with the strong magnetic field and

Fig. 7.3 Schematic representation of components of atomic emission spectroscopy

106 7 Atomic Emission Spectroscopy

Fig. 7.4 Schematic

representation of inductively
coupled plasma as an
emission source in atomic
emission spectroscopy

forces to move in the surroundings of induction coil with a high speed. Because of
this a high temperature is achieved due to high resistance because of circulating
argon. The topmost of quartz tube gets a very high temperature and therefore, it
should be isolated, then cooled. It may be achieved if argon is passed tangentially
around the tube walls. An ICP is usually known as torch plasma. The torch is
produced due to the emission of argon at a very high temperature of the plasma
(Fig. 7.4).

7.4.2 Monochromator

Similar to an AAS, the monochromator acts as a wavelength selector and it separates

different wavelengths and selects the required wavelength. They may be prism
monochromator or grating monochromator.

7.4.3 Detector

The required wavelength from the monochromator is allowed to pass through a

detector which is used to convert light signal into electrical signal. These are the
devices that are responsible to detect the wavelengths which are being absorbed by
the sample. The most commonly used detector in AES is photomultiplier tube which
is actually vacuum photo tube and possess high sensitivity.
7.7 Interferences of AES 107

7.4.4 Readout Device

It is used to display the absorption spectrum and absorbance at a specific wavelength.

Nowadays, the read out devices have microprocessor-controlled electronics that
deliver outputs compatible with computers and printers. Thus, it minimizes the
risk of operator error while transferring data.

7.5 Working of AES

Instrumentation of atomic emission spectroscopy is almost the same as that of atomic

spectroscopy with a slight difference. Light source is not required. Atomization
source is most important component here which is required for the atomization of
sample and excitation of free atoms. When atoms reached in their excited state they
move to the ground state with the emission of light which passes through the
monochromator which isolates the specific wavelength for a specific element. This
specific wavelength then enters in detector that converts light signal into electrical
signal. At the end, the absorption spectrum is displayed on the screen of the readout
device.

7.6 Comparison Between AAS and AES

Comparison between AAS and AES has been described in Table 7.1.

7.7 Interferences of AES

1. Ionization Interference: During high flame temperature, metal such as potassium

may completely lose an electron that ultimately reduces the observed emission
from the sample.
2. Viscosity interference: Organic substances may either decrease or increase the
rate with which it draws towards flame compared to a standard solution. For
example, sucrose usually decreases the rate, consequently giving false negative

Table 7.1 Comparison of atomic absorption spectroscopy with atomic emission spectroscopy
AAS AES
It depends upon the number of atoms present in It depends upon the number of atoms present in
ground state excited state
It measures the radiations absorbed by the It measures the radiations emitted from atoms
atoms in ground state in excited state
Light source is present Light source is absent
The temperature of the atomizer is adjusted for The temperature of the atomizer is high enough
the atomization of atoms of analyte present in in order to atomize the atoms of analyte and
the ground state only excite them to a greater energy level
108 7 Atomic Emission Spectroscopy

results, on the other hand, ethanol may increase the rate, consequently giving a
false positive result.
3. Anionic interference: Anions like sulfates and phosphates form non-volatile salts
by reacting with metal ions and decrease the reading of a given sample solution.
The anions may be removed with the addition of lanthanum chloride, it
participates them out and replaces them with the chloride anions.

7.8 Applications of AAS

7.8.1 Analysis of Pharmaceuticals

1. For quantitative analysis of alkali metals in alkali metal salts, dialysis solutions,
and infusions.
2. For determination of metallic impurities in some inorganic salts that are used in
the preparation of many solutions.
3. To detect lithium, sodium, and potassium in some raw materials of
pharmaceuticals.
4. To detect metals during IPQC and final dosage forms of pharmaceuticals.

7.8.2 Biomedical Applications

1. Detection of metals in biological fluids.

2. Detection of metals (Cu) in brain.
3. Estimation of sodium salts in breast milk.

7.9 Advantages

This technique has the following advantages:

1. High accuracy.
2. High resolution.
3. Low stray light.
4. Wide dynamic range.
5. High precision.
6. Highly reproducible.
7. Preferred over atomic absorption spectroscopy as all the atoms in a sample are
detected at the same time.
Further Reading 109

7.10 Disadvantages

This technique has the following disadvantages:

1. Expensive.
2. Destroying the sample.
3. Used mainly for metals.

Further Reading
Labmate Online. Atomic emission spectroscopy. Accessed 12 Sept 2019
LibreTextsTM. Atomic emission spectroscopy. Accessed 12 Aug 2019
Metcalfe E (1987) Atomic absorption and emission spectroscopy. Wiley, Hoboken
Sudha PC (2012) Pharmaceutical analysis. Pearson Education India, Chennai
Waters Corporation. Atomic emission spectroscopy. Accessed 15 Aug 2019
Watson DG (2015) Pharmaceutical analysis E-book: a textbook for pharmacy students and phar-
maceutical chemists. Elsevier Health Sciences, Amsterdam
Molecular Emission Spectroscopy
8

Abstract
In this chapter, the steps involved in molecular emission spectroscopy have
been completely described. The types of fluorescence are schematically
discussed. There are multiple components of molecular emission spectroscopy
that are needed for proper functioning. The factors that affect the fluorescence
have been described along with quenching. At the end of chapter, application of
AES has also been discussed.

Keywords
Types of luminescence · Types of fluorescence · Fluorescence intensity ·
Quenching

8.1 Introduction

Molecular emission spectroscopy is an analytical technique that is used for

measurement of the emission from excited atoms of elements present in samples
by quantitative. The energy to excited atoms is provided by the radiation source.
It is the phenomena in which the molecules of element present in sample solutions
absorb the radiation of specific wavelength (at ground state having low energy level)
and get excited (at excited state having low energy level). At excited state, the
molecules are unstable and emit the radiation. The emitted radiation is estimated
from the excited molecules in the sample after the absorption of radiations of specific
wavelength which is referred as excitation wavelength. The measurement of the
emitted radiation at a longer wavelength is known as fluorescence or emission
wavelength. Molecular emission spectroscopy is a type of spectroscopy in which
the atoms and/or molecules during the transition from an excited state to ground
state, emit the radiation of specific wavelength which can be measured (Fig. 8.1).
What happens after a molecule has absorbed light? (Fig. 8.2). A large number of
substances are known which can absorb UV or VIS light energy and lose excess

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112 8 Molecular Emission Spectroscopy

Fig. 8.1 Schematic

representation of emission of
light by the molecule
and/or atom

Fig. 8.2 Schematic representation of absorption of light by molecule

energy as heat through collision with neighboring atoms or molecules. But some
substances also lose only part of this excess energy as heat and emit the remaining
energy as ER wavelength longer than that absorbed. This process of emitting
radiation is known as luminescence or photoluminescence and the substance
which exhibits such characteristics is known as luminescent.

8.2 Types of Luminescence

8.2.1 Fluorescence

When a substance absorbs radiation and immediately emits the radiation after
the radiation absorption, this phenomenon is called as fluorescence. The substances
which exhibit phenomenon of fluorescence are called as fluorescent. The phenome-
non of fluorescence happens instantaneously and begins immediately after the
absorption of radiation of light and terminates quickly as the incident light radiations
are cut off.
8.3 Theory 113

8.2.2 Phosphorescence

When a substance absorbs the radiation and subsequently emits the radiation contin-
uously after the absorption of radiation is cut off. This type of phenomenon is
called as phosphorescence. The substances exhibit such characteristic are called
as phosphorescent substances.

8.2.3 Chemiluminescence

During the process of chemiluminescence, light is produced from chemical reaction.

During chemical reaction, the light is emitted because this reaction produces electri-
cally excited molecules. When these excited molecules return to their ground state,
the emit light. In many biological systems, the chemiluminescence reactions occur
where this phenomenon is known as bioluminescence. One of the distinguished
characters of chemiluminescence is that it does not need any sophisticated instru-
mentation. Additionally, no external radiation source is also required for excitation
purpose. The instrument may contain only two things, one is reaction vessel and the
other one is a photomultiplier tube. The devices that are used for the selection of
wavelength are not needed because the source of radiation is only chemical reaction.
Moreover, there is no need of excitation source. Chemical reaction delivers the
energy in order to excite the molecules. In its simplest form, chemical reaction can
be represented as follows:

A þ B ! C ! C þ hν:

Chemiluminescence has wide range of applications. It can be used for the

detection of arsenic in water and can also be applied to fabricated microarrays on
a flow chip, allowing for patterned biosensor applications.

8.3 Theory

Following steps (Fig. 8.3) are involved in molecular emission spectroscopy:

1. Vibrational relaxation: There is a transfer of surplus energy from vibrationally

excited specie to the solvent molecules. This process happens within very short
time span of approximately 1015 s and solvent molecules return back in the low
vibrational energy state from an electronically excited state. The molecules that
are found in the singlet excited lose the energy comparatively easier due to the
collision with surrounding molecules of solvent.
2. Internal conversion: When the lower and upper electronic states of the
excited singlets have same multiplicity, this phenomenon is known as internal
conversion.
114 8 Molecular Emission Spectroscopy

Fig. 8.3 Schematic representation of electronic levels and transitions in a fluorescence and
phosphorescence

3. Photon emission: The molecules in the singlet excited state come back to the
ground state and cause the emission of the photon that is called as fluorescence.
4. Energy transfer: The energy is transferred from singlet state to the triplet
state when the molecule come back to the ground state which is also called as
intersystem crossing.

Figure 8.4 briefly describes how fluorescence excitation-emission cycle takes

place on emission spectroscopy.

8.4 Principle

When one electron from an electron pair present in a molecule is get excited
(Fig. 8.5) to a high level of energy then may be a singlet or triplet state produced.
During the excited singlet state of molecule, the spinning movement of the excited
electron remains opposite to that of the left-over ground state electron. However,
during the triplet state, the spinning movement of both electrons becomes parallel
8.5 Types of Fluorescence 115

Fig. 8.4 Schematic representation of fluorescence excitation-emission cycle

Fig. 8.5 Schematic representation of states of the electrons in molecule in ground and excited
states

and unpaired. The excited singlet state is comparatively more energetic as compared
to its corresponding unpaired singlet state.

8.5 Types of Fluorescence

Following are the important types of fluorescence as shown in Fig. 8.6:

116 8 Molecular Emission Spectroscopy

Fig. 8.6 Schematic representation of types of fluorescence

Fig. 8.7 Schematic representation of components of atomic emission spectrophotometer

8.6 Instrumentation

Following are the important components of AES which have been schematically
illustrated in Fig. 8.7:

8.6.1 Radiation Source

The plasma can be generated by applying the potential difference between the two
electrodes. The generation of plasma by this way is known as direct current plasma.
For this purpose, plasma supported gas is needed. An argon is commonly used for
this purpose. In spectroscopic analysis, for desolvation and vaporization of sample,
heat may also be provided by the flame.
8.7 Factors Affecting the Fluorescence Intensity 117

8.6.2 Filters

Monochromator or filter is a very essential part in an atomic emission spectrometer.

It is used for the separation of all the thousands of lines. If good monochromator is
not used, the efficiency of spectrometer is severely compromised.

8.6.3 Detectors

In atomic emission spectroscopy, the most commonly used detector is

photomultiplier tube (PMT). PMT is used for the detection of weak signals. In
photomultiplier tube, photons are absorbed which result in the emission of electrons.
PMT is highly sensitive for UV, VIS, and near-IR regions of EMR.

8.6.4 Amplifiers

It amplifies the signals received from the detector and then transfer to the read-out
device.

8.6.5 Read-out Device

It is used to display the spectrum. Read-out devices work in conjunction with

multiprocessor-controlled electronics that deliver the output to computer and printer.
It minimizes the risk error by operator. The most widely used read-out devices are:

1. Digital voltmeter.
2. Simple galvanometer.
3. Potentiometer.
4. Computer.

8.7 Factors Affecting the Fluorescence Intensity

Following are the most important factors that commonly influence the working
of AES:

1. Temperature
2. Viscosity
3. Nature of solvent
4. pH
5. Presence of solutes
6. Presence of functional groups
7. Nature of the molecule
118 8 Molecular Emission Spectroscopy

8. Structure of the molecule

9. Light

8.8 Quenching

The decrease in fluorescence intensity is known as quenching.

8.8.1 Reasons of Quenching

1. Concentration of the molecule

2. pH
3. Presence of chemical substances
4. Temperature
5. Viscosity

8.8.2 Types of Quenching

1. Self-quenching
2. Collisional quenching
3. Static quenching
4. Chemical quenching

8.9 Applications

1. Used for the determination of inorganic ions.

2. Widely used in the field of pharmaceuticals.
3. Estimation of metals in biological fluids.
4. Estimation of vitamins.

Further Reading
Bonchin SL, Zoorob GK, Caruso JA (1999) Atomic emission, methods and instrumentation.
In: Lindon JC (ed) Encyclopedia of spectroscopy and spectrometry. Elsevier, Oxford, pp 42–50
Cullen M (2004) Atomic spectroscopy in elemental analysis. Taylor & Francis, Milton Park
Hareland WA (1997) Atomic emission spectroscopy. In: Analytical instrumentation handbook,
vol 2. CRC Press, Milton Park, p 221
http://elchem.kaist.ac.kr/vt/chem-ed/spec/atomic/aes.htm
https://chem.libretexts.org/Bookshelves/Analytical_Chemistry/Book%3A_Analytical_Chemistry_
2.0_(Harvey)/10_Spectroscopic_Methods/10.7%3A_Atomic_Emission_Spectroscopy
Lagalante AF (2004) Atomic emission spectroscopy: a tutorial review. Appl Spectrosc Rev
34(3):191–207
Further Reading 119

Moore GL (2012) Introduction to inductively coupled plasma atomic emission spectrometry.

Elsevier, Amsterdam
Pavia DL, Lampman GM, Kriz GS, Vyvyan JA (2014) Introduction to spectroscopy.
Cengage Learning, Boston
Sudha PC (2012) Pharmaceutical analysis. Pearson Education India, Chennai
Varma A (1991) Handbook of inductively coupled plasma atomic emission spectroscopy.
CRC Press, Milton Park
Watson DG (2015) Pharmaceutical analysis E-book: a textbook for pharmacy students and
pharmaceutical chemists. Elsevier Health Sciences, Amsterdam
Mass Spectrometry
9

Abstract
In this chapter, we have described the instrumentation of mass spectrometer along
with comprehensive description of sources of ionization mechanism of ionization
and types of analyzers. Moreover, the description of peaks in the mass spectrum
has been given in this chapter. At the end of this chapter, application along with
advantage and disadvantages has been given.

Keywords
Electron ionization · Chemical ionization · Atmospheric pressure ionization ·
MALDI · Mechanism of ionization

9.1 Introduction

A mass spectrometer is a device that determines the weight of the molecules by

estimating the mass to charge ratio (m/e) of ions. In this technique, molecules are
bombarded with electrons; high energized positive ions are produced which are
further breakdown to produce small fragments. These ions are separated in magnetic
or electric field according to their mass to charge ratio. Figure 9.1 illustrates how
mass spectrometer works.
This technique is used for studying the effect of ionization energy on the
molecules. It depends upon which type of chemical reaction takes place in the
sample which exist in the form of gaseous phase. The function of mass spectrometer
is associated with its components. Such as ion source is responsible for the produc-
tion of ions. The mass analyzer separates the ions on the basis of their mass to charge
ratio. Detector measures the separated ions and the results are displayed on mass
spectrum. The mass spectrum is plotted between ions abundance and mass to charge
ratio. This is used for the detection of isotopes on the basis of their mass. Nowadays,
it is used in combination with the gas chromatograph for the estimation of quantities
of the contaminants and toxins.

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 122

12 units
0 units

Number of counts

Number of counts
14 units
12 units
8 9 10 11 12 13 14 15 16 8 9 10 11 12 13 14 15 16
mass mass
9

Number of counts
12 units
8 9 10 11 12 13 14 15 16
mass

Fig. 9.1 Schematic representation of how mass spectrophotometer works?

Mass Spectrometry
9.3 Instrumentation 123

9.2 Principle

When compound is bombarded with an electron, the compound has tendency to lose
one electron and form the metastable ions as represented in the following equation:

e
M ! M þ þ e :

The increase in energy will lead to the production of cations which are determined
by the mass spectrometer which is based on their m/e of positive ions. Mass to
charge ratio (m/e) is the mass of samples divided by the charge of the sample. Ions
give information about the structure and nature of the sample molecule. The ions
which has high mass to charge ratio, they are heavier isotopes and vice versa. The
molecular mass of these separated ions is also determined. The separated ions on the
basis of m/e ratio are determined in proportion to their abundance. The result will be
obtained in the form of mass spectrum. The mass spectrum is plotted between the
ion’s abundance and m/e ratio. By this way, a mass spectrum of the molecule is thus
produced. It displays the result in the form of a plot of ion abundance versus m/e
ratio.

9.3 Instrumentation

Following are the main components of mass spectrometer. A schematic representa-

tion of components of mass spectrometry has been illustrated in Fig. 9.2:

Fig. 9.2 Schematic representation of components of mass spectrophotometer

124 9 Mass Spectrometry

9.3.1 The Inlet System

The sample is placed in mass spectrometer at an atmospheric pressure. There are two
ways by which the sample can be introduced into the mass spectrometer.

9.3.1.1 Direct Introduction

Initially, the sample is introduced in the probe of mass spectrometer and then placed
into the ionization source.

9.3.1.2 Direct Infusion

The sample capillary is used to introduce the sample into the ionization source.

9.3.2 The Ion Sources

It is a mechanical device which is used to convert the sample into ions. It is process
of charging a molecule. In mass spectrometry, generally, three types of samples are
used for the analysis of solid, liquid, and/or gas samples. Following methods are
used to charge the molecules and its schematic representation has been illustrated in
Fig. 9.3:

Fig. 9.3 Schematic representation of sources of ionization. CI chemical ionization, EI electrical

ionization, ESI electrospray ionization, APPI atmospheric pressure photo-ionization, APCI atmo-
spheric pressure chemical ionization, MALDI matrix-assisted laser desorption ionization
9.3 Instrumentation 125

9.3.2.1 Electron Ionization

It is the most commonly used ionization process that is used in mass spectrometry. It
can be used for variety of gaseous phase molecules. It is mostly used for spectral
libraries. It produces the large number of fragments that is why all the molecular ions
are not observed. Fragmentation is responsible for providing the information about
the structure by interpreting the unknown spectra. For the purpose of ionization, the
electrons are required. These electrons can be produced by passing the current
through a filament. The number of electrons produced by filament is controlled by
the amount of current. These electrons move due to electric field present in the
source region. As a result, a high energy electrons beam is produced. When this
high energy beam passes through a sample, ions are formed because electrons are
removed from the sample. After the ionization of molecules, the spectrum is
produced. The spectrum shows the separation of the product ions. The ions which
have less molecular energy act as intact molecular ions. The molecular ion that has
high energy undergoes the process of fragmentation. The group of these resulting
fragments is known as product ion. These product ions can be determined by
ionization energy and kinetics of fragmentation pathway. The change in kinetic
energy causes the distribution of different fragment ions. With the help of distribu-
tion, information can be interpreted in the form of mass spectra.

9.3.2.2 Chemical Ionization

In this process, ionization of analyte in gaseous state happens by a gaseous phase ion
molecule reaction instead of the field ionization by direct electron impact and photon
impact. The electron ionization of gas is usually occurred due to ion molecule
reaction. Due to this reaction, ions are formed. The gas which is present in excess
amount is neutral for the production of chemical ionization. The collision of these
reagent ions with analyte to produce ionic character in the analyte. Then, analyte
which is formed initially passes through the fragmentation process and sometimes
these may react further with reagent gas for the formation of array of ions. These
arrays of ions produce the mass spectrum of analyte by using specific gas.

9.3.2.3 Electrospray Ionization

Electrospray ionization is a very soft ionization method that is particularly used for
the determination of molecular weight of peptides, proteins, and other biological
fluids. This technique is very helpful especially when we analyzing the sample
having high molecular weight. Fragmentation of sample into small charged particle
is not involved in this technique. In this process, the macromolecules are converted
into small droplets by ionization. These small droplets undergo desolvation process
and very small droplets are produced. These very small droplets produce molecules
having the attached protons. These molecular ions after the protonation and
desolvation process pass through the mass analyzer and then to detector. The mass
of the interested sample can be estimated by using this technique.
126 9 Mass Spectrometry

9.3.2.4 Atmospheric Pressure Ionization

The source of atmospheric pressure ionization (API) can ionize the sample at
atmospheric pressure and these ions are then transferred to the mass spectrometer.
API is used for temperature senstive samples such as polymers, proteins, and
peptides. By dissolving the sample into an appropriate solvent, a solution is formed.
This newly formed solution is placed into mass spectrometer. Due to conventional
inlet, solvent produces more pressure in the source zone of mass spectrometer. When
the liquid enters into a vacuum, the solent freezes due to Joule Thomson effect. This
reduces the efficiency due to the formation of cluster. To avoid this problem, analyte
must pass through a series of pumps. In this process, solvent passes through the
nozzle which converts solvent into small droplets and also get charged. The charge
density of droplet increases due to evaporation of solevent that caused the shrinkage
of droplet and charge density increase on the droplets. These droplets eventually
reached at a point where the surface tension that is responsible for holding these
droplets is less than repulsion from an electric charge. Aa a result, droplet explodes
to produce the multiple ions. The spectra will represent the molecular ions having the
different charge numbers.

9.3.2.5 Atmospheric Pressure Photo-Ionization

A way in which ionization is done by photo-ionization. In this process, a dopant
absorbs the light then it is added to a sample for the ionization of the sample. The
estimation of small molecules in biological mixture becomes very crucial part for the
drug discovery.

9.3.2.6 Atmospheric Pressure Chemical Ionization

This method uses the source of electrospray ionization. But voltage is applied on a
needle that produces corona charge at atmospheric pressure. Due to this discharge,
the ions are produced such as hydronium ion or water cluster. The sample is
discharged through the spray which is produced when heated gas is mixed with
liquid and as a result sample is evaporated. Transfer of the proton from water cluster
or hydronium ion causes the production of ions. These ions are passed through the
opening of vacuum.

9.3.2.7 Matrix-Assisted Laser Desorption Ionization

Matrix-assisted laser desorption ionization (MALDI) is based on the ionization
which can be done by the bombardment of sample molecules along a laser light
for the ionization of sample. MALDI is a soft ionization technique in which a laser
strikes with a matrix of many small molecules in order to make a gaseous state from
the sample molecule without causing the fragmentation or decomposition in sample.
Some high molecular weight biomolecules can be decomposed by heating and
some conventional techniques can also cause the fragmentation or destruction of
macromolecules. MALDI is suitable to analyze the biomolecules like saccharides,
lipids, peptides, or many other organic macromolecules. The sample is mixed with a
very high absorbing matrix compound (X) and then dried on a plate. An analyte is
embedded in a very large amount of the matrix compound that is deposited on a solid
9.3 Instrumentation 127

Fig. 9.4 Schematic representation of mode of action of MALDI in mass spectrometry

surface known as target. The target is constructed of a conducting metal and having
many spots. After a very brief laser pulse, the irradiated spot excited vibrationally
due to heating. The matrix molecules energetically excited from the sample surface
and absorbed the laser energy, as a result the analyte molecules are converted into the
gaseous phase. During the excitation process, the analyte molecules are getting
ionized due to protonation or deprotonation with the nearby matrix molecules. A
schematic representation of mode of action of MALDI has been illustrated in
Fig. 9.4.

9.3.2.8 Mechanism of Ionization

Protonation
When a proton is added to a molecule then it increases the positive charge.

M þ Hþ ! MHþ :

It is used in MALDI, electrospray ionization and, APCI. The samples used are
carbohydrates.

Deprotonation
A proton is removed from a molecule which causes production of cations.

M  Hþ ! ðM  HÞþ :

It can be used in MALDI, APCI, and electrospray ionization. The sample used in
this process is salicylic acid.
128 9 Mass Spectrometry

Cationization
This can be done by the addition of cation into the molecule along with an
ammonium or alkali. This is a very stable method as compared to that of protonation.

M þ cation ! Mcationþ :

It is used in MALDI, APCI, and electrospray ionization. The sample used is

D-galactose.

Charge Transfer
This is also called as desorption. In this method the sample solution is converted into
gas state. It is particularly used for charged complexes. This method cannot be used
for many other compounds.

Mþ ðsolutionÞ ! Mþ ðgasÞ:

It is used in MALDI and electrospray ionization. The sample used is tetraphenyl

phosphine.

Electron Ejection
This can be done by removing the electron from molecule to form the positively
charged molecule. It is used in the process of electron ionization. The sample used is
anthracene.

e
M ! Mþ :

Electron Capture
Addition of electrons to the sample molecule by absorption or capture.

þe
M ! M :

9.3.3 Electrostatic System

The cations produced from the ionization source are allowed to pass through the
electric field. Electric field is produced between the repeller plate and the accelerator
plate which causes the acceleration of the ions of masses m1, m2, and m3 to their final
velocities.

Energy eV ¼ ½m1 v1 2 ¼ ½m2 v2 2 ¼ ½m3 v3 2 :

9.3 Instrumentation 129

9.3.4 Ion Separator

This is commonly called as analyzer in which sample molecules are separated on the
basis of their masses.

9.3.4.1 Types of Analyzers

Single Focusing Analyzer (FSA)

At an applied voltage, all the ions which are ionized have the same energy. In this
instrument, single magnet sector is used to generate the magnetic field. The ions are
separated on the basis of mass to charge ratio values by the generation of magnetic
field. It consists of horse shaped glass tube, small inlet, source for bombarding the
electrons and accelerated plates on one end and collector slit on another end. The
curvature tube provides electric and magnetic field. The analyte in the vaporize form
allows to pass through the inlet and bombarded with electrons. As a result, one
electron escapes from each molecule and consequently becomes a positive charge.
By getting charge, they are accelerated by plates and moves in a straight path. Due to
electric and magnetic field, the ions are separated on the basis of charge to mass
ration. The mass spectrum is obtained by these fragments.

Double Focusing Analyzer (DFA)

It is specifically used for achieving the high resolution. Two ion beams are allowed
to pass through and at the end, these are detected by two separate collectors. In this
instrument, an electric sector is interposed to provide the double focusing analyzer.
This has high resolution than single focusing analyzer. It is used to differentiate the
small differences in the segment.

Time of Flight Analyzer (TOF)

By changing their direction, the ions can be separated. These separated ions travel
through an air-free zone, the time utilized by these ions to travel this distance is
known as the flight tube. It has poor mass resolution. Heavier ions move slowly than
the lighter ones. A schematic illustration of TOF has been represented in Fig. 9.5.
MALDI is based on the bombardment of sample molecules with a laser light to
bring about sample ionization. The sample is pre-mixed with a highly absorbing
matrix compound. The matrix transforms the laser energy into excitation energy for
the sample, which leads to sputtering of analyte and matrix ions from the surface of
the mixture. In MALDI-TOF mass spectrometry, the ion source is MALDI and the
mass analyzer is time of flight (TOF) analyzer (Fig. 9.6).

Quadrupole Analyzer
Quadrant of four parallel circular tungsten rods filter the ions by focusing on the ions
through an oscillation with the help of radiofrequency. Quadrupole mass analyzers
are constructed of four rods with a hyperbolic or circular cross section (Fig. 9.7).
Each pair of opposing rods has positively or negatively charged. The ions in the
analyzer are separated on the basis of mass to charge ratio of ions which depend
130 9 Mass Spectrometry

Fig. 9.5 Schematic representation of time of flight (TOF) analyzer

Fig. 9.6 Schematic representation of MALDI-TOF

upon their trajectories when these ions are exposed to the electric field in the space
present between the rods. Oscillate ions travel in an applied electric field (the
quadrupole field) between the paired rods of the quadrupole. By altering the
9.3 Instrumentation 131

Fig. 9.7 Schematic representation of quadrupole analyzer in mass spectroscopy

characteristics of the field, manipulation of ions and molecules having the specific
m/z ratio (molecule A+) will start to oscillate with a harmonic ion trajectory by
creating an ion beam that navigates the quadrupole. All other ions (molecule B+) can
be filtered out of the ion beam.

Fourier Transform Ion-Cyclotron Resonance (FT-ICR)

It is also called as penning ion trap in which magnetic field is used to trap and store
the ions.

9.3.5 Ion Collector

It is also known as ion receiver. Following are the types of ion collector:

1. Photographic plates.
2. Electron multipliers.
3. Electrometers.
4. Faraday cylinders.

9.3.6 Vacuum System

In this system, mercury and oil diffusion pumps are most commonly used.

• Inlet vacuum ¼ 0.015 Torr.

• Ion source ¼ 105 Torr.
• Analyzer ¼ 107 Torr.
132 9 Mass Spectrometry

Fig. 9.8 Schematic representation of types of peaks in mass spectroscopy

9.4 Types of Peaks

There are several types of peaks that can be observed during analysis (Fig. 9.8). The
types of these peaks have been described as follows:

9.4.1 Molecular Peak

It is also called as parent peak. This peak can be produced by the bombardment of the
sample to lose one electron and then this peak can be obtained.

9.4.2 Fragment Peak

This peak is observed when the fragment ions produce when energy is supplied to
the molecular ions.

9.4.3 Rearrangement Ion Peak

This peak is obtained due to the rearrangement of the fragment ions.

9.5 Types of Mass Spectrometry 133

9.4.4 Metastable Ion Peak

The ion produced from the source and analyzer is called as metastable ion and the
peak which is obtained is called as metastable ion peak. These obtained peaks are
broader having the low intensity.

9.4.5 Multicharged Ion Peak

The peaks produced from ions which may have more than one charge are called as
multicharged ion peaks, e.g., CO2, N2, CO.

9.4.6 Base Peak

The largest peak in the spectrum is called as base peak.

9.4.7 Negative Ion Peak

After the increase of energy positive ions are formed and the negative ions also
exhibit the peaks, but these peaks are negligible in MS.

9.5 Types of Mass Spectrometry

Following are the most important types of mass spectrometry:

9.5.1 Gas Chromatography-Mass Spectrometry (GC-MS)

When GC is combined with MS, it increases the sensitivity of the identification and
structure elucidation of the compounds. GC separates the semi-volatile and volatile
compounds, but it does not identify these compounds whereas, MS does this job.

9.5.1.1 Modes of Operation of GC-MS

Following three modes are used:

1. Spectral mode.
2. Total ion current mode.
3. Selective ion monitoring mode.

9.5.1.2 Advantages
1. Highly sensitive.
2. Powerful instrument for qualitative and quantitative analysis.
134 9 Mass Spectrometry

9.5.1.3 Disadvantages
1. Time consuming.
2. Non-volatile compounds cannot be used.
3. Thermolabile compounds cannot be used.

9.5.2 Liquid Chromatography-Mass Spectrometry (LC-MS)

This is the combination of LC and MS.

9.5.2.1 Advantages of LC-MS

1. Most advanced technique than GC-MS because no heating is required.
2. Non-volatile and thermolabile can be used for analysis.
3. Mainly used for structural and molecular weight determinations.
4. Retention time is less than that of GC-MS.
5. Highly sensitive than that of GC-MS.
6. Operating is comparatively easy than that of GC-MS.

9.5.3 Chemical Ionization Mass Spectrometry (CI-MS)

CI-MS is commonly used for the physicochemical characterization of the molecules

such as ions strike with the molecules which are present in the ion source. Chemical
ionization is the basic phenomenon for CI-MS.

9.5.4 Field Ionization Mass Spectrometry (FI-MS)

The molecules having less parent ion can be determined using this technique. FI-MS
has foil type field ionization source which is attached to the mass analyzer and the
data is recoded by using the detector. The modern technique of FI-MS has been
introduced which is FD-MS. In FD-MS, the sample is evaporated by field ion emitter
and then introduced in high electric field.

9.5.5 Fast Atom Bombardment Mass Spectrometry (FAB-MS)

In this technique there is bombardment of the compound with high energy neutral
particles. For example, argon and xenon. This technique is commonly used for the
estimation of vitamins, nucleotides, and peptides.
9.8 Disadvantages 135

9.6 Applications

1. In the field of biotechnology, it is used for the analysis of proteins, peptides,

oligonucleotides.
2. In the field of pharmaceutical industry, it is used for the drugs discovery,
combinatorial chemistry, pharmacokinetics, drug metabolism, purity of
compounds, drug stability, drug–polymer interaction.
3. In the field of clinical and medical sciences, it is used for neonatal screening,
hemoglobin analysis, drug testing, detection of steroids, disease diagnosis, deter-
mination of anesthetics.
4. In the field of environmental sciences, it is used for the detection of various types
of pollutants in atmosphere, environment, and water.
5. In the field of polymer chemistry, it is used for the characterization of polymers.
6. In the field of food chemistry, it is used for the find out the adulteration in food
stuff, detection of toxicants in food.
7. In the field of medicinal chemistry, it is used for the determination of:
• Accurate molecular weight can be measured.
• Purity of sample, unknown sample, and other side products.
• Reaction monitoring.
• Protein digestion, chemical modification, enzyme activity.
• Structural elucidations.
• By the fragmentation of unknown products and natural products.
• Mechanism of actions of the compounds can be identified.
• The compounds having either non-covalent or the covalent interactions.
• Active site identifications.
• Quantitative analysis.
• Impurity profiling for the bulk drugs.

9.7 Advantages

1. It is high sensitivity.
2. It requires the small size of sample.
3. It is time saving.
4. When it used in combination with other methods, it exhibits high sensitivity and
acceptability.
5. It can be used for differentiation the isotopes of the atom.

9.8 Disadvantages

1. Only pure compounds can be handled readily.

2. Compounds that are non-volatile in nature cannot be handled by MS.
136 9 Mass Spectrometry

Further Reading
Duncan MW, Roder H, Hunsucker SW (2008) Quantitative matrix-assisted laser desorption/
ionization mass spectrometry. Brief Funct Genomic Proteomic 7(5):355–370
Fuchs B, Schiller J (2009) Application of MALD – TOF mass spectrometry in lipidomics. Eur J
Lipid Sci Technol 111(1):83–98
Gauglitz G, Moore DS, Vo-Dinh T (2014) Handbook of spectroscopy. Wiley, Hoboken
Gross JH (2006) Mass spectrometry: a textbook. Springer, Berlin
Guerrera IC, Kleiner O (2005) Application of mass spectrometry in proteomics. Biosci Rep 25
(1–2):71–93
Kemp W (2017) Organic spectroscopy. Macmillan, London
Kenny DJ, Brown JM, Palmer ME et al (2006) A parallel approach to post source decay MALDI-
TOF analysis. J Am Soc Mass Spectrom 17(1):60–66
Labmate Online. Mass spectrometry. Accessed 12 Sept 2019
LibreTextsTM. Analytical methods in spectroscopy. Accessed 2 Oct 2019
Sudha PC (2012) Pharmaceutical analysis. Pearson Education India, Chennai
Waters Corporation. Mass spectrometry. Accessed 15 Sept 2019
Watson DG (2015) Pharmaceutical analysis E-book: a textbook for pharmacy students and phar-
maceutical chemists. Elsevier Health Sciences, Amsterdam
Nuclear Magnetic Resonance Spectroscopy
10

Abstract
Nuclear magnetic resonance (NMR) spectroscopy is usually combined with
infrared (IR) spectroscopy for the complete analysis of the structure of an
unknown molecule. IR spectroscopy is used to detect a functional group in the
sample, whereas NMR spectroscopy detects number of atoms and their type in
sample. NMR technique can detect many nuclei but mostly identifies carbon-
hydrogen frameworks. In this chapter, we have comprehensively discussed the
NMR spectroscopy, its types, basic mechanism along with its instrumentation,
applications, advantages, and disadvantages.

Keywords
Nuclear shielding · NMR spectra · Components of NMR spectroscopy · Types of
NMR spectra

10.1 Introduction

NMR spectroscopy also known as magnetic resonance spectroscopy (MRS) is the

most powerful analytical technique among all spectroscopic techniques. It visualizes
single atom and molecule in various media, both in solution state and solid state.
NMR is a nondestructive technique and it gives molar response that can be used for
structure elucidation and quantification. Magnetic interactions which occur between
the active nuclei and NMR along with covalent bonds result into spin–spin coupling.
We can detect the space interactions by using the effect of nuclear Overhauser
enhancement (NOE). These interactions are used for the elucidation of three-
dimensional structure. However, 1-dimensional and 2-dimensional NMR data can
also be collected. The 1-D NMR experiments are 1H, 13C, 19F, and 31P. The 1-D
NMR techniques are used to study the chemical shift, spin–spin coupling, and
intensities. Information regarding protons and its environment can be obtained
with the help of chemical shift. Nuclei closer to each other exert an effect on other’s

# Springer Nature Singapore Pte Ltd. 2020 137

M. S. H. Akash, K. Rehman, Essentials of Pharmaceutical Analysis,
https://doi.org/10.1007/978-981-15-1547-7_10
138 10 Nuclear Magnetic Resonance Spectroscopy

Fig. 10.1 Schematic representation of distribution of nuclear spins

effective magnetic field. This effect is shown in NMR spectrum when the nuclei are
non-equivalent. If the distance between the non-equivalent nuclei is less than or
equal to three bond lengths then this effect can be observed, the effect is called
indirect spin–spin coupling. Radio waves are the energy source in NMR that has
longer wavelengths, and hence has lesser energy and frequency. When low-energy
radio waves interact with any molecule, then they change the nuclear spins of some
elements, including 1H and 13C. The nuclear spins distribution is mostly random if
there is no external magnetic field (Fig. 10.1a). But when an external magnetic field
is applied then it aligns the nuclear magnetic moments with the applied field either in
parallel or anti-parallel manner (Fig. 10.1b). If parallel alignment happens then
nuclear magnetic moments will be slightly more (Fig. 10.1c).
Resonance: It is the process of amplification which occurs when the frequency of
the applied force shows harmony with the system’s natural frequency. It is related to
the alteration in nuclear spin of systems from lower energy state to a higher energy
state by the process of energy absorption. This can be done by the creating a
magnetic field around the nuclei.
Spin: it is a number which is associated with the quantum mechanical property of
nuclei. Its value must be an integer if mass number is even or half integer if mass
number is odd.
Spin-lattice relaxation: Lattice is a term used for the nuclei which is held in the
framework, whereas lattice field is generated when a magnetic field is created by
vibration of sample nuclei. Magnetic field which shows equilibrium with the ground
state energy field is known as spin-lattice relaxation.
Spin–spin relaxation: It is simply the interaction between the neighboring nuclei
having same frequencies but having different magnetic quantum. In this state, the
nuclei can exchange their quantum state with the nucleus which is excited in the
lower energy state and with the excited nucleus which is relaxed to lower energy
state.
Spin–spin coupling: It is the effect of spin state of one nucleus on the energy of
another nucleus which is responsible for peak splitting. This effect is transmitted by
intervening bonding electrons. Because of this effect, lines of NMR spectra are split.
Nuclear Overhauser enhancement (NOE): When spectrum of proton-decoupled
13
C is obtained then intensity of resonance of some carbon atoms is increased
significantly than those observed in proton-coupled experiment. Carbon atoms
which are attached directly to the hydrogen atoms are enhanced the most and
10.3 Intensities of Resonance Signals 139

when more hydrogen atoms are attached (via saturation) this enhancement is
increased. This effect is known as nuclear Overhauser effect and the degree of
enhancement in the signal is called NOE.
Nuclear Shielding: The applied magnetic field is not equal to the magnetic field
around the nucleus because the electrons present around the nucleus shield it from
the applied field. The difference of both these magnetic fields is known as nuclear
shielding whereas

Chemical shift ¼ nuclear shielding=applied magnetic field

10.2 Types of Nuclear Shielding

1. Local shielding: it is the field created by local electrons on that nucleus.

2. Low range shielding: it is a field which I created by π-electrons that are not
associated with the nucleus.

10.3 Intensities of Resonance Signals

These are of two types which are as follows:

10.3.1 1H NMR Signal Intensities

1
H-NMR is used to detect the type and number of H atoms in a molecule. The
intensity or integral of a signal in the spectrum is thought to be the area under that
signal. The ratios of protons present in a molecule of the compound can be deter-
mined with the comparison of signal intensities in the spectrum. If spectrum shows
multiple readings then whole group of peaks should be integrated separately. The
signal intensity is an important parameter to determine the structure of molecule and
for quantitative analysis of molecule.

13
10.3.2 C NMR Signal Intensities
13
C-NMR is used to detect the type of carbon atoms in the molecule (Fig. 10.2).
13
C-NMR signal is valuable in determining the total number of C-atoms responsi-
ble for the signal. Practically, low abundance and less sensitivity of the 13C isotope
will have an effect on the quantification of number of carbon atoms in the
molecule. Therefore, the signals of carbon are usually not integrated in the
spectrum of 13C NMR. The quantification of 13C signal can be made possible
140 10 Nuclear Magnetic Resonance Spectroscopy

Fig. 10.2 A schematic representation of 13C spectrum of methyl propionate obtained from NMR

with suppression of the NOE, high digital resolution, rate of pulse repetition which
should not be too fast, high pulse power, and small spectral width.

10.4 One-Dimensional NMR Spectroscopy

The spectrum of 1D NMR spectroscopy has 2 dimensions, x-axis which is the

frequency axis and y-axis which corresponds the signal intensities.

10.5 Two-Dimensional NMR Spectroscopy

In the spectrum of 2D NMR both x-axis and y-axis represent frequency and intensity
is shown on the z-axis. In the spectrum of 2D J- resolved NMR, the chemical shifts
are present along x-axis and coupling constants are plotted along y-axis. If both of
the axes correspond to chemical shifts, then it is known as 2D (shift) correlated NMR
spectrum. The correlations can be homo nuclear (1H–1H) or it can be hetero nuclear
(1H/13C).

10.6 NMR Spectra

It is actually a graph between the intensity of peak and its chemical shift which is
measured in ppm.
10.7 Components of NMR Spectroscopy 141

Fig. 10.3 Schematic representation of components of NMR spectrophotometer

10.7 Components of NMR Spectroscopy

General representation of basic components of NMR spectrophotometer has been

illustrated in Fig. 10.3 and the detail description of individual component of NMR
has been discussed in the following sub-sections.

10.7.1 The Magnet

In the absence of magnetic field, all nuclear spin states are well populated and
therefore there is no net polarization. Therefore, an external magnetic field is applied
to achieve a preferential population of nuclear energy spin states. A higher magnetic
field leads to greater separation of energy levels and greater polarization at equilib-
rium. The magnet may be a powerful permanent magnet or a cryogenically cooled
superconducting electromagnet. Both of these magnets align the nuclear spin in the
sample.

10.7.2 A Radiofrequency Oscillator

It consists of radiofrequency synthesizers and amplifiers. They generate a pulse

sequence containing radiofrequency pulses of specific frequency, phase, amplitude,
shape, and time duration. Multiple radiofrequency oscillators are required as some
142 10 Nuclear Magnetic Resonance Spectroscopy

NMR experiments need simultaneous application of radiofrequency pulses having

different frequencies.

10.7.3 The Sample Holder

It is used to load the sample under analysis. Mostly a glass tube is employed for the
holding of both liquid and solid sample.

10.7.4 A Radiofrequency Receiver

It consisted of some components that are preamplifier, amplifier, mixer, and a

converter which converts analog into digital.

10.7.5 A Recorder

A computer is used to display the results on its screen and record the data.

10.8 Solvents Used in NMR Spectra

The solvents should be:

• Chemically inert.
• Show magnetic isotropy.
• Must be volatile.
• And hydrogen atoms should be absent.

Most commonly used solvents are cadmium chloride, carbon tetrachloride, deu-
terium oxide, carbon disulfide, and hexa deuteriobenzene.

10.9 How to Interpret NMR Spectra

Before the interpretation of NMR spectra, it is very important to understand the role
of chemical shift and reference peak during the interpretation of NMR spectra. Role
of chemical shift and reference peak has been briefly elaborated in Fig. 10.4a.
Similarly, how the chemical shift is moved across the NMR spectra, has been
described in Fig. 10.4b.
10.10 Types of NMR Spectra 143

Fig. 10.4 The schematic representation of interpretation of NMR spectra

10.10 Types of NMR Spectra

There are many types of NMR spectra (Fig. 10.5) that depend on many factors
including (a) type of the instrument being used, (b) nucleus and its type that is
involved, (c) physical state of the analyte, (d) environment around the sample
nucleus, and (e) object of data collection. Commonly, NMR spectra are of two types.
144 10 Nuclear Magnetic Resonance Spectroscopy

Fig. 10.5 Schematic

representation of types of
NMR spectra

10.10.1 Wide-Line Spectra

This is the spectra in which the bandwidths between lines are so large that we can
elucidate the fine structure of the analyte because of chemical environment. Each
species shows its own single peak.

10.10.2 High-Resolution Spectra

Most NMR spectrums have a high resolution and they are collected through
instruments which differentiate very small frequency differences that can be of
0.01 ppm or less. For example, in the lower-resolution spectrum of ethanol,
3 peaks can be seen which formed due to the absorption by the protons of CH3,
CH2, and OH, whereas in the higher resolution spectrum, 2 out of 3 peaks are
resolved into the additional peaks.

10.11 Advantages

1. High resolution.
2. High flexibility.
3. Non-destructive method.
4. Analytically tractable.
5. Highly predictable for small molecules.
Further Reading 145

10.12 Disadvantages

1. Low accuracy.
2. Highly expensive.
3. Not able to differentiate the same compounds.
4. Time consuming.

10.13 Applications

1. In the field of natural product chemistry, NMR can be used for the structural and
chemical elucidation of isolated compounds.
2. In the field of synthetic and organic chemistry, it can be used as an analytical
tool of choice by synthetic and organic chemists.
3. It can be used for the determination of study of dynamic processes like reaction
kinetics and study of equilibrium.
4. It is also used for 3-dimensional studies of proteins, protein-ligand complexes,
polysaccharides, DNA < RNA and protein-DNA complexes.
5. In the field of drug design, it is also used for the determination of structure
activity relationship.
6. In the field of medicine, it is also used for the detection of tumors, amino acids,
proteins. RNAs and DNAs. It is also used in metabolic fingerprints from
biological fluids.
7. It can also be used for the purity determination of any compound provided that
molecular weight of structure of that compound is known.
8. It can also be used for diagnostic purposes, e.g., to determine the metabolic
products in body fluids.
9. It is widely used for the study liposomes.
10. This technique allows easy and non-destructive of many components involved
in biodiesel standardization, e.g., water, phosphorus, alcohol, and glycerol
content.
11. In the field of food sciences, this technique is used to analyze moisture content,
solid fat content, etc.

Further Reading
Beckett A, Stenlake J (1997) Practical pharmaceutical chemistry, part II, vol 1. CBS Publications
and Distributors, New Delhi, pp 275–300
Bovey FA, Mirau PA, Gutowsky H (1988) Nuclear magnetic resonance spectroscopy. Elsevier,
Amsterdam
Bruch M (1996) NMR spectroscopy techniques. CRC Press, Boca Raton
Creaser CS, Davies AMC (1988) Analytical applications of Spectroscopy. Blackwell Science,
Oxford
Ernst RR, Bodenhausen G, Wokaun A (1987) Principles of nuclear magnetic resonance in one and
two dimensions. Clarendon Press, Oxford
146 10 Nuclear Magnetic Resonance Spectroscopy

Gauglitz G, Dakin JP (2017) Spectroscopic analysis. In: John PD, Robert GWB (eds) Handbook of
optoelectronics, vol 2. CRC Press, Boca Raton, pp 569–600
Gauglitz G, Moore DS, Vo-Dinh T (2014) Handbook of spectroscopy. Wiley, Hoboken
Holzgrabe U (2017) NMR spectroscopy in pharmaceutical analysis. Elsevier, Amsterdam
Ionin B, Ershov BA, Kol’tsov A (1983) NMR spectroscopy in organic chemistry, vol 167.
Khimiya, Leningrad
Jackman L (2012) Dynamic nuclear magnetic resonance spectroscopy. Elsevier, Amsterdam
Lambert JB, Mazzola EP, Ridge CD (2019a) Nuclear magnetic resonance spectroscopy: an
introduction to principles, applications, and experimental methods. Wiley, Hoboken
Lambert JB, Mazzola EP, Ridge CD (2019b) Nuclear magnetic resonance spectroscopy: an
introduction to principles, applications, and experimental methods. Wiley, Hoboken
Lehmann T (2018) Nuclear magnetic resonance spectroscopy. Multidisciplinary Digital Publishing
Institute, Basel
LibreTexts™. Analytical methods in spectroscopy. Accessed 2 Oct 2019
LibreTexts™. Nuclear magnetic resonance spectroscopy. Accessed 22 Aug 2019
Mooney EF (1970) Annual reports on NMR spectroscopy. Academic Press, Cambridge
Myers RJ (1973) Molecular magnetism and magnetic resonance spectroscopy. Prentice-Hall,
New York
Nöth H, Wrackmeyer B (2012) Nuclear magnetic resonance spectroscopy of boron compounds.
Springer, Berlin
Reichenbächer M, Popp J (2012) Challenges in molecular structure determination. Springer, Berlin
Waters Corporation. Nuclear magnetic resonance spectroscopy. Accessed 21 Aug 2019
Introduction to Chromatographic
Techniques 11

Abstract
In this chapter, we have described the brief overview of the most important
analytical technique i.e., chromatography which is used for the separation,
identification, and purification of the components present in a complex mixture.
We have briefly discussed in detail the basic principle involved in chromatogra-
phy during the phenomenon of separation, identification, and purification of the
components present in complex mixture. Moreover, various types of chromatog-
raphy have also been discussed here.

Keywords
Principle of chromatography · Types of chromatography · Mechanism of
separation

11.1 Introduction

Chromatography is one of the most important analytical techniques that is used for
the separation, identification, and purification of the components present in a com-
plex mixture that contains different sizes and/or molecular weights of individual
components. Chromatography is used for both qualitative and quantitative analysis
of the analyte present in complex mixture. Chromatography is also known as the
science and art of separation of components and/or analyte present in complex
mixture which involves the physical separation of component of interest present in
mixture into its individual components. The components and/or substances that are
going to be separated, identified, and/or quantified should be physically mixed
together, but not chemically combined. Here are some of the most common
examples of physical mixtures:

• Water suspended in air is a physical mixture of fog.

• Mixture of gases present in the universe is the physical of air.

# Springer Nature Singapore Pte Ltd. 2020 147

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148 11 Introduction to Chromatographic Techniques

• Mixture of cereal and milk is known as bowl of cereal.

• Mixture of water, soda syrup, and CO2 gas is known as soda pop.
• Mixture of sugar, water, and flavor crystals is known as kool-aid.

11.2 Principle of Chromatography

Chromatography is based on the principle where the molecules, components, and/or

substances present in mixture are applied onto or into the solid surface and/or
sometimes fluid stationary phase and then separated from each other while moving
from one side of the solid surface to another side with the aid of a mobile phase. The
factors that may effect on this separation phenomenon include molecular
characteristics related to the adsorption of substance and/or components that are
being separated, partition coefficient and affinity among the molecular weights
and/or size of the components that are being separated. Because of the differences
in the molecular weights and/or sizes of the individual components present in the
mixture, some components of the mixture move slowly in chromatographic system
and stay longer on stationary phase, while the others pass rapidly into the mobile
phase and leave the chromatographic system faster.

11.3 Terms Used in Chromatography

11.3.1 Mobile Phase

A phase in which the sample is dissolved is known as mobile phase. It is always

composed of gas, liquid, or supercritical fluid. It is very important that mobile phase
should be compatible with all the components present in the mixture that are going to
be separated.

11.3.2 Stationary Phase

The phase in which the mobile phase is forced through is known as stationary phase.
This phase is always composed of a “solid” phase or “a layer of a liquid adsorbed on
the surface a solid support.”

11.3.3 Supporting Medium

A solid surface on which the stationary phase is bounded or coated is known as

supporting medium. When the mobile phase is allowed to pass on it, the analyte will
11.4 Types of Chromatography 149

distribute itself between the two phases based on the affinity and/or partition
coefficient.

11.3.4 Eluate

It is that component which is obtained after the completion of chromatographic

process.

11.3.5 Eluent

It is the carrier portion of the mobile phase. It moves the analytes through the
chromatographic apparatus.

11.3.6 Elution

It is the phenomenon of extracting a substance and/or component that is adsorbed on

another surface by moving it with the solvent.

11.3.7 Chromatogram

A graph showing the detectors response as a function of elution time, band’s shapes,
position, resolution, is known as chromatogram.

11.3.8 Retention Time

The time needed after the injection of sample into the column for an individual solute
to reach to the detector is known as retention time.

11.4 Types of Chromatography

11.4.1 Based on the Nature of Mobile Phase

11.4.1.1 Liquid Chromatography

This technique is used to separate the components and/or substances present in liquid
samples with a liquid solvent that is known as mobile phase and a column composed
of solid beads that is also known as stationary phase.
150 11 Introduction to Chromatographic Techniques

Fig. 11.1 Schematic representation of partition chromatography

11.4.1.2 Gas Chromatography

This chromatographic technique is used to separate vaporized samples with the help
of a carrier gas which acts as mobile phase and a column that is composed of a liquid
or of solid beads (stationary phase).

11.4.1.3 Paper Chromatography

This chromatographic technique is used to separate the dried liquid samples with the
help of liquid solvent that acts as mobile phase and a paper strip that acts as
stationary phase.

11.4.1.4 Thin Layer Chromatography

This chromatographic technique is used to separate the dried liquid samples with the
help of liquid solvent that acts as mobile phase and a glass plate that is covered with a
thin layer of alumina or silica gel that acts as a stationary phase.

11.4.2 Based on the Mechanism of Separation

11.4.2.1 Partition Chromatography

In this type of chromatographic technique, the components and/or substances of
interest are distributed more likely into two mobile phases because of the differences
in partition coefficients. Molecules will partition into the stationary phase based on
their affinity towards the stationary phase and eventually partition into the mobile
phase again. A schematic representation of partition chromatography has been
described in Fig. 11.1.
11.4 Types of Chromatography 151

Fig. 11.2 Schematic

representation of adsorption
chromatography

11.4.2.2 Adsorption Chromatography

Adsorption chromatography is based on the principle of adsorption phenomenon. In
adsorption chromatography, the separation of the components present in mixture is
based on the interaction of the adsorbate with the adsorbent. The adsorbent is the
solid surface of stationary phase and the adsorbates are the molecules of interest
present in the sample mixture and are getting adsorbed on the adsorbent (solid
stationary phase). The phenomenon of adsorption chromatography is very similar
to that of the partition chromatography. Adsorption of components and/or
substances of interest present in mixture, just take place on the solid surface of the
stationary phase. A schematic representation of adsorption chromatography has been
described in Fig. 11.2.

11.4.2.3 Ion-Exchange Chromatography

It is the type of chromatographic technique, the polar molecules and/or ions are
separated based on their affinity to the ion exchanger (that acts as a stationary phase).
Ion-exchange chromatography is used for the separation of almost any kind of
charged molecule including large amino acids, small nucleotides, and proteins.
Ion-exchange chromatography is used for the separation of either cations or anions.
The separation phenomenon involved in ion-exchange chromatographic technique is
based on the relative strength of ionic bond. A schematic representation of
ion-exchange chromatography has been described in Fig. 11.3.
152 11 Introduction to Chromatographic Techniques

Fig. 11.3 Schematic representation of ion-exchange chromatography

11.4.2.4 Molecular Exclusion Chromatography

This chromatographic technique is also known as gel permeation chromatography size
exclusion chromatography and/or gel filtration chromatography. The phenomenon of
separation of the components of interest present in the sample mixture is based on the
size and/or molecular weight of individual component of interest present in the mixture
by the mechanism of filtration through the gel which acts as stationary phase consisting
of heterosporous (pores of different sizes) cross-linked polymeric gels or beads. Small
molecules get trapped into the pores of stationary phase more rapidly as compared to
that of the large molecules that do not entered into the gel easily and cover the longer
distance in stationary phase. The flow of the component of interest through the gel
(stationary phase) is retarded according to their size. The large molecules are eluted out
(cannot enter into the gel or stationary phase due to their large size or molecular weight).
It is applied to separate the large molecular weight macromolecular complexes such as
proteins and industrial polymers for semi-preparative purifications and various analyti-
cal assays. This technique is used for the purification and studies of desalting, protein-
ligand, protein-folding, and copolymerization phenomenon. A schematic representa-
tion of molecular exclusion chromatography has been described in Fig. 11.4.

11.4.2.5 Gel Electrophoresis

Gel electrophoresis is the type of chromatographic technique which is used to
separate protein molecules, DNA, and RNA according to their molecular sizes. In
this technique, the molecules that are going to be separated are pushed by the
involvement of an electrical field through a gel that contains small pores and acts
as stationary phase. The samples containing the components of interest are loaded
into the wells at the one end of a gel and an electric current is applied to pull them
through the gel. The fragments of the components of interest are negatively charged,
so they move towards the positive electrode when an electric current is applied.
Because all fragments of component of interest have same amount of charge per
11.4 Types of Chromatography 153

Fig. 11.4 Schematic representation of molecular exclusion chromatography

Fig. 11.5 Schematic representation of gel electrophoresis apparatus

mass ratio, therefore, the small fragments move through the gel faster and more
efficiently as compared to that of the large ones as shown in Fig. 11.5.
154 11 Introduction to Chromatographic Techniques

Fig. 11.6 Schematic representation of affinity chromatography

11.4.2.6 Affinity Chromatography

It is the type of chromatographic technique which is based on the specific binding
interaction in adsorption that depends on the specific affinity between an
immobilized ligand that is fixed in the separation material and its binding partner
(desired component present in the mixture). The interaction between the mobile
phase and stationary phase is typically reversible and purification and/or separation
is achieved through a biphasic interaction with the ligand that is immobilized on a
stationary surface while the target that is in mobile phase as part of a complex
mixture. The most common examples that may be separated and/or purified by this
technique are antibody–antigen, enzyme–substrate, and enzyme–inhibitor
interactions. Affinity chromatography is used for selective purification of a molecule
from complex mixtures that is based on highly specific biological interaction
between the two molecules. A schematic representation of affinity chromatography
technique has been described in Fig. 11.6.

11.5 Applications

1. In pharmaceutical industry, chromatographic techniques are used to analyze the

samples for the presence of trace elements, separation of compounds based on
their molecular weight and element composition, detection and identification of
unknown compounds and purity of mixture.
2. In hospitals, chromatography is one of the best tools for the detection of narcotics
and alcohols in blood and/or urine sample.
3. Environmental protection agencies use chromatographic tools for the detection of
pollutants present in the environment.
4. In food industry, chromatographic techniques are used for the detection of
spoilage, presence of additives in food and quality of nutritional components
present in food.
Further Reading 155

5. In forensics, chromatographic techniques are used for crime scene testing,

forensic pathology, and arson investigation.
6. In molecular biology, chromatographic techniques are used to study the redox
reactions that involve various bioorganic molecules, characterization of reaction
mixtures during the study of biomolecules.
7. In metabolomics, chromatographic techniques are used in mimicking the bio-
transformation reactions, such as phase I oxidative reactions in drug metabolism
studies and in the study of active pharmaceutical ingredients.
8. In proteomics, chromatographic techniques are used to analyze the oxidation of
biomolecules such as proteins and peptides. It is also used for the purification of
monoclonal antibodies, hormones, vaccines, and plasma proteins as part of their
development.
9. Chromatographic techniques are also used in nucleic acids research for the
identification of oxidation products of nucleosides, nucleotides, and nucleobases.

Further Reading
Cheriyedath S. Life science applications of chromatography. News-Medical. https://www.news-
medical.net/life-sciences/Life-Science-Applications-of-Chromatography.aspx. Accessed
14 Aug 2019
Coskun O (2016) Separation techniques: chromatography. Northern Clin Istanbul 3(2):156–160
Drabik A, Bodzoń-Kułakowska A, Silberring J (2016) Gel electrophoresis. In: Ciborowski P,
Silberring J (eds) Proteomic profiling and analytical chemistry, 2nd edn. Elsevier, Boston,
pp 115–143
Garrett RH, Grisham CM (2013) Biochemistry. In: Brooks/Cole, 5th edn. Cengage Learning,
Belmont, p 108. ISBN 9781133106296. Chromatography on SephadexG-100
https://chromatography.conferenceseries.com/events-list/applications-of-chromatography.
Accessed 14 Aug 2019
Jones M Jr (2000) Organic chemistry, 2nd edn. W. W. Norton & Company, New York
Lehman JW (2002) Operational organic chemistry, 3rd edn. Prentice Hall, Upper Saddle River
LibreTexts™. Chromatography. Accessed 21 Aug 2019
Marjeta U, Dan S, Kate Z (2009) Affinity chromatography: general methods. In: Burgess RR,
Deutscher MP (eds) Methods in enzymology, vol 463. Academic Press, Cambridge,
pp 417–438
Mayers GL, van Oss CJ (1998) Affinity chromatography. In: Delves PJ (ed) Encyclopedia of
immunology, 2nd edn. Elsevier, Oxford, pp 47–49
Müller MB, Schmitt D, Frimmel FH (2000) Fractionation of natural organic matter by size
exclusion chromatography – properties and stability of fractions. Environ Sci Technol 34
(23):4867–4872
Paul-Dauphin S, Karaca F, Morgan TJ, Millan-Agorio M, Herod AA, Kandiyoti R (2007) Probing
size exclusion mechanisms of complex hydrocarbon mixtures: the effect of altering eluent
compositions. Energy Fuel 21(6):3484–3489
Skoog DA, Holler FJ, Crouch SR (2007) Principles of instrumental analysis, 6th edn. Thomson
Higher Education, Belmont
Striegel AM (2013) Size-exclusion chromatography. In: Fanali S, Haddad PR, Poole CF,
Schoenmakers P, Lloyd D (eds) Liquid chromatography. Elsevier, Amsterdam, pp 193–223
156 11 Introduction to Chromatographic Techniques

Sudha PDC (2013) Pharmaceutical analysis, 1st edn. Dorling Kindersley, Noida
Wade LG Jr (2006) Organic chemistry, 6th edn. Prentice Hall, Upper Saddle River
Waters Corporation. History of chromatography. Accessed 21 August 2019
Werner MH, Clore GM, Gronenborn AM, Kondoh A, Fisher RJ (1994) Refolding proteins by gel
filtration chromatography. FEBS Lett 345(2–3):125–130
Zhang C, Rodriguez E, Bi C, Zheng X, Suresh D, Suh K, Li Z, Elsebaei F, Hage DS (2018) High
performance affinity chromatography and related separation methods for the analysis of
biological and pharmaceutical agents. Analyst 143(2):374–391
Thin Layer Chromatography
12

Abstract
Thin layer chromatography (TLC) is a type of chromatographic technique which
is used for the separation of the components present in mixture using thin
stationary phase. Separation of the components depends on the competition
between the adsorption of solute on stationary phase and its desorption by the
solvent needed to wash out from the stationary phase. In this chapter, we have
discussed the basic principle of TLC, components of TLC, its working and
applications.

Keywords
Nature of phases · Principle of TLC · Types of TLC · Applications of TLC

12.1 Introduction

Thin layer chromatography (TLC) is a type of chromatographic technique which is

used for the separation of the components present in mixture using thin stationary
phase (supported by an inert backing). It is a solid–liquid technique in which there
are two phases: one is stationary phase and the other is mobile phase. Separation of
the components depends on the competition between the adsorption of solute onto
the solid surface (stationary phase) and its desorption by the solvent needed to wash
out from the stationary phase. In TLC, stationary phase is always in solid state while
the mobile phase is always in liquid state.

# Springer Nature Singapore Pte Ltd. 2020 157

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https://doi.org/10.1007/978-981-15-1547-7_12
158 12 Thin Layer Chromatography

12.2 Principle

In 1949, the two scientists, namely Mein Hard and Hall proposed TLC technique
using starch as a binder to separate the inorganic ions present in the mixture. While,
Izmailov and Shraiber proposed TLC principle and Stahl designed the TLC equip-
ment. TLC is based on the following principle:

1. Separation of individual component present in sample mixture is dependent on

the relative affinity of individual component with stationary phase and the mobile
phase.
2. If the affinity of individual component present in a sample mixture is high with
mobile phase, then it travels over the surface of the stationary phase eluted out.
Whereas, if the affinity of individual component present in sample mixture is
higher with stationary phase, then it travels slowly and retained in the stationary
phase. Thus, the separation of components in the mixture is achieved.
3. Once the separation occurs, individual components of interest are visualized as
spots appeared at different points on TLC plate.

12.3 Types of Thin Layer Chromatography

12.3.1 Based on Nature of Phases

Based on the nature of phases used in TLC, it can be classified into the following two
types which have been briefly described in Table 12.1.

12.3.2 Based on Purpose of Use

TLC can also be classified into the following two types based on the purpose of the
application:

12.3.2.1 Analytical Chromatography

When the purification of small amount of the compound present in the mixture is
required, then this type of TLC is known as analytical TLC.

Table 12.1 Difference between normal and reverse phase TLC technique
Sr. # Parameters Normal phase TLC Reverse phase TLC
1 Stationary phase Polar Non-polar
2 Mobile phase Non-polar Polar
3 Component that eluted at first Non-polar Polar
4 Component that eluted at last Polar Non-polar
12.5 Working on Thin Layer Chromatography 159

12.3.2.2 Preparative Chromatography

When the purpose is to monitor any progress in chemical reaction occurring in the
mixture, then this type of TLC is known as preparative TLC.

12.4 Components of Thin Layer Chromatography

Following are the components of TLC:

12.4.1 Developing Chamber

It is used to create and maintain the environment for TLC.

12.4.2 Solid Support

It is used to support TLC film coated on stationary phase.

12.4.3 Stationary Phase

It is used to provide facility for the adsorption of analyte on TLC film.

12.4.4 Mobile Phase

It is a solvent system that carries the analyte present in mixture.

12.4.5 Pipette

It is used to load the samples on the stationary phase of TLC film.

12.4.6 Forceps

They are used to handle the TLC plates during and after experiment.

12.5 Working on Thin Layer Chromatography

The following steps are involved while working on TLC:

160 12 Thin Layer Chromatography

Fig. 12.1 Schematic representation of preparation of TLC plate

12.5.1 Preparation of TLC Plates

At first, the suspension or slurry of coating material is prepared and then applied on
the solid surface that acts as stationary phase by one of the following methods. A
schematic representation of preparation of TLC plates has been briefly described in
Fig. 12.1.

12.5.1.1 Pouring
In this technique, TLC plate is kept on a level surface and the measured amount of
slurry of adsorbent is put on plate. Then the plate is tilted back and forth to spread
slurry of adsorbent (Fig. 12.1).

12.5.1.2 Dipping
This method was developed in 1962 by Peifer. In this technique, TLC plate is dipped
in slurry of adsorbent (Fig. 12.1).
12.5 Working on Thin Layer Chromatography 161

12.5.1.3 Spraying
This method was proposed by Resitsema, but nowadays this technique is not used. In
this technique, pointer sprayer is used to distribute slurry of adsorbent on TLC plate
(Fig. 12.1).

12.5.1.4 Spreading
In this technique, the slurry of adsorbent is placed on applicator and the applicator is
either moved on the stationary phase or it is kept static and TLC plate is pushed
and/or pulled through the applicator (Fig. 12.1).

12.5.1.5 Activation of Adsorbent

Once the TLC plate is prepared, it is dried by keeping it in air for 30 min and then, it
is kept in oven for another 30 min at 110  C for the complete evaporation of solvent.
This drying technique activates the adsorbent layer on TLC plate. The most com-
monly used active layers on TLC plates are silica gel and alumina. These adsorbents
on TLC plate can be activated by heating at 150  C for 4 h.

12.5.1.6 Purification
Sometimes, the adsorbent material may contain any impurity, e.g., silica gel may
contain iron as an impurity. It is very important that the adsorbent material must be
purified.

12.5.1.7 Preparation of Mobile Phase

The mixture of two or more solvents of different polarity with different proportions
is often used for the separation of components present in mixture as compared to that
of chemically homogenous solvents.

12.5.1.8 Marking on TLC Plate

A line about 1 cm is marked below the top of TLC plate and then a small point is
marked on either side of spotting point about 2 cm from the bottom. Care must be
taken during the marking on TLC plate so that the adsorbent material should not be
removed from the sides, top, or bottom of TLC plate. A schematic representation of
marking on TLC plate has been described in Fig. 12.2.

Fig. 12.2 Schematic

representation of marking on
TLC plate
162 12 Thin Layer Chromatography

Fig. 12.3 Schematic

representation of TLC
chamber

12.5.1.9 Preparation of Sample and Standard Solutions

Before the preparation of sample and standard solutions for TLC experiments, stock
and dilutions are prepared and calibrated. One dosage unit or composite of dosage
units of analyte is placed in small container, ground to make its power and transfer to
a suitable vessel. Then, an appropriate solvent is used to dissolve the active ingredi-
ent (analyte).

12.5.1.10 Spotting on TLC Plate

For spotting the sample and standard solution on TLC plate, capillary tube or
micropipette or calibrated glass tube or alga micro syringe is used. It is very
important that the solvent used for the preparation of sample must be non-polar
and volatile in nature so that after spotting the sample, the solvent may evaporate
from the spots before developing.

12.5.1.11 Development
Mobile phase is added in TLC chamber (Fig. 12.3) in such a way that the bottom is
covered to a height of at least 1 mm by the mobile phase.

12.5.1.12 Drying
Once the development of TLC chamber is done, the solvent (mobile phase) is
allowed to reach a proper distance which is also known as solvent front. This
phase may take 20–40 min. After the development of chromatogram, TLC plate is
taken out and solvent front is marked on TLC plate. Then TLC plate is allowed to be
air dried.

12.5.1.13 Detection
It can be done either by specific or non-specific methods. If the analyte is colored,
then it can be visually detected easily (Fig. 12.4) but for the detection of colorless
analyte on TLC plate, two types of techniques are used:
12.6 Advantages 163

Fig. 12.4 Schematic representation of reading of TLC slide and detection of analyte on TLC slide

Table 12.2 Color formation of amino acids with ninhydrin and stannus chloride on TLC slide
Sr. # Type of amino acid on TLC slide Color observation of TLC slide
1 Glycine Reddish brown
2 Leucine Reddish orange
3 Glutamine Pink
4 Proline Yellow
5 Tryptophan Pinkish violet

Destructive Technique
In this technique, specific reagent, e.g., ninhydrin and stannus chloride is used in the
form of spray on TLC slide. Ninhydrin and stannus chloride react with analyte and
produce specific color depending on the nature of analyte (Table 12.2).

Non-destructive Technique
In this technique, different methods are used. For example, for the detection of
radioactive materials, Geiger Muller counter is used, whereas for the detection of
fluorescent compounds, UV chamber is used. Iodine chamber is also used for the
detection of analyte on TLC slide.

12.6 Advantages

1. TLC is a cheaper chromatographic technique.

2. The purity standards of the given sample can be easily assessed.
3. The separation process is faster and the selectivity for components is higher.
4. TLC helps to isolate the most of the components.
164 12 Thin Layer Chromatography

5. TLC helps with the visualization of separated components spots easily.

6. TLC is a simple process because of its short development time.
7. TLC helps to identify the individual components easily.

12.7 Disadvantages

1. It cannot differentiate between the enantiomers and some isomers.

2. Stationary phase in TLC is no longer stable.
3. Results produced by TLC are very difficult to be reproduced.
4. Only soluble components of interest present in mixture can be separated.
5. By TLC, only qualitative analysis is possible.
6. As TLC operates in open system, environmental factors like temperature and
humidity may interfere with the overall results.

12.8 Applications

1. It is used for the separation of inorganic ions from the sample solution.
2. It is widely used for the determination of molecular weight of unknown
compounds.
3. It is widely used in pharmaceutical and natural product analysis.
4. It is used for the separation of active pharmaceutical ingredients (API) from the
mixture and sample solutions.
5. It is also used for the identification of API from the mixture during the synthesis
of API.
6. It is also used in the investigation of pharmacokinetic studies of pharmaceutical
products.
7. It is also used for the determination of essential oils in herbal drugs and assay of
herbal medicines.
8. It is widely used in environmental analysis for the detection of various pollutants
present in environment.
9. It is widely used in qualitative and quantitative analysis purpose.
10. It is also for the isolation and identification of carotenoid pigments.
11. It is also used for the detection of proteins and peptides.
12. It is also used for the identification of pesticides and toxins present in food items.
13. It is also used for the separation of carbohydrates, vitamins, lipids, etc. present in
food items.
14. It is used for the screening and detection of drug abuse in urine samples.
15. In clinical laboratories, TLC is widely used for separation of carbohydrates in
biological samples.
16. TLC is the most widely used for the routine analysis of porphyrins.
17. TLC is also used for the detection of homocysteine, neopterin, lipids, and
protein antigens.
Further Reading 165

18. TLC is also used for the detection and separation of heavy metals and their
complexes.

Further Reading
Ashworth MRF, Stahl E (2013) Thin-layer chromatography: a laboratory handbook. Springer,
Berlin
Basak B, Bhattacharyya UK, Laskar S (1993) Spray reagent for the detection of amino acids on thin
layer chromatography plates. Amino Acids 4(1–2):193–196
Bladek J, Neffe S (2003) Application of thin-layer chromatography in clinical chemistry. Sep Purif
Rev 32:63–122
Carr PW, Grinberg N (2017) Advances in chromatography, vol 55. CRC Press, Boca Raton
Laskar S, Sinhababu A, Hazra KM (2001) A modified spray reagent for the detection of amino acids
on thin layer chromatography plates. Amino Acids 21(2):201–204
LibreTexts™. Think layer chromatography. Accessed 21 Aug 2019
Reich E, Schibli A (2007) High-performance thin-layer chromatography for the analysis of
medicinal plants. Thieme, New York
Sharma L, Desai A, Sharma A (2006) A thin layer chromatography laboratory experiment of
medical importance. Biochem Mol Biol Educ 34(1):44–48
Sinhababu A (2013) Modified ninhydrin reagent for the detection of amino acids on TLC plates. J
Appl Nat Sci 5(1):125–127
Skarkova J, Ostry V (2000) An HPTLC method for confirmation of the presence of ultra-trace
amounts of aflatoxin M1 in human urine. J Planar Chromatogr-Mod TLC 13(1):42–44
Skoog DA, Holler FJ, Crouch SR (2007) Principles of instrumental analysis, 6th edn. Thomson
Higher Education, Belmont
Touchstone JC (1995) Thin-layer chromatographic procedures for lipid separation. J Chromatogr B
Biomed Sci Appl 671(1–2):169–195
Waters Corporation. Chromatography. Accessed 21 Aug 2019
Waters Corporation. History of chromatography. Accessed 21 Aug 2019
Column Chromatography
13

Abstract
Column chromatography is a common chromatographic technique. It is a type of
adsorption chromatography that is widely used for the separation of individual
components of interest present in mixture. This technique can be used on small as
well as on large scale for the isolation and purification of components of interest.
This chapter briefly describes the basic principle involves in it, its, working,
factors that may affect on its working. Moreover, advantages, disadvantages, and
applications of column chromatography have also been discussed in this chapter.

Keywords
Principle of column chromatography · Working of column chromatography ·
Applications of column chromatography

13.1 Introduction

An American chemist D.T Day was the first scientist who introduced this technique
in chemical analysis in 1900 while, in 1906, Polish botanist M.S. Tswett investigated
some plant pigments using adsorption columns. A schematic representation of this
technique has been illustrated in Fig. 13.1. This chromatographic technique is widely
used for both the separation and purification of solid and liquid components of
interest present in mixture. Column chromatography is actually a solid–liquid
technique, where solid is the stationary phase and liquid is the mobile phase.

13.2 Principle

A mixture or compound that needs to be separated is dissolved first in mobile phase

which is then introduced from the top of the column. Components present in the
mixture then move at different rates depending upon their relative affinities towards

# Springer Nature Singapore Pte Ltd. 2020 167

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168 13 Column Chromatography

Fig. 13.1 Schematic representation of column chromatography

the stationary phase. The components having lower adsorption rate and less affinity
with stationary phase will move faster as compared to those components having
more adsorption and more affinity with stationary phase. The components moving
faster are eluted out first, whereas those moving slowly are removed last. From the
distance travelled by solute, a retardation factor is calculated:

Rf ¼ Distance travelled by solute=total distance travelled by the solvent:

13.3 Types of Column Chromatography

Following are the most common types of column chromatography. These techniques
have been briefly described in Chap. 11.

1. Absorption chromatography
2. Partition chromatography
3. Gel chromatography
4. Ion exchange chromatography
13.4 Components of Column Chromatography 169

13.4 Components of Column Chromatography

Following are the main components of column chromatography:

13.4.1 Stationary Phase

Stationary phase is solid in column chromatography which should have good

adsorption property. Stationary phase should be selected properly to achieve the
success of column chromatography. It depends on the following conditions:

1. Removal of impurities present in the compound.

2. Number of components to be separated.
3. Affinity differences between the components.
4. Length of the column used.
5. Quantity of adsorbent used.

A stationary phase should have the following properties:

1. Should have uniform shape and size of particles

2. Mechanically stable
3. Chemically inert
4. Allow free flow of the solvent
5. Should be colorless
6. Inexpensive
7. Free availability

13.4.1.1 Adsorbents
Silica, calcium phosphate, calcium carbonate, starch, and magnesia are the most
commonly used adsorbents in column chromatography. For less polar compounds’
alumina is preferred. Silica gel also gives good results for compounds having polar
functional groups. Adsorbents should have the following properties:

1. Particles should be uniform in size and have spherical shapes.

2. High mechanical stability.
3. Chemically inert.
4. Useful for the separation of many compounds.
5. Inexpensive and freely available.

13.4.1.2 Mobile Phase

Mobile phase is liquid in case of column chromatography which dissolves the
mixture and transfer it to column. It acts as a,
170 13 Column Chromatography

1. Solvent: to introduce the sample mixture into the column.

2. Developing agent: to separate the components of interest present in mixture in the
form of bands.
3. Eluting agent to remove the separated components out of the column.

The solvent is chosen on bases of the solubility properties of the mixture. Low
boiling point and polarity of the solvents are the important factors in the selection of
a solvent in column chromatography. The mostly used solvents are carbon tetrachlo-
ride, petroleum ether, ether, esters, cyclohexane, acetone, toluene, benzene, and
water.

13.4.2 Column

It is used to hold the adsorbent or stationary phase. It is made up of neutral glass

which should be of good quality so that it cannot affect the solvent. Usually, a burette
is used as a column having length and diameter ratio of 10:1, 30:1, or 100:1.
Selection of the column’s dimensions depends upon the number of components in
the sample, type of stationary phase, sample quantity under analysis, and
components affinity towards the stationary phase. A narrow column is preferred
over the short and thick column to achieve better separation.

13.4.2.1 Preparation of Column

Column is first washed properly with purified water and then with acetone. Then, it is
dried properly to remove the impurities. Then column is hanged along a stand with
the help of clamp in such a way that its outlet facing should be downward. Column
should be packed from the bottom with a cotton wool, glass wool, filter paper, or
asbestos pad so that adsorbent will not fall out. After packing, a paper disc is placed
on the top of the column so that the adsorbent layer is not disturbed when the sample
or mobile phase is introduced.

13.4.2.2 Packing of Column

Two techniques are used for the packing of column. One is dry packing or dry filling
and second one is wet packing or wet filling.

Dry Packing of Column

Column is firstly filled with the adsorbent in dry form in this method and then the
solvent is flushed through the column until equilibrium is reached. Air bubbles may
be entrapped between the mobile phase and stationary phase. Cracks or void space
may appear in the adsorbent layer. To address these problems tapping is done while
packing of the column. A schematic representation of dry packing of column has
been represented in Fig. 13.2.
13.5 Working of Column Chromatography 171

Fig. 13.2 Schematic representation of dry packing of column

Fig. 13.3 Schematic representation of wet packing of column

Wet Packing of Column

Formations of air bubbles and cracks are the drawbacks in dry packing. Therefore,
wet packing is preferred in which a slurry made up of adsorbent and solvent is
generally added to the column in portions. Stationary phase (adsorbent) settles
uniformly and no cracks are formed in the column. The solid settle down while
the solvent remains upward. The solvent is then removed and again a cotton plug is
placed in the bottom of column. A schematic representation of dry packing of
column has been represented in Fig. 13.3.

13.5 Working of Column Chromatography

Column is firstly packed either by dry packing or wet packing. Then the sample is
dissolved in minimum quantity of mobile phase and is introduced into the column at
once. Then mobile phase is flushed through the column until 1/3 length of column is
filled with solvent. By the process of elution, the components of interest are
separated out from the column. A schematic representation of working of column
172 13 Column Chromatography

Fig. 13.4 Schematic representation of working of column chromatography

chromatography has been described in Fig. 13.4. There are two techniques which are
involved in the separation process.

13.5.1 Isocratic Elution Technique

Solvents having the same composition and same polarity are used in this technique
throughout the process of separation. One of the most commonly used solvents for
isocratic elution technique is chloroform.

13.5.2 Gradient Elution Technique

Solvents having gradually high polarity or high strength of elution are used in this
technique during the process of separation. For example, initially benzene, then
chloroform, then ethyl acetate then chloroform.

13.6 Detection of Components

Detection of components of interest can be done visually if colored bands are

appeared as separated components. But if the components of interest to be separated
appear as colorless bands then small fractions of the eluent are collected in
labeled tubes and then TLC is performed on each section separately to detect the
composition of each fraction.
13.10 Applications 173

13.7 Factors Affecting on Column Chromatography

1. Column’s dimension, length of the column should be more than its width.
Normally 10:1, 30:1, or 100:1 ratios of length and diameter are used.
2. Particle size of the adsorbent should be small.
3. Proper activation of the adsorbent is needed.
4. Column’s temperature should be managed properly as high temperature can
enhance the process of elusion.
5. Column should be properly packed with the adsorbent and bottom should be also
filled with cotton wool or anything else used for this purpose.
6. Less viscous solvents give better results.

13.8 Advantages

1. It is simple and easy technique.

2. Mixture of any type or quantity can be easily separated using this technique.
3. Many types of solvent/mobile phase can be used in this technique.
4. Automation is possible when using this technique.
5. It is an inexpensive technique.
6. This technique can be used both on small and on large scale.

13.9 Disadvantages

1. It is a time consuming process, especially when components show colorless

bands.
2. In this technique, a large amount of the solvent is required for the proper elusion.
3. It is a simple technique but if automated then it becomes more complex and hence
more expensive.

13.10 Applications

1. It is used to separate a mixture of compounds into its components of interest.

2. It is used for purification process.
3. The active constituents of many drugs can be isolated by using this technique.
4. It is used to determine the drug estimation in drug formulations.
5. It is also used to isolate many metabolites from the biological fluids like blood or
serum.
6. Primary and secondary glycosides in digitalis leaf can be isolated by using this
technique.
174 13 Column Chromatography

7. This technique is also used for the separation of diastereomers.

8. This technique is best employed for the separation of active principles of plant
materials like alkaloids, glycosides, resins, tannins, and flavonoids.
9. Multistage column chromatography can be used to study the nucleotide
sequences in RNA.

Further Reading
Carr PW, Grinberg N (2017) Advances in chromatography, vol 55. CRC Press, Boca Raton
Coskun O (2016) Separation techniques: chromatography. Northern Clin Istanbul 3(2):156–160
https://bitesizebio.com/29947/basics-chromatography-column/
https://orgchemboulder.com/Technique/Procedures/Columnchrom/Procedure.shtml
Jones M Jr (2000) Organic chemistry, 2nd edn. W. W. Norton & Company, New York
Lehman JW (2002) Operational organic chemistry, 3rd edn. Prentice Hall, Upper Saddle River
LibreTexts™. Liquid chromatography. Accessed 21 Aug 2019
Poole CF, Schuette SA (2012) Contemporary practice of chromatography, vol 5. Elsevier,
Amsterdam
Sharma L, Desai A, Sharma A (2006) A thin layer chromatography laboratory experiment of
medical importance. Biochem Mol Biol Educ 34(1):44–48
Skoog DA, Holler FJ, Crouch SR (2007) Principles of instrumental analysis, 6th edn. Thomson
Higher Education, Belmont
Wade LG Jr (2006) Organic chemistry, 6th edn. Prentice Hall, Upper Saddle River
Waters Corporation. History of chromatography. Accessed 21 Aug 2019
High Performance Liquid Chromatography
14

Abstract
High performance liquid chromatography (HPLC) is a form of liquid chromatog-
raphy which is used to separate the individual components of interest present
in mixture and/or dissolved in sample solution. It is based on pumping of
mobile phase through the packed column under high pressure. The basic principle
involved in HPLC is based on the phenomenon of column chromatography in
which the mobile phase is pumped through a packed column by applying
high pressure. In this chapter, the different types of HPLC techniques on the
basis of mode of chromatography, principle of separation, scale of operation, and
the type of analysis have been discussed. The comprehensive instrumentation
has also been discussed. At the end of the chapter, advantages and disadvantages
along with its applications have been described.

Keywords
Types of HPLC · Components of HPLC · HPLC column · Sample injector

14.1 Introduction

High performance liquid chromatography (HPLC) was first proposed by Kirkland

and Huber. HPLC method was developed in 1970s based on the principle of column
chromatography. HPLC is a form of liquid chromatography which is used to
separate the individual components of interest present in mixture and/or dissolved
in sample solution. It is based on the pumping of mobile phase through packed
column under high pressure (approximately 3000 psi). It is also called as “High
Pressure Liquid Chromatography.” The components of the mixture are separated
when the sample is injected into the column. The components of the sample mixture
are passed through the column at different rate depending upon the behavior of
individual component of interest present in mixture and/or sample solution.

# Springer Nature Singapore Pte Ltd. 2020 175

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176 14 High Performance Liquid Chromatography

14.2 Principle

The basic principle involved in HPLC is column chromatography in which the

mobile phase is pumped through a packed column by applying high pressure.
The stationary phase is present at the bottom end of the column, while the other
end of the column is attached with the source of pressurized mobile phase.

14.3 Types of HPLC Techniques

HPLC techniques can be classified into the following types:

14.3.1 Based on Mode of Chromatography

1. Normal phase HPLC mode: In this type of HPLC technique, stationary phase is
polar (e.g., silica gel), whereas the mobile phase is non-polar. In this technique,
non-polar components of interest present in the mixture travel fast and eluted first
as compared to that of polar components of the mixture. This is because of the
less affinity of the non-polar components of interest with stationary phase.
Whereas, the polar components of interest present in mixture are retained longer
period of time in the column because of high affinity with stationary phase and
eluted late as compared to those of non-polar components of the mixture.
2. Reverse phase HPLC mode: In this type of HPLC technique, the stationary phase
which is used in this type of HPLC is mostly non-polar, whereas the mobile
phase is usually polar. In this mode, the polar components of the mixture are
eluted first, whereas the non-polar compounds retained in the column take more
time to be eluted out from the column. As most of the pharmaceutical drugs
are polar in nature, they do not retain in the column for longer period of time and
eluted out rapidly.

14.3.2 Based on Principle of Separation

Following are the most important types of HPLC techniques that are based on the
phenomenon of principle of separation. The principles of these techniques have been
described in Chap. 11.

1. Adsorption chromatography.
2. Ion exchange chromatography.
3. Partition chromatography.
4. Size exclusion chromatography.
5. Affinity chromatography.
14.5 Components of HPLC System 177

14.3.3 Based on Scale of Operation

The following two types are used on the basis of scale of operation:

1. Analytical HPLC: In analytical HPLC, analysis of the given sample is performed

but sample recovery is impossible.
2. Preparative HPLC: In this analysis, the fraction collector is used in order to
collect the components of the sample mixture. In this analysis the collectors
are reused.

14.3.4 Based on the Types of Analysis

The following two techniques are used based on the purpose of analysis:

1. Qualitative HPLC: The qualitative analysis is performed to determine the

characteristics of the components of a sample mixture.
2. Quantitative HPLC: The quantitative analysis is performed to determine the
amount of the components of the sample mixture. This analysis is performed
when peaks have been integrated and identified.

14.4 HPLC System

A schematic representation of HPLC system has been described in Fig. 14.1.

14.5 Components of HPLC System

Following are the major components of HPLC system:

14.5.1 Solvent Reservoir

The mobile phase that is used in HPLC mostly consists of a mixture of polar
and non-polar liquid components. These liquid components have different
concentrations depending on the composition of the sample used for analysis.
Therefore, the components of mobile phase are separately present in a glass
reservoir. This is mainly used to reserve the mobile phase. This is made up of
any inert material such as glass and it does not allow the entry of the microbes.
It is placed in the tray present in HPLC because if this is kept directly on the system
then may be due to any spillage of solvent damage the electronic system.
178 14 High Performance Liquid Chromatography

Fig. 14.1 Schematic representation of HPLC system

14.5.2 Pump

Pump is used for aspiration of the mobile phase from the solvent reservoir. Pumps
force it through a column at a specific flow rate (expressed in mL/min). Normal flow
rates in HPLC are in the range of 1–2 mL/min. Pump can deliver either an isocratic
or gradient mobile phase. The pump should have the following characteristics:

1. It should be non-corrosive.
2. It should have constant flow rate.
3. It should be compatible with mobile phase.
4. It should have the ability to bear the applied pressure for the flow of mobile phase.

Whereas the following factors may affect the efficacy (notably retention time,
reproducibility, and detector sensitivity) of the pump:

1. Detector compatibility.
2. Viscosity.
3. Boiling point.
4. Flammability.
14.5 Components of HPLC System 179

14.5.2.1 Types Pumps

There are two main types of pumps that are used in HPLC technique:

1. Constant pressure pumps: This type of pumps has some limitations such as
limited reservoir and it allows non-pulsating flow. The example is displacement
type pumps. It provides the continuous flow rate of sample by using the gas which
is present in the cylinder.
2. Constant volume pumps: This type of pumps allow the flow of mobile phase into
the column under high pressure. The examples are reciprocating type pumps,
pressure vessel pumps, and syringe type pumps.

14.5.3 Sample Injector

Injector is used in HPLC for the introduction of the liquid sample. Care must be
taken when sample is injected, it must not disturb the column packing. In this way,
the liquid sample is directly entered into the flow stream of mobile phase in column.
Injector is of two types that can be used in HPLC. One is single injection and other is
an automated injection system. The injector must have the ability to withstand the
high pressure that may be developed in the liquid system. Typical sample volumes
of 5–20 μL can be injected.

14.5.3.1 Manual Injector

There are many components of the manual injector. It is a device that controlled
the metered amount of the sample liquid and holds it prior to the injection. A valve
which is used to maintain the path of the sample when there is direct injection of
the sample liquid into the eluent stream. The injection is placed after the pump
head. The sample is introduced in the load mode with the help of microsyringe
under the high controlled conditions. The solution which is injected is stored in the
sample loop. The injection valve is used in the sampling system in order to introduce
the calculated amount of the sample and without changing of pressure of sample.
When the knob is rotated to switch on the injection mode then sample is flow out
from the loop. The sample is entered into the eluent stream. A schematic representa-
tion of operating systems of manual sample injector system has been described
in Fig. 14.2.

14.5.3.2 Autosampler
Injection by an autosampler usually consists of the automatic execution of the
operations. This can be done by using a microsyringe. This principle is based on
a computer-controlled program. This operation is performed manually by the analyst
with the help of machine. This type of sampling has the advantage that a large
amount of the sample is injected.
180 14 High Performance Liquid Chromatography

Fig. 14.2 Schematic representation of operating system of manual sample injector

Fig. 14.3 Schematic representation of pressure injection method for sample injection into the
column

Pressure Injection Method

With this method, after a specific amount of sample is aspirated from the sample vial
and conveyed to the sample loop attached to a 6-port valve, the valve is switched
so that eluent is delivered into the loop and the sample is consequently conveyed
to the column. In other words, some of the operations performed by an analyst
using a manual injector are performed by a machine. A schematic representation of
pressure injection method for sample injection into the column has been described
in Fig. 14.3.
14.5 Components of HPLC System 181

Total-Volume Injection Method

In this method, a predetermined amount of the sample is introduced from the sample
vial into a tube. The eluent is directly transferred into this tube. As a result, the
sample is transferred into the column. The main advantage of this method is eluent
flows inside the needle but not during injection operation. Another main advantage is
that it can aspirate a volume greater than that conveyed to the column and there is a
very little loss of sample. This may be impossible to aspirate the accurate amount of
the sample if there are small air bubbles in the passage of sample from the measuring
pump into the column needle tip. To avoid this situation, the fluid must be degassed
that is filled. A schematic diagram of total-volume injection method for sample
injection into the column has been described in Fig. 14.4.

14.5.4 Column

Columns are usually made of polished stainless steel and/or heavy glass materials.
The reason to use stainless steel or glass material is to withstand the high pressure.
Columns are usually narrow tubes that are packed with 25 μm particles. The column
internal portion should be uniform and smooth. The average internal diameter of
the column should be 4.6 mm, whereas the average length of the column should
be 10–25 cm long and their internal diameter should be between 2 and 5 mm. The
columns are most commonly filled with a stationary phase. For porous packing,
silica is most commonly used with a particle size of 3–10 μm whereas, for pellicular
packing, non-porous glass or polymer beads with 30–40 μm diameter are used.

Fig. 14.4 Schematic representation of total-volume injection method for sample injection into the
column
182 14 High Performance Liquid Chromatography

Columns having the internal diameters approximately less than 2 mm are mostly
referred to use as microbore columns. The temperature of the mobile phase and the
column phase should be kept constant during the whole experiment. For HPLC
techniques, the most commonly used columns are as follows:

1. Analytical columns: These types of columns are used for quantitative analysis of
the samples and they are often used in combination with UV-VIS detectors.
2. Preparative columns: These types of columns are used for the individual fractions
of the components present in the sample mixture.
3. Narrow bar columns: These columns are 1–2 mm in length and are used when
more sensitivity for the separation of the component is desired. These columns
are used in combination with UV-VIS, fluoresce, and/or MS detectors.
4. Capillary columns: These columns are 0.3 mm in length and are made up of fused
silica capillaries. They are exclusively used with alternative detection methods
such as MS detectors.
5. Guard columns: These columns are short in length and are used to increase the
life of the columns. These columns are placed between the sample injector and
column and protect the column from the loss of efficiency and damage caused
by particular matter and/or strongly adsorbed substance in the sample or mobile
phase.

14.5.5 Detector

The detector can detect the individual components that come out from the column so
that the analyst can quantitatively analyze the components of interest that have
been separated from the sample. Detector then provides an output to the recorder
or computer that interprets the results in the form of chromatogram. The most
commonly used detectors in HPLC are as follows:

1. UV/Visible detector.
2. Photodiode array detector.
3. Refractive index detector.
4. Fluorescence detector.
5. Evaporative light scattering detector.
6. Amperometric detector.
7. Potentiometric detector.
8. Electrochemical detector.

14.5.6 Computer System

The computer system not only controls the overall modules of HPLC system but it
also receives the signals from the detector and uses it to determine the time of elution
(retention time) of the sample components for qualitative analysis and the amount of
sample for quantitative analysis.
14.9 Applications 183

14.6 Factors Affecting on HPLC

Following are the most important factors that affect the efficiency of HPLC
technique:

1. Particle size of the stationary phase packed in column.

2. Thickness of the stationary phase.
3. Length of the column.
4. Internal diameter of the column.
5. Flow rate of the mobile phase.
6. Nature of the mobile phase.
7. Viscosity of the mobile phase.
8. Affinity of the component of interest (analyte) with mobile phase and stationary
phase.
9. Column packing.
10. Sample size.
11. Pump pressure.
12. Retention time.
13. Injection volume of sample.
14. Method of sample injection.

14.7 Advantages

1. Highly sensitive technique.

2. High performance.
3. Rapid process and hence time saving.
4. Can be used for qualitative as well as quantitative estimation.
5. Can be used for both analytical and preparative purpose.

14.8 Disadvantages

1. Irreversibly adsorbed components of the sample mixture cannot be detected.

2. Complexity may occur.
3. Costly technique.
4. Sometimes, the sensitivity of some specific compounds is low.
5. Co-elution of some compounds is difficult to detect.

14.9 Applications

1. It is used in the separation of inorganic ions, e.g., fluoride, chloride, bromide,

phosphate, nitrite, magnesium, lead, copper, cadmium, zinc ions.
2. It is used for the detection of intoxicants and poisons in human blood.
184 14 High Performance Liquid Chromatography

3. It is used for the detection of addictive drugs, e.g., cocaine, heroin, morphine,
alcohol, opioid drugs.
4. It is used for the separation of alkaloids present in plants.
5. It is used for the identification of illicit drugs.
6. It is exclusively used in forensic science for the investigation purpose.
7. It is used for the analysis of explosives.
8. It is used for the identification of lipids.
9. It is used for the identification of steroid hormones.
10. It is used for the separation and identification of bile acids.
11. It is exclusively used in nucleic acid research.
12. It is used in metabolic profiling.
13. It is exclusively used in the preparation, separation, and identification of natural
products.
14. It is exclusively used in the analysis of pharmaceutical products.
15. It is used in the separation and identification of antibiotics.
16. It is used for the separation and identification of plant-based bioactive
compounds.

Further Reading
Carr PW, Grinberg N (2017) Advances in chromatography, vol 55. CRC Press, Boca Raton
Chaithanya Sudha PD (2013) Pharmaceutical analysis, 1st edn. Dorling Kindersley, India
http://rxpharmaworld.blogspot.com/2016/12/high-performance-liquid-chromatography.html
https://laboratoryinfo.com/hplc/
Huber JF (2011) Instrumentation for high performance liquid chromatography. Elsevier,
Amsterdam
Kar A (2014) Pharmaceutical analysis, vol II, 1st edn. CBS Publishers and Distributors, Chennai
LibreTexts™. High performance liquid chromatography. Accessed 21 Aug 2019
LibreTexts™. Chromatography. Accessed 21 Aug 2019
Michaelis AF, Cornish DW, Vivilecchia R (1973) High pressure liquid chromatography. J Pharm
Sci 62(9):1399–1416
Poole CF, Schuette SA (2012) Contemporary practice of chromatography. Elsevier, Amsterdam
Skoog DA, Holler FJ, Crouch SR (2007) Principles of instrumental analysis, 6th edn. Thomson
Higher Education, Belmont
Snyder LR, Kirkland JJ, Dolan JW (2011) Introduction to modern liquid chromatography. Wiley,
Hoboken
Snyder LR, Kirkland JJ, Glajch JL (2012) Practical HPLC method development. Wiley, Hoboken
Swadesh JK (2001) HPLC: practical and industrial applications
Waters Corporation. History of chromatography. Accessed 21 Aug 2019
Gas Chromatography
15

Abstract
In this technique, the separation of the components present in mixture is based on
the partition between the gaseous mobile phase and liquid stationary phase. In this
chapter, types of gas chromatography are given. The advantages and
disadvantages have also been described. The instrumentation of gas chromatog-
raphy along with its working has been described. The factors that affect the GC
have also been discussed. At the end of this chapter, advantages and
disadvantages along with its application have been given.

Keywords
Principle of gas chromatography · Components of gas chromatography

15.1 Introduction

This chromatographic technique was invented by Martin in which he suggested that

liquid mobile phase which is used in liquid chromatography can be replaced by using
a suitable gas as mobile phase. As he used gaseous mobile phase instead of liquid
mobile phase for separation purpose of the components of the sample, therefore,
the term was named as gas chromatography. It is a process of separation of the
components of the given sample such as crude substances by using a gaseous mobile
phase. The more classy form of gas chromatography was developed by James and
Martin in 1955. In gas chromatography, the mobile is in gaseous form, whereas the
stationary phase is in solid or liquid form. If the component is more soluble in
stationary phase, then it travels slower in the column, whereas if the component is
less soluble in stationary phase, then it travels faster. Therefore, the components
present in the sample mixture are separated according to their partition co-efficient
between the component of interest and stationary phase.

# Springer Nature Singapore Pte Ltd. 2020 185

M. S. H. Akash, K. Rehman, Essentials of Pharmaceutical Analysis,
https://doi.org/10.1007/978-981-15-1547-7_15
186 15 Gas Chromatography

15.2 Principle

In this technique, the sample is first vaporized by heating and then it is injected into
the head of the chromatographic column. The sample is transferred into the column
by the flow of inert gaseous mobile phase. The column has a liquid stationary phase
which is adsorbed on the surface of an inert solid. It has same principle as chroma-
tography, separation of the components due to partition between stationary phase
and mobile phase.

15.3 Types of Gas Chromatography

Based on the nature of the stationary phase, gas chromatography has the following
two main types:

15.3.1 Gas–Solid Chromatography

When the stationary phase (adsorbent) is solid in nature, then it is known as gas–
solid chromatography (GSC). The most common examples of stationary phase used
in GSC are active carbon, silica, alumina, etc. The principle of separation in GSC is
adsorption. One of the main advantages of GSC is that the column life is long, while
the main disadvantage of this technique is that there may be chances of catalytic
changes in the chemical composition of the components present in sample mixture.

15.3.2 Gas–Liquid Chromatography

If the stationary phase is liquid in the gas chromatography, then it is referred as gas–
liquid chromatography (GLC). The solid surface which may be polymer is coated
with the immobilized liquid. The principle in GLC is partition. Nowadays, GLC is
abundantly utilized in the form of capillary column.

15.3.2.1 Advantages of GLC

1. It provides high resolution capacity for the complex mixtures. For example,
separation of methyl esters of fatty acids.
2. As capillary column is abundantly used in GLC, only few microliter samples are
enough for the complete analysis.
3. The seed of analysis is quite fast because the mobile phase has the ability to attain
the rapid equilibrium between the mobile phase and stationary phase.
4. Sensitivity of detection is quite high while using different types of detectors.
5. It simultaneously allows qualitative and quantitative analysis of analyte.
15.4 Components of Gas Chromatography 187

15.3.2.2 Disadvantages of GLC

The major disadvantage of GLC is that the immobilized liquid is slowly run out from
the solid surface.

15.4 Components of Gas Chromatography

The main components of gas chromatography have been illustrated in Fig. 15.1.

15.4.1 Carrier Gas

The carrier gas must be chemically inert. The most common gases that are used
include nitrogen, helium, argon, and carbon dioxide. Hydrogen has better thermal
conductivity but it also has disadvantage that it often reacts with the unsaturated
compounds and compounds that are inflammable in nature. Thermal conductivity of
helium is excellent but it is too much expensive. Nitrogen is inexpensive but it has
reduced sensitivity. The selection of carrier gas mostly depends upon the type of
detector which is used. The carrier gas system also contains a molecular sieve to
remove water and other impurities. Following characteristics should be present in
carrier gas:

1. It should be chemically inert in nature.

2. It should be suitable for the detector and must enable the detector to respond in a
sufficient adequate manner.

Fig. 15.1 Schematic representation of components of gas chromatography

188 15 Gas Chromatography

3. The carrier gas should have the high purity.

4. It should be easily available.
5. It should be cheap.
6. It should be non-inflammable.
7. It is responsible for giving the best column performance.
8. It should be free from metallic particles.

15.4.2 Columns

The fundamental role of column is to separate the individual components present in

sample mixture. There are two main types of columns that are used in gas chroma-
tography technique.

15.4.2.1 Packed Columns

These columns contain glass or metallic tubes that are coated with immobilized
liquid stationary phase. Most packed columns are 1.5–10 m in length and have an
internal diameter of 2–4 mm.

15.4.2.2 Capillary Columns

These columns have very small internal diameter in millimeters but their length is
between 25 and 60 m. These capillary columns have immobilized stationary phase
which is liquid. The stationary phase is coated with the glass or silica in the inner
sides of columns. These are also called as open tubular columns and have the
following types:

1. Wall-coated open tubular columns: The inner walls of these columns are coated
with inert active material which acts as a support on which the immobilized liquid
stationary phase is adsorbed. Therefore, these columns are also known as support-
coated open tubular columns.
2. Support-coated open tubular columns: Support-coated open tubular columns are
usually coated with layer of the support material having micron size. This layer is
further coated by the thin film of immobilized liquid stationary phase. These types
of columns usually have more capacity for sample as compared to that of wall-
coated open tubular columns.

15.4.2.3 Factors Affecting on Column Efficacy

Following are the important factors that may influence the overall efficacy of the
column:

1. Column breakage: This column may break due to many reasons such as if coating
of the column is done by the weak coating material or may be coating is not done
properly. Due to variation in the temperature of the column which may increase or
decrease depending upon the conditions is also a reason for column breakage. If
the diameter of the column is large then breakage may occur due to this reason.
15.4 Components of Gas Chromatography 189

2. Thermal damage: Higher temperature of the column during heating may also
cause the degradation of stationary phase. Due to presence of oxygen during the
whole operation thermal degradation may be increased. Similarly, the presence of
chemical compounds, such as non-volatile compounds, acids (HCl, H2SO4,
HNO3), bases (KOH, NaOH), organic compounds (perfluoro acids), may also
damage the column.
3. Column contamination: It may be either because of the non-volatile or semi-
volatile contaminants.

15.4.3 Temperature Programmer

The column of GLC is interposed in the thermostatic oven. It is temperature-

controlled device which monitors and regulates the overall temperature of the
column. It is recommended to use thermostatically controlled oven for efficient
separation. Sometimes, preheaters are also used to convert the sample into its
vapor form. These preheaters are present along with injecting devices. There are
two main types of temperature programmers which as follows:

1. Isothermal programming: During the whole experiment, the temperature is kept

constant.
2. Gradient programming: During the whole experiment, the temperature varies. In
this case temperature may be increased or decreased depending upon the
conditions.

15.4.3.1 Factors Affecting on Temperature Programming

During the operation of GC, temperature programming should be performed. The
factors that affect the temperature programming are of the following:

1. Flow rate of the mobile phase: It affects the elution rates of the components of
interest present in sample. When the flow rate of mobile phase in the column is
high, then components remain for the shorter period of time in column as a result
broadening the peak.
2. Stability of the stationary phase: If the sample shows the solubility within
stationary phase, then residential time of sample in column is increased and better
results can be achieved.
3. Stability and solubility of analyte: Temperature affects the stability and solubility
of analyte. If the temperature increases, the solubility of the gas in liquid
decreases and it reduces the retention time of the sample in the column.
4. Volatility of analyte: If the sample evaporates rapidly by applying the heat, then
components of the sample are eluted rapidly.
190 15 Gas Chromatography

15.4.4 Sample Injector

The sample is injected into the carrier gas flow to the column of HPLC by using
sample injector. For the analysis of the gaseous samples, mostly rotary valve is used.
This rotary valve is mostly used in gas chromatography but volume of sample is kept
small than used in HPLC. In the case of the liquid sample, the sample is introduced
into the heated lash by using the gas syringe. The sample evaporated and the carrier
gas is responsible for the transferring of the vaporized sample from the injection site
to the column. In the case of the solid samples, the sample is initially heated at a high
temperature for the volatilization of the sample. As a result, the sample is converted
into the volatile derivatives substances.

15.4.5 Detectors

There are many types of detectors which can be used in gas chromatography.
Detectors can be grouped into the following two main types:

1. Concentration-dependent detectors: The signal detected by a concentration-

dependent detector is directly related to the amount of solute in the detector.
This type of detector cannot cause the destruction of the sample. The detector
response may be lower due to dilution with make-up gas.
2. Mass flow-dependent detectors: This type of detectors mostly destroy the sample.
The rate at which solute components enter into the detector affects the signals.
The make-up gas has no influence on the efficiency of mass flow-dependent
detectors.

Different kinds of detectors exhibit the different level of selectivity. The

non-selective type of detectors can respond to all types of the components of the
sample mixture except for the carrier gas. The selective type of detectors responds to
a wide range of compounds having common physical characteristics. Wide range of
detectors are used in gas chromatography, the most commonly used detectors are as
follows:

1. Flame ionization detector (FID): It is highly sensitive detector. Two different

kinds of gases are placed in the cylinder, which are used for the ignition of the
flame. The effluent of the column is directed towards the flame and the potential
difference is detected.
2. Thermal conductivity detector (TCD): This detector contains the heated filament.
When the carrier gas is passed through the cell, change in the filament current
which is compared with the reference cell. Due to difference in current a signal is
produced.
3. Electron capture detector (ECD): It specifically detects the halogen containing
compound. The sample is ionized due to radioactive material. This ionization
current is quenched by the compounds containing halogen compounds.
15.5 Factors Affecting on Gas Chromatography 191

4. Nitrogen-phosphorus detector: This is used for the detection of the compounds

having nitrogen and phosphorus. Its principle is same as flame ionization
detectors.
5. Flame photometric detector (FPD): It is used for the detection of the sulfur and
phosphorus. The eluent is passed through the flame as a result excited species are
formed and the light is produced in the flame.
6. Photo-ionization detector (PID): This type of detector is used for the detection of
the aromatic compounds. The eluted molecules are photoionized by the ultravio-
let radiation. The compounds that have low ionization energy are detected.

15.4.6 Recorder and Read-Out Device

Signals for separated fractions of vaporized components, received from the detectors
are recorded by recorder and read-out device interpret the input responses received
from detector into results.

15.4.6.1 Features of Detectors

Following are the ideal characteristics of the detector:

1. It should be easily handled easily.

2. The decomposition of the sample may not occur due to the detectors.
3. It can measure the wide range of temperature.
4. The sensitivity and reproducibility of the detectors must be high.
5. The detectors must have high stability.
6. It should not produce noise.
7. Small volume of the sample can be used in order to avoid peak broadening.

15.5 Factors Affecting on Gas Chromatography

1. Volatility of compounds: The components of the sample that have low boiling
point will travel faster than the compounds having high boiling components
through the column.
2. Polarity of the compounds: If the polar column is used, then the components of
the sample that are polar in nature will move more slowly and vice versa.
3. Column temperature: If the temperature is increased, then the components of the
sample will eluted very rapidly.
4. Column packing polarity: All the components of the compounds will move
slower in polar column as compared to that of non-polar column.
5. Flow rate of mobile phase: Speeding up the flow rate of mobile phase also
increases the speed of all compounds to be moved with mobile phase.
6. Length of column: If the column have longer length then it will take longer time
for elution of all the components present in mixture and as a result better will be
the separation.
192 15 Gas Chromatography

15.6 Advantages

1. This technique has strong power for the separation of even complex mixture that
can be resolved into its constituents.
2. The method is highly sensitive.
3. Small sample is required for analysis.
4. It contains high sensitivity detector system.
5. It has good precision and accuracy.
6. The operation is completed in a very short interval of time.
7. Linearity is good.
8. The instrument cost is relatively low.
9. It has generally longer life.
10. The technique is relatively suitable for routine analysis.
11. Precision is high.
12. Easy to handle the equipment.

15.7 Disadvantages

1. Sensitivity is low.
2. Volatilization is required for the analysis and there may be a chance for the
degradation of the sample.
3. It cannot be used for analysis of biological sample because of the high tempera-
ture of the column.

15.8 Applications

1. It is widely used for analysis of gaseous samples.

2. It is used for the estimation of amount of CO2 present in the fuel gases.
3. It is used for the estimation of organometallics.
4. It is used for the estrogens analysis.
5. In pharmaceutical industry, it is widely used for the analysis of the following
drugs:
a. Anti-tuberculosis drugs.
b. Antibiotics.
c. Antiviral drugs.
d. Anti-neoplastic agents.
e. Ointments.
f. Anticonvulsants.
g. Steroids.
6. In food industry, it is also used for the analysis of dairy products.
7. It is used for the determination of pesticides in aquaculture products.
8. It is used for the diagnosis of certain diseases like cancer.
9. It is used for the analysis of plant-based bioactive compounds and essential oils.
10. It is used for the detection of narcotics and alcohols in blood.
Further Reading 193

Further Reading
Skoog DA, Holler FJ, Crouch SR (2007) Principles of instrumental analysis, 6th edn. Thomson
Higher Education, Belmont
Swadesh JK (2001) HPLC: practical and industrial applications
Waters Corporation. History of chromatography. Accessed 21 Aug 2019
LibreTexts™. Gas chromatography. Accessed 22 Aug 2019
Chaithanya Sudha PD (2013) Pharmaceutical analysis, 1st edn. Dorling Kindersley, Noida
Kar A (2014) Pharmaceutical analysis, vol II, 1st edn. CBS Publishers and Distributors, Chennai
http://rxpharmaworld.blogspot.com/2016/12/gas-chromatography.html
LibreTexts™. Chromatography. Accessed 21 August 2019
Waters Corporation. Chromatography. Accessed 21 Aug 2019
Carr PW, Grinberg N (2017) Advances in chromatography, vol 55. CRC Press, Boca Raton
Poole CF, Schuette SA (2012) Contemporary practice of chromatography. Elsevier, Amsterdam
Hübschmann H-J (2015) Handbook of GC-MS: fundamentals and applications. Wiley, Hoboken
Karasek FW, Clement RE (2012) Basic gas chromatography-mass spectrometry: principles and
techniques. Elsevier, Amsterdam
Scott RP (2017) Introduction to analytical gas chromatography, revised and expanded. CRC Press,
Boca Raton
Littlewood A (1970) Gas chromatography: principles, techniques, and application. Academic Press,
Cambridge
McNair HM, Miller JM, Snow NH (2019) Basic gas chromatography. Wiley, Hoboken
Water J, Eric M, Philip S (1997) Analytical gas chromatography. Academic Press, Cambridge
Introduction to Thermal Analysis
16

Abstract
Thermal analysis is an analytical technique in which characteristics of the
materials which involves the temperature and/or heat are studied. Usually, the
measurements are made either by increasing or decreasing the temperature.
Broadly speaking, any measuring and/or analytical technique can be made as
thermal analysis technique which involves the temperature and/or heat as a key
function for analysis. In this chapter, we have briefly introduced thermal analysis
and its basic principle. Moreover, we have also discussed the types and
applications of thermal analysis.

Keywords
Types of thermal analysis · Principle of thermal analysis · Applications of thermal
analysis

16.1 Introduction

Thermal analysis is a branch of physicochemical science where the properties of

materials are studied and quantified as they change with respect to the change in
temperature and/or heat. According to International Union of Pure and Applied
Chemistry, thermal analysis is defined as a group of techniques in which a physico-
chemical property of the material is measured as the function of temperature, while
the sample is subjected to a controlled temperature programs like cooling, heating,
and/or isothermal process. Thermal analysis is basically a series of techniques that
are used to study the characterization and/or properties of material with respect to
change in temperature and/or heat. Thermal analysis is basically used to study the
physical properties of the material such as mass, enthalpy, dimension, dynamic
characteristics, freezing temperature, boiling temperature, melting temperature, cur-
ing rates for adhesives, heat of fusion, heat of vaporization, etc. Thermal analysis
cannot be used for structure analysis of a compound. Thermal analysis is widely used

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https://doi.org/10.1007/978-981-15-1547-7_16
196 16 Introduction to Thermal Analysis

in different disciplines notably from pharmaceutical sciences to polymer sciences

and materials chemistry where the changes in the behavior of sample with respect to
basic characteristics of materials along with its wide range of applications in acade-
mia and quality control in industry, in research and development are monitored
under temperature-controlled conditions.

16.2 Principle

The basic principle in all types of thermal analysis techniques is the same. To study a
sample, its reference is also used. Both the reference and sample are heated at an
identical temperature, even when a thermal event occurs in the sample. The energy
required to obtain a zero temperature is measured precisely. Following are the most
important components of thermal analysis that are mandatory for the basic principle
of thermal analysis:

1. Sample holder and/or compartment to hold the sample and/or reference during
thermal analysis. In modern equipment, mostly two compartments/pans are
present. One for the reference material and the other one for the sample or analyte.
Sample holders are mostly made up of platinum and/or aluminum.
2. A heater on which the pans are placed. The heaters are attached with a computer
whose function is to switch on the heaters and let them to heat at a specific rate.
Computer makes sure that heating rate remains the same throughout the process.
3. Sensors to measure the property of the sample and/or note the temperature
difference between the reference and sample material. There are separate sensors
for the reference and for the sample. The sensors used are mostly platinum
resistance thermocouples.
4. An enclosure and/or insulator within which the experimental parameters are
controlled.
5. Read-out device to record the data collection and processing. The recorder
presents the result in the graphical representation in the form of calibration curve.

16.3 Types of Thermal Analysis

Depending upon the physical properties of the material to be measured, the most
commonly used thermal analytical techniques are as follows:

1. Differential Scanning Calorimetry (DSC): It measures the heat which is being

absorbed or released during the process of heating or cooling. DSC is used to
measure the heat of reaction, melting point, heat capacity, and glass transition.
2. Differential Thermal Analysis (DTA): Used for thermal investigation or analysis
where the thermal change can be studied and investigated. It is used to determine
the oxidation process, decomposition, and loss of water or solvent.
16.4 Applications 197

3. Thermo Gravimetric Analysis (TGA): It measures the change in weight of the

sample during the process of heating or cooling. It is used to measure the phase
changes, glass transition, and melting point.
4. Thermo Mechanical Analysis (TMA): It measures the change in dimensions
during the process of heating or cooling. It can be used to analyze the sample
expansion, penetration, contraction, softening, and glass transition.

16.4 Applications

Thermal analysis is mainly used in the field of research and development but
nowadays, common materials including pharmaceutical products, foods, polymers,
ceramics, electronic materials, organic compounds, inorganic compounds, and even
biological organisms can be studied through thermal analysis. Thermal analysis is
being widely used as a testing standard in the following areas:

1. Quality control of pharmaceutical products.

2. In process quality control during the manufacturing of pharmaceutical products.
3. Inspection of raw materials that are used for the manufacturing of pharmaceuti-
cal products.
4. Determination of glass transition temperature of materials used for the
manufacturing of pharmaceutical products.
5. Melting points determination.
6. Crystallization time and temperatures of polymers and polymeric materials that
are used for the manufacturing of pharmaceutical products.
7. Heat of melting and crystallization of polymers and polymeric materials.
8. Determination of impurities in the polymers by examining thermograms and
plasticizers can also be detected.
9. Oxidative stabilities of pharmaceutical products.
10. Compositional analysis of raw materials and pharmaceutical products.
11. Heat capacity determination.
12. Determination of impurities that may be present in polymers and polymeric
materials.
13. Determination of compatibility of active pharmaceutical ingredients with that of
excipients that are used for the manufacturing of pharmaceutical products.
14. Used to review the kinetics of solid state of API including decomposition,
accelerated stability, and aging effect on various formulations.
15. Determination of nature polymorphism and/or crystallinity of the compound.
16. Physicochemical properties of the polymers, polymeric materials, excipients,
and active pharmaceutical ingredients.
17. Used for accelerated stability studies.
18. For the analysis of the results of lyophilization.
19. Estimation of moisture contents during in process quality control of pharmaceu-
tical products.
20. Determination of denaturation of proteins.
198 16 Introduction to Thermal Analysis

21. Determination of protein folding.

22. In food sciences to determine water dynamics as change in water distribution
may be correlated with texture changes.
23. Along with X-ray diffraction and IR spectroscopy used for the screening of
compatibility of drugs with excipients.

16.5 Advantages

1. Solid, semi-solid, or liquids samples can be analyzed

2. Small sample size is required only
3. Sample preparation is minimum
4. Quantitative analysis of multiple mass loss thermal events
5. Can separate and analyze multiple overlapping mass loss events

16.6 Disadvantages

1. Evolved products can be identified only evolved gas analyzer is connected.

2. Uncontrolled temperature can destroy the sample.

Further Reading
Abraham J, Mohammed AP, Kumar AMP, George SC, Thomas S (2018) Chapter 8—
Thermoanalytical techniques of nanomaterial. In: Mohan Bhagyaraj S, Oluwafemi OS,
Kalarikkal N, Thomas S (eds) Characterization of nanomaterials. Woodhead Publishing,
Sawston, pp 213–236
Brown ME (2001) Introduction to thermal analysis: techniques and applications. Springer, Berlin
Brown ME, Gallagher PK (2011) Handbook of thermal analysis and calorimetry: recent advances,
techniques and applications. Elsevier, Amsterdam
Feist M (2015 February 24) Thermal analysis: basics, applications, and benefit. ChemTexts 1(1):8
Gabbott P (2008) Principles and applications of thermal analysis. Wiley, Hoboken
http://www.excilight.com/sites/excilight.eu/files/uploads/r_lygaitis_2.pdf
Menczel JD, Judovits L, Prime RB, Bair HE, Reading M, Swier S (2009) Differential scanning
calorimetry (DSC). Ther Anal Poly Fundam Appl:7–239
Wunderlich B (2001a) Thermal analysis. In: Buschow KHJ, Cahn RW, Flemings MC, Ilschner B,
Kramer EJ, Mahajan S et al (eds) Encyclopedia of materials: science and technology. Elsevier,
Oxford, pp 9134–9141
Wunderlich B (2001b) Thermal analysis. In: Buschow KHJ, Cahn RW, Flemings MC, Ilschner B,
Kramer EJ, Mahajan S et al (eds) Encyclopedia of materials: science and technology. Elsevier,
Oxford, pp 9134–9141
Wunderlich B (2005) Thermal analysis of polymeric materials. Springer, Berlin
Differential Scanning Calorimetry
17

Abstract
Differential scanning calorimetry (DSC) is a thermal analysis technique which
involves the measurement of temperature difference between the sample and the
reference material as a function of the temperature while sample and reference
both are subjected to a controlled temperature program. This technique is espe-
cially used for qualitative and quantitative determination of change in temperature
in terms of exothermic, endothermic, and heat capacity. In this chapter we have
discussed the principle and types of DSC. The comprehensive instrumentation,
working of the DSC, and the factors affecting the DSC curve have also been
discussed in detail. The interpretation of the result through the curve is also
elaborated. At the end of this chapter, advantages and disadvantages along with
application have been described.

Keywords
Principle of DSC · Types of DSC · Sample preparation for DSC

17.1 Introduction

DSC is a thermo-analytical technique which involves the measurement of the

difference in the amount of heat required to increase the temperature of the sample
and reference as a function of temperature. Sample and reference both are
maintained at almost the same temperature throughout the experiment. Calorimetry
is the process of measuring the amount of heat released or absorbed during a
chemical reaction and the device is known as calorimeter. Watson and O’Neill
developed this thermal analysis in 1960 and then introduced it in 1963 commercially
at Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy.

# Springer Nature Singapore Pte Ltd. 2020 199

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200 17 Differential Scanning Calorimetry

17.2 Principle

The basic principle involved in DSC is that this technique is used to study what is the
effect of heating on the polymers/samples. It examines the thermal transitions of
polymer or sample when heated. For example, this technique can be employed to
study the effect of heating on a crystalline polymer, glass transitions, and crystalli-
zation. The sample and reference materials are heated by separate heaters at the same
temperature throughout the experiment. The energy which is required to obtain zero
temperature difference between sample and reference is measure.

17.3 Types of Differential Scanning Calorimeter

There are two methods involved in DSC which have been described as follows:

17.3.1 Heat Flux DSC

It is a technique where temperature of sample and reference is changed in a specific

program. The difference of the temperature between sample and reference is
measured as a function of heat.

17.3.2 Power Compensated DSC

It is a technique where difference of thermal energy applied to sample per unit time is
measured between the sample and reference as function of temperature.

17.4 Instrumentation and Working

The main components of DSC have been illustrated in Fig. 17.1. Two pans are used
in DSC, one for the sample and other for reference. In sample pan, sample (polymer)
is placed while the reference pan is kept empty as shown in Fig. 17.1. Each pan has a
heater underneath which are connected with a computer which turns on heaters and
heat the two pans at a specific rate, usually 10
C per min. The computer is used to
regulate a constant heating rate during the whole experiment. But the heaters do not
provide the heat at a constant rate. That is because both pans are different, one
contains polymer while other does not. Therefore, an extra heat is required for the
sample pan to keep a zero temperature difference between the sample and reference.
So, the heater underneath the sample pan has to do more work than the other heater.
DSC experiment measures this extra heat of that heater under sample pan by plotting
a graph in which x-axis corresponds to temperature and y-axis corresponds to
difference in temperatures of both heaters.
17.4 Instrumentation and Working 201

Fig. 17.1 Schematic representation of DSC apparatus

17.4.1 Sample Preparation for DSC Analysis

Accurately weighed amount of sample (usually ~3 to 20 mg) is placed in sample pan.

Sample pan is usually made of inert or treated metals, e.g., aluminum, platinum,
nickel, etc. In the DSC analysis, aluminum pans are mostly used if the sample does
not react with Al and the temperature of sample do not exceed to 600
C. Pans are
made up of different kinds of material, e.g., hermetically sealed (airtight) pans,
pinhole, or open pans. It is very important that for a single experiment, the sample
and the reference pan should made up of same kind of material. It is also important
that the material should be placed to cover the bottom of the pan completely to
ensure good thermal contact. It is recommended to avoid overfilling of the pan to
reduce the thermal lag from the bulk of the sample to the sensor. This may be
happened because resolution increases due to sample having less masses and low
heating rates.

17.4.2 Purge Gases

Sample may react with air and oxidize or burn. This problem is overcome by using
inert gases. Most commonly used inert gases are nitrogen, helium, argon, etc.
Nitrogen increases the sensitivity of the experiment. Its typical flow rate is 50 mL/
min. Helium increases the resolution of the peaks and its flow rate is 25 mL/min.
Sometimes, the air and/or oxygen are deliberately used to analyze the oxidative
effects of the sample. In that case the flow rate of oxygen is 50 mL/min.
202 17 Differential Scanning Calorimetry

17.4.3 Heating Rate

It is an important factor that may influence the accuracy of the results. Faster heating
rate may increase the sensitivity but it will decrease the resolution. While, slow
heating rate may decrease the sensitivity but it will increase the resolution. There-
fore, the ideal heating rate is 10
C/min in which the accuracy and sensitivity do not
change.

17.5 Calibration of DSC

It depends upon the estimation of the thermal resistance of both the sample and
reference sensors and the measurements which are made over the interested temper-
ature range. For the calibration of DSC, firstly the temperature difference is
measured between the two empty pans and then thermal response is determined
for the sample material Because DSC is used to measure the difference in heat flow
between a sample pan and reference pan. If there is a difference in heat capacity of
the sample and reference, then the baseline stabilizes faster. This is retained small by
adding more weight (same material on pan) to the reference pan so that it is similar in
total weight to the sample pan.

17.6 DSC Curves

The result of a DSC experiment is displayed in the form of a graph or a curve which
is known as DSC curve. This graph is plotted between heat flux and temperature or
time. There are two different conventions, exothermic and endothermic. If exother-
mic reactions take place then the sample shows a positive peak or negative peak.
This peak depends upon the type of technology used in the experiment. This curve is
used to measure the enthalpies of transitions. This can be measured by integrating
the peak corresponding to a given transition. The enthalpy of transition can be
expressed by using equation:

ΔH ¼ KA:

17.6.1 Factors Affecting DSC Curve

Following are the main factors that may influence the overall results of the DSC
analysis:

17.6.1.1 Instrumental Factors

Following are the important types of instrumental factors that may affect the results
of DSC:
17.8 Disadvantages 203

1. Furnace heating rate: Heating rate should be in the range of 5–10
C/min.
2. Recording or chart speed.
3. Furnace atmosphere: Samples are normally put under an inert gas usually N2 or
He, in order to avoid the oxidation or corrosion.
4. Composition of the sample holder.
5. Sensors location.
6. Sensitivity and accuracy of the recoding system.

17.6.1.2 Sample Characteristics

Sample is also an important factor that may also influence the results of DSC. Here
are the main factors of the sample that may influence on results of DSC:

1. Amount of sample: In order to get accurate results, the sample mass should be in
the range of 3–15 mg, because different amount can give rise different results.
2. Sample nature.
3. Sample preparation.
4. Solubility of evolved gases in the sample.
5. Particle size and shape of the sample.
6. Thermal conductivity.
7. Heat of reaction.

17.7 Advantages

1. A small amount of sample can be used in this technique.

2. The sample present in any form can be tested.
3. It does not require more time for completion.
4. It can be used for studying many types of reactions.
5. It does not require calibration for the entire range of temperature.

17.8 Disadvantages

1. It is not suitable for two-phase mixture.

2. It does not detect the gas generation.
3. There is uncertainty in determined values of heat of transfusion and transition
temperature.
4. It can be used for thermal screening of isolated products and intermediates.
5. The test sample containing solvent may be volatile. So, it is very difficult to
prepare test sample.
6. Interpretation of result is very difficult.
7. It cannot optimize the sensitivity and resolution in a single experiment.
204 17 Differential Scanning Calorimetry

17.9 Applications

1. Study of liquid crystals: Some materials can change their state from solid to
liquid and then pass through a third state anisotropic state, which exhibit the
properties of both solid and liquid phases. This state is called as a liquid
crystalline state or mesomorphous state. DSC can be used to observe these
changes in all these states.
2. Study of oxidative stability: An airtight sample chamber is used to study the
stability to oxidation of samples. By altering the atmosphere of the sample, the
test is done isothermally. First, the sample temperature is set at the desired
temperature under an inert atmosphere that is achieved by inserting the nitrogen
gas. Then, oxygen gas is inserted into the system to achieve oxidation. The
oxidation is observed as a deviation in the baseline. The study of oxidation
process helps in estimating the stability and determining the optimum storage
conditions for a drug compound.
3. Study of drug analysis: Used to study tablet coating. DSC is used to determine
moisture content in drug. DSC is used to study solid drug dispersion. DSC is
used to determine the drying temperature of different excipients. DSC with
support of X-ray diffraction is used as screening technique for the compatibility
testing of drug with excipient. DSC is a valuable tool in choice of
suppository base.
4. Study of chemical analysis: DSC can be used to monitor melting point depres-
sion. This is possible because the temperature range over is dependent on the
relative amounts of the sample. Consequently, less pure compounds will exhibit
a broadened melting dip that begins at lower temperature than a pure compound.
Freezing point depression can be used as a purity analysis tool. This is possible
because the temperature range over which a mixture melt is dependent on their
relative amount. Less pure compounds will exhibit a broadened melting dip that
begins at lower temperature than a pure compound.
5. Study of polymer characteristics: DSC is also used for analyzing polymers to
check their composition. Melting points and glass transition temperatures for
most polymers are available from standard compilations, and the method can
show up possible polymer degradation by the lowering of the expected melting
point, which depends on the molecular weight of the polymer, so lower grades
will have lower melting points than the expected. Impurities in polymers can be
determined by examining thermograms for anomalous peaks, and plasticizers
can be detected at their characteristic boiling points.
6. Study of food analysis: In food sciences, DSC and any other thermal analytical
techniques are used in combination to analyze the water dynamics.
7. Study of crystallization phenomenon: DSC can also tell us about the crystallinity
nature of a polymer. If we know the latent heat of melting, ΔHm, we can figure
out the answer. The first thing we have to do is to measure the peak area for the
melting of a polymer.
8. Determination of heat capacity: It can be used for the determination of heat
capacity. When a certain amount of heat is transferred to the sample, its
Further Reading 205

temperature increases by a certain amount, and the amount of heat it takes to get
a certain temperature increase is called the heat capacity, or Cp, it can be figured
up from the DSC plot.
9. Determination of glass transition temperature (Tg): Tg is an important charac-
teristic of non-crystalline and semi-crystalline materials, but Tg is a particularly
significant property of many common polymers. At a temperature below Tg,
amorphous and semi-crystalline polymers tend to be hard and brittle because the
polymer chains are locked in a tangled, coiled position. Above Tg, the poly-
meric chains are able to more easily rotate and slip past each other, and the
polymer becomes softer and more ductile.
10. Study of proteins: DSC is used to characterize the stability of a protein. It does
this by measuring the heat changes associated with the molecule’s thermal
degradation when heated at constant rate.
11. Qualitative and quantitative analysis mineral: DSC is used for detection of any
mineral in a sample.
12. Study of antibody domains: DSC use to study antibody domains.
13. Study in drug development: DSC is used to determine the status of any process
during the drug development.
14. Study of enzyme kinetics: DSC is used to study the reaction time of enzyme
degradation and/or kinetics.
15. Binding studies: DSC is a valuable approach for studying binding between a
biological macromolecule and a ligand such as another polymer.

Further Reading
Abraham J, Mohammed AP, Kumar AMP, George SC, Thomas S (2018) Chapter 8—
thermoanalytical techniques of nanomaterials. In: Mohan Bhagyaraj S, Oluwafemi OS,
Kalarikkal N, Thomas S (eds) Characterization of nanomaterials. Woodhead Publishing,
Sawston, pp 213–236
Brown ME (2001) Introduction to thermal analysis: techniques and applications. Springer, Berlin
Brown ME, Gallagher PK (2011a) Handbook of thermal analysis and calorimetry: recent advances,
techniques and applications. Elsevier, Amsterdam
Brown ME, Gallagher PK (2011b) Handbook of thermal analysis and calorimetry: recent advances,
techniques and applications. Elsevier, Amsterdam
Charsley E, Price D, Hunter N, Gabbott P, Kett V, Gaisford S et al (2019) Principles of thermal
analysis and calorimetry. Royal Society of Chemistry, London
Feist M (2015) Thermal analysis: basics, applications, and benefit. ChemTexts 1(1):8
Höhne G, Hemminger WF, Flammersheim H-J (2013) Differential scanning calorimetry. Springer,
Berlin
Lopez MM, Makhatadze GI (2002) Differential scanning calorimetry. Calcium-binding protein
protocols. In: Methods and techniques, vol 2. Springer, Berlin, pp 113–119
Menczel JD, Judovits L, Prime RB, Bair HE, Reading M, Swier S (2009) Chapter 2: differential
scanning calorimetry (DSC). In: Menczel JD, Prime RB (eds) Thermal analysis of polymers:
fundamentals and applications. Wiley, Hoboken, pp 7–239
Riga A, Collins R (2006) Differential scanning calorimetry and differential thermal analysis. In:
Encyclopedia of analytical chemistry: applications, theory and instrumentation. Wiley,
Hoboken
206 17 Differential Scanning Calorimetry

Schick C, Lexa D, Leibowitz L (2002) Differential scanning calorimetry and differential thermal
analysis. In: Characterization of materials. Wiley, Hoboken, pp 1–13
Verdonck E, Schaap K, Thomas LC (1999) A discussion of the principles and applications of
modulated temperature DSC (MTDSC). Int J Pharm 192(1):3–20
Wunderlich B, Analysis T (2001) In: Buschow KHJ, Cahn RW, Flemings MC, Ilschner B, Kramer
EJ, Mahajan S et al (eds) Encyclopedia of materials: science and technology. Elsevier, Oxford,
pp 9134–9141
Differential Thermal Analysis
18

Abstract
Differential thermal analysis (DTA) is a thermo-analytical technique which is
used for thermal analysis where thermal changes can be studied. It is used to
determine the oxidation process, decomposition, and loss of water or solvent. In
this chapter we have discussed the basic principle of DTA along with its instru-
mentation, working, factors affecting DTA curve, advantages and disadvantages,
and its applications.

Keywords
Principle of DTA · Instrumentation of DTA · Working of DTA

18.1 Introduction

DTA is a type of thermo-analytical technique. Robert Austen was the first scientist
who worked on this technique by introducing two thermocouples in its apparatus in
1899. Then, in 1909, Burgess modified this technique followed by Norton (1939),
Kerr (1948), Kauffman (1950), Grim (1951), and Fold Vari (1958). However,
differential thermocouple arrangement which was suggested by Robert-A is still
widely used in thermal analysis and therefore this technique is known as DTA or
“Thermography.” In DTA, the temperature difference is measured between the
sample/analyte and the reference as a function of temperature while the sample
and the reference are heated at a controlled temperature program. A plot (curve or
thermogram) is then plotted between the differential temperature and time or tem-
perature. Then changes observed in the sample, due to the absorption or evolution of
heat, are detected relative to the reference.

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208 18 Differential Thermal Analysis

18.2 Principle

DTA is a thermal analysis which involves the comparison between the temperatures
of sample under investigation and a thermally inert material/reference which may be
α-alumina (it is densified alumina in α-phase and occurs in hexagonal structures) and
this comparison is then recorded with the furnace temperature as the sample is heated
or cooled at uniform rate. There is a constant temperature difference (ΔT) between
the sample and the reference as they both have different heat capacities. But when
the sample undergoes the endothermic or exothermic changes then ΔT becomes
different. The basic principle involves the measurement in temperature changes
which are associated with the physical changes or chemical changes of the sample
during the gradual heating process.

18.3 Instrumentation

DTA contains the following basic components. Schematic representation of overall

equipment of DTA has been illustrated in Figs. 18.1 and 18.2.

18.3.1 Furnace Assembly

It works as a temperature programmer. There are many furnaces which are used
depending upon the sample material and the rate of heating, e.g., nichrome furnace
which is made up of Nickel and Chromium alloy is used when rate of heating is up to
1300  C. Platinum furnace which is made of Pt and its alloys is employed when rate
of heating is up to 1750  C and the furnace which is made of molybdenum is
employed for higher temperature ranges that are up to 2000  C.

Fig. 18.1 Schematic representation of components of DTA

18.3 Instrumentation 209

Fig. 18.2 Schematic representation of characteristics of DTA

18.3.2 Sample and Reference Holder with Temperature Detector

In DTA apparatus, two compartments/pans are present, one for the sample and other
one for the reference. The holders are designed in a manner that they can accommo-
date even a small quantity of the sample or reference material and give maximum
thermal effect. Mostly, they are made up of platinum, stainless steel, nickel, silver,
and alloys such as platinum-rhodium. Many ceramic materials, e.g., silica, sintered
alumina, fire clay, heat resistant glass, and graphite are recommended as holders for
the sample and reference (like α-alumina). The holders are connected with tempera-
ture detector which measures the difference in temperatures of the sample and
reference material as a function of time or temperature when the temperature is
rising at the constant linear rate. In simple words, both pans are different, one is
sample pan which loads polymer means extra material and other one is the reference
does not has polymer. Due to extra material more heat is required to keep the sample
pan’s temperature increasing at a same rate as that of the reference pan.

18.3.3 Furnace Temperature Programmer

Electronic temperature regulators are used to ensure a constant rate of heating of the
furnace. For temperature regulation, thermocouples made up of rare metal alloy such
as platinum (Pt-10-13% Rh) are mostly employed for the measurements of tempera-
ture up to 2000  C. A thin thermocouple is usually inserted in both holders. When
few samples are to be studied, visual galvanometric observations are employed
210 18 Differential Thermal Analysis

though inconvenient. But for accurate results and convenience automatic pen and ink
electronic recorder are used mostly.

18.3.4 Amplifier

Its function is to convert the heat signal into electrical signal.

18.3.5 Read-Out Device

It is used to display the results in the form of a thermogram. Nowadays, the read-out
devices have microprocessors that deliver outputs compatible with computers and
printers thus minimizing the risk of operator errors.

18.3.6 Insulator for Furnace and Sample Holder

It a block of ceramic or other insulating material enclosing the furnace and sample
holders which does not readily allow the passage of heat.

18.4 Working of DTA

The sample/analyte under investigation is loaded into a container. This container is

then placed onto the sample pan and it is marked as S (means sample). Same quantity
of inert material (like alumina) as that of sample is placed in another container which
is then placed onto the reference pan and it is marked as R (means reference). In
order to heat the sample pan and the reference pan at an identical rate, the dimensions
of these two pans should be nearly identical; moreover, the sample and the reference
should have equal weights, thermally matched and should be arranged symmetri-
cally with the furnace. The metal block which surrounds the pans acts as a heat sink
whose temperature is increased slowly by using an internal heater. The sink then
heats the sample and reference material simultaneously. Two pairs of thermocouples
are used, one pair is in contact with the sample and the second pair is in contact with
the reference. Thermocouple is attached with an amplifier which amplifies the result
of differential thermocouple and sent this result to the read-out device which displays
the results in the form of DTA curve or thermogram as a function of the sample
temperature, reference temperature or time. No signal is generated if no temperature
difference is observed even though the actual temperatures of both the sample and
reference are increasing. When there is a physical change in the sample then heat is
absorbed or released. For example, when a metal carbonate is decomposed then
carbon dioxide is released. This is an endothermic reaction where the heat is
absorbed and the temperature of the sample is decreased. Now the sample is at a
18.6 Factors Affecting on DTA Curve 211

lower temperature than that of the reference. This temperature difference between
sample and reference produces a net signal, which is then recorded.

18.5 Characteristics of DTA Curve

The DTA curve or thermogram is a plot between differential temperature (ΔT) and
temperature of reference (T). DTA curve may be endothermic (downward plot) or
exothermic (upward plot). In exothermic reaction, the sample temperature is higher
than that of the reference, whereas in endothermic reactions, the sample temperature
is less than that of the reference material and if there is no reaction happening in the
sample material, then the sample temperature remains the same as that of the
reference material. As shown in Fig. 18.2, the value of ΔT is zero along the line
AB which indicates no reaction in the sample material. At point B the curve rises
from the baseline due to the exothermic reaction and it forms a peak BCD with a
maximum temperature or heat value at point C. At this point, the rate of evolution of
heat is almost same to that of the difference between the heat evolution of the sample
and the reference. However, it does not show the maximum rate of heat evolution or
completeness of reaction. So, point C is not important in DTA curves. At point C, the
process of heat is completed and after this, heat is going to be decreased up to D
point. The peak temperature is the characteristic of the sample material. BCD area
has a direct relation with the amount of reacting material. Identification of the sample
material, heat of reactions, sample mass (m), heat of reactions (H ) sample geometry,
and thermal conductivity are the prospects which can be determined using DTA
curves.

18.6 Factors Affecting on DTA Curve

Differential thermal analysis is not a dynamic thermal analytical technique. Its value
can deviate because of many factors which can be divided into four major groups.

18.6.1 Sample Factors

1. Amount of the sample.

2. Packing density.
3. Particle size of the sample material.
4. Degree of crystallinity.
5. Heat capacity.
6. Thermal conductivity.
7. Dilutes of the diluents.
8. Swelling of the sample.
9. Shrinkage of the sample.

All these sample factors can affect the DTA curve or thermogram.
212 18 Differential Thermal Analysis

18.6.2 Instrumental Factors

1. Size or shape of the holders.

2. Material of the sample holder.
3. Recording system sensitivity.
4. Rate of heating of the sample.
5. Atmosphere around the sample.
6. Thermocouple location in the sample.
7. Instrumental design.

18.6.3 Physical Factors

They can be divided into two further groups. Some physical factors affect the
exothermic curve or some affect the endothermic curve. Exo-thermogram may be
affected by one of the following factors:

1. Adsorption.
2. Change in the crystal structure.
3. Crystallization.

Whereas endo-thermograms may be affected by one of the following factors:

1. Desorption.
2. Change in the crystal structure.
3. Melting.
4. Vaporization.
5. Sublimation.

18.6.4 Chemical Factors

They can also be divided into two further groups. Some chemical factors affect the
exothermic curve or some affect the endothermic curve. Chemical factors which may
affect the exothermic reactions are as follows:

1. Oxidation.
2. Breakdown reactions.
3. Chemisorption.
4. Solid state reactions.

Chemical factors which may affect the endothermic reactions are as follows:

1. Reduction.
2. Breakdown reactions.
3. Solid state reactions.
Further Reading 213

18.7 Advantages

1. DTA apparatus can be operated at very high temperature ranges.

2. Highly sensitive technique.
3. Both exothermic and endothermic reactions can be determined accurately.

18.8 Disadvantages

1. There is lot of uncertainty in transition reactions and heat of fusions.

18.9 Applications

1. Used to identify the minerals both qualitatively and quantitatively.

2. Polymers characteristics can be easily characterized.
3. Degree of crystallinity can be measured.
4. Degree of polymerization can be assessed.
5. Many of the biological materials can be analyzed.
6. Melting point, boiling point, and temperatures of decomposition of organic
compounds can be determined.
7. Have wide applications for the quality control (QC) of many substances such as
soil, cement, glass, etc.
8. Also used to determine the thermal stability of many inorganic compound s and
complexes.

Further Reading
Abraham J, Mohammed AP, Kumar AMP, George SC, Thomas S (2018) Chapter 8—
thermoanalytical techniques of nanomaterials. In: Mohan Bhagyaraj S, Oluwafemi OS,
Kalarikkal N, Thomas S (eds) Characterization of nanomaterials. Woodhead Publishing,
Sawston, pp 213–236
Brown ME (2001) Introduction to thermal analysis: techniques and applications. Springer, Berlin
Brown ME, Gallagher PK (2011) Handbook of thermal analysis and calorimetry: recent advances,
techniques and applications. Elsevier, Amsterdam
Charsley E, Price D, Hunter N, Gabbott P, Kett V, Gaisford S et al (2019) Principles of thermal
analysis and calorimetry. Royal Society of Chemistry, London
Laye P (2002) Differential thermal analysis and differential scanning calorimetry. In: Principles of
thermal analysis and calorimetry, vol 52. Royal Society of Chemistry, London
Menczel JD, Judovits L, Prime RB, Bair HE, Reading M, Swier S (2009) Chapter 2: differential
scanning calorimetry (DSC). In: Menczel JD, Prime RB (eds) Thermal analysis of polymers:
fundamentals and applications. Wiley, Hoboken, pp 7–239
Smykatz-Kloss W (2012) Differential thermal analysis: application and results in mineralogy.
Springer, Berlin
Thermo Gravimetric Analysis
19

Abstract
In this thermo gravimetric analysis (TGA) technique, sample is heated at a
given temperature with controlled heating rate to measure the change in the
weight of a sample substance as a function of temperature. The results can be
interpreted from TGA curve. Thermogram is plotted between the change in
mass and temperature. In this chapter, principle of TGA and the instrumentation
along with its comprehensive working have been described. The calibration
procedure is also given in this chapter. The factors affecting the results along
with advantages, disadvantages, and applications have also been described in
this chapter.

Keywords
Principle of TGA · Working of TGA · Types of TGA

19.1 Introduction

A technique in which the mass of a substance is measured as a function of tempera-

ture, while the substance is subjected to a controlled temperature program is
described. This technique is studied under thermal analysis and is used for the
estimation of those materials which undergo the mass change when subjected to
the thermal events such as decomposition, reduction, and oxidation. It is very
important to consider the factors which affect the change in the mass of a sample.
The thermobalance is used in TGA. Results are obtained by plotting a graph
between the mass change and the temperature. The instrument which is mostly
used for TGA is known as “thermobalance.” Data obtained from TGA is recorded
in the form of curve which is known as “thermogram.” The furnace is used to raise
the temperature as high as 1000  C and it is constructed of quartz.

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216 19 Thermo Gravimetric Analysis

19.2 Principle

TGA determines the amount and the rate of weight change of a substance with
respect to temperature or time in controlled conditions. TGA is a technique in
which a change in the weight of a substance is recorded as a function of temperature
or time. Change in the temperature affects the sample by changing the mass of
the sample. The crystallization, desorption, sublimation, evaporation, oxidation,
reduction, and decomposition bring drastic change in the mass of the sample.

19.3 Types of Thermogravimetry

There are three types of TGA, which are as follows:

19.3.1 Dynamic TGA

In this technique, the continuous linear increase in the sample temperature with
respect to time is occurred.

19.3.2 Isothermal TGA

In this type of analysis, the temperature of sample is kept constant for a period
of time and change in weight is measured.

19.3.3 Quasistatic TGA

In this analysis, the sample is heated until the constant weight is obtained when
temperature is increasing in a series.

19.4 Instrumentation

TGA is constructed of the following three important parts and its schematic
representation has been illustrated in Fig. 19.1:-

19.4.1 Microbalance

A balance that is designed to measure the very small weights such as parts per
millions of a gram.
19.4 Instrumentation 217

Fig. 19.1 Schematic representation of components of thermo gravimetric analysis

19.4.2 An Auto Sampler

A device that is used for automatic loading of the collected sample into a laboratory
instrument. It is usually widely used in spectroscopic and thermal analysis.

19.4.3 Thermocouple

An electronic device that is used for measuring the temperature. It has a pair of wires
that is made up of different metals which are joined together and their free ends
are attached to a voltmeter that is used to measure the potential difference created
between the joining point of the metals.

19.4.4 Furnace

It is an enclosed structure which provides heat to the sample and the sample can be
heated at a high temperature.

19.4.5 Temperature Programmer

The temperature of the system is adjusted by entering the pre-planned data.

The temperature of the system may increase or decrease step by step.
218 19 Thermo Gravimetric Analysis

19.4.6 Recording System

The output obtained from the furnace and microbalance is recorded in the form of
chart or using a microcomputer. Nowadays, the recording system is associated with
a software, which allows the data to be stored and plot the graph for interpretation
of the result efficiently.

19.5 Working of TGA

The sample is loaded on to the microbalance with the help of auto sampler. The
thermocouple is placed right above the sample. The temperature of furnace is raised
slowly. Then the change in the mass corresponding to the temperature is measured.
The thermocouple is used to measure the temperature of sample and change in the
weight is measured by finding the beam deflection. During the whole experiment,
it is avoided direct contact of sample with thermocouple while the sample is placed
in platinum pan. TGA is used to measure the changes in the mass of sample and its
scanning is performed in a highly controlled atmosphere. The weight of sample is
measured as a function of temperature by thermobalance. The sample is placed in
a furnace by hanging with balance and sample is thermally isolated from the furnace.
The following points should be considered while working on TGA:

19.5.1 Sample Preparation

For obtaining good data, sample preparation is very important. By increasing the
surface area of a sample which is placed in the pan, the resolution and reproducibility
of weight loss temperatures can be improved. If the weight of the sample is reduced,
then it affects the accuracy of weight measurements. Usually, 10–20 mg of sample
is used in most of the applications. If the sample is volatile in nature, then the
weight of the sample should be 50–100 mg. It is to be considered that most
TGA instruments usually have the baseline drift value of 0.025 mg which is
equal to 0.25% of a 10 mg sample substance.

19.5.2 Heating Rate

In most of the cases, the sample is heated at the rate of 20  C/min. The resolution of
overlapping weight loss is improved by decreasing the heating rate. The variable
heating rates can also be used in TGA. For this purpose, TGA instrument should be
constructed in such a way that it enhances the resolution automatically by decreasing
the heating rate during the interval of weight loss.
19.6 Calibration 219

19.5.3 Purge Gas

Nitrogen is inert in the nature. That’s why it can be used to purge samples in TGA
because of its nature. Helium is used to provide the baseline. The difference in the
oxidative stability of sample components has effect on the result obtained from
TGA. Its resolution can be enhanced by using air. When sample components are
volatile in nature, then vacuum can be used which improves the separation of
components from the start of the decomposition and the volatiles formed from the
sample at lower temperature in vacuum.

19.6 Calibration

The instrument is calibrated by using the following steps:

19.6.1 Blank Test

The sample is not placed in pan when the calibration is performed. Only air is
passed through the pan and temperature is raised up to 1000  C at a heating rate of
10  C/min. The general condition of apparatus should be checked before doing this
blank test. When the temperature is increased, then a slight drift in TGA curve is
observed. This is responsible for change in convection and buoyancy. Noise can
be occurred in TGA curve due to the following reasons. The contact between the
sample dish and thermocouple, quartz suspension wire and purge gas feed pipe and
weight pan and arid glass cap. The vibration and shock are also responsible for
producing noise. A slight decrease in curve is observed in TGA curve when sample
pan or suspended wire is contaminated with decomposition product.

19.6.2 Calibration of Mass Changes

TGA is usually used for measuring the weight of the sample by the rate of the weight
change of sample. That’s why the calibration of absolute weight value of sample is
very essential. A 20 mg weight of the sample is calculated with the precision of 10 μg
by using a precision balance, and the average value is calculated. The instrument
balance control is adjusted to set the automatic zero when furnace is put on and
TGA signals are stabilized.

19.6.3 Calibration of Temperature

The temperature of TGA can be calibrated in two ways. One way is the use of the
melting point of a pure metal and the second is to use Curie point temperature.
In the first procedure, one of the metals is handled in a ribbon shape and then this
220 19 Thermo Gravimetric Analysis

metal is placed on TGA suspension wire. The weight of approximately 100 mg is

attached with suspended wire tip. By heating, the pure metal is fused and as a result,
the weight is reduced. This drop-in weight can be observed in TGA curve. In Curie
point temperature technique, the standard substances are verified by International
Congress on Thermal Analysis (ICTA). The standard substance which can be used in
TGA are ferromagnets and their Curie temperature is different. So, it is necessary to
calibrate it by estimating the apparent weight changes at Curie temperatures by
permanent magnet. The composition of sample materials and their thermal stability
can be predicted from TGA data. It depends upon the mass of the sample changes
due to oxidation, dehydration, evaporation, and decomposition. These phenomena
may occur when temperature is as high as 1000  C. The typical example is calcium
oxalate hydrate. When it is heated up to 1000  C, then decomposition takes place
which is observed from the curve which shows decomposition occur in three steps.
The data of weight loss is measured after half second during the whole experiment.

19.7 Factors Affecting the TG Curve

The factors which may affect the results of TGA are categorized into two major
groups which are as follows:

19.7.1 Instrumental Factors

Instrumental factors that affect the TGA curve are as follows:

19.7.1.1 Furnace Heating Rate

The temperature at which the substance (or sample) degrades depends upon
the heating rate. When the heating rate is so high, then the decomposition of the
substance is also at a high rate. For reproducible and reliable TGA, heating rate
of 3.5  C/min is mostly used. If the heat is endothermic and exothermic, it will be
responsible for the sample temperature and increase or decrease in the furnace
temperature.

19.7.1.2 Furnace Atmosphere

The temperature at which the sample is decomposed is affected by the atmosphere
inside the furnace surrounding the sample. A pure nitrogen gas is passed from
cylinder to the furnace for creating highly inert atmosphere.

19.7.2 Sample Characteristics

The characteristics of the sample which affect the experimental procedure are as
follows:
19.10 Applications 221

19.7.2.1 Weight of Sample

In this technique, usually small weight of the sample is used. Due to the small
amount of the sample, temperature gradient will reduce through the sample. If large
amount of the sample is used, then deviation from the linearity occurs especially in
exothermic reaction.

19.7.3 Particle Size of Sample

The small and uniform shaped particles are usually used in this technique. If large
particle or crystal is used, then it may cause rapid weight loss during heating.

19.8 Advantages

1. Any type of solid can be analyzed with the minimal sample preparation.
2. It has high accuracy of balance, high precision of temperature controlling system
and atmospheric conditions.
3. It is a convenient and time-saving technique because it has no too much
construction requirements.
4. The sample and sample holder can be easily changed.
5. Fast heating rate with good resolution can be maintained.

19.9 Disadvantages

1. Limitation for sample volume.

2. Interpretation of the result is very difficult.
3. It is very sensitive to any change.
4. It cannot optimize the sensitivity and resolution in a single experiment.
5. The quantitative analysis of the individual process is impossible.
6. During the experiment, decomposition of pharmaceuticals occurs. This renders
the products which are practically insoluble and mostly sticky in nature and
stick inside the wall of a DSC cell.
7. Due to these products, the life of DSC cell is reduced.

19.10 Applications

There is a wide range of applications of TGA which are as follows:

1. It is used in the composition of multi-component system.

2. It can be used for thermal stability of polymers.
3. The oxidative stability studies of polymers can be done by this method.
222 19 Thermo Gravimetric Analysis

4. The shelf-life of the finished good product can also be estimated by this
technique.
5. The decomposition kinetics of many polymers can also be studied by this
method.
6. The effect of corrosive and reactive atmosphere on polymers can be studied.
7. It can be used for determination of moisture and volatile contents in the sample
materials.
8. It can also be used for determination of the bound and unbound water in the
suspension of milk of magnesia (MoM) which is used as a laxative.
9. The comparison of the generic and a brand MoM can also be studied by this
method.
10. In thermal analysis method, it is always preferable to perform a TGA experiment
on unknown given sample before performing a DSC experiment (especially for
pharmaceuticals).
11. The change in the state of catalyst is determined by using this technique.

Further Reading
Brown ME (2001) Introduction to thermal analysis: techniques and applications. Springer, Berlin
Brown ME, Gallagher PK (2011) Handbook of thermal analysis and calorimetry: recent advances,
techniques and applications. Elsevier, Amsterdam
Charsley E, Price D, Hunter N, Gabbott P, Kett V, Gaisford S et al (2019) Principles of thermal
analysis and calorimetry. Royal Society of Chemistry, London
Feist M (2015) Thermal analysis: basics, applications, and benefit. ChemTexts 1(1):8
https://www.pharmatutor.org/articles/thermogravimetry
Mantheni DR (2012) Novel solid state properties of drugs, polymers and various chemicals by
thermal and analytical techniques. ETD Arch 192 https://engagedscholarship.csuohio.edu/
etdarchive/192
Menczel JD, Judovits L, Prime RB, Bair HE, Reading M, Swier S (2009) Differential scanning
calorimetry (DSC). In: Thermal analysis of polymers: fundamentals and applications. Wiley,
Hoboken, p 7
Wunderlich B (2001) Thermal analysis. In: Buschow KHJ, Cahn RW, Flemings MC, Ilschner B,
Kramer EJ, Mahajan S et al (eds) Encyclopedia of materials: science and technology. Elsevier,
Oxford, pp 9134–9141