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RECENT TRENDS IN ANALYTICAL CHEMISTRY
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RECENT ADVANCEMENTS IN ANALYTICAL CHEMISTRY
About the Book
Analytical chemistry is a diverse branch of chemistry playing a pivotal role in modern
society. Considering the rising importance of the branch, we are writing this book,
“Recent advancements in analytical chemistry” with multiple chapters dedicated to the
latest methods like hyphenated techniques and conventional methods like Titrimetry.
This book covers titrimetry, gravimetry, organic functional group analysis, spectroscopy,
crystal analysis, and chromatography techniques. This book, with valuable chapters from
eminent academicians and researchers, gives insight into modern and conventional
analytical techniques that are useful in modern analytical chemistry
About the Editors

Dr. N Padmaja, Head of Department & Academic Coordinator, Chemistry –
PG Center Lal Bahadur College, Warangal, A doctorate in Inorganic Chemistry
from Kakatiya University, her field of study and expertise include metal
complexes and coordinate chemistry. She is currently working as Associate
Professor at the Department of Chemistry, Post Graduate center, Lal Bahadur
College Warangal, Telangana, India, and has 28 years of teaching experience. She
is the head of the chemistry department, academic coordinator, and cultural
convener at her college and handles numerous educational and cultural events
by coordinating with various institutes. She has two patents to her credit so far.
She also has multiple research publications and book chapters, both international and nationally, to her
credit. She has published seven research papers in reputed national and International Journals and
three book chapters. She is also a member & contributor to the international congress of chemistry &
pharmacy.
RECENT ADVANCEMENTS IN
Dr. Vishnu Kiran Manam A doctorate in Applied Microbiology - Botany with
specialization in Nanotechnology from the University of Madras, his field of
study and expertise include Nano-biotechnology, Algal Research, Aquaculture,
Vaccine Research, Bio-Remediation and Drug Discovery Services. He has rich
experience in Research & Development and Academics for more than a decade,
ANALYTICAL CHEMISTRY
He has also practical experience in Marketing & Corporate Communications,
Human resources, and Project Management for more than 5 yrs. He has been
certified with Six Sigma [Yellow Belt, Green Belt & Black Belt]. He has bagged the
BEST SCIENTIST AWARD –IARDO, YOUNG SCIENTIST AWARD – ELSEVIER SSRN, RESEARCH
EXCELLENCE AWRAD - RES and BEST RESEARCHER AWARD - ISCAW – ESM for the year 2021. He
has 25 patents to his credit so far. He has also various research publications, book chapters both
PADMAJA I MANAM
internationally and nationally to his credit. He has been a book editor [6 Books] in various disciplines
such as Nanotechnology, Chemical Sciences, Aquaculture, etc. He has actively taken part in various
research programs conducted nationally and internationally. He has been a part of the editorial board
member in various International journals and a member of various research forums. DR N PADMAJA
DR VISHNU KIRAN MANAM
ISBN 978-93-94766-13-6
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<<<<<<<<<<<<<<<<<<
Copyright Editors
Title: RECENT ADVANCEMENTS IN ANALYTICAL CHEMISTRY

Editors: DR N PADMAJA & DR VISHNU KIRAN MANAM
All rights reserved. No part of this publication may be reproduced or transmitted, in any
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First Published, 2022
ISBN: 978-93-94766-13-6
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Disclaimer: The views expressed in the book are of the authors and not necessarily of
the publisher and editors. Authors themselves are responsible for any kind of plagiarism
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<<<<<<<<<<<<<<<<<<
ii
<<<<<<<<<<<<<<<<<<0.75
PREFACE
Analytical chemistry has a vital role in every stage of life. It is the science of obtaining,
processing, and communicating information about the composition and structure of matter.
In other words, it is the art and science of determining what matter consists of and
measuring the same. By applying analytical chemistry, we have gained insight into the
origin and evolution of the universe and life on our planet. Through these insights, we can
improve the material characteristics of natural resources and industrial materials to benefit
humankind. Today we cannot think of even a single product of commercial use which has
not been tested using analytical techniques before clearance from consumption.
There are two essential aims of analytical chemistry – the first is to attain analytical
information of the highest quality with the lowest possible uncertainty, and the second is to
solve analytical problems derived from biochemical details of different areas. This way,
analytical chemistry plays an enormous role in various fields such as drug manufacturing,
process control, medical diagnostics, environmental monitoring, food production, and
forensic surveys. In our modern world, without analytical chemistry, we would not be able
to make any important decisions such as soil remediation, limiting values for environmental
pollution, choosing the correct dosage of medicines and food for patients, etc.
Analytical chemistry is also focused on improvements in experimental design,
chemometrics, and the creation of new measurement tools. It has broad applications in
medicine, science, and engineering. It is poised to make more significant contributions to
improving life and understanding new materials. It will lead the way to developing new
materials with desired features and detection at levels that could not be imagined before.
Analytical chemistry has evolved dramatically over the past few decades from the
traditional notion held for centuries to that of a modern, active discipline of chemistry. It
produces quality (bio) chemical information of global and partial type from natural and
artificial objects and systems to solve analytical problems derived from information needs.
Analytical chemistry is an information discipline and is highly essential to modern society.
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iii
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iv
<<<<<<<<<<<<<<<<<<0.75
CONTENTS
Preface iii
Sr. Tittle Page

No No
1 INTRODUCTION TO UV VISIBLE SPECTROSCOPY IN 1-10
ANALYSIS OF ORGANIC COMPOUNDS
Dr. Ranvijay Pratap Singh
2 BASIC CONCEPTS AND APPLICATIONS OF 11-18

CONDUCTOMETRIC TITRATIONS
Dr. K Vidya
3 RECENT CHROMATOGRAPHIC DEVELOPMENTS IN 19-24

THE SEPARATION OF LEAF PIGMENTS
Dr. Ravinder Manchal
4 A FACILE SYNTHESIS OF 2-CYCLOPROPYL-3- 25-33

PYRAZOLO-1,8-NAPHTHYRIDINE DERIVATIVES
Dr. Laxminarayana Eppakayala, Dr. Shashikala Kethireddy,
Dr. Bhaskar Pittala
5 GROWTH, SURFACE, THERMAL AND MECHANICAL 34-41

PROPERTIES OF NONLINEAR OPTICAL POTASSIUM
THIOUREA CHLORIDE
Dr. D Nagaraju
6 ROLE OF INFRARED (IR) SPECTROSCOPY IN 42-51

ANALYSIS OF ORGANIC COMPOUNDS
Dr. Ranvijay Pratap Singh
7 SEPARATION METHODS: CHROMATOGRAPHY 52-60

Dr. Sridevi Sunakari
<<<<<<<<<<<<<<<<<<
v
<<<<<<<<<<<<<<<<<<
8 ULTRA PERFORMANCE LIQUID CHROMATOGRAPHY 61-66

Dr. K Srivani
9 QUANTITATIVE AND QUALITATIVE CHEMICAL 67-75

ANALYSIS
Dr. Padma Raviteja, Dr. Sravanthi
10 CHROMATOGRAPHY: A VERSATILE TECHNIQUE FOR 76-87

SEPARATION
Dr. Anita S Goswami Giri, Dr. Geeta S Ingawale
11 A REVIEW ANALYSIS OF DIFFERENT ORGANIC 88-94

COMPOUNDS THROUGH VARIOUS INSTRUMENTAL
METHODS WITH THE ADVANCEMENT OF SCIENCE
AND TECHNOLOGY
Dr. Sumanta Bhattacharya, Arkadyuti Seth
<<<<<<<<<<<<<<<<<<
vi
Chapter
INTRODUCTION TO UV-VISIBLE SPECTROSCOPY IN
1 ANALYSIS OF ORGANIC COMPOUNDS
RANVIJAY PRATAP SINGH*
Department of Applied Science & Humanities, Faculty of Engineering & Technology,

University of Lucknow, Lucknow-226031 (U.P.), India
*Corresponding author: Ranvijay Pratap Singh, Email: rsingh0777@gmail.com
ABSTRACT
This chapter aims to introduce readers to the role of Ultraviolet-visible spectroscopy in the analysis of
organic compounds. UV-visible spectroscopy is used in the determination of double bond or
conjugated double bond, aromatic conjugation, and electronic transition within the molecules. This
chapter provides an overview of the fundamental theories of UV-visible spectroscopy. These provide
useful information about the applications of this technique for chemical analysis. The most common
applications of UV-visible spectroscopy are also briefly discussed.
KEYWORDS: UV-visible spectroscopy, Electronic transition, Woodward-Fieser rules,

Chromophores, Auxochromes.
INTRODUCTION
Spectroscopy is a branch of science that deals with the study of the interaction of
electromagnetic radiations with matter. Electromagnetic spectrum ranges all types of
electromagnetic radiations. When electromagnetic radiation interacts they give rise to
electronic transition in UV, molecular vibration in IR, and nuclear spin orientation in NMR.
1. Ultraviolet and visible spectroscopy also known as electronic spectroscopy is used to

measure the number of conjugation of the double bond and aromatic conjugation
within the molecule.
Responsibility of contents of this book rests upon the authors and not upon the Editor & Publisher
2 RECENT TRENDS IN ANALYTICAL CHEMISTRY
2. It involves the promotion of electrons from HOMO to LUMO (HOMO means Highest
Occupied Molecular Orbital whereas LUMO means Lowest Unoccupied Molecular
Orbital).
3. The HOMO-LUMO gap decrease as the conjugation increase.
1. The ultraviolet region corresponds to 400-200 nm and the visible region to 800-400 nm.
2. UV-visible spectroscopy is based on Beer-Lambert law.
3. ⁄
4. Where,
5. Io = intensity of incident light
6. I = intensity of transmitted light
7. = molar absorptivity
8. l = path length of sample
9. c = concentration of sample
10. A = Absorbance
11. Absorbance (A): It is reciprocal of Transmittance
12.
13. Transmittance (T): The fraction of incident light transmitted is known as Transmittance.
14.
Electronic transitions
When a molecule is getting excited by the absorption of electromagnetic radiation in the
UV-visible region then its electrons are promoted from the ground state to excited state or
from bonding orbital to anti-bonding orbital.
Types of electronic transitions
1. * Transition: transition of an electron from bonding sigma orbital ( ) to anti-
bonding sigma orbital ( *), is represented by * transition. For example, alkanes
because in alkane all the atoms are held together with a sigma bond.
2. * & * Transition: Transitions from non-bonding molecular ( ) orbital to anti-
bonding sigma orbital or anti-bonding pi orbital ( *), are represented by * or *
transition respectively. These transitions required less energy than * transition.

For example, alkyl halide, aldehydes, ketones, etc.
3. * Transition: This type of transition generally show in unsaturated molecules like
alkenes, alkynes, aromatics, carbonyl compounds, etc. This transition required less
energy as compared to the * transition.
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RECENT TRENDS IN ANALYTICAL CHEMISTRY 3
n-    n-*  *

Energy
Absorption, intensity shift & UV spectrum

1. Bathochromic shift: It is also known as the Red shift, in this case, absorption shifts
towards a longer wavelength ( max).
2. Hypsochromic shift: It is also known as the Blue shift, in this case, absorption shifts
towards a shorter wavelength ( max).
3. Hyperchromic shift: Intensity of absorption maximum ( max) increases.
4. Hypochromic shift: Intensity of absorption maximum ( max) decreases.
Absorption & Intensity Shift
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UV Spectrum of an Unknown Compound
Chromophores & Auxochromes

1. Chromophores: Chromophore is a covalently unsaturated group that absorbs
electromagnetic radiation in the UV-visible region and imparts color to the compound.
For example, C=C, C C, benzene, NO2, etc.
2. Auxochromes: An auxochrome is a saturated group (containing lone pair of an
electron), when auxochrome attach to the Chromophore, absorption shift towards a
longer wavelength along with an increase in the intensity of absorption. For example, -
OH, -SH, -NH, -NR2, etc.
Factor affecting absorbance & intensity

1. Conjugation
Absorption shifts towards a longer wavelength, if double bonds (chromophore) present in
the molecule are in conjugation. For example,
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In compound 'A', double bonds are in conjugation, therefore 'A' possesses a higher
wavelength ( max) as compared to compound 'B' (non-conjugated derivative).
We know that,
E=
As the conjugation increase, transition energy (E) between the orbitals is decrease, and
therefore wavelength ( max) increase.
2. Effect of solvent
In polar solvent, * transitions shift towards longer wavelength (Red shift), because
dipole interactions with polar solvent molecules lower the energy of excited state ( *) than
that of the ground state ( * orbitals are stabilized by hydrogen bonding with polar solvent).
Whereas in a polar solvent, * transitions shift towards lower wavelength (Blue shift),
because dipole interactions with polar solvent molecules lower the energy of ground state
( ) than that of the excited state ( orbitals are stabilized by hydrogen bonding with polar
solvent).
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Explanation of terminology used in Woodward-Fieser rules

1. Homo-annular and hetero-annular conjugated double bond: if a conjugated double
bond is present in the same ring then it is known as homo-annular when a conjugated
double bond is present in the different ring then it is known as a hetero-annular
conjugated double bond.
2) Exocyclic and endocyclic conjugated double bond: Exocyclic double bonds are
conjugated double bonds formed by any carbon atom of any ring but present outside
the ring. An endocyclic double bond is present inside the ring.
3) Ring residue/alkyl group: Ring residue is a carbon-carbon bond that is not a part of a
conjugated system. It is attached to an anyone carbon atom of a conjugated double bond
system.
4). Extending conjugation: Extra double bond present at the end of the conjugated system
(two double bonds).
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Table. 1: The Woodward-Fieser Rules (Calculation of max (nm) in Conjugated Dienes)
max (nm)
Base value:
Acyclic or heteroannular dienes 214
Homoannular dienes 253
Increments for:
Double bond extending the conjugation 30
R alkyl substituent or ring residue 5
Exocyclic double bond 5
Polar groupings:
-OCOCH3 0
-OR 6
-Cl, -Br 5
-NR2 60
Table. 2: The Woodward-Fieser Rules (Calculation of max (nm) in α,β-Unsaturated Carbonyl

Compounds)
max (nm)
Base value:
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Acyclic or 6-membered cyclic ketone 215

5-membered cyclic ketone 202
Aldehyde 210
Increments for:
Homocyclic diene component 39
Double bond extending the conjugation 30
R alkyl substituent or ring residue
Exocyclic double bond 5
Polar groupings:
-OH
-OR
-Cl,
-Br
-NR2 95
-OCOCH3
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Application of UV spectroscopy
1. UV absorption spectroscopy can be used in qualitative analysis of unknown compounds
with the spectrum of a known compound.
2. Kinetics of reaction rates can be determined using UV-visible spectroscopy. If a
material/reagent of a reaction absorbs radiation in the UV-visible region then we
measure the frequency as a fraction of time.
3. The extent of conjugation: As the double bond increase, absorption shifts towards a
longer wavelength.
4. Study of keto-enol tautomerism:

Carbonyl compounds containing -hydrogen undergo keto-enol tautomerization. UV
spectra show absorption bands which are characteristic of both keto and enol forms. For
example in the case of ethyl acetoacetate (CH3COCH2COOC2H5) in ethanol, a strong band at
245 nm with * transition (due to hydrogen bond) is observed for enol form, and a weak
( *) band at 275 nm is observed in case of keto form.
5. The distinction between conjugated and non-conjugated compounds:

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Both the above compound A & B are isomers of each other. Due to * transition,
compound A appears at a longer wavelength than compound B.
6. Study of geometric isomerism:
In isomer A, due to steric hindrance, absorption shifts towards a lower wavelength. We

know that steric hindrance destabilizes the compound means the energy of the molecule
increase which decreases the wavelength.
7. Used in the detection of impurities: Due to the presence of impurities in the compound,
give additional peaks are observed in the UV spectrum.
REFERENCES
1. Pavia, D. L., Lampman, G.M., Kriz, G.S., Introduction to Spectroscopy, Ed. 3 rd, Thomson books,
Singapore, 2001.
2. Schirmer, R.E., Analytical Chemistry, Ed. 6th, John Wiley and Sons, Inc., Singapore, 2004.
3. J Assoc Arab Univ Basic Appl Sci 2014; 15:53-60.
4. Kalsi, P.S., Spectroscopy of organic compounds, New Age International Publishers, Ed. 6th, New
Delhi, 2004.
5. H. H. Jaffe’ and M. Orchin, Theory and Applications of Ultraviolet Spectroscopy, Wiley, New
York, 1962.
6. R. M. Silverstein, G. C. Bassler, and T.C. Morrill, Spectrometric Identification of Organic
Compounds, 3rd Edition, Wiley, New York, 1974.
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Chapter
BASIC CONCEPTS AND APPLICATIONS OF
2 CONDUCTOMETRIC TITRATIONS
K. VIDYA
Assistant Professor ,Department of Chemistry, JNTUH University College of Engineering,

Nachupally, Kondagattu, Jagtial, Telangana-505501, India
Corresponding author: K. Vidya, Email: vidyaorg@yahoo.com
ABSTRACT
This article describes the basic concepts and applications of conductometric titrations.
Conductometric titration is a basic analytical technique that is very popular due to its fast analysis
and wide range of applications. Conductometric titrations because of their simplicity and numerous
applications. We use these techniques for many purposes in scientific research, medicine, agriculture.
The pharmaceutical industry, environmental protection, clinical diagnostics, food processing,
detection of
INTRODUCTION
Conductivity is the ability to carry current. It is reciprocal of resistivity. Electrolytic
conductivity1 is a measure of the ability of a solution to carry an electric current, Solutions
of electrolytes conduct an electric current by migration of ions under the influence of an
electric field.
According to ohms law, the current I (amperes) flowing in a conductor is directly
proportional to the resistance R (ohms) of the conductor.
I = E/R Where
I= Current strength
E = Potential difference
R= Resistance
Let us consider a cell in which electrodes are l (cm) apart and have A(cm2) as an area of a
cross-section of either electrode. Then resistance (R) offered by the solution taken in this cell
is found to be directly proportional to l and inversely proportional to A.
Where  is constant and depends on the nature of the conductor. It is known as the Specific
resistance of a conductor.
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If l= 1cm and A = 1cm2 the  =R

At a given temperature, the conductivity of an electrolytic solution depends upon the type
of ions present and their concentrations.
Specific Conductance
The inverse of specific resistance is known as specific conductance. It is designated by letter
K (Kappa)
-1 cm-1
× Conductance where l = 1 cm, and A=1cm2
K= C
Specific Conductance or specific conductivity can also be defined as conductance of a one-
centimeter cube of a solution of an electrolyte.
To get the value of specific conductance, the measured conductance has to be multiplied by
a certain factor known as cell constant.
In the above equation is cell constant (i.e by ), Value of specific conductance will be
obtained.
K= Cell constant× measured conductance
To determine cell constant, the KCl solution is taken as the standard solution. The value of
specific conductance of 0.02 N K Cl solutions is 0.002768 at 298K. By knowing the value of
conductance as indicated by Conductivity Bridge, cell constant is calculated as.
If the temperature is not 298 K, then the corresponding value of specific conductance of that
temperature is used in the above relationship.
This cell constant is the ratio of the distance between two electrodes and the area of the
electrode. Its units are cm-1
Equivalent conductance
It is defined as conducting power of all the ions produced by one gram equivalent of an
electrolyte solution placed between two parallel electrodes which are at a unit distance
apart
It is represented by ^
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Equivalent Conductance of a solution is the product of specific conductance of a solution

and the volume in milliliters which contain one gram equivalent of the solute.
^ = Specific conductance (K) ×Volume in ml per gm
Equivalent of the electrolyte
2 eq-1
Molar Conductance
It is defined as conducting power of all the ions produced by one gram mole of an
electrolyte solution placed between two parallel electrodes which are at a unit distance
apart. It is represented by μ
μ = K×V
2 mol-1
Measurement of Conductivity
The electrolytic conductance of an electrolyte is measured in conductivity cells.
Conductivity cell is made of pyrex glass. It has two electrodes with a square shape and is
coated with finely divided platinum wires fused in two glass tubes these tubes are firmly
fixed in the ebonite cover of the conductivity cell so that the distance between the electrodes
remains constant during its experimental measurement. The electrolyte whose conductivity
is to be measured is taken either infused state or in aqueous solution
To measure the conductivity of the solution, the solution is placed in a conductivity cell of
which the cell constant ( ) has been determined by calibration with a solution of accurately
known conductivity. A standard KCl solution is usually employed for this purpose.
Conductometric Titrations
Conductometric titration2 is a type of volumetric titration which is based on the change in
conductance of the solutions at the endpoint or equivalent point during the titration.
Conductometric titrations are based on the fact that the conductance of aqueous solutions
which contain an electrolyte depends on the number of free ions in the solution, charge on
the free ions, and mobility of free ions in the solution.
During the titration of titrant to a titrate the number of free ions in the solution changes, the
identity of ions also changes by which the conductance of electrolytic solution also changes.
When there is a change in conductance the equivalence point is rapid and it can be utilized
for the detection of an endpoint.
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During the ionic reactions, the Conductance may either increase or decrease depending on
the nature of the electrolyte involved. The substitution of ions of one conductivity by the
ions of another conductivity is the basis of conductometric titrations
Let us illustrate with a few specific cases:-
1 Acid-Base Titrations:-
(i) Strong Acid (HCl) and strong base (NaOH) Titration:-
Conductometric titrations help us to identify the equivalence point of strong acid and
strong base titrations easily because the conductance values of hydroxide and hydronium
ions are very high when compared with the conductance of other ions
In a strong acid (HCl) and strong base (NaOH) titration, Strong acid (HCl) is taken in a
beaker, and conductivity cell is dipped in it. After appropriate calibration initial
conductance is measured using a conductivity meter. NaOH solution is taken in a burette
and is gradually added to HCl solution. After each addition of NaOH to the HCl solution,
its conductance is measured. The titration is carried out at a constant temperature and when
we plot conductance against the volume of NaOH. We get a graph as in fig1.
Before the start of titration, we have strong HCl in the beaker which has a high conductance
value which is represented as x in the figure which is due to the high mobility of hydrogen
ions and little contribution from cl-ions once when we start the titration i.e. addition of
NaOH solution from a burette to HCl beaker the highly mobile H+ are replaced by less
mobile Na+ ions which lead to decrease in the conductance values.
H+ Cl- + Na+ OH-→Na+ Cl- + H2O
S. Acid S. Base Salt Water
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The conductance goes on decreasing as we add NaOH from the burette. And at a particular
point y is the equivalence point which contains Na+ and Cl- only and has minimum
conductivity.
After the equivalence point ‘y’ the conductance again increases along yz as we go on adding
NaOH from the burette which is due to the high mobility of OH- ions added.
Hence, we get a v shape graph for titration of a strong acid –strong base. The decrease in
conductance is because of the decrease in H+ ion concentration i.e. along and increase in
conductance is due to an increase in OH- ion concentration along yz
But in actual practice, the lines may be curved slightly because of variation in temperature
due to heat of neutralization and ionic effect
Despite all these, the exact equivalence point can be easily located because the inflection is
sharp enough.
Titration of strong acid (HCl) and Weak Base (NH4OH)
In a strong acid (HCl) and weak Base NH4OH titration, strong acid HCl is taken in a beaker
and a conductivity cell is dipped in it after appropriate calibration, initial conductance
offered by strong acid HCl is measured using a conductivity cell, now gradually add
NH4OH from the burette and conductivity values are taken for each addition of NH4OH.
The titration is carried out at a const temperature and when we plot conductance against
the volume of NH4OH. We get a graph as shown in figure below.
At the start of titration, the acid solution HCl has high conductance 'x' because of the high
mobility of H+ ions and little contribution from chloride ions. Once when we start the
titration i.e. addition of weak base NH4OH from the burette the conductivity decreases
along xy in fig 2 because of the replacement of highly mobile H+ ions by slowly moving
NH4+ ions.
H+ Cl- + NH4+ OH-→NH4+ Cl- + H2O
S. Acid W. Base Saltwater
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This trend is continued till the equivalence point y is reached. The addition of NH4OH after
the equivalence point does not cause any appreciable change in conductance. This is
because NH4OH is poorly ionized and has a very small conductivity compared with that of
the acid or its salt.
Titration of a weak acid (CH3COOH) and strong Base (NaOH)
In a weak acid (CH3COOH) and strong Base (NaOH) titration, weak acid CH3COOH is
taken in a beaker and a conductivity cell is dipped in it. After appropriate calibration, initial
conductance is measured using a conductivity meter. NaOH solution is taken in a burette
and is gradually added to the CH3COOH solution. After each addition is carried out at a
constant temperature and when we plot conductance against the volume of NaOH. We get
a graph as in Figure below
At the start of titration, the conductance offered by CH3COOH is low and decreases owing
to the common ion effect in suppressing the ionization of the acid.
The conductance slightly increases up to the equivalence point. This is due to an increase in
the concentration of highly ionized salt. After the equivalence point, we observe a sudden
increase in OH-ions. The titration curve is shown in Fig3.
Titration of a Mixture of strong Acid (HCl) and Weak Acid (CH3COOH) with a strong
base (NaOH)
In the mixture of acids and strong Base titration, we take an equal amount of strong acid
HCl and weak acid CH3COOH in a beaker, and the conductivity cell is dipped in it. After
appropriate calibration, initial conductance is measured using a conductivity meter. NaOH
is taken in a burette and is gradually added to the mixture of acids in the beaker.
After each addition of NaOH to the HCl solution, its conductance is measured using a
conductivity meter. The titration is carried out at a constant temperature and when we plot
conductance against the volume of NaOH we get a graph as in figure below.
Before we start Titration, strong acid HCl and weak acid CH3COOH are present in the
beaker. But in this case, the strong acid HCl is titrated first because it undergoes complete
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ionization and shows high conductance due to the high mobility of H+ ions we observe a
decrease in conductance values up to point y once when all the Hydrochloric acid has been
completely neutralized by NaOH solution (point y) the next drop of NaOH starts reacting
with CH3COOH present in the mixture. But as acetic acid is a weak acid the rate of
ionization is very less so the conductance offered by it is also very less. But with the further
addition of NaOH, The conductance gradually increases due to an increase in the
concentration of highly ionized salt. Point z corresponds to the equivalence for the
neutralization of CH3COOH
C
O
Eq. point of
N
Strong acid
D
U
C Eq. point of a
T weak acid
A
N
C
VOL. OF BASE
At point 'Z' complete neutralization of the mixture of acids takes place and after the
equivalence point 'z' the conductance again increases along z as we go on adding NaOH
from the burette which is due to the high mobility of OH- ions added. Thus from fig 4, we
can see that it is the combination of curves shown in fig.1 and 3 obtained when a mixture of
strong acid and weak acid is titrated with a strong Base.
APPLICATIONS
1. Conductometric titrations are used to determine the purity of water samples.
2. It is used to examine the salinity of sea water3 and the alkalinity of fresh water and
freshwater bodies.
3. It is proved to be the most cost-effective technique for drug analysis which offers simple,
sensitive, and rapid analytical procedures which involve measurement of conductance of
solution which varies due to interaction between ionic species before and after endpoint.
Here the functional groups present in the selected drugs gets ionized in suitable solvents
resulting in the formation of negatively charged ions i.e bases which are capable of
binding with lewis acids i.e metal ions, where the principle of drug metal ion interaction4
is applied, and in the course of titration the drug species was made to bind to meta
cations which leads to change in conductivities of the solution. It is an eco-friendly
method and is used for routine analysis of selected drugs in quality control laboratories.
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4. It is used in analyzing various biological and chemical pollutants in environmental

matrices. Conductometric enzyme biosensors5,6,7,8,9are based on the thin-film integrated
electrode which is based on the fact that all enzymatic reactions involve either
consumption or production of charged species which lead to change in the ionic
composition of the tested samples which helps in monitoring environmental pollutants.
5. It is used to analyze the conductivities of polymeric materials that can be used as a
membrane in fuel cells10 and water electrolyzers which are energy converters. Polymeric
materials used as a membrane in these devices are with high ionic conductivities.
6. It is used in food microbiology11 for tracing microorganisms.
7. It is used to estimate ash content in sugar juices12.
REFERENCES
1. An introduction to electrochemistry – Samuel Glasstone
2. Vogel AJ. Textbook of quantitative inorganic analysis. 4th edition.
3. Anderson, L. J. (1948). Conductometric Titration of Chloride in Sea Water and Marine Sediments.
Analytical Chemistry, 20(7), 618-619.
4. Alhazmi, H. A., Nasib, A. A. B., Musleh, Y. A., Hijri, K. Q., ur Rehman, Z., Khuwaja, G., ...
&Arbab, I. A. (2020). Application of drug–metal ion interaction principle in conductometric
determination of imatinib, sorafenib, gefitinib, and bosutinib. Open Chemistry, 18(1), 798-807.
5. Jaffrezic-Renault, N., &Dzyadevych, S. V. (2008). Conductometric micro biosensors for
environmental monitoring. Sensors, 8(4), 2569-2588.
6. Dzydevich, S. V., Shul'ga, A. A., Soldatkin, A. P., NyamsiHendji, A. M., Jaffrezic-Renault, N., &
Martelet, C. (1994). Application of conductometric for sensitive detection of pesticides biosensors
based on cholinesterases. Electroanalysis, 6, 752-758.
7. Shul'ga, A. A., Soldatkin, A. P., El'skaya, A. V., Dzyadevich, S. V., Patskovsky, S. V., &Strikha, V. I.
(1994). Thin-film conductometric biosensors for glucose and urea determination. Biosensors and
Bioelectronics, 9(3), 217-223.
8. Watson, L. D., Maynard, P., Cullen, D. C., Sethi, R. S., Brettle, J., & Lowe, C. R. (1987). A
microelectronic conductimetric biosensor. Biosensors, 3(2), 101-115.
9. Dzyadevych, S. V., Arkhypova, V. N., Korpan, Y. I., Anna, V., Soldatkin, A. P., Jaffrezic-Renault,
N., &Martelet, C. (2001). Conductometric formaldehyde sensitive biosensor with specifically
adapted analytical characteristics. Analytica Chimica Acta, 445(1), 47-55.
10. Lavorante, M.J., Franco, J.I. Conductometric titration as a technique to determine variation in
conductivity in perfluorosulfonic acid materials for fuel cells and electrolyzers. Int J Energy Environ
Eng 8, 123–134 (2017). https://doi.org/10.1007/s40095-017-0230-z
11. Ali, A. A., Altemimi, A. B., Alhelfi, N., & Ibrahim, S. A. (2020). Application of biosensors for
detection of pathogenic food bacteria: a review. Biosensors, 10(6), 58.
12. Kolthoff, I. M. (1930). Conductometric titrations. Industrial & Engineering Chemistry Analytical
Edition, 2(3), 225-230.
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Chapter
RECENT CHROMATOGRAPHIC DEVELOPMENTS IN
3 THE SEPARATION OF LEAF PIGMENTS
RAVINDER MANCHAL*
Department of Chemistry, Chaitanya (Deemed to be University), Kishanpura,

Hanamkonda, Telangana 506001.
*Corresponding author: Ravinder Manchal, Email: ravinder@chaitanya.edu.in
ABSTRACT
Plant pigments are coloured materials that have good market potential. The plant pigments mainly
consist of chlorophylls, carotenoids, and xanthophylls. Due to antimicrobial properties and radical
scavenging activities of plant pigments have been beneficially used in many industries like cosmetics,
coloring, and textiles. Many chromatographic methods such as paper, thin-layer, column, and high-
performance liquid chromatography have been developed for the separation of pigments into
individual components. Column chromatography is one of the familiar analytical techniques for the
bulk separation of pigments mixture. Thus, recent developments on this column chromatography are
reviewed in this chapter.
KEYWORDS: Pigments; Separation; Column chromatography; Chlorophylls; Carotenoids.

INTRODUCTION
The organic pigments market is projected to grow from USD 5.5 billion in 2021 to USD 6.7
billion by 2026. Organic pigments are colored materials made of carbon chains with
pigment properties. They are mainly obtained from natural and synthetic sources. Natural
organic pigments are generally derived from either vegetables (plants) or animals. Some of
the natural organic pigments are rose or crimson dyes from madder root, deep red carmine
made from the dried bodies of female wingless scale insects, a dark red resin exuded from
the fruit of the rattan palm, dark blue indigo made from the fermented gray-green leaves
and flowering stalks of over 30 related plant species, and many others.
Photosynthesis in plants occurs in the chloroplasts, which contain a number of colored
pigments and which fall into two categories such as chlorophylls and carotenoids.
Chloroplasts also contain several oxygen- containing derivatives of carotenes called
xanthophylls. The most abundant plant pigments are chlorophyll a and chlorophyll b which
occur in a ratio (a:b) of approximately 3:1. Chlorophyll-a is consists of a methyl group,
whereas chlorophyll b has an aldehyde. The chlorophylls serve as the primary
photosynthetic pigments in the living plant cell. The carotenoids can be classified into two
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different types, (1) the carotenes which contain only C and H, and (2) the xanthophylls
which contain C, H, and O atoms in the form of various functional groups (epoxide or
hydroxyl). The structural information of some important pigments is mentioned in Fig. 1 [1,
2].
Fig. 1: Important Pigments of Red and Green Leafs (Adopted from Ref. 1)
The chlorophyll absorbs light in the red (long wavelength) and the blue (short wavelength)
regions of the visible light spectrum. The chlorophylls a and b are reflected green and thus
these pigments make plants look green. Carotenoids are part of a larger collection of plant-
derived compounds called terpenes, which consist of 40 carbon atoms (tetraterpenes) with
conjugated double bonds of 8 isoprenoid units. Carotenoids absorb light in the blue-green
and violet region and reflect the longer yellow, red, and orange wavelengths [3].
Carotenoids have consist of very efficient physical and chemical quenchers of singlet
oxygen (O2) and act as important natural antioxidants. Moreover, carotenoids are suggested
to play a protective role in a number of disorders, such as i.e, cardiovascular diseases,
several types of cancer or neurological, as well as photosensitive or eye-related disorders
[4].
Plant pigments are important in controlling the photosynthesis, growth, and development
of plants. Pigments act as visible signals to attract insects, birds, and animals for pollination
and seed dispersal. Pigments also protect plants from damage caused by UV and visible
light [5]. Natural and synthetic pigments are widely used as food colorants and dyes in
textile industries. Synthetic pigments have many properties like oxygen stability, excellent
vibrancy, and can be produced at lower costs. Due to these reasons, it had been used in
many formulated foods like confectionaries and baked products [6]. Pigments are also used
in many industries as color stearin candles, leather, waxes and resins, pomades and other
cosmetic preparations, salves, ointments, Vaseline, and to hide the color of mineral oils [7].
Various spectroscopic and chromatographic methods have been most frequently used for
plant pigments measurement because they provide a quick, accurate, and inexpensive
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estimation of various pigments concentrations. However, conventional chromatographic

methods are used for the bulk separation of photosynthetic pigments. The paper, thin-layer,
column, and high-performance liquid chromatography (HPLC) are more commonly used
for the identification and extraction of leaf pigments [8, 9].
Recent developments in chromatographic methods
Amalya et al. reported extraction of carotenoid pigments from the leaves and flowers of
Peltophorum pterocarpum by column chromatography and analysis of their pigments by
Nuclear Magnetic Resonance Spectroscopy and Mass Spectrometry. In this study, the
yellow carotene band is eluted with 100% hexane mobile phase using silica gel as stationary
phase [4].
Latowski and his research group disclosed extraction of carotenoid diatom pigments such
as fucoxanthin, diadinoxanthin, and diatoxanthin from phaeodactylum tricornutum by the
combination of two methods column chromatography followed by HPLC. Initially, the
fucoxanthin is isolated with the help of column chromatography using petroleum ether:
acetone: n-propanol (60:40:3) as mobile phase and neutral calcinated Al2O3 as stationary
phase. After fucoxanthin separation from normal column chromatography, the
diadinoxanthin and diatoxanthin is separated using HPLC columns with a purity of
diadinoxanthin exceeded 99.7% and that of diatoxanthin reached 99.5% [10].
Ranjith et al. identified different pigments including six species of carotenoids, chlorophyll
a, chlorophyll b, and xanthophylls from various plants extracts such as Ocimum sanctum,
Lawsonia inermis, and Piper nigrum using thin-layer chromatography. The highest distance
travelled pigments are carotenoids followed by chlorophyll b in the Ocimum sanctum plant,
which means carotenoids are non-polar in nature as compared with other pigments and
which elute first during column chromatography [11].
Katayama et al. reported a thin-layer chromatography for identification and separation of
various pigments (carotenes, chlorophyll a, chlorophyll b, chlorophyll c, lutein,
violaxanthin, and fucoxanthin) from various algae species including Camellia japonica, a
terrestrial green plant, the frond of Ulva pertusa (green algae), the frond of Sargassum
horneri (brown algae), and Grateloupia filicina (red algae). The mentioned pigments are well
separated using a 60 TLC plastic sheet and 7:3 mixture of petroleum ether: acetone mobile
phase [12].
McDougal and his students developed a column chromatographic method in a green way
to separate the various pigments (β-carotene, xanthophylls, and chlorophyll a) from
Spinach leaf. In this work used hexane and acetone as mobile phase and alumina as
stationary phase, the results from this study are mentioned in Table 1. More importantly,
the used mobile phase (acetone and hexane) were recycled by fractional distillation, and this
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fraction was further used for the next cycle of separation. Moreover, the used alumina in
column chromatography was washed with recycled acetone and was also used for the next
cycle of pigments separation with similar results. The recycle and reuse of mobile and
stationary phase in this reveals minimize waste and this concept is good practice for the
young generation [13].
Table. 1: Average Rf values and observed color for spinach pigment extracts collected by
columnchromatography and identified by TLC
Spinach Pigment Rf value Color
β-Carotene 0.94-0.98 Yellow to orange
Xanthophyll 0.80-0.86 Yellow
Chlorophyll a 0.43-0.48 Blue-green
Limantara and her research group have been reported separation of photosynthetic
pigments by HPLC using different columns. The HPLC method has several advantages
such as speed, high resolution, and sensitivity. The separation of the pigments mainly
depends on the column (stationary phase). Different reverse-phase columns (C8, C18, C18
monolithic, naphtylethyl group bonded silica, and cholester) were studied in this work to
separate the pigments of Pleomele angustifolia leafs at several fixed conditions of mobile
phase and temperature. From the studied columns, the cholester was shown superior
separation even structurally similar pigments (α-carotene and β-carotene). The work helps
in the selection of columns for HPLC analysis of leaf pigments [14].
An eco-friendly procedure was developed by Dias and Ferreira to separate the various plant
pigments including β-carotene, xanthophylls, chlorophylls a and b, and flavonoids from
green and red leaves. In this study introduced ‚supermarket column‛ such as potato starch
for better separation of the above-mentioned pigments using a mixture of petroleum
ether/acetone as mobile phase. The potato starch column initially separated the β- carotene
and xanthophylls in presence of petroleum ether only, further eluted the xanthophylls,
chlorophyll a and b using a mixture of petroleum ether/acetone 90:10 (Fig. 2). The authors
disclosed that the present study (potato starch as a column) is eco-friendly and which helps
broaden the learning experience of this activity and allows teachers to design laboratory
pedagogies for exercising student critical thinking [1].
Recently Ciaccio and Hassan have been reported a simple, rapid, reproducible, and small-
scale method for extraction of photosynthetic pigments using pipet microcolumn
(containing as little amount of silica gel) from dried herbs (spinach, cilantro, parsley, and
dill) that affords a dark-green extract solution. The carotenes are eluted first from the
microcolumn within 20 min by gravity using 9:1 (v/v) heptane: ethyl acetate as eluent;
chlorophylls are then eluted within seconds using air pressure and ethyl acetate as eluent
(Fig. 3). In this study used as such crude dark green pigments mixture, no need for
additional extraction, filtration, drying, or evaporation steps [15].
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Fig. 2: (a) Separation of β-Carotene and Xanthophylls on a Potato Starch Column by

Elution with Petroleum ether;
(b and c) Separation of Xanthophylls, Chlorophyll a and b on the Same Column by
Elution with Mixture ofPetroleum Ether/Acetone 90:10; (d) all the Fractions
Collected from Leaves. (Adopted from Ref. 1)
Fig. 3. (a and b) Separation of Carotenes and Chlorophylls; (c) Column Fractions, Yellow
Carotenes (left) andGreen Chlorophylls (right). (Adopted from Ref. 15)
CONCLUSION
This chapter deals with the recent findings in the separation of leaf pigments using column
chromatography. The important developments are summarized below. The recycle and
reuse of mobile and stationary phase have been disclosed by McDougal et al., this study
reveals minimize waste and this concept is good practice for the graduate students.
Different reverse-phase columns (C8, C18, C18 monolithic, naphtylethyl group bonded
silica, and cholester) were introduced by Limantara and her research group for the
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separation of pigments in HPLC method. An eco-friendly ‚supermarket column‛ such as

potato starch has been introduced by Dias and Ferreira for better separation of pigments
using a mixture of petroleum ether/acetone as a mobile phase. Extraction of photosynthetic
pigments using pipet microcolumn (containing as little amount of silica gel) from crude
dark green pigments mixture is revealed Ciaccio et al. research group. These mentioned
successful stories in this chapter may help graduate/postgraduate students for the better
separation of leaf pigments.
REFERENCES
1. A.M. Dias, M.L.S. Ferreira, Journal of Chemical Education 92 (2014) 189-192.
A. Oren, in: F. Rainey, A. Oren (Eds.), Methods in Microbiology, Academic Press, 2011, pp. 261-282.
2. R.F. Boyer, Biochemical Education 18 (1990) 203-206.
3. J.T.A.J.a.J.H.S. V, Unique Research Journal of Chemistry 02 (03) (2014) 11-17.
4. W.S. Choo, in: L. Melton, F. Shahidi, P. Varelis (Eds.), Encyclopedia of Food Chemistry, Academic
Press,Oxford, 2019, pp. 117-123.
5. S. Muthusamy, S. Udhayabaskar, G.P. Udayakumar, G.B. Kirthikaa, N. Sivarajasekar, Properties
and Applications of Natural Pigments Produced from Different Biological Sources—A Concise
Review, Springer Singapore, Singapore, 2020, pp. 105-119.
6. F.M. Schertz, Industrial & Engineering Chemistry 19 (1927) 1152-1153.
7. Xueyun Hu, Ayumi Tanaka, R. Tanaka, Plant Methods (2013) 9:19.
8. M.H. Anwar, Journal of Chemical Education 40 (1963) 29.
9. W. Tokarek, S. Listwan, J. Pagacz, P. Lesniak, D. Latowski, Acta biochimica Polonica 63 (2016)
443-447.
10. Ranjith N.P, Noorbina E, M.K.S. Veerabhadra Swamy A.L, International Research Journal of Plant
ScienceVol. 12(2) (2021) 1-7.
11. N. Katayama, Y. Kanaizuka, Y. Yokohama, Journal of Biological Education 37 (2003) 186-189.
A. Johnston, J. Scaggs, C. Mallory, A. Haskett, D. Warner, E. Brown, K. Hammond, M.M.
McCormick,
12. O.M. McDougal, Journal of Chemical Education 90 (2013) 796-798.
13. Indriatmoko, Y. Shioi, T.H.P. Brotosudarmo, L. Limantara, Procedia Chemistry 14 (2015) 202-210.
14. J.A. Ciaccio, K. Hassan, Journal of Chemical Education 97 (2020) 2362-2365.
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Chapter
A FACILE SYNTHESIS OF 2-CYCLOPROPYL-3-
4 PYRAZOLO - 1,8-NAPHTHYRIDINE DERIVATIVES
SHASHIKALA KETHIREDDY1, BHASKAR PITTALA2 &

LAXMINARAYANA EPPAKAYALA3*
Geethanjali College of Engineering and Technology, Keesara, Rangareddy-501301

1
2 Nalla Narasimha Reddy Education Society’s Group of Institutions Integrated Campus

(Autonomous), Korremula ‘X’ Road,
Chowdariguda, Ghatkesar, Medchal, Hyderabad. – 500088
3Sreenidhi Institute of Science and Technology (Autonomous) Yamnampet, Ghatkesar,
Hyderabad -501301.
*Corresponding author: Laxminaryana Eppakayala, Email: elxnkits@yahoo.co.in
ABSTRACT
An efficient method was developed to synthesize 2-Cyclopropyl-3-pyrazolo- 1,8-
naphthyridine derivatives through winder bromide reaction. The compounds synthesized
were characterized by IR, NMR and Mass spectral analysis.
INTRODUCTION
Pyrazole is an unsaturated five membered heterocyclic compound with molecular formula
C3H4N2. This aromatic ring comprises 3 carbon atoms and 2 nitrogen hetero atoms at
adjacent positions. The first natural pyrazole was isolated from watermelon seeds. Pyrazole
framework plays an essential role in medicines as well as in dyes (Alessandro B, et al.,2006;
John M F,et al.,2006; Michael G C, et al.,2006; Thomas D P, et al.,2006; Manuela V, et al.,2006;
Abid M & Azam A, et al.,2006). It has been proven to be a fertile source of medicinal agents
such as antibacterial, antifungal, antiviral, anti-inflammatory, antitubercular,
antiandrogenic, antiamoebic, estrogen receptor ligand etc. (Amr G, et al., 2006; Garge H G &
Chandraprakash, et al., 1971; Singh A, et al., 2000; Klimova E I., et al., 1999; Padmavathi V,
et al., 1999) Some of these compounds have also exhibited antidiabetic, analgesic,
anaesthetic and antiparasitic properties. The presence of pyrazole moiety in
pharmacologically potential compounds such as difenamizole (an analgesic), celecoxib (an
anti-inflammatory), amino phenazone (a pyrazalone with analgesic, anti-inflammatory and
antipyretic properties), pyrazofurin (antiviral), fezolamide (the antidepressant agent),
CDPPB (antipsychotic), phenylbutazone (antipyretic and anti-inflammatory mainly used in
Reiter's disease, osteoarthritis, rheumatoid arthritis and spondylitis), rimonabant (anti-
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obesity drug). (Bhaskar reddy D,et al.,1998;Hartfiel U, et al., 1995;Vadim A Soloshonok,et

al.,2005;Reddy K R,et al.,2006;Wu F,et al.,2006; Bensa D,et al.,2004 ;Ooi T,et al., 2005)
Lonazolac and ramifenazone (nonsteroidal anti-inflammatory drugs, NSAID) and betazole
(a H2-receptor agonist) depicts the significance of pyrazole in medicinal chemistry. Dyes
containing pyrazole have also been used as sensitizing agents in colour photography, as
they are found to be luminescent and fluorescent agents. Tartrazine is a synthetic yellow
azo dye containing pyrazole moiety and used as food colour.
Fig. 1: Activity and Examples of the Drugs Containing Pyrazole Moiety
In addition, pyrazoles are also used extensively as useful synthon in organic synthesis. It is
interesting to note that fused bis-pyrazoles are reported as well-known pharmacophores.
The general synthetic procedures used in the preparation of pyrazole derivatives involve
 The cyclo condensation of 1,3- dicarbonyl compounds with hydrazine derivatives.
 The condensation of phenyl hydrazine with ethyl aceto acetate.
 The reaction of nitro pyrimidine with aryl hydrazines.
 Metal catalysed reaction of diols with phenyl hydrazine.
Pyrazoles are not reactive to nucleophiles. But they are reactive to electrophiles, even much
more reactive than benzene, due to the presence of 6 pi electrons. Pyrazoles are not as active
as pyrroles, as the additional nitrogen atom of pyrazole reduces the electron density of
carbon Anti-obesity Ex: Rimonabant Anti-Viral Ex: Pyrazofurin Anti-Cancer Ex: Criotinib
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Anti-inflammatory Ex: Lonazolac, Celecoxib Anti-pyretic Ex: Phenyl butazone Anti-

psychotic Ex: CDPPB atoms of ring. Electrophilic addition takes place on nitrogen atom but
electrophilic substitution at 4th position of the 5 membered ring. Usually, they are resistant
to oxidation and reduction. They undergo catalytic reduction in presence of H2 in palladium
Fig: 2: Structures of the Drugs Containing Pyrazole Moiety
Another significant nitrogen containing heterocyclic compounds are 1,8 Naphthyridines,

which were known to be excellent anti-microbial, anti- convulsant, analgesic, anti-cancer
and anti-viral agents. Apart from these, they show anti-psychotic, anti-depressant, anti-
Alzheimer’s, antioxidant, anti-hypertensive and antimalarial activities. 1,8 Naphthyridines
are also used as CB2 receptor agonists and pesticides. Nalidixic acid (anti-biotic), vorelaxin
(anti-cancer), tosuflaxacin (an anti-bacterial drug used to treat against respiratory, eye and
dental infections), gemfloxacin (anti-bacterial used against pneumonia), enoxacin (anti-
bacterial used to treat against urinary tract infections) are some of the drugs containing 1,8
Naphthyridines scaffold.
Most of the synthetic methods for the preparation of 1,8 naphthyridines, such as, Doebner,
Skraup, Knorr use 2-amino pyridine as the starting material. It was stated in the literature
that, Doebner reaction gives 2-substituted 1,8-naphthyridine-4- carboxylic acids with 2-
amino pyridine and from which 1,8 naphthyridines were obtained upon decarboxylation.
But the reaction needs confirmation, as it may even give rise to o-1,4 a - diazanaphthalene
derivatives having a shared nitrogen atom.
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Fig. 3: Structures of the Drugs Containing 1,8 Naphthyridine Scaffold
In Knorr method, 2,6-diaminopyridine was treated with acetylacetone to give 7-amino2,4-

dimethyl-1,8-naphthyridine.
1,8-naphthyridine derivatives are also synthesized by hetero annulation reaction of
aromatic aldehyde with ene hydrazino ketone and a dimer malononitrile, in the presence of
piperidine. The reaction of 2-aminonicotinaldehyde, Meldrum's acid, and alcohols in the
presence of anhydrous FeCl3 produces 2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxylates
in 56– 80% yield.
The synthesis of 2,3-disubstituted 1,8-naphthyridines proceeds smoothly in water, when the
reaction of the methyl vinyl ketone with 2-aminonicotinaldehyde, in the presence of
Lithium hydroxide (LiOH) as a catalyst. The reaction is called Green Friedländer reaction.
Keeping in view of the importance of both pyrazole and 1,8 Naphthyridine derivatives in
medicinal chemistry, authors explained the best method of synthesis of pyrazolo 1,8
naphthyridine derivatives in this chapter
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Scheme. I R= H, 4-Bromo, 4-Methoxy, 4-Methy, 4-Nitro
RESULTS AND DISCUSSION

2-Cyclopropyl-1,8-naphthyridine-3-carbonitrile reacts with aq. NaOH to obtain 2-
cyclopropyl-1,8-naphthyridine-3-carboxylic acid, which on winder bromide reaction offered
N-methoxy-N-methyl-1,8-naphthyridine-3-carboxamide. Further it on reaction with
DIBALH, gave 2-cyclopropyl-1,8-naphthyridime-3-carbaldehyde, which on reaction with
different aromatic acetophenones provided yielded 2-cyclopropyl 3-(3-phenyl-1H-pyrazol-
5-yl)-18- naphthyridines. All the compounds synthesized were characterized by spectral
analysis.
EXPERIMENTAL SECTION
2-Cyclopropyl-1,8-naphthyridine-3-carboxylic acid (2)
2-Cyclopropyl-1,8-naphthyridine-3-carbonitrile (0.5 g, 2.70 mmol) was dissolved in 10 ml of
aq NaOH solution (5 N), and was heated at 1000C for about 8hrs. The resulting reddish
colour suspension was neutralized with hydrochloric acid (2N) and adjusted the pH to 4-5.
Then precipitated solid was filtered off, dried and recrystallised from ethanol. (400 mg,
Yield: 72.6 %).
IR (KBr) 3427, (OH) cm-1, 1607 (C=O) cm-1;
1H NMR in DMSO-d6 δ = 12.52 (s, 1H, OH), 8.62 (d, 1H, J=10Hz CH), 8.41, (s, 1H, CH), 8.31
(d, 1H, J=12Hz CH), 7.32 (t, 1H, J=12Hz CH), 3.17, (m, 1H, CH), 1.12 (m, 4H, 2CH2).
Mass at m/z 215.3 [M+1]
2-Cyclopropyl-N-methoxy-N-methyl-1,8-naphthyridine-3-carboxamide (3)
To a Solution of 2-cyclopropyl-1,8-naphthyridine-3-carboxylic acid (2), (400 mg, 1.96 mmol)
in DMF (10 mL) EDCI (0.562 g, 2.94 mmol), HOBt ( 0.39 g, 2.94 mmol) and N,O-dimethyl
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hydroxylamine hydrochloride ( 0.22 g, 2.35 m mol) were added at 0oC. To this suspension
NMM (0.79 g, 7.84 mmol) was added slowly at 0oC, then the resulting solution was stirred
for about 4h at RT. After completion of starting material, H2O (10 mL) was added and
extracted with EtOAc (2 X 25 Ml), dried over Na2SO4. The resulting crude compound was
purified by column chromatography by eluting 5-15 % methanol in DCM to obtain the
brown colour solid (250 mg, 51.6 % yield).
IR (KBr) 3108 (CH) cm-1, 1653 (C=N) cm-1, 1121 (C-N) cm-1;
1H NMR in DMSO-d6 δ = 8.68 (d, 1H, J=10Hz CH), 8.51, (s, 1H, CH), 8.33 (d, 1H, J=12Hz
CH), 7.29 (t, 1H, J=12Hz CH), 3.66,(s, 3H, CH3), 2.46,(s, 3H, CH3), 3.16,(m, 1H, CH), 1.14 (m,
4H, 2CH2).
Mass at m/z 258 [M+1].
2-Cyclopropyl-1,8-naphthyridine-3-carbaldehyde (4)
To a solution of 2-cyclopropyl-N-methoxy-N-methyl-1,8-naphthyridine-3-carboxamide (3) (
250 mg, 1.01 mmol) in THF (10 mL) DiBAL-H (1M) (0.43 g, 3.03 mmol) was added dropwise
at -78oC and stirred for about 2h at -78oC. The resulting solution is quenched with saturated
NH4Cl solution (30 mL), filtered, extracted with EtOAc (3 x 30mL), and dried over Na2SO4.
The resulting crude compound was purified by column chromatography by eluting 20-40 %
ethyl acetate in hexane to obtain the light yellow colour solid (130 mg, 68% yield).
IR (KBr) 1658 (C=O) cm -1;
1H NMR in DMSO-d6 δ = 9.82 (s, 1H, CHO), 9.02 (d, 1H, J=10Hz CH), 8.42, (d, 1H, CH),
8.31 (s, 1H, J=12Hz CH), 7.57 (t, 1H, J=12Hz CH), 2.57, (m, 1H, CH), 1.06-1.21 (m, 4H,
2CH2).
MS: [M+1] peak at m/z 199.1.
3-(2-Cyclopropyl-1,8-naphthyridin-3-yl)-1-phenylprop-2-en-1-one (5a)
To a solution of 2-cyclopropyl-1,8-naphthyridine-3-carbaldehyde (4) (100 mg, 0.53 mmol) in
DMF NaOH ( 31.9 mg, 0.79 mmol) and acetophenone (76.3 mg, 0.63 mmol) was added at
0oC and stirred for about 1h. Then water was added and extracted with DCM (2 X 15 mL),
dried over Na2SO4 and evaporated under reduced pressure. The resulting crude compound
was purified by column chromatography by eluting 20-40% ethyl acetate in hexane to
obtain the light yellow colour solid (92 mg, 60 % yield).
IR (KBr) 3057 (CH) cm-1, 1661 (C=O) cm -1;
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1H NMR in DMSO-d6 δ = 9.12 (s, 1H, CH), 8.51-8.61 (m, 4H, J=18Hz 4CH), 8.23, (m, 3H,
3CH), 7.81 (dd, 2H, J=18Hz 2CH), 7.62 (m, 2H, J=14Hz 2CH), 2.61, (m, 1H, CH), 1.12-1.28 (m,
4H, 2CH2),
Mass [M+1] peak at m/z 301.
1-(4-Bromophenyl)-3-(2-cyclopropyl-1,8-naphthyridin-3-yl)prop-2-en-1-one (5b)
IR (KBr) 1632 (C=O) cm -1.
1H NMR in DMSO-d6 δ = 9.22 (s, 1H, CH), 8.69 (s, 1H, 1CH), 8.23, (d, 1H, 1CH), 7.71 (d, 2H,
J=12Hz 2CH), 7.62 (m, 4H, 4CH), 2.52, (m, 1H, CH), 1.14-1.29 (m, 4H, 2CH2),
Mass at m/z 380 [M+1].
3-(2-Cyclopropyl-1,8-naphthyridin-3-yl)-1-(4-methoxyphenyl)prop-2-en-1-one (5c)
1H NMR in DMSO-d6 δ = 9.24 (s, 1H, CH), 8.71 (s, 1H, 1CH), 8.33, (d, 1H, J=12Hz, 1CH), 7.69
(d, 2H, J=12Hz 2CH), 7.62 (m, 4H, 4CH), 2.48, (m, 1H, CH), 1.16-1.27 (m, 4H, 2CH2).
Mass [M+1] peak at m/z 331. IR (KBr) 1642 (C=O) cm -1.
3-(2-Cyclopropyl-1,8-naphthyridin-3-yl)-1-p-tolylprop-2-en-1-one (5d)
(d, 2H, J=14Hz 2CH), 7.63 (m, 4H, 4CH), 1.19-1.28 (m, 4H, 2CH2), 2.53 (m, 1H, CH),
3-(2-Cyclopropyl-1,8-naphthyridin-3-yl)-1-(4-nitrophenyl)prop-2-en-1-one (5e)
(d, 2H, J=14Hz 2CH), 7.63 (m, 4H, 4CH), 1.19-1.28 (m, 4H, 2CH2), 2.53 (m, 1H, CH),
2-Cyclopropyl-3-(3-phenyl-1H-pyrazol-5-yl)-1,8-naphthyridine (6a)
To a solution of 3-(2-cyclopropyl-1,8-naphthyridin-3-yl)-1-phenylprop-2-en-1-one (5a)
(90mg, 0.31mmol) in ethanol (5mL) hydrazine hydrate 99 % (2mL) was added. The resulting
solution was refluxed for about 12hrs. After completion of starting material, evaporated the
ethanol completely under reduced pressure then the title compound was recrystallized
from diethyl ether to obtain the brown colour solid. (40mg, 42% yield).
IR (KBr) 3523 (NH) cm-1 3143 (CH) cm-1, 1579 (C=N) cm-1;
1H NMR in DMSO-d6 δ = 10.81 (s, 1H, NH), 9.11 (s, 1H, CH), 8.42 (m, 2H, 2CH), 7.69 (m, 4H,
4CH), 7.19 (d, 1H, J=10Hz CH), 6.59, (m, 2H, 2CH), 6.31 (m, 1H, J=12Hz CH), 5.41(s, 1H,
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J=12Hz CH), 5.21(s, 1H, J=12Hz CH), 2.21,(m, 1H, CH), 1.22 (m, 2H, CH2), 1.16(m, 2H, CH2).
MS at m/z 313 [M+1].
3-(3-(4-Bromophenyl)-1H-pyrazol-5-yl)-2-cyclopropyl-1,8-naphthyridine (6b)
IR (KBr) 3288 (NH) cm -1.
1H NMR in DMSO-d6 δ = 10.69 (s, 1H, NH), 8.39 (d, 1H, CH), 8.19, (m, 2H, 2CH), 1.21(m,
2H, CH2), 1.28 (m, 2H, CH2), 2.37,(m, 1H, CH), 6.21(s, 1H, J=12Hz CH), 7.32(s, 1H, CH), 7.41
(m, 4H, J=12Hz 4CH),
MS: [M+2] at m/z 392.
2-Cyclopropyl-3-(3-(4-methoxyphenyl)-1H-pyrazol-5-yl)-1,8-naphthyridine (6c)
IR (KBr) 3288 (NH) cm -1.
2H, CH2), 1.28 (m, 2H, CH2), 2.37, (m, 1H, CH), 6.21(s, 1H, J=12Hz CH), 7.32(s, 1H, CH), 7.41
(m, 4H, J=12Hz 4CH),
MS: [M+2] at m/z 392.
2-Cyclopropyl-3-(3-p-tolyl-1H-pyrazol-5-yl)-1,8-naphthyridine (6d)
IR (KBr) 3288 (NH) cm -1.
2H, CH2), 1.28 (m, 2H, CH2), 2.37, (m, 1H, CH), 6.21(s, 1H, J=12Hz CH), 7.32(s, 1H, CH), 7.41
(m, 4H, J=12Hz 4CH),
MS: [M+2] at m/z 392.
2-Cyclopropyl-3-(3-(4-nitrophenyl)-1H-pyrazol-5-yl)-1,8-naphthyridine (6e)
IR (KBr) 3247 (NH) cm-1.
1H NMR in DMSO-d6 δ = 11.19 (s, 1H, NH), 8.24 (d, 1H, J=12Hz, CH), 8.12, (m, 2H, 2CH),
7.27 (m, 4H, 4CH), 7.15(s, 1H, CH), 6.18 (s, 1H, CH), 2.20,(m, 1H, CH), 1.31 (m, 2H, CH2),
1.18 (m, 2H, CH2), MS at m/z 358 [M+1].
CONCLUSION
In conclusion, we have synthesized 2-cyclopropyl 3-(3-phenyl-1H-pyrazol-5-yl)-18-
naphthyridines using commercially available intermediates. All the compounds synthesized
were characterized by spectral analysis. The yield of the compounds was high and the
compounds obtained purity was 80-90%. The conversion of carboxylic acid into aldehyde
was carried out by Winder bromide reaction.
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REFERENCES
1. Abid M & Azam A,(2006) Bioorg Med Chem Lett, 16(10), 2812
2. Alessandro B, Maria A, Mauro M, Mariangela M, Maria B, Luciano O & Franco D,(2006), Bioorg
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Constantieux T and Rodriguez J, (2004), Synthesis, 923.
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8. John M F, Joseph C, Joseph B J, Karen A R, Robert K M, Joseph M L, Pancras C W, Stephen A B &
Ruth R W,(2006), Bioorg Med Chem Lett, 16, 3755.
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13. Ooi T, Ohara D, Fukumoto K and Maruoka K,(2005), Org. Lett., 7, 3195 Padmavathi V, Sumathi R
P, Chandrasekhar B N & Bhaskarreddy D,(1999), J Chem Research, 610.
14. Reddy K R and Kumar N S, (2006), Synlett, 6,224.
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16. Thomas D P, Albert K, Barbara B C, Mark A R, Mark L B, Yaping W, Tiffany D V, Wayne E, Mary
B F & Sandra K F,(2006), Bioorg Med Chem Lett, 16, 3156
17. Vadim A Soloshonok, Chaozhong Cai, Takeshi Yamada, Hisanori Ueki, Yasufumi Ohfune and
Victor J. Hruby ,(2005), J. Am. Chem. Soc., 43,127.
18. Wu F, Li L, Hong R and Deng L, (2006), Angew. Chem. Int. Ed. 45,947.
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Chapter GROWTH, SURFACE, THERMAL AND MECHANICAL

5 PROPERTIES OF NONLINEAR OPTICAL POTASSIUM
THIOUREA CHLORIDE
DR. D. NAGARAJU*
*Department of Physics, P.G. Centre, Lal Bahadur College, Warangal - 506 007, India
*Corresponding author: Dr. D. Nagaraju, Email: nagaraju.1512@gmail.com
ABSTRACT
Potassium thiourea chloride (PTC) is a nonlinear semi-inorganic material. The fine quality
of the crystals was grown in a period of 15 days, with an average growth rate of <100> is 0.2
mm/day. Most of the as-grown faces of the PTC reveal typical striations, conforming to the
2D growth mechanism of the crystals. The crystal is optically transparent in the entire
visible region with 75%. Thermal analysis of the crystal shows the melting point of the
crystals is 228 oC For the first time, load-independent hardness, reverse indentation size
effect, and yield strength of the crystal were estimated to assess the mechanical strength of
the crystal. Further, the anisotropy in hardness was studied using Knoop indentation
studies.
KEYWORDS: Crystal growth; Surface; Optical; Mechanical
INTRODUCTION
Nonlinear optical (NLO) single crystals have gained huge importance because of their
potential application in electro-optic and optoelectronic devices, frequency mixing, etc.
During the last few years, many researchers have tried to develop many new NLO
materials for the above applications. Thiourea is one of the simple organic compounds
having high crystallographic symmetry. Because of its large dipole moment and the
formation of an extensive network of hydrogen bonds in its molecule, it can be used as an
inorganic matrix modifier [1]. It is also known that metals with d10 configuration like zinc,
cadmium, mercury readily combine with thiourea resulting in stable compounds with high
optical nonlinearity [2], good physicochemical behaviour, and hence have applications in
electro-optics and piezoelectricity. Further, thiourea, which is Centro symmetric yields
excellent non- Centro symmetric materials when it is incorporated into the respective
inorganic salts [3]. Because of these properties, thiourea complexes have attracted
significant attention. A variety of crystals of this class have been studied recently by several
researchers viz. zinc tris thiourea sulfate (ZTS) [4], bis thiourea cadmium chloride (BTCC)
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[5], bis thiourea zinc chloride (BTZC) [6], bis thiourea bismuth chloride (BTBC) [7],
potassium thiourea bromide (PTB) [8] and bis thiourea cadmium formate (BTCF) [9]. In the
present study, we have initiated the growth of potassium thiourea chloride single crystal.
Although there are some reports available on growth and optical studies of these crystals
[10], systematic hardness study is
lacking. Given this, we have emphasized the results of surface, thermal microhardness, and
anisotropy of the PTC crystal in the present communication.
EXPERIMENTAL SYNTHESIS
PTC was synthesized by dissolving thiourea and potassium chloride in the ratio of 4:1[10].
The chemical reaction is as follows
KCl + 4[CS (NH2) 2] →K[CS (NH2)2]4 Cl
The reactants were thoroughly dissolved in double distilled water and stirred well using a
temperature-controlled magnetic stirrer to yield a homogenous mixture of solution. The
saturated solution was prepared at 35oC then it was allowed for slow evaporation. After the
period of 15 days, good crystals were obtained with the size of 10×6×3 mm3. The average
growth rate along <100> is 0.2 mm/day. Figure 1 shows the photographs of PTC crystals.
Fig. 1: As-Grown PTC Crystals
CONFIRMATION STUDIES
The crystal structure and physicochemical properties were studied by powder X-ray
diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) to confirm the
formation of the grown crystal PTC. The observed results of powder XRD (PW1830 Philips
analytical powder X-ray diffractometer) indicate that the crystal belongs to a tetragonal
crystal system and the determining unit cell parameters are a=20.4458 Å, b=20.4552 Å
c=9.0021 Å, and α=β=γ=90o volume =3764.88 Å3. The functional groups of the synthesized
compound have been estimated from the FTIR analysis using the KBr pellet technique in the
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range from 400 cm-1 to 4000 cm-1. To come to this decision, a Perkin-Elmer BX I system
spectrophotometer was used. These results are in good agreement with the earlier report
[10].
RESULTS AND DISCUSSIONS
SURFACE STUDIES
In order to understand the growth mechanism, the as-grown faces of PTC crystals are
observed under a transmission microscope (Magnus MLX). It is observed that most of the
faces of these crystals reveal typical striations (Figure 2) which are parallel to the shorter
edge of the crystal. Sangwal [11] pointed out that the cause of the striations could be due to
fluctuations in growth conditions such as sudden changes in temperature, cooling rate,
convection of the solution or melt and concluded that these features are mostly due to two-
dimensional growth mechanism. Prasad [12] attributed different types of striations
observed on FeS2 crystals to two-dimensional (2D) growth nucleation. In the present
studies, we have not observed any growth spirals except striations. Hence, under the
present conditions, it appears reasonable to attribute the growth mechanism of PTC crystals
to a two-dimensional growth mechanism
Fig. 2: Sriations in the PTC Single Crystal Grown from

Aqueous Solutions.
OPTICAL STUDIES
The optical property of the PTC compound has been assessed by using a Perkin-Elmer
Lambda spectrometer in the range of 200 - 650 nm. It was observed from the spectrum
(Figure 3) that PTC has a wide optical transmission window. It has a good optical
transparency of 75% and the lower cut-off wavelength of the crystal is found to be 220 nm.
From this study, it is clear that PTC crystals have reasonably good transmittance, without
any remarkable absorption peaks in the entire visible region. Hence, the large transmission
window along with a low UV cut-off wavelength suggests their suitability for SHG devices
or other optical applications in the blue region [9].
<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<
<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<
Fig. 3: UV-Vis. Spectrum for PTC.
THERMAL ANALYSIS
DSC studies have been carried out using Mettler Toledo (DSC 822e) in the temperature
range 25 to 500 oC at a heating rate of 100 C/min in the N2 atmosphere. The DSC curve
(Figure 4) indicates that PTC is thermally stable up to 180 oC. A broad endothermic peak at
228o C may be attributed to the melting point of the PTC crystal. The absence of peaks
before the melting point indicates better chemical stability of the samples. Further, a broad
endotherm indicates a slow change in heat capacity.
Fig. 4: DSC Curve of PTC Crystal

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<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<
MICROHARDNESS
The mechanical strength of the crystals is usually assessed by microhardness is a measure of
the resistance offered by the crystal to local deformation caused by indentation. It is well
known that the hardness of solids depends on the applied indentation test load, termed as
Indentation Size Effect (ISE). Such dependence of hardness can observe with normal ISE
and reverse ISE. In the case of normal ISE, the hardness value initially decreases with the
load then attains load-independent whereas in reverse ISE, increases with the load and
attains constant. To study such variation of hardness on (100) face of crystals a Leitz-
Wetzler microhardness tester was employed. The Vickers number (Hv) was calculated using
the relation.
Hv = 1854.4 (P/d2) -----------1
where P is the load applied on the indenter in g and d is the mean diagonal length of the
square impression formed on the crystal surface in µm. The behaviour in the load variation
of hardness on (100) face of PTC crystal is depicted in figure 5a. From the figure, it is
observed that the hardness value increases with load up to 35 g and with further increase in
load, almost reaches a load-independent hardness value of 77 kg/mm2. It is worthwhile to
mention that most of the crystals show cracks at low loads (P = 25 g), which suggests that
these crystals are brittle and plastic deformation occurs even at low loads. Further, the
initial increase in hardness with applied load shows the reverse ISE of these crystals. A
similar trend in load variation of hardness was observed in ammonium sulphate [13],
bismuth telluride [14], sulphanilic acid crystals [15], and most of the recently studied
organic and semi-organic NLO crystals.
The load variation can also be interpreted by using Meyer's law,
P = Adn -----------------2
Where A is a constant and n is Meyer's index (or work hardening co-efficient). In the case of
normal ISE behaviour n<2 and for reverse ISE n>2, when n = 2 the hardness is independent
of applied load, which is given by Kick’s law. The plot (figure 5b) of a linear regression
between ln p vs ln d gives the n value of 2.53 shows the reverse ISE behaviour of the grown
crystals. According to the concept of Onitsch [16] and Hanneman [17], the value of n is 1-1.6
for hard materials and more than 1.6 for soft ones. Hence, the n value for the PTC crystal
suggests that it belongs to the soft material category. Further, from the hardness values, the
yield strength (y) can be calculated [18] using the relation
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<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<
and if n < 2, the eq. (3) is reduced to σy = Hv/3 [19]. The estimated value of σy for PTC
crystals is 46.86MPa.
Fig. 5: a) Variation of Vickers Hardness with Load and b)

Plot of ln P Against ln d.
KNOOP HARDNESS
The Vickers microhardness measurements are not so sensitive to studying the anisotropic
nature of hardness in crystals. Hence, indentation studies were also carried out using
Knoop indenter to understand the anisotropic nature of conventional grown PTC crystal.
The Knoop hardness Hk was calculated using the expression
Hk = 14230(P/a2) -----------4
where Hk is
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<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<
in kg/mm2 and a is the longer indentation diagonal length in µm. These studies have been
made by mounting the samples on a laboratory-made mini-circular stage with which the
angular position could be determined with an accuracy of 0.5o. Measurements were made at
various orientations of θ ranging from 0 to 180o, where θ is the angle made by the long axis
of the Knoop indenter with the longer edge of the crystal. Further, measurements were
made at P = 50 g, which gives a Knoop impression of reasonable dimensions (Figure 6a).
Figure 6b shows the Knoop hardness anisotropy on PTC crystals. No distortion in the shape
of the indentation has been observed with crystal orientation. From this plot, it is interesting
to note that the hardness value reaches a maximum (86 kg/mm2) and minimum value (65
kg/mm2) for crystal orientation. The crystal structure and slip system play an important role
in the observed variation of hardness with crystal orientation [20]. The variation of the size
of the impression with orientation appears to depend on the participation of the slip system
at different angles. The larger impressions appear when the resolved shear stress is
sufficiently high and smaller impressions when it is low. These studies suggest the
anisotropic nature of the PTC crystal face. Similar observations were made on a variety of
crystals viz. lanthanum hexaboride [21], rubidium halides [22], ZTS [23], L-arginine
hydrochloride monohydrate, and L-arginine hydrobromide monohydrate [24].
Fig. 6: (a) Knoop Impression on the (100) face of PTC and (b) Variation of
Knoop Hardness with Indenter Orientation.
CONCLUSIONS
Good quality crystals of PTC were grown at room temperature. The average growth rate
along <100> is 0.2 mm/day. XRD and FTIR studies confirm the formation of the grown
crystal. The observation of striations indicates that these crystals are grown by a 2D growth
process, particularly by a layer growth mechanism. The transmittance percentage of PTC
crystal was found to be 75 %. The sample is stable up to 180 oC Further, the melting point of
the crystal is 228 oC Vickers hardness studies illustrate that the Hv value increases initially
up to a load of 35 g and becomes almost load independent (77 Kg/mm2) exhibiting the
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<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<
reverse indentation size effect. The yield strength of the material is 46.86 MPa. The
calculated n value indicates that PTC belongs to the soft material category. The anisotropy
in hardness is observed with the Hk value reaching a maximum of 86 kg/mm2 and a
minimum of 65 kg/mm2.
REFERENCES
1. Hellwege KH, Hellwege AM, Landolt-Bornstein Group II, 1982; 1: 584.
2. Venkataramanan V, Srinivasan MR, Bhat HL, J. Raman Spectroscopy, 1994; 25: 805-811.
3. Kannan V, Rajesh NP, Bairava Ganesh R, Ramasamy P, J. Cryst. Growth, 2004; 269: 565-569.
4. Venkataramanan V, Dhanaraj G, Wadhawan VK, Sherwood JN, Bhat HL, J. Cryst. Growth, 1995;
154: 92-97.
5. Ushasree PM, Muralidharan R, Jayavel R, Ramasamy P, J. Cryst. Growth, 2000; 218: 365-371.
6. Angeli Mary PA and Dhanuskodi S, Cryst. Res. Technol. 2001; 36: 1231-1237.
7. Sankar R, Raghavan CM, Jayavel R, Cryst. Growth and Design, 2007; 7: 501-505.
8. Marcy HO, Warren LF, Webb MS, Ebbers CA, Velsko SP, Kennedy GC, Catella GC, Appl.
Opt.,1992; 31:5051-5060.
9. Selvakumar S, Ravi Kumar SM, Rajarajan K, Joseph Arul Pragasam A, Rajasekar SA,
Thamizharasan K,Sagayaraj P, Cryst. Growth and Design, 2006; 6: 2607-2610.
10. Selvaraju K, Valluvan R, Kumararaman S, Material Lett. 2007; 61: 751-753.
11. Sangwal K, Prog. Cryst. Growth Charact.1989; 19: 237-239.
12. Prasad PBV, Cryst. Res. Technol. 1986; 21: K68-K70.
13. Susmita Karan, Sen Gupta S, Sen Gupta SP, Mater. Chem. Phys. 2001; 69: 143.
14. Augustine S, Mathai E, Mater. Char. 2004; 52: 253-262.
15. Mythili P, Kanagasekaran T, Gopalakrishnan R, Cryst. Res. Technol. 2007; 42: 791-799.
16. Onitsch EM, Mikroskopia. 1947; 2: 131-151.
17. Hanneman M, Metallurgia Manchu. 1941; 23: 135-139.
18. Chaoon JP, Broughton WH, Katzuk AR, Metall. Trans.1971; 2: 1979-1984.
19. Wyatt DH, Metals, Ceramics and Polymers, Cambridge University Press, London, 1974.
20. Susmita Karan and Sen Gupta SP, Mater. Sci. Eng. 2005; A398:198-203.
21. Li H, Bradt R, Mater. Sci. Eng.1991; A142: 51-61.
22. Thermal Rao T, Sirdeshmukh DB, Cryst. Res. Technol. 1991; 26: K53-K59.
23. Venkataramanan V, Dhanaraj G, Wadhawan VK, Sherwood JN, Bhat HL, J. Cryst. Growth, 1995;
154: 92- 97
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Chapter
ROLE OF INFRARED (IR) SPECTROSCOPY IN
6 ANALYSIS OF ORGANIC COMPOUNDS
RANVIJAY PRATAP SINGH*
Department of Applied Science & Humanities, Faculty of Engineering

& Technology, University of Lucknow-226031 (U.P.), India
*Corresponding author: Ranvijay Pratap Singh, Email: rsingh0777@gmail.com
ABSTRACT
This chapter aims to introduce readers to the role of Infrared (IR) spectroscopy in the analysis of
organic compounds. IR spectroscopy is used in the identification of functional groups present in
organic compounds as well as the purity of the compound. This chapter provides an overview of the
fundamental theories of Infrared spectroscopy. These provide useful information about the
applications of this technique for chemical analysis. The most common factors which affect
vibrational frequency and applications of Infrared spectroscopy are also briefly discussed.
KEYWORDS: IR spectroscopy, Hooke’s law, Fermi resonance, Fingerprint region, Fundamental

vibrations.
INTRODUCTION
Spectroscopy is a branch of science that deals with the study of the interaction of
electromagnetic radiations with the matter. Electromagnetic spectrum ranges all types of
electromagnetic radiations. When electromagnetic radiation interacts they give rise to
electronic transition in UV, molecular vibration in IR, and nuclear spin orientation in NMR.
1. Infrared spectroscopy is used in the identification of functional groups in pure
compounds.
2. Infra-red (IR) does not have sufficient energy to induce electronic transition as seen in
UV spectroscopy. When a molecule absorbed electromagnetic radiation in the IR region,
undergoes vibrational or a rotational transition which causes a net change in the dipole
moment in the molecule (IR active, for example, HCl, CO, etc), if the dipole moment does
not change in molecules then they are IR inactive (for example O2, H2, N2, etc.) means
they do not absorb IR radiation. IR region ranges from 4000-400 cm-1.
3. If the frequency of IR radiation matched the vibrational frequency of the molecule, then
the molecule absorbs radiation.
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<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<
4. IR spectroscopy is based on Hooke’s law, suppose two atoms or masses are connected
through spring(bond), then the frequency of vibration can be represented by the
following equation
FUNDAMENTAL VIBRATIONS
These vibrations are arising when the molecule is promoted from the ground state to the
lower excited state. The fundamental vibrations for linear and non-linear molecules are
determined by following way:
Molecule Degree of freedom
Linear 3n-5
Non-linear 3n-6
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<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<
Where 'n' is the number of atoms present in the molecule

The vibrations discuss below are fundamental vibrations.
Stretching vibration: Distance between two atom increase and decreases but bond angle
remains constant.
Types of stretching vibrations
Symmetric stretching vibration: In this case, both the atoms stretched or compressed in the
same direction.
Asymmetric stretching vibration: In this vibration one atom undergoes stretching and
another atom undergoes compression and vice versa.
Bending vibrations: Distance between two atoms remains constant but the bond angle
changes.
These vibrations can occur either in-plane or out of a plane. Types of bending vibrations
In-plane bending vibrations:
7. Scissoring: both atoms move towards each other just like a scissor.
8. Rocking: both the atoms move in the same direction, either on the left side or right side.
Out of plane bending vibrations:

9. Wagging: both the atom moves up and down with respect to the central atom.
10. Twisting: one
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<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<
OVERTONES AND COMBINATION BANDS

When molecule absorbed electromagnetic radiation in IR region, and then molecule
promoted from ground state to second, third or even fourth vibrational excited state. These
bands are known as Overtones. The intensity of these bands is very weak. It is helpful in the
characterization of aromatic compounds.
When two fundamental vibrational frequencies (ν1 + ν2) in a molecule couple to give rise
to a new vibrational frequency within the molecule, it is known as a combination band.
COUPLED VIBRATIONS
The coupled vibrations are observed in a group like –CH2, NH2, etc. In these groups, the
same atoms are attached to the central atom. When –CH2 undergoes vibration by the
absorption of IR radiation, due to internal perturbation, the energy of one C-H bond is
transferred to the neighbouring C-H bond which enhances its vibrational frequency.
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<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<
Therefore two stretching vibrational frequencies for the –CH2 group are observed at 2950
cm-1 (asymmetric stretching) and 2860 cm-1 (symmetric stretching).
FERMI RESONANCE
When fundamental vibration is coupled with overtones or a combination band, the coupled
vibration is called Fermi resonance or when a molecule absorbs IR radiation then it transfers
its energy or intensity from fundamental vibration to overtones, then Fermi resonance is
observed. As we know that the intensity of the overtones band is very weak as compared to
fundamental vibrations. But, due to the transfer of energy, the strong band is observed for
overtones along with the fundamental frequency. Fermi resonance is generally observed in
carbonyl groups. For example, in benzoyl chloride –C=O stretching vibration observed at
1790 cm-1 and 1745 cm-1. The lower frequency band at 1745 cm-1 is observed due to the
combination of overtones of CH bending vibration at 875 cm-1 with the fundamental
vibration of C=O stretching.
FINGERPRINT REGION
The region from 1500-600 cm-1 in the IR spectrum is known as the Fingerprint region. In
this region number of bending vibrations is more than the number of stretching vibrations
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<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<
The number of molecules contains the same functional group & shows a similar peak above
1500 cm-1 but they show a different peak in the fingerprint region. Therefore we can say
that every molecule has a unique peak or band which is observed in the fingerprint region,
it is just like the fingerprint of a human.
Table 1: Some Important IR Absorption Bands
Types of vibration Frequency (cm-1)
Alkane C-C stretching 1200
C-H stretching 3000-2840
CH2 bending 1465
-CH3 bending 1375
CH2 rocking 720
Alkenes C=C stretching 1650
=C-H stretching 3095-3010
=C-H bending 1000-650
Alkynes C C stretching 2100

C-H stretching 3300
C-H bending 700-600
Aromatic C=C stretching 1600, 1500, 1450

=C-H stretching 3040-3010
=C-H bending below 900
C=O stretching Amide 1680
Carboxylic acid 1710
Ketone 1715
Aldehyde 1725
Ester 1735
Acid chloride 1800
Anhydride 1760 (I), 1810
(II) C-O Stretching 1300-1000
O-H Alcohol, phenol 3600
Free 3400-3200
H-bonded 3400-2400
Carboxylic acid
Amine N-H stretching 3440 (as), 3350 (s)
-N-H bending 1650-1580
C-N stretching 1350-1000
Amide N-H stretching 3370 (as), 3150 (s)
-N-H bending 1650-1560
Nitriles C N 2250
Imines C=N 1690-1640
<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<
<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<
Nitro group (NO2) N=O 1550 (as), 1350 (s) Alkyl halide
(C-X)
C-F 1350
C-Cl 750
Factor affecting the vibrational frequency

a) Conjugation: As the conjugation increase, stretching frequency decreases, because force
content decrease due to conjugation.
b) Inductive effect and resonance effect

Oxygen is more electronegative than nitrogen, therefore nitrogen easily donates electrons or
lone pair of nitrogen undergoes delocalization with a C=O bond. Due to delocalization
double bond of C=O changes into a partial double bond therefore force constant decreases
which decreases the C=O stretching frequency.
c) Hydrogen bonding
Intermolecular hydrogen bonding weakens the O-H bond, thereby shifting the band to a
lower frequency. For example, in neat solution O-H stretching vibration of phenol was
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<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<
observed in the range from 3400-3300 cm-1. When a solution is dilute then O-H frequency-
shifted towards a higher frequency at 3600 cm-1.
Whereas in the case of methyl salicylate, intramolecular hydrogen bonding lowers the
stretching frequency of O-H at 3200 cm-1. Intramolecular hydrogen bonding does not
change its frequency even in a very dilute solution because upon dilution structure of the
compound does not change.
d) Ring strain: As the size of the ring decrease, the vibrational frequency of C=O increases.
For example
Application of IR Spectroscopy
a) Identification of different functional groups: As organic compounds possess different
functional groups having different force constant and reduce mass, therefore they absorb
characteristic frequency in the IR spectrum.
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<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<
b) Distinction between intermolecular and intramolecular hydrogen bonding:

Absorption band increase as concentration increase due to intermolecular hydrogen
bonding increase whereas intramolecular hydrogen bonding remains unchanged.
c) Study of keto-enol tautomerism: IR spectra show absorption bands that are
characteristic of both keto and enol forms. for example in the case of ethyl acetoacetate
(CH3COCH2COOC2H5) in ethanol, a band at approx. 3300 cm-1 and 1645 cm-1 for
O-H and C=O stretching frequency respectively observed due to hydrogen bond for
enol form whereas in keto form C=O stretching frequency for carbonyl and ester group is
observed at 1710 cm-1 and 1733 cm-1 respectively.
d) Identification of purity of the compound: If the compound is impure then additional

peaks are observed in the IR spectrum.
e) Identification of geometrical isomers (cis-trans): When molecules absorbed
electromagnetic radiation in the IR region, undergoes vibrational or rotational transitions
which cause a net change in the dipole moment in the molecule, are IR active. In the case
of disubstituted alkene, cis isomer exhibit a C=C absorption peak at 1645 cm-1 whereas
trans isomer does not show C=C absorption peak. This is due to the cis isomer having a
non-zero dipole moment whereas the Trans isomer has zero dipole moment.
f) Study of chemical reaction: As in the following reaction, in cyclohexanone C=O

stretching frequency observed at 1710 cm-1 when this undergoes reduction,
cyclohexanol form in which O- H frequency observed at 3300 cm-1 in IR spectrum,
confirms the reduction of C=O group into O- H group.
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<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<
g) Identification of unknown compound: In this case fingerprint region is used to identify

the functional unknown compound with the spectrum of the known compound.
REFERENCES
1. Pavia, D. L., Lampman, G.M., Kriz, G.S., Introduction to Spectroscopy, Ed. 3 rd., Thomson books,
Singapore, 2001.
2. Schirmer, R.E., Analytical Chemistry, Ed. 6th, John Wiley and Sons, Inc., Singapore, 2004. [3] J
Assoc Arab Univ Basic Appl Sci 2014; 15:53-60.
3. Kalsi, P.S., Spectroscopy of organic compounds, New Age International Publishers, Ed. 6th, New
Delhi, 2004.
4. H. H. Jaffe’ and M. Orchin, Theory and Applications of Ultraviolet Spectroscopy, Wiley, New
York, 1962.
5. R. M. Silverstein, G. C. Bassler, and T.C. Morrill, Spectrometric Identification of Organic

Compounds, 3rd. Edition, Wiley, New York, 1974.
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Chapter
7 SEPARATION METHODS: CHROMATOGRAPHY
SRIDEVI SUNKARI
* University Arts and Science College, subedari

Kakatiya University, Hanamkonda, India
*Corresponding author: Sridevi Sunkari, Email: sridevisunkari9@gmail.com
ABSTRACT
Chromatography is now about 116 years since (1903, 1906) M. S. T Sweet (1906) described his classical
experiment on the separation of coloured pigments of leaves and termed the process
chromatography. Although earlier experiments by D. T. Day on changes in the composition of crude
petroleum when in contact with rocks displaying adsorptive activity can now be defined as
chromatographic experiments. In 1941 martin and synge introduced partition and paper
chromatography. During the next decade the routine use of chromatography as a separation
technique became universal and extended to several areas like chemistry, biology and medicine. A
part from use in analysis, it is now widely used in the preparation of very pure compounds in
pharmaceutical industry.
Chromatography is a physical method of separating mixtures of organic compounds, amino acids,
sugars organic and inorganic salts etc by distribution or partition between mobile and stationary
phases. These two phases are in contact with each other and the mobile phase moves through the
stationary phase. The separation is based on the difference in the partition or distribution coefficients.
The compounds (or) ions in the mixture do not undergo any chemical changes during
chromatographic separation.
KEY WORDS: Column chromatography, Stationary phase, Mobile phase, Adsorbent,

Solvent.
INTRODUCTION
STATIONARY PHASE: A phase doesn’t move. The stationary phase may be solid (or)
liquid.
MOBILE PHASE: A phase that moves. A phase may be liquid (or) gas.
CLASSIFICATION OF CHROMATOGRAHY: Chromatographic methods are classified
either based on the Stationary phase (or) Mobile phase.
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Classification based on the stationary phase

No. Stationary phase Mobile phase Name of the technique
Column chromatography
Liquid Thin- layer chromatography

Solid
1. Ion-exchange chromatography
Gas Gas chromatography
Solid matrix Liquid Gel Permeation Chromatography
Paper chromatography
Liquid
2. Liquid High performance liquid chromatography
Gas Gas liquid chromatography
1. ADSORPTION CHROMATOGRAPHY: The techniques that use a solid as the

stationary phase are called adsorption chromatography .It is based on the principle that
different strength of adsorption chromatography to the stationary phase.
Adsorbent (solid) Ex: Silica gel, Alumina, Charcoal, Magnesium Silicate, Sucrose etc.
2. PARTITION CHROMATOGRAPHY: The techniques that use a liquid as the stationary
phase are called partition chromatography It is based on the principle that different
compounds in the mixture have different distribution coefficients values in a two phase
system: Corasil, Isoctane.Isoprpanol.
Column chromatography: Column chromatography is a widely used technique for the
quantitative separation of complex mixtures of organic compounds (GRAM-KILOGRAM).It
is mainly used in industry for separation and purification of reaction products.
In column chromatography, is finely divided porous solid. Such as Silica gel or Alumina et.
al. Which constitute the stationary phase are packed into a glass tube commonly called the
column. Now, clamp the column vertically and carefully. A concentrated solution of the
mixture prepare mixture of the compound dissolve in a very small volume of organic
solvent n-hexane, benzene, chloroform constituting the mobile phase, is allowed to flow
through the stationary phase by gravity. The compound is the mixture gets adsorbed to the
stationary phase particle surface and the different compounds adsorbed with different
strengths. Therefore, the compounds in the mixture when allowed to move down the
column, move with different speeds, weakly adsorbed compounds move faster than the
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strongly adsorbed compounds. The different speeds of movement of compounds in the

column is known as the differential migration. This process is continued, as a result the
materials are practically separated and adsorbed in the various parts of column. The initial
separation of the various compounds can be improved by passing either the original or
some other suitable solvent slowly through the column. The various bands present in the
column become more defined. The banded column of adsorbent is termed a chromatogram,
and the operation is expressed of as development of chromatogram. The portion of a
column which is occupied by a particular substance is called its zone. The narrower the
zones, the large number of substances which can be separated in a column of a limited
length and the more concentrated are the elutes. (Fig 1)
In order to separate the various constituents, two procedures are used

i) After development, the column of adsorbent may be pushed out of the tube, the various
zones are cut with a cutter at boundaries and the substance present in zones extracted
with a suitable solvent. This process of recovery of constituents from the chromatogram
is known as elute.
ii) After development, the column may be washed with more solvent, now termed the
eluent and each compound is collected separately as it reaches the end of the column
released
Methods of chromatogram development:
Chromatographic separations are performed through development of three methods these
are:
i) Frontal analysis
ii) Elution development
iii) Displacement development
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1. FRONTAL ANALYIS: This consists of the continuous addition of the sample mixture
(A+B) on to the column filled with a suitable adsorbent. After a certain time the mixture
is adsorbed completely and the column is saturated (based on the distribution
coefficient) on continuing the addition of mixture (A+B), the less strongly adsorbed A
elute first, then most strongly adsorbed B elute later. The height of front is proportional
to the concentration of the component in the mixture being introduced. (Fig 2)
2. ELUTION DEVELOPMENT: In this technique. A small amount of mixture (A+B) is

introduced at the top of the column. This is eluted with c which has lesser affinity for
the stationary phase then A and B. The components migrate at a rate determined by the
relative affinity for the stationary phase but at a rate slower than the eluant. The
components are eluated in the order of their affinities but their migration is determined
by the mobile phase.
3. DISPLACEMENT DEVELOPMENT: This involves the development by an eluant C.

This will have a greater affinity for the stationary phase than mixture components (A and
B). The mixture (A+B) is introduced first at the top of the column. This stick to the
stationary phase. Elution occurs when c is passed through the column displacing the
components A and B on the column. (Fig-4)
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COMPOUND IDENTIFICATION PARAMETERS

The components separated in a chromatographic experiment are usually identified by any
of the following parameters
1. Retention time tR
2. Retention volume VR
3. Retardation factor RF
1. Retention time tR: The time taken by a component to travel the length of the column L is
called Retention time
tR=length/velocity of the component
2. Retention volume VR: The volume of the mobile phase require to carry the component
solute molecules through the system to the detector is called retention volume
3. Retardation Factor RF: This is the ratio of the distance travelled by the component and
that travelled by the solvent front measured from the point of application of the mixture
on the column.
RF=Distance a component travelled from origin/distance the solvent travelled from origin
ABOUT ADSORBENTS: The adsorbents should have following characteristics
1. Adsorbents are finely-divided, porous particles with large surface areas
2. Adsorbents are mechanical stability must be great enough to prevent the formation of
fine dust which might be deposited in the channels of the packing
4. They should not react with chemically either with the eluting solvents (or) with the
sample composition
5. Adsorbents should be catalytically inactive.
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Some of the common adsorbents for Chromatography-Silica gel, Alumina, Magnesium

silicate, Kieselguhr, Charcoal, Sucrose, Starch
1. SILICA GEL: Silica (or) Silicic acid formula SiO₂. XH₂O. The Silica gel surface is weakly
acidic nature (PH=3-5)
It is a highly polar, solid, stationary phase. The surface of the silica gel has hydroxyl groups
that is responsible for the selective adsorption properties of silica gel.
2. ACIDIC ALUMINA(ACTIVATED ALUMINA): PH=4 On heating hydrated alumina
Al₂O₃, XH₂O to 300-400 degree Celsius most of the adsorbed water is drawn off, with
the remained of the water reading with the surface Al₂O₃ to form hydroxyl groups.
Acidic alumina is also a highly polar, solid, stationary phase.
3. BASIC ALUMINA: On heating hydrated alumina Al₂O₃, XH₂O to 800-1000 degree
Celsius removes the totally molecules to give hydroxyl free Al₂O₃ the oxide ions now
give the basic properties to Alumina basic alumina also a polar, solid, stationary phase.
4. CHARCOAL: Charcoal is a good adsorbent due to the it has large surface area and fine
porus, it is cheaper easier to be used and recyclable.
NATURE OF ADSORPTION FORCES BETWEEN STATIONARY PHASE AND
COMPOUND
Adsorption is the dependence of the nature of the compound and nature of the stationary
phase between the forces are purely physical in nature, between different types of forces,
Dipole forces, Hydrogen bonding.
Polar compound bind strongly to a polar stationary phase and less polar or non-polar
compounds bind weakly to a polar stationary phase.
1. VANDER WAAL’S FORCES: These are intermolecular forces which hold non-polar
molecules together in the liquid (or) solid state between do not form any chemical bond
only physical in character are weak adsorption forces .are called Vander Waal’s forces.
EXAMPLE: Consider a mixture of CH₃(CH₂) 10CH₂-OH (Designated A) and CH₃(CH₂) taken
15 CH₂-OH (Designated B) to be separated by a column chromatography using silica gel. B
has a larger alkyl surface area than A. Therefore, B is more adsorbent than A on silica gel
surface due to the Vander Waal’s force and interaction between B and silica gel surface is
greater than that between A and silica gel surface.
2. DIPOLE FORCES: In compound which have charged or polar functional groups
induces a dipole in stationary phase, C=O, C-O.C-Cl, C-NO2etc.The carbon with
heteroatom bond has strong permanent dipole. The binding strength depends on the
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strength of the dipole thus different compounds in the mixture different functional
groups have different binding strengths to the stationary phase.
3. HYDROGEN BONDING FORCES: These binding forces stronger than dipole and
Vander Waal’s forces due to the compounds with polar functional groups such as- OH,-
NH₂,-COOH etc. Bind with the stationary face surface group by hydrogen bonding.
In general, The differences in their adsorption strength is the basis for their
chromatographic separation when a column containing the mixture applied as a band at the
front end, is eluted and developed with the mobile phase solvent. A weakly adsorbed
compound in the mixture moves faster than that strongly adsorbed compounds. There by
getting separated. Thus in a mixture contain two compounds (1 and 2) where compound 1
is more strongly adsorbed (silica gel) than compound 2 to the stationary phase on column
chromatography .Compound 2 is eluted first then Compound 1.
PACKING OF COLUMN: Clean the column with chromic and mixture. Rinse with distilled
water and dry it. Insert a cotton wool plug at the constricted end of the column. Now
introduce adsorbent and press it down gently but firmly by dropping a glass pestle on to it
several times. Repeat the process column height should be 2/3 of the length of the column
(Dry packing method).Fill the column with solvent (suitable) to half level. Carefully transfer
the slurry into the column with the stop cock slightly open. The slurry slowly settles down,
Drain out any excess solvent, when all the solvent is drained out and the slurry level in the
column is unchanged, close the stop cock. The packed column height should be 2/3 of the
length of the length (wet packing).Now clamp the column vertically and carefully. The
surface of the column should remain covered with solvent throughout the experiment to
avoid the formation of air bubbles.
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SOLVENT USED: The choice of solvent will naturally depends in the first place upon the
solubility relations of substance. Before studying the column chromatographic separation of
a mixture, one should first study of the TLC of this mixture, the TLC indicates which
solvent gives the best separation of the compounds in the mixture and also the number of
compounds in the mixture. The solvent which gives the best separation in the TLC can be
used as the mobile phase (eluent) for column chromatography.
Table. 1 Elutotropic Scale for Alumina
n-Hexane Solvents arranged in the

Cyclo hexane increasing order of
Carbon di Sulphide polarity
Carbon tetra chloride
Toluene
Benzene
Methylene chloride
Chloroform
Tetra hydro furan
Absolute alcohol
Ethyl acetate
Pyridine
Acetone
Ethanol
Methanol
Water
Acetic acid
ELUTOTROPIC SERIES
The arrangement of solvents in the increasing order of polarity is known as the elutotropic
series
The compounds in the mixture have different adsorption strength towards to stationary
phase. Their actual separation can be done only when a mobile phase through the stationary
phase.
First choice any of these solvents can be used as a mobile phase. In general the starting use
lower polarity solvent and gradually the solvent polarity is increased. Example: To start
with n-hexane. After the collection of some fractions of the elute, the eluent polarity is
increased. This increase in polarity of the eluent is continued till all the compounds in the
mixture are eluted. When a less polar solvent is used as the eluent, the less polar
compound(s) of the mixture are eluted first because these are weakly adsorbed to the
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stationary phase while. The more polar compound(s) of the mixture more strongly
adsorbed. These are eluted
later. Thus, in column chromatography where the stationary phase is polar, the starting
eluent is less polar and the eluent polarity is gradually increased. Therefore, the less polar
compound elute first, followed by the more polar compounds.
REFERENCES
1. Biophysical chemistry- Upadhyay and Upadhya
2. Concepts and Theories of Chromatography- Ibrahim Noha Elsayed , Mohamed Yaser Hagag
.Lambert Academic publishing
3. Chromatography: Basic Principles, Sample Preparations and Related Methods- Elsa Lundanes,
Léon Reubsaet, Tyge Greibrok
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Chapter
ULTRA PERFORMANCE LIQUID
8 CHROMATOGRAPHY
DR. K. SRIVANI
Department of chemistry, Lal Bahadur College, Warangal-506007, Telangana, India.

*Corresponding author: Dr. K. Srivani, Email: vani.kandukoori123@gmail.com.
ABSTRACT
Nowadays preparation of new drugs in the pharmacy and chemical industries at low cost is a big
task. For this, we require a new chromatography technique. The novel technique is UPLC. UPLC
denotes ultra-performance liquid chromatography. This is a modified HPLC method consisting of
high pressure and small-sized particles. This UPLC reduces the cost in the development of new drugs
and also increases the selectivity, sensitivity, and resolution for drug detection. It is a time-saving
process and reduces the consumption of solvent also. UPLC shows improvement in the three areas:
resolution, sensitivity, and speed. This chapter gives information regarding the principle,
instrumentation, applications of UPLC.
KEYWORDS: Ultra performance liquid chromatography; High separation efficiency; Cost-effective;

High pressure.
INTRODUCTION
For the segregation of different components in a mixture mostly high-performance liquid
chromatography was used. This is also used in the identification of compounds in drug
development. To increase the resolution, speed, and sensitivity in liquid chromatography a
new technique was discovered by waters in 2004 which is ultra-performance liquid
chromatography. This chromatography is based on small porous particles (2micron). The
principle behind this is the van deemter equation .which is associated with the connection
between linear velocity and plate height. The pressure required for the small particles is
6000 psi. As the particle size decreases then effectiveness increases. Then compared to
HPLC this method reduces the mobile phase volume consumption by at least 80%
compared to HPLC with a shorter runtime of about 1.5 min. To increase resolution, higher
efficiency lower injection volume is required for UPLC. This technique reduces the cost of
preparation of drugs and is also eco-friendly.
PRINCIPLE
The principle of UPLC is based on the van Deemter relationship it gives the correlation
between flow rate and plate height. The van Deemter equation (i) shows that the flow range
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with the smaller particles is much larger in comparison with larger particles for good
results.
Where H represents height equivalent to the theoretical plate (HETP), A, B & C are the
constants and v is the flow rate (linear velocity) of the carrier gas. The target is to reduce
HETP to increase column efficiency. The term A does not depend on velocity and indicates
swirl mixing. It is smaller if the columns are filled with small and uniform-sized particles.
The term B denotes the tendency of the natural diffusion of the particles. At high flow rates,
this effect is smaller, so this term is divided by v.
The term C represents the kinetic resistance to equilibrium during the process of separation.
The kinetic resistance is the time lag involved in moving from the mobile phase to the
stationary phase and back again. The higher the flow rate of the mobile phase, the more a
molecule on the packing material inclines to lag behind molecules in the mobile phase.
Thus, this term is inversely proportional to linear velocity. Consequently, it is likely to
enhance the throughput, and without affecting the chromatographic performance, the
separation can be speeded up. The emergence of UPLC has necessitated the improvement of
the existing instrumentation facility for LC, which takes the benefit of the separation
performance (by decreasing dead volumes) and consistent pressures (about 500 to 1000
bars, compared with 170 to 350 bars in HPLC). Efficiency is proportionate to the length of
the column and inversely proportional to the radius of the particles. Consequently, the
column length can be reduced by a similar factor as the particle radius without affecting the
resolution. The use of UPLC has helped in the enhancement of the quality of separation
spectra and the detection of drug metabolites.
INSTRUMENTATION
The WATERS Acquit UPLC is Equipped with Mass, els, and Diode Array Detection.
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The instrumentation of UPLC includes- sample injection, UPLC columns, and detectors.
A. SAMPLE INJECTION
The injectors inject a small volume of samples in the form of a mobile phase. The injection
should be done accurately. A quick injection cycle time is required to fully avail the speed
afforded by UPLC. The injection process must be comparatively pulse-free. The volume of
the sample in UPLC is usually 2-5 μL. For the biological samples, direct injection
approaches are utilized.
B. UPLC columns
For better efficiency, a resolution is increased in a 1.7 μm particle-packed column. A bonded
phase is compulsory for the separation of components of a sample it provides both
selectivity and retention. Four bonded phases are available for UPLC separations:
(i) ACQUITY UPLCTM BEH C8 (straight-chain alkyl columns),
(ii) ACQUITY UPLCTM BEH C18 (straight-chain alkyl columns),
(iii) ACQUITY UPLC BEH Shield RP18 (embedded polar group column) and
(iv) ACQUITY UPLC BEH Phenyl (phenyl group tethered to the silyl functionality with a
C6 alkyl),
ACQUITY UPLC BEH Phenyl (phenyl group tethered to the silyl functionality with a C6
alkyl).
Each column chemistry gives a different combination of silanol activity hydrophobicity,
chemical interaction with analytes, and hydrolytic stability. ACQUITY UPLC BEH C18 and
C8 columns are considered the universal columns of choice for most UPLC separations by
providing an extensive pH range. They incorporate trifunctional ligand bonding chemistries
which produce superior low pH stability. This low pH stability is combined with the high
pH stability of the 1.7μm BEH particle to deliver the widest usable pH operating range. An
internal dimension (ID) of a 2.1 mm column is used. For maximum resolution, choose a 100
mm length and for faster analysis, and higher sample throughput, choose a 50 mm column.
Half-height peak widths of less than one second are obtained with 1.7μm particles, which
gives significant challenges for the detector. To integrate an analytes peak accurately and
reproducibly, the detector sampling rate must be high enough to capture enough data
points across the peak. The detector cell must have minimal dispersion (volume) to preserve
separation efficiency. Conceptually, the sensitivity increase for UPLC detection should be 2-
3 times higher than HPLC separations, depending on the detection technique. MS detection
is significantly enhanced by UPLC; increased peak concentrations with reduced
chromatographic dispersion at lower flow rates promote increased source ionization
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efficiencies. The ACQUITY UPLC System consists of a binary solvent manager, sample
manager including the column heater, detector, and optional sample organizer.
C. DETECTORS
The detector used in UPLC analysis is UV/Visible detector. Detection of analytes is
conventionally based on absorbance that is concentration sensitivity detectors. In UPLC the
flow cell volume would have to be reduced to maintain concentration and signal. Based on
Beer's Law, smaller volume conventional flow cells would also reduce the path length upon
which the signal strength depends. A reduction in cross-section means the light path is
reduced, and the transmission drops with increasing noise. Therefore, if a conventional
HPLC flow cell were used, UPLC sensitivity would be compromised. The ACQUITY
Tunable UV/Visible detector cell consists of a light-guided flow cell equivalent to an optical
fiber. Light is efficiently transferred down the flow cell in an internal reflectance model that
still maintains a 10mm flow cell path length with a volume of only 500mL. Tubing and
connections in the system are efficiently routed to maintain low dispersion and to take
advantage of leak detectors that interact with the software to alert the user to potential
problems
Detectors Flow Cell.
ADVANTAGES OF UPLC
1. Various advantages of UPLC are as follows:
2. Require less run time and enhance sensitivity.
3. Provides the selectivity, sensitivity, and dynamic range of LC analysis.
4. In chromatogram resolved peaks are obtained.
5. Multi residue methods are applied.
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6. Speedy analysis, quantify accurately analytes and related products.

7. The use of fine particle (2μm) for packing of stationary phase make analysis fast.
8. Time and cost both are reduced.
9. Consumption of solvents is less.
10. More products are analyzed with existing resources.
11. Increases sample throughput and enables manufacturers to produce more material that
consistently meets or exceeds the product specifications, potentially eliminating
variability, failed batches, or the need to re-work material.
12. Delivers real-time analysis in step with manufacturing processes.
13. Assures end-product quality, including final release testing.
DISADVANTAGES OF UPLC
In UPLC analysis the main disadvantage that occurs is the life of columns, during analysis
high pressure developed because of the particle size.
Increased pressure reduces the life of the columns. Increased pressure requires more
maintenance and reduces the life of the columns of these types. Using the stationary phase
of particle size 2μm perform better analysis without the adverse effects of high pressure.
APPLICATIONS OF UPLC
In the pharmaceutical industry, the demand for UPLC analysis is very high, because of the
unique features of UPLC like high resolution in the chromatogram, short-time analysis
which make more analytical work in less time with valuable, reliable, and authentic data.
Scientists can generate more accurate data by UPLC in a faster way.
By this method, the standard of analysis in every respect like qualitative, quantitative, and
complexity of sample can be differentiated in very high standard. The UPLC/MS system is
used to generate data that solved the complexity of the compound. By using MS as a
detector with UPLC the interpretation of analysis is going to depth. Such analysis is very
used fully in the bio-analytical field.
The unique features of UPLC that high resolution and speedy analysis were also very
helpful in pharmacokinetic studies like – adsorption, distribution, metabolism, and
excretion (ADME). ADME studies measure the physical and chemical properties of
compounds. UPLC/MS/ MS method saves time.
UPLC technique is used for the analysis of herbal products. In an analytical laboratory, the
demand for UPLC is very high because the method developed is accurate and précised, and
also this expands the research information of the analyte at the nano level.
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For the drug development and formulation process, profiling, detecting, and quantifying
drug substances and their impurities can be performed very accurately
UPLC Analysis can be done like:
1. Amino acid analysis.
2. Analysis of natural medicine and herbal medicine.
3. Analysis of drugs in human plasma (e.g. Levofloxacin and metabolites).
4. Study of metabolomics.
REFERENCES
1. Taleuzzaman M*, Ali S, Gilani SJ, Imam SS and Hafeez A. Ultra Performance Liquid
2. Chromatography (UPLC) - A Review Austin J Anal Pharm Chem. 2015; 2(6): 1056.
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Chapter
QUANTITATIVE AND QUALITATIVE CHEMICAL
9 ANALYSIS
M. SRAVANTHI1* & DR. PADMA RAVITEJA2*
1*Department
of Chemistry, University College of Science for Women, Osmania
University, Koti, Hyderabad, Telangana State-500 095, India
2Department of Chemistry, Kakatiya University, Warangal-, Telangana State- 500 009,
India
*Corresponding author: M. Sravanthi & Dr. Padma Raviteja,
Email: bandarishravanthi@osmania.ac.in, prtkuc@gmail.com
ABSTRACT
Analytical Chemistry is a branch of chemical science that focuses on the identification, quantification,
and determination of the structure of chemical compounds. The techniques used for determining the
sample chemical composition can be achieved by gaining knowledge through various analytical
techniques. A chemical analysis would be either quantitative (quantity) or qualitative (identification)
type. Further qualitative analysis can be achieved by identifying the chemical species and
quantitative analysis involves conventional methods like gravimetry and titrimetry and instrumental
methods.
KEYWORDS: Chemical Analysis, Qualitative analysis, Quantitative analysis

INTRODUCTION
QUANTITATIVE AND QUALITATIVE ANALYSIS
Analytical chemistry deals with qualitative and quantitative analysis. Qualitative analysis
is concerned with the identification of what elements or compounds are present in a sample
while quantitative analysis deals with the determination of how much quantity of a
particular substance is present in a sample.
GRAVIMETRIC ANALYSIS
The quantitative chemical analysis deals with gravimetric procedures and titrimetric
techniques. The gravimetric analysis involves the determination of the quantity of analyte
based on its precipitation. When the sample solution is treated with a suitable reagent. The
precipitate obtained is filtered, washed, dried in a desiccator, and weighed (Fig.1) [1]. For
example, an excess of dilute sulphuric acid is added to the given solution containing barium
ions. The precipitation of barium sulphate formed is filtered, washed, and weighed. From
the weight of barium sulphate the quantity of barium in the given solution is calculated.
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PRINCIPLE
The principle behind the gravimetric procedure involves the determination of the mass of
an ion of interest in a pure compound.
Fig. 1: Apparatus Used for Gravimetric Analysis
The steps to be followed in Gravimetric analysis are as follows [2]:

1. Solution of a known weight of the sample is prepared.
2. Isolation of the analyte.
3. Determining the weight of the separated/isolated component.
4. The amount of analyte is derived from the determined weight of the separated
component.
TYPES OF GRAVIMETRIC ANALYSIS:
Gravimetric analysis is of four fundamental types.
Volatilization gravimetry
Volatilization gravimetry involves heating or chemically degrading the sample into
separate components from the sample mixture.
Precipitation gravimetry
Precipitation gravimetry involves the isolation of various components from a sample
mixture containing one or more constituents into individual entities using precipitation
reaction.
Electrogravimetry
Electrogravimetry is a classical technique in which a metal ion from the sample mixture is
deposited electrolytically onto an electrode and weighed.
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Thermogravimetry
Thermogravimetry is a sort of thermal analysis that evaluates changes in materials'
physicochemical qualities as a function of temperature or time.
COMMON ION EFFECT AND SOLUBILITY PRINCIPLES
COMMON ION EFFECT
When a salt containing a common ion is added to an ionic equilibrium, the equilibrium
shifts backward [3].
This is referred to as the common ion effect

Definition
It states that if the concentration of any of the ions in an ionic reaction is increased, then the
surplus ions should be eliminated from the solution by mixing them with oppositely
charged ions i.e., according to Le chatlier’s principle. Until the ionic product equals the
solubility product, some of the salt will be precipitated.
SOLUBILITY PRODUCT:
For a sparingly soluble salt, the product of the molar concentration of ions is constant at
constant temperature and this is referred to as the solubility product denoted as "Ksp".
Precipitation is an ionic reaction and when the ionic product exceeds the solubility product
of a sparingly soluble electrolyte, precipitation occurs which is represented as Ksp < ionic
product. If Ksp = ionic product or Ksp > ionic product, then precipitation does not occur 1
FACTORS AFFECTING PRECIPITATION:
1. Acids affect Solubility of a Precipitate: When a strong acid is added, the rate of
solubility of a sparingly soluble salt of strong acid rises. This is due to the presence of a
strong acid, which raises the ionic strength of the solution, lowering the activity
coefficients of the ions of sparingly soluble salt
2. Effect of Temperature on the solubility of a precipitate: The solubility of a sparingly
soluble salt increases as the temperature rises.
3. Effect of the nature of solvent on the solubility of a precipitate: A solvent has a higher
impact on the solubility of a salt that is sparingly soluble. When some inorganic solvents
(such as methanol, dichloromethane, and others) are introduced to water, the solubility
of inorganic compounds is reduced.
4. Supersaturation and precipitation formation: The solubility of precipitate increases as
the particle size of the precipitate decreases. The precipitate particle size is determined
by the solution's Supersaturation. If a solution contains a larger concentration of solute
than expected at equilibrium conditions at a fixed temperature, it is said to be
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supersaturated. With increasing solute concentration, the particle size of the precipitate
decreases.
IMPURITIES ASSOCIATED WITH PRECIPITATES
The precipitate formed must be partially insoluble and devoid of impurities. Because
precipitation often occurs in a solution containing dissolved solids, the initial precipitate
may not be pure. Hence, before determining the mass/weight of the precipitate formed,
remove the impurities.
Physico-chemical interactions may be to blame for the presence of contaminants on the
precipitate's surface. As they contain incomplete coordination spheres, a precipitate is often
a crystal—even if only on a microscopic scale—with a well-defined cations and anions
lattice leading to a positive or negative charge at the precipitate's surface. In the core
interiors of AgCl precipitate, each silver ion is bound to six chloride ions, whereas at the
surface, it is bound to no more than five chloride ions, resulting in a positive charge (Fig.2).
Because of these partial charges, the precipitate's surface becomes an active location for the
physicochemical processes that lead to impurities.
Fig. 2: Ball and stick Diagram Showing the Lattice Structure of Agcl
Inclusion
One of the most common impurities found in precipitates is when an interfering ion with a
similar size and charge to a lattice ion is replaced in the lattice structure and the crystal
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structure is preserved (Fig.3a). The higher the interfering ion concentration compared to the
lattice ion concentration, the greater the chance of producing an inclusion [4]. In the
presence of inclusion, the amount of analyte that precipitates will not change as long as the
precipitant is present in appropriate amounts. As a result, the mass/weight of the precipitate
is always more than planned. Because inclusion is chemically a part of the lattice, removing
it is a difficult operation. By using the re-precipitation technique, one can obtain a
precipitate lattice that is devoid of inclusions.
Fig. 3: Impurities that May form during the Precipitation Process. The Cubic
Frame Signifies formed and the Blue Spots are Impurities: a) Inclusion b)
Occlusions and c) Surface Adsorbates
Occlusions
Within the developing precipitate, the second type of interfering ion becomes trapped. An
occlusion, unlike inclusions, is much more localized, either along faults within the
precipitate lattice or inside aggregates of individual precipitate ions (Fig. 3b). The mass of
the precipitate increases due to occlusion, but it is smaller than that of the precipitate lattice
if the occlusion includes a lower molecular weight analyte. By keeping the precipitate in
equilibrium with its supernatant liquid for an extended period, the digesting process can
reduce occlusions.
Surface adsorbates are the third type of impurity linked with precipitation that forms even
after the precipitation process is completed because the surface continues to draw ions from
the solution (Fig.3c). We can limit surface adsorption by reducing the surface area of the
precipitate, and we can remove adsorbates by washing the precipitate.
The best examples of co-precipitates or soluble particles that coordinate with the precipitate
lattice carrying the analyte ions include inclusions, occlusions, and surface adsorbates.
Another important category of interferent exists as an independent precipitate along with
the analyst species under the analysis conditions. For example, Nickel (Ni+2) precipitates as
Nickel dimethylglyoxime under slight basic pH. The interferent Fe+3 if present in the
sample precipitates as Fe(OH)3 under similar conditions. Thus an impure precipitate would
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result as a consequence of the precipitant reacting similarly with both the analyte and an
interfering species. One can minimize such additional precipitates formation by
maintaining proper solution conditions. By adopting the filtration process, we can
eliminate the interfering precipitate, if it is soluble when compared to that of the analytes
precipitate leaving the analyte in solution. On the other hand, by masking one of the
species either analyte of the interfering ion we can control its precipitation [5].
ADVANTAGES OF GRAVIMETRIC ANALYSIS:
1. Accurate and precise results are obtained
2. Sources of error can be minimized since filtrates can be further tested for completeness
of precipitation reaction
3. If any contaminants are found in the precipitates, they can be examined.
4. It is an absolute/true method
5. Its instrumentation and operation are inexpensive
GRAVIMETRIC ANALYSIS HAS ITS DISADVANTAGES
It allows you to analyze a single element, or a small set of elements, at a time.
VOLUMETRIC/ TITRIMETRIC ANALYSIS
Quantitative analysis in which estimation of quantity (volume) of a particular chemical
reactant i.e., analyte by measuring the volume (in mL) of solution of that reactant in a
suitable solvent (in mL) is called a volumetric method of analysis.
PRINCIPLE:
To the unknown concentration of sample solution(X), a reagent solution (0.01M) of known
concentration is gradually added till the reaction is completed which can be visualized
using a suitable indicator. The concentration of the given sample solution (X) can be
calculated if the volume of the sample, reagent solution, and the concentration of reagent
solution are also known. For example, a known volume of hydrochloric acid solution
(20mL) whose concentration is to be determined is taken in a conical flask and 2-3 drops of
phenolphthalein solution are added as an indicator.
A solution of sodium hydroxide (0.01M) of known concentration is gradually added
through a burette until the solution in the flask becomes pale pink. The volume of sodium
hydroxide solution added is recorded (burette value say 20mL) and from this, the
concentration of the given hydrochloric acid is calculated. The solution whose
concentration is determined is called "titrate" and the solution was taken in a burette whose
concentration is known as "titrant". The process is called titration and the determination is
termed titrimetric determination.
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ADVANTAGES OF TITRIMETRY
1. Robustness
2. Can be automated
3. Accuracy and precision can be achieved
4. Instrumentation does not require expertise and is inexpensive
1. ACID-BASE TITRATION:
Acid-Base titration involves the determination of the strength of acidic solution with that of
a basic solution of known concentration or determination of the concentration of alkali with
a known standard concentration of acid. Acid-Base reactions are otherwise called
neutralization reactions which result in salts and water as products. The endpoint
determination is achieved with the help of suitable indicators which are either weak organic
acids or weak organic bases and their degree of dissociation is highly influenced by any
alteration in the hydrogen ion concentration of the solution. An acid indicator takes the
general formula HIn and the basic indicators as InOH. This type of acid-base titration
depends on the pH changes
Two such indicators are Phenolphthalein and Methyl orange.
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2. REDOX TITRATION
The redox titrations progress through either oxidation or reduction of the analyte ion. Thus
in this type of titration, chemical reactions occur due to the transfer of electrons in the
reacting ions of aqueous solutions.
The redox titrations are categorized after the reagent that is used in are as follows
1. Permanganate titrations
2. Dichromate Titrations
3. Iodimetric and Iodimetric Titrations
Permanganate Titrations/ Permanganometry
In this titration, the potassium permanganate (a self-indicator) acts as an oxidizing agent in
presence of dilute sulphuric acid. The equation is as follows.
Or
Furthermore, before the equivalence point, the solution is colorless. Because potassium
permanganate is a self-indicator, the reaction proceeds without the use of any external
indicators. Potassium permanganate can be used to measure oxalic acid, ferrous salts,
hydrogen peroxide, oxalates, and other compounds. While the potassium permanganate
solution, which is a secondary standard, is always standardized before use.
Dichromate titrations
Dichromate titrations are those in which potassium dichromate is utilized as an oxidizing
agent in acidic media. Dilute sulphuric acid is used to create the acidic medium. The
following is the potential equation:
Or
The potassium dichromate is a primary standard solution hence the solution can be directly
used for the titration. Ferrous salts and iodides can be estimated using this solution
Iodometric and Iodometric Titrations
These redox titrations involve the reduction of free iodide ions and oxidation of iodide ions
to free iodide ions
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The starch solution serves as an indicator. Free iodine is used in the Iodimetric reaction,
while in the Iodometric titration, an oxidation agent is used to react to liberate free iodine.
3. PRECIPITATION TITRATIONS
Precipitation Titration occurs when two reactive species come into contact, resulting in the
development of an insoluble precipitate. When silver nitrate is added to a solution of
ammonium thiocyanate or sodium chloride, for example, it reacts and generates a white
silver thiocyanate or silver chloride precipitate
4. COMPLEXOMETRIC TITRATIONS
Complexometric titration refers to reactions that result in the creation of an undissociated
complex at an equivalence/endpoint. There will be no inaccuracy owing to co-precipitants
because it is greater than the precipitation titrations.
Ethylenediaminetetraacetic acid (EDTA) is an important reagent that forms complexes with

metals.
REFERENCES
1. Daniel C. H., Freeman, W. H. & Co (2003). Quantitative chemical analysis, 6th ed. New York,
1. 2. Jeffery, G. H., Bassett, J., Mendham, J., Denny, R.C. (1989). Vogel's Textbook of quantitative
chemical analysis 5th edition.
2. Chiappe, C., et al. (2010), 1. An unusual common ion effect promotes dissolution of metal salts in
room temperature ionic liquids: a strategy to obtain ionic liquids having organic-inorganic mixed
cations, green chemistry.
3. Dr. Vijaya Laxmi, G. Dr. Aliya Begum, Dr. Pallavi, P., Dr. Tirupati, M., Mr. Raghavendra Prasad,
U.S.H. A Textbook of chemistry (with practical’s).
4. Beamish, F.E., 21(1) (1949). Inorganic gravimetric analysis, analytical chemistry,
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Chapter
CHROMATOGRAPHY: A VERSATILE TECHNIQUE
10 FOR SEPARATION
DR. ANITA S. GOSWAMI-GIRI1 & DR. GEETALI S. INGAWALE2
1 Associate Professor and Head, Department of chemistry, VPMs B N Bandodkar College of

Science (autonomous) Thane-1 MS India.
2Assistant Professor, Department of Humanities and Applied Sciences, VPMs B N
Bandodkar College of Science Thane-1 MS India

* Corresponding authors: Dr. Anita S. Goswami-Giri & Dr. Geetha S. Ingawale,
Email: anitagoswami@yahoo.com, gsingawale@vpmthane.org
INTRODUCTION
Chemical analysis is the concern with the identification and estimation of a component in a
given substance to evaluate the scientific problem. To identify unknown compounds/
elements, proportions/amount is the major concern with using new, easy, and quick
analytical methods and improving existing works. Ancient time quantitative approach in
chemical research and normal experimental is the key role in the development of science.
All branches of chemistry including biological, environmental, pharmaceutical, medicinal,
petroleum chemistry depend on analytical techniques such as chromatographic and
spectroscopic techniques. Hence, in present chapter discussed separation techniques-
different types of chromatography procedures, advantages, and their application.
Chromatography is the biophysical technique based on a principle where molecules in a
mixture applied onto a solid surface or liquid phase are separated from each other by
running with the mobile phase. Chromatography is regarded as a method of separation in
which the separation of solute occurs between stationary and mobile phases. This
separation of solute occurs between the stationary and mobile phases. This separation
technique becomes universal and has been extended in chemistry, biology, medicine, and
pharmaceutical industry in the manufacturing of pure chemicals and bioscience for the
separation of biomolecules. The name chromatography means color, writing. In
chromatography method, it involves the following steps.
1. Adsorption of substance on the stationary phase.
2. Separation of the absorbed substance by the mobile phase.
3. The recovery of the separated substances is called elution.
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4. Quantitative and quantitative analysis of the eluted substance.

Types of Chromatography and its Stationary and Mobile Phase
Sr. No. Chromatography Stationary Phase Mobile Phase
1. Column Chromatography Solid -liquid
2. Partition chromatography liquid liquid
e.g. Paper chromatography liquid liquid
3. Adsorption chromatography
e.g. Thin layer Chromatography (TLC) Solid liquid
High-Pressure Thin layer Chromatography
Solid liquid
(HPTLC)
High-Pressure Liquid Chromatography
non-polar moderately polar
(HPLC)
4. Gas-liquid chromatography liquid Gas
Gas solid Chromatography Solid Gas
5. Iron Exchange Chromatography Solid Gas
Iron Exchange Chromatography Solid liquid
Gel-permeation (molecular sieve) A stagnant liquid
6. liquid
chromatography in porous bead
7. Affinity chromatography Solid Lysate/ liquid
The chapter deals with Column chromatography, Partition chromatography Paper

chromatography, Adsorption Chromatography- a)Thin-layer chromatography (TLC),b)
High-pressure Thin-layer chromatography (HPTLC) and c) High Pressure Liquid
chromatography, Gas Chromatography, and Ion Exchange Chromatography.
COLUMN CHROMATOGRAPHY
The technique in which the stationary phase is alumina or silica gel and the mobile phase is
either gas or liquid is known as adsorption chromatography. These techniques are used for
Identification of the non-identified two substances, in determining the concentration of
products, contaminants in commercial products, in separation and Purification of technical
products, etc.
The principle of chromatography is based on differential adsorption of substances by the
adsorbent.
Factors Affecting Column Efficiency: Following are some of the important factors after
column efficiency.
1. Dimension of the column
Column efficiency has been improved by increasing the length/width ratio of the column
for column preparative separations sample/column packing ratios from 1:20 to 1: 100 in
adsorption and from 1:50 to 1:500 in partition chromatography. Recently length/ width
ratios of 10: 1 to 100: 1 are more satisfactory.
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2. Particle size of column packing

Particle size plays an important part in chromatography is improved by decreasing the
particle size of the adsorbent. This is probably due to the rapid decrease in flow rates. The
recommended particle size used for both adsorption and partition is 100-200 mesh range.
3. Pore diameter of column packing:
Polar adsorbents have been found to have a pore diameter of 20 A0 according to keen a
decrease in average pore diameter from 70-20 A0.
Elution is a chemical process that involves removing a material's ions by ion exchange with
another material. Eluent is a solvent/mobile phase that passes through the column. In liquid
chromatography, the eluent is liquid while in Gas chromatography eluent is a gas carrier.
PARTITION CHROMATOGRAPHY
Paper Chromatography
In partition chromatography, the substances are distributed between the stationary
liquid/stationary phase and the moving liquid/mobile phase. The component of the mixture
to be separate traveled at different rates and appeared as spots at different points on
method. Originally paper chromatography was used to separate a mixture of organic
substances such as dye and amino acids, steroids, vitamins, Pesticides, Pigments but now
this method has been used to separate cation and anion of inorganic substances as well.
Procedure
1. A drop of the test solution is applied to the bottom of a filter paper. After drying the
spot, filter paper is placed in a suitable solvent in such a way that the edge of filter
paper is deep into a solvent called developing solvent.
2. As soon as the filter paper gets the liquid through its capillary axis and reaches to spot
of the test solution, the various substances are moved system at various speeds. When
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the solvent has moved to a suitable height the paper is dried and various spots are
visualized by the suitable reagent called visualizing reagent. The movement of the
substance relative to a solvent is expressed in terms of Retention Factor/ Retardation
Factor (RF values).
R.F. =
Types of paper chromatography: There are 5 types of paper chromatography

1. Descending, b) Ascending, c) Ascending-descending, d) Circular and e) Two-
dimensional
When the development of the paper is done by allowing the solvent to travel up the paper.
It is known as Ascending technique while as the solvent travels down the paper it is known as
the 'Descending technique’. Both ascending and descending techniques have been employed
for the separation of organic and inorganic substances. The advantage of the descending
technique is that the development can be continued even though the solvent runs off at the
other end of the paper.
1. Ascending-descending - This is the hybrid of both of the above techniques. The upper
part of ascending chromatography can be folded over a rod to allow the paper to
become descending after crossing the rod.
2. Circular chromatography –The sample is applied at the center of circular paper after
marking with a pencil. After drying, kept it in a Petri dish by wick of the paper dipped
in the solvent. As solvent rises through the wick, components get separated into
concentric rings.
3. Two-dimensional- on square or rectangular filter paper the sample is applied to one of
the corners and development is performed at a right angle to the direction of the first
run.
Limitation of paper chromatography
1. A large quantity of sample and Corrosive material cannot be applied on paper
chromatography
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2. Complex mixtures cannot be separated

ADSORPTION CHROMATOGRAPHY
Thin Layer Chromatography: TLC was first introduced in 1938. It is used for the isolation
of non-volatile mixtures. It’s also called as TLC is also made by other names such as-
1. Surface chromatography
2. Column chromatography
ADVANTAGE OF TLC OVER OTHER CHROMATOGRAPHY TECHNIQUES

TLC is superior to column and paper chromatography. The main advantage is given below-
1. TLC required simple equipment and methodology.
2. It required less analysis time for development for drying and Ultra-choice of stationary
phase.
3. It requires low solvents can be analyzed numbers of samples.
4. Complex mixtures easily separated based on polarity
5. Required very small quantity of samples
6. Easy recovery: It is possible to remove the powder coating of plates by scraping a knife.
7. Easy equalization of the paper component. Detection of a component under U.V. light
is easier than paper chromate.
8. Chemically inert stationary phase the greatest advantage of this technique is very
strong corrosive reagents such as concentrated can also be used.
APPLICATION OF TLC
Due to its simplicity, sharpness, high sensitivity in separation, and easy recovery, TLC has
found increasing application in all branches of chemistry and its allied branches. The
application of TLC in organic chemistry is as follows.
1. It is used for checking the purity of organic, inorganic, and biological samples
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2. To observe the purified product and also measurement cycles in the purification process
3. Estimation of reactions/progress in reactions/purification method and for the identity of
separated organic compounds such as amino acids, proteins, alkaloids, phospholipids
antibiotics, acids, alcohols, ethers, amine, etc.
LIMITATIONS OF TLC
1. It is used only for small preparative work but researchers have obtained high resolution
by using TLC while using film techniques.
2. Humidity and temperature can affect the result
3. Compounds run streak rather than spot
4. A limited amount of material can be isolated
PROCEDURE FOR TLC
1. TLC plate is be prepared by using silica as a stationary phase with fine and uniform.
2. Mark the line by pencil to the bottom of the plate and upload the sample to be separated
3. A suitable solvent or solvent mixture which is pure and particulate-free can be used as a
mobile phase. Sample loaded TLC plate is placed into TLC chamber in such a way that
the loaded sample should be well above the solvent system/mobile phase and Cover
the chamber with lid
4. After the development of the spot remove the plate and observed the spot under the UV
chamber
5. Mark the spot and calculate RF values of spots.
HIGH-PERFORMANCE THIN LAYER CHROMATOGRAPHY (HPTLC)
The advanced version of TLC is HPTLC, working on the adsorption-based principle for
separation. HPTLC can be used as an alternative technique for high-performance liquid
chromatography (HPLC) and gas chromatography (GC). HPTLC is also called flat-bed
chromatography or planar chromatography.
APPLICATION OF HPTLC
1. It is cost-effective, easy to maintain and Multiple analyses can be done simultaneously
2. No risk of contamination and have a wide range of stationary phases
3. The method is sensitive, rapid, reproducible, precise Required less solvent for
separation
DISADVANTAGE
1. Limited samples can be tested on a plate
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2. Short separation of bed

3. Silica also detected in the test
PROCEDURE
1. Sample preparation- For HPTLC 0.1µl concentration of sample used for transferring the
sample solutions to the thin layer qualitative work.
2. Preparation of chromatographic layers- Layers can be performed on a sheet of glass, plastics,
and or aluminum foil which is coated with a thin layer of adsorbent materials/stationary
phase. Generally, silica gel, alumina oxide, calcium phosphate are used as a coating
material for better plating some binders like gypsum, calcium sulphate, starch are added
to absorbent gypsum is most used.
1. The various methods of preparing layers such as Pouring, dipping, spraying, and
spreading. After preparing slurry in water or solvent, the plate dipped into slurry,
remove and dry it well. The slurry is spread uniformly on the glass surface. After setting
the layer of adsorbent activate the plate by keeping it in an oven at 100 oC for 1 hr.
2. Washing and conditioning- Methanol and also in combination with ethyl acetate wash the
plate. Plates should be handled only at the upper age to avoid contamination. For
quantitative analysis and reproducible results plated need to equilibrate.
3. Sample application- For a sampling of the standard sample, a capillary tube or
micropipette can be used for spotting. The spot can be placed 2cm above the base of the
plate. The plate should be kept into the
4. Selection of mobile phase- It depends on the stationary phase used in the system and
chemical properties of solvents such as Diethyl ether, methylene chloride, and
chloroform combined individually or together with hexane. The Choice of solvent is an
important decision. In practice generally benzene, chloroform, acetone, benzene-
methanol, chloroform-Methanol
5. Chromatographic development: Assembling of chromatography T.L.C. plate is placed in the
development chamber with angle 450 it is imp that development chamber is perfectly
saturated with solvent paper.
6. Detection of spots: The lower edge of the plate is deep into the closed developing chamber.
Due to capillary action, the samples run up to desired distance .depending upon the
vapor pressure in the chamber and composition of components, the stationary phase
absorbs molecules from the gas phase, and migration of components of the mobile phase
is separated. Chamber saturation is more important for the detection of spots
7. Documentation: the developed plate may be digitally documented under UV and white
light
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HIGH-PRESSURE LIQUID CHROMATOGRAPHY (HPLC)

HPLC is a non-destructive method used for the simultaneous analysis method. The
separation of sample ingredients/ analytes between the mobile phase and stationary phase.
The distribution of analytes depends on chemical structure, intermolecular interaction
between molecules, and packing material in the column. Different common packing
materials are for normal phase, reversed phase, size exclusion, ion exchange, affinity chiral,
or hydrophilic interaction for the separation in HPLC. Change in composition of mobile
phase in HPLC the system is known as gradient elusion system.
HPLC is mainly divided into two types.
1. Normal phase HPLC and
2. Reversed-phase HPLC.
PRINCIPLE
Resolving power of chromatography column increases with increase in column length.
There is development in adsorption partition, exchange affinity chromatography resulted in
faster resolution hence HPLC is the most popular, powerful, and versatile form of
chromatography.
EQUIPMENT
Fluent reservoir, de-gasing solvents, Gradient controller and mixing unit, filter, Hinge
press. Pump, filter, pressure gauge injection port (Column), director, recorder, eluent
collector.
This is a schematic representation of high performance eluent-
1. From the solvent reservoir achieves a constant flow of solvent at 100 psi.
2. The flow gets smooth with the dumper.
3. It is possible to measure the inlet press of column with manometer after leaving the
column the sample studs high pressure. The sample is injected through a spectrum in an
injection hole either directly or to the column or on a small plug, employing an
appropriate valve. The fluent form a channel section through the injection pole.
Degassing is used to prevent gas bubbles in the pump detector. The solvent must be
passed through the column at high pressure while as stationary phase particles are
smaller (5-10µ) resistant to the flow of solvent hence high pressure is recommended. A
flow rate of 0.1to 10 ml/min is recommended. The outlet which leads directly onto the
column is highly impure solvents, Urine, whole blood which has preferably been
detected by high resolving power. The separated analytes are recorded by the system in
the form of peaks. Total all peaks are known as a chromatogram. All peaks given the
information about the analytes such as shape, the intensity of the peaks, time required to
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<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<
appear for peaks. The area of peaks that depend on the concentration of analytes is
known as Gaussian bell-shaped curve. Delay time, retention time, peak width, and
Trailing factor /peak symmetry
ADVANTAGES
1. It is simple, rapid, reproducible, Repeatable, and sensitive methods
2. It exhibits accuracy, precision and its stationary phase is chemically inert.
3. Less amount of mobile phase is required in developing chamber.
4. Techniques are important for the validation of the product, quality control studies.
5. It is used for both analytical and preparative purposes.
6. HPLC is used in easy to fractionate and purify
APPLICATIONS OF HPLC
1. It is used to detect, identify, quantify and purify all chemical and biological molecules.
2. HPLC is widely used for the separation of Polar components such as vitamins. Steroids,
polyphenols, Peptides. The separation of some highly polar compounds such as amino
acids can be separated economically by this method.
GAS CHROMATOGRAPHY
It is quite similar to column chromatography except that gas is used as a mobile phase
instead of a liquid. Gas solid chromatography (GSC) and Gas liquid chromate (GCL) is
encountered. The main advantage of gas chromatography are as follows-
1. A complex mixture can be resolved easily.
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<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<
2. It gives good precision and accuracy.

3. The analysis is completed in a short time.
4. The cost of the instrument is relatively low.
5. Its life is generally long for the operation of gas chromatography. It doesn't require. A
highly skilled person for calculation.
PRINCIPLES OF GAS CHROMATOGRAPHY
When the gas of vapors comes in contact with the adsorbent certain amount of it gets
adsorbed on solid. In the system Helium, argon, or nitrogen are used as carrier gas. The
component separated inside the column and detector measure the number of components.
The separation techniques are capable of separating complex mixtures based on physical
constant, polarity, and vapor pressure.
PROCEDURE
Its operation is similar principle of column chromatography where samples are dissolved in
the mobile phase and passed through the stationary phase.
There are 4 steps in the analysis
1. Sample collection- sample is introduced into the stream of gas. A gas sample is collected
and then it is introduced into an inert gas stream called a carrier gas
2. Sample injection- here it’s in hot conditions which allows the solvents and compounds to
evaporate ( liquid sample need to be evaporated before injecting into the carrier)
3. Sample separation- samples moved through the packed column, with the stationary
nonvolatile phase. Samples interacted with the stationary phase very less
4. Sample detection- Samples quantified collected through the detector.
APPLICATIONS
1. The detection of steroid drugs used by international sports competitions.
2. Hazards pollutants such as CO2, formaldehyde, benzene can be monitored by G.C.
3. An analysis of food products like milk, sugar, butane, and added colors and salts
G.C.cab be easily identified.
4. It is also used in drug analysis, identification of plastic, paints, and synthetic polymers,
and various environmental studies
<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<
<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<
ION EXCHANGE CHROMATOGRAPHY

PRINCIPLE
The principle feature underlying this form of chromatography is the attraction between
oppositely charged particles. Ion exchange separations are mainly carried out in a columns
pack with an Ion exchanger. There are two types of ion exchangers, namely caution and
anion exchanges. Cation exchanges possess negatively changed groups which will attract
negatively charged molecules.
The quality of an ion exchange resin is determined by its capacity which in turn depends
upon the total number of ion active groups per unit weight of the material. Greater the
number of ions the greater the capacity of the resin for the exchange process. The efficiency
of the resin has been found to depend upon the degree of cross-linking and the higher the
efficiency of the resin.
Univalent anions, the capacity has been found 1->NO3-1->Br- > CN->C1- > OH->F-
to decrease
Univalent cation the capacity has been found
H->cs+1->Rb+> NH4+>Na+ > Li+
to decrease
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<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<
A glass column fitted with glass wool or ordinary burette with resin provides a large
surface area for contact between the solution and the resin.
APPLICATIONS OF ION EXCHANGERS
Ion exchange is used mostly in inorganic chemistry.
1. Separation of amino acids and sugars from food, actinides
2. Purification of organic compounds
3. Separation of contaminations from water and for analysis of pollutions
REFERENCES
1. Joseph Sherma , Gunter Zweig Paper Chromatography 1971, chapter 3 - Techniques of Paper
Chromatography Pages 30-89
2. Joseph Sherma , Bernard Fried 2003 the Handbook of Thin-Layer Chromatography, Third
Edition Routledge publication
3. Shrivastava, M.M. 2011, An Overview of HPTLC: A Modern Analytical Technique with Excellent
Potential for Automation, Optimization, Hyphenation, and Multidimensional Applications. In:
Shrivastava, M.M. HPTLC. New York: Springer, pp. 3- 24.
4. Patel, R.B. and Patel, M.R. and Patel, B.G.2011, Experimental Aspects and Implementation of
HPTLC. In: Shrivastava, M.M. HPTLC. New York: Springer, pp. 41- 54.
5. McNair H M, Miller J M, Snow N H. Basic gas chromatography. John Wiley & Sons, 2019.
6. Scott R P W. Introduction to Analytical Gas Chromatography, Revised and Expanded. CRC
Press, 20
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<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<
Chapter A REVIEW ANALYSIS OF DIFFERENT ORGANIC

11 COMPOUNDS THROUGH VARIOUS
INSTRUMENTAL METHODS WITH THE
ADVANCEMENT OF SCIENCE AND TECHNOLOGY
1 DR.SUMANTA BHATTACHARYA & 2ARKADYUTI SETH
Research Scholar at MAKAUT, Public–Foreign-Defence Policy Analyst.

1
2M.Sc. in Forensic Science (BHU), MA in Development Studies
*Corresponding author: Dr. Sumanta Bhattacharya, Email: sumanta.21394@gmail.com
ABSTRACT
The analysis of organic compounds is an essential part of Analytical Chemistry due to its widespread
application in Medical Science, Pharmaceutical industry, food processing industries, Forensic Science,
toxicological analysis, etc. It can be analyzed both quantitatively and qualitatively through different
procedures. The spectroscopic analysis facilitates us to determine the molecular structure of the
compounds. The application of information technology in analytical chemistry opens a new branch
called Computational Chemistry which makes the detailed study of the configuration and geometry
of organic molecules easier and more understandable. The coupled techniques like Gas
Chromatography-Mass Spectrometry, Liquid Chromatography-Mass Spectrometry facilitate both the
qualitative and quantitative study simultaneously. In this paper, different methods of the analysis of
organic compounds will be reviewed.
KEYWORDS: Qualitative analysis, quantitative analysis, spectroscopic analysis, computational

chemistry.
INTRODUCTION
Organic compounds are largely known as those compounds that can be derived from living
organisms. However, these compounds can also be synthesized in the laboratory like
inorganic compounds as shown first by Friedrich Wohler in 1824 by preparing oxalic acid,
an organic compound from cyanogen in the laboratory. Chemically organic compounds are
hydrocarbons i.e. the compounds containing carbon and hydrogen atoms and their
derivatives that are formed by the substitution of one or more hydrogen atoms with other
atoms like oxygen, nitrogen, sulfur, etc. Carbon atoms possess a special property known as
the catenation property due to their favorable atomic radius and electronic configuration
which enable the formation of long-chain carbon-carbon bonds. Due to this unusual
capability of the formation of long-chain polymers carbon-containing compounds are
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<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<
largely abundant in living organisms as essential components like proteins, carbohydrates,

nucleotides, etc. These compounds are also largely used in pharmaceutical industries as
proteins and vitamins are the major constituents of medicines. The aromatic compounds
containing carbon-carbon cyclic bonds are used as a major constituent of the perfume
industry due to their characteristic odor. Organic acids and bases are major constituents of
buffer solution due to their lower degree of dissociation which is used in analytical
chemistry to control the pH of the solution. Organic compounds are one of the most
important ingredients of petroleum products which is very essential in our daily life. In
Forensic Science, the detection of petroleum products in burnt debris indicates the
possibility of initiating fire deliberately at a scene of the fire. Plant derivatives containing
useful organic compounds are used to a larger extent in the treatment of serious diseases
like agents in chemotherapeutic treatments of cancer for less possibility of showing side
effects than synthetic inorganic chemicals. Plant fibers and their extracts are now used in
various fields such as the preparation of bio plastics, organic pesticides, composts, dyes,
papers, clothes, and other handicraft products due to their biodegradability and eco-
friendly nature to promote the values of sustainable development goals.
RESEARCH METHODOLOGY
For this exploration, we have used multiple qualitative and quantitative techniques of
organic analysis including different spectroscopic methods. We also reviewed several
scientific journals, books, and, other academic resources.
THE OBJECTIVE OF THE RESEARCH
1. Review of different methods of analysis of organic molecules.
2. To focus on the importance of the analysis of organic molecules and their chemical
study
3. To explore new methods of organic analysis for future prospective.
LITERATURE REVIEW
The carbon, hydrogen, and other atoms like nitrogen, sulfur, phosphorus, and halogens
present in different functional groups of the organic compounds can be analyzed both
qualitatively and quantitatively through different tests in the laboratory. Liebig’s method is
largely used for qualitative analysis of carbon and hydrogen atoms in organic compounds
and nitrogen, sulfur, halogens, and phosphorus, Lassaigne’s test is used. [8] Carbon atom
reacted with cuprous oxide to form carbon-di-oxide which confirms its presence by making
the lime water i.e. calcium hydroxide turbid whereas hydrogen atom converts into the
water which formed blue vitriol while treated with copper sulfate. On the other hand,
organic nitrogen, phosphorus, sulfur, and halogens are converted into ionic elements by
fusing with sodium and then dissolved in water to form sodium fusion extract which is
further treated in the same manner as inorganic analysis. Nitrogen gives Prussian blue
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<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<
precipitate by reacting with an acidic solution of ferrous sulfate, phosphorus is treated with
nitric acid and ammonium molybdate to give a yellow precipitate after oxidizing to
phosphate, sulfur gives black precipitate after reacting with an acidic solution of lead
sulfate, halogens give characteristic precipitate while treating with silver nitrate after
acidification. [2] The percentage volume of carbon, hydrogen, nitrogen, sulfur, and halogens
present in an organic compound can be determined. [6]
The number of different derivatives of bisphenol A released from wastewater treatment
plants is estimated through Liquid Chromatography/Mass Spectrometry and High-
Resolution Mass Spectrometry with recoveries of 50.6-77.9 % and a relative standard
deviation of 4.3-16%. [3]
The computational programs are also helpful in the field of qualitative analysis of organic
compounds as it facilitates the analysts to work on a greater number of samples within a
short period than manual laboratory works. [4, 5] It also indicates that the quantum
mechanics-based semi-empirical analysis is more effective than classical analysis to study
the configuration of complex organic compounds. [1]
FINDINGS
In an unknown sample, organic compounds can be analyzed in three ways viz. qualitative
analysis i.e. detection of the presence of the particular compound in the sample where the
functional groups like carboxylic acid, hydrazine, amine, cyanide, isocyanide, amide,
halide, etc. are detected; quantitative analysis where the quantity of a particular component
like acid, base, carbon, hydrogen, halogen, nitrogen in the sample is determined;
spectroscopic analysis where information regarding the molecular structure of a compound
is obtained and computational analysis where reliable mathematical models and algorithms
are developed to formulate advanced programs that facilitate to evaluate different chemical
and thermodynamic data related to organic compounds.
Traditionally, the presence of carbon, hydrogen, and functional groups in the organic
compounds can be confirmed through the treatment with other chemicals resulting in the
formation of colorful precipitation. However, the precipitates will remain undetected if it
reacts with other chemicals present in the reaction mixture.
Qualitative test of Carbon:
Carbon in the organic compound is treated with cuprous oxide (CuO) to form carbon-di-
oxide (CO2) which makes the lime water (Ca(OH)2) turbid due to the precipitation of
calcium carbonate (CaCO3).
Qualitative test of Hydrogen:

<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<
<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<
Hydrogen in the organic compound is treated with cuprous oxide (CuO) to form water
(H2O) which turns anhydrous copper sulfate solution (CuSO4) blue.
Qualitative Test of Functional Groups
The Qualitative Test of Functional Groups of Organic Compounds (Source: Danielle M. Solano,
Lab 14: Qualitative Organic Analysis, Department of Chemistry & Biochemistry, California State
University, Bakersfield) [7]
<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<
<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<
The percentage volume of carbon and hydrogen present in an organic compound can be
measured by treating the compound with oxygen and absorbing the water and carbon-di-
oxide formed in anhydrous potassium hydroxide and calcium chloride solution
respectively.
CxHy + (x + y/4)O2 x CO2 + (y/2) H2O
Let, m gm of organic compound produce m1 gm of water and m2 gm of carbon-di-oxide.
Then,
The percentage of carbon will be (12 X m2 X 100)/ (44 X m)
The percentage of oxygen will be (2 X m1 X 100)/ (18 X m)
The percentage volume of nitrogen can be determined by heating the compound with
copper oxide and collecting the nitrogen gas formed over potassium hydroxide solution
(Dumas method)
CxHyNz + (2x + y/2) CuO xCO2 + (y/2) H2O + (z/2) N2 + (2x + y/2) Cu
Let, on treating m gm of organic compound V1 ml of nitrogen has been collected at T1K
temperature. Then,
The volume of nitrogen at STP will be (p1V1 X 273)/ (760 X T1), where p1 will be the pressure
of nitrogen.
V ml nitrogen at STP weighs (28 X V)/22400 gm
Percentage of nitrogen will be (28 X V X 100)/ (22400 X m)
However, nitrogen present in hydrazine cannot be determined by this process.
The quantity of halogen in an organic compound can be estimated by heating it with
fuming nitric acid in presence of silver nitrate which resulted in the formation of silver
halides. (Carius Method).
Let, m gm of organic compound produce m1 gm of silver halide.
Therefore, the mass of halogen in silver halide will be (atomic mass of halogen X
m1)/Molecular mass of silver halide
Through chromatographic analysis of unknown samples, the components present therein
can be separated and that can be captured in the detector depending on their absorption
tendency with the stationary phase and mobile phase. Thereby, their mass can be obtained
by further treating them in mass spectrometry. The chromatographic techniques may differ
according to the nature of the stationary phase and mobile phase, such as liquid
<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<
<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<
chromatography, gas chromatography, thin-layer chromatography, paper chromatography,

column chromatography, etc.
Spectroscopic techniques like UV spectroscopy determine the presence of conjugal bonds,
IR spectroscopy determines the presence of functional groups, NMR spectroscopy
determines the number of carbon-hydrogen bonds, etc. [9]
Thermodynamic properties of the bonds of organic compounds like bond energy, enthalpy
of formation, etc. can be measured with the help of software like CHEMICAL,
AVOGADRO, MOPAC, MOLDEN, PyMOL, etc. through computational analysis which
enables us to study the structural configuration and geometry of the organic compounds.
CONCLUSION
Organic compounds are one of the most essential components in our daily life due to their
presence in different medicines, foods, fertilizers, composts, oil, fuels, and other important
ingredients. For this reason, the analysis of organic compounds is one of the most essential
parts of Analytical Chemistry. The application of information technology widens the scope
of this field through the opening of a new branch called Computational Chemistry which
helps in 3D molecular designing. The plant extracts are now used as chemotherapeutic
agents for the treatment of serious diseases like cancer which proves to be more effective
and free from side effects. The demand for organic compounds in different industries like
packaging, pharmaceutical, medical, agricultural, food processing, textile, etc. is continually
boosting due to the gradual shifting of society towards more environmentally friendly
products to combat the challenges of global warming and climate change resulting by the
unscientific and deregulated utilization of synthetic products since the industrial revolution
in 18th Century A.D.
REFERENCE
1. Filhoa, Eloi Alves da Silva; Uliana, F.; Ambrosio, Stener R.; Oliveira, C.; Martin, R.; Goncalves,
Arlan da Silva; 2019; Computational Study of Organic Compounds- An Application for learning in
Chemistry; p. 257-266; REVISTA Ives CIENCIA, Volume 5, Numero 1
2. Organic Chemistry- Some Basic Principals and Techniques; 2019-20; p. 334-372; National Council
of Educational Research and Training, Ministry of Education, Government of India
4. Takazawa, M., Suzuki, S., Nakano, T., Tsuboi, S., Shinomiya, M.; 2017; Quantitative and
Qualitative Analysis of Organic Halogenated Compounds Unintentionally Generated in
Wastewater Treatment Plants using Liquid Chromatography/Mass Spectrometry and High-
Resolution Mass Spectrometry; Journal of Environmental Chemistry, Volume 27, No. 4, p. 137-144
5. Garcia, M., Gujjarlapudi, Rajan B., Hatti, K., Smedley, C.; 2004; Organic Qualitative Expert System;
American Society for Engineering Education, p. 9.971.1-9.971.10
6. Zampella, G., Gioia, Luca De; Computational Organic Chemistry; Organic and Biomolecular
Chemistry, Volume 2, Encyclopedia of Life Support System
7. Jain, R.; 2006; Pharmaceutical Analysis (Instrumental Methods), Volume 2; Nirali Prakashan.
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<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<
8. Solano, Danielle M.; Lab 14: Qualitative Organic Analysis, Department of Chemistry &
Biochemistry, California State University, Bakersfield
9. Furniss, Brian S., Hannaford, Antony J., Smith, Peter W. G., Tatchell Austin R., 5th Edition; Vogel’s
Textbook of Practical Organic Chemistry; Pearson Publication
10.Kalsi, P. S.; 8th Edition; Spectroscopy of Organic Compounds; New Age International Publishers
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<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<
RECENT ADVANCEMENTS IN ANALYTICAL CHEMISTRY
About the Book
Analytical chemistry is a diverse branch of chemistry playing a pivotal role in modern
society. Considering the rising importance of the branch, we are writing this book,
“Recent advancements in analytical chemistry” with multiple chapters dedicated to the
latest methods like hyphenated techniques and conventional methods like Titrimetry.
This book covers titrimetry, gravimetry, organic functional group analysis, spectroscopy,
crystal analysis, and chromatography techniques. This book, with valuable chapters from
eminent academicians and researchers, gives insight into modern and conventional
analytical techniques that are useful in modern analytical chemistry
About the Editors

Dr. N Padmaja, Head of Department & Academic Coordinator, Chemistry –
PG Center Lal Bahadur College, Warangal, A doctorate in Inorganic Chemistry
from Kakatiya University, her field of study and expertise include metal
complexes and coordinate chemistry. She is currently working as Associate
Professor at the Department of Chemistry, Post Graduate center, Lal Bahadur
College Warangal, Telangana, India, and has 28 years of teaching experience. She
is the head of the chemistry department, academic coordinator, and cultural
convener at her college and handles numerous educational and cultural events
by coordinating with various institutes. She has two patents to her credit so far.
She also has multiple research publications and book chapters, both international and nationally, to her
credit. She has published seven research papers in reputed national and International Journals and
three book chapters. She is also a member & contributor to the international congress of chemistry &
pharmacy.
Dr. Vishnu Kiran Manam A doctorate in Applied Microbiology - Botany with
specialization in Nanotechnology from the University of Madras, his field of
study and expertise include Nano-biotechnology, Algal Research, Aquaculture,
Vaccine Research, Bio-Remediation and Drug Discovery Services. He has rich
experience in Research & Development and Academics for more than a decade,
He has also practical experience in Marketing & Corporate Communications,
Human resources, and Project Management for more than 5 yrs. He has been
certified with Six Sigma [Yellow Belt, Green Belt & Black Belt]. He has bagged the
BEST SCIENTIST AWARD –IARDO, YOUNG SCIENTIST AWARD – ELSEVIER SSRN, RESEARCH
EXCELLENCE AWRAD - RES and BEST RESEARCHER AWARD - ISCAW – ESM for the year 2021. He
has 25 patents to his credit so far. He has also various research publications, book chapters both
PADMAJA I MANAM
internationally and nationally to his credit. He has been a book editor [6 Books] in various disciplines
such as Nanotechnology, Chemical Sciences, Aquaculture, etc. He has actively taken part in various
research programs conducted nationally and internationally. He has been a part of the editorial board
member in various International journals and a member of various research forums. DR N PADMAJA
ISBN 978-93-94766-13-6
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RECENT TRENDS IN ANALYTICAL CHEMISTRY

Book · September 2022

Vishnu Kiran Manam

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About the Editors

DR VISHNU KIRAN MANAM

Title: RECENT ADVANCEMENTS IN ANALYTICAL CHEMISTRY

First Published, 2022

Printed in India, by: Sagar Color Scan, New Delhi.

Sr. Tittle Page

2 BASIC CONCEPTS AND APPLICATIONS OF 11-18

3 RECENT CHROMATOGRAPHIC DEVELOPMENTS IN 19-24

4 A FACILE SYNTHESIS OF 2-CYCLOPROPYL-3- 25-33

5 GROWTH, SURFACE, THERMAL AND MECHANICAL 34-41

6 ROLE OF INFRARED (IR) SPECTROSCOPY IN 42-51

7 SEPARATION METHODS: CHROMATOGRAPHY 52-60

8 ULTRA PERFORMANCE LIQUID CHROMATOGRAPHY 61-66

9 QUANTITATIVE AND QUALITATIVE CHEMICAL 67-75

10 CHROMATOGRAPHY: A VERSATILE TECHNIQUE FOR 76-87

11 A REVIEW ANALYSIS OF DIFFERENT ORGANIC 88-94

RANVIJAY PRATAP SINGH*

Department of Applied Science & Humanities, Faculty of Engineering & Technology,

KEYWORDS: UV-visible spectroscopy, Electronic transition, Woodward-Fieser rules,

1. Ultraviolet and visible spectroscopy also known as electronic spectroscopy is used to

bonding sigma orbital ( *), is represented by * transition. For example, alkanes

bonding sigma orbital or anti-bonding pi orbital ( *), are represented by * or *

transition respectively. These transitions required less energy than * transition.

n-    n-*  *

Absorption, intensity shift & UV spectrum

Absorption & Intensity Shift

UV Spectrum of an Unknown Compound

Chromophores & Auxochromes

Factor affecting absorbance & intensity

Explanation of terminology used in Woodward-Fieser rules

Table. 1: The Woodward-Fieser Rules (Calculation of max (nm) in Conjugated Dienes)

Table. 2: The Woodward-Fieser Rules (Calculation of max (nm) in α,β-Unsaturated Carbonyl

Acyclic or 6-membered cyclic ketone 215

4. Study of keto-enol tautomerism:

( *) band at 275 nm is observed in case of keto form.

5. The distinction between conjugated and non-conjugated compounds:

In isomer A, due to steric hindrance, absorption shifts towards a lower wavelength. We

Assistant Professor ,Department of Chemistry, JNTUH University College of Engineering,

If l= 1cm and A = 1cm2 the  =R

Equivalent Conductance of a solution is the product of specific conductance of a solution

4. It is used in analyzing various biological and chemical pollutants in environmental

Department of Chemistry, Chaitanya (Deemed to be University), Kishanpura,

KEYWORDS: Pigments; Separation; Column chromatography; Chlorophylls; Carotenoids.

estimation of various pigments concentrations. However, conventional chromatographic

Fig. 2: (a) Separation of β-Carotene and Xanthophylls on a Potato Starch Column by

separation of pigments in HPLC method. An eco-friendly ‚supermarket column‛ such as

SHASHIKALA KETHIREDDY1, BHASKAR PITTALA2 &

Geethanjali College of Engineering and Technology, Keesara, Rangareddy-501301

2 Nalla Narasimha Reddy Education Society’s Group of Institutions Integrated Campus

obesity drug). (Bhaskar reddy D,et al.,1998;Hartfiel U, et al., 1995;Vadim A Soloshonok,et

Fig. 1: Activity and Examples of the Drugs Containing Pyrazole Moiety

Anti-inflammatory Ex: Lonazolac, Celecoxib Anti-pyretic Ex: Phenyl butazone Anti-

Fig: 2: Structures of the Drugs Containing Pyrazole Moiety

Another significant nitrogen containing heterocyclic compounds are 1,8 Naphthyridines,

Fig. 3: Structures of the Drugs Containing 1,8 Naphthyridine Scaffold

In Knorr method, 2,6-diaminopyridine was treated with acetylacetone to give 7-amino2,4-

Scheme. I R= H, 4-Bromo, 4-Methoxy, 4-Methy, 4-Nitro

RESULTS AND DISCUSSION

bonding sigma orbital ( ), is represented by transition. For example, alkanes

bonding sigma orbital or anti-bonding pi orbital ( ), are represented by or *