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INTRODUCTION
Page 1
CHAPTER-1
INTRODUCTION
INTRODUCTION
Page 2
Section (i): A brief account on analytical chemistry and its applications
to pharmaceutical analysis
Analytical chemistry seeks ever improved means of measuring the chemical
composition of natural and artificially occurring materials in our surroundings and
environment as well as within ourselves, in our tissues and body fluids and also in
pharmaceutical dosage forms. The techniques of this science involve separation,
identification and determination of the relative amounts of the components in simple or
complex systems. The applications of analytical chemistry are extended to all the
branches of science. Recent advances in analytical chemistry are the significant
developments in the field of chemical analysis, which are versatile in nature adopting
newer techniques. The present trend includes the employment of spectrophotometry,
chromatography, polarography, neutron activation analysis, atomic absorption
spectrophotometry, ICP-atomic emission spectrometry etc. Among these,
spectrophotometric methods occupy special position due to their ease in application,
simplicity and accuracy in the determination of trace amounts of chemical compounds,
i.e. drugs, metals and non-metals.
Pharmaceutical chemistry [1-5]
is the science that makes use of general law of
chemistry to study drugs and their preparation, chemical nature, composition, structure,
influence on an organism, the method of quality control and the conditions of their
storage .etc. All around the world, a large number of varieties of organic compounds are
being synthesized daily and some of them are very useful to cure diseases, pain to get
relief from abnormalities of health in humans or animals and these chemical compounds
INTRODUCTION
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are known as pharmaceutical compounds or drugs. A pharmaceutical drug also referred
as medicine, medication or medicament, can be loosely defined as any chemical
substance intended for use in the medical diagnosis, cure treatment or prevention of
disease. [6-7]
Drug is a substance that affects the function of living cells and used in medicine
to diagnose, cure, prevent the occurrence of diseases and disorders and prolong the life of
patients with incurable conditions. Since 1900, the availability of new and more effective
drugs such as antibiotics, which fight bacterial infections, antiviral, which fight viral
infections and vaccines, which prevent diseases caused by bacterial and viruses has
increased the average humans’ life span from about 60 years to about 75 years.
Pharmaceutical drug analysis provide information on the identity, purity, contents
and stability of starting materials, excipients, active pharmaceutical ingredients(APIs)
and also the secondary pharmaceutical products i.e. the dosage forms having either
single or multi-component formulated product.[8]
The quality of the drug product may
deviate from the standard required, but in carrying out an analysis one can confirm that
whether the quality of the product is of the required standard or not [9]
. The fundamental
reasons for the “analysis of drug substances” are perhaps due to the tremendous growth in
the progress of medicinal chemistry towards achieving one ultimate objective which is to
obtain “better drugs for a better world”[10]
.Drug analysis is important in several phases of
drug development, such as formulation, stability studies, and dissolution studies and in
quality control. The importance of reliable analytical methods for the drug determination
in a fast, inexpensive, sensitive and selective way is thus evident. In this context, the
objective of this thesis is to present the main advances in the development and validation
INTRODUCTION
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of analytical methods for the determination of drugs using spectrophotometric
techniques.
QUALITY CONTROL AND ASSURANCE [11]
Quality is important in every product or service but it is vital in medicine as it
involves life. Unlike ordinary consumer goods, there can be no second quality in drugs.
Quality control is a concept, which strives to produce a perfect product by series of
measures designed to prevent and eliminate errors at different stages of production. The
word quality is derived from the Latin “Qualitas”, which means, incidentally, only the
‘nature’ and ‘inherent characteristics’ of a thing. Quality Control (QC) and Quality
Assurance (QA) are integral part of Analytical Research. "The primary aim of the new
analytical method is to produce correct results, not by chance, but at all times and the use
of quality control methods to achieve and demonstrate this." Two yardsticks are used in
QA viz. Accuracy and Precision. QA is thus by choosing an appropriate specific,
sensitive and accurate procedure with precision which involves consideration of several
other practical aspects such as the speed, economy and the skill required. Such
procedures assure both accuracy and precision and should be used by the laboratories
aiming QA and QC. In research laboratories, no compromise can be made on QA, as
today’s novel analytical method is the trendsetter for tomorrow’s routine analysis.
Internal and external quality control measures should be adopted and recorded by each
laboratory. Internal quality control measures by using controls, replications and random
sample check determine the drifts occurring in the daily tests. External quality control
methods measure periodic analysis of unknown samples from reputed QC programmes.
The variance index scores are the measure of the performance.
INTRODUCTION
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QA & QC methods developed follow standard internal operating procedures directed
toward assuring the quality, safety, purity and effectiveness of the drug supply.
In pharmaceutical analysis, a variety of methods have been used for the
estimation of the drugs in bulk and in their formulations. The methods for the estimation
of drugs are broadly divided in to physical, chemical, physicochemical and biological.
Among these four methods, physicochemical and physical methods are the most used
methods for the analysis of drugs. Physical methods of analysis consider the study of
physical properties of substance, i.e. melting, freezing and boiling points, solubility,
transparency or degree of turbidity and specific gravity. Physicochemical methods are
used to study the physical phenomena that occur as a result of chemical reactions. The
most important physicochemical methods are optical and chromatographic methods. The
optical methods are refractometry, polarimetry, photometry (colorimetry),
spectrophotometry (UV, Visible and IR regions), Flow Injection-spectrofluorimetry and
nephelometry. The chromatographic methods are column, paper, thin layer, gas-liquid,
HPLC, UPLC and RP-HPLC. Recently hyphenated techniques like HPLC with different
detectors, LC-MS, GC-MS, and LC-MS/MS are introduced for the analysis of the drugs
for achieving high sensitivity and selectivity with good precision, accuracy and
reproducibility.
Analytical method development and validation play important roles in the
discovery, development and manufacture of pharmaceuticals. The official test methods
that result from these processes are used by quality control laboratories to ensure the
identity, purity, potency and performance of the drug products. The selection of
analytical method may be based on one or more of the following design criteria,
INTRODUCTION
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accuracy, precision, sensitivity, selectivity, robustness, ruggedness, scale of operation,
analysis time, availability of equipment and cost.
Many organic compounds absorb in the ultra-violet region of the spectrum and
pre-treatment involves only separation of interferences. Some elements in the periodic
table absorb strongly in the visible or UVregions, at least in certain oxidation states and
preliminary steps may involve redox reactions as well as separations [12]
. Development of
absorption methods by means of inorganic reagents is occasionally possible. Many of the
colored complexes formed by metal ions with organic reagents are metal-chelates,
offering most impressive variety of spectrophotometric methods, and they are especially
useful in the field of trace analysis in biological fluids and in pharmaceutical dosage
forms. In some regards, the low aqueous solubility of many of the metal chelate
compounds is disadvantageous, but on the other hand, extraction of metals into non
aqueous solvents by means of chelating agents may lead to very powerful analytical
methods. Reasonable absorbance values are generally obtained with chloroform solutions
whose metal concentrations are in the order of a few µg mL-1
. The solvents used in
spectrophotometric methods poses a problem in some regions of spectrum. There is no
solvent which is transparent throughout the infrared region. However, in UV or Visible
region, most of the organic solvents have UV-cutoff points below 210 nm and water is an
excellent solvent in that region, due to transparency throughout the spectrum.
Analytical methods used in analysis
Analytical chemistry is divided into two types such as Classical and Instrumental.
Modern analytical chemistry is dominated by sophisticated instruments. The roots of
analytical chemistry and some of the principles used in modern instruments are from
INTRODUCTION
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traditional techniques many of which are still used today. These techniques also tend to
form the back bone of most undergraduate chemistry educational labs.
1. Classical methods
a) Qualitative analysis
A qualitative analysis determines the presence or absorbance of particular
compounds, but not the mass or concentration.
b) Chemical tests
There are numerous qualitative tests, for example, the acid test for the gold and
the Kastle-Meyer test for the presence of blood.
C) Flame test
It is a test where the certain metal ions especially alkali and alkaline earth metals
are identified based on the characteristic colors they exhibit in flame.
d) Gravimetric analysis
Chemical analysis is based on weighing a final product is called gravimetric
analysis. It involves determining the amount of material present by weighing the sample
before and / or after some transformations.
e) Volumetric analysis
It is refers to any procedure in which the volume of reagent needed to react with
analyte is measured. In titration, increments of reagent solution, the titrant, are added to
analyte until their reaction is complete. Form the quantity of titrant required, we can
calculate the quantity of analyte that must have been present. The most common type
INTRODUCTION
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titrations are based on acid-base, oxidation-reduction, complex formation and
precipitation reactions. In these titrations different types of indicators are used to
determine the equivalence point.
2. Instrumental methods
a) Chromatographic analysis
Chromatography is the collective term for a family of laboratory techniques for
the separation of mixtures. It involves passing a mixture dissolved in a mobile phase
through a stationary phase, which separate the analyte to be measured from other
components in the mixture and allows it to be isolated. There are several different types
of chromatographic techquines currently in use such as Paper Chromatography, Thin
Layer Chromatography (TLC), Gas Chromatography (GC), Liquid Chromatography
(LC), High performance liquid chromatography (HPLC) with different detectors, Ion
exchange chromatography (IEC) and Gel permeation or Gel filtration chromatography.
b) Electrochemical analysis
Electro analytical methods measure the potential (volts) and/or current (amps) in
an electrochemical cell containing the analyte. Electrochemistry measures the interaction
of the material with electric field. These methods can be categorized according to which
aspects of the cell are controlled and which are measured. The three main categories are
potentiometry (the difference in electrode potentials is measured), coulometry (the cell’s
current is measured over time) and voltammetry (the cell’s current is measured while
actively altering the cell’ potential).
INTRODUCTION
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C) Microscopy
The visualization of single molecules, single cells, biological tissues and
nonmaterials is an important activity in analytical science. Microscopy can be categorized
into three different fields, optical microscopy, electron microscopy and scanning probe
microscopy. Recently, this field is rapidly progressing because of the rapid development
of the computer and camera industries.
d) Spectrophotometry and colorimetry
Spectrophotometry involves the use of a spectrophotometer. A spectrophotometer
is a photometer which can measure light intensity as a function of color or more
specifically, the wavelength of light. There are many kinds of spectrophotometers, among
the most important distinctions they used to classify them are the wavelengths they work
with, the measurement technique they use, how they acquire a spectrum and the sources
of intensity variation they are designed to measure. The use of spectrophotometers is not
limited to studies in physics but they are also commonly used in other scientific fields
such as chemistry, biochemistry and molecular biology.
Direct spectrophotometric determinations such as colorimetric analysis or UV
determination are widely used in pharmaceutical analysis. The estimation of an analyte
concentration based on its absorption of ultraviolet or visible radiation is one of the most
frequently encountered quantitative analytical methods. One reason for its popularity is
that many organic and inorganic compounds have strong absorption bands in the UV or
visible radiation. An additional advantage to UV or visible absorption is that in most
cases it is relatively easy to adjust experimental and instrumental conditions so that
Beer’s law is obeyed. The applications of Beer’s law for the quantitative analysis of
INTRODUCTION
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samples in environmental chemistry, clinical chemistry, industrial chemistry, forensic and
in pharmaceutical chemistry are numerous. The scale of operation for molecular UV or
visible absorption is commonly used for the analysis and is generally better than that of
IR absorption. It is routinely used for the analysis of trace analytes in macro and meso
samples. The analysis of a sample by molecular absorption spectroscopy is relatively
rapid, although additional time may be required when it is necessary to use a chemical
reaction to transform a non absorbing analyte into an absorbing form. The cost of UV-
Visible instrumentation is relatively less. The selectivity and sensitivity of analyte
towards the absorption of light can be increased by converting it into a chromogenic
derivative, by adopting a suitable chemical reaction, which also prevents the
interferences.
Classification of functional groups in drugs [13-16]
A feature of organic drug is the presence of some specific functional groups in
their molecules. Knowing the reactions of functional groups, one can easily analyze any
organic molecule with a complicated structure.
Category Functional group Examples
1. Acidic nature
- COOH (Carboxylic) Aspirin, Ibuprofen, Cetirizine
-SH (Sulpha hydryl) Captopril, Penicillamine
OH
(Phenolic Hydroxyl)
Nelfinavir
INTRODUCTION
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(Sulphone)
Dapsone
(Amide)
Mosapride
2. Basic nature
R1 = R
2 = H;
R1 = H; R
2 = alkyl;
R1 = R
2 = alkyl groups
(Primary, Secondary and
Tertiary amino)
Repagladine, Ampicilline, and
Mesalamine,
3. Neither acidic nor
basic nature
RCR1
O
Ketone
Acuvail
(Hydroxy methyl)
Emtricitabine
NO
O Nitro
Metronidazole, Azathioprine and
Nimesulide
(Methoxy)
Albendazole
R-O-R1
(Ether)
Metoprolol
NH
O (Lactam)
Amoxicillin, Penicillin
(Acetylenic)
Norethindrone, Ethynulesteroid
INTRODUCTION
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Selection of reagents for the analysis of solutes [17]
Several papers are being published every year on the reactions and possible
applications of new and old reagents for organic analysis. The selection of an appropriate
reagent for a particular analytical situation is still a challenging problem. The choice of a
particular reagent depends on careful consideration of such factors as the scale and
economy of the reaction, the presence of other functional groups that might be adversely
affected by the reagent, the deactivation of the reaction centre by steric and electronic
effects, the instability or high reactivity of the desired product, the rate of reaction,
position of equilibrium (in the case of a reversible reaction) and other related factors.
The selection of a reagent for the determination of a particular compound is made
after a literature search for methods that have been used in parallel situations elsewhere
or that show reasonable promise for the compound under consideration. If adequate
information is not available in this way, then the reagents that act most rapidly and
stoichiometrically can be chosen after investigation of the performance of several
plausibly selected ones on a pure sample of the compound. Reagent selectivity for a
particular functional group is normally the minimum requirement. Specificity of the
reagent for a single compound containing that functional group is often desirable, not
only to isolate it from other compounds containing the same functional group, but also to
eliminate the effects of interfering compounds.
The general objective of treating a compound with a reagent (for purpose of
determination of the compound) is to get one or more derivatives having a measurable
physical or chemical property that is as completely different as possible from that of any
INTRODUCTION
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of the reactants. Some examples of properties in which a specific change may be brought
about by the reagent are as follows:
1. The reagent may bring about a change in or destroy an acidic, alkaline, oxidizing or
reducing property of the functional group, the amount of change being determined
titrimetrically or spectrophotometrically.
2. The reagent forms a product with a solubility product different from that of original
sample and this property serves as basis for the gravimetric determination or for the
isolation, concentration and purification of the compound for examination by other
analytical techniques.
3. The reagent forms a chromophore or reduces the concentration of an already existing
chromophore, the change being measured by one of the spectrophotometric techniques-
ultraviolet, infrared or visual. Many spot tests for functional groups or spray reagents in
TLC depend on the formation of a colored derivative. Similarly a fluorophore may be
generated or quenched and the change is measured fluorimetrically.
4. The reagent act on the sample to produce a gas measurable nanometrically or one that
can be collected and determined by titrimetric, gravimetric or other types of
techniques.
5. The reaction of the reagent with the sample produces a derivative that is less polar
and/or more volatile than the original sample and is thus more amenable for
determination by high performance liquid or gas liquid chromatography. Many
compounds containing polar functional groups show unfavorable properties such
as low volatility, tailing, irreversible adsorption on to column packing and thermal
INTRODUCTION
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instability. Vast improvements in these respects are easily realized because the polar
nature of the compound promotes derivatization with suitable reagents to replace the
polar group with a less polar one, giving some times a more sensitive detection
response.
6. The reagent forms derivatives with the sample suitable for structural investigation or
estimation by the application of NMR and Mass spectroscopic techniques.
7. Enzymatic treatment of the sample may catalyze a specific reaction and structural
change which may be monitored by one or more of the several analytical techniques.
INTRODUCTION
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Section (ii): SPECTROSCOPY
Spectroscopy is the branch of science that deals with the measurement and
interpretation of electromagnetic radiation (EMR) absorbed or emitted when the
molecules or the atoms or ions of the sample move from one energy state to another. At
ground state, the energy of the molecule is the sum of rotational, vibrational, and
electronic energies. EMR is made up of discrete particles or photons. EMR possess both
wave and particle characteristics. i.e. it can travel in vacuum also. The different regions
of EMR are Visible, Infra-Red, Microwaves, Radio waves, X-rays, Gamma rays, or
Cosmic rays. The energy of the radiation depends upon the frequency and the
wavelength of the radiation.
Energy of EMR:
E = h υ …………… 1.ii.1
Where; E = Energy of radiation
h = Plank’s constant (6.625 × 10 -34
J.sec-1
)
υ = Frequency of radiation
Types of spectroscopy:
1. Based on the study of the spectra obtained that may be at atomic or molecular
level:
(i) Atomic spectroscopy: where the changes in the energy takes place at atomic level.
E.g. Atomic Absorption Spectroscopy, Flame Photometry.
INTRODUCTION
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(ii) Molecular Spectroscopy: where the changes in the energy takes place at molecular
level.
E.g. UV Spectroscopy, Colorimetry, Fluorimetry, I.R. Spectroscopy etc.
2. Based on the study of the spectra obtained that may be due to absorption or
emission of the EMR:
(i) Absorption Spectroscopy: Spectroscopy involving the spectra due to the absorption
of the EMR.
E.g. UV- Visible Spectroscopy, I R-Spectroscopy etc.
(ii) Emission Spectroscopy: Spectroscopy involving the spectra due to the emission
(following the absorption) of the EMR.
E.g. Flame Photometry, Fluorimetry
3. Based on the study carried out at electronic or magnetic levels:
(i) Electronic Spectroscopy:
E.g. U.V Spectroscopy, Colorimetry, Fluorimetry
(ii) Magnetic Spectroscopy:
E.g. NMR Spectroscopy, ESR Spectroscopy.
Absorption of EMR by organic molecules:
If we pass light from a ultra-violet lamp through an organic sample, some of the
light is absorbed. In particular, some of the wavelengths (frequencies) are absorbed and
others are virtually unaffected. We can plot the changes in absorption against wavelength
INTRODUCTION
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and produce an absorption spectrum. A molecule can only absorb a particular frequency,
if there exists within the molecule an energy transition of magnitude (E = h υ).
Although almost all parts of the electromagnetic spectrum are used for studying
the matter, in organic chemistry we are mainly concerned with energy absorption from
three or four regions -Visible, Infra-Red, and Microwave and Radio frequency absorption
which is shown in Fig 1.ii.1.
Fig 1.ii.1 Electro magnetic spectrum:
Table: 1.ii.1- Spectroscopic techniques in organic chemistry:
Radiation absorbed Effect on the molecule
Ultraviolet-Visible levels
λ: 200 – 380 nm and 380 – 760 nm
Changes in the electronic energy with in the
molecules.
Infra-Red (Mid Infra-Red)
λ : 2.5 – 25 µm
Changes in the vibrational and rotational
movements of the molecule
Microwave
λ : 1 mm – 10 cm
Electronic spin resonance and electronic
paramagnetic resonance; induces changes in
the magnetic properties of the unpaired
electrons.
INTRODUCTION
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Principles of UV-Visible spectrophotometry
Basic principles of spectrophotometry [18]
Spectrophotometry is one of the advanced analytical methods used in qualitative
and quantitative analysis. It is based on the absorption of electromagnetic radiation.
Every compound has a unique characteristic relationship with electromagnetic radiation.
This is a direct process which does not change composition and properties of the
experimental solutions. Spectrophotometry is called colorimetry in the visible region of
electromagnetic spectrum. This technique is mainly used for the determination of trace
amounts of metals, non-metals and organic compounds and also used in the
determination of composition of metal complexes and stability of coordination complexes
in solution state.
The measurement of radiation frequency is done experimentally which gives a
value for the change of energy involved. From this, we can conclude the set of possible
discrete energy levels of the matter. The curve which is obtained by plotting the light
transmitted, the height of the ordinate in the plot at a wavelength is directly proportional
to the concentration of the absorbing medium.
Fig 1.ii.2 Energy Transition:
LUMO
HOMO
Excited stateGround state
E
V
h v
= *
=
The lowest energy transition is that between the highest occupied molecular
orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) in the ground
state. The absorption of the EMR excites an electron to the LUMO and creates an excited
state. The more highly conjugated the system, the smaller the HOMO
and therefore, the lower the frequ
radiation. The colors that our eyes see are typically due to highly conjugated organic
molecules. The unit of molecule that is responsible for the absorption is called the
chromophore, of which the most common are C=C (
By the interaction of UV
the energy. Thereby the molecule undergoes transition from ground state to excited state.
The intensity of absorption depends upon the concentration and path length as given by
the Beer-Lambert’s Law.
Fig 1.ii
Laws of photometry:
When the light passes through a homogeneous transparent medium, a part of
incident light is reflected, a part is absorbed by the medium, and the rest is allowed to
INTRODUCTION
The lowest energy transition is that between the highest occupied molecular
orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) in the ground
state. The absorption of the EMR excites an electron to the LUMO and creates an excited
more highly conjugated the system, the smaller the HOMO-
and therefore, the lower the frequency, the longer the wavelength (λ) of the absorbed
The colors that our eyes see are typically due to highly conjugated organic
The unit of molecule that is responsible for the absorption is called the
chromophore, of which the most common are C=C (π → π*) and C=O (n →
By the interaction of UV-visible radiation with matter, only the valence electron absorbs
the molecule undergoes transition from ground state to excited state.
The intensity of absorption depends upon the concentration and path length as given by
Lambert’s Law.
1.ii.3 Spectrophotometer common features:
When the light passes through a homogeneous transparent medium, a part of
incident light is reflected, a part is absorbed by the medium, and the rest is allowed to
INTRODUCTION
Page 19
The lowest energy transition is that between the highest occupied molecular
orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) in the ground
state. The absorption of the EMR excites an electron to the LUMO and creates an excited
-LUMO gap, ∆E,
) of the absorbed
The colors that our eyes see are typically due to highly conjugated organic
The unit of molecule that is responsible for the absorption is called the
*) and C=O (n → π*) systems.
nce electron absorbs
the molecule undergoes transition from ground state to excited state.
The intensity of absorption depends upon the concentration and path length as given by
When the light passes through a homogeneous transparent medium, a part of
incident light is reflected, a part is absorbed by the medium, and the rest is allowed to
INTRODUCTION
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transmit as such. If Io denotes the intensity of incident light, Ir, that of the reflected light,
Ia , that of the absorbed light and It, that of the transmitted light,
We can write
Io = Ia + It + Ir ……..... (1.ii.2)
In spectrophotometric measurements, comparison cells are used for sample and
reference so the Ir is eliminated. Then
Io = Ia + It ………... (1.ii.3)
The fraction of incident radiation passing through a medium is governed by two
laws, known as Lambert’s law and Beer’s law.
Lambert’s law
According to this law, “when a beam of monochromatic light is allowed to pass
through a transparent medium, the rate of decrease of intensity with the thickness of
medium is directly proportional to the intensity of the incident light.”
This law may be expressed mathematically as follows.
���� �� ……………………(1. ii. 4)
or
���� = �� ……………………(1. ii. 5)
Where I denote the intensity of incident light of wavelength λ, ‘l’ denotes the thickness of
the medium and ‘k’ denotes the proportionality factor.
INTRODUCTION
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The negative sign indicates that ‘I’ decreases as ‘l’ increases. On integrating
equation (1.ii.5) and putting I = Io when l = 0, we get
�� ���� = ��⋯⋯⋯⋯⋯⋯(1. ii. 6)
or
�� = ������⋯⋯⋯⋯⋯⋯(1. ii. 7) Changing from natural to common logarithms, we get
�� = ������.������ = ��������
Where
�� = intensity of the incident light
�� = intensity of the transmitted light
�= thickness of the medium
�� = absorption coefficient.
The ratio ���� is termed as the transmittance T, and its reciprocal
���� is termed as
opacity. The ratio � ! " "# is termed as absorbance, A.
A = � ! ���� ⋯ ⋯⋯⋯⋯⋯⋯⋯(1. ii. 8)
INTRODUCTION
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Beer’s law
This law states that “the intensity of a beam of monochromatic light decreases
exponentially with the increase in concentration of the absorbing medium arithmetically.”
This is expressed as
−���& = ���� ⋯⋯⋯⋯⋯⋯(1. ii. 9)
�� = ������.�������& = ���������& ⋯⋯⋯⋯(1. ii. 10)
Where
c = concentration of the absorbing medium
����= constant
By adding Lambert’s and Beer’s laws, we get
)* = )+10�,-. ⋯⋯⋯⋯⋯⋯⋯(1. ii. 11)
log )+)* = acl ⋯⋯⋯⋯⋯⋯⋯(1. ii. 12)
Where
‘a’ is new proportionality constant.
Since, log ���� = 5,
We can rewrite the above expression as
A = acl ⋯⋯⋯⋯⋯⋯⋯(1. ii. 13)
INTRODUCTION
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In the above equation ‘a’ is called absorptivity when the concentration ‘c’ is
expressed in g L-1
. If ‘c’ is expressed in moles L-1
and the thickness of the medium ‘l’ in
cm, we write ‘ε’ in the place of’a’.
Thus
A = ε;< ⋯⋯⋯⋯⋯⋯⋯(1. ii. 14)
Where, ε is molar absorptivity.
The absorbance of a solution is an additive property. If there are more than one absorbing
species present in the solution, the total absorbance is equal to the sum of all the
absorbance values of the components present in the solution.
This can be expressed as follows,
A = =εc> + εc@ + εcA + εcB⋯⋯⋯+ εcCD = EεFcFC
FGH⋯⋯⋯⋯(1. ii. 15)
Where, the path length is kept constant.
Ringbom’s plot [19]
Ringbom introduced a new method of plotting spectrophotometric data in the year
1939. He plotted the percent absorbance (P) against logarithm of concentration (log C).
Absorbance = I - Transmittance (T) and
% Absorbance = 100 - % transmittance.
An S̕ ̕ shaped sigmoid curve would be obtained. Ringbom proposed that the
accuracy of an analytical determination based on the spectrophotometric method is
INTRODUCTION
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greatest when ΔP/(ΔC/C) reaches a maximum value, where ΔP is photometric error and
ΔC/C is relative analysis error.
ΔP/(ΔC/C) Can be equated to ΔP/2.303.
The Ringbom plot has two outstanding features.
1. The analysis error is least in the concentration range of determination. This is the
concentration range corresponding to the nearly linear portion or the steep slope
of the plot.
2. At any concentration, the accuracy of the analysis can be easily evaluated. The
relative analysis error is obtained on dividing 230 by the slope of the curve at that
point.
Sensitivity of spectrophotometric methods
The term sensitivity implies the smallest determinable concentration or the
amount of species concerned. In photometry it refers to the slope of the calibration curve
plotted between the concentration and absorbance value. Numerically, it is expressed as
molar absorptivity at the wavelength of maximum absorbance.
ε = Abc⋯⋯⋯⋯⋯⋯(1. ii. 16)
Where
A= absorbance
b = path length in centimeters,
c = concentration in moles L-1
.
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The molar absorptivity is expressed in L mole-1
cm-1
in CGS units and in
mole-1
nm-1
in SI units.
The methods [20]
for which ε > 1 x 104 are considered to be sensitive while others
with ε < l x 103 is termed as less sensitive methods. As per the quantum theory, εvalues
never exceed 1.5 x 105. In some indirect methods such as amplification methods higher
value of εcan be obtained.
Sensitivity can also be expressed in terms of specific absorptivity (a) which is
related to molar absorptivity (ε) as
a = εAt. wt × 1000 ⋯⋯⋯⋯⋯⋯(1. ii. 17)
Sandell’s [21]
expressed the sensitivity by a quantity S known as Sandell’s
photometric sensitivity.
S = 10�A
a ⋯⋯⋯⋯⋯⋯⋯(1. ii. 18)
The units of Sare µg cm-2
.
INSTRUMENTATION:
1. Source of Radiation:
UV-Visible spectrophotometer uses two light sources, a deuterium (D2 ) lamp for
ultraviolet light and a tungsten (W) lamp for visible light.
2. Collimating System:
It makes the light beam parallel or focused. It includes lenses, mirrors and slits.
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3. Monochromator (Filter):
It converts the polychromatic radiation to monochromatic radiation. It consists of
filters, prisms and gratings.
4. Sample Compartment:
It is to permit radiation to pass through the sample. These are made of quartz,
fused silica glass.
5. Frequency Analyzer:
Which separates out all the individual frequencies generated by the source (the
most familiar being the triangular glass prism as used by Isaac Newton for visible light)
6. Detector:
For measuring the intensity of the radiation at each frequency, allowing the
measurement of how much energy has been absorbed at each of these frequencies by the
sample. e.g. Barrier layer cells, Photo emissive tubes, Photomultiplier tubes etc.
7. Recorder:
A pen recorder or a computerized data station, with a VDU for initial viewing of
the spectrum
INTRODUCTION
Page 27
Fig 1.ii.4- UV/Vis – spectrophotometer optical design
8. Applications:
1. Quantitative determination of chromophore concentrations in the solution.
2. Impurity determination by spectrum subtraction.
3. Structural elucidation of organic compounds (saturation and unsaturation, and the
extent of aromaticity).
4. Determination of pKa values of indicators.
5. Determination of molecular weights of amines.
6. Identification of cis-trans isomers: Trans isomer absorbs less energy than cis isomer.
7. Chemical kinetics: The rate of reaction is calculated as the change in absorbance per
unit time.
8. Qualitative and quantitative analysis of materials used in dye and pharmaceutical
industries.
INTRODUCTION
Page 28
Analytical Method Validation:
Accuracy [23]:
The accuracy of an analytical method is the degree of agreement of results
generated by the method to the true value or a conventional true value. Accuracy can be
assessed by applying the analytical method to samples or mixtures of sample matrix
components to which known amounts of the analyte have been added, above and below
the normal levels expected in the samples. Method accuracy is the agreement between the
differences in the measured analyte concentrations of the fortified (spiked) and
unfortified samples and the known amount of analyte added to the fortified sample.
Comparison of the method’s results can be performed by using an established reference
method, assuming that the latter is free from systematic errors. Second, accuracy can be
measured by analyzing a certified reference material, and comparing the measured value
with the true value as supplied with the material. If such reference material is not
available, a blank sample matrix can be spiked with a known concentration that should
cover the range of concern, including one concentration close to the quantization limit.
The expected recovery depends on the sample matrix, on the sample processing
procedure, and on the analyte concentration.
Linearity:
The linearity of an analytical method is its ability to elicit test results that directly,
or by means of well-defined mathematical transformation, is proportional to the
concentration of analyte in samples within a given range. Frequently, the linearity is
evaluated graphically in addition or alternatively to mathematical evaluation. The
deviations from linearity are minimized by Regression analysis; a statistical technique
INTRODUCTION
Page 29
which provides the means for objectively obtaining a linear line with minimum deviation
between the plot and data. For linear ranges, the deviations should be equally distributed
between the positive and negative values. The minimum concentration for the working
range is known as the Limit of Detection (LOD) where as, the maximum concentration is
called the Limit of Linearity. It is not always possible to obtain a linear graph due to
interferences, signal noise, sample matrix or deviations from Beer’s Law. However,
several programs are commercially available for computing the best fit curve for the data
set.
Significant Figures:
All measurements carry some degree of uncertainty. The degree of uncertainty
depends upon both accuracy of the measuring device and skill of its operator. In order to
determine the number of significant digits in a measurement or number, the following
rules may be applied:
1. If there is no decimal point, the right-most non-zero digit is the least significant digits.
2. In case of numbers that include a decimal point, the right-most non-zero digit is the
least significant digit regardless of its value.
Significant digits should not be considered the same as uncertainty in
measurement systems. They are used for manipulating experimental data, but not for
expressing uncertainty. Significant figures have economic significance simply because
the more the significance numbers expected from the measurement, the more expensive
will be the equipment and higher the quality of reagents required. Care should therefore,
be taken to properly define the optimum number of significant figures when attempting
an analysis.
INTRODUCTION
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Other Performance Parameters:
Sensitivity:
The sensitivity of an instrument is the change in output signal while a change in
the physical parameter is being measured. It is determined with the help of the calibration
curves. The sensitivity is constant over the entire range in a linear response system and is
found to be changing with respect to the concentration in case of non-linear systems. In
case of analytical instruments, the sensitivity is usually expressed as the concentration of
analyte required to cause a given instrument response.
Selectivity:
The selectivity of an analytical method is defined as its ability to accurately
measure an analyte in the presence of interference that may be expected to be present in
the sample matrix.
Range:
The range of an analytical method is the interval between the upper and lower
levels (including these levels) that have been demonstrated to be determined with
precision, accuracy and linearity by using the specified method. The range is normally
expressed in the same units as the test results (e.g. percentage, parts per million etc.)
obtained by the analytical method.
Limit of Detection (LOD):
The LOD is the point at which a measured value is larger than the uncertainty
associated with it. It is the lowest concentration of analyte in a sample that can be
detected but not necessarily quantified.
INTRODUCTION
Page 31
LOD = 3.3σS ⋯⋯⋯⋯⋯(1. ii. 19)
Units for limit of detection is µg mL-1
Where,
σ = Standard deviation of the response.
S = Slope of the calibration curve.
The slope ‘S’ may be estimated from the calibration curve of the analyte.
The estimate of σ may be carried out in a variety of ways.
Limit of Quantification (LOQ):
LOQ =10σS ⋯⋯⋯⋯⋯(1. ii. 20)
Units for limit of Quantification is µg mL-1
INTRODUCTION
Page 32
Section (iii): TYPES OF REACTIONS AND REAGENTS INVOLVED
IN THE PROPOSED SPECTROPHOTOMETRIC
METHODS
Different types of reactions are applied in the present proposed methods for the
determination of the following selected pharmaceutical drugs. A brief discussion of these
reactions and the reagents used to bring about these reactions are presented here.
Table 1.iii.1 Different types of the proposed methods.
S.No Name of the drug
determined
Method Reagent(s) used
1
Atazanavir(AZV)
AZV-BTB Bromothymol blue (BTB); Phthalate buffer
2 AZV-BPB Bromophenol blue(BPB) ; Phthalate buffer
3 AZV-BCG Bromocresol green(BCG) ;Phthalate buffer
4
Emtricitabine
(EMT)
EMT-PDAB P-Dimethylaminobenzaldehyde(PDAB);
H2SO4
5 EMT-MBTH 3-Methyl 2-benzothiazolinone hydrazone
(MBTH) ; FeCl3
6
EMT-HMBA 4-Hydroxy-3-methoxybenzaldehyde
(HMBA) ; H2SO4
7
Eplerenone(EPL)
EPL-BTB Bromothymol blue(BTB); Phthalate buffer
8 EPL- BPB Bromophenol blue(BPB); Phthalate buffer
9 EPL-BCG Bromocresol green(BCG); Phthalate buffer
10
Olopatadine(OLO)
OLO-BTB Bromothymol blue(BTB);Phthalate buffer
11 OLO- BPB Bromophenol blue(BPB);Phthalate buffer
12 OLO-BCG Bromocresol green(BCG); Phthalate buffer
13
Pemetrexed
disodium (PMD)
PMD-PDAC P-Dimethylaminocinnamaldehyde(PDAC)
; H2SO4
14 PMD-HMBA 4-Hydroxy-3-methoxybenzaldehyde
(HMBA); H2SO4
15 PMD-MBTH 3-Methyl2-benzothiazolinone hydrazone
(MBTH) ; cerric ammonium sulphate
INTRODUCTION
Page 33
16
Zolmitriptan(ZOL)
ZOL-PDAC P-Dimethylaminocinnamaldehyde(PDAC);
H2SO4
17 ZOL-PDAB P-Dimethylaminobenzaldehyde(PDAB);
H2SO4
18 ZOL-NQS 1,2-Napthoquinone-4Sulphonic acid
(NQS); NaOH
Oxidative coupling reactions:
Oxidative coupling procedure involving the use of 3-methy1-2-benzothiazolinone
hydrazone (MBTH) in the presence of an appropriate oxidant under acidic, neutral or
alkaline conditions to form highly colored products were explored for the determination
of some of the mentioned active pharmaceutical ingredients possessing amenable
structural features.
MBTH – Oxidant: (EMT and PEM)
The reported applications of 3-Methyl-2-benzothiazolinone hydrazone (MBTH)
include the detection and determination of phenols[24-26],
formaldehyde[27]
and other
aldehydes[28,29]
, polyhydroxy compounds[30,31],
aromatic amines including heterocyclic
amines such as antipyrine and amidopyrine,[32]
iminohetero aromatic compounds, indoles,
carbazoles and phenothiazines[33].
MBTH was also used for the indirect determination of
compounds such as glycerides [34]
and glycosamine glycans [35]
etc., which yield one of
the above mentioned groups on preliminary treatment with certain reagent.
The reaction of MBTH with phenols or amines was found to be based mainly on
the nature of the oxidant and acidic, alkaline or neutral conditions employed. Friested.et
al[36]
initially carried out, the coupling under alkaline conditions with ferricyanide, but a
modification, to use cerric ammonium sulphate in acidic medium was successful and led
to an increased sensitivity.[25]
Later, Umeda [37]
carried out the reaction using a basic
INTRODUCTION
Page 34
solvent (triethanolamine) with phenols. El-Kommos [25]
suggested cerric ammonium
sulphate as an oxidant under acidic conditions for the determination of 17 pharmaceutical
phenols. Ferric chloride has been mostly used for the determination of aromatic and
heteroaromatic amines by Sawicki.et al [33]
(in neutral conditions) and by Pays.et. al [32]
(in acidic conditions). Other oxidants such as periodate (acidic conditions), ammonium
persulphate (alkaline conditions) and potassium dichromate (acidic conditions) were used
for the determination of ethylenic compounds and primary alcohols [38]
(after oxidation
with ruthenium textraoxide) and phenidone [39].
Recently, Sastry and Sastry reviewed
various aspects of MBTH chemistry in pharmaceutical analysis [40].
Under the reaction conditions, on oxidation MBTH loses two electrons and one
proton, forming an electrophilic intermediate which has been postulated to be the active
coupling species.[41-45]
This intermediate undergoes electrophilic substitution with the
drug to form the colored product. It also reacts with phenol or amine by electrophilic
attack on the most nucleophilic site on the aromatic ring of the phenol or amine (i.e.
Para-position to phenolic hydroxyl or amino group, if it is either free or substituted with
electron withdrawing substituent like -Cl, -COOH etc.; where Para-position is substituted
with electron releasing groups, coupling proceeds through ortho-position to phenolic
hydroxyl or amino group) and the oxidant to form the colored species.
The absorption maximum and molar absorptivity values of the colored species are
influenced by the nature (inductive, mesomeric or steric effects) of other substituents
present in the compound besides the desired substitutes, the oxidizing agent used, the
solvent employed and the pH of the medium. Some of the antiviral and anticancer agents
under investigation (Emtricitabine and Pemetrexed disodium) possess structural features
INTRODUCTION
Page 35
that are likely to enable them to react with MBTH. Attempts were made by the present
author to study the suitability of MBTH reagent for the estimation of these two drugs.
The effect of the concentration of the regent (MBTH with different oxidants) pH,
temperature, time and order of addition of reagents with respect to maximum sensitivity,
minimum blank and obedience to Beer’s law have been investigated and the results are
recorded in chapter 4 of EMT and chapter 7 of PEM in the thesis.
Ion association complex formation reactions: (AZV, EPL and OLO)
Ion-association is a chemical reaction where by ions of opposite electrical charge
come together in solution to form a distinct chemical entity. Ions of opposite charge are
naturally attracted to each other by the electrostatic force. This is described by coulomb’s
law.
F=
q1q2
ε r2
F is the force of attraction, q1 and q2 are the magnitudes of the electrical charges,
and ‘ε’ is the dielectric constant of the medium and ‘r’ is the distance between the
ions[46].
Sulphonaphthalein dyes are commonly used as anionic dyes to form ion-pair
complexes with the nitrogenous compound present in positively charged protonated
forms. Ion-pair complexation reaction has been applied for the spectrophotometric
determination of many organic compounds [47-52]
.
The term molecular complex is used to describe a variety of types of association
products of two or more molecules. In recent years, extensive attention has been given to
a large group of complexes formed by weak interaction of certain classes of organic
compounds functioning as electron donors (bases), with others which act as electron
INTRODUCTION
Page 36
acceptors (acids) [53-55]
. The forces which lead to the formation of molecular complex
include physical forces such as dipole and induced dipole interactions, London dispersion
forces, hydrogen bonding and dative bonding interactions. The donor-acceptor complexes
(whose composition can be represented by integral ratio of the components) are, in many
instances, so unstable that they cannot be isolated in the pure state at ordinary
temperatures but exist only in solutions in equilibrium with their components. They can,
however, usually be detected readily because of differences in physical properties (e.g.
Absorption spectra, solubility in organic solvents) from those of the pure components.
The ion-association complex or adduct (commonly known as ion pair, if two ions
are involved, colored, neutral and extractable into organic solvents) is a special form of
molecular complex resulting from two components extractable into organic solvents from
aqueous phase at suitable pH. One component is a chromogen (dye or metal complex)
possessing charge (cationic or anionic in nature) and so it is insoluble in organic solvents.
The second component is colorless, possessing opposite charge to that of chromogen.
Acid dyes such as bromothymol blue, bromophenol blue, bromocresol green,
mordant black-III, solochrome black-T, metanil yellow dye, bromocresol red, eosin,
methyl orange, phenol red, alizarin violet 3B, alizarin brilliaint violet R, and fast green
FCF have been used arbitrarily by various workers for the determination of components
exhibiting basic properties e.g. amines, quaternary ammonium compounds and
heterocyclic compounds[56-62]
. Some basic dyes (parent moiety - xanthene, triphyenyl
methane, azine, oxazine and thiazine) and metal complexes have been tried by some
workers for the determination of components exhibiting acidic properties [63-65].
INTRODUCTION
Page 37
In the present investigations, it was found that atazanavir (AZV), eplerenone
(EPL) and olopatadine (OLO) form ion-association complexes with the bromothymol
blue, bromophenol blue, and bromocresol green, in the presence of phthalate buffer
(pH 2.4).The formed respective colored ion-association complexes are extracted into
chloroform and the analytical results obtained in these methods are reported in
chapters-3, 5 and 6.
Condensation reactions using aromatic aldehydes like PDAC, PDAB and HMBA in
acidic media (EMT, PEM and ZOL)
PDAB, PDAC and HMBA are used as analytical reagents [66-68]
for the detection
and spectrophotometric determination of aromatic and aliphatic primary and secondary
amines. PDAC and PDAB has demonstrated a wide usefulness as a chromogenic reagent
for spectrophotometric analysis of several compounds such as urea, thiourea and their
N-alkyl/aryl derivatives[69]
, aceclofenac[70]
, sodium diclofenac and oxyphenbutazone
[71],
glafenine and metoclopramide[72]
. PDAC and PDAB have also been applied for micro
plate-based assay of p-aminohippuric acid [73]
in plasma and urine samples, and for rapid
testing of benzodiazepines [74]
, nimesulide [75]
, satranidazole [76]
and sympathomimitic
amines in pharmaceuticals. The use of modified dimethylaminocinnamaldehyde has also
been described for the analysis of flavanols in wines. [77]
The majority of the methods
mentioned above involve a reaction in acid medium with heating to produce coloured
compounds (ranging from orange to red or pink).
In the present study, PDAB, PDAC and HMBA form orange red or red colored
chromogens with zolmitriptan, emtricitabine and pemetrexed Disodium. The reaction
between PDAB or PDAC with secondary amino group of zolmitriptan is assumed to take
INTRODUCTION
Page 38
place via condensation of amino group with the carbonyl group of the reagent to produce
Schiff’s bases or enamines [78-82].
The primary amino group of emtricitabine and
pemetrexed disodium is involved in the condensation process with PDAB, HMBA and
PDAC to form Schiff’s base complexes between these drugs in acidic medium.
The attempts were made by the author to study the suitability of the reagents for
the estimation of these three drugs. The effect of the concentration of the regents,
temperature, time and order of addition of reagents with respect to maximum sensitivity,
minimum blank and obedience to Beer’s law have been investigated and the results are
recorded in Chapter-4, Chapter-7 and Chapter-8 which are to a large extent based on
reactions suggested in the literature.
Reaction of 1, 2-naphthoquinone-4-sulfonic acid (NQS) with zolmitriptan (ZOL):
1, 2-Napthaquinone-4-sulphonic acid (NQS) is a reagent widely applied for
analytical determinations of amines and amino acids by using UV-Visible
spectrophotometric method. NQS is readily able to react in basic medium and at
moderate temperatures with both primary and secondary amino groups to form colored
derivatives, which were determined by spectrophotometrically. The reagent (NQS) was
first used as a colorimetric reagent by Folin [83]
for the determination of amino acids in an
alkaline solution to form highly colored complexes. Later on, this reagent was employed
for the spectrophotometric determination of many amino drugs [84-110].
In the present investigation, the author utilized the reagent, NQS to estimate the
drug zolmitriptan in bulk and in its pharmaceutical formulations and the results are
reported in Chapter-8.
INTRODUCTION
Page 39
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