3. project final edit

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Department of Pharmaceutical Analysis Page 1
INTRODUCTION
Analytical chemistry:
Modern analytical chemistry is dominated by instrumental analysis. An effort to develop
a new method might involve the use of a tunable laser to increase the specificity and sensitivity
of a spectrometric method. Analytical chemistry plays an increasingly important role in the
pharmaceutical industry where, aside from QA, it is used in discovery of new drug candidates
and in clinical applications where understanding the interactions between the drug and the
patient are critical.
Analytical chemistry can be split into two main types,
 Qualitative and
 Quantitative
Qualitative inorganic analysis seeks to establish the presence of a given element or inorganic
compound in a sample.
Qualitative organic analysis seeks to establish the presence of a given functional group or
organic compound in a sample.
Quantitative analysis seeks to establish the amount of a given element or compound in a
sample
ANALYSIS
Qualitative
Analysis
Inorganic Organic
Quantitative
Analysis

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Most modern analytical chemistry is quantitative. Quantitative analysis can be further split into
different areas of study. The material can be analyzed for the amount of an element or for the
amount of an element in a specific chemical species. The latter is of particular interest in
biological systems; the molecules of life contain carbon, hydrogen, oxygen, nitrogen, and others,
in many complex structures
The complete analysis of a substance consists of 5 main steps.
1. Sample preparation/sampling.
2. Dissolution of the sample, conversion of the analyze in to a form suitable for
measurement.
3. Measurement.
4. Calculation and interpretation of the measurement.
Techniques:
There are bewildering arrays of techniques available to separate, detect and measure
chemical compounds.
A) Based on suitable chemical reaction:
Eg: Neutralisation (Acid-Base reaction), Complex forming reaction, Precipitation reaction,
Oxidation-Reduction reaction.
B) Appropriate electrical measurement of current, voltage or resistance in relation to the
concentration of a certain species in solution
Eg: Voltametry, Potentiometry, Conductometry.
C) On the emission of radiant energy and the measurement of the amount of energy of a
particular wavelength emitted.
Eg: Visible spectrophotometry, Ultraviolet spectrophotometry, Infrared spectrophotometry.

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D) Chromatography:
For the separation of mixture of substances and also for identification of components.
Eg: Gas Chromatography, HPLC, and HPTLC
E) Mass spectrometry:
It is used to determine the molecular mass, the elemental composition, structure and
sometimes amount of chemical species in a sample by ionizing the analyte molecules and
observing their behavior in electric and magnetic fields.
F) X-Ray methods:
When high speed electrons collide with a solid target, X-rays are produced. From the
emitted X-rays, it is possible to identify certain peaks which are characteristics of elements.
G) Radioactivity:
It involves measurement of radiation from a natural radioactive substance arising from
exposure of sample to a neutral source.
H) Optical methods:
1) Refractometer - Based on measurement of refractive index of liquids
2) Optical rotation - For optically active compounds.
I) Thermal Analysis:
Changes in weight and energy are recorded as a function of temperature.
Eg: Thermogravimetry, Differential Scanning Colorimetry.

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Concept of Electromagnetic radiation:
The electromagnetic spectrum is a continuum of all electromagnetic waves arranged
according to frequency and wavelength. Electromagnetic energy passes through space at the
speed of light in the form of sinusoidal waves. The wavelength is the distance from wave crest to
wave crest.
Wavelength, Frequency and Speed of light:
The distance between two crests is called wavelength of light.
Number of crests passing through a particular point per second is the frequency of light.
Units: Cycles per second or Hertz (Hz).
Light has a constant speed through a given substance.
Light always travels at a speed of approximately 3 x 108
meters per second in vacuum.
This is actually the speed that all electromagnetic radiation travels - not just visible
light.Relationship between wavelength and frequency of a particular color and speed of
light is given by :

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If you increase the frequency, you must decrease the wavelength and vice versa.
The frequency of light and its energy:
Each particular frequency of light has a particular energy associated with it. It is given by
another simple equation:
The higher the frequency, higher is the energy of light.
Electromagnetic spectrum covers an extremely broad range, from radio waves with wave
lengths of a meter or more, down to x-rays with wave lengths of less than a billionth meter. The
visible portion occupies an intermediate position, exhibiting both wave and particle properties in
varying degrees. Like all electromagnetic waves, light waves can interfere with each other,
become directionally polarized, and bend slightly when passing an edge. These properties allow
light to be filtered by wave length.
Diagram of electromagnetic spectrum:

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Visible light:
Above infrared in frequency comes visible light. Visible light (and near-infrared light) is
typically absorbed and emitted by electrons in molecules and atoms that move from one energy
level to another. Electromagnetic radiation with a wavelength between 380 nm and 760 nm
(790–400 terahertz) is detected by the human eye and perceived as visible light. Other
wavelengths, especially near infrared (longer than 760 nm) and ultraviolet (shorter than 380 nm)
are also sometimes referred to as light, especially when the visibility to humans is not relevant.
Violet : 400 - 420 nm
Indigo : 420 - 440 nm
+Blue : 440 - 490 nm
Green : 490 - 570 nm
Yellow: 570 - 585 nm
Orange: 585 - 620 nm
Red : 620 - 780 nm

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Ultraviolet light:
Next in frequency comes ultraviolet (UV). This is radiation whose wavelength is shorter
than the violet end of the visible spectrum, and longer than that of an x-ray. Being very energetic,
UV can break chemical bonds, making molecules unusually reactive or ionizing them, in general
changing their mutual behavior. Sunburn, for example, is caused by the disruptive effects of UV
radiation on skin cells, which is the main cause of skin cancer, if the radiation irreparably
damages the complex DNA molecules in the cells. However, most of it is absorbed by the
atmosphere's ozone layer before reaching the surface.
Spectroscopy:
Spectroscopy was originally the study of the interaction between radiation and matter as a
function of wavelength (λ).
Separation of light by a prism according to wavelength

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Spectrometry is the spectroscopic technique used to assess the concentration or amount of a
given species.
PRINCPLES OF SPECTROSCOPY:
Based on the principle of absorption and emission of light they are classified as:
Absorption spectroscopy uses the range of the electromagnetic spectra in which a
substance absorbs. This includes atomic absorption spectroscopy and various molecular
techniques, such as infrared, ultraviolet-visible and microwave spectroscopy.
Emission spectroscopy uses the range of electromagnetic spectra in which a substance
radiates (emits). The substance first must absorb energy. This energy can be from a
variety of sources, which determines the name of the subsequent emission, like
luminescence. Molecular luminescence techniques include spectrofluorimetry.

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Ultra Violet Spectroscopy:
Principle:
Any molecule has n, π or σ or a combination of these electrons. These bonding (σ & π)
and non-bonding (n) electrons absorb the characteristic radiation and undergoes transition from
ground state to excited state. By the characteristic absorption peaks and the nature of the
electrons present, the molecular structure can be elucidated.
There are three distinct types of electrons involved in organic molecules. These are as follows:
1) σ – Electrons:
These electrons are involved in saturated bonds, such as those between carbon and
hydrogen in olefins. These bonds are known as sigma bonds. As the amount of energy required
to excite sigma electrons is much more than produced by UV light, compounds containing sigma
bonds do not absorb UV radiation. For this reason paraffin compounds are frequently very useful
as solvents.
2) π – Electrons:
These electrons are involved in unsaturated hydrocarbons. Typical compounds with π-
bonds are trienes and aromatic compounds.
3) n – Electrons:
These electrons are not involved in bonding between atoms in molecules. Examples are
organic compounds containing nitrogen, oxygen or halogens. As n- electrons can be excited by
UV radiation any compound that contains atoms like nitrogen, oxygen, sulphur, halogen
compounds or unsaturated hydrocarbons may absorb UV radiation.

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It was stated earlier that π, n and σ electrons are present in a molecule and can be excited
from ground state by the absorption of UV radiation. The various transitions are n→π*, n→σ*,
π→π*, σ→σ*. The different energy states associated with such transitions can be given by the
diagram .
The possible electron jumps that might cause are:

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Types of electro-magnetic transition:
1. n→π*: Of all the types of transitions, n→π* transition requires the lowest energy. The peaks
due to this transition are also called as R-bands. This type of peak can be seen in compounds
where „n‟ electrons (present in S,O, N or halogens) is present in a compound containing double
bond or triple bond (e.g.) aldehydes or ketones , nitro compounds etc
2. π- π*: These types of transition give rise to B, E and K bands.
Type Due to
B-bands (benzenoid bands) Aromatic and Hetero aromatic systems
E-bands (ethylenic bands) Aromatic systems
K-bands (π- π*) Conjugated systems
3. n→σ*: This transition occurs in saturated compounds with hetero atoms like S, O, N or
Halogens. The peaks due to this transition occur from 189nm to 250nm.
e.g.: Methylene chloride, Ethanol, Water, Methanol, Ether…
4. σ→σ*: This is observed with saturated compounds. The peaks do not appear in UV region,
but will occur in vaccum UV region (i.e.) 125-135nm.
Instrumentation
The various components of a UV spectrometer are as follows.
1 Radiation Source:
In ultraviolet spectrometers, the most commonly used radiation sources Are hydrogen or
deuterium lamps, the xenon discharge lamps and mercury arcs. In all sources, excitation is done
by passing electrons through a gas and these collisions between electrons and gas molecules may

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result in electronic, vibrational and rotational excitation in the gas molecules. When the pressure
of the gas is low, only line spectra are emitted. But, if the pressure of gas is high, band spectra
and continuous spectra will be obtained.
The following are requirements of a radiation source.
(i) It must be stable
(ii) It must be of sufficient intensity for the transmitted energy to be detected at the end of
the optical path.
(iii) It must supply continuous radiation over the entire wavelength region in which it is
used.
The various radiation sources are as follows:
The two most common radiation sources are tungsten lamps and hydrogen discharge lamps.
(i) Tungsten lamp:
The tungsten lamp is similar in its functioning to an electric light bulb. It is a tungsten filament
heated electrically to white heat. It has two shortcomings. The intensity of radiation at short
wavelengths (<350 mm) is small. Furthermore, to maintain a constant intensity, the electrical
current to the lamp must be carefully controlled. However, the lamps are generally stable,
robust, and easy to use. Typically, the emission intensity varies with wavelength.
(ii) Hydrogen discharge lamps.
In these lamps, hydrogen gas is stored under relatively high pressure. When an electric
discharge is passed through the lamp, excited hydrogen molecules will be produced which emit
UV radiations. The high pressure in the hydrogen lamps causes the hydrogen to emit a
continuum rather than a simple hydrogen spectrum.
Hydrogen lamps cover the range 3500-1200 Å. These lamps are stable, robust and
widely used. The hydrogen discharge lamp consists of hydrogen gas under relatively high
pressure through which there is an electrical discharge. The hydrogen molecules are excited
electrically and emit UV radiation. The high pressure brings about many collisions between the

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hydrogen molecules, resulting in pressure broadening. This causes the hydrogen to emit a
continuum (broad band) rather than a simple hydrogen line spectrum. The lamps are stable,
robust, and widely used. If deuterium (D2) is used instead of hydrogen, the emission intensity is
increased by as much as a factor of 3 at the short-wavelength end of the UV range. Deuterium
lamps are more expensive than hydrogen lamps but are used when higher intensity is required.
(iii) Deuterium lamps. If deuterium is used in place of hydrogen, the intensity of
radiation Emitted is 3 to 5 times the intensity of a hydrogen lamp of comparable design and
wattage.
(iv) Xenon discharge lamps. In these lamps, xenon gas is stored under pressure in
the range Of 10-30 atmospheres. The xenon lamp possesses two tungsten electrodes separated
by about 8 mm. When an intense arc is formed between two tungsten electrodes by applying a
low voltage, the ultraviolet light is produced.
The intensity of ultraviolet radiation produced by xenon discharge lamp is much greater than
that of hydrogen lamp.
(v) Mercury arc. In this, the mercury vapour is under high pressure, and the
excitation of Mercury atoms is done by electric discharge. The mercury arc, a standard source
for much ultraviolet work, is generally not suitable for continuous spectral studies because of the
presence of sharp lines or bands.
Generally, the low pressure mercury arc is very useful for calibration.
2 Monochromators:
The monochromator is used to disperse the radiation according
to the Wavelength. The essential elements of a monochromator are an entrance slit, a dispersing
element and an exit slit. The entrance slit sharply defines the incoming beam of heterochromatic
radiation. The dispersing element disperses the heterochromatic radiation into its component
wavelengths whereas exit slit allows the nominal wavelength together with a band of
wavelengths on either side of it. The position of the dispersing element is always adjusted by
rotating it to vary the nominal wavelength passing through the exit slit.
The dispersing element may be a prism or grating. The prisms are generally made of
glass, quartz or fused silica. Glass has the highest resolving power but it is not transparent to

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radiations having the wavelength between 2000 and 3000 Å because glass absorbs strongly in
this region. Quartz and fused silica prisms which are transparent throughout the entire UV range
are widely used in UV spectrophotometers.
Fused silica prisms are little more transparent in the short wavelength region than quartz
prisms and are used only when very intense radiation is required.
The mirrors in the optical system are front surfaced because glass starts to absorb in the
ultraviolet region.
3 Detectors
There are three common types of detectors which are widely used in UV
spectrophotometers. These are as follows.
(i) Barrier layer cell.
This cell is also known as photovoltaic cell. A typical barrier cell is shown in Fig. 6.10
The barrier cell consists of a semiconductor, such as selenium, which is deposited on a strong
metal base, such as iron. Then a very thin layer of silver or gold is sputtered over the surface of
the semiconductor to act as a second collector electrode.
The radiation falling on the surface produces electrons at the selenium silver interface. A
barrier exists between the selenium and iron which prevents the electrons from flowing into iron.
The electrons are therefore accumulated on the silver surface. The accumulation of electrons on
the silver surface produces an electrical voltage difference between the silver surface and the
base of cell. If the external circuit has a low resistance, a photocurrent will flow which is
directly proportional to the intensity of incident radiation beam.
The sensitivity of a photovoltaic cell is only moderate and it is generally used for
instruments like photometers which allow a wide band of radiations to strike the detector.
Photovoltaic cell is simple in design. It does not require any external power supply.
However, it can be hooked directly to a micrometer or galvanometer to read its output.

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The response time on a photovoltaic cell is only fair, and thus, it cannot cause the
reduction of noise. With the pace of time, a photovoltaic cell becomes useless because of
transformations of the selenium layer.
(ii) Photocell.
It consists of a high-sensitive cathode in the form of a half-cylinder of metal which is
contained in an evacuated tube. The anode is also present in the tube which is fixed more or less
along the axis of the tube. The inside surface of the photocell is coated with a light sensitive
layer (Fif.6.11).
When the light is incident upon a photocell, the surface coating emits electrons. These
are attracted and collected by an anode. The current, which is created between the cathode and
anode, is regarded as a measure of radiation falling on the detector.
A photocell is more sensitive than photovoltaic cell because high degree of amplification
can be used. If quartz or fused silica windows are used, the range of the photocells can be
increased through the near ultraviolet and into the far-ultraviolet region.
(iii) Photomultiplier tube:
A photomultiplier tube is generally used as a detector in UV
spectrophotometers. A typical photomultiplier is shown in Figure 6.12.
A photomultiplier tube is a combination of a photodiode and an electron-multiplying
amplifier. A photomultiplier tube consists of an evacuated tube which contains one photo-

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cathode and 9-16 electrodes known as dynodes. The surface of each dynode is of Be-Cu, Cs-Sb
or similar material.
When radiation falls on a metal surface of the photocathode, it emits electrons. The
electrons are attracted towards the first dynode which is kept at a positive voltage. When the
electrons strike the first dynode, more electrons are emitted by the surface of dynode ; these
emitted electrons are then attracted by a second dynode where similar type of electron emission
takes place. The process is repeated over all the dynodes present in the photomultiplier tube
until a shower of electrons reaches the collector. The number of electrons reaching the collector
is a measure of the intensity of light falling on the detector. The dynodes are operated at an
optimum voltage that gives a steady signal.
The photomultiplier tube is extremely sensitive as well as extremely fast in response.
The transit time between absorption of the photon and the arrival of the shower of electrons is
typically in the range of 10-100 µsec. For every quantum of light, approximately 106
electrons
are produced.
4 Recording system:
The signal from the photomultiplier tube is finally received by the recording system. The
recording is done by recorder pen. The type of arrangement is only done in recording UV
spectrophotometers.

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5 Sample cells:
The cells that are to contain samples for analysis should fulfill their main conditions:
(i) They must be uniform in construction; the thickness must be constant and surfaces
facing the incident light must be optically flat.
(ii) The material of construction should be inert to solvents.
(iii) They must transmit light of the wavelength used.
The most commonly used cells are made of quartz or fused silica. These are readily
available even in matched pairs where sample cell is almost identical to the reference cell.
6 Matched cells:
Double-beam instrumentation is used, two cells are needed, one for the reference and one
of the sample. It is normal for the absorption by these cells to differ slightly. This causes a
small error in the measurement of the sample absorption and can lead to analytical error. For
most accurate work, matched cells are used. These are cells in which the absorption of each
one is equal to or very nearly equal to the absorption of the other. A large number of these
cells are manufactured at one time and their respective absorptivities measured. Those with
very similar absorptivities are put together and designated as matched cells. Naturally, the
cost of a pair of matched cells is greater than the cost of two unmatched cells. It should also be
noted than if one matched cell is broken, it cannot be used with another matched cell from
another pair, because it is unlikely that their absorptivities will be equal to each other.
At all times when not in use, cells should be kept clean and dry. Any sample left in a cell
will tend to dry out and cause a stain on the cell walls, and this will lead to analytical error and
eventual destruction of the cell.
7 Power Supply :
The power supply serves a triple function.
(i) It decreases the line voltage to the instruments operating level with a transformer.
(ii) It converts A.C. to D.C. with a rectifier if direct current is required by the instrument.
(iii) It smooths out any ripple which may occur in the line voltage in order to deliver a
constant voltage.

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8.Description of a UV Spectrophotometer:
(a) Single-Beam System
In the single-Beam system, UV radiation is given off by the source. A convex lens
gathers the beam of radiation and focuses it on the inlet slit. The inlet slit permits light from the
source to pass, but blocks out stray radiation. The light then reaches the monochromator, which
splits it up according to wavelength. The exit slit is positioned to allow light of the required
wavelength to pass through. Radiation at all other wavelengths is blocked out. The selected
radiation passes through the sample cell to the detector, which measures the intensity of the
radiation reaching it. By comparing the intensity of radiation before end after it passes through
the sample, it is possible to measure how much radiation is absorbed by the sample at the
particular wavelength used. The output of the detector is usually recorded on graph paper.
One problem with the single-beam system is that it measures the total amount of light
reaching the detector, rather than the percentage absorbed. Light may be lost at reflecting
surfaces or may be absorbed by the solvent used to dissolve the sample. Furthermore, the source
intensity may vary with changes in line voltage. For example, when the line voltage decreases,
the intensity of the light coming from the source may decrease unless special precautions are
taken. Consequently, the intensity of radiation may be constantly changing.
Another problem is that the response of the detector varies significantly with the
wavelength of the radiation falling on it. Even if the light intensity is constant at all
wavelengths, if the wavelength is steadily increased from 200 to 750 nm, the signal from the
detector starts at a low value, increases to a value that is steady over a wide range, and then
decreases once more. This relationship between the signal from the detector and the
wavelength of radiation is called the response curve. It indicates the wide variation in signal
that than be expected from the detector even though the light intensity falling on it is constant.
Different detectors respond differently at different wavelengths. For example, the 1P28 is not
useful at 800 nm, but the R136 and gallium arsenide detectors respond in this range. The
detector selected must operate over the desired range of the experiment. The problem of
instrument variation can be largely overcome by using the double-beam system.

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(b) Double-beam System
The basic layout of a double-beam ultraviolet spectrophotometer is shown in Fig. 6.13.
The description of a double-beam ultraviolet spectrophotometer is as follows.
(i) The radiation from the source is allowed to pass via a mirror system to the
monochromator unit. The function of the monochromator is to allow a narrow range
of wavelengths to pass through an exit slit.
(ii) The radiation coming out of the monochromator through the exit slit is received by
the rotating sector which divides the beam into two beams, one passing through the
reference and other through the sample cell.
(iii) After passing through the sample and reference cells, the light beams are focused onto
the detector.
(iv) The output of the detector is connected to a phase sensitive amplifier which responds
to any change in transmission through sample and reference.
(v) The phase sensitive amplifier transmits the signals to the recorder which is followed
by the movement of the pen on chart. The chart drive is coupled to the rotation of the
prism and thus the absorbance or transmittance of the sample is recorded as a function
of wavelength.

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Advantages of Double Beam Instruments :
Although the double beam instruments are more complicated and expensive, they do
offer the following advantages:
(i) It is not necessary to continually replace the blank with the sample or to zero adjust at
each wavelength as in the single beam units.
(ii) The ratio of the powers of the sample and reference beams is constantly obtained and
used. Any error due to variation in the intensity of the source and fluctuation in the
detector is minimized.
(iii) Because of the previous two factors, the double beam system lends itself to rapid
scanning over a wide wavelength region and to the use of a recorder or digital read out.
Applications of Spectroscopy to Organic Compounds
The main applications of ultraviolet spectroscopy are as follows.
1 Detection of conjugation:
It helps to show the relationships between different groups, Particularly with respect to
conjugation; the conjugation may be (a) between two or more carbon-double (or triple) bonds,
(b) between carbon-carbon and carbon-oxygen double bonds or (c) between double bonds and an
aromatic ring.
It can reveal the presence of an aromatic ring itself and the number and locations of
substituents attached to the carbons of the conjugated system.
1. Detection of geometrical isomers. In case of geometrically isomeric compounds, the
trans isomers exhibit λmax at slightly longer wavelengths and have larger extinction coefficients
than the cis isomers. For example, of the two stilbenes (C6 H5 – CH = CH – C6 H5), the trans
isomer show λmax = 294 (ε = 24000) while the cis isomer has λmax = 278 nm (ε = 9350).
2. Detection of functional groups. It is possible to detect the presence of certain
functional groups with the help of UV spectrum. Even the absence of any absorption above 200
nm is of some utility since it shows the absence of conjugation, carbony1 group and benzene
rings in the compound.

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Visible Spectroscopy:
Principle:
Colorimetry is concerned with study of absorption of visible radiation whose
wavelength ranges from 400-800nm.Coloured substances will absorb light of different
wavelength in different manner and hence we get an absorption curve (absorption (vs.)
wavelength) which is characteristic of every colored substance. In this curve the wavelength at
which maximum absorption of radiation takes place is called λ max. λ max is not usually
affected by the concentration of the substance. The absorbance of a solution increases with
concentration of a substance but there is no change in λ max when concentration changes. When
we plot a graph of concentration (vs.) absorbance, we get a calibration or standard curve. The
calibration curve is useful in determining the concentration or amount of substance in the given
sample solution by extrapolation or intrapolation method.
The two laws related to absorption of radiation are:
1. Beer‟s Law (Related to concentration of absorbing species)
2. Lambert‟s Law (Related to thickness or path length of absorbing species)
Beer’s Law:
Beer‟s law states that “The intensity of a beam of monochromatic light decreases
exponentially with increase in concentration of absorbing species arithmetically”.
Accordingly,
I= Io e-kc
Lamberts Law:
“The rate of decrease of intensity with the thickness of the medium is directly proportional
to the intensity of incident light.”
I= Io e-kt

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Beer – Lambert’s Law:
By combining Beers law and Lamberts law we get:
I= Io e-kct
A = log IO/ I = abc
Where
“a” is absorptivity which is a constant.
Molar Absorptivity:
The name and value of “a” depends on the units of concentration. When “c” is in
mole/liter, the constant is called molar absorptivity and has the symbol ε and „b‟ is the path
length (cm). The equation therefore takes the form
A = εbc
Diagram of Beer–Lamberts absorption of a beam of light as it travels through a cuvette of
width ℓ.

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Specific Absorbance:
Specific absorbance A1%
1cm Which is the absorbance of 1g/100ml (1% w/v) solution in a
1cm cell. The Beer-Lamberts equation therefore takes the form:
A = A1%
1cm bc
Where “c” is in g/ml and b is in cm. The units of A1%
1cm are dlg-1
cm-1
. A simple easily
derived equation allows inter conversion of ε and A1%
1cm is:
ε = A1%
1cm x molecular weight
10
Where,
A1%
1cm means the absorbance of 1% w/v solution, using a path length of 1cm.
Chromophore:
Groups in a molecule which absorb light are known as Chromophores. When a molecule
absorbs certain wavelengths of visible light and transmits or reflects others, the molecule has a
color. A chromophore is a region in a molecule where the energy difference between two
different molecular orbital falls within the range of the visible spectrum. Visible light that hits
the chromophore can thus be absorbed by exciting an electrons from its ground state into an
excited state.Chromophores always arise in one of the two forms:
Conjugated pi systems and
Metal complexes.
In the former, the energy levels that the electrons jump between are extended pi orbital created
by a series of alternating single and double bonds, often in aromatic systems. Common examples
include retinal (used in the eye to detect light), various food colorings, fabric dyes (azo
compounds), lycopene, β-carotene, and anthocyanins.
The metal complex chromophores arise from the splitting of d-orbital by binding of a
transition metal to ligands. Examples of such chromophores can be seen in chlorophyll (used by

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plants for photosynthesis), hemoglobin, hemocyanin, and colorful minerals such as malachite
and amethyst.
Validation of Analytical Procedure:
Validation is an act of proving that any procedure, process, equipment, material, activity
or system performs as expected under given set of conditions and also give the required
accuracy, precision, sensitivity, ruggedness etc.
The various validation parameters are:
Accuracy,
Precision (Repeatability and Reproducibility),
Linearity and Range,
Limit of detection(LOD)/ Limit of quantitation(LOQ),
Selectivity/ Specificity,
Robustness/ Ruggedness and
Stability and system suitability studies.
1) Accuracy: -
The accuracy of an analytical method may be defined as the closeness of the test results
obtained by the method to the true value. It is the measure of the exactness of the analytical
method developed. Accuracy may often express as percent recovery by the assay of a known
amount of analyte added. The ICH documents recommend that accuracy should be assessed
using a minimum of nine determinations over a minimum of three concentration levels, covering
the specified range (i.e. three concentrations and three replicated of each concentration).
2) Precision: -
The precision of an analytical method is the degree of agreement among individual test
results when the method is applied repeatedly to multiple samplings of homogenous
samples.Precision may be considered at three levels:

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 Repeatability,
 Intermediate precision,
 Reproducibility.
(a) Repeatability:
Repeatability expresses the precision under the same operating conditions over a short
interval of time. Repeatability is also termed intra-assay precision.
(b) Intermediate precision:
Intermediate precision expresses within laboratory variations in different days,
different analysts & different equipments.
(c) Reproducibility:
Reproducibility means the precision of the procedure when it is carried out under
different conditions-usually in different laboratories-on separate, putatively identical samples
taken from the same homogenous batch of material.
(3) Linearity and range:-
The linearity of an analytical procedure is its ability (within a given range) to obtain
test results which are directly proportional to the concentration (amount) of analyte in the
sample.
The range of an analytical method is the interval between the upper and lower levels
of the analyte (including these levels) that has been demonstrated that the analytical procedure
has a suitable level of precision, accuracy and linearity.

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4) Limit of Detection:-
It is defined as the lowest concentration of an analyte in a sample that can be detected
but not quantified. LOD is expressed as a concentration at a specified signal to noise ratio. A
signal-to-noise ratio of 2:1 or 3:1 is generally accepted.
For spectroscopic techniques or other methods that rely upon a calibration curve for quantitative
measurements, the IUPAC approach employs the standard deviation of the intercept (Sa) which
may be related to LOD and the slope of the calibration curve, b, by
LOD = 3 Sa / b
5) Limit of Quantitation:-
It is defined as the lowest concentration of an analyte in a sample that can be
determined with acceptable precision and accuracy under stated operational conditions of the
method. LOQ is expressed as a concentration at a specified signal to noise ratio. In many cases,
the limit of quantitation is approximately twice the limit of detection.
LOQ = 10. Sa / b
6) Selectivity and Specificity:-
The selectivity of an analytical method is its ability to measure accurately and
specifically the analyte of interest in the presence of components that may be expected to be
present in the sample matrix. Selectivity may be determined by comparing the test results
obtained on the analyte with and without the addition of the potentially interfering materials.
Hence one basic difference in the selectivity and specificity is that, while the former is
restricted to qualitative detection of the components of a sample, the latter means quantitative
measurement of one or more analyte.

28
6) Robustness and Ruggedness:-
The robustness of an analytical procedure is a measure of its capacity to remain
unaffected by small, but deliberate variations in method parameters and provides an indication of
its reliability during normal usage.
The ruggedness of an analytical method is the degree of reproducibility of test results
obtained by the analysis of the same samples under a variety of normal test conditions such as
different laboratories, different analysts, using operational and environmental conditions that
may differ but are still within the specified parameters of the assay.
7) Stability and System suitability tests:-
Stability of the sample, standard and reagents is required for a reasonable time to
generate reproducible and reliable results. For example, 24 hour stability is desired for solutions
and reagents that need to be prepared for each analysis.
Efficient development and validation of analytical methods are critical elements in the
development of pharmaceuticals.

29
DRUG PROFILE
ROPINIROLE HYDROCHLORIDE
Molecular Structure :
.
Molecular Formula : C16H24N2O
Molecular Weight : 260.375 .
CAS Number : 91374-20-8.
Chemical Name : 4-[2-(dipropylamino)ethyl]-1,3-dihydro-2H-indol-2-one.
Brand Names : Requip tiltab (tablet), Ropitor (tablet).
Description : Ropinarole is a light yellow powder.

30
Solubility
1. Freely Soluble in Methanol and Ethanol.
2. Freely Soluble in Water.
Storage
Protect from light and moisture. Close container tightly after each use. Store at
controlled room temperature 20°-25°C (68°-77°F).
Clinical Pharmacology:
Mechanical of Action :
Ropinirole binds the dopamine receptors D3 and D2. Although the precise mechanism of
action of ropinirole as a treatment for Parkinson's disease is unknown, it is believed to be related
to its ability to stimulate these receptors in the striatum. This conclusion is supported by
electrophysiologic studies in animals that have demonstrated that ropinirole influences striatal
neuronal firing rates via activation of dopamine receptors in the striatum and the substantia nigra,
the site of neurons that send projections to the striatum.
Pharmacodynamics :
Ropinirole is a non-ergot dopamine agonist with high relative in vitro specificity and full
intrinsic activity at the D2 subfamily of dopamine receptors, binding with higher affinity to D3
than to D2 or D4 receptor subtypes. The relevance of D3 receptor binding in Parkinson's disease is
unknown. The mechanism of ropinirole-induced postural hypotension is presumed to be due to a
D2 -mediated blunting of the noradrenergic response to standing and subsequent decrease in
peripheral vascular resistance.
Pharmacokinetics
Ropinirole is a non-ergot dopamine D2-agonist with similar actions to those of
bromocriptine. It is used in the management of Parkinson's disease, either alone or as an adjunct
to levodopa.

31
Absorption : Rapidly absorbed from the GI tract after oral admin. Bioavailability about 50%.
Distribution : Widely distributed. Plasma protein binding is 10-40%.
Metabolism : Extensively metabolised in the liver by CYP1A2
Excretion : Excreted in the urine as inactive metabolites; <10% of the oral dose is excreted
unchanged.
Elimination half-life: about 6 hours.
Therapeutic Uses :
Ropinarole is used to cover the lack of dopamine and alleviate symptoms such as
stiffness, poor muscle control, tremors, muscle spasms in the patients with Parkinson's disease.
This medication is also used to treat restless legs syndrome.
Side Effects :
Nausea, somnolence (including sudden sleep onset), abdominal pain/discomfort,
dizziness, headache, constipation; dyskinesia, vomiting, syncope, fatigue, dyspepsia, infections,
pain, sweating, asthenia, edema, postural hypotension, hypertension, changes in heart rate,
pharyngitis, confusion, hallucinations, abnormal vision, aggravated parkinsonism. Advanced
disease (with levodopa): also arthralgia, tremor, anxiety, dry mouth, hypokinesia, paresthesia.
Precaution
Dyskinesia
Ropinirole hydrochloride may potentiate the dopaminergic side effects of L-dopa and
may cause and/or exacerbate preexisting dyskinesia in patients treated with L-dopa for
Parkinson‟s disease. Decreasing the dose of L-dopa may ameliorate this side effect.
Renal Impairment :
No dosage adjustment is needed in patients with mild to moderate renal impairment
(creatinine clearance of 30 to 50 mL/min). The use of Ropinirole hydrochloride in patients with
severe renal impairment has not been studied.

32
Hepatic Impairment
The pharmacokinetics of Ropinirole have not been studied in patients with hepatic
impairment. Since patients with hepatic impairment may have higher plasma levels and lower
clearance, Ropinirole hydrochloride should be titrated with caution in these patients.
Overdosage
Symptoms include nausea, vomiting, visual hallucinations, hyperhidrosis, asthenia and
nightmares. General supportive measures and monitoring of vital signs are recommended. May
consider gastric lavage.
Administration of Ropinirole:
Dosage and direction :
Take exactly as prescribed by your doctor. Do not take more than two doses
of the medication at once. Do not suddenly stop taking of this medication without approval of
your doctor as it may worsen your symptoms such as fever, muscle stiffness, and confusion. It
may take a few weeks till this medication starts to work.
Contraindications :
Ropinarole cannot be used in the patients with hypersensitivity (including urticaria,
angioedema, rash, pruritus) to ropinirole or to any of the excipients. to the medication.
Ropinirole and Pregnancy : Caution when used during pregnancy
Category C: Either studies in animals have revealed adverse effects on the foetus (teratogenic or
embryocidal or other) and there are no controlled studies in women or studies in women and
animals are not available. Drugs should be given only if the potential benefit justifies the
potential risk to the foetus.
Ropinirole and Lactation : Contraindicated in lactation
Ropinirole and Children :Safety and effectiveness in the pediatric population have not been
established.

33
Drug interactions :
Inform your doctor if you currently take narcotic pain medicine, sleeping pills, cold
or allergy medicine, muscle relaxers, and medicine for depression or anxiety, seizures as
concomitant use of Requip may increase drowsiness.
Precautions :
This medication causes unusual sleepiness while you are working eating, talking or
driving. Avoid performing of potentially hazardous activities which require high concentration of
attention until you know they way Requip affects you.. This medication may cause auditory or
visual hallucinations. You may need regular skin exams if you are treated for Parkinson's disease
due to increased risk of skin cancer (melanoma).

34
4. AIM AND PLAN OF WORK
Aim:
Development and Validation of Ropinirole Hydrochloride by UV
spectrophotometric method.
According to literature review there are various UV, HPLC and HPTLC methods
have been reported on the individual drugs as well as in combination with other
drugs. Hence development of sensitive, simple, rapid and accurate UV methods
are needed for estimation of the Ropinirole.
Plan of work:
1. Solubility parameters
2. Determination of λmax
3. Analytical method development
4. Analytical validation
a. Precision
b. Recovery studies
5. Least square method
6. Determination of stability

35
REVIEW OF LITERATURE
1. Krishnan et al 2010 reported a novel stability-indicating gradient reverse phase ultra
performance liquid chromatographic (RP-UPLC) method was developed for the determination of
purity of Ropinirole in presence of its impurities and forced degradation products. The method
was developed using Waters Aquity BEH 100 mm, 2.1 mm, 1.7 μm C-8 column with mobile
phase containing a gradient mixture of solvent A and B. The eluted compounds were monitored
at 250 nm. The run time was within 4.5 min which Ropinirole and its four impurities were well
separated.
2. Yogita Shete et al (2009) reported a simple, sensitive, rapid, accurate and precise
spectrophotometric method has been developed for estimation of ropinirole hydrochloride in
bulk and tablet dosage forms. Ropinirole hydrochloride shows maximum absorbance at 250 nm
with molar absorptivity of 8.703×103
l/mol.cm. Beer's law was obeyed in the concentration range
of 5-35 μg/ml.
3. Jignesh Bhatt et al (2006) reported a rapid and robust liquid chromatography-mass
spectrometry (LC-MS/MS) method for non-ergoline dopamine D(2)-receptor agonist, Ropinirole
in human plasma using Es-citalopram oxalate as an internal standard. The method involves solid
phase extraction from plasma, reversed-phase simple isocratic chromatographic conditions and
mass spectrometric detection that enables a detection limit at picogram levels. The proposed
method was validated with linear range of 20-1,200 pg/ml.The R.S.D.% of intra-day and inter-
day assay was lower than 15%.
4. Onal (2006) reported the method development of a rapud determination of Ropinirole in
tablet dosage form by LC-UV. The assay utilized UV detection at 250 nm and a Luna CN
column (250 × 4.6 mm I.D, 5 μm). The mobile phases were comprised of acetonitrile: 10 mM
nitric acid (pH 3.0) (75:25, v/v). The method was linear over the concentration range of 0.5–
10.0 μg mL−1
. The method showed good recoveries (99.75–100.20%) and the relative standard
deviations of intra and inter-day assays were 0.38–1.69 and 0.45–1.95%, respectively.

36
5. Susheel J et al(2007) reported in the development and analysis of Ropinirole with UV
Spectrosopy at250nm. For the spectrophotometric method, the linearity was found to be in the
range of 5-30 mg/ml. Aliquots of standard Ropinirole solutions ranging from 5-30 µg/ml (from
stock solution of 100 µg/ml) were prepared using ethanol and absorbances were noted at 250 nm.
Calibration curve was drawn by plotting absorbances of ropinirole versus concentration of
respective drug solutions. Twenty tablets of ropinirole were weighed and average weight was
calculated. Quantity of powder equivalent to 10 mg was weighed accurately and transferred to a
100 ml volumetric flask. It is dissolved in ethanol and made upto the mark and filtered. The
filtered solution was further diluted to get requisite concentrations and analyzed for pure sample.
6. Azeem et al (2008) reported in the Development and validation of an accurate, sensitive,
precise, rapid, and isocratic reversed phase HPLC (RP-HPLC) method for analysis of Ropinirole
in the bulk drug and in pharmaceutical preparations. The best separation was achieved on a 250
mm × 4.6 mm i.d, 5-μm particle, C 18 reversed-phase column with methanol-0.05 M ammonium
acetate buffer (pH 7) 80:20 ( v/v) as mobile phase, at a flow rate of 1 mL min −1
. UV detection
was performed at 250 nm. The method was linear over the concentration range 0.2–100 μg mL −1
( r= 0.9998), with limits of detection and quantitation of 0.061 and 0.184 μg mL −1
, respectively.
7. Erin Chambers et al (2007) reported in the development and determination of
Ropinirole in Human plasma by a Rapid and Sensitive SPE-UPLC--MS--MS Method. The assay
was determined to be linear over the required range of 0.02 to 20 ng/mL. For each day of
analysis, calibration curves were analysed in duplicate or triplicate. All calibration curves had an
r2
> 0.996. The combination of UPLC and Oasis μElution SPE provided the sensitivity necessary
to easily achieve a 0.02 ng/mL LLOQ. The elution solvent was modified from 5% NH4OH in
100% MeOH to 5% NH4OH in 90:10 MeOH:H2O to obtain a cleaner final extract for analysis.
8. N. Sreekanth et al(2009) reported in the development a simple and accurate RP-HPLC
method for the estimation of Ropinirole hydrochloride in bulk and pharmaceutical dosage forms
using C18 column 250 x 4.6 mm i.d, 5μm particle size in isocratic mode, with mobile phase
comprising of buffer (pH 6.0) and Acetonitrile in the ratio of 50:50 v/v. The flow rate was
0.5ml/min and detection was carried out by UV detector at 245nm. The proposed method has
permitted the quantification of Ropinirole hydrochloride over linearity in the range of 5-50μg/ml

37
and its percentage recovery was found to be 99.3-100.4%. The intra day and inter day precision
were found 0.27% and 0.26% respectively.
9. Pavel Coufal et al (1999) reported in Separation and quantification of Ropinirole and
some impurities using capillary liquid chromatography. pH, buffer concentration and acetonitrile
content was performed employing an experimental design approach which proved a powerful
tool in method development. The retention factors of the investigated substances in different
mobile phases were determined. Baseline resolution of the six substances on a C18 reversed
stationary phase was attained using a mobile phase with an optimized composition [acetonitrile–
8.7 mM 2-(N-morpholino)ethanesulfonic acid adjusted to pH 6.0 (55:45, v/v)].
10. Ramji et al (1999) reported the disposition and metabolic fate of Ropinirole, in the
mouse, rat, cynomolgus monkey and man, following oral and intravenous administration of
ropinirole hydrochloride. It is a novel compound indicated for the symptomatic treatment of
Parkinson's disease, was studied In all species, nearly all of the p.o. administered dose (94%) was
rapidly absorbed from the gastrointestinal tract following administration of 14C-ropinirole
hydrochloride.
11. Karel tulík et al (1998) reported in the Determination of the dissociation constants of
ropinirole and some impurities and their quantification using capillary zone electrophoresis. The
dissociation constants obtained from the CZE measurements were confirmed by UV
spectrophotometry for some of the test compounds, obtaining a good agreement between the
values. Careful optimization of the running buffer composition permitted base-line resolution of
the six compounds in a borate buffer containing acetonitrile and magnesium sulfate (a 100 mM
borate buffer containing 30 mM MgSO4 and 20 vol.% of acetonitrile). It was shown that CZE
can determine the level of these impurities, down to a level of 0.05% of the main component
within 15 min.

38
EXPERIMENTAL WORK
UV SPECTROPHOTOMETRIC METHOD
Instrument Used
UV- 1601, serial No. A- 1075 Manufacture by Schimadzu Corporation, Japan.
Choice of solvent
Ropinirole HCl was soluble in distilled water, ethanol, methanol 0.1N hydrochloric acid
and aceto-nitrile. Sparingly soluble in distilled water was found to be suitable solvent in the uv
spectrophotometeric method, its absorbance was 249nm which gave individual peak with
maximum absorbance. Hence distilled water was selected as an ideal solvent and used for the
entire experimental work.
Determination of λmax
λmax is the wave length of an absorption maximum. The standard drug of Ropinirole HCl
was dissolved in distilled water to obtain 10µg/ml concentration range. The solution was scanned
between 200-400 nm and found that the peak at 249nm showed maximum absorbance. Further
dilutions were made to get the concentration range.
Determination of molar Absorptivity
Absorptivity constant „a‟ is the ratio of the absorbance of the sample to the product of the
thickness of the medium and concentration of the sample. As the thickness of the medium for
various determination is the same, Absorptivity depends up on the absorbance and concentration
of the sample. Due to increase or decrease in the concentration of sample, the absorbance also
will increase or decrease respectively, which is always a constant.
From the stock solution Ropinirole hydrochloride 10µg/ml to 30µg/ml of a standard
solution were prepared. The absorbance of different concentrations was noted at 249nm using the
formula.

39
A=A/bc
Where
a= Absorptivity
A= Absorbance
b= path length (1cm)
c= concentration
Effect of time on stability of absorbance:
The stability of the solution was checked by measuring the absorbance the regular
intervals of time.
It was observed that the absorbance remained stable for a period of 2 days and then the
absorbance decreased with increase in time.
Preparation of the standard solutions
About 100mg of Ropinirole hydrochloride was accurately weighed and transferred to a
clean dry 100ml calibrated standard flask and dissolved in distilled water. It was shaken for few
minutes and the solution was diluted to 100ml with same. 10ml of this solution was pipetted to
another clean dry calibrated 100ml volumetric flask and the volume was made up with distilled
water. Further resulting solution from 5-15ml to 50ml with distilled water in 50ml standard flask.
The resulting solutions were scanned at 249nm against blank. The absorbances obtained were
plotted against the concentration of the solution and standard graph was obtained.
Preparation of sample solutions
Five tablets were accurately weighed and average weight was taken, weight of sample
equivalent to 10mg of Ropinirole hydrochloride was taken 100ml standard flask and the sample
was dissolved in distilled water, sonicated for five minutes and made up to 100ml with the same.
The solution was then filtered through whatmann filter paper No.1.

40
Then from the above solution 10µg/ml concentration was prepared and scanned at 249nm
against blank. The above procedure was followed for the determined marketed sample and the
absorbance value was recorded.
The amount of Ropinirole hydrochloride per tablet was calculated by comparing
absorbance value of standard and sample at 249nm.
VALIDATION METHOD
PRECISION
Procedure
Standard drug solution was prepared as per procedure given under preparation of
standard absorbance curve. This parameter was validated by assaying the number of aliquots of
homogeneous samples of Ropinirole hydrochloride and estimating its validity using parameters
such as standard deviation (S.D) and relative standard deviation (RSD).
RECOVERY STUDIES
PROCEDURE :
5 Ropinirole hydrochloride tablets were taken and weights of all tablets were found out.
The average weight was 0.481 g.
All 5 tablets were powdered and the following procedures were used to prepare the
sample solutions.
Recovery Studies at 50%
The following procedures were used to prepare the sample solutions for recovery studies:
Weighed accuaretly 400mg of Ropinirole hydrochloride tablet powder equivalent to
10mg of the drug and transferred to calibrated 50ml volumetric flask. Then measured 50mg of
pure drug powder of Ropinirole hydrocholide and transferred it to the same volumetric flask.
Added 50ml of water and sonicatef for 10mins. Then made upto the mark with water. Then
filtered the solution, during the filtration discard the initial 10ml of filtrate 2 times and then
collect the filtrate. Labelled this flask as stock solution.1500µg/ml.

41
Transferred 3.3ml of the above solution to another calibrated 50ml volumetric flask and
made upto the mark with water. Label this flask as dilution 1, 1000µg/ml. Prepared a set of
standard dilution using calibrated 10ml standard flask ( dilution 2 ). 1ml to 10ml , 1.5ml to 10ml,
2ml to 10ml and measured the absorbance of each dilutions at λmax (249nm) of Ropinirole
hydrochloride in water using photometric mode – quantitative mode using the absorbance values
at various concentrations. Calculated the total amount present in the stock solution, amount
recovered and percentage recovery.
Recovery Studies at 100 %
Weigh accurately 400mg of Ropinirole hydrochloride powder equivalent to 100mg of
pure drug and transfer it to the calibrated 50ml volumetric flask. Then measure 100mg of pure
drug powder and transfer it to the same volumetric flask. Add 50ml of water and sonicate for
10mins. Then make upto the mark with water then filter the solution, during filtration discard
initial 10ml filtrate 2 times and then collect the filtrate. Label this flask as stock solution
2000µg/ml solution
Transfer 2.5ml of the above solution to another calibrated 50ml volumetric flask and
make up to the mark with water. Label this flask as dilution 1, 100µg/ml. prepare a set of
standard dilutions using calibrated 10ml standard flask dilution 2. 1ml to 10ml, 1.5ml to 10ml
and 2ml to 10ml. measure the absorbance of each dilution at λmax (249nm) of Ropinirole
at various concentrations. Calculate the total amount present in the stock solution, amount
Recovery Studies at 150 %
Weighed accurately 400mg of Ropinirole hydrochloride powder equivalent to 10mg of
Ropinirole hydrochloride and transferred it to the calibrated 50ml volumetric flask. Then
measure 150mg of pure drug powder and transferred it to the same volumetric flask. Added 50ml
of water and sonicated for 10mins. Then made upto the mark with water then filtered the
solution, during filtration discarded the initial 10ml filtrate 2 times and then collected the filtrate.
Labelled this flask as stock solution 3000µg/ml solution

42
Transferred 1.8ml of the above solution to another calibrated 50ml volumetric flask and
made up to the mark with water. Labelled this flask as dilution 1, 100µg/ml. Prepared a set of
standard dilutions using calibrated 10ml standard flask dilution 2. 1ml to 10ml, 1.5ml to 10ml
and 2ml to 10ml. measured the absorbance of each dilution at λmax (249nm) of Ropinirole
at various concentrations. Calculated the total amount present in the stock solution , amount
Percentage recovery was calculated by using the following formula:
Amount of drug found Amount of drug
% Recovery = in sample after addition of drug - found in sample
________________________________________________ x100
Amount of standard drug added
STATISTICAL ANALYSIS
The quantitative results obtained were subjected to the following statistical analysis
Sample Mean (SM)
SM = X1+ X2+ X3+…………+ Xn
--------------------------------------
n
Standard Deviation (SD)
SD = ∑(X-X) 2
------------
n-1

43
Relative Standard Deviation (%RSD) or Coefficient of Variation (%CV)
SD
RSD = ------------ x 100
Mean
Standard Error of Mean (SE)
SD
SE = ----------
Mean
Statistics of straight line
Correlation coefficient r = (X-X) (Y-Y)
-------------------
√(X-X) (Y-Y)
Where X = ∑x1/n and y = ∑y1/n
Slope of the line = ∑(X-X) (Y-Y)
--------------------
∑(X-X) 2

44
RESULTS AND DISCUSSION
DETERMINATION OF λ MAX
The Ropinirole Hydrochloride standard drug was dissolved in distilled water to obtain
10µg/ml solution. The solution was scanned between 200-400nm and found that the peak at 249
nm showed maximum absorbance. Further 12µg/ml to 30µg/ml concentrations were also
scanned between 230-350 nm.
Fig no: 1

45
DETERMINATION OF ABSORPTIVITY :
Absorptivity constant „a‟ is the ratio of the absorbance of the sample to the product of the
thickness of the medium and concentration of the sample. Due to increase or decrease in the
concentration of the sample, the absorbance also will increase (or) decrease respectively, which
is always a constant.
From the stock solution, Ropinirole Hydrochloride 10µg/ml to 30 µg/ml of standard
solutions was prepared. The absorbance of different concentrations was noted at 249 nm and the
molar absorptivity was determined using the formula.
A=A/bc
Where
a= Absorptivity
A= Absorbance
b= path length (1cm)
c= concentration

46
Absorptivity of Ropinirole hydrochloride
Table no - 1
S.No Concentration Absorbance
at 249nm
= A/bc
(µg/ml) %
1 10 0.0010 0.337 337.0
2 12 0.0012 0.400 333.3
3 14 0.0014 0.463 330.7
4 16 0.0016 0.520 325.0
5 18 0.0018 0.591 328.3
6 20 0.0020 0.650 325.0
7 25 0.0025 0.776 310.4
8 30 0.0030 0.949 316.3

47
EFFECT OF TIME ON STABILITY OF ABSORBANCE:
The stability of Ropinirole hydrochloride solution was checked by measuring the
absorbance the regular intervals of time.
It was observed that the absorbance remained stable for a period of 2 days and then the
absorbance decreased with increase in time.
Effect of time on stability
Table no - 2
S.no Time Absorbance (249nm)
1 0 hrs 0.309
2 6 hrs 0.309
3 12 hrs 0.309
4 18 hrs 0.309
5 24 hrs 0.306
6 30 hrs 0.306
7 36 hrs 0.306
8 42 hrs 0.305
9 48 hrs 0.305

48
Fig no: 2
Stability of Ropinirole Hydrochloride in water – Absorbance Vs Time
0.2
0.22
0.24
0.26
0.28
0.3
0.32
0.34
0.36
0.38
0.4
0 hrs 6 hrs 12 hrs 18 hrs 24 hrs 30 hrs 36 hrs 42 hrs 48 hrs
AxisTitle
Chart Title
Series1

49
DETERMINATION OF OVERLAY
The Ropinirole Hydrochloride standard drug was dissolved in distilled waterto obtain
10µg/ml solution. The solution was scanned between 200-400nm and found that the peak at 249
nm showed maximum absorbance. Further 12µg/ml to 30µg/ml concentrations were also
scanned between 200-400nm in the overlay mode. The overlay of the Ropinirole Hydrochloride
was found to be 249 nm.
Fig no: 3

50
DETERMINATION OF STANDARD ABSORBANCE
The standard drug absorbance was observed at 249nm in the concentration of 10 to
30µg/ml solutions were found to obey Beer‟s law with the correlation coefficient (r) of 0.9998.
Standard absorbance Ropinirole Hydrochloride
Table no : 3
S.No Vol taken
(ml)
Concentration
(µg/ml)
Absorbance (249nm)
1 5 10 0.337
2 6 12 0.400
3 7 14 0.463
4 8 16 0.520
5 9 18 0.591
6 10 20 0.65
7 12.5 25 0.776
8 15 30 0.949

51
Linearity curve of Ropinirole Hydrochloride
Fig no - 4
Graph showing standard absorbance curve of Ropinirole hydrochloride

52
DATA FOR LEAST SQUARE METHOD:
The data for least square method was determined from the absorbance Vs concentration
data as shown in the table - 4 . The β slope and intercept α were calculated. The slope was found
to be 0.0316 and the intercept was found to be 1.1177.
Table no - 4
S.No Conc. µg/ml
X
Absorbance 249nm
Y
xy x2
1 10 0.337 3.37 100
2 12 0.400 4.80 144
3 14 0.463 6.48 196
4 16 0.520 8.32 256
5 18 0.591 10.63 324
6 20 0.650 13.00 400
7 25 0.776 19.40 625
8 30 0.949 28.47 900
∑x = 145 ∑y = 4.686 ∑xy = 94.47 ∑ x2
=2945

53
REPEATABILITY OF ABSORBANCE (249nm) AT 10µg/ml
The repeatability of absorbance values at 249nm 10 µg/ml concentration was tabulated.
Results are shown in table –
The standard deviation of Absorbance was found to be 0.000373 and % RSD was found
to be 0.119 %. LOD was found to be 0.038µg/ml. LOQ was found to be 0.118 µg/ml.
Limit Of Detection was calculated by using the formula (LOD).
LOD = 3.3 X N/β.
Limit Of Quantification was calculated using the formula
LOQ = 10 X N/β.
Where N = SD
Β = Slope
Table showing repeatability
Table no - 5
S.No Concentration µg/ml No. of repetitions Absorbance (249nm)
1 10 1 0.337
2 10 2 0.336
3 10 3 0.337
4 10 4 0.337
5 10 5 0.335
6 10 6 0.336
Mean 0.36333
Standard Deviation 0.000816
% Relative Standard Deviation 0.2245

54
DETERMINATION OF % ASSSAY FROM AMOUNT DETERMINED:
10 µg/ml concentration of Ropinirole Hydrochloride was prepared using sample solution
procedure. The absorbance of the solution was recorded at 249nm from the absorbance value the
amount of Ropinirole Hydrochloride was calculated.
Table showing percentage assay
Table no - 6
S.no Concentration
µg/ml
Absorbance at
249nm
Label Claim
(mg)
Amount
determined (mg)
% Assay
1 10 0.3230 12 11.81 98.5
2 10 0.3340 12 11.82 98.3
3 10 0.3372 12 11.81 98.5
Mean 11.81333 98.4
Standard Deviation 0.005774 0.11547
% Relative Standard Deviation 0.0488 0.1172
RECOVERY STUDIES:
The percentage recovery was calculated for each recovery level at 50%, 100% and 150%
Table no - 7
S.No Label
Claim
(mg)
Target
Concentration
(%)
Known
Amount
(µg/ml)
Amount
Added
(ml)
Amount of
pure drug
added (mg)
Amount
Found
µg/ml
%
Recovery
1 12 50 10 5 15 15.02 101.30
2 12 100 10 10 20 19.60 98.00
3 12 150 10 15 25 24.02 96.00
Mean 98.43

55
Graph showing recovery studies for 50%
Fig no - 5

56
Fig no - 6

57
Fig no - 7

58
SUMMARY & CONCLUSION
The work done involved the development of new, simple spectrophotometric method for
the estimation of Ropinirole HCl in the pure form and its formulation.
The method is based on the absorbance in the UV region. It showed maximum
absorbance at 249 nm. The Ropinirole HCl was stable more than 24 hours. The Beer's law was
obeyed over a range of 10-30 µg/ml with slope (β) 0.0316 and intercepts (α) 1.1177.
The repeatability, precision and accuracy of the method were carried out. The results
confirm the repeatability, precision and accuracy of the method.
Repeatability experiment of 10 µg/ml Ropinirole HCl solution showed an absorbance of
0.337 with a % of RSD 0.119.
Precision study showed percentage assay of Ropinirole HCl as 98.50 %
Recovery study showed percentage recovery between 96.00% - 101.00%
Limit of detection (LOD) = 0.038µg/ml.
Limit of quantitation (LOQ) = 0.118 µg/ml.
Least square method was precisely carried out and the results were confirmed as Ʃ x2
= 2945
The marketed formulations were analyzed by the proposed method and were found that
there was no interference with the excipients incorporated in the tablet formulation as seen from
recovery studies. The method described can be used for the estimation of tablet formulation due
to simplicity in preparation and cost effective.
The results obtained are in close declaration and found to be satisfactory.
The method can be adopted for the confirmation of Ropinirole HCl in pure as well as for
its formulation.

59
Abbreviations
B.P : British Pharmacopoeia
I.P : Indian Pharmacopoeia
U.S.P : United States Pharmacopoeia
µ1 : Micro Liter
mg : Milligram
µg : Micro Gram
S.D : Standard Deviation
R.S. D : Relative Standard Deviation
ml : Milliliter
ICH : International Conference onHarmonization
nm : Nanometer
Hr : Hour
Min : Minute
L.O.D : Limit of Detection
L.O.Q : Limit of Quantification
U.V : Ultra – Violet
API : Active Pharmaceutical Ingredient
Abs. : Absorbance
M : Molar

60
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