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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

Department of Pharmaceutical Analysis Page 1

QualitativeAnalysis

Inorganic

Organic

Quantitative Analysis

http://www.spiritus-temporis.com/quantitative-analysis/

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http://www.spiritus-temporis.com/functional-group/

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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


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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.


http://en.wikipedia.org/wiki/Luminescence

http://en.wikipedia.org/wiki/Microwave_spectroscopy

http://en.wikipedia.org/wiki/Ultraviolet-visible_spectroscopy

http://en.wikipedia.org/wiki/Infrared_spectroscopy

http://en.wikipedia.org/wiki/Atomic_absorption_spectroscopy

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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…


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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

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


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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

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.


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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

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.


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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.

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).


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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-

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.


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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.

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.


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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.

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.


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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.

(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.


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(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.

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.


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(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.

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


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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

Beer – Lambert’s Law:

By combining Beers law and Lamberts law we get:

I= Io e-kct


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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.

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:


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ε = A1%1cm x molecular weight

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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

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:


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http://en.wikipedia.org/wiki/Photosynthesis

http://en.wikipedia.org/wiki/Chlorophyll

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http://en.wikipedia.org/wiki/Transition_metal

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http://en.wikipedia.org/wiki/Carotene

http://en.wikipedia.org/wiki/Lycopene

http://en.wikipedia.org/wiki/Azo_compound

http://en.wikipedia.org/wiki/Azo_compound

http://en.wikipedia.org/wiki/Food_coloring

http://en.wikipedia.org/wiki/Retinal

http://en.wikipedia.org/wiki/Aromatic

http://en.wikipedia.org/wiki/Chemical_bond

http://en.wikipedia.org/wiki/Pi_bond

http://en.wikipedia.org/wiki/Conjugated_system

http://en.wikipedia.org/wiki/Excited_state

http://en.wikipedia.org/wiki/Ground_state

http://en.wikipedia.org/wiki/Electron

http://en.wikipedia.org/wiki/Molecular_orbitals

http://en.wikipedia.org/wiki/Visible_spectrum

26

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:

Repeatability,

Intermediate precision,

Reproducibility.

(a) Repeatability:


http://www.ich.org/

27

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.

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.


28

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.

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.


29

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.

DRUG PROFILE

ROPINIROLE HYDROCHLORIDE

Molecular Structure :


http://www.pharmainfo.net/reviews/introduction-analytical-method-validation

30

.

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.

Solubility1. Freely Soluble in Methanol and Ethanol.

2. Freely Soluble in Water.


http://en.wikipedia.org/wiki/Oxygen

http://en.wikipedia.org/wiki/Nitrogen

http://en.wikipedia.org/wiki/Hydrogen

http://en.wikipedia.org/wiki/Carbon

31

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.

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


32

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.

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.


33

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.

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.


34

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).

4. AIM AND PLAN OF WORK

Aim:


35

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

REVIEW OF LITERATURE

1. Krishnan et al 2010 reported a novel stability-indicating gradient reverse phase ultra


36

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.

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


37

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

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.


38

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.

EXPERIMENTAL WORK

UV SPECTROPHOTOMETRIC METHOD

Instrument Used


http://informahealthcare.com/action/doSearch?action=runSearch&type=advanced&result=true&prevSearch=%2Bauthorsfield%3A(RAMJI%2C+J.+V.)

39

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.

A=A/bc

Where

a= Absorptivity


40

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.

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.


41

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.

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,


42

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

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


43


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

Relative Standard Deviation (%RSD) or Coefficient of Variation (%CV)


44

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

RESULTS AND DISCUSSION

DETERMINATION OF λ MAX


45

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

DETERMINATION OF ABSORPTIVITY :


46

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

Absorptivity of Ropinirole hydrochloride

Table no - 1


47

EFFECT OF TIME ON STABILITY OF ABSORBANCE:


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

48

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

49

Fig no: 2

0 hrs 6 hrs 12 hrs

18 hrs

24 hrs

30 hrs

36 hrs

42 hrs

48 hrs

0.2

0.22

0.24

0.26

0.28

0.3

0.32

0.34

0.36

0.38

0.4

Chart Title

Series1

Axis Title

Stability of Ropinirole Hydrochloride in water – Absorbance Vs Time

DETERMINATION OF OVERLAY


50

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

DETERMINATION OF STANDARD ABSORBANCE


51

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

Linearity curve of Ropinirole Hydrochloride


52

Fig no - 4

Graph showing standard absorbance curve of Ropinirole hydrochloride

DATA FOR LEAST SQUARE METHOD:


53

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

REPEATABILITY OF ABSORBANCE (249nm) AT 10µg/ml


54

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

DETERMINATION OF % ASSSAY FROM AMOUNT DETERMINED:


55

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

Graph showing recovery studies for 50%


56

Fig no - 5


57


Fig no - 6



58

Fig no - 7


59

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 Ʃx2 = 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.


60

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


61

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3. project final edit