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3. project final edit

  1. 1. 1 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
  2. 2. 2 Department of Pharmaceutical Analysis Page 2 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.
  3. 3. 3 Department of Pharmaceutical Analysis Page 3 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.
  4. 4. 4 Department of Pharmaceutical Analysis Page 4 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 :
  5. 5. 5 Department of Pharmaceutical Analysis Page 5 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:
  6. 6. 6 Department of Pharmaceutical Analysis Page 6
  7. 7. 7 Department of Pharmaceutical Analysis Page 7 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
  8. 8. 8 Department of Pharmaceutical Analysis Page 8 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
  9. 9. 9 Department of Pharmaceutical Analysis Page 9 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.
  10. 10. 10 Department of Pharmaceutical Analysis Page 10 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.
  11. 11. 11 Department of Pharmaceutical Analysis Page 11 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:
  12. 12. 12 Department of Pharmaceutical Analysis Page 12 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
  13. 13. 13 Department of Pharmaceutical Analysis Page 13 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
  14. 14. 14 Department of Pharmaceutical Analysis Page 14 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
  15. 15. 15 Department of Pharmaceutical Analysis Page 15 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.
  16. 16. 16 Department of Pharmaceutical Analysis Page 16 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-
  17. 17. 17 Department of Pharmaceutical Analysis Page 17 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.
  18. 18. 18 Department of Pharmaceutical Analysis Page 18 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.
  19. 19. 19 Department of Pharmaceutical Analysis Page 19 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.
  20. 20. 20 Department of Pharmaceutical Analysis Page 20 (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.
  21. 21. 21 Department of Pharmaceutical Analysis Page 21 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.
  22. 22. 22 Department of Pharmaceutical Analysis Page 22 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
  23. 23. 23 Department of Pharmaceutical Analysis Page 23 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.
  24. 24. 24 Department of Pharmaceutical Analysis Page 24 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
  25. 25. 25 Department of Pharmaceutical Analysis Page 25 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:
  26. 26. 26 Department of Pharmaceutical Analysis Page 26  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.
  27. 27. 27 Department of Pharmaceutical Analysis Page 27 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. 28. 28 Department of Pharmaceutical Analysis Page 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. 29. 29 Department of Pharmaceutical Analysis Page 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. 30. 30 Department of Pharmaceutical Analysis Page 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. 31. 31 Department of Pharmaceutical Analysis Page 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. 32. 32 Department of Pharmaceutical Analysis Page 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. 33. 33 Department of Pharmaceutical Analysis Page 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. 34. 34 Department of Pharmaceutical Analysis Page 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. 35. 35 Department of Pharmaceutical Analysis Page 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. 36. 36 Department of Pharmaceutical Analysis Page 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. 37. 37 Department of Pharmaceutical Analysis Page 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. 38. 38 Department of Pharmaceutical Analysis Page 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. 39. 39 Department of Pharmaceutical Analysis Page 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. 40. 40 Department of Pharmaceutical Analysis Page 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. 41. 41 Department of Pharmaceutical Analysis Page 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 hydrochloride in water using photometric mode – quantitative mode using the absorbance values at various concentrations. Calculate the total amount present in the stock solution, amount recovered and percentage recovery. 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. 42. 42 Department of Pharmaceutical Analysis Page 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 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. 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. 43. 43 Department of Pharmaceutical Analysis Page 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. 44. 44 Department of Pharmaceutical Analysis Page 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. 45. 45 Department of Pharmaceutical Analysis Page 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. 46. 46 Department of Pharmaceutical Analysis Page 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. 47. 47 Department of Pharmaceutical Analysis Page 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. 48. 48 Department of Pharmaceutical Analysis Page 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. 49. 49 Department of Pharmaceutical Analysis Page 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. 50. 50 Department of Pharmaceutical Analysis Page 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. 51. 51 Department of Pharmaceutical Analysis Page 51 Linearity curve of Ropinirole Hydrochloride Fig no - 4 Graph showing standard absorbance curve of Ropinirole hydrochloride
  52. 52. 52 Department of Pharmaceutical Analysis Page 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. 53. 53 Department of Pharmaceutical Analysis Page 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. 54. 54 Department of Pharmaceutical Analysis Page 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. 55. 55 Department of Pharmaceutical Analysis Page 55 Graph showing recovery studies for 50% Fig no - 5
  56. 56. 56 Department of Pharmaceutical Analysis Page 56 Graph showing recovery studies for 100% Fig no - 6
  57. 57. 57 Department of Pharmaceutical Analysis Page 57 Graph showing recovery studies for 150% Fig no - 7
  58. 58. 58 Department of Pharmaceutical Analysis Page 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. 59. 59 Department of Pharmaceutical Analysis Page 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. 60. 60 Department of Pharmaceutical Analysis Page 60 BIBILIOGRAPHY 1. Yogita Shete, Nayana Pimpodkar, R. S. Nalawade, Y.V.Pore Spectrophotometric Estimation of Ropinirole Hydrochloride in Tablet dosage forms. Indian J Pharm Sci. 2009 Jan-Feb; 71(1): 61–62 2. Aydogmus.Z Highly sensitive and selective spectrophotometric and spectrofluorimetric methods for the determination of ropinirole hydrochloride in tablets dosage form. Acta A Biomol Spectroscopy Indian Journal of Pharma Research 2008 Jun;70(1):69-78. 3. Monali S. ali, Ajay L. Barhate, Vinit D. Patil, Ajay S. Bhadoriya, Vishnu P. Choudhari* Bhanudas S. KuchekarDer Development and Validation of Area under Curve and First Derivative Spectrophotometric Methods for Ropinirole in Tablet dosage Forms Pharma Chemica, 2010, 2(3):225-229. 4. A. Azeem, Z. Iqbal, F. J. Ahmad, R. K. Khar, S. Talegaonkar Development and validation of a stability-indicating method for determination of ropinirole in the bulk drug and in pharmaceutical dosage forms Acta chromatographica vol 20 No 1 2008. 5. B.Sahasrabuddhey, R. Nautiyal, H. Acharya, S. Khyade, P.K. Luthra and P.B. Deshpande Isolation and characterization of some potential impurities in ropinirole hydrochloride tablet dosage forms. Journal of Pharmaceutical and Biomedical Analysis Volume 43, Issue 4, 12 March 2007, Pages 1587-1593
  61. 61. 61 Department of Pharmaceutical Analysis Page 61 6. Armagan Onal reported in the development of dopaminergic drug Ropinorole used for parkinsonisam by Strophotometric determination Chem .Pharm .Bull; 2007 55 (4): 629-631. 7. B.Jagadeesh, S.Sirish kumar , Revathi Nagalakshmi, Kishore Kumar Hotha, A.Naidu, Ramesh Mullangi., Issue Biomedical Chromatography Volume 23, Issue 5, pages 557–562, May 2009 8. Jignesh Bhatt, Rapid and sensitive liquid chromatography-mass spectrometry method for determination of ropinirole in human plasma. J Pharm Biomed Anal 40:1202-8. 2006. 9. Krishnan reported the development and validation of Ropinirole Hydrochloride by UPLC in API. Journal of Chinese chemical society 57, page no-348-355, 2010. 10. Erin Chambers reported development and determination of Ropinirole in human plasma by SPE UPLC MS method. Chem pharma 55 page no – 128-130. 2009 11. Pavel Coufal reported separation and quantification of Ropinirole and some impurities using capillary liquid chromatography. Journal of chromatography biomedical science and applications vol 732 issue 2, 24th sept pages 437-444. 1999. 12. Karel tulik reported determination of the dissociation constant of Ropinirole and some impurities. Journal of chromatography biomedical science and applicationsvol 720, issue 1-2, 11th Dec, pages 197-204, 1998. 13. Instrumentation R Chatwal, Sham K Anand 2.167-2.172, 2010. 14. Analytical chemistry theory and practise U.N.Das page no – 42 15. UV Spectroscopy by Dr.S.Ravishanker 4th edition page no – 2.2-2.5, 2010. 16.Pharmaceutical drug analysis 2nd edition byAshutoshkar page no – 293.
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