multi-components assay by UV- shreyas goswami


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methods of drug estimation in multicomponents formulation by UV spectrophotometer. presented by: shreyas goswami


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Methods of drug estimation in multicomponent formulation by UV spectrophotometer.: 

Methods of drug estimation in multicomponent formulation by UV spectrophotometer. Prepared by: Shreyas goswami Guided by: Mr. K.N.Patel M.Pharm.(Q.A.), Assistant professor, 1 st sem. Dept. of Q.A. Nootan pharmacy college, visnagar. 1 Shreyas

Introduction UV-Visible spectroscopy : 

Introduction UV-Visible spectroscopy UV-Visible spectroscopy is concerned with the study of radiation whose wavelength ranges from 200nm-800nm . Substances absorb light of different wavelength in different manner and hence we get an absorption curve in a unique pattern for the substance. In this absorption curve, the wavelength at which maximum absorption of the radiation takes place is called as λ max . λ max is characteristic for every substances and this is qualitative aspect, useful in identifying the substances. Absorption of solution is increased with concentration, but there is no change in λ max when concentration changes . 2 Shreyas

Application of UV spectrophotometer:: 

Application of UV spectrophotometer: Qualitative analysis: The UV spectra of most compounds are of limited value for qualitative analysis as compared to IR and Mass spectra. Qualitative analytical use of UV spectra has largely involved λ-max and absorptivities . Quantitative analysis: UV spectroscopy is perhaps the most widely used spectroscopic techniques for the quantitative analysis of chemical substances as pure materials and as components of dosage forms. 3 Shreyas

Introduction to spectrophotometric methods of analysis for drugs in combination:: 

Introduction to spectrophotometric methods of analysis for drugs in combination: Simultaneous estimation of drug combination is generally done by separation using chromatographic methods like HPLC, GC and HPTLC etc. These methods are accurate and precise with good reproducibility, but the cost of analysis is quite high owing to expensive instrumentation, reagent and expertise.  Hence it is worthwhile to develop simpler and cost effective method for simultaneous estimation of drugs for routine analysis of formulations. 4 Shreyas

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Spectrophotometric analysis fulfils such requirement where the simultaneous estimation of the drug combination can be done with similar effectiveness as that of chromatographic methods. The pharmaceutical analyst frequently encounters the situation where the concentration of one or more substances is required in samples known to contain other absorbing substances, which potentially interfere in the assay. 5 Shreyas

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A number of modifications to the simple spectrophotometric procedure are available to the analyst, which may eliminate certain sources of interference and permit the accurate determination of all of the absorbing components. The basis of all the spectrophotometric techniques for multicomponent samples is the property that at all wavelengths the absorbance of a solution is the sum of absorbance of the individual components or the measured absorbance is the difference between the total absorbance of the solution in the sample cell and that of the solution in the reference cell. 6 Shreyas


Methods: Simultaneous equation method Derivative spectrophotometric method Absorbance ratio method(Q-Absorbance method) Difference spectrophotometry Geometric correction method Chemical Derivatization 7 Shreyas

Simultaneous Equation method:: 

Simultaneous Equation method: If a sample contains two absorbing drugs (X and Y) each of which absorbs at the λ-max of the other (λ1 and λ2), it may be possible to determine both the drugs by the simultaneous equations method. 8 Shreyas

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The information required is ·        The absorptivities of X at λ1 and λ2, aX1 and aX2, ·        The absorptivities of Y at λ1 and λ2, aY1 and aY2, ·        The absorbances of the diluted sample at λ1 and λ2, A1 and A2. Let, Cx and Cy be the concentration of X and Y respectively in the sample. The absorbance of the mixture is the sum of the individual absorbances of X and Y 9 Shreyas

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At λ1 A1 = aX1* Cx + aY1* Cy (1) At λ2 A2 = aX2* Cx + aY2* Cy (2) Multiply the equation (1) with aX2 and (2) with aX1 A1 aX2 = aX1 Cx aX2 + aY1 Cy aX2 (3) A2 aX1 = aX2 Cx aX1+ aY2 Cy aX1 (4) A1 aX2 - A2 aX1 = aY1 Cy aX2 - aY2 Cy aX1 A1 aX2 - A2 aX1 = Cy (aY1 aX2 - aY2 aX1) Cy = (A1 aX2 - A2 aX1) / (aY1 aX2 - aY2 aX1) (5) 10 Shreyas

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Same way we can derive Cx = (A2 aY1 – A1 aY2) / (aY1 aX2 - aY2 aX1) (6) These equations are known as simultaneous equations and by solving these simultaneous equations we can determine the concentration of X and Y in the sample. 11 Shreyas


Examples: Estimation of Losartan potassium and Hydrochlorthiazide in tablets. Estimation of Salbutamol and Theophylline from tablets. Estimation of Amlodipine besylate and Enalepril maleate from tablets. Estimation of Ibuprofen and Pseudoephedrine hydrochloride from tablets. Estimation of Salbutamol and Theophylline from tablets 12 Shreyas

Q-Absorbence ratio method:: 

Q-Absorbence ratio method: The absorbance ratio method is a modification of the simultaneous equations procedure. In the quantitative assay of two components in admixture by the absorbance ratio method, absorbances are measured at two wavelengths, one being the λ-max of one of the components (λ2) and other being a wavelength of equal absorptivity of two components (λ1), i.e. an iso-absorptive point. 13 Shreyas

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At λ1 A1 = aX1* Cx + aY1* Cy (1) At λ2 A2 = aX2* Cx + aY2* Cy (2) Now divide (2) with (1) A2/A1 = (aX2* Cx + aY2* Cy) (aX1* Cx + aY1* Cy) 14 Shreyas

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Divide each term with (Cx + Cy) A2/A1 = (aX2* Cx + aY2* Cy) / (Cx + Cy) (Cx + Cy) (aX1* Cx + aY1* Cy) / (Cx + Cy) Put Fx = Cx / (Cx + Cy) and Fy = Cy / (Cx + Cy) A2/A1 = [aX2 Fx + aY2 Fy] / [aX1 Fx + aY1Fy] Where Fx is the fraction of X and Fy is the fraction of Y i.e. Fy = 1-Fx There fore A2/A1 = [aX2 Fx + aY2 (1-Fx)] / [aX1 Fx + aY1(1-Fx)] = [aX2 Fx + aY2 – aY2Fx] / [aX1 Fx + aY1 – aY1Fx] 15 Shreyas

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At iso-absorptive point aX1 = aY1 and Cx = Cy There fore A2/A1 = [aX2 Fx + aY2 – aY2Fx] / aX1 = (aX2 Fx/ aX1) + (aY2/ aX1) –( aY2Fx/ aX1) Let Qx = aX2/aX1 , Qy = aY2/aY1 and absorption ratio Qm = A2/A1 Qm = Fx Qx + Qy - Fx Qy = Fx (Qx-Qy) + Qy Fx = (Qm – Qy) / (Qx – Qy) (3) From the equations (1) A1 = aX1 (Cx + Cy) there fore Cx + Cy = A1 / aX1 There fore Cx = (A1/aX1) – Cy (4) From the equation (3) Cx / (Cx + Cy) = (Qm – Qy) / (Qx – Qy) There fore Cx / (A1 / aX1) = (Qm – Qy) / (Qx – Qy) There fore Cx = [(Qm – Qy) / (Qx – Qy)] X (A1 / aX1) (5) 16 Shreyas


Examples: Estimation of Rifampicin and Isoniazide in pharmaceutical dosage forms. Estimation of Spiranolactone and hydroflumethiazide. Estimation of Nalidixic acid and Metronidazole from tablets. Estimation of Noscapine, Chlorpheniramine Maleate and Ephedrine hydrochloride from tablets. 17 Shreyas

Derivative Spectroscopy:: 

Derivative Spectroscopy: For the purpose of spectral analysis in order to relate chemical structure to electronic transitions, and for analytical situations in which mixture contribute interfering absorption, a method of manipulating the spectral data is called derivative spectroscopy. Derivative spectrophotometry involves the conversions of a normal spectrum to its first, second or higher derivative spectrum. In the context of derivative spectrophotometry, the normal absorption spectrum is referred to as the fundamental, zero order, or D 0 spectrum. 18 Shreyas

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The first derivative D 1 spectrum is a plot of the rate of change of absorbance with wavelength against wavelength i.e. a plot of the slope of the fundamental spectrum against wavelength or a plot of dA/dλ vs. λ. . The maximum positive and maximum negative slope respectively in the D spectrum correspond with a maximum and a minimum respectively in the D 1 spectrum. The λmax in D spectrum is a wavelength of zero slope and gives dA/dλ = 0 in the D 1 spectrum. 20 Shreyas

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The second derivative D 2 spectrum is a plot of the curvature of the D spectrum against wavelength or a plot of d 2 A/ dλ 2 vs. λ. The maximum negative curvature in the D spectrum gives a minimum in the D 2 spectrum, and the maximum positive curvature in the D spectrum gives two small maxima called satellite bands in the D 2 spectrum. The wavelength of maximum slope and zero curvature in the D spectrum correspond with cross-over points in the D 2 spectrum. 21 Shreyas

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For the quantitative estimation of binary mixtures by the derivative spectroscopy, first of all we have to find out the Zero Crossing Points (ZCP) for both the components (A and B). Now select ZCP for A and B so that at that particular ZCP other component shows remarkable absorbance. Now prepare calibration curve of A at the ZCP of B and of B at the ZCP of A. Find out the unknown concentration using calibration curves. 22 Shreyas


Examples: Estimation of Propranolol and Hydrochlorthiazide. Estimation of Phenylpropanolamine, chlorpheniramine and Bromhexine. Estimation of Naphazoline hydrochloride and Chlorpheniramine maleate . 23 Shreyas

Geometric correction method:: 

Geometric correction method: A number of mathematical correction procedures have been developed which reduce or eliminate the background irrelevant absorption that may be present in samples of biological origin. The simplest of this procedure is the three point geometric procedure, which may be applied if the irrelevant absorption is linear at the three wavelengths selected. 24 Shreyas

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If the wavelengths λ 1, λ 2 and λ 3 are selected to that the background absorbances B 1 , B 2 and B 3 are linear, then the corrected absorbance D of the drug may be calculated from the three absorbances A 1 , A 2 and A 3 of the sample solution at λ 1, λ 2 and λ 3 respectively as follows, Let v D and w D be the absorbance of the drug alone in the sample solution at λ 1 and λ 3 respectively, i.e. v and w are the absorbance ratios vD/D and wD/D respectively. B 1 = A 1 – vD, B 2 = A 2 –D and B 3 = A 3 –wD 26 Shreyas

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Let y and z be the wavelengths intervals (λ 2 – λ 1 ) and (λ 3 - λ 2 ) respectively D= y(A 2 -A 3 ) + z(A 2 – A 1 ) / y (1-w) + z(1-v) This is a general equation which may be applied in any situation where A 1, A 2 and A 3 of the sample, the wavelength intervals y and z and the absorbance ratio v and w are known. 27 Shreyas

Difference Spectroscopy:: 

Difference Spectroscopy: Difference spectroscopy provides a sensitive method for detecting small changes in the environment of a chromophore or it can be used to demonstrate ionization of a chromophore leading to identification and quantitation of various components in a mixture. The essential feature of a difference spectrophotometric assay is that the measured value is the difference absorbance (Δ A) between two equimolar solutions of the analyte in different forms which exhibit different spectral characteristics. 28 Shreyas

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The criteria for applying difference spectrophotometry to the assay of a substance in the presence of other absorbing substances are that: A) Reproducible changes may be induced in the spectrum of the analyte by the addition of one or more reagents. B) The absorbance of the interfering substances is not altered by the reagents. The simplest and most commonly employed technique for altering the spectral properties of the analyte properties of the analyte is the adjustment of the pH by means of aqueous solutions of acid, alkali or buffers. 29 Shreyas

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A B A) The Spectrum of compound in A(acid) and B(Base) B) The difference spectrum of B relative to A 30 Shreyas

Chemical Derivatisation:: 

Chemical Derivatisation: Indirect spectrophotometric assays are based on the conversion of the analyte by a chemical reaction to a derivative that has different spectral properties. When an excess of the reagent is used , to ensure complete conversion, the absorbance of the derivative is usually, but not always proportional to the concentration of the analyte. The majority of indirect spectrophotometric procedures involve the conversion of the analyte to a derivative that has a longer λ max and/ or a higher absorptivity. 31 Shreyas

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Chemical derivatisation procedures may be adopted for any of the following reasons. If the analyte absorbs weakly in the uv region, a more sensitive method of the assay is obtained by converting the substance to a derivative with a more intensely absorbing chromophore. For e.g.sugaras which do not absorb significantly above 220 nm can be determined spectrophotometrically by heating with anthore in concentrated sulphuric acid and measuring the absorbance of the coloured derivative at 625 nm. 32 Shreyas

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The interference from irrelevant absorption may be avoided by converting the analyte to a derivative which absorbs in visible region, where irrelevant aborption is negligible. Indirect spectrophotometic methods are also useful to improve the selectivity of the an uv absorbing substance in a sample that contains other uv absorbing components. The adoption of a visible spectrophotometric procedure instead of an uv procedure, may be based on cost considerations. Colorimeters are generally cheaper than ultraviolet –visible spectrophotometers. 33 Shreyas


References: 34 Shreyas