UV-VISIBLE SPECTROSCOPY IN QUANTITATIVE ANALYSIS Seminar on UV-VISIBLE SPECTROSCOPY IN QUANTITATIVE ANALYSIS

BASIS OF UV-VISIBLE SPECTROSCOPY When electromagnetic radiations(UV- Visible radiations) interact with matter, electronic transitions takes place in the molecule and electrons migrates from HOMO to LUMO is the basis of UV- Visible spectroscopy.

UV-Visible ranges Vacuum UV region: Below 200nm. Near UV region: 200nm- 400nm. Visible region: 400nm-800nm.

ELECTRONIC TRANSITIONS

.   * In alkane High-energy, accessible in vacuum UV (max <150 nm). n  * O,N,S,Halogen. Wavelength (max) in the 150-250 nm region. n  * carbonyl compound. Forbidden ,Wavelength 280nm   * In lone pair & multiple bond Alkene,alkyne, azo comp. Wavelength (200-600nm )

BEER-LAMBERT LAW When a beam of light is passed through a transperent cell containing solution of an absorbing substance , reduction in intensity of light may occur. A =log(Io/It)= abc A = Absorbance Io = Intencity of incident light It = Intencity of transmitted light a = Molar absorptivity b = Lenth of sample cell/path lenth. c = Concentration of sample

UV-VISIBLE SPECTROSCOPY IN QUANTITATIVE ESTIMATION Ultraviolet–visible spectrophotometry is perhaps the most widely used spectrophotometric technique for the quantitative analysis of chemical substances as pure materials and as components of dosage forms.

STEPS FOR ASSAY Step1: Select the Solvent Step2: Prepare the series of known dilutions. Step3: Set λmax in spectrophotometer. Step4: Measure absorbance. Step5: Plot calibration curve.

CALIBRATION GRAPH William Kemp, Organic spectroscopy,third edition,page no.243-252.

METHODS Simultaneous Equation Method. Absorbance Ratio Method. Difference Spectroscopy. Derivative Spectroscopy. Multiwavelength uv spectrophotometry. Dual wavelength spectroscopy. Area Under Curve Method.

Simultaneous equation Method * If a sample contains two absorbing drugs, each of which absorbs at the λmax of the other, then both the drugs can be determine by this technique. * The λmax of two component should be reasonably dissimilar. * The two component should not interact chemically.

The individual absorption spectra of substances X & Y .

EQUATIONS Cx = (A2. ay1 – A1.ay2) / (ax2.ay1 – ax1.ay2) Cy = (A1. ax2 – A2.ax1) / (ax2.ay1 – ax1.ay2) Where Cx = Concentration of Drug X in gms/lit Cy = Concentration of Drug Y in gms/lit A2 =Absorbance at λ2 ; A1 =Absorbance at λ1 ax1= Absorptivity of Drug X at λ1 ay1= Absorptivity of Drug Y at λ1 ax2= Absorptivity of Drug X at λ2 ay2= Absorptivity of Drug Y at λ2 A.H.Becket, J.B. Stenlake; Practical pharmaceutical chemistry, fourth edition-part 2;page no. 284-286

Example Validated spectrophotometric methods for simultaneous estimation of telmisartan and indapamide in pharmaceutical dosage form: Solvent used: Methanol. Drug: Telmisartan and Indapamide λmax: TEL 296 nm IND242 nm

Preparation of stock & std solution: Preparation of std. stock solution: TEL:100 ppm solution. IND: 100 ppm solution. Preparation of std.solution: TEL: 20 ppm solution. IND: 20 ppm solution.

OVERLAIN SPECTRA OF TEL & IND: .

Ramesh L. Sawant et al,Der Pharma Chemica, 2012, 4 (2):633-638. RESULT: Beer's law is obeyed in the concentration range of 5-25 μg/ml for telmisartan and 10- 30 μg/ml for indapamide with correlation coefficient within range of 0.996 -0.998 for both the drugs. Ramesh L. Sawant et al,Der Pharma Chemica, 2012, 4 (2):633-638.

Absorbance Ratio Method For a substance which obeys BEER’S LAW at all the wavelengths, the ratio of absorbance at any two wavelengths is constant value, independent of concentration or path length. For e.g. Cyanocobalamin exhibit 3 λmax at 278 nm, 361 nm, 550 nm A361/A550 = 3.30 ± 0.15 A361/A278 = 1.79 ± 0.09

Wavelength selection for substance X &Y .

Equations Cx = (Qm-Qy). A1 / (Qx-Qy). ax1 Cy = (Qm-Qx). A1 / (Qy-Qx). ay1 Where Qm = A2/ A1; Qx = ax2/ ax1; Qy = ay2/ ay1 A2 =Absorbance at λ2 ; A1 =Absorbance at λ1 ax1= Absorptivity of Drug X at λ1 ay1= Absorptivity of Drug Y at λ1 ax2= Absorptivity of Drug X at λ2 ay2= Absorptivity of Drug Y at λ2 A.H.Becket, J.B. Stenlake; Practical pharmaceutical chemistry, fourth edition-part 2;page no. 286-288.

Example Spectrophotometric method for simultaneous estimation of Atrovastatin and Niacin in tablet dosage form: Solvent: Methanol Drugs: Atrovastatin, Niacin. λmax: ATR:246nm NIA:262nm Isoabsorptive point: 258 nm

Preparation of std. solutions Preparation of std. stock solution: ATR: 100 ppm NIACIN: 100ppm Preparation of std. solution: ATR: 20 ppm NIACIN: 20ppm

Overlain spectra of ATR and NIACIN .

Result This method follows the Beer lambert law within range of 5-25 ppm. The overlain UV absorption spectra of ATR(246nm) and NIA(262nm) shows iso- absorptive point at 258nm in Methanol. Recovery study: ATR100.01±0.0345%. NIA99.97±0.3567.% Sawant Ramesh et al. IJPER 2012,3(5)

Difference Spectroscopy The selectivity and accuracy of spectro- photometric analysis of samples contain- ing absorbing interferents may be markedly improved by this technique. Essential feature is that measured value is ΔA i.e. difference in absorbance between two equimolar solutions of the analyte in the same chemical form having different spectral characteristics. ΔA = A( alkaline ) – A( acid ).

Criteria & Technique Reproducible changes may induced in the spectra by addition of one or more reagents. The absorbance of the interfering substance is not alter by any reagent. Technique for altering the spectral properties of the analyte is the adjustment of the pH.

Example: Solvent: 0.1 N HCL and 0.1 N NaoH Drugs: Ibuprofen Difference spectrophotometric estimation and validation of ibuprofen from bulk and tablet dosage form: Solvent: 0.1 N HCL and 0.1 N NaoH Drugs: Ibuprofen

Preparation of Standard Drug Solutions Standard stock solution of ibuprofen: 100ppm. Standard solution of ibuprofen:10 ppm.

Overlain spectra of Ibuprofen in 0.1 N HCl and 0.1 NaOH

Result Ibuprofen exhibits a substantial difference in absorbance in the two solvents that is in 0.1 N HCL and 0.1 N NaoH at 222 nm. Beer’s law is followed within range of 5 to 40 μg /ml. Hapse S.A. et al., Der Pharmachemica letter, 2011,3(6):18-23

Derivative spectroscopy Derivative spectroscopy involves the conversion of a normal spectrum to it’s first, second or higher derivative spectrum. The first derivative spectrum of an absorption band is characterised by a maximum, a minimum & a cross-over point at the λmax of the absorption band. The second derivative spectrum is characterised by a minimum corresponds to λmax of the fundamental band (Maximum negative curvaure).

Zeroth (a), First(b),&Second (c) derivative spectra .

Example Drugs: Ofloxacin and Satranidazole. Wavelength: OFL-259nm Simultaneous Spectrophotometric Estimation of Ofloxacin and Satranidazole in Tablet Dosage Form: Drugs: Ofloxacin and Satranidazole. Wavelength: OFL-259nm SAT-227nm Solvent: Double distilled water was used as a solvent. Preparation of Standard stock solution: OFL&SAT: 100ppm. Preparation of Standard solution: OFL&SAT: 25ppm.

Overlain first derivative spectra of OFL & SAT.

Result: Beer’s law is followed within range of 5 to 40 μg/ml. % Recovery: OFL-98.74% SAT-100.95% Wankhede SB*et al, Asian J. Research Chem. 1(1): July-Sept. 2008.

Multiwavelength UV – Spectroph- otometric Method. Six mixed standard solutions of drugs in the ratio of 1:1 μg/mL were prepared in specific solvent. All the standard solutions were scanned over the range of 400 - 200 nm, in the multicomponent mode, using two sampling wavelength . The overlay spectra of mix standard solution drawn. The data from these scans were used to determine the concentrations of two drugs in tablet sample solution.

Example Simultaneous Estimation & Validation Of Paracet- amol, Phenylephrine Hydrochloride And Chlorphen- iramine Maleate In Tablets By Spectrophotometric Method: Drugs: Paracetamol Phenylephrine Hydrochloride Chlorpheniramine maleat Wavelength: Paracetamol -256.8nm Phenylephrine Hydrochloride-236.8nm Chlorpheniramine maleat -222.4nm

. Solvent: 0.1NNaOH Preparation of standard Stock solutions: Paracetamol -250ppm. Phenylephrine Hydrochloride -250ppm. Chlorpheniramine maleat -250ppm. Preparation of standard solutions: Paracetamol -20ppm. Phenylephrine Hydrochloride-20ppm. Chlorpheniramine maleat -20ppm.

Overlain spectra of paracetamol, phenyle- phrine hydrochloride, and chlorpheniramine maleate.

Result Beer- Lambert’s is obayed in concentration range of 0-35 μg/mL for paracetamol, phenylephrine hydrochloride and chlorpheni- ramine maleate with coefficient of correlation 0.9990, 0.9990 and 0.9960 respectively. R.Sawant JPRHC Volume 3,Issue 2,Page 23-28.

DUAL WAVELENGTH SPECTR- OSCOPIC METHODS In dual wavelength method, two wavelengths were selected for each drug in a way so that the difference in absorbance is zero for another drug. Dual wavelength spectroscopy offers an efficient method for analyzing a component in presence of an interfering component. For elimination of interferences, dual analytical wavelengths were selected in a way to make the absorbance difference zero for one drug in order to analyse the other drug.

Example: Dual Wavelength Spectrophotometric Method for Simultaneous Estimation of Ofloxacin and Cefpodoxime Proxetil in Tablet Dosage Form: Drugs: Ofloxacin Cefpodoxime Proxetil Wavelength: Ofloxacin 224 nm and 247.4 nm CefpodoximeProxetil278.2nm&320nm Solvent: Methanol.

Preparation of sample solution: Preparation of standard Stock solutions: Ofloxacin -100ppm Cefpodoxime Proxetil -100ppm Preparation of standard solutions: Ofloxacin -10ppm Cefpodoxime Proxetil -10ppm

Overlain Absorption Spectra Of OFL And CEFPO .

Result: Linearity range: ofloxacin 2-14 μg/ml and cefpodoxime proxetil 4-24 μg/ml. LOD and LOQ: OFLO 0.332 and 1.09μg/ml CEFPO 0.30 and 0.99μg/ml Patel Sanket A et al . , Asian Journal of Pharmacy and Life Science Vol. 1 (3), July-Sept, 2011

AREA UNDER CURVE METHOD Principle: Total area under curve of a mixture at a wavelength range is equal to the sum of area under the individual component at that wavelength range. Applicable when the λmax of the two components are reasonably dissimilar. Components do not interact chemically. Components must be soluble in same solvent.

Example: Simultaneous Spectrophotometric Estimation of Ofloxacin and Satranidazole in Tablet Dosage Form: Drugs: Ofloxacin -292.5-282.5nm. Satranidazole -325.0-315.0nm. Solvent: Doubled distilled water. Preparation of standard Stock solutions: Ofloxacin - 100ppm Satranidazole-100ppm Preparation of standard solutions: Ofloxacin - 25ppm Satranidazole-25ppm

Overlain Spectra of OFL and SAT .

Result: In area under curve method, the area under curve in the range of 292.5-282.5 nm (for OFL) and 325.0-315.0 nm (for SAT) were selected for the analysis. Linearity for detector response was observed in the concentration range of 5-40 μg/ml for OFL and SAT. Percent recovery for OFL and SAT, was found in the range of 98.74 % to 100.95 %. Wankhede SB et al. ,Asian J. Research Chem. 1(1): July-Sept. 2008.

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