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

2. 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.

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

4. ELECTRONIC TRANSITIONS

5. .
  *
In alkane
High-energy, accessible in vacuum UV (max <150 nm).
n  *
O,N,S,Halogen.
Wavelength (max) in the nm region.
n  *
carbonyl compound.
Forbidden ,Wavelength 280nm
  *
In lone pair & multiple bond Alkene,alkyne, azo comp.
Wavelength
( nm )

6. 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

7. 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.

8. 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.

9. CALIBRATION GRAPH
William Kemp, Organic spectroscopy,third edition,page no

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

11. 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.

12. The individual absorption spectra of substances X & Y.

13. 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

14. 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

15. 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.

16. OVERLAIN SPECTRA OF TEL & IND:.

17. 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 μg/ml for indapamide with correlation coefficient within range of for both the drugs.
Ramesh L. Sawant et al,Der Pharma Chemica, 2012, 4 (2):

18. 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

19. Wavelength selection for substance X &Y.

20. 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

21. 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

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

23. Overlain spectra of ATR and NIACIN.

24. 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± %
Sawant Ramesh et al. IJPER 2012,3(5)

25. 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 ).

26. 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.

27. 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

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

29. Overlain spectra of Ibuprofen in 0.1 N HCl and 0.1 NaOH

30. 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

31. 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).

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

33. 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.

34. Overlain first derivative spectra of OFL & SAT.

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

36. 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 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.

37. 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 nm
Phenylephrine Hydrochloride-236.8nm
Chlorpheniramine maleat nm

38. .
Solvent: NNaOH Preparation of standard Stock solutions: Paracetamol ppm.
Phenylephrine Hydrochloride -250ppm.
Chlorpheniramine maleat ppm.
Preparation of standard solutions:
Paracetamol ppm.
Phenylephrine Hydrochloride-20ppm.
Chlorpheniramine maleat ppm.

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

40. 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 , and respectively.
R.Sawant JPRHC Volume 3,Issue 2,Page

41. 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.

42. 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 nm
CefpodoximeProxetil278.2nm&320nm
Solvent: Methanol.

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

44. Overlain Absorption Spectra Of OFL And CEFPO.

45. Result:
Linearity range: ofloxacin 2-14 μg/ml and cefpodoxime proxetil μg/ml.
LOD and LOQ: OFLO 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

46. 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.

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

48. Overlain Spectra of OFL and SAT.

49. Result:
In area under curve method, the area under curve in the range of nm (for OFL) and 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 % to %.
Wankhede SB et al. ,Asian J. Research Chem. 1(1): July-Sept

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