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1. UV-Visible spectroscopy: Introduction, Theory, Laws, Instrumentation associated with UV-Visible spectroscopy, Choice of solvents and solvent effect and Applications of UV-Visible spectroscopy, Difference/ Derivative spectroscopy.

2. UV Visible Absorption Spectroscopy

3. Beer Lambert’s law

4. Radiation source Visible radiation Tungsten filament in glass envelope Range- 350- 2000 nm. Life limited by evaporated of tungsten which deposits on inner walls on glass envelope. Alternative: Tungsten- Halogen lamps- Filled halogen gas in quartz envelope. Expensive with

5. Absorption filter: Oxides of metals viz. vanadium, chromium, manganese, iron, nickel and copper Color absorbed is the complement of color of filter Ex. Filter absorbing yellow light appears blue Wide bandwidth Dyes mixed with gelatin and sandwiched between glass plate has narrow BW (25nm) Large errors

6. Interference filter Principle: Interference of light • Narrower BW (15 nm) • Two parallel glass plates, silvered internally and separated by thin film of cryolite or other dielectric material. Relatively less errors as selective in wavelength selection.

7. Gratings

8. Barrier Layer Cell Photon Tubes Cathode Anode

9. Photomultiplier Tubes

10. OPTIMUM CONDITIONS FOR SPECTROPHOTOMETRIC MEASUREMENT 1. Sample Conditions: Solvent Concentration and pathlength

11. Cut-off wavelength

12. Deviation from the Beer- Lambert Law 1. Non-monochromatic Light

13. Instrumental Parameters . Slitwidth- Monochromaticity or spectral purity of the light incident on the sample cell is defined by the spectral bandwidth, which is the width of the triangle (in nm) at one half of the peak intensity. The spectral bandwidth (SBW) is related to the mechanical width of the slit (SW) and the dispersion (D) of the monochromator SBW = SW * D Ex. SW =0.1 mm and D = 3 nm/ mm Then SBW = 0.3 nm An increase in the slitwidth increases the spectral bandwidth and reduces the monochromaticity of the light.

14. The effect that increasing the slitwidth has on a measured absorbance value depends on the width of the absorption band and on whether the wavelength of measurement corresponds with a maximum and minimum absorbance. The width of the absorption band is defined by its natural bandwidth, which is the width of the absorption band at one half the Amax. It has been shown that, if the spectral bandwidth exceeds 1/10 th the natural bandwidth of the substances, the absorbance at the λ max will be less than the true value.

15. This is because lower absorbances at wavelengths on slope of the absorption band, included within a broad spectral bandwidth, contribute to the measured value. Furthermore, as the absorbance increases due to higher concentration or longer pathlengths, the % error increases, resulting in a negative deviation from the Beer- Lambert Law. Positive deviation- Reverse case

16. Scanning Speed

17. Chemical Effect Dissociation, Association, Polymerization, Complex formation

18. Solvent effects

19. Applications Quantitative analysis Single component analysis Multicomponent analysis

20. Single component analysis Use of standard absorptivity value Use of a calibration graph Single point or double point standardization

21. Use of standard absorptivity value Transmittance Absorbance, Absorptivities, Specific absorbance

22. Use of standard absorptivity value No effect of variation of instrumental parameters e.g. slit width, scan speed No need to prepare reference standard solution Reference standard is costly Equations

23. Use of a calibration graph If the absorbance has a nonlinear relationship with concentration If necessary to confirm proportionality of absorbance as function of concentration If the absorbance or linearity is dependent on assay conditions(pH, temp and time of heating Manual vs statistical construction of graph If linear relationship then regression line estimated by method of least squares.

24. Linear regression is a way to model the relationship between two variables. You might also recognize the equation as the  slope formula . The equation has the form Y=a+bX, where Y is the dependent variable (that’s the variable that goes on the Y axis), X is the independent variable (i.e. it is plotted on the X axis), b is the slope of the line and a is the y-intercept. The first step in finding a linear regression equation is to determine if there is a relationship between the two variables.

25. How to Find a Linear Regression Equation: Steps SUBJECT AGE X GLUCOSE LEVEL Y XY X 2 Y 2 1 43 99 4257 1849 9801 2 21 65 1365 441 4225 3 25 79 1975 625 6241 4 42 75 3150 1764 5625 5 57 87 4959 3249 7569 6 59 81 4779 3481 6561 Σ 247 486 20485 11409 40022 Step 1: Make a chart of your data, filling in the columns From the above table, Σx = 247, Σy = 486, Σxy = 20485, Σx2 = 11409, Σy2 = 40022. n is the sample size (6, in our case).

26. Step 2:  Use the following equations to find a and b. a = 65.1416 b = .385225

27. Step 3: Insert the values into the equation. y = a + bx y = 65.14 + .385225x *

28. Correlation coefficient To establish whether there is a linear relationship between two variables x 1 and y 1 , use Pearson’s correlation coefficient, r. What is Pearson Correlation? Correlation between sets of data is a measure of how well they are related. The most common measure of correlation in stats is the Pearson Correlation.  Where n is the number of data points. The value of r must lie between +1 and -1; the nearer it is to ±1, the greater the probability that a definite linear relationship exists between the variables x and y. Values close to +1 indicate positive correlation and values close to -1 indicate negative correlation. Values of r that tend towards zero indicate that x and y are not linearly related.

29. Correlation coefficient formulas are used to find how strong a relationship is between data. The formulas return a value between -1 and 1, where: A correlation coefficient of 1 means that for every positive increase in one variable, there is a positive increase of a fixed proportion in the other. Ex. Absorbance increases with increase in concentration of substance. A correlation coefficient of -1 means that for every positive increase in one variable, there is a negative decrease of a fixed proportion in the other. For example, the amount of gas in a tank decreases in (almost) perfect correlation with speed. Zero means that for every increase, there isn’t a positive or negative increase. The two just aren’t related.

30. Single point or double point standardization Single point standardization Substances obey Beer lambert’s law Reference standard of adequate purity is available

31. Double point standardization A linear but nonpropotional relationship between concentration and absorbanceindicated by a significant positive or negative intercept in a Beer’s Law plot. Two point bracketing standardization Subscripts std1 and std2 refer to the more concentrated and less concentrated standard resp

32. Determination of equilibrium constants

33. Determination of rate constants

34. Multi-component Spectrophotometric Analysis UV-Visible Spectrophotometry

35. Single component analysis A systematic error of less than 1% would normally considered to be to be acceptable Assay of paracetamol in paediatric paracetamol elixir.(3250 times diluted)

36. Assay using absorbance corrected for interference Identity concentration and absorptivity of the absorbing interferents is know

37. Assay after solvent extraction of the sample Eg: Caffeine in Aspirin and caffeine tablets

38. SIMULTANEOUS EQUATION METHOD Vierodt’s Method Sample containing two absorbing drugs each of which absorbs at the λmax of the other. Preliminary requirements : Two absorbing species (x and y), each of which should absorb at the λmax of the other. The λmax of two drugs should be reasonably dissimilar. The two components should not interact chemically.

39. λ max of drugs Fig.: Wavelength selection by Simultaneous equation method A 2 ay 1 – A 1 ay 2 Cx = ---------------------- ax 2 ay 1 – ax 1 ay 2 A 1 ax 2 – A 2 ax 1 Cy = ---------------------- ax 2 ay 1 – ax 1 ay 2 Simultaneous Equations Where Cx and Cy = concentration of x and y respectively, ax1 and ax2 = absorptivities of x at λ1 and λ2 respectively, ay1 and ay2 = absorptivities of y at λ1 and λ2 respectively, A1 & A2 = The absorbances of the diluted sample at λ 1 and λ 2

40. Simultaneous Equation If sample contains two absorbance species (X and Y) each of which absorbs at the λmax of the other, it may be possible to determine both drugs by the technique of simultaneous equation (Vierodt’s method). Criteria for obtaining maximum precision- and Should lie outside the range 0.1 – 2.0 A 2 / A 1 Ax 2 / ax1 Ay2 / ay1 A2 / A1

41. Simultaneous equation Cx = A 2 ay 1 – A 1 ay 2 ax 2 ay 1 – ax 1 ay 2 Cy = A 1 ax 2 – A 2 ax 1 ax 2 ay 1 – ax 1 ay 2

42. Absorbance ratio method This is modification of simultaneous equation method. It depends on the property that, for a substance that obeys Beer’s law at all wavelengths, the ratio of absorbances at any two wavelengths is a constant value independent of concentration or pathlength. Ex. Two different dilutions of the same substance give the same absorbance ratio.

43. Absorbance ratio method…… In USP this value is referred as Q value. BP also uses ratio of substances at specified wavelengths in certain confirmatory tests of identity. For Eg: Cyanocobalamin exhibits three λmax at 278nm, 361nm and 550nm. The A 361 /A 550 is required to be 3.30±0.15 and the A 361 /A 278 is required to be 1.79±0.19. Application: In quantitative assay of two components absorbances taken at two wavelengths- - one being the λmax of one of the components (λ 2 ) and - the other being a wavelength of equal absorptivity of the two components (λ 1 ).

44. Absorbance ratio method Cx = (Q M – Q Y )A1 (Q X – Q Y ) ax 1 Q X = ax 2 ax 1 Q y = ay 2 ay 1 Q M = A 2 A 1

45. Difference spectrophotometry The selectivity and accuracy of spectrophotometric analysis samples containing absorbing interferents may be improved by this technique. Important feature- Measured value is the difference absorbance (ΔA) between two equimolar solutions of the analyte in different chemical forms which exhibit different spectral characteristics. Criteria- Reproducible changes may be induced in the spectrum of the analyte by the addition of one or more reagents’ The absorbance of the interfering substance is not altered by the reagents. Simplest approach- Adjustment of pH.

46. The criteria for applying 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.

47. Technique for altering the spectral properties of the analyte Adjustment of the pH by means of aqueous solutions of acid, alkali or buffers. If the individual absorbances, A alk and A acid are proportional to the concentration of the analyte and path length, the Δ A also obeys the Beer-Lambert law and a modified equation may be derived. Δ A = Δ abc Where Δ a is the difference absorptivity of the substance at the wavelength of measurement.

48. Ex. Phenylephrine (A) 0.1 M NaOH (pH 13) (B) 0.1 M HCl (pH 1)

49. If one or more other absorbing substances is present in the sample which at the analytical absorbance Ax in the alkaline and acidic solutions, its interference in the spectrophotometric measurement is eliminated. Δ A = (Aalk + Ax) – (Aacid + Ax) The selectivity of the Δ A procedure depends on the correct choice of the pH values to induce the spectral change of the analyte without altering the absorbance of the interfering components of the sample. The use of 0.1M sodium hydroxide and 0.1M hydrochloric acid to induce the Δ A of the analyte is convenient and satisfactory

50. Derivative Spectrophotometry Derivative spectrophotometry is a useful means of resolving two overlapping spectra and eliminating matrix interferences. Derivative spectrophotometry involves the conversion of a normal spectrum to its first, second or higher derivative spectrum. Fig. I, II, III Derivative spectra of Substance

51. the number of bands observed is equal to the derivative order plus one. A strong negative or positive band with minimum or maximum at the same wavelength as max of the absorbance band is characteristic of the even-order derivatives.

52. The absorbance of a sample is differentiated with respect to wavelength λ to generate first, second or higher order derivative [A]   Vs (λ ): zero order [dA/dλ ]   Vs (λ ): first order [d 2 A/d λ 2 ]  Vs (λ ): second order The first derivative spectrum of an absorption band is characterized by a maximum, a minimum, and a cross-over point at the λ max of the absorption band. The second derivative spectrum is characterized by two satellite maxima and an inverted band of which the minimum corresponds to the λ max of the fundamental band.

53. The enhanced resolution and bandwidth discrimination increases with increasing derivative order. The important features of derivative technique include enhanced information content, discrimination against back ground noise and greater selectivity in quantitative analysis. It can be used for detection and determination of impurities in drugs, chemicals and also in food additives and industrial wastes.