1. LAB. S2: Spectrophotometric Determination of Indomethacin/Salicylic Acid Mixture

2. Objectives Scan an absorbance spectrum using the spectrophotometer. Deduce the dependence of the absorption coefficient on the wavelength. Choose a suitable wavelength for photometric determination of a mixture from the spectra scans of its components. Calculate the absorption coefficient. Define the additive character of absorbance. Calculate the concentration of the individual components of a mixture spectrophotometrically.

3. Principle The total absorbance of a solution at a given wavelength is equal to the sum of the absorbencies of the individual components present in it. This relationship allows the quantitative determination of the individual constituents of a mixture, even if their spectra overlap.

4. I.Spectral scan preparation Transfer 2.0 mL of the standard indomethacin stock solution into a 100-mL volumetric flask and complete to 100.0 mL with distilled water. Record the absorbance of your standard solution from λ = 230 nm to λ = 330 nm at 5 nm intervals. Do not forget the blanks! Reset the absorbance reading to zero using the blank solution each time you change the wavelength. Draw the absorbance spectrum of indomethacin (absorbance vs. wavelength), use Microsoft Excel™ or graph paper. From the absorbance spectrum select λ max for indomethacin, (λ'). For salicylic acid, repeat the previous steps to prepare a standard solution. Draw an absorbance spectrum and determine its λ max (λ").

5. II.Calibration curves In three 100-mL volumetric flasks, pipette 0.5, 1.0, and 2.0 mL of the standard indomethacin stock solution respectively and complete the volume to 100.0 mL with distilled water. Using these 3 serial dilutions, construct a three point calibration curve (absorbance vs. concentration) at λ' and λ" in order to determine the absorption coefficients of indomethacin at both λ (ε' I, ε" I ), where ε is the slope of the curve. For salicylic acid, in three 100-mL volumetric flasks, pipette 1.0, 2.0, and 3.0 mL of the standard salicylic acid stock solution respectively, complete the volume to 100.0 ml with distilled water & repeat the previous steps to calculate absorption coefficients of salicylic acid at both λ (ε' S, ε" S ). ’

6. III. Test solution Transfer 2.0 ml of the test solution (indomethacin/salicylic acid mixture) into a 100-mL volumetric flask and complete the volume to 100.0 mL with distilled water. Measure the absorbance of the mixture at λ' and λ" then insert the results in the table in your lab report.

7. Simultaneous Equation Using the absorption coefficients of indomethacin at both λ (ε' I, ε" I )and the absorption coefficients of salicylic acid at both λ (ε' S, ε" S ) we will have 2 equations: Substitute with the absorbance of the mixture at both λ ‘ and λ “ and solve for the unknown concentration of Indomethacin and Salicylic acid simultaneously.