Tools

Simultaneous estimation method for multicomponent samples: Principle of additivity of absorbance

  1. Prepare five calibration standards for each analyte: 0.025 mM, 0.020 mM, 0.015 mM, 0.010 mM, and 0.005 mM coumarin 343 solutions and 0.020 mM, 0.015 mM, 0.010 mM, 0.005 mM and 0.0025 mM coumarin 6 solutions in ethanol. Also prepare a solution containing 1:1 (v/v) mixture of 0.025 mM coumarin 343 and 0.020 mM coumarin 6. The solutions to be analyzed are selected from the drop-down menu. The solution concentrations are selected from the concentration scale bars.
  2. Switch on the computer and the instrument powers; wait for 30 minutes for ¡®warm-up¡¯ of the instrument.
  3. In the instrument one can select light sources (UV and visible), choose the slit width, scan speed and %transmittance or absorbance display, wavelength range of interest, etc.
  4. Take two clean and dry quartz cuvettes with a given path length (here we used 1 cm path length cuvette).
  5. Fill up one cuvette with sample blank (ethanol in the present case) and the other cuvette with the lowest concentration coumarin 343 solution.
  6. Place the sample blank in the reference holder and the sample in the sample holder.
  7. Run the scan (A versus λ).
  8. Similarly run spectral scans for all the other samples proceeding from lower concentrations to higher concentrations. Every time one should rinse the cuvette taking a small portion of the solution from the solution that will be analyzed.
  9. Repeat steps 5 to 8 for coumarin 6 solutions.
  10. Run the scan for the multi-component solution (mixture) sample, containing both coumarin 343 and coumarin 6 solutions.
  11. Determine the wavelengths of maximum absorbance (λmax ) for both coumarin 343 and coumarin 6. Now find out the wavelengths, the absorbance at a given λmax for all the solutions and prepare a table containing these data.
  12. Determine the absorbance values of 2.510-5 M coumarin 343, 2.010-5 M coumarin 6 and their 1:1 mixture sample at the wavelengths of maximum absorbance λmax ).
  13. For any λmax , calculate the sum of absorbance values of individual components in the mixture from the relation: A X+Y = (A X0 /C X0 ).C X + (A Y0 /C Y0 ).C Y (see eq 4).
  14. Compare the above sum with the measured absorbance value of the mixture = Amix for the same λmax .
  15. Check A mix = A X+Y relationship for another λ max .
  16. Construct calibration plots by plotting absorbance versus concentration for two wavelengths (say, ¦Ë=420 nm and 463 nm) for both coumarin 343 and coumarin 6 solutions. Determine the slopes of the calibration plots and hence the respective values of ¦Å¡¯s for two different wavelengths for coumarin 343 and coumarin 6.
  17. Determine the absorbance values of 1:1 mixture sample at the above two wavelengths.
  18. Using thevalues of mixture absorbance and ¦Å, determine the concentrations of both coumarin 343 and coumarin 6 in the mixture by solving the Beer Lambert law for the two component system.
  19. Calculate the actual concentrations of coumarin 343 and coumarin 6 in the mixture from their original concentrations and volumes taken for making the mixture. 20 Compare the concentration values determined from absorbance measurements with the concentration values calculated from the actual concentrations taken to prepare the mixture to verify the validity of the principle of additivity of absorbance values.
  20. Do a multi-peak fitting of the spectrum obtained from the solution containing both coumarin 343 and coumarin 6.
  21. Discuss your results.