Tools

Determination of unknown concentration of an analyte by using the Beer-Lambert Law

  1. This method involves measuring the absorbance of the sample solution using a UV-Visible spectrophotometer. One needs to prepare a stock solution and Calibration Standards from this stock solution.
  2. Prepare a standard K 2 Cr 2 O 7 aqueous solution of strength nearly 3x10-3 M in 0.01 N H2SO4 (for the experiments described here we have used a solution of strength = 3.16x10-3 M. This is used as stock solution.
  3. From the above stock solution, prepare five different concentrations of K 2 Cr 2 O 7 solutions: say, 0.158 mM, 0.316 mM, 0.474 mM, 0.632 mM, and 0.790 mM.
  4. Switch on the computer and the instrument powers; wait for 30 minutes for 'warm-up' of the instrument.
  5. 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.
  6. Take two clean and dry glass (only for visible range scan) or quartz cuvettes with a given path length (say, 1 cm).
  7. Fill up one cuvette with sample blank (aq. 0.01 N H2SO4 in the present case) and the other cuvette with aqueous K 2 Cr 2 O 7 solution with lowest concentration.
  8. Place the sample blank in the reference holder and the sample in the sample holder.
  9. Run the scan (A or %T versus λ).
  10. Similarly spectral runs are done for all the other samples starting from the lowest concentrations to next higher concentrations of K 2 Cr 2 O 7. Every time one should rinse the cuvette taking a small portion of the solution to be analyzed next.
  11. Spectral run is carried out for the unknown K 2 Cr 2 O 7 sample.
  12. Now find out the wavelengths of maximum absorbance (λ max ), the absorbance at a given λ max for all the concentrations of K 2 Cr 2 O 7 and make a table containing these data.
  13. Construct a calibration plot by plotting absorbance versus concentration for a given λ max . Thus one construct two calibration plots for two peak positions of K 2 Cr 2 O 7.
  14. Determine the slopes of the calibration plots and hence the respective values of λ's.
  15. Determine the concentration of the unknown K 2 r 2 O 7 solution from two calibration plots.
SNo Concentration K 2 Cr 2 O 7 (mM) λ max (nm) Absorbance λ =350 nm Absorbance anotherwavelength (say,λ max
1. 0.158
2. 0.316
3. 0.474
4. 0.632
5. 0.790
6. Unknown Sample Conc.?