1. 1 Spectroscopic ANALYSIS Part 5 – Spectroscopic Analysis using UV-Visible Absorption Chulalongkorn University, Bangkok, Thailand January 2012 Dr Ron Beckett Water Studies Centre & School of Chemistry Monash University, Melbourne, Australia Email: Ron.Beckett@monash.edu Water Studies Centre

2. 2 UV-Visible Absorption Spectroscopy Absorption of UV and visible light by a molecule causes electronic excitation

3. 3 UV-Visible spectral peaks result from electronic-vibrational transitions Case (b) in the diagram is most common which gives the typical symmetric peak shape

4. 4 Molecular Orbitals Bonding in organic molecules is based on overlap between s and p atomic orbitals. This can give rise to bonding  and  molecular orbitals, nonbonding n molecular orbitals antibonding  * and  * molecular orbitals Two p atomic orbitals overlapping to give a  and a  * molecular orbital

5. 5 Molecular Orbitals + p x **  Two p atomic orbitals overlapping to give a bonding  molecular orbital and a  nonbonding   *  molecular orbital A A B A B B

6. 6 Electronic energy levels of polyatomic molecules  * (antibonding)  n (non-bonding)  (bonding)  Molecular Orbitals and Electronic Jumps   *   * n  * n  *

7. 7 Peak Position and the Type of Electronic Jump Conjugated  bonds

8. 8 Peak Position for Molecules containing Double and Triple Bonds

9. 9 Effect of Conjugation on Peak Position The greater the number of conjugated double bonds the lower the energy jump and higher the wavelength of the UV-visible peak   *

10. 10 Effect of Conjugation on Peak Position Highly conjugated molecules may be coloured if the absorption peak moves into the visible region

11. 11 Question Time ! Fanta has red and green colours ! Will red light pass through each of these solutions or will it be absorbed ? (a) (b) (c)(d)

12. 12 Question Time ! Fanta has red and green colours ! Will green light pass through each of these solutions or will it be absorbed ? (a) (b) (c)(d)

13. 13 Complementary Colours not When white light is absorbed by a chromophore, the eye detects the colours that are not absorbed. This is called the complementary colour to the colour absorbed. V I B G Y O R

14. 14 Colorimetric Analysis Used for determination of the concentration of analytes in solution when: 1.The analyte is a coloured compound 2.The analyte produces a coloured species when a suitable reagent is added

15. 15 Colorimetric Analysis Photometric measurement (a) visual comparison using colour standards Determination of concentration depends on detection of change in colour intensity (absorption) at a particular wavelength. Eye

16. 16 Colorimetric Analysis (b) Colorimeter/Photometer Filters used to select a wavelength range Detection with photosensing device P P o Filter wheel Photodetector

17. 17 Spectrophotometric Analysis (c) Spectrophotometer –Spectral bandwidth ≤ 1 nm, i.e very monochromatic light. –can operate in both the visible and UV ranges sensitive –Colorimetry and spectrophotometry provide sensitive methods of analysis, i.e. ppm to ppb ranges. Prism or Grating Phototube, photomultiplier or photodiode

18. 18 Single Beam Colorimeter Single beam spectrometer

19. 19 Quantifying Light Absorption Incident Light Intensity (P I ) (sometimes I i is used) P I = P r + P a(solvent) + P a(solute) + P P a(solvent) & P a(solute) are absorbed light intensities P r ≈ 4% for air-glass interface P P I b Absorbing solution of concentration,c. Reflected beam P r P a(solvent) P a(solute) Incident beam Transmitted beam

20. 20 Quantifying Light Absorption Transmitted Light (P 0 ) P I = P r + P a(solvent) + P 0 P 0 = P I - P r - P a(solvent) P0P0 P I b Absorbing solvent Reflected beam P r P a(solvent) Incident beam Transmitted beam Intensity lost due to reflection and solvent absorption are removed by measuring the transmitted intensity of a blank containing only solvent

21. 21 Quantifying Light Absorption A = log P0P0 P Absorbance Absorbance is defined as A = log (1/T) = log(100/%T) Transmittance Transmittance defined as T = P P0P0 Thus

22. 22 Relationship between Absorbance and Concentration Beer-Lambert Law A =  l c Where: path length in cm l is the path length in cm concentration c is the concentration in mol/L molarabsorptivity  is the molar absorptivity

23. 23 Applications of the Beer-Lambert Law Analysis of a single analyte 1.Measure absorbance of a series of standard solutions 2.Plot a standard curve (should be a straight line ?) 3.Measure absorbance of unknown samples 4.Use standard curve to measure concentrations Assumptions – At fixed and l,  is constant for a given solute – the chemical matrix of the standards is the same as the sample. A x CxCx 4 3 2 1 023 A A A A A 0 C C 1 C C C 4 Concentration A =  l c

24. 24 Standard Addition Method Used for samples with complex matrix & chemical  interferences. 1.Measure A of sample 2.Repeat with known additions of standard to the sample. Applications of the Beer-Lambert Law Concentration of Standard added (mL) 0 C Add Sample Sample plus standard additions Sample Concentration

25. 25 Limitations of the Beer-Lambert Law Concentration effects – B-L law applies to dilute solutions (negligible interaction between solute ions). – Higher concentrations of analyte (i.e. > 10 -2 M) or high electrolyte concentrations, may produce molecular/ionic interactions which result in  reduced light absorption at some  wavelengths. Concentration Deviation from B-L law (loss of sensitivity) Adherence to B-L law

26. 26 Experimental Considerations Wavelength selection Choose where A is large to obtain best sensitivity. Choose where dA/d = 0 or is small. Absorbance 4 3 2 1 2 3 Wavelength

27. 27 Experimental Considerations Choice of reagents for colorimetric analysis –Should be stable and pure –Should not absorb at of measurement –Should react rapidly with analyte to give a stable coloured compound (chromophore). –Absorptivity,  should not be sensitive to minor changes in pH, Temp., electrolyte changes, etc. –Should be selective for the analyte of interest.