Spectroscop1c Analysis Part 6 – Spectroscopic Analysis using Fluorescence and Atomic Absorption Spectrophotometry

1. Spectroscop1c AnalysisPart 6 – Spectroscopic Analysis using Fluorescence and Atomic Absorption Spectrophotometry Chulalongkorn University, Bangkok, Thailand January 2012 Dr Ron Beckett Water Studies Centre & School of ChemistryMonash University, Melbourne, Australia Email: Ron.Beckett@monash.edu Water Studies Centre

3. Vibrational relaxation Singlet Excited States Triplet Excited State T S 1 1 ENERGY Absorption Fluorescence Ground state S l l l l 2 4 3 1 0 Stokes Shift Fluorescence Analysis Fluorescence • Excitation to higher electronic state by absorption • Loss of vibrational energy as heat • Emission of fluorescence EMR • Results in Stokes shift to longer wavelength (lower energy). • Lifetime of excited singlet state 10-5 - 10-8 s

4. C Scan Incidence EMR Absorbance Signal PI P F Fluorescence Signal Fluorescence Analysis • l

5. Factors Affecting Fluorescence Molecular Structure • Most intense fluorescence from aromatic groups, esp. multi-ring compounds or highly conjugated molecules, e.g. PAH's (polycyclic aromatic hydrocarbons). • Halogen or carboxyl substitution inhibits fluorescence

6. l C PI P F Factors Affecting Fluorescence Relationship between Fluorescence and Concentration of an analyte in solution F = K c

7. Atomic Absorption Spectrometry • AAS was invented in 1955 by Sir Alan Walsh at CSIRO in Melbourne • It is now used extensively around the world for elemental analysis of environmental, industrial and biological samples Sample atoms in gas phase absorb light Atomic emission produces light of the right frequency Detector

8. E2 Excitation DE = hn E1 Intensity n Frequency Origin of an Atomic Emission Peak Excitation to a higher electronic state by heat, EMR, etc. Emission Spectrum

9. Atomic Emission

10. Atomic Emission After Excitation in a Flame Different atoms give rise to characteristic colours that can be used to identify the elements present Na Ba

11. Atomic Emission Spectra Mercury (Hg) Neon (Ne)

12. E2 DE = hn E1 Intensity n Frequency Origin of an Atomic Absorption Peak Energy Transition Absorption Spectrum

13. Atomic Absorption for a Hydrogen Atom Electronic energy levels in a H atom 1s 2s 2p 3s 3p 3d The lowest energy state has the single electron in the 1s orbital 1s1

14. Solar spectrum A brief history of Atomic Absorption Spectrometry • 1802 Wollastone - discovered black lines in Sun's spectrum which were subsequently investigated by Fraunhoffer (1823).

15. A brief history of Atomic Absorption Spectrometry • 1820 Brewster - suggested black lines in solar spectrum due to absorption processes in Sun's atmosphere. • 1859 Kirchhoff and Bunsen - demonstrated Na D line absorption invisible spectra.

16. Solar spectrum • Hydrogen spectrum Mercury spectrum A brief history of AAS Interpretation of Fraunhoffer lines in terms of atomic absorption by elements in the suns atmosphere 1859-1955 Astronomers use atomic absorption to estimate metal concentrations in atmospheres of stars.

17. Sir Alan Walsh (d 1998) A brief history of Atomic Absorption Spectrometry • 1955 - Alan Walsh at CSIRO in Melbourne proposed atomic absorption spectrometry for chemical analysis. • Mid 1960’s - First commercial AAS manufactured in Melbourne by Varian-Techtron.

18. Atomic Absorption Spectrometer

19. Flame Atomic Absorption Instrumentation

20. Principle of Flame AAS • Sample solution is sucked into a spray chamber to produce an aerosol • The aerosol is introduced into a flame with the fuel gas • The solution is evaporated and the elements are atomised but not excited or ionised • A lamp containing the element being analysed is used to produce light of the correct wavelength • Absorbance of this light by the sample atoms is measured

21. Analysis by AAS • Measure the absorbance of a blank solution Abl and a series of standard solutions Ax • Plot the calibration line (Ax – Abl) vs Cx • Measure the absorbance of the unknown solutions Calculate the concentrations of the unknown solutions Where m is the gradient of the calibration line

22. Analysis by AAS Standard Addition Method • Compensates for chemical interferences • Cannot eliminate spectral interferences Concentration of Unknown Sample plus Standard Addition Sample Volume of Standard Added (mL)

23. Use of AAS in Forensic Science

24. Who fired the murder weapon ?

25. Gunshot residue (GSR) • Bullets contain “primer” and “propellant” which result in GSR on the firing hand • Swabbing hands with mild acid will release barium (Ba) and antimony (Sb) • Measure byAtomic Absorption