1. Spectroscop1c Analysis Part 6 – Spectroscopic Analysis using Fluorescence and Atomic Absorption Spectrophotometry 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 1

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

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

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 5

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

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 Atomic emission produces light of the right frequency Sample atoms in gas phase absorb light Detector 7

8. Origin of an Atomic Emission Peak Frequency Intensity Excitation to a higher electronic state by heat, EMR, etc. Emission Spectrum E2E2 E1E1  E = h Excitation 8

9. Atomic Emission 9

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

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

12. Origin of an Atomic Absorption Peak E2E2 E1E1  E = h Frequency Intensity Energy Transition Absorption Spectrum 12

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 1s 1 13

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

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 in visible spectra. 15

16. A brief history of AAS Hydrogen spectrum Solar spectrum Mercury spectrum 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. 16

17. 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. Sir Alan Walsh (d 1998) 17

18. Atomic Absorption Spectrometer 18

19. Flame Atomic Absorption Instrumentation 19

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 20

21. Analysis by AAS 1.Measure the absorbance of a blank solution A bl and a series of standard solutions A x 2.Plot the calibration line ( A x – A bl ) vs C x 3.Measure the absorbance of the unknown solutions Calculate the concentrations of the unknown solutions Where m is the gradient of the calibration line 21

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

23. Use of AAS in Forensic Science 23

24. Who fired the murder weapon ? 24

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 by Atomic Absorption 25