1. Fluorescence

2. Topics Definition Instrumentation Sensitivity (nM-pM) –Contrast UV-vis measurements Derivatization Laser-induced Fluorescence

3. definitions Singlet

4. S0S0 S2S2 S1S1 T1T1 v2v2 v4v4 v1v1 v3v3 v5v5 v1v1 v3v3 v5v5 v1v1 v3v3 v5v5 v7v7 v1v1 v3v3 v5v5 v7v7 Absorption Vibrational relaxation Fluorescence Phosphorescence Internal conversion Intersystem crossing Molecular Fluorescence

5. Competing Rates Absorption occurs on the femptosecond time scale (10 -15 s) Relaxation from the ground state occurs through the fastest available process Most molecules do not fluoresce because the excited vibrational states of S 0 overlap with the S 1 state and relaxation can take place rapidly by vibrational relaxation Generally on the picosecond timescale

6. Molecules that fluoresce Fluorescence- relaxation through the emission of a photon Generally occurs on the ms-ns timescale. Molecules that fluoresce tend to be rigid aromatic compounds that possess limited vibrational freedom Phosphorescence is the emission of a photon from a excited triplet state Phosphorescence is an extremely rare property

7. Instruments Fluorimeter Spectrophorimeter

8. Fluorimeter Tungsten source Low pass filter High pass filter cuvette Photomultiplier tube

9. Spectrofluorimeter Tungsten source Excitation Monochrometer Photomultiplier tube cuvette slit Emission Monochrometer

10. Why fluorescence? More sensitive than UV/vis absorption by a factor of 10-10 5. Measuring against a zero background Biochemical tool; Fluorescent tagging Laser Induced fluorescence –F is proportional to intensity of source –Selectivity of monochromatic light source –Convenient with HPLC

11. Atomic Absorption Spectroscopy Elemental analysis Dissolved metals Source –Hallow Cathode Lamp –Cathode of specific element Sample –Acetylene-air flame, nebulizer Detector –PMT

12. Atomic absorption Gas-phase elemental atoms Narrow absorption lines (±0.01 nm) Element specific source Flame conditions control sensitivity 1-100 ppm (mg/L) varies with element

13. Hollow cathode lamp PMT Flame sample nebulizer Air Acetylene Coating of element AA Spectrometer monochrometer

14. Experiment Aspirate blank, set 100 % T Aspirate sample Matrix effects are common –Standard addition analysis

15. Atomic Emission Spectroscopy

16. Emission of light from exited species ICP source Multi-element analysis –Fancy optics Greater sensitivity for most elements

17. Removal of Atomic Emission Place a chopper before the flame The signal from the source is modulated by the chopper Thus, a AC signal is produced on top of a DC signal that originates from emission in the flame

18. ICP source Ar tourch –Very hot, 10000 K –Abundance of e- inhibits ionization –Lack of O2 inhibits oxide formation –Excited atomic species predominate –5-20 L/min

19. Sample Introduction Nebulizer Electrothermal vaporization Laser ablation In either case Ar gas in used to carry sample into the ICP

21. Multi-channel Instruments Polychromators –60 photomultipliers in fixed positions Array-based –Echelle and prism for 2-D dispersion plane –Charge injection detector with 94672 different detector elements (8.7x6.6mm) –A set of 39 elements makes up a read window for a given element –CCD detectors

22. CCD detectors Array of semiconductor detection elements 512x320 array =163840 individual detectors Charge of impinging photons are stored The charge accumulated is registered one by one by sending the charge to a preamplifier and readout, emptying the registrar Very sensitive

23. Performance Sensitivity: Ranges from 1-300 ng/mL Number of useful lines; 1-24 Calibration curves; almost 3 orders of magnitude Internal standards Self-absorption at higher concentrations