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Fluorescence

Fluorescence. Topics. Definition Instrumentation Sensitivity (nM-pM) Contrast UV-vis measurements Derivatization Laser-induced Fluorescence. definitions. Singlet . v 7. v 7. v 5. v 5. v 3. v 3. v 1. v 1. v 5. v 3. v 1. Molecular Fluorescence. Absorption

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Fluorescence

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  1. Fluorescence

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

  3. definitions • Singlet

  4. v7 v7 v5 v5 v3 v3 v1 v1 v5 v3 v1 Molecular Fluorescence Absorption Vibrational relaxation Fluorescence Phosphorescence Internal conversion Intersystem crossing S2 v5 S1 v3 v1 T1 v4 v2 S0

  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 S0 overlap with the S1 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 cuvette Low pass filter High pass filter Photomultiplier tube Tungsten source

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

  10. Why fluorescence? • More sensitive than UV/vis absorption by a factor of 10-105. • 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. AA Spectrometer PMT Hollow cathode lamp Flame monochrometer Coating of element Air nebulizer Acetylene sample

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

  15. Atomic Emission Spectroscopy

  16. Atomic Emission Spectroscopy • 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

  20. 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

  21. 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

  22. 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

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