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Fluorescence

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					Fluorescence
                 Topics
• Definition
• Instrumentation
• Sensitivity (nM-pM)
  – Contrast UV-vis measurements
• Derivatization
• Laser-induced Fluorescence
            definitions
• Singlet
                           Absorption
Molecular Fluorescence     Vibrational relaxation
                           Fluorescence
                           Phosphorescence
            v7             Internal conversion
           v5              Intersystem crossing
           v3        v7
      S2   v1       v5
                    v3                        v5
            S1      v1                        v3
                      T1                      v1




                                        v5
                                       v4
                                        v3
 S0                                    v2
                                       v1
          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
      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
             Instruments
• Fluorimeter
• Spectrophorimeter
           Fluorimeter

            cuvette


Low pass
                      High pass
filter
                      filter




                                  Photomultiplier
                                  tube


Tungsten
source
                        Spectrofluorimeter
       Excitation Monochrometer     Emission Monochrometer




slit




                         slit
                                  cuvette         slit
Tungsten
source



                                                         Photomultiplier
                                                         tube
         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
  Atomic Absorption Spectroscopy
• Elemental analysis
• Dissolved metals
• Source
  – Hallow Cathode Lamp
  – Cathode of specific element
• Sample
  – Acetylene-air flame, nebulizer
• Detector
  – PMT
            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
                          AA Spectrometer
                                                    PMT

    Hollow cathode lamp
                                     Flame




                                                          monochrometer

Coating of element
       Air
                                        nebulizer

   Acetylene                                         sample
               Experiment
• Aspirate blank, set 100 % T
• Aspirate sample
• Matrix effects are common
  – Standard addition analysis
Atomic Emission Spectroscopy
 Atomic Emission Spectroscopy
• Emission of light from exited species
• ICP source
• Multi-element analysis
  – Fancy optics
• Greater sensitivity for most elements
  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
               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
          Sample Introduction
•   Nebulizer
•   Electrothermal vaporization
•   Laser ablation
•   In either case Ar gas in used to carry
    sample into the ICP
    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
           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
             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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