Fluorescence - PowerPoint PPT Presentation

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Fluorescence

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Most molecules do not fluoresce because the excited vibrational states of S0 ... Molecules that fluoresce tend to be rigid aromatic compounds that possess ... – PowerPoint PPT presentation

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Title: Fluorescence


1
Fluorescence
2
Topics
  • Definition
  • Instrumentation
  • Sensitivity (nM-pM)
  • Contrast UV-vis measurements
  • Derivatization
  • Laser-induced Fluorescence

3
definitions
  • Singlet

4
Molecular Fluorescence
Absorption Vibrational relaxation Fluorescence Pho
sphorescence 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
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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
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