Fluorescence

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Fluorescence
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Topics

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

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definitions

• Singlet

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Molecular Fluorescence
Absorption Vibrational relaxation Fluorescence Pho
sphorescence Internal conversion Intersystem
crossing
S2
v5
S1
v3
v1
T1
v4
v2
S0
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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

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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

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Instruments

• Fluorimeter
• Spectrophorimeter

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Fluorimeter
cuvette
Low pass filter
High pass filter
Photomultiplier tube
Tungsten source
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Spectrofluorimeter
Emission Monochrometer
Excitation Monochrometer
slit
slit
slit
cuvette
Tungsten source
Photomultiplier tube
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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

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Atomic Absorption Spectroscopy

• Elemental analysis
• Dissolved metals
• Source
• Hallow Cathode Lamp
• Cathode of specific element
• Sample
• Acetylene-air flame, nebulizer
• Detector
• PMT

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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

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

• Aspirate blank, set 100 T
• Aspirate sample
• Matrix effects are common
• Standard addition analysis

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Atomic Emission Spectroscopy
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Atomic Emission Spectroscopy

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

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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

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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

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Sample Introduction

• Nebulizer
• Electrothermal vaporization
• Laser ablation
• In either case Ar gas in used to carry sample
  into the ICP

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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

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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

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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