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http://departments.colgate.edu/geology/instruments/aa.htm
Flameless AA
• Sample is heated electrically in closed chamber
• Carbon rod, Tantalum, or Platinum is used to dry the sample
to become ash and atoms
• Sensitivity is higher than Flame AA
FLUOROMETRY
• Molecule with fluorophore absorb light
• Orbital electron go up to excited state (unstable)
• Orbital electron come down to ground state
and emit light at a longer waevlength
• The intensity of light emitted varies linearly
with analyte concentration
Fluorescence spectrophotometer
Components
Light source Mercury or xenon arc lamp
P = Fluorescence polarization
Iv = Intensity in vertical plane
Ih = Intensity in horizontal plane
Applications
• Receptor/ligand studies (e.g. hormone/receptor assays)
• Protein/peptide interactions
• DNA/protein interactions
• Tyrosine Kinase Assays
• Competitive Immunoassays
Example Measurement of serum digoxin
serum + Ag* + Ab measure fluorescence
polarization with ABBOT TDx
+ Ag
Ag* + Ab Ag:Ab + Ag*
No Ag No fluorescence
polarization
Ab:Ag*
P
fluorescence
polarization
Conc
Time-Delayed Fluorescence Spectrophotometry
• Time-resolved fluorescence spectrophotometry
• long-lived fluorophore e.g., lanthanide or europium (Eu3+ )
• fluorescence remain >100 microsec.
• decrease interference/ increase specificity
CHEMILUMINESCENCE PHOTOMETRY
Chemiluminescence: the emission of light with as the result
of a chemical reaction
- Chemical reactions using synthetic compounds
- Light-emitting reactions arising from a living organism,
are commonly termed bioluminescent reactions.
- Light-emitting reactions which take place by the use of
electrical current are designated electrochemiluminescent reactions.
2 H2O2 + Luminol + enhancer peroxidase
Oxidized luminol + 2H2O + light
Ru-tris(bipyridyl)2+ e- + Ru-tris(bipyridyl)3+
Anode
Tripropylamine(TPA) e- + TPA .+ TPA. + H+
Ru-tris(bipyridyl)3+ + TPA. Excited Ru-tris(bipyridyl)3+
Excited Ru-tris(bipyridyl) 3+ Ru-tris(bipyridyl)3+ + light
ECL • Use immunoassay, nucleic acid assay
• High sensitivity
• Detection limit 200 fmol/L
TURBIDIMETRY & NEPHELOMETRY
• Light interact with particle cause light scattering
• Wavelength of scattered light is the same as of the
incident light
• Detect by turbidimeter
(detector is parallel to light source)
or nephelometer
(detector is aligned at an angle 70-75 °)
Interaction of Light with Particles
Small molucules
Light scattered symmetrically
Large particles
Light scattered preferentially
forward (Raleigh –Debye)
• commonly used in immunoassay
• detect AgAb complex particles (diameter = 250-1500 nm)
Limitation