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REFLECTANCE SPECTROPHOTOMETRY

• monochromatic light strike strip or slide with chromophore


• chromophore absorb light
• reflected light reach the detector
• intensity of reflected light is compared with intensity of
light reflected from reference surface
•Kubelka-Munk equation or Clapper-Williams
transformation is used
Reflectance photometer or Reflectometer
1 = Light source 2 = Slit
3 = Filter 4 = Collimating lens
5 = Test surface 6 = Collimating lens or slit
7 = Detector 8 = Readout or meter
FLAME EMISSION PHOTOMETRY
• Heat metal atom to be analyzed
• Orbital electron go to excited state
• Electron come back to ground state and emit light
A+ + e- A0
A0 + heat A*
A* A0 + light
* 1-5 % of atoms are in excited state
Components of flame photometer
Atomizer spray sample as fine mist into flame
Flame heat atoms to excited state
propane, acetylene, or natural gas is used as fuel
Monochromator select emitted light
specific filter for each analyte
Detector photomultiplier tube to detect emitted light
Meter or Readout display the result
ATOMIC ABSORPTION SPECTROPHOTOMETRY
•Heat atoms to be in ground state
• Light from hollow cathode lamp is absorb by atoms
• Transmitted light reach the detector
• The intensity of light absorbed is linearly associated with
concentration
• Absorption line spectrum for each analyte
• use AAS to measure calcium, magnesium, lead, mercury etc.
Components

Hollow cathode lamp Light source


Chopper Open the light from light source
Atomizer Spray sample as fine mist into flame
Flame Heat atoms to become ground state atoms
Monochromator
Detector
Hollow cathode lamp
• Lamp containing inert gas e.g., He, Ar
• Cathode is coated with the metal to be analyzed
• Potential difference between electrodes cause the electron
to collide with inert gas → ionized gas

• Ionized gas collide with metal atoms on cathode


A0 A* A0 + light
http://www.worldoftest.com/img/products/wfx110_1.jpg

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

Primary monochromator Prism or grating /select


excitation light
Cuvette Quartz cuvette
Secondary monochromator Detect emitted light
Detector
Readout or meter
Factors affecting fluorescence
• concentration Should be diluted solution
• pH Affect net charge of molecule
• Temperature High temp, more collision of
molecule → less fluorescence
• Viscosity High viscosity, high fluorescence
• Interfering substance Decrease fluorescence
(quencher)
FLUORESCENCE POLARIZATION SPECTROPHOTOMETRY

• Polarizer provide plane-polarized light


• Fluorescent molecules in solution, excited with plane-
polarized light, will emit light back into a fixed plane (i.e. the
light remains polarized) if the molecules remain stationary
• If the molecule rotates and tumbles out of this plane during
the excited state, light is emitted in a different plane from the
excitation light
Fluorescence polarization spectrophotometer
http://las.perkinelmer.com/content/snps/fp-tdi.asp
Iv - Ih
P = ------------------
I v + Ih

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

• Detect the light by luminometer


• Chemiluminescence substance: luminol, isoluminol,
acridinium ester, dioxetane
• Use in immunoassay, label Ag or Ab with
enzyme or chemiluminescence substance
Example Assay of Ag in patient serum

Ab2-E = second Ab labeled with enzyme


ELECTROCHEMILUMINESCENCE
Electrochemiluminescence
or electrogenerated chemiluminescence
• a kind of luminescence produced during electrochemical
reactions in solutions
• excitation is caused by energetic electron transfer
• emission of light by electrochemical intermediate
Cathode

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

Very large molecules


Light mostly scattered forward

Large particles
Light scattered preferentially
forward (Raleigh –Debye)
• commonly used in immunoassay
• detect AgAb complex particles (diameter = 250-1500 nm)

•Rayleigh-Debye light scatter


• use spectrophotometer in turbidimetry

• Light scatter in nephelometry may be measured by end


point or kinetic method
NEPHELOMETER
Light source:
•quartz halogen, xenon arc lamp, laser

Limitation

• Antigen excess: low signal


• Matrix effect: turbid sample causes erroneous result

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