Transcript G Absortion cells and applications

Double Beam spectrometer
• Provides a signal that is largely free of drift
in the source and detector without
requiring really expensive components
• Beam alternates very fast between sample
and reference cells.
• Don’t need to keep zeroing
Sample Cells
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Usually 1 cm pathlength
Glass – visible
Quartz – UV
Plastic disposable – beware solvents
5 cm or 10 cm for dilute samples
Smaller cells for small samples
Flow through cells
Temperature control
Gas cells – longer
Fibre optic probes
Fibre Optics
• Fibres of glass, usually about 120 µm in
diameter.
Fibre Optic Probe
Derivative Spectroscopy
• Can determine flat maxima more precisely
• Isolate shoulders
• Distinguish weak signals from background
Photometric Titration
Solution:
2 x 10-3 M in Bi3+ and Cu2+
Titrant: EDTA
At 745 nm, neither cation,
nor reagent, absorbs
Bi complex forms first –
more stable – but doesn’t
absorb
The cu complex does
absorb at 745 nm
Reaction rates
• Following enzyme kinetics
• Determine enzymes
• Determine substrates
Stop Flow Methods
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For fast reactions
Two syringes driven at same rate
Solutions flow into mixing chamber
When plunger hits stop, measurement
starts
• Generally measure initial rates of reactions
Absorbance ratios and differences
Measure A at two 
Use A1/A2 or A1 – A 2
Plot versus concentration
Use to asses purity of samples – to check
just one component is present
• Eg ratio of A 260 nm: A 280 nm indicates how
pure A sample of DNA you have.
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Applications
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Metal ion analysis
eg iron II or III
React with ligands to get intense colours
Reduce Fe III with hydroxylamine or
hydroquinone etc
• Can extract the complexes into isoamyl
alcohol for a cleanup/preconcentration
step
• 0.1 - 0.005 µg/mL are typical LOD’s
Organic/Biologicals
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Most common application
Many absorb strongly
May need to derivatize
eg alcohol with phenyl isocyanate to give
alkyl carbamates – 280 nm
• Free amino acids react with ninhydrin –
blue/purple – 575 nm – aa analyzers
Automated clinical methods
• Many samples/hour
• Expensive to buy
• Can run many samples unattended
Centrifugal Analysis
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Combines robotic pipettors
Centrifuge
Spectrophotometer
Computer
• Increases sample throughput
• Reduces volume of sample and reagents
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Eliminates chemistry changeover time
No set-up equilibrium time
Used for water quality measurements
Based on standard procedures
Liquids are dispensed into separate
compartments attached to the cuvettes
• Cuvettes are round a rotor. When rotor is
spun, reagents are propelled into cuvettes.
• All reactions start together – good for
kinetics
• Samples and standards are mixed and run
in parallel.
• Identical conditions are ensured for all
cuvettes
• Can analyze 110 samples/hour
• Rotor spins at 2000 revolutions /min
• Get an average of ~ 7 readings/sample
Water Pollution Analysis
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Molybdenum blue method
(NH4)3 P (Mo3O10)4 yellow
Reduce with hydroquinone, Sn II or Fe II
Get a polymer of Mo of different oxidation
states
• Not stoichiometrically well-defined but is
blue
• As, Si interfere – so they can also be
determined this way
Air Pollution Analysis- SO2
• Collect by bubbling through 0.1 M sodium
tetrachloromercurate
• HgCl42- + 2SO2 + 2H2O  Hg(SO3)22- +
4Cl- +
4H+
• Treat with formaldehyde and p-rosaniline
to give red-violet colour 569 nm
• 0.005 ppm by volume
• NO2 interferes above 2 ppm
Advantages Visible
• Less interferences
• Often higher molar absorptivities