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 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 M in Bi 3+ and Cu 2+ 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 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 A 1 /A 2 or A 1 – 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.

Applications 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 µg/mL are typical LOD’s

Organic/Biologicals 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 Combines robotic pipettors Centrifuge Spectrophotometer Computer Increases sample throughput Reduces volume of sample and reagents

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 Molybdenum blue method (NH 4 ) 3 P (Mo 3 O 10 ) 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- SO 2 Collect by bubbling through 0.1 M sodium tetrachloromercurate HgCl SO 2 + 2H 2 O  Hg(SO 3 ) Cl - + 4H + Treat with formaldehyde and p-rosaniline to give red-violet colour 569 nm ppm by volume NO 2 interferes above 2 ppm

Advantages Visible Less interferences Often higher molar absorptivities