UV-vis

Applications Quantitative analysis Organics (if composition is simple and known) Specific binding to chromaphore Metal-ligand absorption (d-orbital splitting), (Co2+ experiment in gen chm)

Beer’s Law A = -logT = log(P 0 /P) =  bc T = P solution /P solvent = P/P 0 Works for monochromatic light Compound x has a unique  at different wavelengths cuvette source slit detector

b1cm c M P0P W P500 W P/P T %T50 log(1/T) (-logT) absorbance ebc eb L/mol e Lcm -1 mol -1

Beer’s Law Analysis Choice of wavelength –Typically choose wavelength of maximum absorbance –May deviate from this to avoid an interference

Common UV-vis instuments cuvette Tungsten Filament (vis) slit Photomultiplier tube monochromator Deuterium lamp Filament (UV) slit Scanning Instrument

sources Tungten lamp ( nm) Deuterium ( nm) Xenon Arc lamps ( nm)

Monochromator Braggs law, n = d(sin i + sin r) Angular dispersion, d r/ d = n / d(cos r) Resolution, R = /  nN, resolution is extended by concave mirrors to refocus the divergent beam at the exit slit

Sample holder Visible; can be plastic or glass UV; you must use quartz

Single beam vs. double beam Source flicker

Diode array Instrument cuvette Tungsten Filament (vis) slit Diode array detector 328 individual detectors monochromator Deuterium lamp Filament (UV) slit mirror

Advantages/disadvantages Scanning instrument –High spectral resolution (63000), /  –Long data acquisition time (several minutes) –Low throughput Diode array –Fast acquisition time (a couple of seconds), compatible with on-line separations –High throughput (no slits) –Low resolution (2 nm)

HPLC-UV Mobile phase HPLC Pump syringe 6-port valve Sample loop HPLC column UV detector Solvent waste