1. UV-vis

2. 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)

3. 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

4. b1cm c0.000056M P0P0 1000 W P500 W P/P 0 0.5 T %T50 log(1/T)0.30103 (-logT)0.30103 absorbance0.30103 ebc0.30103 eb5375.536L/mol e5375.536Lcm -1 mol -1

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

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

7. sources Tungten lamp (350-2500 nm) Deuterium (200-400 nm) Xenon Arc lamps (200-1000 nm)

8. 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

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

10. Single beam vs. double beam Source flicker

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

12. 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)

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