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(P0/P) = bc

• T = Psolution/Psolvent = P/P0

• Works for monochromatic light• Compound x has a unique at different

wavelengths

cuvette

sourceslit

detector

b 1 cm

c 0.000056 M

P0 1000  W

P 500  W

P/P0 0.5  

T 0.5  

%T 50  

log(1/T) 0.30103  

(-logT) 0.30103  

absorbance 0.30103  

ebc 0.30103  

eb 5375.536 L/mol

e 5375.536 Lcm-1mol-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 lampFilament (UV)

slit

Scanning Instrument

sources

• Tungten lamp (350-2500 nm)

• Deuterium (200-400 nm)

• Xenon Arc lamps (200-1000 nm)

Monochromator

• Braggs law, n = d(sin i + sin r)

• Angular dispersion, dr/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 detector328 individual detectors

monochromator

Deuterium lampFilament (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 valveSample

loop

HPLC column

UV detector

Solvent waste