L9: Molecular Absorption Spectroscopy

Absorption v. Fluorescence v. Phosphorescence

Beer’s Law and Absorption Measurements

Molecular Absorption Spectroscopy Losses

• reduce the intensity of transmitted radiation, which cause attenuation of the light beam

• To prevent this: the power of radiation transmitted through the analyte is compared to the power transmitter through only the solvent

• The difference is attributed to absorption by the analyte

Limitations of Beer’s Law

• the direct proportionality between absorbance and concentration may occur only for relatively low analyte concentration

• ɛ is dependent on the refractive index

• while applied for polychromatic radiation, beer’s law only applies to monochromatic radiation

• If the analyte molecules dissociate/associate/react with the solvent deviations occur if they cause a different absorption spectrum

Components for UV/VIS/NIR Spectroscopy

source, wavelength selector, sample, radiation transduce, signal processor

Light sources:

• Deuterium and hydrogen lamps (most common light source)

  D2 + Ee → D2* → D‘ + D“ + hν

  Ee=ED‘ +ED“ +hν

• Tungsten filament lamps: most common for VIS and NIR; Energy distribution similar to black body

• Xenon Arc lamps: Passage of current through a xenon atmosphere

• Sample container: Cuvette, Quartz, fused silica, silicate glasses

Single Beam Instruments

Tungsten/deuterium lamp

Measure the intensity of light before and after it passes through the sample, thus avoids

using a reference beam

Probe-type single beam instruments

• Use optical fibers to deliver and collect light directly from the sample.

• A mirror reflects light back through the sample to increase the effective path length.

• Adjustable path lengths (1 mm–10 cm) allow analysis of samples with different concentrations

Double Beam Imstrument

Beam Splitter divides collimated light in beam into equal halves

Separtion of Beams in the double beam instrument separation

• Rotating sector mirror divides the entire beam to reference cell and sample cell

• Pulses of radiation combined by another sector mirror

Diode Array Instruments

• Single beam instrument with a diode array transducer, fixed grating and linear array of several hundreds of photodiodes

• Output onto each diode corresponds to a different wavelength

NanoDrop Spectrometer