Molecular Spectrometry (UV and Visible) Part 1: Absorption

Instrument Evolution Beckman DU

Replaced prism with grating

Turner Spectrometer Spec 20 as well

Dual Beam High resolution Cary and Perkin-Elmer

PDA Hewlett Packard

Fiber Optic Probes Ocean Optics

Signal Expressions E out = Φ λ (Ω/4π) T m T s R λ G Φ λ = source spectral power (W/nm) T m = monochromator throughput (nm) T s = Sample transmittance R λ = Detector Responsivity (A/W) G = Gain of electronics (V/A)

Signal Expressions T m = (WH/A eff ) Δλ eff T op W= slit width (cm) H= slit height (cm) A eff = Effective area of source image (cm 2 ) Δλ eff = Effective bandwitdh of the monochromator (nm) T op = Transmittance of optics

Signal Expressions T s = e -εbc = E sam /E ref E ref = Output voltage for a blank E sam = Output voltage for sample

Signal Expressions A = -log (E sam /E ref ) = -log (T s ) = ε b c Notes: 1. Since UV-Vis absorbance bands are much wider than the mono- chromator bandwidth, Beer’s Law assumes that A does not depend on Δλ eff. 2. ε is wavelength dependent, but does not depend on any other instrumental parameter.

A = -log (E sam /E ref ) = -log (T s ) = ε b c

A = ε b c Design Cell to Maximize b

Deviations from Beer’s Law 1. Optical a) slit width b) wavelength selection c) stray radiation 2. Chemical a) high analyte concentration b) pH dependence c) mixtures

Deviations from Beer’s Law Slit Width

Deviations from Beer’s Law

Wavelength Selection

Deviations from Beer’s Law Stray Radiation Effect No Stray Light 10% Stray Light

Deviations from Beer’s Law High Analyte Concentrations

Deviations from Beer’s Law pH Dependence

Deviations from Beer’s Law Mixtures: Absorbance is Additive

Deviations from Beer’s Law Mixtures: Solve multiple equations!

Deviations from Beer’s Law Mixtures: When all else fails, Separate the mixture components

UV Absorbance Detector for HPLC

Final Note on UV-Vis Signals The Absorbance (Analytical Signal) does not depend on the magnitude of the source signal (except for the deviations discussed above). The Noise in the measurement often DOES depend on the source signal level.