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Molecular Spectrometry (UV and Visible)Part 1: Absorption
InstrumentEvolution
Beckman DU
Replaced prism with grating
Turner Spectrometer
Spec 20 as well
Dual BeamHigh resolution
Cary and Perkin-Elmer
PDAHewlett Packard
Fiber Optic Probes
Ocean Optics
Signal Expressions
Eout = Φλ (Ω/4π) Tm Ts Rλ G
Φλ = source spectral power (W/nm)Tm = monochromator throughput (nm)Ts = Sample transmittanceRλ = Detector Responsivity (A/W)G = Gain of electronics (V/A)
Signal Expressions
Tm = (WH/Aeff) Δλeff Top
W = slit width (cm)
H = slit height (cm)
Aeff = Effective area of source image (cm2)
Δλeff = Effective bandwitdh of the
monochromator (nm)Top = Transmittance of optics
Signal Expressions
Ts = e-εbc = Esam/Eref
Eref = Output voltage for a blank
Esam = Output voltage for sample
Signal Expressions
A = -log (Esam/Eref) = -log (Ts) = ε b c
Notes:1. Since UV-Vis absorbance bandsare much wider than the mono-chromator bandwidth, Beer’s Lawassumes that A does not dependon Δλeff.
2. ε is wavelength dependent, but doesnot depend on any other instrumentalparameter.
A = -log (Esam/Eref) = -log (Ts) = ε b c
A = ε b cDesign Cell to Maximize b
Deviations from Beer’s Law
1. Optical
a) slit widthb) wavelength selectionc) stray radiation
2. Chemical
a) high analyte concentrationb) pH dependencec) mixtures
Deviations from Beer’s Law
Slit Width
Deviations from Beer’s Law
Deviations from Beer’s Law
Wavelength Selection
0
1
2
3
4
5
6
Concentration
Ab
so
rban
ce
Deviations from Beer’s Law
Stray Radiation Effect
No StrayLight
10% Stray Light
0.000
0.100
0.200
0.300
0.400
0.500
0.600
0.700
0.800
0.900
1.000
0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18 0.2
Concentration (mM)
Tra
ns
mit
tan
ceDeviations from Beer’s Law
High Analyte Concentrations
Deviations fromBeer’s Law pH Dependence
Deviations fromBeer’s Law
Mixtures:Absorbance isAdditive
Deviations fromBeer’s Law
Mixtures:Solve multipleequations!
Deviations fromBeer’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) doesnot depend on the magnitude of the sourcesignal (except for the deviations discussedabove).
The Noise in the measurement often DOESdepend on the source signal level.