Chapter 13 An Introduction to UVVIS Molecular Absorption Spectroscopy

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Chapter 13An Introduction to UV-VIS Molecular
Absorption Spectroscopy
b and c can be any units
b must be cm and c must be M
usually C (not c)
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Beer's Law

• A ebC
• best way to determine molar absorptivity?
• Use slope of calibration curve (m eC)
  constructed from several known concentrations
  measured at desired wavelength
• A ebC 0 (ideally)
• y mx b (real world)
• Do not use e A/bC except for rough estimate

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benefits to using slope of best fit
line minimizes error by 1) factoring out
intercept if e A/bC used then each e
would be different because intercept
not factored out. Larger A gives
better e estimate 2) not using
individual points which may or not be on the
line if e (A-b)/bC used for
individual data points e will also
change unless R2 1.00000
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Deviations from Beer's Law

• 1) Real - e is not constant with concentration
  as concentration increases e decreases because
  hsolution increases with concentration
• emeasured (etrue)h/(h22)2

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Deviations from Beer's Law

• 2) chemical - shifts in equilibria between
  species which absorb at different wavelengths
• Example
• 2 CrO42- 2 H 2 HCrO4-
  Cr2O72- H2O
• yellow orange
• How to measure different concentrations of
  chromate?
• How to measure different concentrations of
  dichromate?

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Deviations from Beer's Law

• 3) instrumental - Beer's Law assumes
  monochromatic light which is never the case
• example on board

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Deviations from Beer's Law

• Instrumental deviations from Beer's Law most
  severe if
• 1) large bandpass and
• 2) narrow absorption peak
• if effective bandwidth (bandpass) of
  spectrophotometer is lt 1/10 width of absorption
  peak at half height then no significant
  deviations from Beer's Law

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Stray light effects

• Stray light scattered, reflected, pin hole
  leaks, higher orders

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Effects of instrumental noise (uncertainties) on
spectrophotometric analyses
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k1 0.003
k2 0.003
k3 0.013
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Sources

• black body
• W lamp VIS and near IR
• electric discharge lamps
• D2, H2 lamps primarily UV D2 more intense
  than H2
• Hg, Xe UV and VIS
• Xe lamp is very intense

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Types of Instruments

• 1) single beam
• 2) double beam in space
• 3) double beam in time
• 4) multichannel

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Figure 13-19 (b) Spectronic 20
(photodiode)
detector on older Spectronic 20 models is a
phototube
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Single source double beam spectrophotometer
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Two source double beam spectrophotometer
transparent
front view of chopper wheel
nonreflective
mirror
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transparent
mirror
transparent
From Skoog, Holler, and Crouch, Principles of
Instrumental Analysis, p. 359, 6th edition,
Thomson Brooks/Cole, Belmont, CA, 2007.
Figure 13-22
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sample
sample
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Single Beam vs. Double Beam

• Single Beam
• higher S/N (usually) fewer surfaces means more
  hn transmitted
• fewer photons lost measuring
  dark current
• less noise
• excels at single l measurement (single channel)
• cost (single channel)
• - corrects for slow fluctuations in signal
• Double Beam
• - lower S/N (usually) (more components means more
  hn loss, only ¼ of hn goes through sample, noise)
• - cost
• - more complex
• A vs. l (spectra)
• corrects for all but fastest fluctuations in
  signal

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Agilent Technologies Model 8453 diode-array
spectrophotometer
Figure 13-25
From Skoog, Holler, and Crouch, Principles of
Instrumental Analysis, p. 362, 6th edition,
Thomson Brooks/Cole, Belmont, CA, 2007.
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Benefits of single beam multichannel instrument

• Has all benefits of a double beam instrument
  except

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From Skoog, Holler, and Crouch, Principlesof
Instrumental Analysis, p. 360, 6th edition,
Thomson Brooks/Cole, Belmont, CA, 2007.
Figure 13-23