Ultraviolet and visible Absorption Spectroscopy

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• Chapter 7
• Ultraviolet and visible Absorption Spectroscopy

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Properties of Electromagnetic Radiation

• Electromagnetic Radiation
• energy radiated in the form of a WAVE caused by
  an electric field interacting with a magnetic
  field
• result of the acceleration of a charged particle
• does not require a material medium and can travel
  through a vacuum

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Electromagnetic Radiation
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Electromagnetic Radiation

• vi n li where vi gt velocity n gt
  frequency li gt wavelength

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Electromagnetic Spectrum
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Electromagnetic Spectrum
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Interaction of EMR with Matter
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Jablonski diagram
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Selection Rules

• The electron must be promoted without a change in
  its orientation. ?s 0
• When ?s ? 0 transition is forbidden. It may occur
  with very low probability
• Some other from quantum mechanics

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• Etotal (molecule) Eelectronic Evibrational
  
• Erotational
  Enuclear

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Absorption of Light
Uv Vis
IR
Microwave
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?
?
?
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Molecular and Atomic Absorption
? ?
Less extent
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Collisions between molecules lead to broadening
of absorption bands
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Types of Transitions

• Three types of transitions
• 1. p, s, and n electrons
• 2. d f electrons
• 3. charge transfer electrons

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Electronic transition in Formaldehyde
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Spectroscopy Nomenclature
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Effect of Structure on
? ? vacuum UV
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?max Cl lt ?max Br lt ?max I
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n ? transitions occur at longer
wavelengths
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• is a function of
• 1. Cross sectional area of absorbing species (? )
• 2. Transition probability (P)
• 9X1019 ? P (? 10-15 cm2( about 105
  for the
• 
  average organic molecule

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Transition Multiplicity
Consider two electrons paired in an orbital, and
their possible transitions to an empty orbital.
ground state excited singlet excited triplet

• the ground state has all electrons in the lowest
  energyorbital
• organic compounds almost always have paired
  spins, thus their
• ground state is almost always a singlet
• singlet-triplet transitions are optically
  forbidden - light cannot
• both promote an electron to a new orbital and
  change its spin
• in an organic compound most absorption spectra
  are due to
• singlet-singlet electronic transitions

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Electronic Transitions in Ethylene

• Attention will be restricted to electrons
• involved with carbon-carbon bonding
• The two sp2 electrons form the s-bond,
• while the two pz electrons form the p-bond
• Absorption of a photon will promote one
• of the bonding electrons into an anti-
• bonding orbital, preserving electron spin
• The wavelength of absorbed light will
• follow
• Planck's Law, E hc/l
• The transition energies are
• s ? s gt s ? p, p ? s gt p ? p
• The p ? p transitions are of most
• interest since they give us information
• about the conjugated double bond
• structure of a molecule

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p ? p Transitions in Butadiene

• Each carbon atom has one electron in a
• pz-orbital
• The four pz electrons create two bonding
• p-orbitals and two anti-bonding p-orbitals
• p2 ? p2 absorption is in the deep UV, it has
• an energy similar to that in ethylene
• The longest wavelength absorption is due to
• the p1 ? p1 transition
• intermediate wavelength absorption is due to
• p2 ? p1 and p1 ? p2 transitions
• The long wavelength transition has an
• energy that decreases with the number of
• double bonds

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Rotational Broadening

• Boltzmann's constant is 0.694 cm-1
• kT 200 cm-1 at room temperature
• the spacing of molecular rotational levels
• is a few tenths of reciprocal centimeters
• Thermal energy populates many
• rotational levels giving molecules an
• internal source of energy
• rotational energy available within a
• molecule can add to that of a photon,
• making a range of optical energy that
• can satisfy Planck's Law, DE hn Erot

• the graph shows the energy of thermally
  populated rotational levels the distribution has
  a width of 700 cm-1
• an electronic transition will be broadened by
  this width
• 500 nm transition will be 17 nm wide (491 - 508
  nm)
• 400 nm transition will be 11 nm wide (394 - 405
  nm)
• 300 nm transition will be 7 nm wide (296 - 303
  nm)

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Vibronic Transitions
(a)

• A simultaneous change in vibrational and
• electronic quantum numbers is called a
• vibronic transition
• if the inter-nuclear distances are not affected
  
• when the electron changes orbitals AND the
• transition is symmetry allowed, the spectrum
• will have a single peak and, no or very weak,
• vibronic bands
• If one or more vibrations have different
• equilibrium inter-nuclear coordinates, a
• vibronic sequence will appear in the spectrum -
  this is shown in (a) for a single vibrational
• mode (more than one can be affected)
• If the electronic transition is symmetry
• forbidden, vibronic bands will appear for
• those vibrations that deform the molecule
• into a shape which has an allowed transition
  (b)
• symmetry allowed e 103 - 105 M-1 cm-1
• symmetry forbidden e ? 102 M-1 cm-1

(b)
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State Diagrams and Absorption Spectra
absorption spectrum state diagram
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Chromophores

• They are groups with one element of unsaturation
  (unsaturated linkages or groups) and cause
  coloring to the molecules when they are attached
  to a non-absorbing hydrocarbon chain

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Effect of Multichromophores on Absorption

• More chromophores in the same molecule cause
  bathochromic effect
• (Red shift shift to longer wavelength)
• and hyperchromic effect (increase in
  intensity)
• Hypsochromic effect Blue shift shift to shorter
  wavelengths
• Hypochromic effect decrease in intensity
• In the conjugated chromophores ? electrons are
  delocalized over larger number of atoms causing a
  decrease in the energy of ? to ? transitionsand
  an incrase in ? due to an increase in probability
  for transition

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• Aromatic Hydrocabons
• They absorb at three bands 260, 200 and 180 nm
• Policyclic aromatic (Naphthalene) exhibit
  regular shift towards longer wavelength (Red
  shift)
• Azo Compounds with the linkage NN- show low
  intensity bands in the near Uv and Vis due to n
  to ? transitions
• Azobenzenes absorb at about 445 nm the NN- may
  be conjugated with the ring ? system.

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UV absorption spectra of benzene, naphthalene,
and anthracene
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Auxochromes

• They are groups that do not confer color but
  increase the coloring power of a chromophore.
• They are functional groups that have non-bonded
  valence electrons and show no absorption at ? gt
  220 nm they absorb in the far UV
• -OH and -NH2 groups cause a red shift

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Steric Effect

• Extended conjugation of ? orbitals requires
• coplanarity of the atoms involved in the ?-
• cloud delocalization for maximum resonance
• interaction
• Large bulki groups cause a perturbation of the
  coplanarity of the ? system .
• Thus ?max is usually shifted towards shorter ?
• and ? also decreases

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Linear Polyenes

• As the number of double bonds increases, the
  long wavelength
• absorption shifts to higher values (called a
  red-shift)
• The molar absorptivity increases as the
  molecular orbital size
• increases
• To anticipate the spectrum, use the number of
  conjugated
• double bonds, i.e. CH2CH-CH2-CHCH2 has a
  spectrum closer to
• ethylene than butadiene.

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Linear Fused Aromatics

• As the number of fused rings increases, the long
  wavelength
• absorption shifts to higher values
• The long wavelength transition is forbidden in
  benzene and
• naphthalene, but allowed in anthracene and
  tetracene
• To anticipate the spectrum use the number of
  conjugated
• double bonds, i.e. diphenylmethane has a
  spectrum that
• resembles toluene

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Linear Fused Aromatics
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Non-Linear Fused Aromatics

• The 0-0 band appears at lower wavelengths than
  would be
• predicted by the number of fused rings (379
  for anthracene and
• 479 for tetracene)
• The first three have band positions similar to
  anthracene and
• molar absorptivities similar to naphthalene
• Perylene has properties between anthracene and
  tetracene

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Non-Linear Fused Aromatics
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Linear Polyphenyls

• As the number of conjugated rings increases, the
  0-0 band shifts
• to higher wavelengths
• The increase in wavelength is not as fast as the
  polyenes or
• linear aromatics because of the bond between
  the rings is wisted
• The spectrum is featureless because thermally
  induced
• oscillation about the twist angle adds width
  to the vibronic bands
• The molar absorptivity increases because the
  number of double
• bonds is increasing

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Linear Polyphenyls
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Alkyl Substituents

• Alkyl substituents shift the 0-0 band of the
  parent aromatic a few
• nanometers to the red, and increase the molar
  absorptivity a
• small amount
• Multiple substituents will increase the shift by
  smaller increments
• The vibronic pattern in the spectrum will change
  because of the
• new vibrations

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Alkyl Substituents
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Substituents with Lone-Pairs of Electrons

• When atoms with lone-pairs of electrons are
  attached to
• aromatic compounds they can effectively
  increase the size of the
• ring system
• An increase in the size of the ring system
  shifts the parent
• spectrum to the red
• Lone-pairs often break the symmetry of a
  molecule, converting
• a forbidden transition into a moderately
  allowed transition
• When a transition is made more allowed, there is
  an increase in
• the molar absorptivity
• When aromatic compounds with hydroxyl or amine
  substituents
• are dissolved in hydrogen bonding solvents,
  the absorption
• bands become broad and vibronic structure is
  decreased or lost

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Halogen Substituents

• Halogen substituents shift the 0-0 band to the
  red
• The larger the halogen the larger the shift
• Halogens can break symmetry to make a transition
  more allowed
• Multiple substituents will increase the red shift

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Halogen Substituents
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Hydroxide and Amine Substituents

• Hydroxide and amine substituents shift the 0-0
  band to the red
• Both substituents create broad bands when the
  compound is
• dissolved in a hydrogen bonding solvent
• Both substituents can break symmetry to make a
  transition
• more allowed
• Molar absorptivities are in the range of 1,000 -
  6,000

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Hydroxyl and Amine Substituents
cyclohexane cyclohexane
ethanol
ethanol methanol
ethanol
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• Ultraviolet absorption spectra for
  1,2,4,5-tetrazine (a.) in the vapor phase, (b.)
  in hexane solution, and (c.) in aqueous solution.

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Effect of polar solvents on transitions
Polar solvents stabilize both non-bonding
electrons in The ground state and ? elctrons in
the excited state
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Absorption
Excited state
Incident beam
Transmitted beam
Loss of energy as radiation, heat, etc ...
Sample
Absorption
Absorption along radiation beam
Ground state
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Beers law (Beer-Lambert Law)

• Empirical relationship between transmitted
• intensity and number of absorbers.
• A. Beer, 1852. See H. G. Pfeiffer and H. A.
• Liebhafsky, J. Chem. Ed. 1951, 28, 123-
• 125,The origins of Beers law.
• The incident radiation is monochromatic.
• The absorbing units (atoms, molecules, ions)
• act independently of one another.
• The absorption is limited to a volume of uniform
  
• cross section.

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Beers law (The Beer-Lambert Law)

• Exponential attenuation with
• concentration
• sample thickness (optical path length)
• Assumes
• sample is non-turbid (Non-scattering)
• Scattering effects
• create losses out of the side of the sample
• apparent absorption is greater than actual
  absorption
• optical path-length is now no longer simply the
  length of the cuvette
• lead to requirement for model of light
  propagation (diffusion theory, etc)

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?bc
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?bc
?bc

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Beers Law Example
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Example
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known
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Example
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Solvents for UV-Visible Regions

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Analysis of Mixtures of Absorbing Substances

• Absorption spectrum of a two-component mixture

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• Solution of Binary Mixture
• Schematic representation of the absorption
  spectra of solutions containing
• (1) c1 moles per liter of substance 1
• (2) c2 moles per liter of substance 2
• (3) c1 moles per liter of substance 1 and
• c2 moles per liter of substance 2

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Solution of Binary Mixture

• Wavelength 1
• Am,l1 a1,l1bc1 a2,l1bc2
• Am,l1 (a1,l1c1 a2,l1c2)b
• Wavelength 2
• Am,l2 a1,l2bc1 a2,l2bc2
• Am,l2 (a1,l2c1 a2,l2c2)b

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Solution of Binary Mixture

• let A1 Am,l1 A2 Am,l2
• D1 a1,l1 D2 a1,l2
• ?1 a2,l1 ?2 a2,l2
• then A1 (D1c1 ?1c2)b
• A2 (D2c1 ?2c2)b

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• solve for c2
• A2/b (D2c1 ?2c2)
• A2/b - D2c1 ?2c2
• ?2c2 A2/b - D2c1
• c2 (A2/(b ?2) - (D2c1)/ ?2

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• then
• A1 (D1c1 ?1((A2/(b ?2)-(D2c1)/ ?2))b
• A1/b (D1c1 ?1((A2/(b ?2)(D2c1)/ ?2))
• A1/b (c1(D1 - D2(?1/ ?2))(?1/ ?2)(A2/b))
• A1/b - (A2/b)(?1/ ?2) c1(D1-D2(?1/ ?2))

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• thus
• (A1/b - (A2/b)(?1/ ?2))
• c1 -------------------------------
• (D1 - D2(?1/ ?2))
• and
• C2 (A2/(?2b) - (D2c1)/ ?2

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Example
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Example
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Deviation from Beers Law

• It is a deviation from direct proportionality
  between A and C.
• It is either ve (upward curvature) or Ve
  (downwod curvature) deviation
• Sources are Real, Instrumental , or Chemical
  factors

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Real Deviations

• high concentration - particles too close
• Dependence of absorptivity on refractive index of
  solution

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Real Factors

• Derivation did not take into consideration the
• changes in Refractive index of the solution
• due to concentration changes.
• Refractive index increase as concentration
  increases
• Consequently the proportionality constant is not
  ? but ?n / (n22)2 where n is the refractive
  index of the medium

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Instrumental Factors

• Alterations in power supply voltage, light source
  or
• detector response. Others include

• Polychromatic Radiation
• Assume a radiation consisting of two
  wavelengths
• ? and ? and Beers Law applies at each

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At ? the absorbance will be given as follows
Ac ? b c when ? ?
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• Departure increases by increasing the difference
  between ? Values of the polychromatic
  radiation due to the increase in the difference
  between ? and ?
• The steeper the absorption (A Vs ?) curve
  the greater the error

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Overlap of Sample with Optical Beam

• As long as the optical beam is narrower than the
  
• sample cell width (cases A and B in the
  figure)
• measured absorption is constant.
• When the optical beam is larger than the sample
• cell width (case C) light misses the sample
  and
• cannot be absorbed.
• The equation describing transmission when light
  
• misses the cell is given below. In the
  example, the
• true absorption is 0.30, the incorrect
  absorption is
• 0.22

• This situation can occur when trying to make
  absorption
• measurements in capillary liquid
  chromatography.

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Monochromator Stray Light
All gratings and mirrors scatter a small fraction
of the input light. The scattered radiation gets
spread throughout the monochromator, with some
reaching the exit slit. The scatter is composed
of all frequencies entering the monochromator.
The scatter is then transferred by lenses or
mirrors though the sample and to the detector.
The expression for transmission has to be
modified to take this into account.
Since the stray light occurs at all wavelengths
it will be absorbed to a different extent than ?.
Usually a large fraction of stray light is not
absorbed at all. This makes it difficult to
measure absorbance values above 2.
High-performance spectrophotometers are
ordinarily constructed with double monochromators
to reduce the amount of stray light.
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3. Stray Light
The stray light striking detector is a potential
source of error. Apparent A is decreased as a
result

• Thus a negative deviation is expected
• Deviations are expected near the limits of the
  instrument
• Components
• Visible radiation is the most serious stray light
  problem
• For Uv-Vis spectrometers

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Sources of Stray Light and Its Effect of on
Absorbance
Source M. R. Sharpe, Anal. Chem. 56, 339A-356A
(1984).
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Apparent deviation due to stray radiation
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Sample Fluorescence

• When a sample fluoresces, some of the emitted
  photons
• reach the detector. This gives an abnormally
  high
• transmission.

• The effect of fluorescence can be reduced by
  moving the
• sample away from the detector.
• The effect of fluorescence can be eliminated by
  using two
• monochromators that scan synchronously.

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Sample Scatter

• Samples that scatter light lose radiation in
  addition to
• that absorbed. This causes an abnormally low
  
• transmission.

• Since most scattered
• light is in the forward
• direction (toward the
• detector), its effect can
• be reduced by moving
• the detector very close
• to the sample.
• Scattered light is
• proportional to 1/?4.
• This makes it easy to
• identify.

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Sample Cell Reflections

• For a quartz sample cell each air/quartz
  interface reflects
• 3.5 of the light, while each quartz/water
  interface
• reflects 0.2. These reflective losses
  represent light
• that never reaches the detector. The measured
• transmission is lower than expected.

• The effect of reflection can be reduced by using
  a
• reference cell that contains only solvent.
  The two cells
• must have identical optical properties (called
  matched
• cells) for the reflective losses to cancel.
• This problem is sufficiently acute that
  expensive
• spectrophotometers have a mechanism for
  "flattening"
• and zeroing the baseline when running solvent
  versus
• solvent.

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Source Width

• The spectral width of the source determines the
  shape of
• the measured absorption spectrum. The two
  curves are
• "convoluted" mathematically (source original
  spectrum
• measured spectrum)

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Source Width (2)

• Beer's Law only holds for monochromatic light.
  With
• non-monochromatic light, the slope of the
  calibration
• curve decreases. In addition the curve is
  non-linear for
• high absorption values.

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Slit Width

• Spectral slit width it is the spread of the
  image
• along the frequency, wavenumber or wavelength
  scale
• It is proportional to the mechanical slit width
• If the absorption band is sharp or if the
  measurement
• is made at the steep slope of the spectral band
  ? may
• be different over the spectral band width and
• deviation may be noticed
• Typical bandwidth of a spectrometer is of the
  order of 1 nm
• Molecular absorption bands are broader than 1
  nm thus the
• Effect of spectral bandwidth is negligible
  when A is measured
• at ?max

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• Effect of Bandwidth on Spectral Detail
• Absorbance increases (significantly)
• as slit width decreases.
• However, decrease in slit width leads to a
• (second-order) reduction in power of radiant
• energy so at very narrow slit widths, high
• S/N can lead to loss of spectral detail.

Source Skoog, Holler, and Nieman, Principles of
Instrumental Analysis, 5th edition, Saunders
College Publishing.
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Chemical Deviations from Beer's Law

• A chemical system in equilibrium will often
• create deviations from Beer's Law.
• Examples might be dimerization, acid/base
• reactions, redox reactions, and reversible
• reactions with molecular oxygen.
• Consider a weak base in an aqueous
• solution, where A- is monitored by
• absorption. There are three unknowns and
• three equations. They can be solved for
• the concentration of A?.

• This equation shows that A- is not directly
  proportional
• to CB, unless CB gtgt KB. As a result, a plot
  of absorbance
• versus CB will not be linear.

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Deviation Due to Chemical Factors

• Sources are Dissociation, association, complex
  formation,
• polymerization or solvolysis.
• For example dissociation of benzoic acid and
  potassium
• dichromate