An introduction to Ultraviolet/Visible Absorption Spectroscopy

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An introduction to Ultraviolet/Visible Absorption
Spectroscopy

• Chapter 13

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• In this chapter, absorption by molecules, rather
  than atoms, is considered. Absorption in the
  ultraviolet and visible regions occurs due to
  electronic transitions from the ground state to
  excited state. Broad band spectra are obtained
  since molecules have vibrational and rotational
  energy levels associated with electronic energy
  levels. The signal is either absorbance or
  percent transmittance of the analyte solution
  where

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An Introduction to Ultraviolet/VisibleMolecular
Absorption Spectrometry

• Absorption measurements based upon ultraviolet
  and visible radiation find widespread application
  for the quantitative determination of a large
  variety species.
• Beers Law
• A -logT logP0/P ?bc
• A absorbance
• ? molar absorptivity M-1 cm-1
• c concentration M
• P0 incident power
• P transmitted power (after passing through
  sample)

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• Measurement of Transmittance and Absorbance
• The power of the beam transmitted by the analyte
  solution is usually compared with the power of
  the beam transmitted by an identical cell
  containing only solvent. An experimental
  transmittance and absorbance are then obtained
  with the equations.
• P0 and P refers to the power of radiation after
  it has passed through the solvent and the
  analyte.

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Beers law and mixtures

• Each analyte present in the solution absorbs
  light!
• The magnitude of the absorption depends on its e
• A total A1A2An
• A total e1bc1e2bc2enbcn
• If e1 e2 en then simultaneous determination
  is impossible
• Need nls where es are different to solve the
  mixture

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Limitations to Beers Law

• Real limitations
• Chemical deviations
• Instrumental deviations

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• 1.      Real Limitations
•  
• a.       Beers law is good for dilute analyte
  solutions only. High concentrations (gt0.01M) will
  cause a negative error since as the distance
  between molecules become smaller the charge
  distribution will be affected which alter the
  molecules ability to absorb a specific
  wavelength. The same phenomenon is also observed
  for solutions with high electrolyte
  concentration, even at low analyte concentration.
  The molar absorptivity is altered due to
  electrostatic interactions.

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• b.      In the derivation of Beers law we have
  introduced a constant (e). However, e is
  dependent on the refractive index and the
  refractive index is a function of concentration.
  Therefore, e will be concentration dependent.
  However, the refractive index changes very
  slightly for dilute solutions and thus we can
  practically assume that e is constant.
• c.       In rare cases, the molar absorptivity
  changes widely with concentration, even at dilute
  solutions. Therefore, Beers law is never a
  linear relation for such compounds, like
  methylene blue.

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• 2.      Chemical Deviations
•  
• This factor is an important one which largely
  affects linearity in Beers law. It originates
  when an analyte dissociates, associates, or
  reacts in the solvent. For example, an acid base
  indicator when dissolved in water will partially
  dissociate according to its acid dissociation
  constant

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• Chemical deviations from Beers law for
  unbuffered solutions of the indicator Hln. Note
  that there are positive deviations at 430 nm
  and negative deviations at 570 nm. At 430 nm, the
  absorbance is primarily due to the ionized In-
  form of the indicator and is proportional to the
  fraction ionized, which varies nonlinearly with
  the total indicator concentration. At 570 nm, the
  absorbance is due principally to the
  undissociated acid Hln, which increases
  nonlinearly with the total concentration.

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Calculated Absorbance Data for Various Indicator
Concentrations
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• 3.      Instrumental Deviations
•  
• a.       Beers law is good for monochromatic
  light only since e is wavelength dependent. It is
  enough to assume a dichromatic beam passing
  through a sample to appreciate the need for a
  monochromatic light. Assume that the radiant
  power of incident radiation is Po and Po while
  transmitted power is P and P. The absorbance of
  solution can be written as

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• The effect of polychromatic radiation on Beers
  law. In the spectrum at the top, the absorptivity
  of the analyte is nearly constant over Band A
  from the source. Note in the Beers law plot at
  the bottom that using Band A gives a linear
  relationship. In the spectrum, Band B corresponds
  to a region where the absorptivity shows
  substantial changes. In the lower plot, note the
  dramatic deviation from Beers law that results.

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• Therefore, the linearity between absorbance and
  concentration breaks down if incident radiation
  was polychromatic. In most cases with UV-Vis
  spectroscopy, the effect small changes in
  wavelengths is insignificant since e differs only
  slightly especially at the wavelength maximum.

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• b.      Stray Radiation
•  
• Stray radiation resulting from scattering or
  various reflections in the instrument will reach
  the detector without passing through the sample.
  The problem can be severe in cases of high
  absorbance or when the wavelengths of stray
  radiation is in such a range where the detector
  is highly sensitive as well as at wavelengths
  extremes of an instrument. The absorbance
  recorded can be represented by the relation
• A log (Po Ps)/(P Ps)
• Where Ps is the radiant power of stray radiation.

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Instrumental Noise as a Function in Transmittance
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• Therefore, an absorbance between 0.2-0.7 may be
  advantageous in terms of a lower uncertainty in
  concentration measurements. At higher or lower
  absorbances, an increase in uncertainty is
  encountered. It is therefore advised that the
  test solution be in the concentration range which
  gives an absorbance value in the range from
  0.2-0.7 for best precision.
• However, it should also be remembered that we
  ended up with this conclusion provided that sT is
  constant. Unfortunately, sT is not always
  constant which complicates the conclusions above.

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EFFECT OF bandwidth WIDTH

• Effect of bandwidth on spectral detail for a
  sample of benzene vapor. Note that as the
  spectral bandwidth increases, the fine structure
  in the spectrum is lost. At a bandwidth of 10 nm,
  only a broad absorption band is observed.

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• Effect of slit width (spectral bandwidth) on peak
  heights. Here, the sample was s solution of
  praseodymium chloride. Note that as the spectral
  bandwidth decreases by decreasing the slit width
  from 1.0 mm to 0.1 mm, the peak heights increase.

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Effect of Scattered Radiation at Wavelength
Extremes of an Instrument

• Wavelength extremes of an instrument are
  dependent on type of source, detector and optical
  components used in the manufacture of the
  instrument. Outside the working range of the
  instrument, it is not possible to use it for
  accurate determinations. However, the extremes of
  the instrument are very close to the region of
  invalid instrumental performance and would thus
  be not very accurate. An example may be a visible
  photometer which, in principle, can be used in
  the range from 340-780 nm. It may be obvious that
  glass windows, cells and prism will start to
  absorb significantly below 380 nm and thus a
  decrease in the incident radiant power is
  significant.

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B UV-VIS spectrophotometer A VIS
spectrophotometer
EFFECT OF SCATTERED RADIATION

• Spectrum of cerium (IV) obtained with a
  spectrophotometer having glass optics (A) and
  quartz optics (B). The false peak in A arises
  from transmission of stray radiation of longer
  wavelengths.

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• The output from the source at the low wavelength
  range is minimal. Also, the detector has best
  sensitivities around 550 nm which means that away
  up and down this value, the sensitivity
  significantly decrease. However, scattered
  radiation, and stray radiation in general, will
  reach the detector without passing through these
  surfaces as well as these radiation are
  constituted from wavelengths for which the
  detector is highly sensitive. In some cases,
  stray and scattered radiation reaching the
  detector can be far more intense than the
  monochromatic beam from the source. False peaks
  may appear in such cases and one should be aware
  of this cause of such peaks.

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• Instrumentation
• Light source
• ? - selection
• Sample container
• Detector
• Signal processing
• Light Sources (commercial instruments)
• D2 lamp (UV 160 375 nm)
• W lamp (vis 350 2500 nm)

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SourcesDeuterium and hydrogen lamps (160 375
nm)

• D2 Ee ? D2 ? D D h?

Excited deuterium molecule with fixed quantized
energy
Dissociated into two deuterium atoms with
different kinetic energies
Ee ED2 ED ED hv
Ee is the electrical energy absorbed by the
molecule. ED2 is the fixed quantized energy of
D2, ED and ED are kinetic energy of the two
deuterium atoms.
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Sources
Deuterium lamp UV region

• (a) A deuterium lamp of the type used in
  spectrophotometers and (b)
• its spectrum. The plot is of irradiance E?
  (proportional to radiant power) versus
• wavelength. Note that the maximum intensity
  occurs at 225 m.Typically,
• instruments switch from deuterium to tungsten at
  350 nm.

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Visible and near-IR region

• (a) A tungsten lamp of the
• type used in spectroscopy
• and its spectrum (b).
• Intensity of the tungsten
• source is usually quite low
• at wavelengths shorter than about 350 nm. Note
  that the intensity reaches a maximum in the
  near-IR
• region of the spectrum
• (1200 nm in this case).

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• The tungsten lamp is by far the most common
  source in the visible and near IR region with a
  continuum output wavelength in the range from
  350-2500 nm. The lamp is formed from a tungsten
  filament heated to about 3000 oC housed in a
  glass envelope. The output of the lamp approaches
  a black body radiation where it is observed that
  the energy of a tungsten lamp varies as the
  fourth power of the operating voltage.

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• Tungsten halogen lamps are currently more popular
  than just tungsten lamps since they have longer
  lifetime. Tungsten halogen lamps contain small
  quantities of iodine in a quartz envelope. The
  quartz envelope is necessary due to the higher
  temperature of the tungsten halogen lamps (3500
  oC). The longer lifetime of tungsten halogen
  lamps stems from the fact that sublimed tungsten
  forms volatile WI2 which redeposits on the
  filament thus increasing its lifetime. The output
  of tungsten halogen lamps are more efficient and
  extend well into the UV.

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SourcesTungsten lamps (350-2500 nm)

• Why add I2 in the lamps?
• W I2 ? WI2

• Low limit 350 nm
• Low intensity
• Glass envelope

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• 3. Xenon Arc Lamps
•  
• Passage of current through an atmosphere of high
  pressured xenon excites xenon and produces a
  continuum in the range from 200-1000 nm with
  maximum output at about 500 nm. Although the
  output of the xenon arc lamp covers the whole UV
  and visible regions, it is seldom used as a
  conventional source in the UV-Vis. The radiant
  power of the lamp is very high as to preclude the
  use of the lamp in UV-Vis instruments. However,
  an important application of this source will be
  discussed in luminescence spectroscopy which will
  be discussed later

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Sample Containers

• Sample containers are called cells or cuvettes
  and are made of either glass or quartz depending
  on the region of the electromagnetic spectrum.
  The path length of the cell varies between 0.1
  and 10 cm but the most common path length is 1.0
  cm. Rectangular cells or cylindrical cells are
  routinely used. In addition, disposable
  polypropylene cells are used in the visible
  region. The quality of the absorbance signal is
  dependent on the quality of the cells used in
  terms of matching, cleaning as well as freedom
  from scratches.

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• Instrumental Components
• Source
• ? - selection (monochromators)
• Sample holders
• Cuvettes (b 1 cm typically)
• Glass (Vis)
• Fused silica (UVVis)
• Detectors
• Photodiodes
• PMTs

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• 1. Single beam
• Place cuvette with blank (i.e., solvent) in
  instrument and take a reading ? 100 T
• Replace cuvette with sample and take reading ?
  T for analyte (from which absorbance is calcd)

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Instrumentation

• Most common spectrophotometer Spectronic 20.

• On/Off switch and zero transmission adjustment
  knob
• Wavelength selector/Readout
• Sample chamber
• Blank adjustment knob
• Absorbance/Transmittance scale

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• End view of the exit slit of the Spectronic 20
• spectrophotometer pictured earlier

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• Single-Beam Instruments for the
  Ultraviolet/Visible Region

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• Single-Beam Computerized Spectrophotometers

Inside of a single-beam spectrophotometer
connected to a computer.
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Types of Instruments

• Instrumental designs for UV-visible photometers
• or spectrophotometers. In (a), a single-beam
  instrument is shown. Radiation from the filter
  or monochromator passes through either the
  reference cell or the sample cell before
  striking the photodetector.

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• 2. Double beam (most commercial instruments)
• Light is split and directed towards both
  reference cell (blank) and sample cell
• Two detectors electronics measure ratio (i.e.,
  measure/calculate absorbance)
• Advantages
• Compensates for fluctuations in source intensity
  and drift in detector
• Better design for continuous recording of spectra

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General Instrument Designs Double Beam In - Space
Needs two detectors
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General Instrument Designs Double Beam In - Time
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• Merits of Double Beam Instruments
• Compensate for all but the most short term
  fluctuation in radiant output of the source
• Compensate drift in transducer and amplifier
• Compensate for wide variations in source
  intensity with wavelength

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Location of Sample cell

• In all photometers and scanning
  spectrophotpmeters described above, the cell has
  been positioned after the monochromators. This is
  important to decrease the possibility of sample
  photodecomposition due to prolonged exposure to
  all frequencies coming from the source. However,
  the sample is positioned before the monochromator
  in multichannel instruments like a photodiode
  array spectrophotometer. This can be done without
  fear of photodecomposition since the sample
  exposure time is usually less than 1 s.
  Therefore, it is now clear that in UV-Vis where
  photodecomposition of samples can take place, the
  sample is placed after the monochromators in
  scanning instruments while positioning of the
  sample before the monochromators is advised in
  multichannel instruments.

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• 3. Multichannel Instruments
• Photodiode array detectors used (multichannel
  detector, can measure all wavelengths dispersed
  by grating simultaneously).
• Advantage scan spectrum very quickly snapshot
  lt 1 sec.
• Powerful tool for studies of transient
  intermediates in moderately fast reactions.
• Useful for kinetic studies.
• Useful for qualitative and quantitative
  determination of the components exiting from a
  liquid chromatographic column.

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Multi-channel Design
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• A multichannel diode-array spectrophotometer, the
  Agilent Technologies 8453.

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4. Probe Type Instruments

• These are the same as conventional single beam
  instruments but the beam from the monochromators
  is guided through a bifurcated optical fiber to
  the sample container where absorption takes
  place. The attenuation in reflected beam at the
  specified wavelength is thus measured and related
  to concentration of analyte in the sample.
• A fiber optic cable can be referred to as a light
  pipe where light can be transmitted by the fiber
  without loss in intensity (when light hits the
  internal surface of the fiber at an angle larger
  than a critical angle). Therefore, fiber optics
  can be used to transmit light for very long
  distances without losses. A group of fibers can
  be combined together to form a fiber optic cable
  or bundle. A bifurcated fiber optic cable has
  three terminals where fibers from two separate
  cables are combined at one end to form the new
  configuration.

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Fiber optic probe
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Double Dispersing Instruments

• The instrument in this case has two gratings
  where the light beam leaving the first
  monochromators at a specified wavelength is
  directed to the second grating. This procedure
  results in better spectral resolution as well as
  decreased scattered radiation. However, double
  dispersing instruments are expensive and seem to
  offer limited advantages as compared to cost
  especially in the UV-Vis region where exact
  wavelength may not be crucial.

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• Optical diagram of the Varian Cary 300
  double-dispersing spectrophotometer. A second
  monochromator is added immediately after the
  source.

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Molar absorptivities

• e 8.7 x 10 19 P A
• A cross section of molecule in cm2 (10-15)
• P Probability of the electronic transition (0-1)
• Pgt0.1-1 ? allowable transitions
• Plt0.01 ? forbidden transitions

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Molecular Absorption

• M hn ? M (absorption 10-8 sec)
• M ? M heat (relaxation process)
• M ? ABC (photochemical decomposition)
• M ? M hn (emission)

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Visible Absorption Spectra
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• The absorption of UV-visible radiation generally
  results from excitation of bonding electrons.
• can be used for quantitative and qualitative
  analysis

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• Molecular orbital is the nonlocalized fields
  between atoms that are occupied by bonding
  electrons. (when two atom orbitals combine,
  either a low-energy bonding molecular orbital or
  a high energy antibonding molecular orbital
  results.)
• Sigma (?) orbital
• The molecular orbital associated with single
  bonds in organic compounds
• Pi (?) orbital
• The molecular orbital associated with parallel
  overlap of atomic P orbital.
• n electrons
• No bonding electrons

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Molecular Transitions for UV-Visible Absorptions

• What electrons can we use for these transitions?

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MO Diagram for Formaldehyde (CH2O)
H
C
O
H
s
p
n
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Singlet vs. triplet

• In these diagrams, one electron has been excited
  (promoted) from the n to ? energy levels
  (non-bonding to anti-bonding).
• One is a Singlet excited state, the other is a
  Triplet.

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Type of Transitions

• s ? s
• High energy required, vacuum UV range
• CH4 ? 125 nm
• n ? s
• Saturated compounds, CH3OH etc (? 150 - 250 nm)
• n ? ? and ? ? ?
• Mostly used! ? 200 - 700 nm

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Examples of UV-Visible Absorptions
LOW!
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UV-Visible Absorption Chromophores
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Effects of solvents

• Blue shift (n- p) (Hypsocromic shift)
• Increasing polarity of solvent ? better solvation
  of electron pairs (n level has lower E)
• ? peak shifts to the blue (more energetic)
• 30 nm (hydrogen bond energy)
• Red shift (n- p and p p) (Bathochromic shift)
• Increasing polarity of solvent, then increase the
  attractive polarization forces between solvent
  and absorber, thus decreases the energy of the
  unexcited and excited states with the later
  greater
• ? peaks shift to the red
• 5 nm

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UV-Visible Absorption Chromophores
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Typical UV Absorption Spectra
Chromophores?
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The effects of substitution
Auxochrome function group
Auxochrome is a functional group that does not
absorb in UV region but has the effect of
shifting chromophore peaks to longer wavelength
as well As increasing their intensity.
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Now solvents are your container

• They need to be transparent and do not erase the
  fine structure arising from the vibrational
  effects

Polar solvents generally tend to cause this
problem
Same solvent must be Used when comparing absorptio
n spectra for identification purpose.
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Summary of transitions for organic molecules

• s ? s transition in vacuum UV (single bonds)
• n ? s saturated compounds with non-bonding
  electrons
• l 150-250 nm
• e 100-3000 ( not strong)
• n ? p, p ? p requires unsaturated functional
  groups (eq. double bonds) most commonly used,
  energy good range for UV/Vis
• l 200 - 700 nm
• n ? p e 10-100
• p ? p e 1000 10,000

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List of common chromophores and their transitions
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Organic Compounds

• Most organic spectra are complex
• Electronic and vibration transitions superimposed
• Absorption bands usually broad
• Detailed theoretical analysis not possible, but
  semi-quantitative or qualitative analysis of
  types of bonds is possible.
• Effects of solvent molecular details complicate
  comparison

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Rule of thumb for conjugation
If greater then one single bond apart - e are
relatively additive (hyperchromic shift) - l
constant CH3CH2CH2CHCH2 lmax 184 emax
10,000 CH2CHCH2CH2CHCH2 lmax185 emax
20,000 If conjugated - shifts to higher ls
(red shift) H2CCHCHCH2 lmax217 emax
21,000
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Spectral nomenclature of shifts
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What about inorganics?

• Common anions n?p nitrate (313 nm), carbonate
  (217 nm)
• Most transition-metal ions absorb in the UV/Vis
  region.
• In the lanthanide and actinide series the
  absorption process results from electronic
  transitions of 4f and 5f electrons.
• For the first and second transition metal series
  the absorption process results from transitions
  of 3d and 4d electrons.
• The bands are often broad.
• The position of the maxima are strongly
  influenced by the chemical environment.
• The metal forms a complex with other stuff,
  called ligands. The presence of the ligands
  splits the d-orbital energies.

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Transition metal ions
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Charge-Transfer-Absorption

• A charge-transfer complex consists of an
  electron-donor group bonded to an electron
  acceptor. When this product absorbs radiation, an
  electron from the donor is transferred to an
  orbital that is largely associated with the
  acceptor.
• Large molar absorptivity (emax gt10,000)
• Many organic and inorganic complexes

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