AN INTRODUCTION TO
ULTRAVIOLET/VISIBLE ABSORPTION
SPECTROSCOPY
CHAPTER 13, 14
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PROBLEM SET
Chapter 13
13-5, -7, -8, -10, -11, -12, -22
Chapter 14
14-1, 14-6, 14-7, 14-11
Due Tues., May 1, 2012
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FIRST LONG EXAMINATION
Ch 135 A/B/C
May 3, Thursday. 1:30 – 4:00 PM, C114
Coverage: Chapters 1, 6-10, 13-17 (5th Ed., Skoog)
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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.
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AN INTRODUCTION TO ULTRAVIOLET/VISIBLE
MOLECULAR ABSORPTION SPECTROMETRY
Absorption measurements based upon ultraviolet and
visible radiation find widespread application for the
quantitative determination of a large variety species.
Beer’s Law:
A = -log T = 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.
Psolution
P
T 

Psolvent
P0
Psolvent
P0
A  log
 log
Psolution
P
P0 and P refers to the power of radiation after it has passed
through the solvent and the analyte.
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BEER’S LAW AND MIXTURES
 Each analyte present in the solution absorbs light!
 The magnitude of the absorption depends on its 
A total = A1+A2+…+An
A total = 1bc1+2bc2+…+nbcn
 If 1 = 2 = n then simultaneous determination is
impossible
 Need nl’s where ’s are different to solve the
mixture
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LIMITATIONS TO BEER’S LAW
 Real limitations
 Chemical deviations
 Instrumental deviations
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REAL LIMITATIONS
 Beer’s law is good for dilute analyte solutions only. High
concentrations (>0.01M) 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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REAL LIMITATIONS
In the derivation of Beer’s law we have introduced a constant
(). However, e is dependent on the refractive index and the
refractive index is a function of concentration.
Therefore,  will be concentration dependent. However, the
refractive index changes very slightly for dilute solutions and
thus we can practically assume that  is constant.
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REAL LIMITATIONS
In rare cases, the molar absorptivity changes widely with
concentration, even at dilute solutions. Therefore, Beer’s law is
never a linear relation for such compounds, like methylene blue.
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CHEMICAL DEVIATIONS
This factor is an important one which largely affects linearity
in Beer’s 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 Beer’s 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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INSTRUMENTAL DEVIATIONS
Beer’s law is good for monochromatic light only since  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 Beer’s law. In the
spectrum at the top, the absorptivity of the analyte is nearly
constant over Band A from the source. Note in the Beer’s
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 Beer’s 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  differs only
slightly; especially at the wavelength maximum.
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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.
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EFFECT OF BANDWIDTH
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
 l - 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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SOURCES
Deuterium 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 Eλ (proportional to radiant power) versus
wavelength. Note that the maximum intensity occurs at ~225 nm.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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SOURCES
Tungsten 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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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
l - selection (monochromators)
Sample holders

Cuvettes (b = 1 cm typically)
1.
2.

Glass (Vis)
Fused silica (UV+Vis)
Detectors


Photodiodes
PMTs
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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 calc’d)
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INSTRUMENTATION
Most common spectrophotometer: Spectronic 20.
1. On/Off switch and zero
transmission adjustment
knob
2. Wavelength
selector/Readout
3. Sample chamber
4. Blank adjustment knob
5. Absorbance/Transmittanc
e 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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DOUBLE BEAM (most commercial instruments)
o Light is split and directed towards both reference cell
(blank) and sample cell
o Two detectors; electronics measure ratio (i.e.,
measure/calculate absorbance)
o 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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




MULTICHANNEL INSTRUMENTS
Photodiode array detectors used (multichannel detector,
can measure all wavelengths dispersed by grating
simultaneously).
Advantage: scan spectrum very quickly “snapshot” < 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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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 doubledispersing
spectrophotometer. A
second monochromator
is added immediately
after the source.
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MOLAR ABSORPTIVITIES
 = 8.7 x 10 19 P A
 A: cross section of molecule in cm2 (~10-15)
 P: Probability of the electronic transition (0-1)
 P>0.1-1  allowable transitions
 P<0.01  forbidden transitions
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MOLECULAR ABSORPTION
M + h  M* (absorption 10-8 sec)
 M*  M + heat (relaxation process)
 M*  A+B+C (photochemical decomposition)
 M*  M + h (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
H
C
=
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O
=
n=
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
 σ → σ*
High energy required, vacuum UV range
CH4: l = 125 nm
 n → σ*
Saturated compounds, CH3OH etc (l = 150 - 250 nm)
 n → * and  → *
Mostly used! l = 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- *) (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- * and  –*) (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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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
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Same solvent must be
Used when comparing
absorption spectra for
identification purpose.
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SUMMARY OF TRANSITIONS FOR ORGANIC MOLECULES
  * transition in vacuum UV (single bonds)
n  * saturated compounds with non-bonding electrons
l ~ 150-250 nm
 ~ 100-3000 ( not strong)
n  *,   * requires unsaturated functional groups (eq. double
bonds) most commonly used, energy good range for UV/Vis
l ~ 200 - 700 nm
n  * :  ~ 10-100
  *:  ~ 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 semiquantitative 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
-  are relatively additive (hyperchromic
shift)
- l constant
CH3CH2CH2CH=CH2
lmax= 184
max = ~10,000
CH2=CHCH2CH2CH=CH2
lmax=185
max = ~20,000
If conjugated
- shifts to higher l’s (red shift)
H2C=CHCH=CH2
lmax=217 max97 = ~21,000
SPECTRAL NOMENCLATURE OF SHIFTS
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WHAT ABOUT INORGANICS?
 Common anions n  * 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
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the ligands splits the d-orbital energies.
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 (εmax >10,000)
• Many organic and inorganic complexes
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