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Course on Analytical Methods
Electronic Spectroscopy
Ultraviolet and visible spectroscopy
Scope
Some applications
Some features of measurements
Identification of organic species
Quantification of Inorganic species
Colorimetric analysis
The origin of the analytical signal
Excitation of atom or molecule by ultra violet or visible radiation
190-900 nm
PHOTON IN PHOTON OUT
The essential features
• Count the number of photons (intensity)
• Energy analysis
• Analyze other effects (polarizations)
Where in the spectrum are these transitions?
Electronic Excitation by UV/Vis Spectroscopy :
X-ray:
core electron
excitation
UV:
valance
electronic
excitation
IR:
molecular
vibrations
Radio waves:
Nuclear spin states
(in a magnetic field)
Ultraviolet (UV) Spectroscopy – Use and Analysis
Of all the forms of radiation that go to make up the electromagnetic
spectrum UV is probably the most familiar to the general public
(after the radiation associated with visible light which is, for the
most part, taken for granted).
UV radiation is widely known as something to be aware of in hot
weather in having a satisfactory effect of tanning the skin but which
also has the capacity to damage skin cells to the extent that skin
cancer is a direct consequence of overexposure to UV radiation.
This damage is associated with the high energy of UV radiation
which is directly related to its high frequency and its low wavelength
(see the equations below).
E = energy; c = speed of light;  = wavelength;
 = frequency; h = Planck’s constant
c = 
E = h
E = (hc)/ 
E  1/ 
Ultraviolet (UV) Spectroscopy – Use and Analysis
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When continuous wave radiation is passed through a
prism a diffraction pattern is produced (called a
spectrum) made up of all the wavelengths associated
with the incident radiation.
When continuous wave radiation passes through a
transparent material (solid or liquid) some of the
radiation might be absorbed by that material.
Spectrum with ‘gaps’ in it
Spectrum
Diffraction prism
Transparent material that
absorbs some radiation
Radiation source
If, having passed through the material, the beam is diffracted by passing through a prism it will produce a light
spectrum that has gaps in it (caused by the absorption of radiation by the transparent material through which is passed).
The effect of absorption of radiation on the transparent material is to change is from a low energy state (called the
ground state) to a higher energy state (called the excited state).
The difference between all the spectroscopic techniques is that they use different wavelength radiation that has
different associated energy which can cause different modes of excitation in a molecule.
For instance, with infra red spectroscopy the low energy radiation simply causes bonds to bend and stretch when a
molecule absorbs the radiation. With high energy UV radiation the absorption of energy causes transition of bonding
electrons from a low energy orbital to a higher energy orbital.
The energy of the ‘missing’ parts of the spectrum corresponds exactly to the energy difference between the orbitals
involved in the transition.
Ultraviolet (UV) Spectroscopy – Use and Analysis
n
Occupied

Energy
Levels

Increasing energy
Unoccupied*
Energy Levels
*
The bonding orbitals with which you are familiar are the -bonding orbitals typified
by simple alkanes. These are low energy (that is, stable).
Next (in terms of increasing energy) are the -bonding orbitals present in all
functional groups that contain double and triple bonds (e.g. carbonyl groups and
alkenes).
Higher energy still are the non-bonding orbitals present on atoms that have lone
pair(s) of electrons (oxygen, nitrogen, sulfur and halogen containing compounds).
All of the above 3 kinds of orbitals may be occupied in the ground state.
Two other sort of orbitals, called antibonding orbitals, can only be occupied by an
electron in an excited state (having absorbed UV for instance). These are the * and
* orbitals (the * denotes antibonding). Although you are not too familiar with the
concept of an antibonding orbital just remember the following – whilst electron
density in a bonding orbital is a stabilising influence it is a destabilising influence
(bond weakening) in an antibonding orbital.
Antibonding orbitals are unoccupied in the ground state
UV
A transition of an electron from occupied to an unoccupied energy level can be
caused by UV radiation. Not all transitions are allowed but the definition of which
are and which are not are beyond the scope of this tutorial. For the time being be
aware that commonly seen transitions are  to * which correctly implies that UV is
useful with compounds containing double bonds.
A schematic of the transition of an electron from  to * is shown on the left.
Ultraviolet (UV) Spectroscopy – The Instrumentation
The instrumentation used to run a UV is shown below. It involves two lamps (one for visible light and one for UV
light) and a series of mirrors and prisms as well as an appropriate detector. The spectrometer effectively varies the
wavelength of the light directed through a sample from high wavelength (low energy) to low wavelength (high
energy).
As it does so any chemical dissolved in a sample cell through which the light is passing may undergo electronic
transitions from the ground state to the excited state when the incident radiation energy is exactly the same as the
energy difference between these two states. A recorder is then used to record, on a suitable scale, the absorption of
energy that occurs at each of the wavelengths through which the spectrometer scans.
The recorder assembly
The spectrometer itself – this houses the lamps, mirrors,
prisms and detector. The spectrometer splits the beam of
radiation into two and passes one through a sample and
one through a reference solution (that is always made up
of the solvent in which you have dissolved the sample).
The detector measures the difference between the sample
and reference readings and communicates this to the
recorder.
The samples are dissolved in a solvent which is transparent to UV light and put into sample cells called cuvettes.
The cells themselves also have to be transparent to UV light and are accurately made in all dimensions. They are
normally designed to allow the radiation to pass through the sample over a distance of 1cm.
Ultraviolet (UV) Spectroscopy – The Output
The output from a UV scanning spectrometer is not the most informative looking piece of data!! It looks like a series
of broad humps of varying height. An example is shown below.
A = .c.l
Increasing absorbance *
Beer Lambert Law
*Absorbance has no units
– it is actually the
logarithm of the ratio of
light intensity incident on
the sample divided by the
light intensity leaving the
sample.
Decreasing wavelength in nm
There are two particular strengths of UV (i) it is very sensitive (ii) it is very useful in determining the quantity of
a known compound in a solution of unknown concentration. It is not so useful in determining structure although it
has been used in this way in the past.
The concentration of a sample is related to the absorbance according to the Beer Lambert Law which is described
above.
A = absorbance; c = concentration in moles l-1; l = pathlength in cm ;  = molar absorptivity (also known as
extinction coefficient) which has units of moles-1 L cm -1.
Ultraviolet (UV) Spectroscopy – Analysing the Output
Absorbance
1.0
0.5
Beer Lambert
Law
A = .c.l
Handling samples of known concentration
If you know the structure of your compound X and you wish
to acquire UV data you would do the following.
Prepare a known concentration solution of your sample.
Run a UV spectrum (typically from 500 down to 220 nm).
From the spectrum read off the wavelength values for each of
the maxima of the spectra (see left)
Read off the absorbance values of each of the maxima (see
left).
Then using the known concentration (in moles L-1 ) and the
0.0
450 known pathlength (1 cm) calculate the molar absorptivity ()
350
400
wavelength (nm)
for each of the maxima.
Determining concentration of samples with
Finally quote the data as follows (for instance for the largest
known molar absorptivity ().
peak in the spectrum to the left and assuming a concentration
Having used the calculation in the yellow box to
of 0.0001 moles L-1 ).
work out the molar absorptivity of a compound you
max = 487nm A= 0.75
can now use UV to determine the concentration of
 = 0.75 /(0.001 x 1.0) = 7500 moles-1 L cm -1
compound X in other samples (provided that these
sample only contain pure X).
Simply run the UV of the unknown and take the absorbance reading at the maxima for which you have a known value
of . In the case above this is at the peak with the highest wavelength (see above).
Having found the absorbance value and knowing  and l you can calculate c.
This is the basis of your calculation in Experiment 4 of CH199 and also the principle used in many experiments to
determine the concentration of a known compound in a particular test sample – for instance monitoring of drug
metabolites in the urine of drug takers; monitoring biomolecules produced in the body during particular disease states
UV / visible Spectroscopy
Abs
Abs
 / nm
 / nm
UV / visible Spectroscopy
UV / visible Spectroscopy
• Electronic transitions involve the
promotion of electrons from an occupied
orbital to an unoccupied orbital.
• Energy differences of 125 - 650 kJ/mole.
UV / visible Spectroscopy
• Beer-Lambert Law
A = log(IO/I) = cl
UV / visible Spectroscopy
A = log(IO/I) = cl
– A = Absorbance (optical density)
– IO = Intensity of light on the sample cell
– I = Intensity of light leaving the sample cell
– c = molar concentration of solute
– l = length of sample cell (cm)
  = molar absorptivity (molar extinction
coefficient)
UV / visible Spectroscopy
• The Beer-Lambert Law is rigorously
obeyed when a single species is present
at relatively low concentrations.
UV / visible Spectroscopy
• The Beer-Lambert Law is not obeyed:
– High concentrations
– Solute and solvent form complexes
– Thermal equilibria exist between the ground
state and the excited state
– Fluorescent compounds are present in solution
UV / visible Spectroscopy
• The size of the absorbing system and the
probability that the transition will take place
control the absorptivity ().
• Values above 104 are termed high intensity
absorptions.
• Values below 1000 indicate low intensity
absorptions which are forbidden transitions.
UV / visible Spectroscopy
• Organic Spectroscopy
• Transitions between
MOLECULAR ORBITALS
UV / visible Spectroscopy
• Highest occupied molecular orbital
HOMO
• Lowest unoccupied molecular orbital
LUMO
UV / visible Spectroscopy
UV / visible Spectroscopy
• Not all transitions are observed
• There are restrictions called
Selection Rules
• This results in
Forbidden Transitions
UV / visible Spectroscopy
• The characteristic energy of a transition
and the wavelength of radiation absorbed
are properties of a group of atoms rather
than of electrons themselves.
• The group of atoms producing such an
absorption is called a
CHROMOPHORE
UV / visible Spectroscopy
UV / visible Spectroscopy
UV / visible Spectroscopy
• It is often difficult to extract a great deal
of information from a UV spectrum by
itself.
• Generally you can only pick out
conjugated systems.
UV / visible Spectroscopy
UV / visible Spectroscopy
ALWAYS
use in conjunction with
nmr and infrared spectra.
UV / visible Spectroscopy
• As structural changes occur in a
chromophore it is difficult to predict
exact energy and intensity changes.
• Use empirical rules.
Woodward-Fieser Rules for dienes
Woodward’s Rules for enones
UV / visible Spectroscopy
1. Bathochromic shift (red shift)
– lower energy, longer wavelength
– CONJUGATION.
2. Hypsochromic shift (blue shift)
– higher energy, shorter wavelength.
3. Hyperchromic effect
– increase in intensity
4. Hypochromic effect
– decrease in intensity
Spectroscopic Techniques and
Chemistry they Probe
UV-vis
UV-vis region
bonding electrons
Atomic Absorption
UV-vis region
atomic transitions (val. e-)
FT-IR
IR/Microwave
vibrations, rotations
Raman
IR/UV
vibrations
FT-NMR
Radio waves
nuclear spin states
X-Ray Spectroscopy
X-rays
inner electrons, elemental
X-ray Crystallography
X-rays
3-D structure
Spectroscopic Techniques and Common Uses
UV-vis
UV-vis region
Quantitative
analysis/Beer’s Law
Atomic Absorption
UV-vis region
Quantitative analysis
Beer’s Law
FT-IR
IR/Microwave
Functional Group Analysis
Functional Group
Analysis/quant
Raman
IR/UV
FT-NMR
Radio waves
X-Ray Spectroscopy
X-rays
Elemental Analysis
X-ray Crystallography
X-rays
3-D structure Anaylysis
Structure determination
Different Spectroscopies
• UV-vis – electronic states of valence e/d-orbital
transitions for solvated transition metals
• Fluorescence – emission of UV/vis by certain
molecules
• FT-IR – vibrational transitions of molecules
• FT-NMR – nuclear spin transitions
• X-Ray Spectroscopy – electronic transitions of
core electrons
Quantitative Spectroscopy
• Beer’s Law
Al1 = el1bc
e is molar absorptivity (unique for a given
compound at l1)
b is path length
c concentration
Beer’s Law
slit
cuvette
source
detector
•
•
•
•
A = -logT = log(P0/P) = ebc
T = Psolution/Psolvent = P/P0
Works for monochromatic light
Compound x has a unique e at different
wavelengths
Characteristics of
Beer’s Law Plots
• One wavelength
• Good plots have a range of absorbances
from 0.010 to 1.000
• Absorbances over 1.000 are not that valid
and should be avoided
• 2 orders of magnitude
Standard Practice
•
•
•
•
•
Prepare standards of known concentration
Measure absorbance at max
Plot A vs. concentration
Obtain slope
Use slope (and intercept) to determine the
concentration of the analyte in the
unknown
A
Typical Beer’s Law Plot
1.2
1
0.8
0.6
0.4
0.2
0
y = 0.02x
0.0
20.0
40.0
concentration (uM)
60.0
UV-Vis Spectroscopy
• UV- organic molecules
– Outer electron bonding transitions
– conjugation
• Visible – metal/ligands in solution
– d-orbital transitions
• Instrumentation
Characteristics of UV-Vis spectra of
Organic Molecules
• Absorb mostly in UV unless highly
conjugated
• Spectra are broad, usually to broad for
qualitative identification purposes
• Excellent for quantitative Beer’s Law-type
analyses
• The most common detector for an HPLC
Molecules have quantized energy levels:
ex. electronic energy levels.
energy
energy
hv
}
= hv
Q: Where do these quantized energy levels come from?
A: The electronic configurations associated with bonding.
Each electronic energy level
(configuration) has
associated with it the many
vibrational energy levels we
examined with IR.
Broad spectra
• Overlapping vibrational and rotational
peaks
• Solvent effects
Molecular Orbital Theory
• Fig 18-10
*
*
2p
n
2p


*
2s
2s

Ethane
C C
*
*
hv



H
C C
H
H
HH
H
*
max = 135 nm (a high energy transition)
Absorptions having max < 200 nm are difficult to observe because
everything (including quartz glass and air) absorbs in this spectral
region.
C C
*
*
*
*
= hv
=hc/
hv





*
Example: ethylene absorbs at longer wavelengths:
max = 165 nm = 10,000
C O
*
*
*
*
n
hv
n




n
*
The n to pi* transition is at even lower wavelengths but is not
as strong as pi to pi* transitions. It is said to be “forbidden.”
Example:
Acetone:
n* max = 188 nm ; = 1860
n* max = 279 nm ; = 15
C C
*
135 nm
C C
*
165 nm
n*
183 nm
weak
*
n*
n*
150 nm
188 nm
279 nm
weak
H
C O
C O
180 nm
C O
A
279 nm

Conjugated systems:
C
C
LUMO
HOMO
Preferred transition is between Highest Occupied Molecular Orbital
(HOMO) and Lowest Unoccupied Molecular Orbital (LUMO).
Note: Additional conjugation (double bonds) lowers the HOMOLUMO energy gap:
Example:
1,3 butadiene:
max = 217 nm ; = 21,000
1,3,5-hexatriene
max = 258 nm ; = 35,000
Similar structures have similar UV spectra:
O
O
O
max = 238, 305 nm
max = 240, 311 nm
max = 173, 192 nm
Lycopene:
max = 114 + 5(8) + 11*(48.0-1.7*11) = 476 nm
max(Actual) = 474.
Metal ion transitions
E
Degenerate
D-orbitals
of naked Co
D-orbitals
of hydrated Co2+
Octahedral Configuration
Octahedral Geometry
H2O
H2O
H2O
Co2+
H2O
H2O
H2O
Instrumentation
• Fixed wavelength instruments
• Scanning instruments
• Diode Array Instruments
Fixed Wavelength Instrument
• LED serve as source
• Pseudo-monochromatic light source
• No monochrometer necessary/ wavelength selection
occurs by turning on the appropriate LED
• 4 LEDs to choose from
sample
beam of light
LEDs
photodyode
Scanning Instrument
Scanning Instrument
monochromator
Tungsten
Filament (vis)
slit
slit
Deuterium lamp
Filament (UV)
Photomultiplier
tube
cuvette
sources
• Tungten lamp (350-2500 nm)
• Deuterium (200-400 nm)
• Xenon Arc lamps (200-1000 nm)
Monochromator
• Braggs law, nl = d(sin i + sin r)
• Angular dispersion, dr/d = n / d(cos r)
• Resolution, R = /=nN, resolution is
extended by concave mirrors to refocus
the divergent beam at the exit slit
Sample holder
• Visible; can be plastic or glass
• UV; you must use quartz
Single beam vs. double beam
• Source flicker
Diode array Instrument
mirror
Diode array detector
328 individual detectors
Tungsten
Filament (vis)
slit
slit
cuvette
Deuterium lamp
Filament (UV)
monochromator
Advantages/disadvantages
• Scanning instrument
– High spectral resolution (63000), /
– Long data acquisition time (several
minutes)
– Low throughput
• Diode array
– Fast acquisition time (a couple of
seconds), compatible with on-line
separations
– High throughput (no slits)
– Low resolution (2 nm)
HPLC-UV
HPLC
Pump
Mobile
phase
Sample
loop
6-port
valve
HPLC
column
UV
detector
syringe
Solvent
waste
UV / visible Spectroscopy
• The radiation which is absorbed has an energy
which exactly matches the energy difference
between the ground state and the excited state.
• These absorptions correspond to electronic
transitions.
Why should we learn this stuff?
After all, nobody solves structures with UV any longer!
Many organic molecules have chromophores that absorb UV
UV absorbance is about 1000 x easier to detect per mole than NMR
Still used in following reactions where the chromophore changes. Useful
because timescale is so fast, and sensitivity so high. Kinetics, esp. in
biochemistry, enzymology.
Most quantitative Analytical chemistry in organic chemistry is conducted
using HPLC with UV detectors
One wavelength may not be the best for all compound in a mixture.
Affects quantitative interpretation of HPLC peak heights
Uses for UV another aspect
Knowing UV can help you know when to be skeptical of quant results. Need
to calibrate response factors
Assessing purity of a major peak in HPLC is improved by “diode array” data,
taking UV spectra at time points across a peak. Any differences could
suggest a unresolved component. “Peak Homogeneity” is key for purity
analysis.
Sensitivity makes HPLC sensitive
e.g. validation of cleaning procedure for a production vessel
But you would need to know what compounds could and could not be detected
by UV detector! (Structure!!!)
One of the best ways for identifying the presence of acidic or basic groups, due
to big shifts in  for a chromophore containing a phenol, carboxylic acid,
etc.
“hypsochromic” shift
“bathochromic” shift

The UV Absorption process
–   * and   * transitions: high-energy, accessible in
vacuum UV (max <150 nm). Not usually observed in molecular
UV-Vis.
– n  * and   * transitions: non-bonding electrons (lone
pairs), wavelength (max) in the 150-250 nm region.
– n  * and   * transitions: most common transitions
observed in organic molecular UV-Vis, observed in compounds
with lone pairs and multiple bonds with max = 200-600 nm.
– Any of these require that incoming photons match in energy the
gap corrresponding to a transition from ground to excited state.
– Energies correspond to a 1-photon of 300 nm light are ca. 95
kcal/mol
What are the nature of these absorptions?
Example:   * transitions
responsible for ethylene
UV absorption at ~170 nm calculated
with ZINDO semi-empirical excitedstates methods (Gaussian 03W):
π*
π*
π*
n
π
π
-*; max=218
=11,000
π*
π*
π*
n
π
π
π*
π*
π*
n
π
π
Example
for a
simple
enone
n-*; max=320
=100
h 170nm photon
HOMO u bonding molecular orbital
LUMO g antibonding molecular orbital
How Do UV spectrometers work?
Rotates,
Matched quartz cuvettes
to achieve scan
Sample in solution at ca. 10-5 M.
System protects PM tube
from stray light
D2 lamp-UV
Tungsten lamp-Vis
Double Beam makes
it a difference technique
Experimental details
What compounds show UV spectra?
Generally think of any unsaturated compounds as good candidates.
Conjugated double bonds are strong absorbers
Just heteroatoms are not enough but C=O are reliable
Most compounds have “end absorbance” at lower frequency.
Unfortunately solvent cutoffs preclude observation.
You will find molar absorbtivities  in L•cm/mol, tabulated.
Transition metal complexes, inorganics
Solvent must be UV grade (great sensitivity to impurities with double
bonds)
The NIST databases have UV spectra for many compounds
An Electronic Spectrum
1.0
Visible
Absorbance
UV
0.0
200
400
Make solution of
concentration low
enough that A≤ 1
(Ensures Linear Beer’s
law behavior)
Even though a dual
beam goes through a
solvent blank, choose
solvents that are UV
transparent.
Can extract the  value
if conc. (M) and b (cm)
are known
UV bands are much
broader than the
photonic transition
event. This is because
vibration levels
800are
superimposed on UV
Wavelength, , generally in nanometers (nm)
Solvents for UV (showing high
energy cutoffs)
•
•
•
•
•
•
•
•
Water
CH3CN
C6H12
Ether
EtOH
Hexane
MeOH
Dioxane
205
210
210
210
210
210
210
220
•
•
•
•
•
•
THF
CH2Cl2
CHCl3
CCl4
benzene
Acetone
220
235
245
265
280
300
Organic compounds (many of
them) have UV spectra
• One thing is clear
• Uvs can be very nonspecific
• Its hard to interpret except at
a cursory level, and to say
that the spectrum is
consistent with the structure
• Each band can be a
superposition of many
transitions
• Generally we don’t assign
the particular transitions.
From Skoog and West et al. Ch 14
The Quantitative Picture
• Transmittance:
T = P/P0
• Absorbance:
A = -log10 T = log10 P0/P
P0
(power in)
P
(power out)
B(path through sample)
•The Beer-Lambert Law (a.k.a. Beer’s Law):
A = ebc
Where the absorbance A has no units, since A = log10 P0 / P
e is the molar absorbtivity with units of L mol-1 cm-1
b is the path length of the sample in cm
c is the concentration of the compound in solution, expressed in mol L-1 (or M, molarity)
Beer-Lambert Law
Linear absorbance with increased concentration-directly proportional
Makes UV useful for quantitative analysis and in
HPLC detectors
Above a certain concentration the linearity
curves down, loses direct proportionality--Due
to molecular associations at higher
concentrations. Must demonstrate linearity in
validating response in an analytical procedure
Polyenes, and Unsaturated Carbonyl groups;
an Empirical triumph
R.B. Woodward, L.F. Fieser and others
Predict max for π* in extended conjugation
systems to within ca. 2-3 nm.
Homoannular, base 253 nm
Acyclic, base 217 nm
heteroannular, base 214 nm
Attached group increment, nm
Extend conjugation
+30
Addn exocyclic DB
+5
Alkyl
+5
O-Acyl
0
S-alkyl
+30
O-alkyl
+6
NR2
+60
Cl, Br
+5
Interpretation of UV-Visible Spectra
•Transition metal complexes;
d, f electrons.
•Lanthanide complexes –
sharp lines caused by
“screening” of the f electrons
by other orbitals
• One advantage of this is the
use of holmium oxide filters
(sharp lines) for wavelength
calibration of UV
spectrometers
See Shriver et al. Inorganic Chemistry, 2nd Ed. Ch. 14
Quantitative analysis
Great for non-aqueous
titrations
Example here gives detn
of endpoint for
bromcresol green
Binding studies
Form I to form II
Isosbestic points
Single clear point, can exclude
intermediate state, exclude light scattering
and Beer’s law applies
More Complex Electronic Processes
• Fluorescence: absorption of
radiation to an excited state,
followed by emission of radiation
to a lower state of the same
multiplicity
• Phosphorescence: absorption of
radiation to an excited state,
followed by emission of radiation
to a lower state of different
multiplicity
• Singlet state: spins are paired, no
net angular momentum (and no
net magnetic field)
• Triplet state: spins are unpaired,
net angular momentum (and net
magnetic field)