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An introduction to Ultraviolet/Visible
Absorption Spectroscopy
Chapter 13
1
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/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 = -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.
Psolution P
T

Psolvent P 0
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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1.
a.
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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.
b.
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, 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, Beer’s 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 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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3.
a.
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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 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
•
•
•
•
•
•
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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)
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 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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Sources
Tungsten lamps (350-2500 nm)
Why add I2 in the lamps?
W + I2 → WI2
Low limit: 350 nm
1)Low intensity
2)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
•
l - selection (monochromators)
•
Sample holders
•
Cuvettes (b = 1 cm typically)
1. Glass (Vis)
2. Fused silica (UV+Vis)
•
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 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
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Inside of a
single-beam
spectrophot
ometer
connected
to a
computer.
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
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• Better design for continuous recording
of spectra
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
1.Compensate for all but the most short term
fluctuation in radiant output of the source
2.Compensate drift in transducer and amplifier
3.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” < 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 doubledispersing
spectrophotomet
er. 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
C
O
H
=
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=
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
has89the effect of shifting chromophore peaks to longer wavelength as well
As increasing their intensity.
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
absorption spectra for
identification purpose.
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Summary of transitions for organic
molecules
•
•
•
•
•
•
•
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  * 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
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.
•94 Effects of solvent & molecular details complicate comparison
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
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lmax=217 max = ~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 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.
1) Large molar absorptivity (εmax >10,000)
2) Many organic and inorganic complexes
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