CHAPTER 5

UV-VIS MOLECULAR
ABSORPTION SPECTROMETRY
 UV-Vis Molecular Absorption
Spectroscopy
• Based on EMR in wavelength region of
190 – 800 nm
• Widely used in the quantitative
determination of
– Inorganic
– Organic species
– Biological
 Beer’s Law (revisited)

Based on measurement of T or A of
solutions contained in a transparent
cells having a pathlength of b cm

P
A   logT   log  εbc
P0
Measurement of Transmittance
and Absorbance
Table 13-1: Terms and Symbols for
Absorption Measurements
 Measurement of T and A
• T and A as defined in Table 13-1 cannot
be measured directly
– The analyte must be held in a transparent
container or cell
 Reflection and scattering losses
• Reflection occurs at
the
– Two air-wall interfaces
– Two wall-solution
interfaces
• Attenuation of beam
may occur as a result
of:
Resulting beam  Scattering by large molecules
attenuation is  Absorption of the container
Substantial (fairly large) walls
To compensate those effects:
P is compared with Po, one that pass acrosses an identical cell containing only the
solvent (blank)

Psolution P
T 
Psolvent Po
Po Psolvent P Psolution
A   log   log
Blank
Po Psolvent
Po and P refer to power of a
Po Psolution beam that has passed
through cells containing the
Solution with blank (solvent) and analyte
Absorbing species
respectively
 Attenuation of the initial radiant
power

Radiation of initial radiant power Po is attenuated to

transmitted power P by solution containing c mole per
L of absorbing solution with a pathlength of b cm
 Quantitative analysis using Beer’s Law

Analysis carried out at one wavelength – usually at the

maximum absorbance (max) in spectrum
• The peak maximum corresponds to the maximum value of
the molar absorptivity (max)and hence the sensitivity of the
analysis
• The Beer-Lambert Law is strictly valid only where the molar
absorptivity () changes little with wavelength
• The linearity of the Beer-Lambert law is limited by chemical
and instrumental factors
 Limitations to Beer’s Law
Types of deviations
• Real deviation
– Fundamental and represent real limitations of
the law
Deviations from Ameasured  c frequently occur when b is a constant

• Instrumental deviation
– How the A measurements are made
• Chemical deviation
– A result of chemical changes that occur when
concentration changes
 Real limitation to Beer’s Law
• Beer’s law describes absorption behavior of
relatively low analyte concentration in a
solution
– A limiting law
At high concentrations (> 0.01 M)
• Solute-solvent interaction
• Solute-solute interaction
• H bonding
– Affect analyte environment and its
absorptivity
Intermolecular Interactions
Beer’s Law is strictly a limiting law for dilute solutions.
At high concentrations ( ≈ > 0.01 M) the average distance between the
analyte molecules is become small enough that the charge distribution
around one affects that around another.

Sometimes high concentrations of inert electrolytes can themselves alter

the absorptivity of a species present even at low concentrations (not
necessarily a problem for linearity, but calibrations are important).

Some large organic molecules can show deviations even at 10-6 M

concentrations.

Need to be aware of concentration linearity

and confirm its validity.
Solutions at high concentrations

• Average distances between the molecules

or ions responsible for absorption are
decreased
• Each particle affects the charge
distribution of its neighbors
– Solute-solute interaction alter the ability of the
analyte species to absorb at a given 
 Solutions with low absorber
concentration but high
concentrations of other species
(electrolytes)

Ions are close to the absorber

• Changes the molar absorptivity of
absorber by electrostatic interactions
• The effect is lessened by dilution
 Real deviation
• The absorbance-concentration relationship will
flatten or level off at high concentrations.
• As the concentration of the solution increases,
less radiation reaches the detector.
• At sufficiently high concentrations, the amount of
radiation reaching the detector generates
insignificant amount of current compared to
noise.
• Increasing concentration past this point cannot
generate any higher absorbance.
 Measuring absorbances
Errors may be made during the measurement
of absorbances
• At low concentrations
– A small change in c can result in large change
in %T
• At high concentrations
– Changes in %T are very small
• To minimize measurement errors
– Best to stay in the range of 80 – 20%T
 Measuring absorbances

small ∆conc – large ∆%T

small ∆%T – large ∆conc

 Measuring absorbances

Relative error vs. %T

Relative error

80% 20%

%T
 Chemical Deviations
When an analyte
• Dissociates or
• Associates or
• Reacts with a solvent
producing a product with a different
absorption spectrum than the analyte
Chemical deviations for unbuffered
solutions of an indicator

• Positive deviations
at 430 nm
• Negative deviations
at 570 nm
 Instrumental deviations

• Polychromatic radiation
• Stray radiation
• Mismatched cells
 Effects of polychromatic radiation
• Beer’s law applies to monochromatic radiation

In practice, use of
• a continuous source
• a monochromator
produces a very narrow band
with an effective bandwidth
Effects of polychromatic radiation
on Beer’s Law
At Band A
• The A is nearly constant
• constant  value
At Band B
• A changes with 
•  shows substantial (fairly large)
changes

Band A; a linear relationship

Band B; deviation from Beer’s Law
Effects of polychromatic radiation
on Beer’s Law
As Δ
between ɛ’
and ɛ”
increases,
the
deviation
from
linearity
increases
 Presence of stray radiation
• Radiation exiting from monochromator is
usually contaminated with scattered or stray
radiation
– Radiation from the instrument that is outside
the nominal wavelength band chosen for the
determination
– Result of scattering and reflection of the surfaces
of components of monochromator
– Different from principal radiation, Po
– May not have been passed through the sample
 Apparent deviation from Beer’s Law caused by
stray radiation
• Observed A < theoretical A
• Leads to neg. absorbance
errors
• A become even with
concentration at high stray
light levels
• stray-light limits max A that
can be obtained
- when A is high, radiant
power transmitted through
P  Ps the sample is comparable
A'   log or lower than stray-light
Po  Ps level
A’ is the observed A in the presence of stray radiation
Ps is the power of nonabsorbed stray radiation
 Mismatched cells
Cells that are NOT
• of equal pathlength
• equivalent in optical characteristics
– an intercept k will occur in the calibration
curve
A = bc + k
 Mismatched cells
To reduce the errors
• Ensure of matched cells
• Use linear regression procedure to
calculate the slope and intercept of the
calibration curve
– Best strategy
– An intercept can also occur if the blank
solution does not completely compensate for
interferences
 Deviations from Beer’s Law
• deviations in  values at high concentrations (> 0.01
M) due to electrostatic interactions between molecules
in close proximity
• scattering of light due to particulates in the sample
• fluoresecence or phosphorescence of the sample
• changes in refractive index at high analyte
concentration
• shifts in chemical equilibria as a function of
concentration
• non-monochromatic radiation, deviations can be
minimized by using a relatively flat part of the
absorption spectrum such as the maximum of an
absorption band
• stray radiation and • mismatched cells
 Principles
• Many molecules and solid compounds absorb UV or VIS
radiation
The absorbed energy region:

ΔE = h = hc
λ constant (6.626 x 10
where,h = Planck J s)
-34

c = speed of light in vacuum (2.998 x 108 m s-1)

UV: ΔE =  190 to  400 nm (6.53 to 3.1 eV)
VIS: ΔE =  400 to  800 nm (3.1 to 1.55 eV)
 Electronic transitions
The absorption of UV or visible radiation
corresponds to the excitation of outer
electrons.
Three types of electronic transitions
involving:
• π, σ, and n electrons
• charge-transfer electrons
• d and f electrons
When an atom or molecule absorbs energy,
• electrons are promoted from their ground
state to an excited state.
In a molecule, the atoms can rotate and
vibrate with respect to each other
• These vibrations and rotations also have
discrete energy levels,
• which can be considered as being packed
on top of each electronic level.
 Electronic transitions

Transitions are governed by Selection Rules

• symmetry considerations
• some transitions are “allowed”
• others may be “forbidden”.
 Absorption spectrum
The electronic spectra of absorbing species
• are complex
Superimposition of vibrational transitions on
the electronic transitions
– Lead to intricate combination of overlapping
lines
– The result is a broad band
– Appears to be continuous
 Molecular absorption

Complex nature of the spectra makes detail

theoretical analysis difficult or impossible
For a particular Amax, the magnitude of 
• depends on the capture cross section of the
species and the probability of the energy
absorbing transition to occur

  = 8.7 x 1019 PA
• where P is the transition probability and A is the
cross section target area in cm2 per molecule
 Applications of UV-vis molecular
absorption spectrometry
 values
• Ranges from zero to 105 L mol-1 cm-1 are
observed in UV-vis molecular absorption
spectrometry
For quantum mechanically allowed
transition,
• P values range 0.1 to 1
– leads to strong absorption bands
 max = 104 to 105 L mol-1 cm-1
 max < 103
– Low intensity
– Result from forbidden transition
 Absorbing species
• A two-step process
M + h  M*
• M* has a brief half life
• Several relaxation process leads to
deexcitation of M*
• Most common involves conversion of
excitation energy to heat
M*  M + heat
Absorption by organic compounds
All organic compounds
• are capable of absorbing EMR
• contain valence electrons that can be
excited to higher energy levels
 Transitions involving σ, π, and n
electrons
• are due to absorption of ultraviolet (UV)
and visible (VIS) radiation by electrons
that are in π, σ, or n-type molecular
orbitals.
 Energy levels of molecular orbitals

In general, the following energy order applies to electronic

states: σ < π < n < π∗ < σ∗
 Electronic transitions
• From the the highest occupied molecular
orbital (HOMO) in the ground state to the
lowest unoccupied molecular orbital
(LUMO)
 Order of transitions based on
energy difference
n  *
  *
n  * increasing energy
  *
 *
  *
 Organic compounds
Most spectroscopic measurements involve
 > 185 nm
Based on transitions of
• n  * and   *
– require the presence of unsaturated functional
group to provide  orbitals
• The energies for the processes are in the
UV/vis regions
– 200 to 700 nm
 Chromophore
• Molecules containing unsaturated
functional groups and capable of
absorbing UV/vis radiation
 Absorption characteristics of some
Table 14-1
common chromophores
 Saturated organic compound

Ethane
H3C-CH3

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.
 max and max
max and max values in the Table 14.1 and
14.2
• serve only a rough guide
• a characteristic of each molecule
Both are influenced by
– solvent effects
– other structural details of the molecule
– Ex: conjugation between 2 or more
chromophores can cause shifts in absorption
maxima
 max
Vibrational effects broaden absorption bands
• difficult to determine max precisely
 max values
n  *
• Low (10 to 100 L mol-1 cm-1)

  *
• Large (1000 to 15,000 L mol-1 cm-1)
 Organic compounds containing
heteroatoms with n electrons
Saturated organic compounds containing
O, N, S, or halogens (heteroatoms)
• can be excited by UV radiation (170 to
250 nm)
• n  *
 n  *
• The excitation energies are high
• Absorption occurs in vacuum UV region
( < 185 nm)
  values are low to intermediate
• Between 100 and 3000 L mol-1 cm-1
• Most studies of organic compounds
involved  > 185 nm
• Experimental difficulties in vacuum UV region
Absorption by organic compounds
containing heteroatom with nonbonding
electrons
Some compounds (example alcohols and
ethers)
• are common solvents
• absorption in the 180 to 200 nm region
– Use to determine halogen and sulfur containing
compounds
– Prevents absorption measurements of analytes
dissolved in these compounds at wavelengths shorter
than 180 to 200 nm region
 279 nm

C C 180 nm
  * 135 nm

C C  * 165 nm

H n  * 183 nm weak
C O

  * 150 nm
C O n  * 188 nm
n* 279 nm weak
 Auxochrome
The attachment of substituent groups (in
place of H) on a basic chromophore
structure changes
• Position
and
• Intensity
of an absorption band of the chromophore
 Auxochrome
The substituent groups
• May not absorb UV radiation
• Their presence modifies the absorption of
principle chromophore
– Increase the intensity of absorption
– Increase the absorption 
Examples: methyl, hydroxyl, alkoxy, halogen
and amino groups
 Effects on absorption by
auxochromes
• Bathochromic shift (red shift)
– A shift to a lower energy or longer wavelength
• Hypsochromic shift (blue shift)
– A shift to a higher energy or shorter wavelength
• Hyperchromic effect
– An increase in intensity
• Hypochromic effect
– A decrease in intensity
   *
 
 
C C

hv E = h
= hc
  λ

 
 
Example: Ethylene absorbs at longer wavelengths
max = 165 nm
 = 10,000
ABSORPTION
SPECTRA FOR
TYPICAL ORGANIC
COMPOUNDS
 max
Conjugation between two or more
chromophores tends to cause shifts in
max to longer wavelengths
 * is lowered
   * is lower
 E is lower
• corresponding to longer 
 Conjugated systems
C C

LUMO

HOMO

Preferred transition: HOMO to

LUMO
Additional conjugation (double bonds) lowers the HOMO-LUMO
energy gap, examples
1,3 butadiene max = 217 nm  = 21,000
1,3,5-hexatriene max = 258 nm  = 35,000
Absorption characteristics of aromatic
compounds
 Electronic transitions
in molecules with π-orbitals
• n → π* transitions will be lower in energy
than π → π* transitions in similar
molecules
• conjugated molecules with non-bonding
electrons will be more highly colored than
similar molecules without them
 Electronic transitions
in molecules with π-orbitals

White in color Yellow in color

 Conjugated double bonds
Lycopene
Lycopene:

max = 114 + 5(8) + 11*(48.0-1.7*11) = 476 nm

max(actual) = 474 nm
n =number of conjugated double bonds
 Structures having similar UV spectra

O
O

max = 238, 305 nm  max = 240, 311 nm  max = 173, 192 nm

For more than 4 conjugated double bonds:

max = 114 + 5 (no. of alkyl groups) + n (48.0 -1.7 n)
Natural organic pigments
Absorption of inorganic species
Inorganic anions show UV absorption
• excitation of n electrons
Ions max, nm
Nitrate 313
Carbonate 217
Nitrite 360 and 280
Azido 230
Trithiocarbonate 500
 Transition series
Ions or complexes from first and second
transition series
• absorb broad bands of visible region in at
least one of their oxidation states
– are colored
 Absorption of d electrons
• Absorption involves transition of filled and
unfilled d orbital
The energy changes, ( max’s) depend on
• position of element in the periodic table
• its oxidation state
• the nature of ligands bonded to the metal
ions
 Absorption of d electrons
d-d transitions
• often occur in the visible region
• are therefore associated with a variety of
colors.
Examples:[Cr(NH3)6]3+, [CrCl(NH3)5]2+
ABSORPTION SPECTRA OF
AQUEOUS SOLUTIONS OF
TRANSITION METAL IONS
Transition metal ions spectra
 Absorption of f electrons
Lanthanides and actinides have narrow,
well defined, characteristic absorption
Peaks because;
• 4f and 5f electrons are shielded from
external influences by electrons occupying
larger principal quantum numbers
ABSORPTION SPECTRA OF
AQUEOUS SOLUTIONS OF
RARE EARTH IONS
 Charge–transfer absorption

’s are large, ( > 10,000)

• particularly important for quantitative
purposes
– leads to high sensitivity
Inorganic and organic complexes show this
type of absorption thus known as:
• Charge transfer complexes
 Charge-transfer complex
• Electron donor group bonded to an
electron acceptor
When the product absorbs radiation
• The e- from the donor is transferred to an
orbital that is largely associated with the
acceptor
• Contrary to the Organic chromophores:
-The excited e- is in a molecular orbital
shared by two or more atoms
 Charge transfer complexes
One species is an electron donor and the
other is an electron acceptor
• The resulting complex forms a ‘resonance’
like structure that can absorb
– Absorbs in the visible region
• Many analytical methods are based on
forming this type of complex
Charge transfer complexes
-possess different colours due to charge transfer mechanisms

1,10-phenanthroline
ferroin
• red
iron(III)/thiocyanate
complex

• 1,10-phenanthroline
complex of iron (II)

• Blue iodide complex

 Charge transfer complexes

Most involve
• metal ions
– the e- acceptor
– Except 1,10-phenanthroline complexes (pg 371)

• Organic compounds form complexes that

absorb in the visible region
 Biochemical Species
• Many species of biochemical importance exhibit
strong absorption in the UV/visible region

Examples
– NADH absorbs at 340 nm
– proteins are strongly absorbing in the UV
region near 280 nm
– molecules containing heme groups often
show strong absorption in the 400 nm region
 Characteristics of UV-Vis spectra
of Organic Molecules
• Absorb mostly in UV unless highly
conjugated
• Spectra are broad, usually too broad for
qualitative identification purposes
– Overlapping vibrational and rotational peaks
– Solvent effects
• Excellent for quantitative Beer’s Law-type
analyses
• The most common detector for an HPLC
Solvents for the UV and Vis regions
Characteristics of UV/Vis Methods

• Wide applicability to organic and inorganic

systems
• Sensitivities to 10-4 to 10-7 M
• Moderate to high selectivity
• Good accuracy
– about 1 - 3% relative uncertainty
• Ease and convenient data acquisition
 Procedural details
Relationship between A and analyte
concentration must be
• Reproducible
• Linear
• Selection of wavelength
• Cleaning and handling of cells
• Variables that influence A must be known
– Nature of solvent
– pH of solution
– Temperature
– Electrolyte concentration
– Presence of interfering substances
Effect of solvent on the absorption
spectrum of Acetaldehyde
 Quantitative Analysis by Absorption
Measurements
Analysis of mixtures of absorbing
substances

• Total absorbance of a solution at a given

wavelength is equal to sum of the
absorbances of individual components in
the solution
Application of Beer’s Law to Mixtures
Multiple species do not
interact with each other
Amixture = A
Atotal = A1 + A2 +……. + An
= 1bc + 2bc + ….+ nbc

At ’ and ’’, from known  and b

A’ = M1’ bcM + N1’ bcN
A’’ = M2’’ bcM + N2’’ bcN
 Derivative spectra
Plot the first or higher order derivative of A
(A/) with respect to wavelength as a function
of wavelength
1.Reveal spectral detail that is lost in ordinary
spectra (A vs. )
2.Concentration of an analyte is more easily or
more accurately measured
• In the presence of interference
• For simultaneous determination of two or more
components in a mixture
 Other applications
• Spectroscopic titrations
• Kinetic studies
• Studies of complex ions
 Instrumentation
Capable of
• Spectral scanning at selectable resolution
• Measurement in the UV and visible
regions
• Compensate for source intensity
fluctuations
– Double beam
• Other features
 Effects of instrumental noise
• Instrumental noise on Transmittance
• Slit width on Absorbance (Skoog pg 346)
Types and Sources of Uncertainties
in Transmittance measurements
 Instrument components
1. One or more radiation sources
2. Wavelength selectors
3. Sample containers
4. Radiation transducers
5. Signal processors and readout devices
 Sources of Radiation
• For molecular absorption measurements, a
continuum source is required whose radiant
power does not change sharply over a
considerable range of wavelength
• Stable light source of good intensity and as
large wavelength range as possible

The reality
In practice most sources do not produce a
continuous spectrum of constant intensity
 Sources of Radiation
• UV/Vis spectrophotometers utilize two light
sources
– a deuterium arc lamp for the UV range (190 to
380 nm)
– tungsten-halogen lamp for the visible spectrum
(380 to about 800 nm).
• Some spectrophotometers use xenon flash
lamps
– offer intensity over both the UV and visible
regions.
Sources of Radiation
Blackbody radiation curves
Tungsten filament lamp

• Most common source of

visible and near IR
radiation.

• Useful for the wavelength

region between 350 and
2500 nm.
 Tungsten halogen lamp
• contains a small amount of iodine within the quartz
envelope that houses the filament
• allows the filament to be operated at a
temperature of about 3500 K
• leads to higher intensities
• extends the range of the lamp into UV region
• lifetime is more than double than ordinary tungsten
lamp
• in the presence of iodine, the sublimed tungsten
reacts to give gaseous WI2 molecules, which then
diffuse back to the hot filament where they
decompose and redeposit as W atoms
 Tungsten halogen lamp
Tungsten filament

Tungsten halogen

• extended wavelength
range
• high intensity
• long life
 UV region
• D2 and H2 lamps
• Produce outputs in the range of 160 – 800
nm
• A continuum spectra in the UV region
• At longer wavelengths, > 400 nm
– No more continua: Consist of emission lines
and bands superimposed on a weak
continuum
D2 lamp

A continuum spectrum in the UV

region is produced by electrical
excitation of deuterium at low
pressure.
 Sample containers
• To hold the sample and solvent
• Must be constructed of material that is
transparent in the UV/vis region
Materials for cells, window, lenses and
prisms
 Sample containers
• To minimize reflection losses
– Cells are placed perpendicular to the direction
of the beam
• Most common path length is 1 cm
 Sample cells
• Glass/Disposable plastic
– Visible absorbance measurements but it absorbs UV
• Quartz:
– UV absorbance measurement
Keep the clear surfaces of
the cells clean
Fingerprints scatter and
absorb radiation
• Quality of absorbance data depends
critically on the way the cells are used and
maintained
• Fingerprints, grease and other deposits on
the walls change the transmission
characteristics of a cell
 Handling of cells
• Cells must be cleaned thoroughly before and
after use
• Surface of windows must not be touched during
handling
• Matched cells should never be dried by heat
(oven or flame)
– Cause physical damage and/or a change in path
length
• Cells should be regularly calibrated against each
other with a standard absorbing solution
 Wavelength selector
• Czerny-Turner Grating monochromator
 Monochromator bandwidth
Monochromator
• To provide a beam of radiant energy of a
given  and spectral bandwidth
• Adjustment of the energy throughput
– Radiation emerging from exit slit can be
varied by adjusting the slit width

Wide slits - Large spectral bandwidth

May deviate from Beer’s law

Narrow slits - Narrow bandwidth

Minimize deviation from Beer’s
Law radiation throughput and decreasing S/N ratio
Effects of slit width and max

As slit width is
increased
• A increases
• Peaks are broader
 max determination
is less precise
 Effect of slit width (spectral
bandwidth) on peak heights

• Sample: PrCl3 solution

• Slit width decreases (from 1.0 mm to 0.1 mm)
– Spectral band width decreases
 Effect of slit width on peak height

• Peak heights and separation are distorted at

wider bandwidths
• Spectra for quantitative applications should be
measured with optimum slit widths
 Photon Transducers
• Radiation  electric current
 Types of spectrometers

• Single beam
• Double beam
• Photo Diode Array
Single beam instruments

• simpler
• lower cost
• greater S/N.
Double beam in space instrument

Two beams are formed by a V-shape mirror

– A beam splitter
Double beam in time instrument
Double beam UV-Vis spectrometer

Figure 13-21
 Double-beam spectrophotometer
• A cuvette or cell with the sample solution and a
second cell with pure solvent are placed in the
cell holders.
• Two equal beams of light are passed, one
through the solution of the sample, one through
the pure solvent.
• The intensities of the transmitted light are then
compared over the selected wavelength range.
 Double-beam spectrophotometer
• Free from drift, can use lower quality
electronic components.
• Suitable for continuous recording of
absorption spectra
• More complex components (and noise
from beam switching components)
PE Lamda 40 UV-vis double beam
scanning spectrometer
 Double beam instruments
Advantages
• Compensate for all but the most short-term
fluctuations in the radiant output of the source
• Compensate for drift in the transducer and
amplifier
• Compensate for wide variations of source
intensity with wavelength
 UV-vis with PDA detector
PDA
 Advantages of PDA detector
• transmitted radiation is simultaneously recorded at
multiple wavelengths
– quick
• provides sample absorption spectrum
• provides λmax after reading
• more precision, more sensitivity and more reproducible
results
• can analyze mixtures
• instruments are relatively small and robust

suitable as UV/vis absorption detector for HPLC

Diode array vs conventional
Single-beam spectrophotometer
Involves three successive steps:
1) 0% T with a shutter in place
2) measurement of P0 or adjustment to
100% T
3) measurement of P.
Reliability depends on constancy of
instrument during the time required for each
of these three steps.