MOLECULAR

SPECTROSCOPY
Matter is a molecule
UV-VIS
Fluorescence
FTIR
Raman
MS
NMR

UV-Vis Molecular Absorption

Spectroscopy
Based on EMR in wavelength region of
190 800 nm
Widely used in the quantitative
determination of
Inorganic
Organic
Biological

species

Beers 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 bc
P0

Measurement of T and A
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

Resulting beam
attenuation is
substantial

Scattering by large
molecules
Absorption of the
container walls

To compensate those effects:

P is compared with Po, one that traverses an identical cell
containing only the solvent (blank)

Psolution
P
T

Psolv ent Po
Po

Psolvent

Blank

Po

Psolution

Solution with
Absorbing species

Psolution
P
A log
log
Po
Psolv ent

Po and P refer to power of a

beam that has passed
through cells containing the
blank (solvent) and analyte
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 Beers 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 Beers Law

Deviations from Ameasured c frequently
occur when b is a constant

Limitations to Beers Law

Types of deviations
Real deviation
Fundamental and represent real limitations of
the law

Instrumental deviation
How the A measurements are made

Chemical deviation
A result of chemical changes that occur when
concentration changes

Real limitation to Beers Law

Beers 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
Beers Law is strictly a limiting law for dilute solutions.
At high concentrations ( > 0.01 M) the average distance between the
analyte molecules is becomes 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

Relative error vs. %T

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 438 nm
Negative deviations
at 570 nm

Instrumental deviations
Polychromatic radiation
Stray radiation
Mismatched cells

Effects of polychromatic radiation

Beers 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 Beers Law
At Band A
The A is nearly constant
constant value
At Band B
A changes with
shows substantial changes

Band A; a linear relationship

Band B; deviation from Beers Law

Effects of polychromatic radiation

on Beers Law

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 Beers Law

caused by stray radiation
Observed A < theoretical A
Leads to neg. absorbance
errors
A levels off 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
Po Ps
or lower than stray-light
A is the observed A
level

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 Beers 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:

hc
E = h =

where,h = Planck constant (6.626 x 10-34J s)

c = speed of light in vacuum (2.998 x 108 m s-1)
UV: E = 190 to 400 nm (6.53 to 3.1 eV)
VIS: E = 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:
, , 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 , , and n

electrons
are due to absorption of ultraviolet (UV)
and visible (VIS) radiation by electrons
that are in , , or n-type molecular
orbitals.

Energy levels of molecular orbitals

In general, the following energy order applies to electronic

states: < < n < <

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

Saturated organic compound

Absorption characteristics of some

common chromophores

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

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
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

C C
H

180 nm

135 nm

165 nm

n *

183 nm

weak

*
n *
n*

150 nm
188 nm
279 nm

weak

C O

C O

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

E = h
= hc

hv

C C

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

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 -orbitals
n * transitions will be lower in energy
than * 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 -orbitals

White in color

Yellow in color

Structures having similar UV spectra

O
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)

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

Natural organic pigments

Absorption of inorganic species

Inorganic anions show UV absorption
excitation of n electrons

Ions

max, nm

Nitrate
Carbonate
Nitrite
Azido
Trithiocarbonate

313
217
360 and 280
230
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
involves transition of filled and unfilled d
orbital
Es ,therefore maxs 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
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

Chargetransfer absorption
s are large, ( > 10,000)
particularly important for quantitative
purposes
leads to high sensitivity

Inorganic and organic complexes show this

type of absorption
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
The excited e- is in a molecular orbital
shared by two or more atoms (unlike
organic chromophores)

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

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

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 Beers 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

Analysis of mixtures of
absorbing substances
Atotal of a solution at a given wavelength is
equal to A of individual components in
the solution

Application of Beers 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 + N2 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 T
Slit width on A

Types and Sources of Uncertainties

in Transmittance measurements

Instrument components
1.
2.
3.
4.
5.

One or more radiation sources

Wavelength selectors
Sample containers
Radiation transducers
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
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 Beers law

Narrow slits - Narrow bandwidth

Minimize deviation from Beers
Low 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 max 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.