Chapter 4

Ultraviolet and visible Absorption

Spectroscopy
 Properties of Electromagnetic Radiation

Electromagnetic Radiation
– energy radiated in the form of a WAVE
caused by an electric field interacting with
a magnetic field
– result of the acceleration of a charged
particle
– does not require a material medium and
can travel through a vacuum
Electromagnetic Radiation
 Electromagnetic Radiation

vi = n li

where vi => velocity

n => frequency
li => wavelength
Electromagnetic Spectrum
 Electromagnetic Spectrum
Type of Frequency Wavelengt
Type of Transition
Radiation Range (Hz) h Range
gamma-
1020-1024 <1 pm nuclear
rays
X-rays 1017-1020 1 nm-1 pm inner electron
400 nm-1
ultraviolet 1015-1017 outer electron
nm
750 nm-
visible 4-7.5x1014 outer electron
400 nm
near- 2.5 µm-750 outer electron
1x1014-4x1014
infrared nm molecular vibrations
25 µm-2.5
infrared 1013-1014 molecular vibrations
µm
microwav 1 mm-25 molecular rotations,
3x1011-1013
es µm electron spin flips*
radio
<3x1011 >1 mm nuclear spin flips*
waves
Interaction of EMR with Matter
Jablonski diagram:
 Selection Rules
• The electron must be promoted without
a change in its orientation. s = 0
• When s ≠ 0 transition is forbidden. It
may occur with very low probability
• Some other from quantum mechanics
Etotal (molecule) = Eelectronic + Evibrational +
Erotational + Enuclear
Absorption of Light

Uv & Vis

IR
Microwave


 *
Molecular and Atomic Absorption

 * Less extent
Collisions between molecules
lead to broadening of
absorption bands
 Types of Transitions
Three types of transitions
1. , , and n electrons
2. d & f electrons
3. charge transfer electrons
Electronic transition in Formaldehyde
Spectroscopy Nomenclature
Effect of Structure on

 * vacuum UV
lmax Cl < lmax Br < lmax I
n * transitions occur at longer wavelengths
 is a function of
1. Cross sectional area of absorbing species ( )
2. Transition probability (P)

 = 9X1019  P ( = 10-15 cm2 ( = about 105 for the

average organic molecule
 Transition Multiplicity

Consider two electrons paired in an orbital, and their

possible transitions to an empty orbital.

S   si
i
M  2 S 1

ground state excited singlet excited triplet

• the ground state has all electrons in the lowest energyorbital

• organic compounds almost always have paired spins, thus their
ground state is almost always a singlet
• singlet-triplet transitions are optically forbidden - light cannot
both promote an electron to a new orbital and change its spin
• in an organic compound most absorption spectra are due to
singlet-singlet electronic transitions
 Electronic Transitions in Ethylene

• Attention will be restricted to electrons H H

involved with carbon-carbon bonding C C
• The two sp2 electrons form the -bond, H H
while the two pz electrons form the -
bond *
• Absorption of a photon will promote one
of the bonding electrons into an anti-
bonding orbital, preserving electron spin
*
• The wavelength of absorbed light will
follow pz pz
Planck's Law, E = hc/l sp 2  sp 2
• The transition energies are:
  * >   *,   * >   *
• The   * transitions are of most
interest since they give us information 
about the conjugated double bond
structure of a molecule
   * Transitions in Butadiene

H H
C C H
• Each carbon atom has one electron in a H C C
pz-orbital H H
• The four pz electrons create two bonding
-orbitals and two anti-bonding *-orbitals 2*
• 2  2* absorption is in the deep UV, it has
an energy similar to that in ethylene
• The longest wavelength absorption is due to 1*
the 1  1* transition
• intermediate wavelength absorption is due to
pz pz pz pz
2  1* and 1  2* transitions
• The long wavelength transition has an
energy that decreases with the number of 1
double bonds
2
 Rotational Broadening

• Boltzmann's constant is 0.694 cm-1 298 K

• kT = ~200 cm-1 at room temperature 1

population (relative)
• the spacing of molecular rotational levels
is a few tenths of reciprocal centimeters
• Thermal energy populates many 0.5
rotational levels giving molecules an
internal source of energy
• rotational energy available within a 0
0 1000 2000
molecule can add to that of a photon, energy (cm-1)
making a range of optical energy that
can satisfy Planck's Law, E = hn + Erot

• the graph shows the energy of thermally populated rotational

levels; the distribution has a width of ~700 cm-1
• an electronic transition will be broadened by this width

500 nm transition will be 17 nm wide (491 - 508 nm)

400 nm transition will be 11 nm wide (394 - 405 nm)
300 nm transition will be 7 nm wide (296 - 303 nm)
 Vibronic Transitions
• A simultaneous change in vibrational and (a)
electronic quantum numbers is called a 35000

vibronic transition
• if the inter-nuclear distances are not affected 30000

when the electron changes orbitals AND the

transition is symmetry allowed, the spectrum 25000

will have a single peak and, no or very weak,

vibronic bands 20000

• If one or more vibrations have different

equilibrium inter-nuclear coordinates, a 15000

vibronic sequence will appear in the spectrum

- this is shown in (a) for a single vibrational
10000

mode (more than one can be affected) 5000

• If the electronic transition is symmetry
forbidden, vibronic bands will appear for 0
those vibrations that deform the molecule
0 2 4 6
internuclear distance (Angstroms)
into a shape which has an allowed transition
(b) (b)
• symmetry allowed:  = 103 - 105 M-1 cm-1
symmetry forbidden:   102 M-1 cm-1
State Diagrams and Absorption Spectra
absorption spectrum state diagram
1  2*
with a E (cm-1)
change in
30000
internuclear
distance (333 nm) 3
2
1
25000
S2* v= 0
(400 nm)
3
symmetry forbidden 2
1  1* 20000 1
(500 nm) S1* v= 0

lmax
15000 excited singlet
(667 nm) states
long wavelength 0-0
absorption band transition
10000
(1 m)

infrared 5000
(2 m) ground state
absorption

v=1
0 cm-1 S0 v = 0
(nm)
 Chromophores
• They are groups with one element of
unsaturation (unsaturated linkages or
groups) and cause coloring to the
molecules when they are attached to a
non-absorbing hydrocarbon chain
 Effect of Multichromophores on Absorption
• More chromophores in the same molecule
cause bathochromic effect
(Red shift: shift to longer wavelength)
and hyperchromic effect (increase in
intensity)
• Hypsochromic effect: Blue shift: shift to
shorter wavelengths
• Hypochromic effect: decrease in intensity
• In the conjugated chromophores * electrons
are delocalized over larger number of atoms
causing a decrease in the energy of  to *
transitionsand an incrase in  due to an
increase in probability for transition
•Aromatic Hydrocabons
They absorb at three bands: 260, 200
and 180 nm

•Policyclic aromatic (Naphthalene):

exhibit regular shift towards longer
wavelength (Red shift)
•Azo Compounds with the linkage –N=N-
show low intensity bands in the near Uv
and Vis due to n to * transitions
•Azobenzenes absorb at about 445 nm the –
N=N- may be conjugated with the ring 
system.
UV absorption spectra of benzene, naphthalene, and anthracene
 Auxochromes
• They are groups that do not confer
color but increase the coloring power
of a chromophore.
• They are functional groups that have
non-bonded valence electrons and
show no absorption at l > 220 nm; they
absorb in the far UV
• -OH and -NH2 groups cause a red shift
 Steric Effect

•Extended conjugation of  orbitals requires

coplanarity of the atoms involved in the -
cloud delocalization for maximum resonance
interaction
•Large bulki groups cause a perturbation of
the coplanarity of the  system .
•Thus lmax is usually shifted towards shorter 
and  also decreases
 Linear Polyenes

CH CH
n
n name lmax (nm) max (M-1 cm-1)
1 ethylene 163 ?
2 butadiene 217 21,000
3 hexatriene 268 35,000
4 octatetraene 304 ?
5 decapentaene 328 120,000

• As the number of double bonds increases, the long wavelength

absorption shifts to higher values (called a red-shift)
• The molar absorptivity increases as the molecular orbital size
increases
• To anticipate the spectrum, use the number of conjugated
double bonds, i.e. CH2=CH-CH2-CH=CH2 has a spectrum closer to
ethylene than butadiene.

14.2 : 2
 Linear Fused Aromatics

structure name lmax (nm) max (M-1 cm-1)

benzene 255 220

naphthalene 315 320

anthracene 357 10,000

tetracene 471 10,000

• As the number of fused rings increases, the long wavelength

absorption shifts to higher values
• The long wavelength transition is forbidden in benzene and
naphthalene, but allowed in anthracene and tetracene
• To anticipate the spectrum use the number of conjugated
double bonds, i.e. diphenylmethane has a spectrum that
resembles toluene
Linear Fused Aromatics
 Non-Linear Fused Aromatics

structure name 0-0 band (nm) 0-0 (M-1 cm-1)

3,4-benzo
370 170
phenanthrene

chrysene 360 800

pyrene 370 120

perylene 437 3,700

• The 0-0 band appears at lower wavelengths than would be

predicted by the number of fused rings (379 for anthracene and
479 for tetracene)
• The first three have band positions similar to anthracene and
molar absorptivities similar to naphthalene
• Perylene has properties between anthracene and tetracene
Non-Linear Fused Aromatics
 Linear Polyphenyls

n name 0-0 band (nm) lmax(nm) max (M-1 cm-1)

1 benzene 264 255 220

2 biphenyl 288 248 1,600
3 p-terphenyl 320 276 3,300

4 p-quaterphenyl 340 294 4,000

• As the number of conjugated rings increases, the 0-0 band shifts

to higher wavelengths
• The increase in wavelength is not as fast as the polyenes or
linear aromatics because of the bond between the rings is wisted
• The spectrum is featureless because thermally induced
oscillation about the twist angle adds width to the vibronic bands
• The molar absorptivity increases because the number of double
bonds is increasing
Linear Polyphenyls
 Alkyl Substituents

name 0-0 band (nm) max (M-1 cm-1)

benzene 264 220

toluene 269 290

ethylbenzene 269 260

propylbenzene 268 230

anthracene 377 9,800

9-methylanthracene 387 10,200

• Alkyl substituents shift the 0-0 band of the parent aromatic a few
nanometers to the red, and increase the molar absorptivity a
small amount
• Multiple substituents will increase the shift by smaller increments
• The vibronic pattern in the spectrum will change because of the
new vibrations
Alkyl Substituents
 Substituents with Lone-Pairs of Electrons

N
H
H

•When atoms with lone-pairs of electrons are attached to

aromatic compounds they can effectively increase the size of the
ring system
• An increase in the size of the ring system shifts the parent
spectrum to the red
• Lone-pairs often break the symmetry of a molecule, converting
a forbidden transition into a moderately allowed transition
• When a transition is made more allowed, there is an increase in
the molar absorptivity
• When aromatic compounds with hydroxyl or amine substituents
are dissolved in hydrogen bonding solvents, the absorption
bands become broad and vibronic structure is decreased or lost

14.3 : 4
 Halogen Substituents

name 0-0 band (nm) max (M-1 cm-1)

benzene 264 220
fluorobenzene 266 1,100
toluene 269 290
p-chlorotoluene 278 570
anthracene 377 9,800
9,10-dichloroanthracene 402 14,300

• Halogen substituents shift the 0-0 band to the red

• The larger the halogen the larger the shift
• Halogens can break symmetry to make a transition more allowed
• Multiple substituents will increase the red shift
Halogen Substituents
 Hydroxide and Amine Substituents

name 0-0 band (nm) max (M-1 cm-1)

benzene 264 220

hydroxybenzene (phenol) 278 2,400

aminobenzene (aniline) ~313 1,450

naphthalene 315 320

1-hydroxynaphthalene 325 5,400

2-hydroxynaphthalene 328 2,700

• Hydroxide and amine substituents shift the 0-0 band to the red
• Both substituents create broad bands when the compound is
dissolved in a hydrogen bonding solvent
• Both substituents can break symmetry to make a transition
more allowed
• Molar absorptivities are in the range of 1,000 - 6,000
 Hydroxyl and Amine Substituents
cyclohexane cyclohexane ethanol

ethanol methanol ethanol

“Ultraviolet absorption spectra for
1,2,4,5-tetrazine (a.) in the vapor phase,
(b.) in hexane solution, and (c.) in
aqueous solution.”
Effect of polar solvents on transitions

Polar solvents stabilize both non-bonding electrons in

The ground state and * elctrons in the excited state
 Absorption

Excited state

Incident Transmitted
beam beam Loss of energy as
Sample
Absorption radiation, heat, etc ...

Absorption
along radiation
beam

Ground state
 Beer’s law (Beer-Lambert Law)
•Empirical relationship between transmitted
intensity and number of absorbers.

•A. Beer, 1852. See H. G. Pfeiffer and H. A.

Liebhafsky, J. Chem. Ed. 1951, 28, 123-
125,“The origins of Beer’s law.”

•The incident radiation is monochromatic.

•The absorbing units (atoms, molecules, ions)
act independently of one another.
•The absorption is limited to a volume of uniform
cross section.
 Beer’s law (The Beer-Lambert Law)

• Exponential attenuation with

– concentration
– sample thickness (optical path length)
• Assumes
– sample is non-turbid (Non-scattering)
• Scattering effects:
– create losses out of the side of the sample
» apparent absorption is greater than actual
absorption
– optical path-length is now no longer simply the
length of the cuvette
– lead to requirement for model of light
propagation (diffusion theory, etc)
 dx
Po P

Absorbing medium

x
0 b
= bc
bc

= bc
Beer’s Law Example
Example
known
Example
Solvents for UV-Visible Regions
 Analysis of Mixtures of Absorbing Substances
Absorption spectrum of a two-component mixture
Solution of Binary Mixture
Schematic representation of the absorption spectra
of solutions containing
(1) c1 moles per liter of substance 1
(2) c2 moles per liter of substance 2
(3) c1 moles per liter of substance 1 and
c2 moles per liter of substance 2
 Solution of Binary Mixture
Wavelength 1
Am,l1 = a1,l1*b*c1 + a2,l1*b*c2
Am,l1 = (a1,l1*c1 + a2,l1*c2)*b

Wavelength 2
Am,l2 = a1,l2*b*c1 + a2,l2*b*c2
Am,l2 = (a1,l2*c1 + a2,l2*c2)*b
 Solution of Binary Mixture

let A1 = Am,l1 A2 = Am,l2

D1 = a1,l1 D2 = a1,l2
1 = a2,l1 2 = a2,l2

then A1 = (D1*c1 + 1*c2)*b

A2 = (D2*c1 + 2*c2)*b
solve for c2
A2/b = (D2*c1 + 2*c2)

A2/b - D2*c1 = 2*c2

2*c2 = A2/b - D2*c1

c2 = (A2/(b* 2) - (D2*c1)/ 2

then
A1 = (D1*c1 + 1((A2/(b* 2)-(D2*c1)/ 2))*b

A1/b = (D1*c1 + 1((A2/(b* 2)(D2*c1)/ 2))

A1/b = (c1(D1 - D2*(1/ 2))+(1/ 2)*(A2/b))

A1/b - (A2/b)*(1/ 2) = c1(D1-D2*(1/ 2))

thus
(A1/b - (A2/b)*(1/ 2))
c1 = -------------------------------
(D1 - D2*(1/ 2))

and

C2 = (A2/(2*b) - (D2*c1)/ 2
Example
Example
 Deviation from Beer’s Law

•It is a deviation from direct proportionality

between A and C.

•It is either +ve (upward curvature) or –Ve

(downwod curvature) deviation

•Sources are: Real, Instrumental , or

Chemical factors
 Real Deviations

• high concentration - particles too

close

• Dependence of absorptivity on
refractive index of solution
 Real Factors

•Derivation did not take into consideration the

changes in Refractive index of the solution
due to concentration changes.
Refractive index increase as
concentration increases

•Consequently the proportionality constant is

not  but n / (n2+2)2 where n is the refractive
index of the medium
 Instrumental Factors

•Alterations in power supply voltage, light source or

detector response. Others include:

1. Polychromatic Radiation

Assume a radiation consisting of two wavelengths:

l and l’ and Beer’s Law applies at each
At l the absorbance will be given as follows

Ac =  b c when  = ’
• Departure increases by increasing the difference
between l Values of the polychromatic radiation
due to the increase in the difference between 
and ’
• The steeper the absorption (A Vs l) curve
the greater the error
 Overlap of Sample with Optical Beam

• As long as the optical beam is narrower than the

sample cell width (cases A and B in the figure)
measured absorption is constant.
C
B
• When the optical beam is larger than the sample A
cell width (case C) light misses the sample and
cannot be absorbed.

• The equation describing transmission when light

misses the cell is given below. In the example, the
true absorption is 0.30, the incorrect absorption is
0.22

I (inside)  I 0(outside) 0.5 0.5  0.1

T Ttrue  Terror   0.6
I 0(inside)  I 0(outside) 1.0 0.9  0.1

•This situation can occur when trying to make absorption

measurements in capillary liquid chromatography.
 Monochromator Stray Light

All gratings and mirrors scatter a small fraction of the

input light. The scattered radiation gets spread
throughout the monochromator, with some reaching the
exit slit. The scatter is composed of all frequencies
entering the monochromator. The scatter is then
transferred by lenses or mirrors though the sample and to
the detector. The expression for transmission has to be
modified to take this into account.

I l  I stray
T
I 0l  I 0stray
Since the stray light occurs at all wavelengths it will be
absorbed to a different extent than l. Usually a large
fraction of stray light is not absorbed at all. This makes it
difficult to measure absorbance values above ~2.

High-performance spectrophotometers are ordinarily

constructed with double monochromators to reduce the
amount of stray light.
 3. Stray Light
The stray light striking detector is a potential source of error.
Apparent A is decreased as a result

•Thus a negative deviation is expected

•Deviations are expected near the limits of the instrument
Components
•Visible radiation is the most serious stray light problem
•For Uv-Vis spectrometers
Sources of Stray Light and Its Effect of on Absorbance

Source: M. R. Sharpe, Anal. Chem. 56, 339A-356A (1984).

Apparent deviation due to stray radiation
 Sample Fluorescence

• When a sample fluoresces, some of the emitted photons

reach the detector. This gives an abnormally high
transmission.
I l  I fluor
T
Il
•The effect of fluorescence can be reduced by moving the
sample away from the detector.

•The effect of fluorescence can be eliminated by using two

monochromators that scan synchronously.

sample cell
wavelength

selector #1
source detector
 Sample Scatter
• Samples that scatter light lose radiation in addition to
that absorbed. This causes an abnormally low
transmission.
I l  I scatter
T
I 0l
• Since most scattered 0.35
light is in the forward
direction (toward the 0.3
detector), its effect can
be reduced by moving 0.25

the detector very close

absorbance

0.2
to the sample.
0.15
• Scattered light is
proportional to 1/l4. 0.1

This makes it easy to 0.05

identify.
0
200 250 300 350 400 450 500 550 600 650 700 750 800
wavelength (nm)
 Sample Cell Reflections

• For a quartz sample cell each air/quartz interface reflects

~3.5% of the light, while each quartz/water interface
reflects ~0.2%. These reflective losses represent light
that never reaches the detector. The measured
transmission is lower than expected.
l
I I reflect
T
l
I0
• The effect of reflection can be reduced by using a
reference cell that contains only solvent. The two cells
must have identical optical properties (called matched
cells) for the reflective losses to cancel.

• This problem is sufficiently acute that expensive

spectrophotometers have a mechanism for "flattening"
and zeroing the baseline when running solvent versus
solvent.
 Source Width
• The spectral width of the source determines the shape of
the measured absorption spectrum. The two curves are
"convoluted" mathematically (source; original spectrum;
measured spectrum)
_spectrum 35 _source  12
_spectrum 35 _source  5
1
1

absorption
absorption

0.5
0.5

0
400 450 500 550 600
0
400 450 500 550 600 wavelength (nm)
wavelength (nm)
_spectrum 35 _source  35 _spectrum 35 _source  70

1 1
absorption

absorption

0.5 0.5

0 0
400 450 500 550 600 400 450 500 550 600
wavelength (nm) wavelength (nm)
 Source Width (2)

• Beer's Law only holds for monochromatic light. With

non-monochromatic light, the slope of the calibration
curve decreases. In addition the curve is non-linear for
high absorption values.
1.2

1
Atrue

0.8 source << peak

absorption

0.6 Atruep/ (p2 + s2)1/2

0.4 Ameasured

0.2
source = peak

0
0 0.00002 0.00004 0.00006 0.00008 0.0001
concentration (M)
 Slit Width
• Spectral slit width : it is the spread of the image
along the frequency, wavenumber or wavelength scale

• It is proportional to the mechanical slit width

• If the absorption band is sharp or if the measurement

is made at the steep slope of the spectral band  may
be different over the spectral band width and
deviation may be noticed

• Typical bandwidth of a spectrometer is of the order of 1 nm

Molecular absorption bands are broader than 1 nm thus the
Effect of spectral bandwidth is negligible when A is measured
at lmax
 Effect of Bandwidth on Spectral Detail

• Absorbance increases (significantly)

as slit width decreases.

• However, decrease in slit width leads to a

(second-order) reduction in power of radiant
energy; so at very narrow slit widths, high
S/N can lead to loss of spectral detail.

Source: Skoog, Holler, and Nieman, Principles of Instrumental Analysis, 5th edition, Saunders College Publishing.
 Deviation Due to Chemical Factors
•Sources are: Dissociation, association, complex formation,
polymerization or solvolysis.
•For example dissociation of benzoic acid and potassium
•dichromate
 Determination of Stoichiometry
•Complexes of low stability can be studied by
spectroscopy.

•Consider the formation of a complex MnLp,

where M is a metal ion and L is a ligand:

nM + pL MnLp
•The molar ratio of the two components of a complex
is important.

•In a quantitative determination, an excess of ligand

should be added to force the equilibrium toward
completion.
 Mole-
Ratio
Method

Conc. Of one
component (M) is
kept constant and
that of the other is
varied to get a
series of [L]/[M]
ratios
Molar-ratio method, showing different
curves.
(a) Component L does not absorb at the
wavelength of maximum absorption for the
complex, for example, Fe(III)- Tiron.

(b) Component L absorbs slightly at the

wavelength of maximum absorption for the
complex, for example, Zn-Pan.

(c) An excess of component L causes a

decrease in absorbance of the complex, for
example, Bi-xylenol orange .
 Continuous Variation (Job’s( Method
• Conc. of both components is varied.
• But the total number of moles of both components is
kept constant

• This causes a change in mole ratios

mL of 2.5 m M P mL of 2.5 mM X X/P mol X/(mol X + mol P)

1 9 9/1 0.9
2 8 4/1 0.8
Job curves of thio-Michler's ketone comlexes of
mercury and of palladium
Studies of Chemical Equilibria
[HA] Can be determined spectrophotometrically
[A ]
If HA and A- are known. These can be determined
by converting both forms into any of them through
addition of acid or base

When [HA] = [A-]; pka = pH

The peak belongs to A- since intensity is reduced by decreasing pH
 We may write the equation:

•This gives the segmoid curve shown above.

•This curve can be obtained without prior knowledge of Ka
or  by measuring the absorbance of HA in various buffer
solutions