CHEM F313: Instrumental Methods of Analysis

Lecture 03
UV-Vis Spectrometry
8/18/2023
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Outline
Signal to Noise Ratio
Error
Precision and Accuracy
Electromagnetic waves

Today’s Class
 Width of Spectral Lines
 Fundamental Law
 Types of absorption band
 MO involved in electronic structure
 Solvent Effect
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Representation of spectrum

Y-axis :
Intensity of
Absorption/
Transmission/
Emission

X- axis : λ (wavelength) or related entity

such as wavenumber, frequency etc.
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Width of Spectral Lines
Irrespective of the improvement of the instrumentation (thin slit) the
line width of the spectra are often blurred. Natural line width is
governed by,
• Collision Broadening: Due to continual motion in liquid & gas phase,
molecules/atoms collide frequently. Vibrational & rotational spectra are
usually affected. More effective in liquids than gases.
• Doppler Broadening: Due to collisions. Random motion creates both higher
and lower frequencies. More effective in gases than liquids
• Heisenberg’s Uncertainly Principle: The energy levels are infinitely sharp.
If a system exists in an energy state for a limited time Δt seconds, then
the energy of that state will be uncertain (fuzzy) to an extent ΔE.
ΔE x Δ t = ħ/2 ~ 10-34 J.s , which means that lowest energy state is
most certain.
E.g. lifetime of excited electronic state is ~ 10-8 s, so, ΔE ~ 10-26 J
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 Intensity of Spectral Lines
Three important factors controlling the intensity of lines
• Transition Probability: Governed by the selection rules, easy to understand
pictorially
• Population of States: Higher the population of the lower energy state,
higher is the intensity of the line.
• Path Length of sample: The relation can be expressed as
I/I0 = e-kcl This is certainly non-linear expression.

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UV-Vis: Instrumentation

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 Fundamental Laws
If a monochromatic radiation of radiant power P0 travels an
infinitesimal distance dx, of an absorber then, at a constant
concentration,

• The number of absorbent is proportional to the distance but

independent of P
• k’ depends on the wavelength

or, Lambert’s Law

Also, the relation between radiant power and concentration can be

found out at a constant distance travelled and a fixed wavelength,
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Fundamental Laws

 Beer’s Law

• The number of absorbent is proportional to the concentration, C

Combining, and converting to base 10,
or,

“ε” is called absorptivity [Lg-1cm-1] if C is in g.L-1 and l is in cm

Or, molar absorption coefficient [Lmol-1cm-1] if C is in molL-1 and l is in
cm
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 Fundamental Laws
Limitation of Beer Lambert Law:

• When Different forms of absorbing molecules are in equilibrium as

in keto-enol tautomers.
• When fluorescent compounds are present.
• When solute and solvent form complexes through some sort of
association.
We will solve Numerical in the next class!!!

• Electronic spectroscopy majorly deals with the UV-Vis region :

[A] Visible region 750-400 nm

[B] near UV region 400-200 nm
[C] Far UV region <200 nm
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In chemical terms
• Scope of use

Identification of Conjugated systems, aromatic nature, functional

groups.
Understanding of electronic states
Understanding reaction kinetics and mechanisms
Chromophores: Originally applied to moieties imparting color to the
molecule. E.g. Azo group [Ar-N=N-Ar] is responsible for the color in azo
dyes, -nitro group [-NO2] imparts color to nitrobenzene etc.

Later the term refers to any group that absorbs UV-Vis radiation

Definition: It is an isolated covalently bonded group that shows a

characteristic absorption in the ultraviolet or the visible region.
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List of Chromophores
Chromophore Wavelength,
nm, λmax
C=C 175
C≡C 175, 195, 223
C=O 160, 185, 280
R-NO2 200, 274
C≡N 165
C=C-C=C 217
C=C-C=O 220, 315
C=C-C≡C 220, 230
C6H6 184, 204, 255
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In chemical terms
Auxochromes: Originally applied to moieties enhancing color
capability of the chromophore in the molecule without being a
chromophore. e.g. –OR, -NH2, -NR2 groups etc. Later refers to any
group that contributes to extend conjugation by sharing of non-
bonding electrons.
Definition: Any group that does not itself act as Chromophore but
whose presence brings about a shift in the absorption band towards
the red end of the spectrum.

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MOs involved in electronic transitions

s*

p*

s*

n p*
n
n

s s*

p p*
p

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Orbital participations
In general, the electron in the majority of the compounds can be
categorized as,
1. σ-electrons: Present in σ-bonds, strongly bound, requires high energy
to excite
2. π-electrons: Present in π-bonds, weakly bound, easier to excite
3. n-electrons: Non-bonding, most easy to transition
Far-UV Near-UV Visible

p p*

s s* n p* n p*

n s* Heteroatoms/
conjugation involved

100 200 300 400 500 600 700 800 nm

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Orbital participations
Intensity of absorption is directly proportional to the transition
probability

Different transitions may occur depending on

1. the selection rule
2. the energy of the incident radiation

Selection Rule:
1. Transition which involve the change in the spin quantum number of
an electron during the transition do not occur. Thus singlet to triplet
transitions are forbidden.

2. The transition between orbitals of different symmetry do not occur.

For example, n π* transition is symmetry forbidden.
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Types of Absorption Bands
Four different types of bands are recognized in UV-Vis spectra of organic
molecules
K-Bands (K originates from German word “Konjugierte”)
• Owing to the π-π* transitions
• High molar absorptivity, εmax >104
Compound Transition λmax εmax
Butadiene 1,3 π-π* 217 21000
Pentadiene 1, 3 π-π* 223 22600

R-Bands (R originates from German word “Radikalartig”)

• Owing to n-π* transitions
• Low molar absorptivity, εmax < 102
Compound Transition λmax εmax
Acetone n-π* 270 15
8/18/2023 Acetaldehyde n-π* 293 12 16
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Types of Absorption Bands
B-Bands (B originated from “Benzenoid”)
• π-π* transitions; characteristic of aromatic and heteroaromatic molecules
• Substituents on ring gives B-bands at longer λ
• Multiple peaks or fine vibrational sub-bands
e.g. Benzene has λ max at 230-270 nm, while Styrene has λmax = 244 nm (εmax =
12 x103) & B-band at λmax = 282 nm (εmax = 450)
Compound Transition λmax εmax
Benzene π-π* 255 215
Phenol π-π* 270 1450

E-Bands (E originates from “Ethylenic” bonds in benzenoid system)

• Owing to the oscillations in electron of ring systems/ aromatic
e.g. Benzene has two E-bands at 180 nm (E1) and 200 nm (E2)
Compound E1-band E2-band
Benzene 184 50000 204 7900
8/18/2023 Napthalene 221 133000 286 9300 17
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Solvent Effects
• Dilute solutions are required
Solvents LOWER
Wavelength, 1. Careful handling and sample
limit, nm, lmax preparation
2. Effect of solvent does not
Water 205
nullify completely even after
Ethanol 210 using reference beam –
Hexane, Cyclohexane, 210 strongly absorbing solvents
Methanol, Diethyl will leave small cross-section
ether, Acetonitrile available for sample
THF 220 3. Fineness of spectrum is
affected by solvent
DCM 235
4. Variation of solvent polarity
Chloroform 245 leads to differences in UV-Vis
CCl4 265 absorption
C 6 H6 280 18
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Case Study:α,β -unsaturated ketones
in different solvents
• Stabilization of orbitals in the medium is crucial in understanding the shift of
the spectral band
Solvents Wavelength,
shift, nm In water,
1. n-π* moves to lower
Water +8
wavelength, ΔE
Methanol 0 non-bonding orbitals are better
Chloroform -1 stabilized in H-bonding solvents

Dioxan -5 2. π-π* moves to higher

Diethyl ether -7 wavelength, ΔE
π* orbitals better stabilized than
Hexane -11
π in polar medium
Cyclohexane -11

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Case Study:α,β -unsaturated ketones
in different solvents
Shift towards higher wavelength = red shift or bathochromic shift
Shift towards lower wavelength = blue shift or hypsochromic shift

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Conclusions
 Width of Spectral Lines
 Fundamental Law
 Types of absorption band
 MO involved in electronic structure
 Solvent Effect

 Next Class
 Spectra-structure Relationship
 Use of electronic spectra
 Solvent Effect

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Numerical problem

Q1. An ,β-unsaturated ketone of relative molecular mass 110, has

an absorption band with λmax at 215 nm and ε of 10000. A
solution of this showed absorbance A = 2 L mol-1 cm-1 in a 1 cm
cell. Calculate the concentration of the solution in gL-1

Q2. An unknown compound (0.01g) was dissolved in ethanol in a

250 mL flask, its absorption spectrum was recorded in a 1 cm cell.
The Absorbance at the maximum of one absorption band was
found as 2.2. If the relative molecular mass of the molecule is
186, calculate ε at this λmax .

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