THEORY OF

ULTRAVIOLET
SPECTROSCOPY
 SPECTROSCOPY

 It is the branch of science dealing with the study of interaction of

electromagnetic radiation with matter.

 The ordinary light consists of electromagnetic radiation of a varying

wavelength which can be divided into three parts:-

 Ultraviolet light

 Visible light or white light

 Infrared light
 ULTRA VIOLET SPECTROSCOPY

 The Ultra violet region extends from 1000 – 4000 Å or 100

– 400 nm.

 Ultraviolet region is subdivided into :

Far ultraviolet region

Near ultraviolet region

Far ultraviolet region / vacuum ultraviolet region

 Extending from 10 – 200nm.

 In this region the molecules of air absorb radiation and thus

this region is accessible only with special vacuum equipment.

Near ultraviolet region extends from 200 – 400 nm.

 This spectra is an analytical tool has increased in recent year.

 Because of commercial availability of recording ultraviolet

spectrophotometers
ELECTRON PRESENT IN ORGANIC MOLECULES.

There are three type of electron present

 Sigma (σ) electron

 Pi ( л ) electron

 Non – bonding (n) electron

Sigma (σ) electron:

 Associated with the saturated bonds.

 The σ electrons are tightly held because of strong bonds.

 Compounds containing σ bonds do not absorb in near UV

region but absorbed in vacuum region because energy produced by near
UV region is insufficient to excite the electron.

 Example: the electron in the single valence bonds between C-C, C-H,
and O-H.
Pi ( л ) electron

 These electrons are involved in unsaturated compounds

 Electrons are localized in a direction perpendicular to the nuclear axis.
 These bonds are weak bonds, easily excited by UV rays.
>C=C< -C – N
 Example: alkenes, alkynes and aromatic compounds.

Non – bonding (n) electron

 These electrons are less firmly held.

 Found in Nitrogen, oxygen, sulphur and halogen.
 Involved in the bonding between the atoms in molecules.
LAWS GOVERNING ABSORPTION OF RADIATION

The two laws related to the absorption of radiation are:

 Beer’s law ( related to concentration of absorbing species)

 Lambert’s law (related to thickness/path length of absorbing species)

These two laws are applicable under the following condition:

I = I a + It
I = Intensity of incident light
Ia = Intensity of absorbed light
It =Intensity of transmitted light and
No reflection/scattering of light takes place
Beer’s law
“The intensity of a beam of monochromatic light decreases exponentially
with increase in the concentration of absorbing species arithmetically

Accordingly, - dI / dc α I
(The decrease in the intensity of incident light (I) with concentration c is
proportional to the intensity of incident light (I))
-dI / dc = kI
(removing and introducing the constant of proportionality ‘k’)
-dI / I = k dc (rearranging terms)

-In I = kc + b ……Equation (1)

(on integration , b is constant of integration)
When concentration = 0, there is no absorbance. Hence I= Io
Substituting in equation 1, -In Io =
k*0 + b
-In Io = b
Substituting the value of b, in equation 1,

-In I = kc –InIo

In Io – In I = kc

In Io/I = kc (since log A-log B = log A/B)

Io / I = e kc (removing natural logarithm)

I / Io = e –kc (making inverse on both sides)

I = Io e -kc ….Equation (2) (equation of Beer’s law)

Lambert’s law

“The rate of decrease of intensity (monochromatic light)

with the thickness of the medium is directly proportional to
the intensity of incident light”

i.e. –dI / dt α I

This equation can be simplified similar to equation 2 to get

the following equation (by replacing ‘c’ with ‘t’)

I = Io e –kt ….. Equation (3)

[equation of Lambert’s law]
BEER – LAMBERT,S LAW

Equations (2) and (3) can be combined to get

I= Io e –kct
I = Io 10 –kct
(converting natural algorithm to base 10 & K = k * 0.4343)

I / Io = 10 –kct (rearranging terms)

Io / I = 10 kct (inverse on both side Log

Io / I = kct (taking log on both sides) ….. Equation 4

It can be learnt that transmittance (T) = I / Io and Absorbance

(A) = log 1 / T

Hence A = log 1 / I/ Io

A = log Io /I ……. Equation 5

 Using Equation 4 & 5 ,

Since A= log Io /I

and log Io /I = Kct

we can infer that

A= Kct (instead of K, we can use ε)

A= ε ct (Equation of beer – Lambert’s law)

Where:

A – Absorbance or optical density or extinction co- efficient.

ε – Molecular extinction coefficient
c – Concentration of the drug (mol/lit)
t – Path length (normally 10mm or 1cm)
UV Spectroscopy

Introduction
B. The Spectroscopic Process
1. In UV spectroscopy, the sample is irradiated with the
broad spectrum of the UV radiation
2. If a particular electronic transition matches the energy
of a certain band of UV, it will be absorbed
3. The remaining UV light passes through the sample and
is observed
4. From this residual radiation a spectrum is obtained
with “gaps” at these discrete energies – this is called
an absorption spectrum

15
 




16
UV Spectroscopy

Introduction
C. Observed electronic transitions
• The lowest energy transition (and most often obs.
by UV) is typically that of an electron in the
Highest Occupied Molecular Orbital (HOMO) to the
Lowest Unoccupied Molecular Orbital (LUMO)

• For any bond (pair of electrons) in a molecule, the

molecular orbitals are a mixture of the two
contributing atomic orbitals; for every bonding
orbital “created” from this mixing (s, ), there is a
corresponding anti-bonding orbital of symmetrically
higher energy (s*, *)

17
UV Spectroscopy

• The lowest energy occupied orbitals are

typically the s; likewise, the corresponding
anti-bonding s orbital is of the highest
energy

• -orbitals are of somewhat higher energy,

and their complementary anti-bonding
orbital somewhat lower in energy than s*.

• Unshared pairs lie at the energy of the

original atomic orbital, most often this
energy is higher than  or s (since no bond
is formed, there is no benefit in energy) 18
UV Spectroscopy

Introduction
C. Observed electronic transitions
6. Here is a graphical representation

s
Unoccupied levels


Atomic orbital Atomic orbital

Energy n

Occupied levels


s
Molecular orbitals

19
UV Spectroscopy

Introduction
C. Observed electronic transitions
7. From the molecular orbital diagram, there are several
possible electronic transitions that can occur, each of a
different relative energy:

s
s s alkanes

s  carbonyls

  unsaturated cmpds.
Energy
n
n s O, N, S, halogens

n  carbonyls


s
20
UV Spectroscopy

Introduction
C. Observed electronic transitions
7. Although the UV spectrum extends below 100 nm (high energy), oxygen
in the atmosphere is not transparent below 200 nm

8. Special equipment to study vacuum or far UV is required

9. Routine organic UV spectra are typically collected from 200-700 nm

10. This limits the transitions that can be observed:

s s alkanes 150 nm

s  carbonyls 170 nm

  unsaturated cmpds. 180 nm √ - if conjugated!

n s O, N, S, halogens 190 nm

n  carbonyls 300 nm √
21
ELECTRONIC TRANSITION IN UV REGION.

There are four type f transitions:

 σ → σ*

 n →σ*

 л → л*

 n → л*
 σ → σ* transition

• Transition of an electron from a bonding sigma orbital of a

molecule to the higher energy antibonding sigma orbital.

• Energy required for this transition is very high because sigma

bonds are strong bonds.

• This transition occurs in those compounds only in which all the

electrons are involved in sigma bonds & there is no lone pair of
electrons.

• This transition is studied in vacuum ultra violet region below

200nm because it requires very short wavelength.
Types of Electronic Transitions
σ → σ* transition
л → л* transition

 The transition corresponds to the promotion of an electron from a bonding л

orbital to an antibonding л* orbital and available in compounds with unsaturated
centers such as alkenes, carbonyl compounds etc.

 The excitation of л electrons require lesser energy then n → л* transition.

 These types of transition occur at longer wave length.

 Compounds containing double bonds or triple bonds undergo л → л*. Such as

aromatic compounds, alkenes, alkynes, carbonyl compounds such as aldehyde
and ketones.

 Bands attributed to л → л* transition is also called as K – bands.

Types of Electronic Transitions
л → л* transition
n → л* transition

 Bands attributed to n → л* transition is also called as

R – bands .

 In n → л* transition an electron of unshared electron pair on a hetro

atom such as oxygen, nitrogen or sulphur is excited to л* antibonding
electron.

 This transition requires least amount of energy then all other transitions.

 This transition gives rise to an absorption band at longer wavelength.

Types of Electronic Transitions
n → л* transition
n →σ* transition

 n →σ* transition is seen in saturated compounds containing atoms with

unshared electron pair ( such as sulphur , oxygen, nitrogen, halogen or
non –bonding electrons.

 n →σ* transition needs less energy then σ → σ* transition.

 This transition is studied in near ultraviolet region. (150 – 250 nm).

B – Bands or benzenoid bands are characteristic of spectra of

aromatic or hetro aromatic molecules.
Ex – benzene.

E – Bands or ethylenic bands are characteristic of aromatic

structure.
Types of Electronic Transitions
n →σ* transition
Limitations of the Beer-Lambert law
 The linearity of the Beer-Lambert law is limited
by chemical and instrumental factors.
Causes of nonlinearity include:
 deviations in absorptivity coefficients at high
concentrations (>0.01M) 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 light
 DEVIATION FROM BEER’S LAMBERT LAW:

A system is said to obey beer’s Lambert law, when a plot of

concentration vs. absorbance give a straight line.

There are two type of deviation :

 POSITIVE DEVIATION:
When a small change in concentration produces a greater change in
absorbance.

 NEGATIVE DEVIATION:
When a large change in concentration produces a smaller change in
absorbance.
REASONS FOR DEVATION FROM BEER’S LAMBERT
LAW

 Instrumental deviation
 Physicochemical change in solution

Instrumental deviation:

Factors like stray radiation, improper slit width, fluctuation in single

beam and when monochromatic light is not used can influence the
deviation.

Physicochemical change in solution:

Factors like association, dissociation, ionization (change in pH), faulty

development of colour (incompletion of reaction) refractive index at
high concentration, can influence such deviation.
Example:

 Association:
Methylene blue at a concentration 10 -5 M exists as monomer and
has λmax of 660nm. But methylene blue at concentration above 10-4
M exist as a dimer or trimer , but has a λmax of 600.

 Dissociation:
Potassium dichromate at high concentration exist as a orange
solution (λmax of 450nm).but on dilution, dichromate ions are
dissociated into chromate ions which is yellow in colored (λmax of
410nm)
Cr2O7 2- + H2O → 2H+ + 2CrO4
( orange ) (yellow)
 SOME OF THE IMPORTANT TERMS USED IN
SPECTROSCOPY
CHROMOPHORE

A chromophore is the part (or moiety) of a molecule responsible

for its color.

 When a molecule absorbs certain wavelengths of visible light and

transmits or reflects others, the molecule has a color.

 A chromophore is a region in a molecule where the energy

difference between two different molecular orbitals falls within
the range of the visible spectrum. Visible light that hits the
chromophore can thus be absorbed by exciting an electron from its
ground state into an excited state.
TYPES OF CHROMOPHORES.
Independent Chromophores :

 when a single chromophore is sufficient to impart colour to the

compound.
 For example, azo group, -N = N- , nitroso group, -NO, and 0- and
p- quinonid group etc are independent chomophores.

Dependent Chromophores :

 When more than one chromophore is required to produce color in

the chromogen.

 For example, > C=O group, >C=C < group etc.

AUXOCHROME

 This is a group of atoms attached to a chromophore which

modifies the ability of that chromophore to absorb light.

 Example: -OH , - NH2 , Aldehydes Attached group which

enables the attached of the coloured molecule Add a chemical
group to the molecule of the dye so as to enable its attachment to
the tissue structures while without changing its colour or stability
BATHOCHROMIC SHIF / RED SHIFT

 It is a change of spectral band position in the absorption,

reflectance, transmittance, or emission spectrum of a molecule to a
longer wavelength (lower frequency).

 This can occur because of a change in environmental conditions

 A series of structurally related molecules in a substitution series

can also show a bathochromic shift.

 Bathochromic shift is a phenomenon seen in molecular spectra,

not atomic spectra.
HYPSOCHROMIC SHIFT / BLUE SHIFT

 It is a change of spectral band position in the absorption,

reflectance, transmittance, or emission spectrum of a molecule to a
shorter wavelength (higher frequency).

 This can occur because of a change in environmental conditions

 Hypsochromic shift is a phenomenon seen in molecular spectra,

not atomic spectra
HYPERCHROMIC EFFECT

 An increase in absorption intensity

 If structural modification leads to an increase in the molar extinction

coefficient for a particular chromophoric group it is said to have brought
about a hyperchromic effect.

HYPOCHROMIC EFFECT

 An decrease in absorption intensity

 If structural modification leads to a decrease in the molar extinction

coefficient for a particular chromophoric group it is said to have brought
about a hypochromic effect.
 Red shift :- λmax shifted towards longer wavelength
Bathochromic shift. λmax shifted towards longer
wavelength due to
i) Presence of an auxochrome.
ii) By change of solvent.
n -π* transition for carbonyl compounds experience red
shift when polarity of solvent is decreased
 Hyperchromic
e Hypsochromic Bathochromic
Hypochromic

200 nm 800 nm
Blue shifts: -
λmax shifted towards shorter wavelength or hypsochromic shift or effect. λ max
shifted towards shorter wavelength due to
i) removal of conjugation
ii) by changing polarity of solvent.
E.g. The aniline experienced a blue shift by removal of conjugation in acidic
medium.

+
NH2 NH3

Aniline Anilinium cation

λmax= 280nm λmax = 203nm
 Hyperchromic effect:-
 increases in the intensity of absorption
maximum. Ie.Є max is increased. The
introduction of auxochrome usually increases
intensity of absorption

N CH3
N
Pyridine 2 methyl pyridine
λmax= 257nm λmax = 262nm
εmax = 2750 εmax = 3650
 Hypochromic effect:-
decreases in the intensity of absorption
maximum. i.e. Є max is decreased.

Hyperchromic
e Hypsochromic
Hypochr
Bathochromic
omic

200 nm 800 nm
SOLVENTS USED IN UV SPECTROSCOPY
 UV Solvents are manufactured by high efficiency distillation and
non-distillation processes.
 Ideal characteristic of the UV solvents.
 Freedom from extraneous peaks for more reliable sample
identification.
 Enhanced sensitivity due to greater UV transparency at 200-
230 nm.
 Reproducible absorption curve throughout the entire spectrum
 Acetone Acetonitrile Benzene
 Chloroform Cyclohexane Cyclopentane
 Diethyl ether Dioxane Acetonitrile
 Ethanol Hexane Methanol
 2-Methylbutane Methyl formate 1-Octanol
 Pentane 1-Propanol 2-Propanol
 Pyridine Toluene Water