UV / Vis Spectroscopy

Mr. Z. Clarke
ORIGIN OF ABSORPTION
Molecular absorption of UV and Visible
wavelengths can form electronic absorption bands



Electronic absorption bands consist of closely
spaced discrete lines`


ORIGIN OF ABSORPTION
1. Discrete lines arise from the transition of an
electron from the ground state to the one of many
vibrational or rotational energy levels in an excited
electronic energy levels

ORIGIN OF ABSORPTION
2. Wavelength where maximum absorption (tallest
peak in the absorption band) occurs is
max





ORIGIN OF ABSORPTION
3. Organic functional groups can be identified by
their typical
max
values




ORIGIN OF ABSORPTION
4. Concentration of a solution can be determined by
the amount of light absorbed by a solution



Amount of light absorbed depends on the colour
intensity of the solution which is related to the
concentration of the solution
ion concentrat Absorbance
ORIGIN OF ABSORPTION
The darker the colour of a solution is the higher the
concentration of the solution with respect to analyte
of interest

ABSORPTION IN ORGANIC COMPOUNDS
NOT ALL organic molecules absorb in the UV-Vis
region of the spectrum



Wavelength of absorption depends on how tightly
electrons are bonded in the compound
ABSORPTION IN ORGANIC COMPOUNDS
Electrons in unsaturated bonds (multiple bonds)
and non-bonded pairs (lone pairs) absorb in the
UV-Vis region



Electrons are loosely held and easily excited in
these compounds

MOLECULAR ORBITALS
When molecules are formed, atomic orbitals form
molecular orbitals


Electrons occupy sigma (), pi () or non-bonding
(n) orbitals.


When or bonds are formed a higher
unfavourable energy level (anti-bonding orbital) is
formed associated with the bonding orbital
MOLECULAR ORBITALS
Bonding orbitals are and



Anti-bonding orbitals are
*
and
*




Non-bonding orbitals are n
MOLECULAR ORBITALS
Molecules in the ground state have electrons in
bonding and non-bonding orbitals (, , n)



Absorption of energy can result in promotion of an
electron from a filled , and n orbital to an anti-
bonding
*
or
*
orbital



ELECTRONIC TRANSITIONS IN MOLECULAR
ORBITALS
UV-VIS TRANSITIONS
Only
*
, n
*
, n
*
transitions normally
produce absorption in the UV-Vis region


Only molecules with and or n electrons give UV-
Vis spectra


Molecules with only sigma () bonds show no
absorption in the UV-Vis region

UV-VIS TRANSITIONS & CHROMOPHORES

*
,
*
transitions require greater energy
that fall outside of the UV range



Organic molecules possess structural features
(chemical groups) which absorb in the UV-Vis
region called chromophores





CHROMOPHORES
Chromophore Typical
max
Transition
Alkene C=C 175
*

Conjugated
alkene
C=CC=C 220
*

Alkyne CC 180 (large
max
)
225 (small
max
)

*

Carbonyl C=O 185
280

*

n
*

Carboxyl COOH 205 n
*

Amide CONH 215 n
*

Azo N=N 340 n
*

Nitro NO
2
280 n
*

Nitrate NO
3
270 n
*

Alcohol OH 180 n
*

CONJUGATED SYSTEMS
Increasing the extent of delocalization in a system
containing double bonds, increases the intensity of
absorption, and shifts absorption to a longer
wavelength



Conjugated compounds have alternating double
and single bonds (less energy for
*

transitions)


CONJUGATED SYSTEMS
Absorption can shift to the visible region of the
spectrum (coloured compounds)

C=C bond in ethene absorbs at 175 nm while
-carotene has eleven C=C bonds and absorbs at
450 nm

Diagram -carotene & Absorption shift with
conjugation

CONJUGATED SYSTEMS
ABSORPTION IN TRANSITION METAL
COMPLEXES
Transition metal have a wide range of colours

Transition metals form complexes when surrounded
by coordinating groups called ligands

[Co(H
2
O)
6
]
2+
, [Fe(CN)
6
]
4
, [Ni(NH
3
)
6
]
2+

Interaction between the ligands and the d orbitals of
the metal ion cause the d orbitals to have different
energies (splitting)
DORBITAL SPLITTING
Energy required for the d-d transitions fall in the
visible region

Colour of the complex is complement of the
absorbed colours from visible light
DORBITAL SPLITTING
Ions with d
0
and d
10
electronic configuration have
no possible d-d transitions and are colourless

Ions with d
5
electronic configuration dont have
allowable d-d transitions
UV-VIS APPLICATION COLOURLESS
COMPOUNDS
Colour intensity is used to determine the
concentration of a sample in UV-Vis spectroscopy

Colourless compounds do not absorb visible light
and cannot be determined directly

Colourless samples are reacted with colouring
agents to form coloured complexes which absorb in
the visible region
IRON ANALYSIS
A colouring agent used in the analysis of iron is
1,10 phenanthroline





Iron sample is dissolved in acid and reduced to
Fe
2+
and then added to 1,10 phenanthroline to form
red intense red coloured complex which absorbs at
512 nm
N N
1,10 phenanthroline
IRON ANALYSIS
N N
Fe
2+
N
N
N
N
N
N
3 + Fe
2+
1,10 phenanthroline
red complex
PHOSPHATE ANALYSIS
Phosphates are usually colourless and their
concentration can be determined using UV-Vis
spectroscopy


Ammonium molybdate [(NH
4
)
6
Mo
7
O
6
.4H
2
O] can
react with colourless phosphate to form a blue
complex
UV-VIS SPECTROPHOTOMETER
BEERLAMBERT LAW
The intensity of light exiting a solution (I) is less
than the intensity of the light entering the solution
(I
0
) because solute molecules absorb some of the
energy
BEER LAMBERT LAW
ABSORBANCE & TRANSMITTANCE
Energy absorbed can be expressed in terms of
either transmittance (T) or absorbance (A).

Transmittance is the ratio of the exiting and
incoming radiation


Transmittance expressed as percentage
100 x
I
I
T %
I
I
T
0
0
=
=
BEER LAMBERT LAW
ABSORBANCE & TRANSMITTANCE
Transmittance is not proportional to the
concentration of the absorbing species

Fortunately, Absorbance is proportional to the
concentration of the absorbing species and this is
used

Absorbance is related to transmittance as follows
(

= =

I
I
log logT - A
ion concentrat A
0
BEER LAMBERT LAW
ABSORBANCE & TRANSMITTANCE
When
%T =100, A = 0
%T = 0, A =

BEERLAMBERT LAW
Beers law stated that Absorbance is proportional to
concentration


Lambert law states that Absorbance is proportional
to path length


length) (path A
tion) (concentra c A
l

BEERLAMBERT LAW
Beer-Lambert Law states that the degree of
absorption at a given wavelength of an absorbing
species in a non-absorbing solvent depends on the
concentration of the compound and the path length
of the radiation


A = absorbance
= molar absorptivity or molar extinction coefficient
c = concentration
l = path length







cl c = A
BEERLAMBERT LAW







I
o
= intensity of the incident light
I = intensity of the emerging light
(

= =
I
I
log A
0
cl c
ABSORBANCE AND CONCENTRATION
Plot of Absorbance vs Concentration






Cell length = 1 cm
compound absorbing of ion concentrat absorbed Light
MOLAR ABSORPTIVITY
Slope =
Indicates sensitivity of the method
Known used to determine concentration of species
from absorbance

BEERLAMBERT LAW
ASSUMPTIONS & LIMITATIONS
Assumptions
Absorbing species behave independently of solvent &
neighbouring molecules

Limitations
Generally obeyed for dilute solutions (Conc <0.01 M)

Concetrations >0.01 M leads to interactions between
neighbouring molecules

Monochromatic light (Single wavelength) or narrow
bands of wavelengths


SPIKING
Determine concentration of analyte in complex
mixture
E.g. Biological fluids or soil samples
Contain interferences

Method
Addition of set of standard solution (analyte) to the
sample
Monitor instrument response
Each spike sample used to plot calibration curve

SPIKING
Assumptions
Change in instrument response is due only to the
change in analyte concentration

APPLICATIONS OF UV-VIS SPECTROSCOPY
Qualitative Analysis

Detect chromophoric groups

Spectrum compared with molecules containing various
chromophoric groups

Supplemented with IR Spectroscopy, Mass
Spectroscopy
APPLICATIONS OF UV-VIS SPECTROSCOPY
Quantitative Analysis

Determine the concentration of organic and inorganic
molecules e.g. Glucose, Urea, Iron, Cyanide

Highly sensitive e.g. ppm

Selective eg. Unique wavelength of analyte species

Good accuracy

Easy to perform modern instruments