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 λ
max





ORIGIN OF ABSORPTION
3. Organic functional groups can be identified by
their typical λ
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 (σ), pi (π) or non-bonding
(n) orbitals.


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



 Anti-bonding orbitals are σ
*
and π
*




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



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



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


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


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

UV-VIS TRANSITIONS & CHROMOPHORES
 σ  π
*
, σ  σ
*
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 λ
max
Transition
Alkene C=C 175 π  π
*

Conjugated
alkene
C=C–C=C 220 π  π
*

Alkyne C≡C 180 (large ε
max
)
225 (small ε
max
)
π  π
*

Carbonyl C=O 185
280
π  π
*

n  π
*

Carboxyl COOH 205 n  π
*

Amide CONH 215 n  π
*

Azo N=N 340 n  π
*

Nitro NO
2
280 n  π
*

Nitrate NO
3
270 n  π
*

Alcohol OH 180 n  σ
*

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 π  π
*

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
β-carotene has eleven C=C bonds and absorbs at
450 nm

 Diagram β-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)
D–ORBITAL 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
D–ORBITAL 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
BEER–LAMBERT 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 = ∞

BEER–LAMBERT LAW
 Beer’s 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 ·
·
BEER–LAMBERT 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
 ε = molar absorptivity or molar extinction coefficient
 c = concentration
 l = path length







cl c = A
BEER–LAMBERT 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 = ε
 Indicates sensitivity of the method
 Known ε – used to determine concentration of species
from absorbance

BEER–LAMBERT 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

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