UV-VIS Spectrophotometry

The Electromagnetic Spectrum

Radio waves

Microwave

Infra-red

Visible

Ultraviolet

X-rays

Gamma rays
Long Wavelength Short
Low Energy High
Low Frequency High
 EFFECT OF ENERGY ON A MOLECULE

ELECTROMAGNETIC SPECTRUM
ENERGY
1.2 x105 1.2 x107 12000 310 150 0.12 0.0012
( kJ/mol)

Electronic excitation
e-

FREQUENCY
(Hz) 1020 1018 1016 1014 1012 108

visible
Cosmic γ x Ultra Radio
Infrared Microwave
rays rays rays violet waves
WAVELENGTH
(m) 10-12 10-11 10-9 10-6 10-3 10-1
 The UV Absorption process

• → * and  → * transitions: high-energy, accessible in vacuum UV (max

<150 nm). Not usually observed in molecular UV-Vis.

•n → * and  → * transitions: non-bonding electrons (lone pairs), wavelength

(max) in the 150-250 nm region.
•
•n → * and  → * transitions: most common transitions observed in organic
molecular UV-Vis, observed in compounds with lone pairs and multiple bonds
with max = 200-600 nm.

•Any of these require that incoming photons match in energy the gap
corresponding to a transition from ground to excited state.
 What are the π* π* π* Example
π* π* π* for a
simple
nature of these π* π* π* enone
n n n
absorptions? π π π
π
π π
-*; max=218 n-*; max=320
=11,000 =100
Example:  → * transitions responsible for ethylene UV absorption at ~170 nm
calculated with ZINDO semi-empirical excited-states methods (Gaussian 03W):

h 170nm photon

LUMO g antibonding molecular orbital

HOMO u bonding molecular orbital
 UV / VISIBLE SPECTROSCOPY - THEORY
INCIDENT LIGHT TRANSMITTED LIGHT
254nm 254nm
SAMPLE
Intensity (I o ) Intensity (I t )

• If a particular wavelength of UV or Visible radiation can be isolated from the

source and passed through a sample which can ABSORB some of the radiation
then the TRANSMITTED light intensity (It ) will less than the INCIDENT light
intensity (Io).

• The amount of light transmitted with respect to the incident light is called
TRANSMITTANCE (T) ie.,
T= t
I

Io

• Sample can absorb all or none of the incident light and therefore transmittance
often quoted as a percentage eg.,

It
% T= X 100
Io
 UV / VISIBLE SPECTROSCOPY - THEORY

ABSORBANCE A = - log10 T
It
A = - log10
Io

A = log10 Io
It
•Absorbance is logarithmic, meaning small changes in transmittance can result
in large changes in absorbance.
•As absorbance increases, transmittance decreases, indicating more light is
being absorbed by the sample.
For example:
•If T = 1 (100% transmittance), no light is absorbed, so A = 0.
•If T = 0.1 (10% transmittance), then A = 1.
•If T = 0.01 (1% transmittance), then A = 2.
• By plotting Absorbance vs wavelength an ABSORBANCE SPECTRUM is generated.

• With the advantage that absorbance measurements are usually linear with
Concentration, absorbance spectra are now used
 THE LAWS OF SPECTROPHOTOMETRY

LAMBERTS LAW – ABSORBANCE (A) proportional to the PATHLENGTH (l)

of the absorbing medium.

BEERS LAW - ABSORBANCE (A) proportional to the CONCENTRATION (c)

of the sample.

BEER- LAMBERT LAW - ABSORBANCE (A) proportional to c x l

A  cl
A = Ecl (A is a ratio and therefore has no units)
The constant E is called the MOLAR EXTINCTION COEFFICIENT
 UV / VISIBLE SPECTROSCOPY - THEORY
UNITS OF THE MOLAR EXTINCTION COEFFICIENT
• CONCENTRATION (c) - Moles litre-1
• PATHLENGTH (l) - cm

A = Ecl Hence E= A
cl

E = 1 ˛

mole litre-1 x cm
E = mole-1 litre x cm -1
But 1 litre = 1000cm3
E = 1000 mole -1 cm3 x cm -1
Hence Units of E = 1000 cm2 mole -1
 UV / VISIBLE SPECTROSCOPY - THEORY

IMPORTANCE OF THE BEER LAMBERT LAW

A = Ecl but if E and l are constant

ABSORBANCE  CONCENTRATION and should be linear relationship

Prepare standards of the analyte to be quantified at known concentrations

and measure absorbance at a specified wavelength.

Prepare calibration curve.

ABSORBANCE AT 300nm
From measuring absorbance of sample x
x
Concentration of analyte in sample
can be obtained from the calibration curve x
x
E can be obtained from the slope of the
calibration curve for a given wavelength () x
CONCENTRATION (moles litre-1 )
 Instrumentation
1. Radiation source
2. Collimating system
3. Sample holder/container
4. Detector
5. Amplifier/Read-out unit
 Radiation Source (for UV radiations)
• Ideal characteristics of radiation source-
– Should be stable and no Fluctuations
– Provide high intensity incident radiations
– Should emit continuous spectrum/uniform intensity
– Inexpensive
– Shouldn’t show fatigue on continuous usages

1. Hydrogen Lamp
– A pair of electrodes in glass tube filled with Hydrogen at
low pressure. When current is applied at high voltage,
discharge of electrons occurs which excites Hydrogen gas
to high energy states. The electrons return to ground state
emitting radiation in 180-350nm range.
2. Deuterium Lamp
Radiation Source (for Visible radiations)

• Tungsten Filament lamp:

– Glass enclosed tungsten filament incandescent lamp;
inexpensive and most commonly used .
– Filament is heated by stabilized power supply.
– The tungsten lamp emits continuous radiations in
range 350-2500 nm.
– Only 15% are in visible range, rest are in near IR range.

• Mercury Discharge lamp:

– Emits radiation in 350-800 nm range
 Collimating System
• Radiations emitted by source must be made parallel by
lenses, mirrors and slits.

• Lens materials used must be transparent to radiation

being used (< 0.2% absorbance).
1. Ordinary silicate glass for visible light (350-3000 nm)
2. Quartz and fused silica for UV light (< 350 nm)

• Mirrors to reduce the light losses/to minimize scattering

• Slits widths for resolving polychromatic radiation into

monochromatic radiations.
 Monochromator
• To resolve wide band of radiations into a narrow
band of monochromatic radiations.
1. Filters
– Glass filters
– Gelatin filters
– Interference filters

2. Prism
– Glass or quartz

3. Grating
– Large number of parallel grooves/lines (15000-30000
per inch) on highly polished aluminium surface.
 Sample Holder
• Cells or cuvettes or test tubes are used for
handling liquid samples.

• For visible radiations-

– Good quality, clear glass which shows uniform
transmittance.
– Rectangular or cylindrical

• For UV radiations
– Rectangular cuvettes (with or without lid) of glass with
quartz or fused silica windows
 Detector
• The intensity of transmitted radiation by sample is collected on
detector and is measured.

• Most detectors generate electrical current after receiving

radiations.

• The generated current is amplified and passed on to

meter/recorder.

• Different types of detectors generate different types of signals.

– PMT
– PDA
– CCD
 Ideal Detector

• Should give quantitative response

• Should have high sensitivity and low noise level

• Should have short response time

• Should provide signal in wide spectrum of

radiations

• Should generate sufficient signal to be measured

 Advantages/limitations of UV-VIS
Photo-spectrometry
• IR Spectroscopy vs UV-VIS Photo-spectrometry

• Applications of UV-VIS Photo-spectrometry

– Qualitative vs Quantitative analysis

– Destructive vs Non-destructive analysis

– Preliminary vs Confirmatory analysis