UV-VIS Spectroscopy
Dr. AKM Shafiqul Islam
Spectrometer Experiment
Irradiance in
Irradiance out
Pathlength
Irradiance, P, is measured in W·m-2
Transmittance, T, is the fraction of original light not
P
absorbed by the sample, or T   10kb
P0
Where po = is power of incident light, p = power of
transmitted light and b = path length
• A light source can be a lamp, laser, or even a
light bulb. A monochromator (“one color”) is
used to select a particular wavelength. This is
typically a grating, but can be a prism or filter.
The light then passes through the sample,
containing the analyte. Afterwards, the light is
detected.

Putting this in logarithmic form
P
log T  log  kb
P0


In 1852, Beer and Bernard each stated
that a similar law holds for the
dependence of concentration, c
P
 k c
T   10
P0
Where k’ is a new constant
P
log T  log  k c
P0

Combining these two laws which describes the
dependence of T on both the path length and
concentration
P
T   10  abc
P0

Where a is a combined constant of k and k’
P
log T  log  abc
P0


It is more convenient to remove negative sign on right
hand side, we get
P0
1
A   log T  log  log  abc
T by P
The transmittance is given
P
%T  100
P0

or,
T  %T / 100

Now, we can write
100
A  log
 log 100  log %T
%T
Absorbance and Beer’s Law

Absorbance, A, the amount light absorbed by
the sample is related to transmittance:
P 
A  log  0    log T
P

Beer’s law relates the absorbance of a chemical
to its concentration:
A    bc
b is the pathlength, typically in cm, and c is the
concentration of the chemical species in M
 is the molar absorptivity, the unit that tells
how much light is absorbed for a given
wavelength.  has units of M-1 cm-1
Wavelengths and Color
Beer’s Law Assumptions


The light being shined on the sample must be
monochromatic (one color or wavelength)
The analyte must not be participate in a concentration
dependent equilibrium
 This isn’t a good technique for many weak acid
systems, as dilution increases dissociation and HA and
A- probably don’t have the same absorbance
To do a Spectroscopic Analysis


You need:
 A continuous light source
 A wavelength selector
 A sample cell
 A detector
The sample cell is called cuvet and can be made of
many substances
 Glass (good for visible)
 Quartz (UV-vis)
 NaCl/KBr (IR)
Spectroscopic Procedure


You may have a single-beam or double beam
 Single-beam instrument has one sample holder, you
must swap blank and sample
 Double-beam instrument splits light output between
two holders so you can measure blank and sample
 A baseline spectrum is a spectrum of a reference
solution (solvent or reagent blank)
We try to do an analysis at the λmax if we can
 Sensitivity is greatest at maximum absorbance
 Curve is relatively flat in case the monochromator drifts
and is off by a little in wavelength
The Single-Beam Spectrometer
How Do UV spectrometers work?
Rotates, to
achieve scan
Matched quartz cuvettes
Sample in solution at ca. 10-5 M.
System protects PM tube from
stray light
D2 lamp-UV
Tungsten lamp-Vis
Double Beam makes it a
difference technique
Two photomultiplier inputs,
differential voltage drives
amplifier.
Optics of the Grating Monochromator
The polychromatic light is separated into
monochromatic wavelengths by diffraction.
n = d(sin  + sin )
Optics of the Grating Monochromator
In the equation n = d(sin  + sin ) n is the order of
the diffraction n = 1, 2, 3 etc, d is the number of
lines etched on the grating,  is the angle of the
incident beam and  is the angle of the emerging
beam.
The photodiode array detector
The photodiode array detector
UV Instrumentation
Key components:
(1) Light Source
(2) Monochromator
(3) Sample/reference holder
(4) Radiation detection
(5) Readout device
Spectroscopic Procedure


We should always try to keep the absorbance reading of
our sample below 1.
 Because % transmittance is related logarithmically with
concentration, it means that from 1-99% transmittance
we can detect ~ 2 orders of magnitude in analyte
concentration.
 Any orders of magnitude greater than that will be
detected in the range of 0-1% T.
In order to maximize accuracy, you should dilute the
solution if you have to so that the transmittance reading is
not maxed out in that region.
Spectroscopic Procedure


You should always try to keep the absorbance reading of
your sample below 1.
 Because % transmittance is related logarithmically with
concentration, it means that from 1-99% transmittance
you can detect ~ 2 orders of magnitude in analyte
concentration.
 Any orders of magnitude greater than that will be
detected in the range of 0-1% T.
In order to maximize accuracy, you should dilute the
solution if you have to so that the transmittance reading is
not maxed out in that region.
An Electronic Spectrum
Make solution of
concentration low enough
that A≤ 1
(Ensures Linear Beer’s
law behavior)
1.0
maxwith certain
extinction 
UV
Visible
Even though a dual beam
goes through a solvent
blank, choose solvents
that are UV transparent.
Absorbance
Can extract the  value if
conc. (M) and b (cm) are
known
UV bands are much
broader than the photonic
transition event. This is
because vibration levels
are superimposed on UV.
0.0
200
400
Wavelength, , generally in nanometers (nm)
800
Ultraviolet Spectroscopy



200-400 nm photons excite electrons from a 
bonding orbital to a * antibonding orbital.
Conjugated dienes have MO’s that are closer in
energy.
A compound that has a longer chain of conjugated
double bonds absorbs light at a longer wavelength.
=>
Absorption Characteristics of Some Common Chromophores
Chromophore
Alkene
Example
Solvent
Type of
transition
177
13,000
*
n-Heptane
178
196
225
10,000
2,000
160
*
_
_
O
n-Hexane
186
280
1,000
16
CH3CCH3
O
ns*
n*
n-Hexane
180
293
Large
12
Ethanol
204
41
n*
Water
214
60
n*
Ethanol
339
5
n*
n*
C5H11C
Carbonyl
Amido
max
n-Heptane
C6H13HC
CH2
Alkyne
Carboxyl
max (nm)
C
CH3
CH3CH
O
CH3COH
O
ns*
n*
CH3CNH2
Azo
H3CN
NCH3
Nitro
CH3NO2
Isooctane
280
22
Nitroso
C4H9NO
Ethyl ether
300
665
100
20
n*
270
12
n*
Nitrate
C2H5ONO2
Dioxane
_
Solvents for UV (showing high
energy cut-offs)
Water
205
THF
220
CH3CN
210
CH2Cl2
235
C6H12
210
CHCl3
245
Ether
210
CCl4
265
EtOH
210
benzene
280
Hexane
210
Acetone
300
MeOH
210
Dioxane
220
Various buffers for
HPLC, check before
using.
Deviation from Beer’s Law




Beer’s law is only valid for low concentration, up to 10
mM;
The intermolecular distances in a given solution will
decrease, eventually reach a point at which neighboring
molecules mutually affect the charge distribution of the
other affect 
Chemical processes such as the reversible associationdissociation of analyte molecules, or the ionization of a
weak acid in an unbuffered solvent.
Instrumentation limitation-incident beam may be
polychromatic .
Background Correction
-Processes other than analyte absorption
result in significant decrease in the power
of the incident beam;
- Reference cell is used to correct
these processes;
- Reference cell is often prepared by
adding distilled water to an absorption
cell;
- The reference cell is then placed in the
path of the light beam, and the power
of the radiation exiting the reference cell
is measured and taken as P0 for the sample cell.
Calibration Curves
• Linear calibration curve;
• Nonlinear calibration
An Example--Pulegone
Frequently
plotted as
log of
molar
extinction

So at 240 nm,
pulegone has
a molar
extinction of
7.24 x 103
Antilog of 3.86
O
Example

A solution containing a compound of
formula weight 280 g/mol absorbed 65.0%
of the light with 450 nm wavelength in a
2.00 cm cell at a concentration of 15.0 ×
10-3 g/L. Calculate its molar absorptivity at
450 nm.