CVB222 UV-vis Absorption and Fluorescence Lecture

Faculty of Science and Engineering
Absorption and Fluorescence
Lecture
Dr Mark Selby
E413D (GP)
m.selby@qut.edu.au

Spectrochemical Analysis
• In spectrochemical analysis, the electromagnetic
spectrum of radiation is used to identify and/or quantify
chemical species.
• A "spectrum" is a plot of some measurable property of
the radiation, as a function of the frequency f(ν), or
wavelength, f(λ) , of the radiation .
For instance, the Near-infrared
absorbance
f(λ), spectrum of
chloroform over the
wavelength (λ) range
from 1100 nm to about
1700 nm shown

The Star Trek Tricorder
• The perfect biochemical scanner!
We don’t have a
tricorder – BUT
we do have UV-vis
absorption
and Fluorescence
Spectrometry!

• For a photon of electromagnetic radiation, the frequency
(ν) is related to the energy by the Planck equation:
E = h ν
where E is the energy of the photon, ν is its frequency and
h is the Planck constant (6.624 × 10-34 J s).
• Since ν λ = c (where c is the speed of light in vaccuum
and λ is the wavelength then:
1
E  h  
hc hc

c
 

Absorption Spectrophotometry
• If a beam of radiation is sent into a chemical
sample, it is possible that the sample will
absorb some portion of that radiation, as shown
Thickness
b
P
o Chemical Sample
P
Concentration, c
• The incident radiant power of the beam being
Po and that transmitted being P.

Fundamental Laws For Absorption
of Radiation
• The transmission of electromagnetic radiation
through a sample depends upon the number of
encounters between photons and species
capable of absorbing them. This is turn
depends upon:
(i) the power of the radiation;
(ii) the concentration of the sample species and
(iii) the thickness of the sample container.

of Radiation
Thickness
b
P
o Chemical Sample
P
Concentration, c
• The relationship between radiant power,
concentration and rate of absorption is known
as the Beer-Lambert law, or often, simply as
Beer's Law:
A = log(I0/I) = εbc

of Radiation
• where I0 is the power of the incident radiation, I is the power of the
transmitted radiation, A is the absorbance, b is the thickness of the cell, c is
the concentration (in mol L-1) of the sample and is the molar absorptivity
constant (in units of mol-1 L cm-1).
• If the concentration, c, of the sample is expressed in g L-1, then Beer's Law
can be written as:
A = log(I0/I) = abc
• where A is the absorbance (as before) and a is the absorptivity in
g-1 L cm-1.

of Radiation
• The ratio I/I0 is called the transmittance,
T, whereas 100T is the percent
transmittance (%T).
• Instruments for absorption spectro-photometry
are generally calibrated in
terms of both transmittance and
absorbance:
• A = log(I0/I) = log(1/T) = log(100/%T).

Absorption and Transmittance
• Absorption (NOT absorbance) and
transmittance are complementary:
absorption = 1 – T
This is usually expressed as a percentage:
% absorption = 100 - %T

Analytical Working Curves
• It is seldom safe to assume adherence to
Beer's law. In general, a number of
calibration standards should be prepared
and measured in turn. The concentration
of an unknown sample is then determined
from an analytical working curve (also
known as a calibration curve).

Analytical Working Curves
• Example: The determination of formaldehyde by the addition of
chromatropic acid and conc. sulfuric acid recording the absorbance
on a spectrophotometer at 570 nm.

Deviations from Beer's Law
Beer’s Law Obeyed
A
Conc. of absorbing species
A
Deviations from
Beer’s Law
c1
Conc. of absorbing species
• Generally, the data over a wide range of concentrations will
deviate from Beer's law, similarly to the plot above. This indicates
that Beer's law is only applicable up to a concentration of c1.

Deviations from Beer's Law
• Nevertheless, it is still possible to determine the
concentration of the absorbing substance from
such a curve.
• The most common reason for departures from
Beer's law is the use of non-monochromatic light.
Beer's law is rigorously applicable only for
absorption of radiation at a single frequency.
• In practice, therefore, some deviation from Beer's
law will generally be found in instrumental
systems

EFFECT OF POLYCHROMATIC
UV-vis Spectroscopy -
Dr Mark Selby
RADIATION
• In the diagram below, the Beer’s Law
linear relationship is maintained for Band
A but not for Band B

Single-beam Spectrophotometer
• Instruments with a continuous source have a dispersing element
and aperture or slit to select a single wavelength before the light
passes through the sample.
• Either type of single-beam instrument, the instrument is
calibrated with a reference cell containing only solvent to
determine the I0 value.
UV-vis Spectroscopy - Dr Mark Selby
The simplest
instruments use a
single-wavelength
light source, such
as a light-emitting
diode (LED), a
sample container,
and a photodiode
detector.

Double-beam Spectrophotometer
•The double-beam design greatly simplifies this process by
simultaneously measuring I and I0 of the sample and reference
cells, respectively. Most spectrometers use a mirrored rotating
chopper wheel to alternately direct the light beam through the
sample and reference cells. The detection electronics or software
program can then manipulate the I and I0 values as the
wavelength scans to produce the spectrum of absorbance or
transmittance as a function of wavelength.
UV-vis Spectroscopy - Dr Mark Selby

LUMINESCENCE
SPECTROSCOPY
Absorption first -
Followed by emission
in all directions, u sually
at a lower frequency

LUMINESCENCE
SPECTROSCOPY
• Collectively, fluorescence and
phosphorescence are known as
photoluminescence.
• A third type of luminescence -
Chemiluminescence - is based upon
emission of light from an excited species
formed as a result of a chemical reaction.

Jablonski Diagram
(energy levels)
s2
SINGLET STATES TRIPLET STATES
Ground
State
   
T
s1 T
1
2
INTERSYSTEM
CROSSING
VIBRATIONAL
RELAXATION
FLUORESCENCE PHOSPHORESCENCE
INTERNAL
INTERNAL
CONVERSION CONVERSION

Fluorescence and
Phosphorescence - 1
• Following absorption of radiation, the
molecule can lose the absorbed energy by
several pathways. The particular
pathway followed is governed by the
kinetics of several competing reactions.
(Note: in the next slides 1- 10 you need to
identify each slide with its place with the
energy level diagram from the previous
slide)

Fluorescence and
Phosphorescence - 2
• One competing process is vibrational
relaxation which involves transfer of
energy to neighbouring molecules which
is very rapid in solution (10-13 sec).
– In the gas phase, molecules suffer fewer
collisions and it is more common to see the
emission of a photon equal in energy to that
absorbed in a process known as resonance
fluorescence.
(Energy level diagram)

Fluorescence and
Phosphorescence - 3
• In solution, the molecule rapidly relaxes
to the lowest vibrational energy level of
the electronic state to which it is excited
(in this case S2). The kinetically favoured
reaction in solution is then internal
conversion which shifts the molecule
from S2 to an excited vibrational energy
level in S1.

Fluorescence and
Phosphorescence - 4
• Following internal conversion, the
molecule loses further energy by
vibrational relaxation. Because of
internal conversion and vibrational
relaxation, most molecules in solution
will decay to the lowest vibrational
energy level of the lowest singlet
electronic state before any radiation is
emitted.

Fluorescence and
Phosphorescence - 5
• When the molecule has reached the
lowest vibrational energy level of the
lowest singlet electronic energy level then
a number of events can take place:

Fluorescence and
Phosphorescence - 6
• the molecule can lose energy by internal
conversion without loss of a photon of
radiation, however, this is the least likely
event;

Fluorescence and
Phosphorescence - 7
• the molecule can emit a photon of
radiation equal in energy to the difference
in energy between the singlet electronic
level and the ground-state, this is termed
fluorescence;

Fluorescence and
Phosphorescence - 8
• the molecule can undergo intersystem
crossing which involves and electron spin
flip from the singlet state into a triplet
state. Following this the molecule decays
to the lowest vibrational energy level of
the triplet state by vibrational relaxation;

Fluorescence and
Phosphorescence - 9
• the molecule can then emit a photon of
radiation equal to the energy difference
between the lowest triplet energy level
and the ground-state in a process known
as phosphorescence.

Fluorescence and
Phosphorescence - 10
• In fluorescence, the lifetime of the
molecule in the excited singlet state is
10-9 to 10-7 sec.
• In phosphorescence, the lifetime in the
excited singlet state is 10-6 to 10 sec
(because a transition from T1 to the
ground state is spin forbidden).

Quantum Efficiency
• Fluorescence, phosphorescence and
internal conversion are competing
processes. The fluorescence quantum
efficiency () and the phosphorescence
quantum efficiency are defined as the
fraction of molecules which undergo
fluorescence and phosphorescence
respectively.
,

. ,

.

CONCENTRATION AND
FLUORESCENCE INTENSITY
• The power of fluorescent radiation, F, is
proportional to the radiant power of the
excitation beam absorbed by the species able to
undergo fluorescence:
F = k(I0 - I)
where I0 is the power incident on the sample, I
is the power after it traverses a length b of the
solution and k is a constant which depends upon
experimental factors and  the quantum
efficiency of fluorescence.

CONCENTRATION AND
• Beer's law can be rearranged to give:
I/I0 = 10-bc
where A = bc is the absorbance.
Substitution gives:
F = kI0(1 - 10- bc)
• This is the fluorescence law
• Unlike Beer’s Law fluorescence isn’t in
general linear with concentration.

CONCENTRATION AND
For low concentration this simplifies to:
F = kI0 bc
which demonstrates two important points:
– that at low concentrations fluorescence
intensity is proportional to concentration;
– that fluorescence is proportional to the
incident power in the incident radiation at the
absorption frequency.

CONCENTRATION AND
F
c1
Conc. of fluorescing species
For a
concentration
above c1 the
calibration
curve is no
longer linear.

INSTRUMENTATION
Schematic Diagram of Fluorescence Spectrometer. M1 =
excitation monochromator, M2 emission monochromator,
L light source. s = sample cell, PM photo multiplier
detector.

INSTRUMENTATION
• The fluorescence is often viewed at 90°
orientation (in order to minimise interference
from radiation used to excite the fluorescence).
• The exciting wavelength is provided by an
intense source such as a xenon arc lamp
(remember F  I0).
• Two wavelength selectors are required - filters
(in fluorimeters) or monochromators (in
spectrofluorometers).

Types of Fluorescent Molecules
• Experimentally it is found that fluorescence is
favoured in rigid molecules, eg.,
phenolphthalein and fluorescein are structurally
similar as shown below. However, fluorescein
shows a far greater fluorescence quantum
efficiency because of its rigidity.
•
phenolphthalein

Types of Fluorescent Molecules
• It is thought that the extra rigidity
imparted by the bridging oxygen group in
Fluorescein reduces the rate of
nonradiative relaxation so that emission
by fluorescence has sufficient time to
occur.
Fluorescein

APPLICATIONS
A. Determination of polyaromatic hydrocarbons
– Benzo[a]pyrene is a product of incomplete
combustion and found in coal tar.

APPLICATIONS
• Benzo[a]pyrene, is a 5-
ring polycyclic aromatic
hydrocarbon that is
mutagenic and highly
carcinogenic
• It is found in tobacco
smoke and tar
• The epoxide of this
molecule intercalates in
DNA, covalently
bonding to the guanine
base nucleotide

APPLICATIONS
Excitation and fluorescence
spectra for benzo(a)pyrene
in H2SO4. In the diagram
the solid line is the
excitation spectrum (the
fluorescence signal is
measured at 545 nm as the
exciting wavelength is
varied). The dashed line is
the fluorescence spectrum
(the exciting wavelength is
fixed at 520 nm while the
wavelength of collected
fluorescence is varied).
Benzo(a)pyrene

APPLICATIONS
B. Fluorimetric Drug
Analysis
– Many drugs possess
high quantum
efficiency for
fluorescence. For
example, quinine can
be detected at levels
below 1 ppb.
Quinine

APPLICATIONS
• In addition to ethical
drugs such as
quinine, many drugs
of abuse fluoresce
directly. For
example lysergic
acid diethylamide
(LSD) whose
structure is:

APPLICATIONS
Because LSD is active in minute quantities (as little as 50
g taken orally) an extremely sensitive methods of analysis
is required. Fluorimetrically LSD is usually determined in
urine from a sample of about 5mL in volume. The sample
is made alkaline and the LSD is extracted into an organic
phase consisting of n-heptane and amyl alcohol. This is a
"clean-up" procedure that removes potential interferents
and increases sensitivity. The LSD is then back-extracted
into an acid solution and measured directly using and
excitation wavelength of 335 nm and a fluorescence
wavelength of 435 nm. The limit of detection is
approximately 1 ppb: An old method – but still a
goodie in certain circumstances!

CVB222 UV-vis Absorption and Fluorescence Lecture

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CVB222 UV-vis Absorption and Fluorescence Lecture