Transcript Spectrofluorimetry.ppt

Faculty of Science
Spectrofluorimetry Lecture
notes
Faculty of Science
Copyright Statement
• Images used in this work are distributed
under the GNU Free Documentation
License, Version 1.2 or any later
version published by the Free Software
Foundation;
• Solution structure of a trans-opened
(10S)-dA adduct of +)-(7S,8R,9S,10R)7,8-dihydroxy-9,10-epoxy-7,8,9,10tetrahydrobenzo[a]pyrene in a DNA
duplex is by Richard Wheeler
(Zephyris) 2007 under the same license.
Faculty of Science
LUMINESCENCE
SPECTROSCOPY
• The emission of radiation from a species
after that species has absorbed radiation.
FLUORESCENCE
LUMINESCENCE
SPECTROSCOPY
PHOSPHORESCENCE
CHEMILUMINESCENCE
Faculty of Science
LUMINESCENCE
SPECTROSCOPY
Absorption first -
Followed by emission
in all directions, usually
at a lower frequency
Faculty of Science
LUMINESCENCE
SPECTROSCOPY
• In favorable cases, luminescence methods
are amongst some of the most sensitive
and selective of analytical methods
available.
• Detection Limits are as a general rule at
ppm levels for absorption
spectrophotometry and ppb levels for
luminescence methods.
Faculty of Science
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.
Faculty of Science
LUMINESCENCE
SPECTROSCOPY
• Most chemical species are not naturally
luminescent.
• Derivatisation reactions are often
available to form luminescent derivatives
of non-luminescent compounds.
• However, this extra step lessens the
attractiveness of luminescence methods.
Faculty of Science
LUMINESCENCE
SPECTROSCOPY
• Fluorimetry is the most commonly used
luminescence method. Phosphorimetry
usually requires at liquid nitrogen
temperatures (77K).
• The terms fluorimetry and fluorometry are
used interchangeably in the chemical
literature.
• Chemiluminescence won’t be further
discussed in PCB314
Faculty of Science
Energy Level Diagram
SINGLET STATES  
s2
TRIPLET STATES  
VIBRATIONAL
RELAXATION
T2
s1
T1
INTERSYSTEM
CROSSING
FLUORESCENCE
PHOSPHORESCENCE
INTERNAL
CONVERSION
Ground
State
INTERNAL
CONVERSION
Faculty of Science
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)
Faculty of Science
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.
Faculty of Science
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.
Faculty of Science
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.
Faculty of Science
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:
Faculty of Science
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;
Faculty of Science
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;
Faculty of Science
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;
Faculty of Science
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.
Faculty of Science
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).
Faculty of Science
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.
Faculty of Science
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'(P0 - P)
where P0 is the power incident on the sample, P
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.
Faculty of Science
CONCENTRATION AND
FLUORESCENCE INTENSITY
• Beer's law can be rearranged to give:
P/P0 = 10-bc
where A = bc is the absorbance.
Substitution gives:
F = K'P0(1 - 10- bc)
• This is the fluorescence law
• Unlike Beer’s Law fluorescence isn’t in
general linear with concentration.
Faculty of Science
CONCENTRATION AND
FLUORESCENCE INTENSITY
• This expression can be expanded (Taylor series):


(2.3bc) (2.3bc)
F = K P0 2.3bc 
 
2!
3!


2
3
• To a good approximation if bc is small (< 0.05) the
higher-order terms are nearly zero, we have:
F = 2.3K'bcP0
Faculty of Science
CONCENTRATION AND
FLUORESCENCE INTENSITY
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.
Faculty of Science
CONCENTRATION AND
FLUORESCENCE INTENSITY
F
c1
Conc. of fluorescing species
For a
concentration
above c1 the
calibration
curve is no
longer linear.
Faculty of Science
INSTRUMENTATION
SOURCE
SAMPLE
EXCITATION
WAVELENGTH
SELECTOR
EMISSION
WAVELENGTH
SELECTOR
DETECTOR
Faculty of Science
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  P0).
Faculty of Science
INSTRUMENTATION
• Because An intense monochromatic light
source is required ...
• Lasers are an almost ideal light source for
fluorimetry (laser-induced fluorescence) but are
too expensive and/or impractical for most
routine applications.
• Two wavelength selectors are required filters
(in fluorimeters) and monochromators (in
spectrofluorometers).
Faculty of Science
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
Faculty of Science
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
Faculty of Science
APPLICATIONS
A. Determination of polyaromatic hydrocarbons
– Benzo[a]pyrene is a product of incomplete
combustion and found in coal tar.
Faculty of Science
APPLICATIONS
• Benzo[a]pyrene, is a 5ring 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
Faculty of Science
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
Faculty of Science
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
Faculty of Science
APPLICATIONS
• In addition to ethical
drugs such as
quinine, many drugs
of abuse fluoresce
directly. For
example lysergic
acid diethylamide
(LSD) whose
structure is:
Faculty of Science
APPLICATIONS
• Because LSD is active in minute quantities (as little as
50 g taken orally) an extremely sensitive methods of
analysis is required. Fluorimetricaly 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: