1. Spectrofluorimetry Lecture
notes

2. 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-7,8,9,10-tetrahydrobenzo[a]pyrene in a DNA duplex is by Richard Wheeler (Zephyris) 2007 under the same license.

3. LUMINESCENCE SPECTROSCOPY
The emission of radiation from a species after that species has absorbed radiation.

4. LUMINESCENCE SPECTROSCOPY

5. 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.

6. 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.

7. 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.

8. 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

9. Energy Level Diagram
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 

10. 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 you need to identify each slide with its place with the energy level diagram from the previous slide)

11. 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.

12. 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.

13. 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.

14. 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:

15. 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;

16. 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;

17. 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;

18. 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.

19. Fluorescence and Phosphorescence - 10
In fluorescence, the lifetime of the molecule in the excited singlet state is 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).

20. 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.

21. 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.

22. 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( bc)
This is the fluorescence law
Unlike Beer’s Law fluorescence isn’t in general linear with concentration.

23. CONCENTRATION AND FLUORESCENCE INTENSITY
This expression can be expanded (Taylor series):
To a good approximation if bc is small (< 0.05) the higher-order terms are nearly zero, we have:
F = 2.3K'bcP0

24. 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.

25. CONCENTRATION AND FLUORESCENCE INTENSITY
For a concentration above c1 the calibration curve is no longer linear.

26. INSTRUMENTATION

27. 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).

28. 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).

29. 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

30. 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

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

32. 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

33. 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).

34. 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

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

36. 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: