1. Dr. Hisham E Abdellatef ezzat_hisham@yahoo.com
2. Spectrofluorimetry
Dr. Hisham E Abdellatef

2. Instruments for Measuring Absorption of Light….

3. Fluorescence and Phosphorescence
Excitation Beam
Emitted Beam
Detector

5. Resonance Fluorescence
Usually atomic
Emitted light has same E as excitation light
Simpler, atomic systems with fewer energy states (vs molecules) undergo resonance fluorescence
Not as widely used in analytical chemistry as non-resonance fluorescence
Hg analysis is one example
Excitation Beam
Emission (identical E)

6. Non-resonance Fluorescence
Typical of molecular fluorescence
Large number of excited states
rotational
vibrational
etc..
Molecules relax by ‘stepping’ from one state to another
Resulting emitted light “shifts” to lower energies
longer wavelengths = lower energy
Excitation Beam
Emission (lower E, longer  )

8. Energy diagram and basic concepts Fluorescence quantum yield
Chapter 15 Molecular Luminescence
Important topics in this chapter:
Energy diagram and basic concepts
Fluorescence quantum yield
Fluorescence instrumentation
Homowork in Chapter 15: 1, 2, 3, 4, 6, 7

9. Luminescence: Energy diagram and basic concepts
2. The factors affect fluorescence
3. Excitation and emission spectra
4. Instrumentation
5. Applications

10. Diamagnetic: no net magnetic field due to spin paring. The
Singlet: all electron spins are paired; no energy level splitting occurs when the molecule is exposed to a magnetic field;
Triplet: the electron spins are unpaired and are parallel; excited triplet state is less energetic than the corresponding singlet state.
Diamagnetic: no net magnetic field due to spin paring. The
electrons are repelled by permanent magnetic fields.
Paramagnetic: magnetic moment and attracted to a magnetic field (due to unpaired electrons).
Ground
Single state
Excited
Single state
Excited
triplet state

11. Partial energy diagram for a photoluminescent system

12. Deactivation processes for an excited state:
Vibrational relaxation: fluorescence always involves a
transition from the lowest vibrational states of an excited electronic state; electron can return to any one of the vibrational levels of the ground state; s;
Internal conversion: intramolecular processes by which a molecule passes to a lower-energy electronic state without emission of radiation.
External conversion: interaction and energy transfer between the excited molecule and the solvent or other molecules.
Intersystem crossing: the spin of an excited electron is reversed and a change in multiplicity of the molecule results.
Phosphorescence: an excited triplet state to give radiative emission. emission: a photon is emitted.

13. Fluorescence and Phosphorescence

14. Comparison of Fluorescence and Phosphorescence
Fluorescence Phosphorescence
life time short, < 10-5s long, several seconds
electron spin no yes
excited states singlet triplet
quantum yield high low
temperature most temperature low temperature more likely
Resonance fluorescence: absorbed radiation is re-emitted without a change in frequency.
Stokes shift: molecular fluorescence bands are shifted to wavelengths that are longer than the resonance line.

15. Luminescence: Energy diagram and basic concepts
2. The factors affect fluorescence
3. Excitation and emission spectra
4. Instrumentation
5. Applications

16. Variables that affect Fluorescence and phosphorescence
Quantum yield:
the ratio of the number of molecules that
luminescence to the total number of excited molecules.
f = kf/ (kf + ki + kec + kic + kpd + kd)
kf: Fluorescence constant
ki: Intersystem crossing constant
kec: External conversion constant
kic: Internal conversion constant
kpd: Predissociation constant
kd: Dissociation constant
p ® p* transitions: high quantum efficiency

17. Quantum yield can be close to unity if the radiationless kf
F = kf
+ knr
Quantum yield = kf / (kf + ki + kec + kic + kpd + kd)
Quantum yield can be close to unity if the radiationless
decay rate is much smaller the the radiative decay.
High quantum yield molecules: rhodamine, fluorescein etc
Effect of structural rigidity: Molecules with rigid structures
have high fluorescence yield.
Nonrigid molecule can undergo low-frequency vibrations. kic

19. I/I0 = 10-ebc Effect of Concentration on Fluorescence Intensity
Power of fluorescence emission F
F = K’ (I0 –I)
I0 and I are the intensities of excitation lights before and after absorbed by the analytes. K’ is the constant
related to the quantum yield
I/I0 = 10-ebc
F = K’ I0 (1–10-ebc)
F = 2.3 K’ I0 ebc, (when ebc<0.05)

20. luminescence in quantitative analysis:
inherent sensitivity (usually three orders of magnitude better than absorption methods;
Better selectivity than absorption spectroscopy;
The precision and accuracy of photoluminescence method is usually poorer than spectrophotometer by a factor of two to five.
Less widely applicable than absorption spectroscopy;

21. t = Luminescence Lifetime:
average time the molecule spends in the excited state prior to return to the ground state 1
t =
Kf + Knr
determines the time available for the fluorophore to interact
with or diffuse in its environment, and hence the information
available from its emission.

22. Lifetime measurements:
ps or fs lasers used for lifetime measurements;
fluorescence lifetime refers to the mean lifetime of the
excited state, i.e., the probability of finding a given molecule that has been excited still in the excited state after time t is exp(-t/t0):
I = I0 e(-t/t0)
precise measurement of the observed lifetime is important since it can be used to calculate the natural lifetime t0 (life time in the absence of nonradiative processes, also called intrinsic lifetime).
For a single exponential decay, 63% of the molecules have decayed prior to t=t0.

23. Luminescence: Energy diagram and basic concepts
2. The factors affect fluorescence
3. Excitation and emission spectra
4. Instrumentation
5. Applications

24. Mirror images of absorption and fluorescence spectra:
vibrational levels in the ground and excited states have
similar energy gaps, thus absorption and fluorescence spectra have mirror images (Fig. 15-1).

25. Figure l.3. Absorption and fluorescence emission spectra of perylene and quinine. Emission spectra cannot be correctly presented on both the wavelength and wavenumber scales. The wavenumber presentation is correct in this instance. Wavelengths are shown for convenience. See Chapter 3. Revised from Ref. 5.
Internal conversion: excitation by l1 and l2 produces the same fluorescence l3.
Qunnine: two absorption bands: 250 nm and 350 nm;
fluorescence at 450 nm.

26. Figure 15-2 Fluorescence excitation and emission spectra for a solution of quinine.

27. Figure Fluorescence spectra for 1 ppm anthracene in alcohol: (a) excitation spectrum; (b) emission spectrum.

28. Figure Spectra for phenanthrene: E, excitation; F, fluorescence; P, phosphorescence. (From W. R. Seitz, in Treatise on Analytical Chemistry, 2nd ed., P. J. Elving, E. J. Meehan, and I. M. Kolthoff, Eds., Part I, Vol. 7, p New York: Wiley, Reprinted by permission of John Wiley & Sons, Inc.)

29. Luminescence: Energy diagram and basic concepts
2. The factors affect fluorescence
3. Excitation and emission spectra
4. Instrumentation
5. Applications

30. Components of a fluorometer:
sources;
wavelength selection: two wavelength selection devices;
detectors;
sample cell.

31. Figure 15-4 Components of a fluorometer of a spectro-fluorometer.

32. Figure 15-6 A typical fluouometer. (courtesy of Farrand Optical Co
Figure A typical fluouometer. (courtesy of Farrand Optical Co., Inc.)

33. Figure 15-7 A spectrofluorometer. (Courtesy of SLM Instruments, Inc
Figure A spectrofluorometer. (Courtesy of SLM Instruments, Inc., Urbana, IL.)

34. Figure (a) Schematic of an optical system for obtaining a total luminescence spectrum with a two-dimensional charge-coupled device. (b) Excitation and emission spectra of hypothetical compound. (c) Total luminescence spectrum of compound in b.

35. Figure 15-9 Schematic of a device for alternately exciting and observing phosphorescence.

36. Luminescence: Energy diagram and basic concepts
2. The factors affect fluorescence
3. Excitation and emission spectra
4. Instrumentation
5. Applications

37. Fluorescence Sensing
sensing is based on changes in fluorescence signal
either in intensity or in spectrum.
Fluorophore based sensors:
Enzyme based sensors:
Ion sensors
DNA/RNA sensors
neurotransmitter sensors
environmental sensors

38. Ion Sensors

39. phosphorimetric methods:
better selectivity;
poorer precision; lower temperature;
heavy atom results in strong phosphorescence
room temperature methods:
deposit analytes on surface: rigid matrix minimize deactivation of the triplet state by external and internal conversions;
Using micelles: micelles increase the proximity between heavy metal ion and the phosphur, thus enhance phosphorescence.

40. Chemiluminescence
chemiluminescence is produced when a chemical reaction yields an electronically excited species, which emits light as it returns to its ground states.
A + B ® C* + D
C* ® C + hv
NO + O3 ® NO2* +O2
NO2* ® NO2 + hv
Measurements of chemiluminescence is simple:
only detector, no excitation necessary

41. Figure 15-11 Chemiluminescence emission intensity as a function of time after mixing reagents.

42. Preview:
Laser Chapter 7
Homework:
Chapter 7: 6

43. Instruments for Measuring Absorption of Light….

44. Fluorescence and Phosphorescence
Right angle
Excitation Beam
Emitted Beam
Detector

45. Filter = flurometer
Prism and grating =spectroflurometer

46. Fluorescence and Phosphorescence
Excited single state S1 or S2
Excited triplet state
phosphorescence
Ground state
Fluorescence

47. Factors influencing intensity of fluorescence
Concentration of fluorescing species F
Presence of other solutes pH
Temperature
Photocomposition of sample due to sunlight
viscosity

48. Disadvantages of fluoremetry
Dilute solution are less stable
Adsorption on the surface of container
Oxidation of fluorescence sample
Photodecomposition
Quenching (even traces of non fluorescent can quench a fluorescent one in S1 state)
It does not exhibit very high precision or accuracy (2 – 10%)

50. Difference between fluorometry and spectrophotometry
Measuring absorption
Measuring emission
nature
Microgram
Nanogram scale ( times sensitive)
sensitivity
Single or double beam
Only one
Single beam
Use 2 filter monochromatic
instrumentation
Less selective
more
Selectivity
Absorption only
Absorption and emission
Lambda maximum
A = ε. B.C
F=2.3 QIε. B.C
equations
Potassium chromate in H2O
Quinine in dilute H2SO4
Calibration