2. spectrofluorimetry dr. hisham e abdellatef [email protected]
TRANSCRIPT
Instruments for Measuring Absorption of Light….
Fluorescence and Phosphorescence
Excitation Beam
Emitted Beam
Detector
Resonance Fluorescence• 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)
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 )
Important topics in this chapter:
Energy diagram and basic concepts
Fluorescence quantum yield
Fluorescence instrumentation
Chapter 15 Molecular Luminescence
Homowork in Chapter 15: 1, 2, 3, 4, 6, 7
Luminescence:
1. Energy diagram and basic concepts
2. The factors affect fluorescence
3. Excitation and emission spectra
4. Instrumentation
5. Applications
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).
GroundSingle state
ExcitedSingle state
Excitedtriplet state
Partial energy diagram for a photoluminescent system
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; 10 -12 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.
Fluorescence and Phosphorescence
Comparison of Triplet and Singlet
Singlet Tripletmagnetic effect diamagnetic paramagnetic
electron transition foremission
more probable less probable(unlikely)
radiation inducedexcitation
more probable less probable
luminescence Fluorescence Phosphorescencelife time short, < 10-5 s to
10-9 slong, 10-5 s to several
seconds or longer
Comparison of Fluorescence and Phosphorescence
Fluorescence Phosphorescence life time short, < 10-5s long, several seconds electron spin no yesexcited states singlet tripletquantum yield high lowtemperature 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.
Luminescence:
1. Energy diagram and basic concepts
2. The factors affect fluorescence
3. Excitation and emission spectra
4. Instrumentation
5. Applications
Quantum yield:
the ratio of the number of molecules that luminescence to the total number of excited molecules.
= kf/ (kf + ki + kec + kic + kpd + kd)
kf: Fluorescence constantki: Intersystem crossing constantkec: External conversion constantkic: Internal conversion constantkpd: Predissociation constantkd: Dissociation constant
* transitions: high quantum efficiency
Variables that affect Fluorescence and phosphorescence
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
Quantum yield = kf / (kf + ki + kec + kic + kpd + kd)
= kf
kf + knr
Effect of structural rigidity: Molecules with rigid structureshave high fluorescence yield.
Nonrigid molecule can undergo low-frequency vibrations. kic
Effect of Concentration on Fluorescence Intensity
F = K’ (I0 –I)
Power of fluorescence emission F
I0 and I are the intensities of excitation lights before and after absorbed by the analytes. K’ is the constantrelated to the quantum yield
I/I0 = 10-bc
F = K’ I0 (1–10-bc)
F = 2.3 K’ I0 bc, (when bc<0.05)
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;
Luminescence Lifetime:
average time the molecule spends in the excited state prior to return to the ground state
determines the time available for the fluorophore to interact with or diffuse in its environment, and hence the information available from its emission.
1
Kf + Knr
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.
Luminescence:
1. Energy diagram and basic concepts
2. The factors affect fluorescence
3. Excitation and emission spectra
4. Instrumentation
5. Applications
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).
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 1 and 2 produces the same fluorescence 3.
Qunnine: two absorption bands: 250 nm and 350 nm;fluorescence at 450 nm.
Figure 15-2 Fluorescence excitation and emission spectra for a solution of quinine.
Figure 15-5 Fluorescence spectra for 1 ppm anthracene in alcohol: (a) excitation spectrum; (b) emission spectrum.
Figure 15-3 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. 169. New York: Wiley, 1981. Reprinted by permission of John Wiley & Sons, Inc.)
Luminescence:
1. Energy diagram and basic concepts
2. The factors affect fluorescence
3. Excitation and emission spectra
4. Instrumentation
5. Applications
Components of a fluorometer:
sources;wavelength selection: two wavelength selection
devices;detectors;sample cell.
Figure 15-4 Components of a fluorometer of a spectro-fluorometer.
Figure 15-6 A typical fluouometer. (courtesy of Farrand Optical Co., Inc.)
Figure 15-7 A spectrofluorometer. (Courtesy of SLM Instruments, Inc., Urbana, IL.)
Figure 15-8 (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.
Figure 15-9 Schematic of a device for alternately exciting and observing phosphorescence.
Luminescence:
1. Energy diagram and basic concepts
2. The factors affect fluorescence
3. Excitation and emission spectra
4. Instrumentation
5. Applications
Fluorescence Sensing
sensing is based on changes in fluorescence signaleither in intensity or in spectrum.
Fluorophore based sensors:
Enzyme based sensors:
Ion sensors
DNA/RNA sensors
neurotransmitter sensors
environmental sensors
Ion Sensors
phosphorimetric methods:
better selectivity;poorer precision; lower temperature; heavy atom results in strong phosphorescenceroom 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.
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* + DC* C + hv
Measurements of chemiluminescence is simple:
only detector, no excitation necessary
NO + O3 NO2* +O2
NO2* NO2 + hv
Figure 15-11 Chemiluminescence emission intensity as a function of time after mixing reagents.
Preview:Laser Chapter 7
Homework:
Chapter 7: 6
Instruments for Measuring Absorption of Light….
Fluorescence and Phosphorescence
Excitation Beam
Emitted Beam
Detector
Right angle
Filter = flurometer
Prism and grating =spectroflurometer
Fluorescence and Phosphorescence
Excited single state S1 or S2
Ground state
Excited triplet state
phosphorescence
Fluorescence
Factors influencing intensity of fluorescence
1. Concentration of fluorescing species F
2. Presence of other solutes
3. pH
4. Temperature
5. Photocomposition of sample due to sunlight
6. viscosity
Disadvantages of fluoremetry
1. Dilute solution are less stable2. Adsorption on the surface of container3. Oxidation of fluorescence sample4. Photodecomposition5. Quenching (even traces of non fluorescent
can quench a fluorescent one in S1 state)6. It does not exhibit very high precision or
accuracy (2 – 10%)
Difference between fluorometry and spectrophotometry
difference Fluorometry spectrophotometry
nature Measuring emission Measuring absorption
sensitivity Nanogram scale (102 -104 times sensitive)
Microgram
instrumentation Single beam
Use 2 filter monochromatic
Single or double beam
Only one
Selectivity more Less selective
Lambda maximum
Absorption and emission Absorption only
equations F=2.3 QIε. B.C A = ε. B.C
Calibration Quinine in dilute H2SO4 Potassium chromate in H2O