Luminescence Spectroscopy
Internal conversion
- crossing of e⁻ to lower-energy electronic state (of the same multiplicity: singlet-singlet or triplet-triplet) without emission of radiation ∙allows e⁻ to continue releasing vibrational energy - S1 to S0 would also happen . - very efficient, so it is the reason why most compounds do not fluoresce (aliphatic) - especially probable if vibrational levels of two electronic states overlap, can lead to predissociation or dissociation.
Vibrational relaxation
- λ emission > λ excitation (Stokes shift) - vibrational relaxation is efficient and goes to lowest vibrational level of electronic state within 10⁻¹²s or less. - significantly shorter life-time than electronically excited state - fluorescence is emitted from lowest vibrational level of electronic excited state, and the e⁻ can the jump to vibrational level of the ground state.
How many wavelengths are needed for fluorescence?
two: one each for excitation and emission
What is resonance fluorescence?
type of fluorescence in which the absorbed radiation is reemitted without a change in frequency
How does chemical structure affect fluorescence?
Aromatic compounds have strong fluorescence that increases with structural rigidity. Quantum yield increases with # rings and degree of condensation
Why are luminescence methods less widely applicable for quantitative analysis than absorption methods?
Because excited states are quite susceptible to being deactivated by collisions and other processes, many molecules do not fluoresce or phosphoresce at all. Because of such deactivation processes, quantitative luminescence methods are often subject to serious interference effects. Many more species absorb UV-Vis radiation than exhibit photoluminescence when radiation is absorbed in this region
What two processes may occur during internal conversion?
DISSOCIATION: direct excitation (absorption) to vibrational state with enough energy to break a bond PREDISSOCIATION: relaxation to vibrational state of a lower electronic state with enough energy to break a bond
Phosphorescence
Deactivation from a triplet electronic state to the ground state, producing a photon
What wavelength selectors are used with fluorescence spectrophotometers?
Filters (cheap equip.) Monochromators
How does pH affect fluorescence?
Fluorescence is pH dependent for compounds with acidic/basic substituents, with fluorescence occurring mostly at only the basic pH (bc acidic states have resonance structures that stabilize the excited state)
Why is spectrofluorometry potentially more sensitive than spectrophotometry?
For spectrofluorometry, the analytical signal F is proportional to the source intensity P₀ and the transducer sensitivity. In spectrophotometry, the absorbance A is proportional to the ratio of P₀ to P. Increasing P₀ or the transducer sensitivity to P₀ produces a corresponding increase in P or the sensitivity to P. Thus the ratio does not change. As a result, the sensitivity of fluorescence can be increased by increasing P₀ or transducer sensitivity, but that of absorbance does not change.
What is the difference between a fluorescence emission spectrum and a fluorescence excitation spectrum?
In a fluorescence emission spectrum, the excitation wavelength is held constant and the emission intensity is measured as a function of the emission wavelength. In an excitation spectrum, the emission is measured at one wavelength while the excitation wavelengths are scanned.
What is a triplet state?
In the excited triplet state, the spins of the two electrons have become unpaired and are thus parallel, making the molecule paramagnetic. The excited triplet state is less energetic than the excited singlet state.
What are the smaller peaks next to the resonance fluorescence?
Non-resonance fluorescence
Why do some fluorescence bands NOT exhibit a Stokes shift?
Overlap occurs only for the resonance peak involving transitions between the lowest vibrational level of the ground state and the corresponding level of an excited state.
What effect does dissolved O₂ have on fluorescence?
O₂ is an excellent quencher that decreases fluorescence by oxidizing fluorescing species. Its paramagnetic property promotes intersystem crossing (spin flipping) and conversion of excited molecules to a triplet state
What detectors are used with fluorescence spectrophotometers?
Photomultipliers CCD cameras
What cells and sample compartments are used with fluorescence spectrophotometers?
Quartz cells Light tight compartments to minimize stray light
What variables determine the luminescence process that a sample ultimately undergoes?
Solvent Environment Type of molecule
How do you explain the Stokes shift exhibited by fluorescence bands?
Stokes shift is a consequence of the efficiency of vibrational relaxation.
How do fluorescence and phosphorescence differ?
The electronic energy transitions responsible for fluorescence do not involve a change in electron spin, so the excited states involved in fluorescence are short-lived (<10⁻⁵s)
Which fluorescence spectrum, emission or excitation, more closely resembles an absorption spectrum and why?
The excitation spectrum closely resembles an absorption spectrum since the emission intensity is usually proportional to the absorbance of the molecule.
How does the power of fluorescence emission relate to the radiant power of the excitation beam absorbed by the system?
The power of fluorescence emission (F) is proportional to the radiant power of the excitation beam (P₀) that is absorbed by the system.
What is the cause of the self-absorption contributing to deviations in linearity between fluorescence and concentration?
This happens when the wavelength of emission overlaps the absorption band. The emission traverses the solution and gets reabsorbed by other molecules such as analyte or other species in the solution, which leads to a decrease in fluorescence.
How is vibrational relaxation involved in the mechanism of fluorescence from solution?
Vibrational relaxation is so efficient that the average lifetime of a vibrationallv excited molecule is 10⁻¹²s or less, a period significantly shorter than the average lifetime of an electronically excited state. As a consequence fluorescence from solution always involves a *transition from the lowest vibrational level of an excited electronic state*. Several closely spaced emission lines are produced, however, and the *transition can terminate in any of the vibrational levels of the ground state*
When is predissociation observed?
When compounds have bonds that easily rupture and can then absorb excitation energy and go through internal conversion. Therefore, the relative intensity of fluorescence and fluorescent wavelength is not observed. -fluorescence seldom results from absorption of ultraviolet radiation of wavelengths shorter than 2S0 nm because such radiation is sufficiently energetic to cause deactivation of the excited states by predissociation or dissociation. For example, 200-nm radiation corresponds to about 140 kcallmol. Most organic molecules have at least some honds that can be ruptured by energies of this magnitude.
What is a singlet state?
When one of a pair of electrons of a molecule is excited to a higher energy level, a singlet or a triplet state is formed. In the excited singlet state, the spin of the promoted electron is still paired with the ground-state electron and the molecule remains diamagnetic.
Why is fluorescence a much faster transition than phosphorescence?
With fluorescence, the singlet e⁻s still work as a pair bc there is no net magnetic field effect. With phosphorescence, the unpaired spins in the triplet create a net mag field that slows the return to ground state and while emitting light
Why is a larger Stokes shift preferred and resonance fluorescence avoided?
a Stokes shift of at least 20nm is preferable in order to distinguish between excitation and emission spectra (overlapping in resonance)
How do transition probabilities differ with singlet and triplet states?
a singlet-to-triplet transition (or the reverse), which also involves a change in electronic state, is a significantly less probable event than the corresponding singlet-to-singlet transition.
How do we calculate fluorescence at low concentrations (A<0.05) and why do deviations occur at higher concentrations?
at low concentrations: F= 2.3K'εbcP₀ Deviations at higher concentrations (A >0.05) can be attributed to absorbance becoming a significant factor and by self-quenching or self-absorption.
Why is fluorescence rarely with σ*→σ transitions?
bc the high energy needed to achieve the excited state σ* ruptures the bond
Why must phosphorescence be performed at very low Temps?
bc the triplet state is very unstable, photoluminescence is limited to systems incorporating structural and environmental features that cause the rate of radiationless relaxation or deactiva- tion processes to he slowed to a point where the emission process can compete kinetically.
Why are quartz cells used with fluorescence spectrophotometers?
bc we are using wavelengths around 250nm
Why does fluorescence seldom occur from absorbance of UV wavelengths less than 250nm?
because radiation at this wavelength has sufficient energy to deactivate the electron in the excited state by predissociation or dissociation. The bond of some organic molecules would rupture at 140 kcal/mol, which corresponds to 200nm of radiation. For this reason, σ→σ∗ transition in fluorescence are rarely observed. Instead, emissions from the less energetic transition will occur which are either π∗→π or π∗→n transition
Why is the greatest quantum efficiency of fluorescence observed with π*→π transitions?
because such excited states exhibit relatively short average lifetimes (k, is larger) and because the deactivation processes that compete with fluorescence are less likely to occur.
How is fluorescence affected by decreased temperature and/or increased viscosity?
both will increase fluorescence by minimizing deactivation through collisions
How is quantum yield (Φ) maximized?
by decreasing factors that promote other deactivation processes
How do absorbance and fluorescence differ in their response to increasing the power of light?
cannot increase absorbance simply by increasing the power of light, but you CAN increase fluorescence by increasing the power of light
What happens in chemiluminescence?
chemical reaction yields an electronically excited species that emits light as it returns to ground state A+B→C*→C+hv
What happens to EM energy when you have absorbance?
deactivation process by which molecule returns to the ground or lower energy state (10⁻⁸ to 10⁻⁹s) via a non-radiative transition such as vibration, collision with other molecules, etc. M*→ M + heat
What is chemiluminescence?
emission of radiation by an excited species formed during a chemical reaction.
How are fluorescence and phosphorescence alike?
excitation is brought about by absorption of photons
What is the relationship between concentration and fluorescence?
if (2.303εbc=A)< 0.05, then fluorescence is proportional to concentration (linear relationship)
What determines whether relaxation is called absorbance, flurorescence, or phosphorescence?
it is determined by what happens to absorbed EM energy
Why is fluorescence used for quantitative analysis?
it is sensitive to the nM level
What are the energy conditions under which phosphorescence may occur?
kisc > kF + kec + kic + kpd + kd
What is luminescence?
light emission
How do luminescence methods compare to absorption spectrometry?
luminescence spectrometry has better sensitivity by a factor of up to 3 orders of magnitude and also has much larger linear concentration ranges
Which exhibits the larger Stokes shift, fluorescence or phosphorescence?
phosphorescence always occurs at a longer wavelength and with lower intensity
What is the function of a grating system in monochromators?
they can generate wavelength spectra continously
Intersystem crossing
process in which a molecule in one spin state changes to another spin state with nearly the same total energy; only way for e⁻ to go from singlet to triplet ∙enhanced if vibrational levels overlap ∙more common if molecule contains heavy atoms (I, Br)
External conversion
radiationless process in which a molecule loses electronic energy while transferring energy to the solvent or another solute
What is Quantum Yield (Φ)?
ratio of the number of molecules that luminesce to the total number of excited molecules → efficiency
What do the transition times for energy relaxing processes related to?
relative transition times indicate probability of the transition, with faster times indicating higher probability of that transition occurring over slower ones
What is the heavy atom effect?
the "heavy atom effect" suggests that the probability of intersystem crossing (leading to phosphorescence) increases as the size of the molecule increases. (ex.) a halogen substitution decreases fluorescence as the molar mass of the halogen increases. -In heavy atom substitution such as nitro derivatives or heavy halogen substitution such as iodobenzene, the compounds are subject to predissociation.
What is the Stokes shift?
the difference between the excitation and emission wavelengths resulting from loss of energy via collisions
For qualitative and quantitative analysis, what value does luminescence measure?
the emission energy hv₂
For an excited molecule, what is the favored route to the ground state?
the one that minimizes the lifetime of the excited state
Define fluorescence.
the process by which a molecule, excited by the absorption of radiation, emits a photon while undergoing a transition from an excited single electronic state to a lower state of the same spin multiplicity (singlet→singlet transition)
Define phosphorescence.
the process by which a molecule, excited by the absorption of radiation, emits a photon while undergoing a transition from an excited triplet state to a lower state of a different spin multiplicity (triplet→singlet transition)
What does a total fluorescence spectrum show?
the relationship between excitation and emission intensity
When do you use fluorescence vs UV for analyte detection?
use fluorescence whenever you can bc the signal is much stronger (more sensitive) than UV
What types of transitions produce fluorescence?
π*→π (great quantum efficiency) π*→n
What can be done to decrease collisions in order to increase fluorescence or phosphorescence?
↓Temp ↑Viscosity ↓conc of quencher (Q)
In what ways does an analyte participate in chemiluminescence?
∙The *excited species can be the product of a reaction between the analyte and a suitable reagent* (usually a strong oxidant such as ozone or hydrogen peroxide), resulting in an emission spectrum characteristic of the oxidation product of the analyte or the reagent rather than the analyte itself. ∙Sometimes the analyte is not directly involved in the chemiluminescence reaction. Instead, the *analyte inhibits or has a catalytic effect* on a chemiluminescence reaction.
List the deactivation processes of the Jablonski diagram. slide 9
∙Vibrational relaxation (solvent collisions) ∙Internal conversion (dissociation/predissociation) ∙External conversion ∙Intersystem crossing ∙Phosphorescence
What are the main features of a high quality spectrofluorometer?
∙Xe lamp (sensitivity increases with lamp intensity) ∙both excitation and emission spectra ∙two grating monochromators (wavelength selectors) ∙quantitative analysis
How is quantum yield (Φ) determined?
∙determined by the relative rate constants (kx) of deactivation processes Influenced by environment: ∙ext/int conversion (S₁→S₀) ∙intersystem conversion (S₁→T₁) Chemical structure-dependent: ∙predissociation/dissociation ∙fluorescence
What is photoluminescence?
∙light emission from any form of matter after the absorption of photons (EM radiation) ∙initiated via excitation by photons (hence the prefix photo-). ∙includes both fluorescence and phosphorescence ∙occurs at longer wavelengths than that of the excitation radiation
What light sources are used with fluorescence spectrophotometers?
∙low pressure Hg lamp (vapor gives 254, 302, 313nm lines) ∙high pressure Xe arc lamp (wide spectrum) ∙lasers
What extra features do spectrofluorometers have for phosphorescence?
∙sample cell in cooled Dewar flask with liquid nitrogen ∙delay between excitation and emission
What happens to EM energy when you have fluorescence?
∙transition from ground (singlet) state to singlet state and back (10⁻⁵ to 10⁻⁸s) ∙Some energy is lost through various processes (e.g. non- radiative transitions) and then light is given off.
What happens to EM energy when you have phosphorescence?
∙triplet to singlet transition (10⁻⁴ to 10⁰s) ∙The molecule transitions from an excited triplet state to a lower energy singlet state and gives off light at a much longer wavelength that in fluorescence. ∙Non-radiative transitions intervene
How does a total fluorescence instrument work?
∙uses an array detector (CCD) to collect total fluorescence spectrum ∙all wavelengths of light go to the sample so that all can examined simultaneously