7. Fluorescence Spectroscopy

i) 6.1433·1014Hz

ii) 2770.1 nm

iii) 4.0706-20J

= x eV = 24.5414 kJ/mol= x kcal/mol

Absorption can be described using Lambert-Beer’s Law:

Via irradiation with light of UV/Vis wavelengths, valence electrons are excited, leading to transitions to orbitals of higher energy.

Depending on the quantum numbers of the electron and the energy of the radiation, different transitions occur with different likelihoods.

UV/Vis absorption spectroscopy measures the amount of UV and visible light that is absorbed by the sample.

Molecules absorb light when the dipol moment is not zero. Most biochemically relevant molecules absorb in UV/Vis wavelengths.

The absorption wavelength depends on the chain length and the number of delocalized electrons.

Energy of pi-electrons in the system:

Absorption wavelength:

The major advantage of UV/Vis spectroscopy is its sensitivity.

The phosphorescence peak cannot be at a higher wavelength than the fluorescence peak because reaching the triplet state (necessary for phosphorescence) requires more energy than staying in the singlet state (leading to fluorescence).

Relaxation types can be seen in the Jablonski diagrams:

(only pi-orbitals are shown)

Non-radiative transitions

• Vibrational relaxation (VR)

  • relaxation of the excited electronic state to its lowest vibrational level

  • heat release

• Internal conversion (IC)

  • relaxation to a lower electronic state, often followed by VR

• Intersystem crossing (ISC)

  • transition to a state with different spin multiplicity

  • often followed by phosphorescence

Radiative transitions (emission)

• Fluorescence (singlet state)

• Phosphorescence (triplet state)

Time scales:

Absorption is very fast, basically instantaneously (fs).

IC is almost as fast.

VR is much slower compared to IC, but still very fast compared to radiative transitions (ps).

Fluorescence happens in ns timescale.

Phosphorescence is quite slow (ms).

The wavelength of emission is greater (—> lower in energy) than the absorption wavelength because some energy is “lost” during the non-radiative relaxations.

The probability of an electron returning to a certain vibrational level in the ground state is similar to the probability of the electron to be in this position before excitation.

The emission spectrum is independent of the excitation wavelength. If we excite only with one particular wavelength, the emission spectrum still looks like the mirror image of the absorption spectrum of the compound.

Exceptions:

Quinine

The absorption spectrum of quinine has peaks for the transition of S0 to S1 and of S0 to S2. The emission spectrum only shows a peak for the transition of S1 to S0 because the transition of S2 to S1 has occurred non-radiatively (e.g., internal conversion).

Fluorescence lifetime is the average time that a molecule spends in its excited singlet state S1 before spontaneous emission occurs.

kf: radiative rate constant

knr: non-radiative rate constant

Fluorescence lifetime is measured by determining the phase modulation:

For fluorophores with short lifetimes, the resulting emission wave has a small phase shift and small decrease in amplitude.

For fluorophores with long lifetimes, the resulting emission wave has a large phase shift and large decrease in amplitude. Reason: The longer the electron stays in the excited S1 state, the more energy is lost to vibrational transitions.

This is measured by Time-Correlated Single-Photon Counting (TCSPC).

• pH

• pressure

• viscosity

• temperature

• quenchers

• electric potential

• ions

• polarity

Depending on the solvent, a fluorophore can have different emission wavelengths.

• depends on the dielectric constant and the hydrogen bonding capacity

As a result, the size of the gap between excitation and emission wavelength changes.

Depending on the polarity of the solvent, some energy of the excited state is “lost” to the solvent.

Reason: When excited, the dipol moment of the fluorophor and the solvent are mismatched. Relaxation means that the dipol moments of the solvent molecules align with the one of the fluorophor. This process requires some energy.

This effect is smaller for more unpolar fluorophores.

There are two types of quenching:

• collisional quenching: fluorophore in excited state collides with quencher and returns to ground state without emitting radiation

• static quenching (or contact quenching): fluorophore in the ground state is bound by the quencher and cannot be excited anymore

In collisional quenching, quenching is increased when temperature rises because collision of the quencher and the fluorophor is more likely.

In static quenching, quenching is decreased when temperature rises because the quencher-fluorophor aggregates are more likely to break apart.

The type of quenching and the Stern-Vollmer constant can be calculated using the Stern-Vollmer equation and the Stern-Vollmar plot:

Collisional quenching:

Only when collisional quenching occurs, this will be true:

Static quenching:

tao_0: lifetime of the unquenched fluorophor

Fluorescence anisotropy: Fluorophors show different emission intensities in different polarizations.

This effect increases with the size of the molecule and the viscosity of the solvent. The fluorescence polarization increases as the mobility of the emitting species decreases.

Fluorescence anisotropy (how much the movement of the fluorophores is restricted) can be measured by exciting with polarized light and putting a polarization filter in front of the detector:

This can then be used to measure how many molecules are in the same orientation as the polarizer (left) and how many have undergone rotational diffusion.

This can be used in biochemical context to determine the rotational freedom of biomolecules:

When anisotropy is low, the small molecule of interest is most likely free to rotate fast. When anisotropy is high, it might be bound to a larger structure.

Anisotropy is temperature-dependent and decreases with higher T. In the case of membranes, the addition of cholesterol ensures stability of anisotropy even at higher temperatures.

Light source (ideally emits light over multiple wavelengths in the same intensity)

Excitation monochromator (to get the wavelength you want)

Sample chamber (where sample is illuminated with light of the desired wavelength)

In a 90° angle: lenses etc. to direct the fluorescing light where we need it

Emission monochromator

Detector (photomultiplier tube, must detect photons of all wavelength with the same efficiency)

A photomultiplier tube is a type of detector for light.

Incoming photons are directed throught a series of charged plates (dynodes) where electrons are converted to photons (the initial photons are multiplied).

This increases the number of photons so you can get a signal from very low initial number of photons.