- Observing fluorescence
If a solution contains a fluorescent substance (a fluorochrome)
it emits fluorescence when illuminated with light of appropriate
colour (wavelength) - the excitation light. Fluorescence is best
observed at a right angle to the axis of the excitation light because
this avoids looking into the excitation lamp. In a fluorescence
spectrometer, a light detector(PMT = photomultiplier tube) is ususally
mounted at a right angle to the excitation light path. Excitation
light and fluorescence can also be separated using optical filters.
- What is fluorescence?
The processes that lead from the
absorption of excitation light by a fluorochrome to the emission of
fluorescence can be illustrated by energy diagrams named after the
polish physicist Alexander Jablonski. A Jablonski diagram depicts the
various electronic energy levels of a fluorochrome molecule. Many
fluorochromes have aromatic ring structures. Such molecules possess
delocalized electrons in so-called pi-orbitals. Electrons in pi orbitals
interact readily with the environment. Through absorption of a photon
such an electron can be boosted into a higher orbital. Electrons in pi
orbitals usually have anti-parallel spin, characterizing the singlet
states S0, S1, and S2. In the dark, electrons sit in the resting state
S0. When absorbing a photon of the right wavelength, the electrons reach
the excited states S1 or S2. This is a very fast process, developing
within 10-15 s. Excited electrons may hop from S2 to S1
without emitting anything (internal conversion). However, when returning
to the ground state S0 (typically after a few nanoseconds), the energy
difference is released as a fluorescence photon. The energy of the
fluorescence photon is always lower than the energy of the absorbed
excitation photon. Therefore, the wavelength of fluorescence light is
always longer than that of the excitation light (Stokes shift).
In some compounds
electrons can reach a triplet state (T1) when jumping back from an
excited singlet state. This process (intercombination) requires a spin
reversalof the excited electron, and also the jump back to the ground
state requires spin reversal. Such a process occurs with very low
probability, and emission rates are very low (1 - 1000 per second).
Compounds showing this property are phosphorescent. The low rates of
interconversion and emission in these substances cause a slow decline of
light emission, causing a persistent "glow" in the dark.