Chemistry Reference
In-Depth Information
Fluorimetry
Fluorimetry is an analytical technique that relies on the emission of electro-
magnetic energy by molecules. The chromophore of the molecule must be
capable of absorbing light (usually in the ultraviolet region of the electro-
magnetic spectrum) and emitting it again (usually in the visible portion of
the spectrum) to be measured by a detector. To do this, the chromophore
(sometimes called the
fluorophore
) must be shielded from the normal
processes that account for energy loss in the excited state (e.g. collision
between molecules).
The light that is emitted by the sample is always of
longer wavelength (i.e. lower energy) than the light absorbed by the
molecule
. This is known as Stokes' law (after the Irish physicist Sir George
Gabriel Stokes) and applies because the energy transfer process occurring
within the excited state of the molecule is not 100% efficient. Some of the
absorbing energy is lost (e.g. in vibrational transitions) and so the light
emitted as fluorescence is of lower energy than the light absorbed.
The instruments used to measure fluorescence, spectrofluorimeters,
require a high-energy light source (usually a xenon arc lamp) to deliver the
energy required to excite the molecule, and the detector of the instrument
is usually aligned at 90
to the source to minimise detection of light directly
from the light source. A spectrofluorimeter also requires two monochroma-
tors, one to select the wavelength of excitation light and the other to select
the wavelength of light emitted by the sample. Analytical spectrofluor-
imetry is widely used in pharmaceutical analysis, particularly for the assay
of highly potent drugs present in medicines in tiny amounts.
There are two main advantages in the use of fluorimetry over ultraviolet
spectroscopy:
The presence of two monochromators, and the fact that not all molecules with a
chromophore fluoresce, means that fluorimetry is more specific than ordinary
ultraviolet spectroscopy. This allows drugs that fluoresce to be assayed in the
presence of other compounds that would interfere in an ultraviolet assay.
1.
Fluorimetry is approximately 100 times more sensitive than ultraviolet spec-
troscopy and is ideal for the analysis of very small amounts of potent drugs.
Examples are the steroids digoxin in Digoxin Tablets BP and the contraceptive
agent ethinylestradiol, which is present at levels of only 30 lg per tablet.
2.
Quenching
This phenomenon, as its name suggests, is a reduction in the intensity of
light emitted during fluorescence. There are two types: self-quenching and
quenching by other, non-fluorescent agents.
