Documenti di Didattica
Documenti di Professioni
Documenti di Cultura
Biniam P 1
Objectives
At the end of this session the students will be able
To define florescence spectroscopy. To discuss structural requirements of fluorescent
compounds.
Biniam P
Emission spectroscopy
Emission spectroscopy is a spectroscopic technique which examines the wavelengths of photons emitted by atoms or molecules during their transition from an excited state to a lower energy state.
Biniam P
Luminescence
Luminescence is the emission of light from any substance, and occurs from electronically excited states. Luminescence is divided into two categoriesfluorescence and phosphorescence. The emission rates of fluorescence are typically 108 s1, so that a typical fluorescence lifetime is near 10 ns. The emission rates of phosphorescence are slow (103 to 100 s1), so that phosphorescence lifetimes are typically milliseconds to seconds. Fluorescence is much more widely used for chemical analysis than phosphorescence.
Biniam P 4
The first observation of fluorescence from a quinine solution in sunlight was reported by Sir John Frederick William Herschel in 1845.
Quinine
The quinine in tonic water is excited by the ultraviolet light from the sun. Upon return to the ground state the quinine emits blue light with a wavelength near 450 nm.
Biniam P 5
Thus fluorescence emission is typically shifted by 50150 nm (Stokes shift) to the longer wavelength in comparison to the wavelength of the radiation used to produce excitation.
Biniam P 8
Relaxation processes
Once the molecule is excited to E1 or E2 several processes can occur that cause the molecule to lose its
excess energy.
Two of the most important of these mechanisms,
Biniam P
10
Vibrational relaxation involves transfer of the excess energy of a vibrationally excited species to molecules of the solvent. This process takes place in less than 10-15 s and leaves the molecules in the lowest vibrational state of an electronic excited state.
Vibrational relaxation depicted by the short wavy arrows between vibrational energy levels takes place during collisions between excited molecules and molecules of the solvent.
Biniam P 12
Biniam P
13
Fluorescent species
It is not entirely possible to predict how strongly fluorescent a molecule will be. For example adrenaline and noradrenaline differ in their structure by only a single methyl group but nor adrenaline exhibits fluorescence nearly 20 times more intensely than adrenaline. Generally, flurescence is associated with an extended chromophore or auxochrome and a rigid structure.
Biniam P
16
and pyrrole, do not exhibit molecular fluorescence, but fusedring structures containing these rings often do for example quinoline, isoquinoline, indole.
Biinam P 17
Substitution on an aromatic ring causes shifts in the wavelength of absorption maxima and corresponding changes in the fluorescence peaks.
Biniam P
18
The influence of rigidity also explains the increase in fluorescence of certain organic chelating agents when they are complexed with a metal ion. For example, the fluorescence intensity of 8hydroxyquinoline is much less than that of the zinc complex.
Biniam P
20
Biniam P 21
Fluorescence spectroscopy
Fluorescence spectroscopy (fluorometry or spectrofluorometry), is a type of electromagnetic spectroscopy which analyzes fluorescence from a sample. It involves using a beam of light, usually ultraviolet light, that excites the electrons in molecules of certain compounds and causes them to emit light of a lower energy, typically, but not necessarily, visible light. This shift to longer wavelength is called the Stokes shift. Devices that measure fluorescence fluorometers or fluorimeters.
Biniam P
are
called
22
Instrumentation
The light from an excitation source passes through a filter or monochromator, and strikes the sample. A portion of the incident light is absorbed by the sample, and some of the molecules in the sample fluoresce. The fluorescent light is emitted in all directions. Some of this fluorescent light passes through a second filter or monochromator and reaches a detector, which is usually placed at 90 to the incident light beam to minimize the risk of transmitted or reflected incident light reaching the detector.
Biniam P 23
Biniam P
24
Light source
Xenon lamps
Biniam P
26
2. Heavy atoms in solution quench fluorescence by colliding with excited molecules so that energy is dissipated, e.g. chloride or bromide ions in solution cause collisional quenching. 3. Formation of chemical complex with other molecules in solution can change fluorescence behavior, e.g. the presence of caffeine in solution reduces the fluorescence of riboflavin. 4. In most molecules, fluorescent property decreases with increasing temperature because the increased frequency of collision at elevated temperatures increases the probability of collisional relaxation. A decrease in solvent viscosity leads to the same result.
Biniam P 28
Application
1. Determination of fluorescent drugs in low-dose formulations in the presence of non-fluorescent excipients. 2. In carrying out the limit tests where the impurity is fluorescent. 3. Useful for studying the binding of drugs to component in complex formulations. 4. Widely used in bioanalysis for measuring small amounts of drug and for studying drug-protein binding.
Biniam P 29
Reading Assignments
What is structural requirements of fluorescent compounds and What are the factors affecting fluorescence intensity Reference: Read it from Kenneth A. Connors, A text book of Pharmaceutical analysis page 213-214.
Biniam P 30