(Bright,
1988) fluorescence, theory, applications, HPLC, high performance liquid chromatography, technique, luminescence, method, detection, detector, spectroscopy, bioanalytical, biology, analytical, energy, photon, ground state, excited state, state, excited, ground, spins, DNA, RNA, enzymatic methods, protein-ligand interaction, protein, ligand, enzyme, analysis, chemistry, analytical chemistry, university of kentucky, college of pharmacy, promotion
HPLC is only one of many assaying techniques that utilizes
luminescence as a method of analysis and detection. Fluorescence
spectroscopy is therefore a versatile method that can be employed
in many fields. This section attempts to explain the theory of
fluorescence and mentions its usefulness with regard to certain
bioanalytical applications.Most compounds and molecules occupy
the lowest energy state, which is referred to as the
"ground" state. These species can in turn absorb
electromagnetic radiation as long as the incident photon contains
an energy level equal to the difference between two
"allowed" energy states. This results in the promotion
to a higher energy or "excited" state. Each of these
energy states can be classified as singlet or triplet states,
where the difference lies in the spin of the electrons. Each
electron has either a -1/2 or +1/2 spin. In the ground state, the
electrons are paired and each has opposite spins. The orbital
angular momentum of any energy state is predicted by the equation
M = 2S + 1, where M is multiplicity and S is total spin. Thus,
for ground state molecules where the electrons are usually
paired, M = 2(-1/2 +1/2) + 1 = 1. When the spin of one electron
reverses, M becomes equal to 3. States where M = 1 are singlet,
whereas those when M = 3 are triplet states. The latter is
attained when a compound is raised to an excited singlet state
and then "crosses" to a triplet state.(Bright,
1988)
1) DNA/RNA. Fluorescence spectroscopy is useful when quantifying the amount of DNA or RNA in a sample. The reagents Hoechst-33258 and ethidium bromide are commonly used (Bright, 1988) . The former is a fluorescent dye that increases the fluorescent intensity of DNA (excitation 355 nm, emission 460 nm) without significantly accomplishing the same in RNA. Using this, the chromatographer can determine the amount of DNA in a tissue sample. Ethidium bromide can be used to determine DNA or RNA because it enhances the signal in both nucleic acids (excitation 530 nm, emission 620 nm). To determine RNA only, RNAase is added to one sample but not to a control sample. Both samples are incubated at 37 deg. C for 30 minutes. The RNAase should enzymatically destroy the RNA and the difference in intensity between the experimental (DNA alone) and control (RNA + DNA), the amount of RNA may be determined.2) Enzymatic Methods. Enzyme assays are widely employed by fluorescent techniques. There are several ways to implement a fluorometric analyzer; for example, some procedures measure the appearance or disappearance of fluorescent products formed from the interaction of enzyme and substrate.3) Protein-Ligand Interaction. A protein's functionality and biochemistry can be determined by studying the binding of the protein to a certain compound or chemical species. It is worth mentioning that fluorescence spectroscopy is becoming one of the leading procedures used to elucidate this interaction. The following reference gives more detailed information: (Haugland, 1985)
Haugland, R.P., Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes Inc.; Eugene, Oregon, 1985.
Bright, F.V. Analytical Chemistry , 1988, Vol. 60, pp. 1031-1039.
Exit to the University of Kentucky College of Pharmacy
fluorescence, theory, applications, HPLC, high performance liquid chromatography, technique, luminescence, method, detection, detector, spectroscopy, bioanalytical, biology, analytical, energy, photon, ground state, excited state, state, excited, ground, spins, DNA, RNA, enzymatic methods, protein-ligand interaction, protein, ligand, enzyme, analysis, chemistry, analytical chemistry, university of kentucky, college of pharmacy, promotiono