2019-FluorescenceSpectroscopy-Home-KSUFacultyMember…荧光光谱法-家居-堪萨斯州立大学教员…-文

1、Fluorescence SpectroscopyPrinciples Interaction of photons with molecules results in promoti on of vale nee electr on sfrom ground state orbitals to high energy levels The molecules are said to be in excited state Molecules in excited state do not remain there long but spontaneously relax to more st

2、able ground state The relaxation process is brought about by collisi onal en ergy tra nsferto solve nt or other molecules in the solution. Some excited molecules however return to the ground state by emitting the excess energy as light This process is called fluoresce nee.Energy-level diagram outlin

3、ing the fluorescence process.Excited state, S/Ground state, GDistance between atoms in a moleculeFluorescence spectroscopy -FAIIIII:iII*IExcitation-The process (1)Euerav exclianse with suiToundiiia moleculesEmission The emitted light has two important characteristics :It is usually of Ion ger wavele

4、 ngth (lower energy) than the excited light This is because part of the energy associated with S state is lost as heat energy.2. The emitted light is composed of many wavelengths which results in fluorescence spectrum.Quantam yield Q The fluoresce nee inten sity is described in terms of quantum yiel

5、d The quantum yield Q is the 如/o 屮加 number of photons emitted to the number of photons absorbedIntrinsic Fluors Some biomolecules are intrinsic fluors ie.z they are fluoresce nt themselves The amino acids with aromatic groups eg3henylalamine, tyrosine, tryptophan are luorescent Hence proteins contai

6、ning these ami no acids have intrinsic fluoresce nee. The purine and pyrimidine bases and some coenzymes eg NAD and FAD are also intrinsic fluors Intrinsic fluoresce nee is used to study protei n conformation changes and to probe the location of active site and coenzymes in enzymesExtrinsic Fluors T

7、hese are fluoresce nt molecules that are added in biochemical system under study Extri nsic fluoresce nee has bee n used to study the binding of fatty acids to serum albumin, to characterize the binding sites for cofactors and substrates in enzyme molecules and to study the intercalaUon of small mol

8、ecules into the DNA double helix.Fluoresce nt molecules useful in biochemical studies.n(ch3)2o=s=o1 -Anili no8 naphthale nesulfonate (ANS)Dansyl chlorideFluoresceinAmino methyl coumarin(AMC)2H2NAcridine orange ANS, dansyl chloride, fluorescein are used for protein studies Ethidium, proflavine and ac

9、ridines are used for nucleic acid characterization. Ethidium bromide has enhan ced fluoresce nee when bound to double stranded DNA but not single stranded DNA.Not all compounds fluoresce. Fluorophores requiie Aiomatic rmg Tlie potential for increased extended conjugation At least one electron donati

10、ng group -OH. -NH? attached to the aromatic ring (note : electron withdrawing groups can diniinish or often destroy fluorescenceFluorescentOHFluoi'osceiiiIl ElectrqDlnhcii Linkms functional eroup sFluorosceiii isothiocyanate (FITC)Instrumentation The basic instrument is a spectrofluorometer. It

11、contains a light source, two monochromators, a sample holder and a detector. There are two monochromators, one for selection of the excitation wavelength, another for an alysis of the emitted light The detector is at 90 degrees to the excitation beam Upon excitation of the sample molecules, the fluo

12、resce nee is emitted in all directio ns and is detected by photocell at right angles to the excitation light beam The lamp source used is a xenon arc lamp that emits radiation in the UV, visible and n ear-i nf rared regio ns. The light is directed by an optical system to the excitation monochromator

13、, which allows either preselection of wavelength or seanning of certain wavelength range. The exciting light then passes into the sample chamber which contains fluoresce nee cuvette A special fluoresce nt cuvette with four translucent quartz or glass sides is used When the excited light impinges on

14、the sample cell, molecules in the solution are excited and some will emit light Light emitted at right an gles to the in comi ng beam is analyzed by the emissionmono chromator. The wavelength analysis of emitted light is carried out by measuring the intensity of fluoresce nee at preselected wavele ngth. The analyzer monochromator directs emitted light of the preselected wavelength to the detector. A photomultiplier tube serves as the detector to measure the intensity of the light The output