spectrofluorimetry ppt

instrumentation

1. spectrofluorimetry Presenter :-Seema Bisht Moderator :- Dr. kanchan singh

2. History The term fluorescence comes from the mineral fluorspar (calcium fluoride) when Sir George G. Stokes observed in 1852 that fluorspar would give off visible light (fluorescence) when exposed to electromagnetic radiation in the ultraviolet wavelength.

3. Introduction Fluorescence is the phenomenon where by a molecule, after absorption radiation, emits radiation of a longer wavelength A compound absorbs radiation in the UV –region and emits visible light. Absorption of uv /visible radiation causes transition of electrons from ground state(low energy) to excited state (high energy ). This increase in wavelength is k/a the Stokes shift.

4. Principle Fluorescence is an emission phenomenon where an energy transition from a higher to a lower state is accompained by radiation. Only molecules in their excited forms are able to emit fluorescence ; thus they to be brought into a state of higher energy prior to the emission phenomenon.

5. Important Terms :- Singlet ground state : state in which electrons in a molecule are paired. Singlet excited state : state in which electrons are unpaired but of opposite spins . Triple state : state in which unpaired electrons of some spin are present . Excitation process : absorption of energy or light followed by conversion from ground state to excite state . Relaxation process : process by which atom or molecule losses energy &returns to ground state .

6. Basic concept :- It is an analytical device depends on the fluorescence phenomenon which is a short –lived type of photoluminescence created by electromagnetic excitation. That is ,fluorescence is generated when a molecule transmits from its ground state S o ,to one of several vibrational energy levels in the first excited electronic state S1 , or Second electronic excited state S2 , both of which are singlet states. Relaxation to the ground state from these excited states occur by emission of energy through heat or photons.

7. The difference between the excitation and emission wavelengths is called the Stokes shift. stokes’ studies of fluorescent substances led to the formulation of Stokes’ Law ,which states that the wavelength of fluorescent light is always greater than that of the exciting radiation. Thus , for any fluorescent molecule, the wavelength of emission is always longer than the wavelength of absorption .

8. What can spectroflurometer do ? It has been used for the direct or indirect quantitative and qualitative analysis by measuring the fluorescent intensity “F” . It is relatively inexpensive and sensitive (approximately 1,000 times greater than absorption spectrophotometric methods ) .

9. Fluorescent intensity (F) is dependent on both intrinsic properties of the compound (fluorescence quantum yield φ f ) , and on readily controlled experimental parameters including :- * intensity of the absorbed light Io *molar absorption coefficient ɛ *path length of the cell b *concentration of the fluorophor in solution c F= φ Io(1- e`ɛbc)

10. * At low concentrations of fluorophore ,the fluorescence intensity of a sample is essentially linearly proportional to concentration. * However ,as the concentration increases , a point is reached at which the intensity increases is progressively less linear, and the intensity eventually decreases as concentration increases further .

11. What is the fluorescence quantum yield ( φ f) ? It is the quantitative expression of the fluorescence efficiency ,which is the fraction of excited molecules returning to the ground state by fluorescence . Quantum yields range from 1, when every molecule in an excited state undergoes fluorescence , to when fluorescence does not occurs.

12. A molecule’s fluorescence quantum yield is influenced by external variables such as Temperature Viscosity of solvent pH . Increase temperature generally decrease Φ f because more frequent collisions between the molecule and the solvent increases external conversion . Decrease the solvent’s viscosity decreases φ f for similar reasons . For an analyte with acidic or basic functional groups, a change in pH may change the analyte’s structure and, therefore, its fluorescent properties .

13. Components of spectroflurometer Light source , Monochromator , Cuvettes , Detector , Amplifier , Read out device (Recorder).

14. Schematics of a spectrofluorimeter with ‘T’ geometry (90ͦdegree)

15. excitation emission

16. Light sources : -xenon arc lamp or mercury lamp * A high pressure xenon arc lamp is used for spectrometer ,which provides intense ,stable and continuous beam in UV and visible region. * Filter fluorimeters generally use low –power mercury vapour lamp which emits distinct line spectra in the UV and visible region .

17. Monochromator Two sets of monochromator are used :- primary monochromator , secondary monochromator . Primary monochromator :- it includes slit and dispersive device to isolate the wavelength for excitation of sample . Secondary monochromator :- isolates the wavelength of emitted fluorescence .

18. * Cuvettes :- usually quartz cuvettes are used . * Detector :- A phototube, photovoltaic cell or photomultiplier tube is used . * Read out device (Recorder) :- A galvanometer or a potentiometer is used as read out device .

19. Application of spectroflurometer For the chemical modification such as oxidation ,reduction ,hydrolysis, couping and self condensation . The determination and comparison of both excitation and fluorescence spectra of a compound may help to identify it . The assay of vitamin in foodstuffs, NADH in mitochondria , microorganism , hormones' like cortisol , oestradiol ,drugs (pharmaceuticals ) ,cholesterol ,and porphyrins . Enzyme assay and kinetic analysis . Study of protein structure .

20. Fluorescent indicators Mainly used in acid base titration. Intensity of colour of the fluorescence of many substances depend upon the pH of solution. These are called as fluorescent indicators and are generally used in acid base titration . Eg. – pH (3.0-4.0) eosin -colourless to green . Fluorescien –pH(4.0-6.0) colourless to green . Quinine sulphate-: blue – violet. Acridine : green – violet .

21. Advantages Fluoremetric techniques have a high degree of specificity . Nuclear research ( uranium salts). Precision upto 1% can be achieved easily . The tests based on fluorimetry are highly sensitive . It is possible to determine concentrations of fluorescing species down to nanogram and attogram rang . As both excitation & emission wave length are characteristic ,it is more specific than absorption methods . Qualitative and quantitative analysis of organic aromatic compounds present in cigarette smoke, air pollutants, automobile exhausts etc.

22. Disadvantages The susceptibility to environmental conditions and the virtual impossibility of predicting whether a compound will fluorescence . The other major problem is quenching, whereby the energy, transferred to the other molecules . Contamination can quench the fluorescence and hence give false /no result .

23. References Principle and techniques of BIOCHEMISTRY and MOLECULAR BIOLOGY-keith Wilson and john walker. TIETZ-fundamentals of clinical chemistry. Wikipedia . Google .