Fluorimetry presentation

presentation of fluorimetry

1. Fsn 503 food analysis(1+2) Presentation on “ Fluorimetry and its Application

2. Definition Principle Classification Instrumentation Parameters Applications Pros and Cons LEARNING OUTCOMES

3. Fluorescence: When a beam of light passes through a substances, the particles would absorbs some form of energy and emits the radiation. It is phenomenon in which the excited singlet electrons(the electrons are paired) emits emission radiation when brought down to ground level at the certain wavelength. Fluorescence occurs at (10-9 – 10-6 sec). DEFINITIONS

4. It is a type of electromagnetic spectroscopy that analyses the fluorescence from the sample. It involves the use of beam of light, usually ultra violet light that excites the electrons in the molecules of certain compounds (fluorescence substances) and that produces a light of lower energy FLUORIMETRY:

5. The principle involved is Vibrational relaxation. Molecules have bonding ( σ ) electrons and ( π )electrons and n non bonding electrons. When the excited electrons absorbs the radiant energy, the bonding electrons gets down to anti bonding orbital. Has high energy but less stable. it is a instantenous process PRINCIPLE:

6. The factors effecting fluorescence intensity are Concentration Quantum yield of fluorescence Intensity of incident light Adsorption Oxygen pH Temperature & Viscosity Photodecomposition Quenchers Scatter FACTORS INVOLVED

7. It is the process of reduction of fluorescence intensity by the presence of substances in the sample other than fluorescent analyte . Forms of quenching Self quenching Collision quenching Static quenching Inner fluorescent effect Quenching

8. Contd., Self quenching: Fluorescence intensity with concentration. Static quenching : Due to complex formation between fluorescent analyte and substances. Collision quenching: Collision between fluorescent substances and halide ions. Inner fluorescent effect: Absorption of incident light by filters decreases the fluorescent intensity.

9. Test solutions prepared for fluorescence spectrophotometer are usually 10 times to 100 times less concentrated than those used in absorption spectrophotometer. Concentrations of 10-5-10-7 mol/l are frequently used. Blank should be not less than 0.40 and not more than 2.50. PREPARATION OF SOLUTION

10. FLUORIMETER

11. The basic components of fluorimeter includes An excitation light source An excitation monochromator A cuvette An emission monochromator A detector A data analyser INSTRUMENTATION

12. Instrumentation

13. Mercury lamps are the most commonly employed light sources. They have the property that their spectral output depends upon the pressure of the filler gas. Mercury arc lamp-radiation @254 nm Xenon arc lamp- spectrum over the range between 250nm-600nm Tungsten lamp- intensity of lamp is low Turn able dye laser- radiation about 360nm-650nm Light source

14. Monochromator  produces monochromatic light by removing unwanted wavelengths from the source light beam. The  function of the monochromator  is to isolate a single atomic resonance line from the spectrum of lines emitted by the hollow cathode lamp. Excitation monochromators : Isolates only the radiation absorbed by the molecule Emission monochromators : Isolates only the radiation emitted by the molecule Monochromators

15. The proper selection of filters requires familiarity with the emission spectrum and the excitation spectrum. Primary filter: It absorbs the visible light and transmits UV light. Secondary filter: It absorbs the UV light and transmits visible light. Filters

16. Cuvettte are cylindrical or rectangular cells fabricated of silica or glass. They are meant for holding the diluted samples. Path length is about 10mm or 1cm Cells and other glassware used for fluorimetric analysis should be carefully cleaned, preferably by boiling in 50% nitric acid followed by thorough rinsing in distilled water. Cuvette

17. Detectors are device or instrument designed to detect the presence of a particular object or substance. Three forms of detectors are Photovoltaic cell Phototube Photomultiplier tube. Detectors

18. A  photomultiplier tube is a photo emissive device in which the absorption of a photon results in the emission of an electron . These detectors  work  by amplifying the electrons generated by a photocathode exposed to a photon flux. Cathode voltage is 75-100 V. Amplification of 10^6 is obtained. Electron_Multiplier_Continuous_Dynode.mp4 Photomultiplier tube

19. TYPES OF FLUORIMETER SINGLE BEAM FLUORIMETER DOUBLE BEAM FLUORIMETER In single beam fluorimeter , all the light waves pass through the sample. Primary filter– absorbs visible light. Secondary filter– absorbs UV light. Reference and sample cant be analyzed simultaneously In double beam fluorimeter the light  beam  splits into two parts and only  one  part passes through the sample. Primary filter– absorbs the light and passes to the sample. Secondary filter – absorbs the light from the sample and sent to detector. Reference and sample can be analyzed simultaneously

20. Contd., Single beam fluorimeter Double beam fluorimeter

21. The fluorescence intensity is proportional to the initial radiation times abc . log( Fo / Fo -F)= abc Fo-100 (standard) F- fluorescence measured b- cell length c- concentration a- proportionality constant B y Simplification, abc =2-log(100-F ) InShot_20191128_123357970.mp4 Quantitative analysis

22. Determination of inorganic substances; Determination of thiamine Determination of indoles , phenols, phenothiazines . Napthols ,proteins ,plant pigment and steroids can be determined. Detection of impurities at nanogram quantities. Detection of respiratory tract infection. Determines phenyltoin . Applications

23. Case study 1

24. Objective: Analyses of free glucose and glucose 6 phosphates in cow’s milk using fluorimetry . Materials and methods: Reagents- tris buffer The samples were excitated with 544 nm monochromatic light and read at 590 nm light.

25. The present enzymatic- fluorometric methods for determinationof total glucose and glucose 6-P in milk are reliable analytical methods This method matches the indigenous methods for the glucose analysis. The free glucose level in milk obtained in the present study, mean 331 lmol /l, inter percentile 143–529 lmol /l. Conclusion

26. Case study 2

27. Objective: Determination of Ag(I) in Some Food and Water Samples using fluorimeteric detection. Methods and materials: Ag(I) stock solutions (1000 mg/L) DKMS complex– fluorimetric reagent Rhodamine B hydrazide (RH) + ferrocenecarboxaldehyde (1 mmol , 0.21 g) in absolute ethanol (50 mL ) in the presence of catalysis acetic acid at refluxing temperature for 24 h.

28. GRAPHICAL REPRESENTATION

29. RESULTS

30. The procedure is highly sensitive, as well as selective, because it does not require the preparation of samples. Rapid response and high specificity The new sensitive system is inexpensive Has a low LOD (micrograms per liter). Easy in construction. Allows wide sample variation because it is interference-free. This system minimizes analysis time and the manpower needed CONCLUSION

31. Fifield&kealey ., principlkes and practice of analytical chemistry (2000) Syed&haddad ., textbook of analytical chemistry,(2011) https://www.slideshare.net/sreevidyavemuri/fluorimetry-45064390 http://www.au thorstream.com/Presentation/nalinisahoo- 2331005-fluorimetry/ https://www.chem.uci.edu/~dmitryf/manuals/Fundamentals/Fluorescence%20Spectroscopy.pdf http://srmbiotech25.yolasite.com/resources/Applications_of_Fluorimetry_and_Phosphorimetry.pdf https://www.nature.com/subjects/fluorescence-spectrometry https://link.springer.com/journal/10895 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2662353/ REFERENCES