SPECTROPHOTOMETER (2).pptx measurement by using different wavelenght of light and uv rays

it is instrument used to measure the cocentration of solution

1. Spectrophotometer (SHIMADZU UV-1601) Presenter: Dr.Harshit Tiwari (JR3) Moderator: Dr Shakeel Ahmad( SR) Peer support: Dr. Savitha A K (JR2) Department of Pharmacology and Therapeutics King George’s Medical University, Lucknow, UP, India- 226003 Email: [email protected]

2. Contents Introduction Principle Parts of Instrument & components inside Working mechanism Standard operating procedure Applications Limitations Summary References

3. Specific Learning objectives At the end of this session, the audience will be able to: 1) Identify and describe the key components of the instrument 2) Comprehend the principle of operation and functionality of the spectrophotometer 3) Operate the spectrophotometer 4) Utilize the spectrophotometer effectively for various laboratory applications

4. Introduction Arnold O. Beckman in 1940 Measures the amount of light absorbed by a sample Estimation of transmittance or reflectance of a solution Scientific fields - biochemistry, chemical engineering, molecular biology

5. Beer-Lambert law Absorbance of light is directly proportional to both the concentration of the absorbing medium and the thickness of the medium. Thickness of the medium is called ‘ pathlength ’ A = ε bC =Log 10 I /I A – absorbance ε – molar absorptivity b - length of light path C- concentration Principle I = Intensity of the incident light I = Intensity of transmitted light

6. SHIMADZU UV-1601 Spectrophotometer

7. Requirements A pair of cuvette Beaker Sample solution Reference solution Plastic pipette

8. Parts of Instrument Front View Sample compartment Keyboard LCD unit

9. Parts of instrument LCD unit keyboard

10. Parts of instrument Sample cell holder Reference cell holder Sample compartment

11. Parts of instrument Left side view Power Switch button O - OFF I - ON Ground Terminal Power cable

12. Parts of instrument Right side view

13. Parts of instrument Deuterium lamp Tungsten halogen lamp Mirror Light source compartment

14. Components 1. Light source: High output of polychromatic light Tungsten lamp, hydrogen or deuterium lamps and xenon flash lamps. 2. Dispersion devices : Monochromator It consists of three parts: entrance slit, dispersion device , exit slit 3. Focusing devices : Combination of lenses, slits and mirrors

15. 4. Absorption cells (Cuvettes): Cell sealed at one end, made of plastic, glass or optical grade quartz Hold samples for spectroscopic experiments 5. Detectors: Photosensitive device - detector of radiant energy Produce voltage signals proportional to the intensity of the light striking them 6. Display devices : Data displayed by a readout device Analog meter (light beam reflected on a scale)or a digital display or LCD

16. Working Mechanism https://www.researchgate.net/figure/Schematic-diagram-illustrate-the-essential-features-of-UV-Vis-spectrophotometer_fig44_343366480

17. Standard Operating Procedure Turn on the power switch Wait for the instrument to initialize After initialization done ,LCD will show main mode screen Initialization done

18. Main mode screen Standard Operating Procedure Press button ‘2’ on the keyboard to access the Spectrum mode

19. Set the measurement mode and scanning range Standard Operating Procedure

20. For the first run: must do blank solution scan (Both the reference and sample cell contain solvent only) Open the lid of sample chamber Wipe the transparent side of cuvette with tissue paper(Avoid Fingerprints) Insert the cuvette in such a way that the transparent sides are on side ways i.e., between light source and photo diodes Standard Operating Procedure

21. Press ‘Auto zero’ key for blank correction Takeout the sample cuvette  Discard the solution Rinse the cuvette Fill the cuvette with sample solution and place it in sample cell holder Close the lid Standard Operating Procedure

22. Press start button on keyboard Graph will start running and it will show highest peak (Maximum absorption at specific wavelength) Used for Identification/Analysis of compounds Standard Operating Procedure

23. Pharmaceutical applications Drug analysis Quality control In Chemical analysis Quantification of concentrations Reaction kinetics Environmental monitoring Water quality testing Air quality monitoring Applications

24. Biochemical & Biomedical research Protein quantification DNA/RNA quantification Enzyme activity assays Clinical diagnostics Hemoglobin measurement Bilirubin testing Industrial applications Quality control in manufacturing Applications

25. Limitations Dependence of chromophores Limited sensitivity for low concentrations Spectral Limitation Light scattering by sample impurities Sample preparation requirements Need for Calibration curve

26. Summary Spectrophotometer is an analytical instrument measures the amount of light absorbed or transmitted by the analyte Follows Beer Lambert law Different modes has different practical applications In Pharmacology ,it is used in drug analysis and quality control

27. References Sarkar S, Srivastava V, Mohanty M.Postgraduate pharmacology. 1st ed.Paras Medical Publisher;2020 Shimadzu Corporation. UV-1601 Shimadzu Recording Spectrophotometer User’s System Guide. Japan: Shimadzu Corporation; 1994 https://youtu.be/pxC6F7bK8CU?si=Hsx-emnfG31aDs3Y https://youtu.be/V0Ht3uTAU2E?si=G26ofzKdujeIE3Wb

28. Questions What is the working mechanism of Spectrophotometer? Define Beer Lambert law Describe parts of the instrument What are the applications of Spectrophotometer? What are the limitations of this instrument?

29. THANK YOU