Uv visible spectroscopy

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1. UV-VISIBLE SPECTROSCOPY Prepared By 1 Asmita S. Satao , 2 Bhupeshkumar Nanhe 1 M.Sc. Sem - 2 (Analytical Chemistry), Fergusson College, Pune (MH) 2 Assistant Professor, Dept. of Forensic Science, Shri Shivaji College, Akola (MH)

2. CONTENT 2/24 Introduction Electromagnetic Radiations Electromagnetic Spectrum UV-Visible Spectroscopy Principle UV-Visible Spectrophotometer Radiation source Monochromator Sample cell Detector Recorder Sample preparation Applications Advantages Limitations

3. Spectroscopy is the interaction of electromagnetic radiation with matter. After interaction, may variation in intensity of electromagnetic radiation with frequency. The device which is used to measure variation of EMR is called as spectrophotometer or sometime called as spectrometer. Spectroscopy is an scientific tool which is used for the study of atomic as well as molecular structure of matter. Introduction 3 /24

4. Explain EMR The radiation constitutes electric and magnetic field is known as electromagnetic radiation (EMR) . EMR may be produced by changing electric and magnetic field simultaneously which act at right angle to each other and also perpendicular to the direction of propagation of electromagnetic waves. Electromagnetic radiation Properties of Electromagnetic Radiations Electromagnetic waves are transverse in nature. These waves can propagate in vacuum also. They may produced optical effects. They can be polarized. 4/24

5. Electromagnetic SPECTRUM The electromagnetic spectrum is bunch of energies which are arranged in order of increasing wavelength and decreasing frequency . With increasing wavelength, gamma rays situated in first position with low wavelength and highest frequency and it has more energy as compare to other electromagnetic waves. Cosmic rays having shorter wavelength and highly energetic. Radio waves are longer wavelength, hence less energetic. 5 /24

6. Diagram of electromagnetic radiation Electromagnetic SPECTRUM 6 /24

7. UV visible spectroscopy. UV-visible spectroscopy uses electromagnetic radiations of UV and Visible region. UV rays ranging from 180 to 380 nm and visible region ranging from 380 to 720 nm of electromagnetic spectrum. UV-Visible spectroscopy refers to absorption spectroscopy. Molecules containing bonding and non-bonding electrons can be absorbed in the form of UV or visible light and gets excited into higher molecular orbital. It is also knows as electron spectroscopy. Uv -visible spectroscopy 7/24

8. Principal Many molecules absorb UV visible light. The problems of solution increases as attenuation of the be am increases. Absorbance directly proportional to the path length (l) and con centration (c) of absorbing species . The intensity of absorption is governed by two fundamental laws of absorption known as B eer and Lambert law . B eer ' s and Lambert ‘s law : when a beam of light passes through a transparent sample cell containing a solution of absorbing substance the intensity of light reduces . PRINCIPLE 8/24

9. This is due to :- reflection of the inner and outer surface of cell. scattering by particle of solution. absorption of light by molecule in the solution . (Note: Since it is not possible to directly measure the amount of radiation absorbed by partially transparent substance it may be measured by calculating the difference in intensity between original and transmitted radiation falling on the sample ). I absorbed = I o – I t ( Where as I o is the original intensity falling on the cell and I t total intensity) Continue… 9/24

10. Beers law :- When rays of monochromatic light passes through and absorbing medium, its intensity decreases export exponentially as the concentration of absorbing material increases . Lambert’s law :- When the ray of monochromatic light passes through an absorbing medium its intensity decreases exponentially as a length of absorbing material increases . Continue… 10/24

11. UV visible spectro photometer is a devices which is used to measure absorbance of samples . Spectrophotometer available in market with two type; one is single beam in which only one sample can be investigate and another is double beam spectrophotometer in which two samples can be investigate (questioned & reference sample). It consist of following parts; 1) R adiation source . 2) M onochromat or . S ample cell . D etector . D ata output and recording device . Uv -visible spectrophotometer 11/24

12. Uv -visible spectrophotometer: Block Diagram 12/24

13. The radiation source is D euterium lamp used for UV region ha ving wavelength range of approximately 190-450 nm . For the visible region T ungsten filament bulb is used having wavelength range from about 332 – 850 nm . Xenon arc lamp also is used in entire UV visible region. The condensing mirror is rotated manually to focus the light emitted from other source into the entrance slit of the monochromator Radiation source 13 /24

14. It is a device for isolating monochromatic or narrow band of radiant energy from the source . It will allow to pass radiant energy of particular wavelength . Quartz prism was used in earlier photometer but now they are completely replaced by diffraction grating . It mainly consists two part s; 1 ) S lit 2 ) D ispersion Monochromator 14 /24

15. Pyrex glass in visible region is satisfactory material for sample cell but below 350 nm (UV region) wavelength quartz i.e. glass of pure silicon dioxide must be used . Sample cell Fig. Various types of Sample Cells 15 /24

16. D e t ec t o r The detector measures the quantity of radiation that passes through the sample converting it in to the electric signal . The most common detector used are vacuum photo cells and this is based on photoelectric effect . Receiving signal is obtained when a photon of sufficient energy strikes the metals of face cause the election of an electron . T his is determined by quantitative measurement of amount of light passing through instrument by means of detector . Detector 16/24

17. Another type of detector is photomultiplier tube (PMT) which is frequently use for the detection of absorbance in spectroscopy. Continue… Fig. Photomultiplier tube 17 /24

18. Recorder Sample signal output can be analog absorbance or transmittance meter where the data can be read, recorded and processed. Some spectrophotometer having attached computer for monitoring the instrument and record the absorbance. 18 /24

19. Sample preparation UV-Visible spectroscopy allows transparent solid, liquid and gaseous sample. Very small amount such as microgram or microliter sample is sufficient for detection. There are some point to be consider while preparing the sample; High purity of transparent solvent should be used. Aqueous system often requires for the use of buffer system. Sample clean up may be required before investigation. Turbid sample should be filtered to reduced light scattering. 19 /24

20. Application(s) UV-Visible spectroscopy used to detect conjugations. It also used to detects isomers. Detection of functional group can be possible in this technique. It is also use for identification and quantification of organic as well as inorganic compound. It is also use for research and development of science and technology . It also play key role in ink and fibers in Forensic science. UV-Visible spectroscopy is widely used as a significant tool for both qualitative and quantitative drug analysis . 20/24

21. Advantages UV-Visible spectroscopy is an quick and inexpensive technique available for identification of certain class of material. It can detect minute amount of sample such as microliter or microgram. Quantity of sample can be done precisely and reproducibly. 21/24

22. limitations Extensive sample preparation is required. The sample should be purify before analysis. Broad band obtained in this technique. Sometime definite identification is not possible for this band broadening. 22/24

23. Conclusion UV-Visible spectroscopy is one of the most important tool used for the purpose of identification and quantification. It has wide applications in various field such as Chemistry, Forensic Science, Biochemistry, Pharmaceutical industry, Cosmetics Industry, Petroleum Industry, Food Industry and many more. It has wide used because most of the molecules present in the universe shows absorption in UV-Visible region of electromagnetic spectrum. It acts as a screening technique in most of the cases to identify molecule before performing other highly sophisticated technique of that sample molecule. Other advantages with this technique is that it is simple to perform and inexpensive. But sometime sample preparation is difficult and it will depends upon the nature of sample. 23/24

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