Seminar on applications of uv
- 1. Seminar on Applications of U.V. Spectroscopy Submitted to: Dr PREETI N TALLUR Submitted by: VAISHNAVI NAIK
- 2. Introduction to UV spectroscopy UV spectroscopy is type of absorption spectroscopy in which light of ultra-violet region (200-400 nm.) is absorbed by the molecule. Absorption of the ultra-violet radiations results in the excitation of the electrons from the ground state to higher energy state. The energy of the ultra-violet radiation that are absorbed is equal to the energy difference between the ground state and higher energy states. In all the compounds (other than alkanes), the electrons undergo various transitions. Some of the important transitions with increasing energies are: nonbonding to pie*, nonbonding to sigma*, pie to pie*, sigma to pie* and sigma to sigma*.
- 4. Principle of UV spectroscopy – UV spectroscopy obeys the Beer-Lambert law, which states that: when a beam of monochromatic light is passed through a solution of an absorbing substance, the rate of decrease of intensity of radiation with thickness of the absorbing solution is proportional to the incident radiation as well as the concentration of the solution. The expression of Beer-Lambert law is- A = log (I0/I) = Ecl Where, A = absorbance I0 = intensity of light incident upon sample cell I = intensity of light leaving sample cell C = molar concentration of solute L = length of sample cell (cm.) E = molar absorptivity From the Beer-Lambert law it is clear that greater the number of molecules capable of absorbing light of a given wavelength, the greater the extent of light absorption. This is the basic principle of UV spectroscopy.
- 6. Applications of UV spectroscopy 1. Detection of Impurities 2. Structure elucidation of organic compounds. 3. QUANTITATIVE ANALYSIS 4. QUALITATIVE ANALYSIS 5. CHEMICAL KINETICS 6. DETECTION OF FUNCTIONAL GROUPS 7. Estimation of extent of conjugation 8. EXAMINATION OF POLYNUCLEAR HYDROCARBONS 9. MOLECULAR WEIGHT DETERMINATION 10. AS HPLC DETECTOR 11. Determination of the purity of a substance- . 12.CLINICAL ANDPRE-CLINICAL APPLICATION IN CANCER 13.Determination of configurations of geometrical isomers
- 7. 1. Detection of Impurities UV absorption spectroscopy is one of the best methods for determination of impurities in organic molecules. Additional peaks can be observed due to impurities in the sample and it can be compared with that of standard raw material. By also measuring the absorbance at specific wavelength, the impurities can be detected.
- 8. 2. Structure elucidation of organic compounds. UV spectroscopy is useful in the structure elucidation of organic molecules, the presence or absence of unsaturation, the presence of hetero atoms. From the location of peaks and combination of peaks, it can be concluded that whether the compound is saturated or unsaturated, hetero atoms are present or not etc
- 9. 3. QUANTITATIVE ANALYSIS UV absorption spectroscopy can be used for the quantitative determination of compounds that absorb UV radiation. Many drugs are in the form of raw material or in the form of formulation. They can be assayed by making a suitable solution of drug in a solvent and measuring the absorbance at specific wavelength. Diazepam tablet can be analyzed by 0.5% H2SO4 in methanol at wavelength 284 nm.
- 10. 4. QUALITATIVE ANALYSIS Qualitative analysis through spectrophotometric methods achieves fast and accurate results using only small sample quantities. This fast and effect instrumentation has become an essential tool in the pharmaceutical industry thanks to its adaptability and economic value. Qualitative analysis has proven highly useful in many major forms of organic compounds UV absorption spectroscopy is generally used for characterizing aromatic compounds and aromatic olefins.
- 11. 5. CHEMICAL KINETICS – Kinetics of reaction can also be studied using UV spectroscopy. The UV radiation is passed through the reaction cell and the absorbance changes can be observed. – Spectrophotometry is a commonly used method to experimentally determine the reaction rates . The UV- VIS spectroscopy can measure the change of the concentrations of reactants or products over time
- 12. 6. DETECTION OF FUNCTIONAL GROUPS – UV spectroscopy is used to detect the presence or absence of chromophore in the compound. This is technique is not useful for the detection of chromophore in complex compounds. The absence of a band at a particular band can be seen as an evidence for the absence of a particular group. – f the spectrum of a compound comes out to be transparent above 200 nm than it confirms the absence of – a) Conjugation – b) A carbonyl group C) Bromo or iodo atoms.
- 13. 7. Detection of extent of conjugation – The extent of conjugation in the polyenes can be detected with the help of UV spectroscopy. – With the increase in double bonds the absorption shifts towards the longer wavelength. – If the double bond is increased by 8 in the polyenes then that polyene appears visible to the human eye as the absorption comes in the visible region.
- 14. 8. EXAMINATION OF POLYNUCLEAR HYDROCARBONS Benzene and Polynuclear hydrocarbons have characteristic spectra in ultraviolet and visible region. Thus identification of Polynuclear hydrocarbons can be made by comparison with the spectra of known Polynuclear compounds. Polynuclear hydrocarbons are the Hydrocarbon molecule with two or more closed rings; examples are naphthalene, C10H8, with two benzene rings side by side, or diphenyl, (C6H5)2, with two bondconnected benzene rings. Also known as polycyclic hydrocarbon
- 16. 9. MOLECULAR WEIGHT DETERMINATION Molecular weights of compounds can be measured spectrophotometrically by preparing the suitable derivatives of these compounds. For example, if we want to determine the molecular weight of amine then it is converted in to amine picrate. Then known concentration of amine picrate is dissolved in a litre of solution and its optical density is measured at λmax 380 nm.
- 17. After this the concentration of the solution in gm moles per litre can be calculated by using the following formula. "c" can be calculated using above equation, the weight "w" of amine picrate is known. From "c" and "w", molecular weight of amine picrate can be calculated. And the molecular weight of picrate can be calculated using the molecular weight of amine picrate.
- 18. 10. AS HPLC DETECTOR A UV/Vis spectrophotometer may be used as a detector for HPLC. HPLC UV detectors are used with high performance liquid chromatography to detect and identify analytes in the sample. A UV visible HPLC detector uses light to analyze samples. By measuring the sample's absorption of light at different wavelengths, the analyte can be identified.
- 19. 11.UV-VISIBLE SPECTROSCOPY FOR CLINICAL AND PRE- CLINICAL APPLICATION IN CANCER – Methods of optical science and engineering have been developed for cancer detection and diagnosis and more recently to assess response to therapy in a variety of tissue sites for applications in both preclinical and clinical studies . The interaction of light with complex media such as biological tissues, is characterized by processes that depend on the physical nature of the light and the specific tissue morphology and composition .
- 21. 12. Determination of the purity of a substance – Purity of a substance can also be determined with the help of UV spectroscopy. The absorption of the sample solution is compared with the absorption of the reference solution. The intensity of the absorption can be used for the relative calculation of the purity of the sample substance.
- 22. 13.Determination of configurations of geometrical isomers- – It is observed that cis-alkenes absorb at different wavelength than the trans- alkenes. The two isomers can be distinguished with each other when one of the isomers has non-coplanar structure due to steric hindrances. – The cis-isomer suffers distortion and absorbs at lower wavelength as compared to trans- isomer.
- 23. THANK YOU