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Premium member Presentation Transcript SRI RAMACHANDRA UNIVERSITY PORUR, CHENNAI – 600 116 DEPARTMENT OF HUMAN GENETICS : Mr. RUPENDRA SHRESTHA SRI RAMACHANDRA UNIVERSITY PORUR, CHENNAI – 600 116 DEPARTMENT OF HUMAN GENETICS RAMAN SPECTROSCOPY I can determine modes of molecular motions, especially vibrations .PowerPoint Presentation: What is Spectroscopy? study of how 'species' (i.e., atoms, molecules, solutions) behave, like: react to light. Light absorbs by an atom. electromagnetic radiation absorbed, emitted or scattered by the molecule is analyzed . Laser a beam of radiation passed through a sample radiation exiting the sample is measured. e.g. Raman , depend on a molecule's vibrations in reaction to the light. Department of Human Genetics/2012/ Rupendra ShresthaSir Chandrashekhara Venkata Raman: Sir Chandrashekhara Venkata Raman November 7, 1888-November 21, 1970 Won the Noble Prize in 1930 for Physics Discovered the "Raman Effect" Besides Discovering the Raman Effect, He studied extensively in X-Ray Diffractions, Acoustics, Optics, Dielectrics, Ultrasonic, Photo electricity, and colloidal particles. Department of Human Genetics/2012/ Rupendra ShresthaWhy Raman?: Why Raman? In Raman spectroscopy, by varying the frequency of the radiation, a spectrum can be produced, showing the intensity of the exiting radiation for each frequency. This spectrum will show which frequencies of radiation have been absorbed by the molecule to raise it to higher vibrational energy states. Department of Human Genetics/2012/ Rupendra Shrestha Raman Spectroscopy: Raman Spectroscopy When radiation passes through a transparent medium, the species present scatter a fraction of the beam in all directions. In 1928, the Indian physicist C. V. Raman discovered that the visible wavelength of a small fraction of the radiation scattered by certain molecules differs from that of the incident beam the shifts in wavelength depend upon the chemical structure of the molecules responsible for the scattering. Department of Human Genetics/2012/Rupendra ShresthaPowerPoint Presentation: Raman Spectroscopy The theory of Raman scattering shows that the phenomenon results from the same type of quantized vibrational changes that are associated with infrared absorption. the difference in wavelength between the incident and scattered visible radiation corresponds to wavelengths in the mid-infrared region. advantage of Raman spectra over infrared, Raman spectra can be obtained from aqueous solutions (no interference of water). glass or quartz cells can be employed, thus avoiding the inconvenience of working with sodium chloride or other atmospherically unstable window materials. Department of Human Genetics/2012/Rupendra ShresthaPowerPoint Presentation: THEORY OF RAMAN SPECTROSCOPY Raman spectra are acquired by irradiating a sample with a powerful laser source of visible or near-infrared monochromatic radiation . During irradiation, the spectrum of the scattered radiation is measured at some angle (often 90 0 ) with a suitable spectrometer. At the very most, the intensities of Raman lines are 0.001 % of the intensity of the source; as a consequence, their detection and measurement are somewhat more difficult than are infrared spectra. Department of Human Genetics/2012/Rupendra ShresthaPowerPoint Presentation: Excitation of Raman Spectra A Raman spectrum can be obtained by irradiating a sample of carbon tetrachloride with an intense beam of an argon ion laser having a wavelength of 488.0 nm (20492 cm -1 ). The emitted radiation is of three types: 1. Stokes scattering 2. Anti-stokes scattering 3. Rayleigh scattering Department of Human Genetics/2012/Rupendra ShresthaPowerPoint Presentation: Excitation of Raman Spectra Raman effect is a 2-photon scattering process. These processes are inelastic scattering : Stokes scattering : energy lost by photon: — (( — )) Photon in Photon out No vibration Vibration Anti-Stokes scattering : energy gained by photon: (( — )) — Photon in Photon out Vibration No vibration Department of Human Genetics/2012/ Rupendra ShresthaPowerPoint Presentation: But dominant process is elastic scattering: Rayleigh scattering — — Photon in Photon out No vibration No vibration If incident photon energy E; vibration energy v, then in terms of energy, photon out has energy: E - v Stokes scattering E + v anti-Stokes scattering E Rayleigh scattering Department of Human Genetics/2012/ Rupendra ShresthaStokes vs. Anti Stokes: Stokes vs. Anti Stokes Atoms are at a certain energy level at any given time. As a laser light hits the atom, it is excited and reaches a higher level of energy, and then is brought back down. If an atom is at a given energy level, it can be excited then fall below the original level. Anti-stokes spectrum are mirror spectrums of Stokes Raman Spectrums Department of Human Genetics/2012/ Rupendra ShresthaPowerPoint Presentation: Energy Scheme for Photon Scattering Energy Rayleigh Scattering (elastic) The Raman effect comprises a very small fraction, about 1 in 10 7 of the incident photons. Stokes Scattering Anti-Stokes Scattering h n 0 h n 0 h n 0 h n 0 h n 0 - h n m h n 0 + h n m E 0 E 0 +h n m Raman (inelastic) Virtual State IR Absorption E-hvm Department of Human Genetics/2012/ Rupendra ShresthaPowerPoint Presentation: Raman vs. I.R For a given bond, the energy shifts observed in a Raman experiment should be identical to the energies of its infrared absorption bands, provided that the vibrational modes involved are active toward both infrared absorption and Raman scattering . The differences between a Raman spectrum and an infrared spectrum are not surprising. Infrared absorption requires that a vibrational mode of the molecule have a change in dipole moment or charge distribution associated with it. Department of Human Genetics/2012/Rupendra ShresthaPowerPoint Presentation: Department of Human Genetics/2012/Rupendra ShresthaPowerPoint Presentation: Raman vs. I.R In contrast, scattering involves a momentary distortion of the electrons distributed around a bond in a molecule, followed by reemission of the radiation as the bond returns to its normal state . In its distorted form, the molecule is temporarily polarized; that is, it develops momentarily an induced dipole that disappears upon relaxation and reemission. The Raman activity of a given vibrational mode may differ markedly from its infrared activity. Department of Human Genetics/2012/Rupendra ShresthaPowerPoint Presentation: Sample I 0 ( n ) I( n ) n 0 n 0 - Rayleigh Sample n 0 n M - Raman IR Spectrography - Absorption Raman Spectrography - Scattering Laser detector Laser detector Department of Human Genetics/2012/Rupendra ShresthaPowerPoint Presentation: Intensity of Normal Raman Peaks The intensity or power of a normal Raman peak depends in a complex way upon the polarizability of the molecule, the intensity of the source, and the concentration of the active group . The power of Raman emission increases with the fourth power of the frequency of the source; however, advantage can seldom be taken of this relationship because of the likelihood that ultraviolet irradiation will cause photodecomposition . Raman intensities are usually directly proportional to the concentration of the active species. Department of Human Genetics/2012/Rupendra ShresthaPowerPoint Presentation: Raman Depolarization Ratios Polarization is a property of a beam of radiation and describes the plane in which the radiation vibrates . Raman spectra are excited by plane-polarized radiation. The scattered radiation is found to be polarized to various degrees depending upon the type of vibration responsible for the scattering. Department of Human Genetics/2012/Rupendra ShresthaPowerPoint Presentation: Department of Human Genetics/2012/ Rupendra ShresthaPowerPoint Presentation: Raman Depolarization Ratios The depolarization ratio p is defined as Experimentally , the depolarization ratio may be obtained by inserting a polarizer between the sample and the monochromator . The depolarization ratio is dependent upon the symmetry of the vibrations responsible for scattering. Department of Human Genetics/2012/Rupendra ShresthaPowerPoint Presentation: Raman Depolarization Ratios Polarized band: p = < 0.76 for totally symmetric modes (A 1g ) Depolarized band: p = 0.76 for B 1g and B 2g nonsymmetrical vibrational modes Anomalously polarized band: p = > 0.76 for A 2g vibrational modes Department of Human Genetics/2012/Rupendra ShresthaHow To carry Out a Raman Experiment: How To carry Out a Raman Experiment An insulated conductor was soldered to a piece of pure silver which was the embedded into a chemically resistant resin, leaving one face exposed. 1 micro liter of sample solution was placed on the roughened surface. The Sample is put through an Oxidation Reduction Cycle. Department of Human Genetics/2012/Rupendra ShresthaPowerPoint Presentation: Raman Instruments Department of Human Genetics/2012/ Rupendra ShresthaPowerPoint Presentation: Modern Raman Spectroscopy Instrumentation for modern Raman spectroscopy consists of three components: A laser source, a sample illumination system and a suitable spectrometer. L aser source The sources used in modern Raman spectrometry are nearly always lasers because their high intensity is necessary to produce Raman scattering of sufficient intensity to be measured with a reasonable signal-to-noise ratio . Because the intensity of Raman scattering varies as the fourth power of the frequency, argon and krypton ion sources that emit in the blue and green region of the spectrum have and advantage over the other sources. Department of Human Genetics/2012/Rupendra ShresthaPowerPoint Presentation: Department of Human Genetics/2012/ Rupendra ShresthaPowerPoint Presentation: Department of Human Genetics/2012/Rupendra ShresthaPowerPoint Presentation: Sample Illumination System Sample handling for Raman spectroscopic measurements is simpler than for infrared spectroscopy because glass can be used for windows, lenses, and other optical components instead of the more fragile and atmospherically less stable crystalline halides. In addition, the laser source is easily focused on a small sample area and the emitted radiation efficiently focused on a slit. Consequently, very small samples can be investigated. A common sample holder for non-absorbing liquid samples is an ordinary glass melting-point capillary. Department of Human Genetics/2012/Rupendra ShresthaPowerPoint Presentation: Department of Human Genetics/2012/Rupendra ShresthaPowerPoint Presentation: Sample Illumination System Liquid Samples: A major advantage of sample handling in Raman spectroscopy compared with infrared arises because water is a weak Raman scattered but a strong absorber of infrared radiation. Thus, aqueous solutions can be studied by Raman spectroscopy but not by infrared. This advantage is particularly important for biological and inorganic systems and in studies dealing with water pollution problems . Solid Samples: Raman spectra of solid samples are often acquired by filling a small cavity with the sample after it has been ground to a fine powder. Polymers can usually be examined directly with no sample pretreatment. Department of Human Genetics/2012/Rupendra ShresthaPowerPoint Presentation: Special Raman Spectrometers Raman spectrometers were similar in design and used the same type of components as the classical ultraviolet/visible dispersing instruments . Most employed double grating systems to minimize the spurious radiation reaching the transducer. Photomultipliers served as transducers. Now Raman spectrometers being marketed are either Fourier transform instruments equipped with cooled germanium transducers or multichannel instruments based upon charge-coupled devices. Department of Human Genetics/2012/Rupendra ShresthaPowerPoint Presentation: Department of Human Genetics/2012/Rupendra ShresthaPowerPoint Presentation: APPLICATIONS OF RAMAN SPECTROSCOPY Raman Spectra of Inorganic Species The Raman technique is often superior to infrared for spectroscopy investigating inorganic systems because aqueous solutions can be employed. In addition, the vibrational energies of metal-ligand bonds are generally in the range of 100 to 700 cm -1 , a region of the infrared that is experimentally difficult to study . These vibrations are frequently Raman active, however, and peaks with values in this range are readily observed. Raman studies are potentially useful sources of information concerning the composition, structure, and stability of coordination compounds. Department of Human Genetics/2012/Rupendra ShresthaPowerPoint Presentation: Raman Spectra of Organic Species Raman spectra are similar to infrared spectra in that they have regions that are useful for functional group detection and fingerprint regions that permit the identification of specific compounds. Raman spectra yield more information about certain types of organic compounds than do their infrared counterparts. Biological Applications of Raman Spectroscopy Raman spectroscopy has been applied widely for the study of biological systems. small sample requirement, the minimal sensitivity toward interference by water, the spectral detail, and the conformational and environmental sensitivity . Department of Human Genetics/2012/Rupendra ShresthaPowerPoint Presentation: Quantitative applications Raman spectra tend to be less cluttered with peaks than infrared spectra. As a consequence, peak overlap in mixtures is less likely, and quantitative measurements are simpler. In addition, Raman sampling devices are not subject to attack by moisture, and small amounts of water in a sample do not interfere. Raman spectroscopy has not yet been exploited widely for quantitative analysis. This lack of use has been due to high cost of Raman spectrometers relative to that of absorption instrumentation. Department of Human Genetics/2012/Rupendra ShresthaPowerPoint Presentation: Resonance Raman Spectroscopy Resonance Raman scattering refers to a phenomenon in which Raman line intensities are greatly enhanced by excitation with wavelengths that closely approach that of an electronic absorption peak of an analyte. Under this circumstance, the magnitudes of Raman peaks associated with the most symmetric vibrations are enhanced by a factor of 10 2 to 10 6 . As a consequence, resonance Raman spectra have been obtained at analyte concentrations as low as 10 -8 M. Department of Human Genetics/2012/Rupendra ShresthaPowerPoint Presentation: Department of Human Genetics/2012/Rupendra ShresthaPowerPoint Presentation: Application of resonance Raman Spectroscopy To the study of biological molecules under physiologically significant conditions; that is , in the presence of water and at low to moderate concentration levels. As an example, the technique has been used to determine the oxidation state and spin of iron atoms in hemoglobin and cytochrome-c . In these molecules, the resonance Raman bands are due solely to vibrational modes of the tetrapyrrole chromophore. Department of Human Genetics/2012/Rupendra ShresthaPowerPoint Presentation: Surface-Enhanced Raman Spectroscopy (SERS ) Surface enhanced Raman spectroscopy involves obtaining Raman spectra in the usual way on samples that are adsorbed on the surface of colloidal metal particles (usually silver, gold, or copper) or on roughened surfaces of pieces of these metals. For reasons that are not fully understood, the Raman lines of the adsorbed molecule are often enhanced by a factor of 10 3 to 10 6 . When surface enhancement is combined with the resonance enhancement technique, the net increase in signal intensity is roughly the product of the intensity produced by each of the techniques. Consequently , detection limits in the 10 -9 to 10 -12 M range have been observed. Department of Human Genetics/2012/Rupendra Shrestha Surface-Enhanced Raman Spectroscopy (SERS) : Surface-Enhanced Raman Spectroscopy (SERS) Department of Human Genetics/2012/Rupendra Shrestha Applications: receptor binding single molecule studies optical activity identification and characterization of bacteria thrombin detection with aptamer -Au nanoparticles Nanoplex biotags heterogeneous immunoassays pH sensor in live cellsOther Application: Other Application It is predominantly applicable to the qualitative and quantitative analyses of covalently bonded molecules . Extra : -Identification of phases (mineral inclusions, composition of the gas phase inclusions) -Anions in the fluid phase (OH-, HS-, etc.) -Identification of crystalline polymorphs (Sillimanite, Kyanite, andalusite, etc .) -Measurement of mid-range order of solids -Measurement of stress -High-pressure and High-temperature in situ studies -Phase transition and order-disorder transitions in minerals (quartz, graphite) -Water content of silicate glasses and minerals -Speciation of water in glasses Department of Human Genetics/2012/ Rupendra ShresthaPowerPoint Presentation: THANK YOU FOR KIND ATTENTION Department of Human Genetics/2012/Rupendra ShresthaRupendra speaks;" More u read ,the more confusion; confusion triggers our curiosity, curiosity on new things is searching for it that’s called research; researcher are honoured as scientists that’s we should aim in life”: Rupendra speaks;" More u read ,the more confusion; confusion triggers our curiosity , curiosity on new things is searching for it that’s called research ; researcher are honoured as scientists that’s we should aim in life” Department of Human Genetics/2012/Rupendra ShresthaThe first major study of CNTs using Raman Spectroscopy in: SCIENCE VOL. 275 10 JANUARY 1997 (4 years after CNTs were discovered by Iijima): The first major study of CNTs using Raman Spectroscopy in: SCIENCE VOL. 275 10 JANUARY 1997 (4 years after CNTs were discovered by Iijima) You do not have the permission to view this presentation. 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