Raman fluorescencespec

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Raman fluorescencespec

  1. 1. SPECTROSCOPY
  2. 2. Theory of light • Light is electromagnetic radiation that is visible to the human eye, and is responsible for the sense of sight. • Visible light has a wavelength in the range of about 380 nm, or 380×10−9 m, to about 740 nanometres – between the invisible infrared with longer wavelengths and the invisible ultraviolet with shorter wavelengths. • Light is emitted and absorbed in tiny "packets" called photons, and exhibits properties of both waves and particles. This property is referred to as the ”wave–particle duality”. • The study of light, known as optics.
  3. 3. Electromagnetic spectrum
  4. 4. What are the different theories of Light? 1.Corpuscular Theory by Sir Isaac Newton 2.Wave Theory 3.Electromagnetic Theory 4.Photoelectric Theory 5.Dual Property 6.Quantum Theory by Max Planck
  5. 5. What are the basic properties of light? • Light travels in straight lines • Light can be reflected • Light can be refracted ~ bent • Light is a form of energy • Light can be dispersed • Light can be diffracted
  6. 6. Spectroscopy • Spectroscopy pertains to the dispersion of an object's light into its component colours (i.e. energies). • By performing this dissection and analysis of an object's light, physical properties of object such as temperature, mass, luminosity and composition can be inferred. • Spectroscopy is the study of the interaction between matter and radiated energy. • Applications: 1. To determine the molecular structure 2.To estimate the energy levels of the ions and complexes in a chemical system along with the compositions. 3. To understand the structure making and structure breaking processes in solutions 4, To get an idea regarding absorption and emission details of the specimen 5. To understand the intrinsic configuration and relative association and chemical shifts
  7. 7. Spectroscopy – Radiation Terminology • Wavelength (λ) -- length between two equivalent points on successive waves • Wavenumber (n)–– the number of waves in a unit of length or distance per cycle -- reciprocal of the wavelength • Frequency (ν) –– is the number of oscillations of the field per second (Hz) • Velocity (c) –– independent of wavelength –– in vacuum is 3.00 x 1010 cm/s (3.00 x 108m/s) • Photon (quanta) –– quantum mechanics ~ the minimum amount of any physical entity involved in an interaction
  8. 8. Chromophore& Auxochrome • Chromophore : A Chromophore is a group of atoms within a molecule which are responsible for the color of the molecule. • A Chromophore adds color to a molecule because of the nature of the atoms involved and the way they are bonded with each other. • These specialized moieties are also present in atoms inside cells which have a color-related function, including photo pigments and chromatophores. • A classic example of a photo pigment can be found in the human eye, where sensitized cells respond to visible light to provide a picture of the visible world. • e.g. chlorophyll's porphyrin ring, or an azo dye's benzene rings joined by a N=N double bond.
  9. 9. Chromophore& Auxochrome • Auxochrome: functional group that does not absorb radiation itself in the UV range but has a shifting effect on main chromophore peaks to longer wavelength as well as increasing their intensity. OR Auxochromes are functional groups attached to chromophore that alter the shade of colour it emits. • Example: --OH and ––NH2 on benzene chromophore
  10. 10. Quantification terms • Transmittance : The transmittance of a sample is the ratio of the intensity of the light that has passed through the sample to the intensity of the light when it entered the sample T = I₀/I where, I₀= Light passed out I = Light entered Absorbance: the measure of the quantity of light that a sample neither transmits nor reflects (= absorbs) and is proportional to the concentration of a substance in a solution. Absorbance = -log (percent transmittance/100) A = log(1/T) = - log T = log I/ I₀
  11. 11. Beer-Lambert’s Law • The absorbance of light is directly proportional to the thickness of the media through which the light is being transmitted multiplied by the concentration of absorbing chromophore. A = εbc where, A = absorbance ε =molar extinction coefficient b = thickness of the solution c = concentration
  12. 12. Types of Spectroscopy  There are as many different types of spectroscopy as there are energy sources. Few of them are as follows : Astronomical Spectroscopy Electron Paramagnetic Spectroscopy Electron Spectroscopy Infrared Spectroscopy Mass Spectroscopy Laser Spectroscopy Raman Spectroscopy NMR Spectroscopy Fluorescence spectroscopy Gamma-ray Spectroscopy
  13. 13. LASER-Raman Spectroscopy  Introduction: Raman spectroscopy is a spectroscopic technique based on “inelastic scattering” of monochromatic light, usually from a laser source.  Inelastic scattering means that the frequency of photons in monochromatic light changes upon interaction with a sample  Photons of the laser light are absorbed by the sample and then reemitted. Frequency of the reemitted photons is shifted up or down in comparison with original monochromatic frequency, which is called the Raman effect.  This shift provides information about vibrational, rotational and other low frequency transitions in molecules of solid, liquid and gaseous samples.
  14. 14. Origins of Raman Spectroscopy  The Raman effect is based on molecular deformations in electric field E determined by molecular polarizability α. The laser beam can be considered as an oscillating electromagnetic wave with electrical vector E. Upon interaction with the sample it induces electric dipole moment P = αE which deforms molecules. Because of periodical deformation, molecules start vibrating with characteristic frequency υm.  Amplitude of vibration is called a nuclear displacement. In other words, monochromatic laser light with frequency υ0 excites molecules and transforms them into oscillating dipoles. Such oscillating dipoles emit light of three different frequencies
  15. 15. Energy level diagram showing the states involved in Raman signal.
  16. 16. A molecule with no Raman-active modes absorbs a photon with the frequency υo. The excited molecule returns back to the same basic vibrational state and emits light with the same frequency υo as an excitation source. This type of interaction is called an elastic Rayleigh scattering. A photon with frequency υo is absorbed by Raman-active molecule which at the time of interaction is in the basic vibrational state. Part of the photon’s energy is transferred to the Raman-active mode with frequency υm and the resulting frequency of scattered light is reduced to υo- υm. This Raman frequency is called Stokes frequency or just “Stokes”. A photon with frequency υo is absorbed by a Raman-active molecule, which, at the time of interaction, is already in the excited vibrational state. Excessive energy of excited Raman active mode is released, molecule returns to the basic vibrational state and the resulting frequency of scattered light goes up to υo+ υm. This Raman frequency is called Anti-Stokes frequency, or just “Anti- Stokes”.
  17. 17. Instrumentation  A Raman system typically consists of four major components: 1. Excitation source (Laser) 2. Sample illumination system and light collection optics. 3. Wavelength selector (Filter or Spectrophotometer). 4. Detector (Photodiode array, CCD or PMT).
  18. 18. 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 of argon and krypton ion sources that emit in the blue and green region of the spectrum have an advantage over the other sources.
  19. 19. Common laser sources for Raman LASER type Wave lengths in nm Argon ion 488.0 – 514.5 Krypton ion 530.9 – 647.1 Helium-neon 632.8 Diode 785 – 830 Nd-YAG 1064
  20. 20. Sample system:  Sample handling for Raman spectroscopic measurements is simple 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.
  21. 21. Liquid Samples: A major advantage of sample handling in Raman spectroscopy arises because water is a weak Raman scatterer 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.
  22. 22. Spectrophotometer/Filters  Since spontaneous Raman scattering is very weak the main difficulty of Raman spectroscopy is separating it from the intense Rayleigh scattering. As rayleigh scattering may greatly exceed the intensity of the useful Raman signal in the close proximity to the laser wavelength.  In many cases the problem is resolved by simply cutting off the spectral range close to the laser line where the stray light has the most prominent effect. Commercially available, interference (notch) filters which cut-off spectral range of ± 80-120 cm-1 from the laser line. This method is efficient in stray light elimination but it does not allow detection of low-frequency Raman modes in the range below 100 cm-1.  Raman spectrometers typically use holographic gratings which normally have much less manufacturing defects in their structure then the ruled ones.
  23. 23. Detectors:  In earlier times people primarily used single-point detectors such as photon-counting Photomultiplier Tubes (PMT). However, a single Raman spectrum obtained with a PMT detector in wave number scanning mode was taking substantial period of time, slowing down any research or industrial activity based on Raman analytical technique.  Nowadays, more and more often researchers use multi- channel detectors like Photodiode Arrays (PDA) or, more commonly, a Charge-Coupled Devices (CCD) to detect the Raman scattered light. Sensitivity and performance of modern CCD detectors are rapidly improving. In many cases CCD is becoming the detector of choice for Raman spectroscopy.
  24. 24. Raman Strength & Limitations  Easy identification of chemical structures  Widely applicable for various materials  Samples can be solid/aqueous  Little or no sample treatment  Remote control with fiber optics  When incidence light goes to blue, Fluorescence is required  Expensive due to cost of laser sources  Low excitation probability  Spatially refrained by optical limits Strength Limitations
  25. 25. Applications  Pharmaceutical and Bio-medical Applications  Material science and Nano-technology  Forensic labs, also used by Anti-terror squad  Gemology, Geology, Mineralogy  Archaeology, Art, Heritage
  26. 26. FLUORESCENCE SPECTROSCOPY  Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation at a longer wavelength than absorbed.  Emission Spectroscopy: A spectroscopic technique that examines the wavelength of photons emitted by atoms or molecules during their transition from an excited state to lower energy state.  Luminescence: Luminescence is emission of light by a substance not resulting from heat; it is thus a form of cold body radiation. It is caused by chemical reactions, electrical energy, subatomic motions, or stress on a crystal.
  27. 27.  Luminescence is basically divided into two categories: Phosphorescence Fluorescence  Emission rates of Fluorescence is typically 10^8 s-1 ; while emission rates of Phosphorescence are slow i.e., 10^3 to 100 s-1.  Life time of a phosphorescence molecule is milliseconds to seconds, while that of a fluorescence is nanoseconds.
  28. 28.  Fluorescence is a spectrochemical method of analysis where the molecules of the analyte are excited by irradiation at a certain wavelength and emit radiation of a different wavelength.  The emission spectrum provides information for both qualitative and quantitative analysis.  when light of an appropriate wavelength is absorbed by a molecule (i.e., excitation), the electronic state of the molecule changes from the ground state to one of many vibrational levels in one of the excited electronic states.
  29. 29. The excited electronic state is usually the first excited singlet state, Once the molecule is in this excited state, relaxation can occur via several processes. Fluorescence is one of these processes and results in the emission of Light. Following absorption, a number of vibrational levels of the excited state are populated. Molecules in these higher vibrational levels then relax to the lowest vibrational level of the excited state (vibrational relaxation). From the lowest vibrational level, several processes can cause the molecule to relax to its ground state. An overall energy balance for the fluorescence process could be written as: Efluor = Eabs − Evib − Esolv.relax Efluor is the energy of the emitted light, Eabs is the energy of the light absorbed by the molecule during excitation, Evib is the energy lost by the molecule from vibrational relaxation. Esolv.relax term arises from the need for the solvent cage of the molecule to reorient itself in the excited state and then again when the molecule relaxes to the ground state.
  30. 30. Fluorescence spectroscopy aka fluorometry or spectrofluorometry, is a type of electromagnetic spectroscopy which analyses fluorescence from a sample. It involves using a beam of light, usually ultraviolet light, that excites the electrons in molecules of certain compounds and causes them to emit light Devices that measure fluorescence are called fluorometers or fluorimeters. The difference between these two wavelengths is known as Stokes Shift
  31. 31.  The light from an excitation source passes through a filter or a monochromator and strikes the sample, Xenon lamps are widely used as a light source.  A portion of the incident light is absorbed by the sample and some by the fluorescent molecules. The fluorescent light is emitted in all directions, some of this light passes through the second filter/ monochromator and reaches the detectors.  These detectors are placed at 90º to the incident light beam to minimize the risk of transmitted or reflected incident light reaching the detector. Commonly diffraction grating is used.
  32. 32.  Determination of fluorescent drugs in low dose formulations  In carrying out limit-tests where Fluorescent compounds are treated as impurities as Fluorescent probes  Useful for studying binding of drugs to component in complex formulations.  Widely used in Bio-analysis for measuring components present in low concentrations  Enzyme assays and kinetic analysis  Fluorescence recovery after bleaching (FRAP)~ Used to study the movement and working of a biological membrane.  Fluorescence resonance energy transfer (FRET) ~ Used to deduce the distance between Protein molecules  Fluorescence activated cell sorter (FACS)~ used in Fluorescent immunoassays.
  33. 33. Thank You . . . Archa Dave M.Sc MB Sem II 12031G1901

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