UV SPECTROSCOPY ppt.pptx
- 1. TOPIC :- UV VISIBLE SPECTROSCOPY Presented by : Ashish J Hingnekar (M.Pharm 1st year QA) Guided by : Mr. Rushikesh Relekar Government College of Pharmacy,Amravati.
- 2. Contents ... • Spectroscopy • Principle of Spectroscopy • Beer – Lamberts’s Law • Electronic Transitions • Absorption & Intensity Shifts • Instrumention of UV Visible Spectroscopy • Different types of Spectroscopy • Applications • References
- 3. SPECTROSCOPY It is a branch of science that deals with the study of interaction of matter with light. OR It is a branch of science that deals with the study of interaction of electromagnetic radiation with matter.
- 5. • Electromagnetic radiation consists of discrete packages of energy which are called as photons. • A photon consists of oscillating electric field (E) & an oscillating magnetic field (M) which are perpendicular to each other. Electromagnetic Radiation
- 6. Frequency (v): It is defined as the number of times electrical field radiation oscillates in one second. The unit for frequency is Hertz (Hz).1 Hz = 1 cycle per second Wavelength (λ): It is the distance between two nearest parts of the wave in the same phase i.e. distance between two nearest crests or troughs. The relationship between wavelength & frequency can be written as: C = v λ As photon is subjected to energy, So E = h v = h c / λ Electromagnetic radiation
- 8. PRINCIPLE OF SPECTROSCOPY • The principle is based on the measurement of spectrum of a sample containing atoms / molecules. • Spectrum is a graph of intensity of absorbed or emitted radiation by sample verses frequency (v) or wavelength (a). • Spectrometer is an instrument design to measure the spectrum of a compound.
- 9. 1. Absorption Spectroscopy: An analytical technique which concerns with the measurement of absorption of electromagnetic radiation. e.g. UV (185 - 400 nm) I Visible (400 to 800 nm) Spectroscopy, IR Spectroscopy (0.76 to 15 um ) 2. Emission Spectroscopy: An analytical technique in which emission (of a particle or radiation) is dispersed according to some property of the emission & the amount of dispersion is measured. e.g. Mass Spectroscopy
- 10. Principle of UV- Visible Spectroscopy • The UV radiation region extends from 10 nm to 400 nm and the visible radiation region extends from 400 nm to 800 nm. Near UV Region: 200 nm to 400 nm Far UV Region: below 200 nm • Far UV spectroscopy is studied under vacuum condition. • The common solvent used for preparing sample to be analyzed is either ethyl alcohol or hexane.
- 12. Beers Lambert’s Law When a monochromatic radiation is passed through a solution, the decrease in the intensity of radiation with thickness of the solution is directly proportional to the intensity of the incident light as well as concentration of the solution. A = ε b c A = Absorbance ε = molar conductivity with units of L/mol.cm b = path length of the sample (cuvette) c = concentration of the compound in solution in mol /L
- 15. The possible electronic transitions are σ – σ* transition ∏ – ∏* transition n – σ* transition n – ∏* transition σ – ∏* transition ∏ – σ* transition
- 16. Terms used in UV- Visible Spectroscopy Chromophore The part of a molecule responsible for imparting color, are called as chromopheres. OR The functional groups containing multiple bonds capable of absorbing radiations above 200 nm due to n -∏ * & m - ∏* transitions. e.g. NO2 , N=0, C=0, C=N, C=N, C=C, C= S, etc Auxochrome The functional groups attached to a chromophore which modifies the ability of the chromophore to absorb light , altering the wavelength or intensity of absorption .OR The functional group with non-bonding electrons that does not absorb radiation in near UV region but when attached to a chromophore alters thewavelength & intensity of absorption. E.g. Benzene λmax (255nm),Phenol (270nm ), Aniline (280nm ).
- 19. Bathochromic shift (Red shift) When absorption maxima (λmax) of a compound shifts to longer wavelength. Hypsochromic shift (Blue shift) When an absorption maxima (λmax) of a compound shifts to a shorter wavelength. Hyperchromic shift When absoprtion intensity (ε ) of a compound is increased Hypochromic shift When absorption intensity (ε ) of a compound is decreased .
- 22. Componentsof UV Spectrophotometer Recorder Detector Sample Compartment Monochromator Filter Source
- 24. Basic Optical Instrument Components 1) Source : A stable source of radiant energy at the desired wavelength or range). 2) Wavelength Selector : A device that isolates a restricted region of the EM spectrum used for measurement(monochromators, prisms & filters). 3) Sample Container : A transparent container used to hold the sample (cells, cuvettes, etc). 4) Detector/Photoelectric Transducer : Converts the radiant energy into a useable signal (usually electrical). 5) Signal Processor & Readout : Amplifies or attenuates the transduced signal and sends it to a readout device as a meter, digital readout, chart recorder, computer, etc.
- 25. LIGHT SOURCES Various UV radiation sources are as follows a. Deuterium lamp b. Hydrogen lamp c. Tungsten lamp d. Xenon discharge lamp e. Mercury arc lamp Various Visible radiation sources are as follows a. Tungsten lamp b. Mercury vapour lamp c. Carbonone lamp
- 26. Wavelength Selectors Wavelength selectors limited, narrow, continuous group of wavelengths called a band. Two types of wavelength selectors: A) Filters B) Monochromators A)Filters - Two types of filters: a) Interference Filters b) Absorption Filters
- 27. B) Monochromators Wavelength selector that can continuously scan a broad range of wavelengths. Used in most scanning spectrometers including UV, visible, and IR instruments. • Refractive • Reflective Prism type • Diffraction • Transmission Grating type
- 28. Sample Compartment Spectroscopy requires all materials in the beam path other than the analyte should be as transparent to the radiation as possible. The geometries of all components in the system should be such as to maximize the signal and minimize the scattered light. The material from which a sample curette is fabricated controls the optical window that can be used. Some typical materials are: Optical Glass - 335 - 2500 nm Special Optical Glass — 320 - 2500 nm Quartz (Infrared) — 220 - 3800 nm Quartz (Far-UV) — 170 - 2700 nm
- 29. Detector After the light has passed through the sample, we want to be able to detect and measure the resulting tight. These types of detectors come in the form of transducers that are able to take energy from light and convert it into an electrical signal that can be recorded, and if necessary, amplified. Requirements of an ideal detector:- a. It should give quantitative response. b. It should have high sensitivity and low noise level. c. It should have a short response time. d. It should provide signal or response quantitative to wide spectrum of radiation received. Three common types of detectors are used a) Barrier layer cells b) Photo emissive cell detector c) Photomultiplier
- 30. 1. Barrier layer cell/Photovoltaic cell: The detector has a thin film metallic layer coated with silver or gold and acts as an electrode. It also has a metal base plate which acts as another electrode. These two layers are separated by a semiconductor layer selenium.
- 31. 2. Photoemissive cell / Phototube Consists of a evacuated glass tube with a photocathode and a collector anode. The surface of photocathode is coated with a layer of elements like cesium, silver oxide or mixture of them. When radiant energy fails on photosensitive cathode, electrons are emitted which are attracted to anode causing current to flow. More sensitive compared to barrier layer cell and therefore widely used.
- 32. 3.Photomultiplier tube The principle employed in this detector is that, multiplication of photoelectrons by secondary emission of electrons. In a vacuum tube, a primary photo-cathode is fixed which receives radiation from the sample. Some eight to ten dynodes are fixed each with increasing potential of 75-I0OV higher than preceding one. Near the last dynode is fixed an anode or electron collector electrode. Photo-multiplier is extremely sensitive to light and is best suited where weaker or low radiation is received.
- 33. Single Beam UV Spectrophotometer
- 34. Double Beam UV Spectrophotometer
- 35. Different UV - visible spectrophotometer methods For multicomponent analysis (a) Simultaneous equation method (b) Absorbance ratio method (c) Geometric correction method (d) Orthogonal polynomial method (e) Derivative spectrophotometry (f) Difference spectrophotornetry
- 36. Applications Qualitative & Quantitative Analysis: It is used for characterizing aromatic compounds and conjugated olefins. It can be used to find out molar concentration of the solute under study. Detection of impurities: It is one of the important method to detect impurities in organic solvents. Detection of isomers are possible. Determination of molecular weight using Beer's law.
- 37. Reference Books 1) Instrumental Method of Analysis : G.R. Chatwal, S K. Anand. 2) Introduction to Spectroscopy : Donald A. Pavia 3) Elementary Organic Spectroscopy : Y.R. Sharma 4) Physical Chemistry : Puri, Sharma & Pathaniya