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Premium member Presentation Transcript ULTRA VIOLET SPECTROSCOPY: ULTRA VIOLET SPECTROSCOPY TANVI DHINGRA M.PHARM (1 ST YR) PHARMACEUTICAL ANALYSISINTRODUCTION: INTRODUCTION SPECTROSCOPY Measurement and interpretation of electromagnetic radiation absorbed, scattered or emitted by atoms, molecules or other chemical species. This absorption or emission results in the change in energy states of interacting chemical species. UV radiation and Electronic Excitations The difference in energy between molecular bonding, non-bonding and anti-bonding orbitals ranges from 125-650 kJ/mole. This energy corresponds to EM radiation in the ultraviolet (UV) region, 100-350 nm, and visible (VIS) regions 350-700 nm of the spectrum. For comparison, recall the EM spectrum:THE SPECTROSCOPIC PROCESS: THE SPECTROSCOPIC PROCESS In UV spectroscopy, the sample is irradiated with the broad spectrum of the UV radiation. Electron undergoes transition from lower to higher energy level, this energy difference given by, E=h ν erg If a particular electronic transition matches the energy of a certain band of UV, it will be absorbed. The remaining UV light passes through the sample and is observed. From this residual radiation a spectrum is obtained with “gaps” at these discrete energies – this is called an absorption spectrum .OBSERVED ELECTRONIC TRANSITIONS: OBSERVED ELECTRONIC TRANSITIONS The lowest energy transition (and most often obs. by UV) is typically that of an electron in the Highest Occupied Molecular Orbital (HOMO) to the Lowest Unoccupied Molecular Orbital (LUMO) For any bond (pair of electrons) in a molecule, the molecular orbitals are a mixture of the two contributing atomic orbitals ; for every bonding orbital “created” from this mixing ( s , p ), there is a corresponding anti-bonding orbital of symmetrically higher energy ( s * , p * ) The lowest energy occupied orbitals are typically the s; likewise, the corresponding anti-bonding s * orbital is of the highest energy p - orbitals are of somewhat higher energy, and their complementary anti-bonding orbital somewhat lower in energy than s *. Unshared pairs lie at the energy of the original atomic orbital, most often this energy is higher than p or s (since no bond is formed, there is no benefit in energy)Slide 7: Although the UV spectrum extends below 100 nm (high energy), oxygen in the atmosphere is not transparent below 200 nm Special equipment to study vacuum or far UV is required Routine organic UV spectra are typically collected from 200-700 nm This limits the transitions that can be observed:Slide 8: SELECTION RULES Not all transitions that are possible are observed. For an electron to transition, certain quantum mechanical constraints apply – these are called “ selection rules. ” For example, an electron cannot change its spin quantum number during a transition – these are “ forbidden. ” Other examples include: the number of electrons that can be excited at one time symmetry properties of the molecule symmetry of the electronic statesCHROMOPHORE: CHROMOPHORE The characteristic energy of a transition and the wavelength of radiation absorbed are properties of a group of atoms rather than of electrons themselves. The group of atoms producing such transitions are called “ chromophores ” ORGANIC CHROMOPHORES Alkanes – only posses s -bonds and no lone pairs of electrons, so only the high energy s s * transition is observed in the far UV. Alcohols, ethers, amines and sulfur compounds – in the cases of simple, aliphatic examples of these compounds the n s * is the most often observed transition; like the alkane s s * it is most often at shorter l than 200 nm Alkenes and Alkynes – in the case of isolated examples of these compounds the p p * is observed at 175 and 170 nm, respectively. Carbonyls – unsaturated systems incorporating N or O can undergo n p * transitions (~285 nm) in addition to p p *Slide 10: The attachment of substituent groups in place of hydrogen on a basic chromophore structure changes the position and intensity of an absorption band of the chromophore . Substituents that increase the intensity of the absorption , and possibly the wavelength, are called auxochromes . Substituents any of four effects on a chromophore Bathochromic shift (red shift) – a shift to longer l ; lower energy Hypsochromic shift (blue shift) – a shift to shorter l ; higher energy Hyperchromic effect – an increase in intensity Hypochromic effect – a decrease in intensityCONJUGATION: CONJUGATION Most efficient means of bringing about a bathochromic and hyperchromic shift of an unsaturated chromophore : Alkenes The observed shifts from conjugation imply that an increase in conjugation decreases the energy required for electronic excitationWOODWARD FIESER RULE: WOODWARD FIESER RULE ● It is used for calculating the absorption maxima ●Woodward (1941) gave certain rules for correlating λ max with the molecular structure ●These rules were modified by Scott & Feiser . ●These rules are used for calculating λ max in conjugated dienes , trienes , polyenes . Homoannular dienes :-Slide 13: HETEROANNULAR DIENES 1.Endocyclic 2.Exocyclic Woodward’s-Fieser rule for conjugated dienes : Woodward’s- Fieser rule for conjugated dienes a)Parent values- 1. acyclic & Heteroannuler conjugated dienes 215 nm 2.Homoannular conjugated dienes 253 nm 3.Acyclic trienes 245 nm b)Increments - 1.Each alkyl substituent or ring residue 5 nm 2.Exocyclic double bond 5 nm 3.Double bond extending conjugation 30 nm 4.auxochromes- -OR 6 nm -SR 30 nm - Cl , Br 5 nm -NR2 60 nm -OCOCH3 0 nmSlide 15: 1,4- dimethyl cyclohex-1,3,-diene Parent value for Homoannular diene = 253 nm Two alkyl substituent's 2 X 5 = 10 nm Two ring residues 2 X 5 = 10 nm Calculated value = 273 nm Observed value = 265 nmSlide 16: Parent value for Heteroannuler diene = 215 nm Four ring residue 4 X 5 = 20 nm Calculated value = 235 nm Observed value = 236 nm Woodward’s-Fieser rule for α,β-unsaturated carbonyl compounds : Woodward’s- Fieser rule for α , β -unsaturated carbonyl compounds a)Parent values:- 1. α , β -unsaturated acyclic or six membered ring ketone 215 nm 2. α , β -unsaturated five membered ring ketone 202nm 3. α , β -unsaturated aldehyde 207nm b)increments:- 1.Each alkyl substituent or ring residue at α , position 10nm at β ,position 12nm at γ ,position 18nm 2.Each Exocyclic double bond 5nm 3.Double bond extending conjugation 30nm 4.Homoannular conjugatated dienes 39nm 5.Auxochromes. Positions α β γ -OH 35 30 50 -OR 35 30 17 -SR - 85 - -OCOCH3 6 6 6Slide 18: Parent value = 215 nm One alkyl substituent in α position = 10 nm Calculated value = 225 nm Observed value = 220 nm Parent value for α , β - unsaturated 6 membered cyclic ketone =215 nm One ring residue at α position = 10 nm 2 ring residue at β - position 2* 12 =24 nm Double bond Exocyclic to 2 ring 2* 5 =10 nm Calculated value = 259nm Observed value =PRINCIPLES OF ABSORPTION SPECTROSCOPY: PRINCIPLES OF ABSORPTION SPECTROSCOPY the number of molecules capable of absorbing light of a given wavelength, the the extent of light absorption. The more effectively a molecule absorbs light of a given wavelength, the greater the extent of light absorption. From these guiding ideas, BEER – LAMBERT LAW may be formulated: Where, A = absorbance I° = intensity of light incident upon sample cell I = intensity of light leaving sample cell c = molar concentration of solute l = length of sample cell Ɛ = molar absorptivitySlide 20: From an experimental point of view, three other considerations must be made: a longer path length, l through the sample will cause more UV light to be absorbed – linear effect the greater the concentration, c of the sample, the more UV light will be absorbed – linear effect some electronic transitions are more effective at the absorption of photon than others – molar absorptivity , e this may vary by orders of magnitude.INSTRUMENTATION: INSTRUMENTATION Components of spectrophotometer Source Monochromator Sample compartment Detector Recorder RADIANT SOURCE WAVELENGTH SELECTOR SOLVENT PHOTO- DETECTOR READOUT SAMPLE Fig.-block diagram of instrumentation of UV-spectrophotometerSlide 22: Light source a)D2 Lamp b)WI Lamp Entrance slit monochromator sample Exit slit Read out amplifier detector Fig.- block diagrammatic representation of UV-spectrophotometerSchematic diagram of U.V spectrophotometer: Schematic diagram of U.V spectrophotometer SINGLE BEAM DOUBLE BEAMRADIATION SOURCE: RADIATION SOURCE →STABLE →SUFFICIENT INTENSITY → CONTINOUS RADIATION TUNGSTEN LAMP HYDROGEN DISCHARGE LAMP: 3500- 1200 A⁰ DEUTERIUM LAMP : Intensity 3-5 times greater than hydrogen lamp XENON DISCHARGE LAMP MERCURY ARC USED TO DISPERSE RADIATIONS A/C TO THE WAVELENGTH . DISPERSING ELEMENTS: PRISM GRATING MONOCHROMATORSSlide 25: PRISM GRATING Blaze angle Normal surface vector Normal to groove faceDETECTORS: DETECTORS BARRIER LAYER CELL PHOTO CELLSlide 27: PHOTOMULTIPLIER TUBERecorder sample cells: Recorder sample cells Virtually all UV spectra are recorded solution-phase Cells can be made of plastic, glass or quartz Only quartz is transparent in the full 200-700 nm range; plastic and glass are only suitable for visible spectra Concentration is empirically determined A typical sample cell (commonly called a cuvet )SOLVENTS: SOLVENTS Solvents must be transparent in the region to be observed; the wavelength where a solvent is no longer transparent is referred to as the cutoff Since spectra are only obtained up to 200 nm, solvents typically only need to lack conjugated p systems or carbonyls Common solvents and cutoffs: acetonitrile 190 chloroform 240 cyclohexane 195 1,4-dioxane 215 95% ethanol 205 n-hexane 201 methanol 205 isooctane 195 water 190APPLICATIONS: APPLICATIONS Detection of Impurities: Benzene appears as a common impurity in cyclohexane . Its presence can be easily detected by its absorption at 255 nm. Structure elucidation of organic compounds: 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. 3. Quantitative analysis : UV absorption spectroscopy can be used for the quantitative determination of compounds that absorb UV radiation. This determination is based on Beer’s law. 4. Qualitative analysis Identification is done by comparing the absorption spectrum with the spectra of known compounds. UV absorption spectroscopy is generally used for characterizing aromatic compounds and aromatic olefins. 5. Dissociation constants of acids and bases. PH = PKa + log [A - ] / [HA] From the above equation, the PKa value can be calculated if the ratio of [A - ] / [HA] is known at a particular PH. and the ratio of [A - ] / [HA] can be determined spectrophotometrically from the graph plotted between absorbance and wavelength at different PH values.Slide 31: 6. 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. 7. Quantitative analysis of pharmaceutical substances : ●widely applicable to both organc and inorganic substances ●typical detection limits ●moderate to high selectivity ●Good accuracy ●Ease and covenience of data accusition For eg .: Diazepam tablet can be analyzed by 0.5% H2SO4 in methanol at the wavelength 284 nm. 8. Molecular weight determination Molecular weights of compounds can be measured spectrophotometrically by preparing the suitable derivatives of these compounds. For example, to determine the molecular weight of amine 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. After this the concentration of the solution in gm moles per litre can be calculated by using the following formula. 9. As HPLC detector A UV/Vis spectrophotometer may be used as a detector for HPLC. The presence of an analyte gives a response which can be assumed to be proportional to the concentration. For more accurate results, the instrument's response to the analyte in the unknown should be compared with the response to a standard; as in the case of calibration curveRECENT DEVELOPMENTS IN U.V: RECENT DEVELOPMENTS IN U.V Trace-level analysis of carbonyl compounds: This new UHPLC/UV method enables the separation, detection and quantitation of parts per billion (ppb) concentrations of low molecular weight carbonyls in complex samples, safeguarding human health and ensuring compliance with industry regulations . VALIMED: Eliminates human error by identifying drugs, from risky antibiotic drips to dangerous narcotics.REFRENCES: REFRENCES SKOOG, HOLLER AND CROUCH – INSTRUMENTAL ANALYSIS; NINTH EDITION; CENGAGE PUBLICATIONS. PAVIA, LAMPMAN, KRIZ, VYVYAN – SPECTROSCOPY, THIRD EDITION; CENGAGE PUBLICATIONS. INTERNET SITES: http://www.scribd.com/doc/52130160/Lecture-9-Chapter-7-9-28-05 CLASS NOTES.Slide 34: Thank you… You do not have the permission to view this presentation. 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