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1、“UV Visible Spectroscopy”Dr. Paras ShahM. Pharm (Q.A.),What is spectroscopy?Spectrum + Scopies“When a beam of light is allowed to pass through a prism or grating, it will dispersed into seven colors from red to violet and the set of colors or band produced is called spectrum” + Examination“Spectrosc
2、opy is the branch of the science dealt with the study of interaction of Electro Magnetic Radiation (EMR) with matter”So the spectroscopy means examination of spectrum. From the type of radiation, which is absorbed, we can get idea about the nature (type) of the compound and from the amount of the ra
3、diation, which is absorbed, we can get idea about the concentration (amount) of the substance. So the spectroscopy is used for qualitative and quantitative analysis.,When a beam of light is passed through a transparent cell containing a solution of an absorbing substance, reduction of the intensity
4、of the light may occur. This is due to 1) Reflection at the inner and outer surfaces of the cell2) Scattered by the particles present in the solution3) Absorption of the light by the molecules in the solution,Interaction of EMR with matter,Classification of Spectroscopy:1) Absorption Spectroscopy: t
5、he type and amount of the radiation, which is absorbed depend upon the structure of the molecules and the numbers of molecules interacting with the radiation. The study of these dependencies is called absorption spectroscopy. (UV, IR, NMR, X-Ray, ESR)2) Emission spectroscopy: if sufficient energy ge
6、ts impinged upon a sample, the outer electrons in the species will be raised from their stable ground state to higher energy level (unstable in nature). These excited species rapidly emits a photon and return to their ground stable energy level. The type and amount of radiation, which is emitted, is
7、 studied, this type of spectroscopy is called emission spectroscopy. (AES, MES, Fluorimetry)3) Scattering spectroscopy: if the incoming radiation strikes with the solid particles suspended in the solution, the light transmitted at an angle other than 1800 from the incident light. This spectroscopy i
8、s called scattering spectroscopy. (turbidimetry, nephelometry),What is EMR?- EMR is a form of energy that is transmitted through space at an enormous velocity- It can travel in space with the same speed at that of light.- As the name implies an EMR is an alternating electrical and associated magneti
9、c force field in space (It contains electrical and magnetic components)- The two components oscillate in planes perpendicular to each other and perpendicular to the direction of propagation of the radiation.- EMR consist of a stream of discrete packets (particles) of pure energy, which is called pho
10、tons or quanta.- The energy of photon is proportional to the frequencyE = h where E= Energy of photons, h= planks constant (6.624 x 10-27 erg. Sec) and =frequency of radiation in cycles/second,-Wavelength (): it is the distance between two successive maxima on an electromagnetic wave. (m, cm, mm, m,
11、 nm, and A0) - Frequency (): is the numbers of waves passing through a given point in unit time. (T-1, sec-1, cycles/second, hertz, fresnel)- Wave numbers (): is the numbers of waves per centimeter in vacuum. ( cm-1)-Velocity (V): is the product of wavelength and frequency ( X =V) (cm/sec, m/sec),Cl
12、assification of EMR: EMR is arbitrarily classified in to different regions according to wavelength.,Energy associated with the molecules:1) The molecule as a whole may move this is called translation and the energy associate with this movement is called transnational energy. (Etrans)2) The part of t
13、he molecules, that is atom or groups of atoms, may move with respect to each other. This motion is called vibration and the associated energy is called vibrational energy. (Evib)3) The molecule may rotate about an axis. And such rotation is characterized by the rotational energy. (Erot)4) Besides th
14、ese modes of movements, the molecule possesses an electronic configuration and the energy associated with this configuration is called electronic energy. (E ele) E total = E trans + E vib + E rot + E ele.,Theoretical principlesIf a molecule is allowed to interact with the EMR of a proper frequency,
15、the energy of the molecule is raised from one level to a higher one; we say that absorption of radiation takes place. In order for absorption to occur, the energy difference between the two energy level must be equal to the energy of the photon absorbedE2 E1 = h where E1 is energy of lower level and
16、 E2 is the energy of upper level- This energy jump from one level to another is called transition- The graph of the light absorption against the frequency is called absorption spectra.- Visible and Ultraviolet light provides enough energy for electronic transition there for called electronic spectra
17、.,On absorption of energy by a molecule in the ultraviolet region, changes are produced in the electronic energy of the molecule due to transitions of valence electrons in the molecule.,E,6* Anti-bonding,* Anti-bonding,n Non-bonding, Bonding,6 Bonding,Types of transitions:1) 6 6*: A transitions of e
18、lectrons from a bonding sigma orbital to the higher energy antibonding orbitals. ( eg. Alkane). Sigma bonds are, in general, very strong, there fore they require high energy for the transitions and this transitions requires very short wavelength (near about 150 nm)2) n 6*: This transition involves s
19、aturated compounds with one hetero atom with unshared pair of electrons (n electrons). Corresponding band appears at 180-200 nm.3) *: This transition is available in compounds with un-saturation (eg. Alkene). Corresponding band appears at 170-190 nm.4) n *: This type of transitions are shown by the
20、unsaturated molecules containing one or more hetero atoms. (O, N, S),5) Conjugated system: In conjugated dienes, the orbitals of the separate alkene group combine to give new orbitals i.e. the two new bonding orbitals which are designated 1 and 2 and new two anti-bonding orbitals designated as 3* an
21、d 4*. So for the 2 3* transition very low energy is requires corresponding to the higher wavelength.,Some important terms:1) Chromophore: It is a group of molecules, which is responsible for the absorption of light by molecules. It is conjugated dienes. It is minimum structural requirements for the
22、absorption of radiation in UV range. 2) Auxochrome: It is a saturated group containing unshared electrons which when attached to a Chromophore changes both intensity as well as the wavelength of the absorption maxima. e.g. OH, NH2, Cl etc.3) -max: It is a wavelength at which there is a maximum absor
23、ption or absorption intensity. It is a physical constant and characteristic of structure and so useful for identification of compounds. It is independent of concentration. 4) Bathochromic shift: The shifting of absorption to a longer wavelength due to substitution or solvent is called as bathochromi
24、c shift. It is also called as Red shift. e.g., max of Ascorbic acid=243nm, max of Ascorbic acid in alkali medium=299nm.,5) Hypsochromic shift (Blue shift): Shifting of max to lower value or left hand side due to substitution, solvent, pH etc is called as Hypsochromic shift. e.g. max of Phenol in bas
25、ic media=297nm, max of Phenol in acidic media=277nm.6) Hyperchromism: Increase in absorption intensity (e) due to solvent, pH or some other factors called hyperchromic effect.7) Hypochromism : Decrease in absorption intensity due to substituent, solvent, pH etc. called hypochromic effect.8) A1%1cm (
26、A one percent one centimeter): Is the absorbance of the solution having concentration 1 gm per 100 ml of the solution.9) Molar absorptivity (): Is the absorbance of the solution having concentration gm.mol.weight/1000 ml of the solution. = (A1%1cm X Mol. Wt.)/1010) Transmittance (T): is the ratio of
27、 IT/I0 and % transmittance (%T) is given by %T=100 IT/I0,1) Absorbance (A): Is the degree of absorption of light by a medium through which the energy passes. It is expressed as the logarithm of the ratio of light transmitted through a pure solvent to the intensity of light transmitted through the me
28、dium. It is the area under the curve. A= log I0/ITA= log I0 - log IT A=2- log % TAbsorption SpectraThe graph of the light absorption against the frequency is called absorption spectra. It is characterized by 1) -max:- Position of spectra 2) Intensity of absorbance:- the amount of the radiation absor
29、bed by the molecule.1) Factors affecting the position of the spectrum (-max)A) Structural factorsi) Substitution: Placing a substituent on a Chromophore may produce change in -max by two mechanisms: introduction of an entirely new transition and/or shifting the wavelength of existing transitions. E.
30、g. each alkyl substituent produce 5 nm bathochromic shift.,ii) Solvent: the solvent effect arises because solvation is frequently different for the ground and excited states. If the ground state is solvated more strongly than the excited state, the energy difference between the levels is increased.
31、The increase in energy difference is reflected in a shift of the absorbance to shorter wavelengths. iii) Geometry: e.g. stilbene. Trans-stilbene absorbs at a longer wavelength than cis-stilbene due to steric effects. Co-planarity is needed for the most effective overlap of the -orbitals. The cis-iso
32、mer is forced in to a non planar conformation due to steric effects. The cis isomer are twisted slightly out of plane by steric interactions so that the degree of conjugation in the system is slightly less than the trans isomers, resulting in greater energy for the transitions.A) Non Structural fact
33、orsi) PH: e.g. Phenolphthalein: in alkaline medium it is pink and in the acidic medium it is colorless,i) Temperature: Temperature provides more energy to ground state. As a result energy required for excitation will be less, so there is bathochromic shift. 2) Factors affecting the intensity of abso
34、rption of radiation.i) Thickness of the medium: Lamberts law: “when a beam of monochromatic light is allowed to pass through a transparent medium, the rate of decrease of intensity with the thickness of medium is directly proportional to the intensity of incident radiation”. It gives relationship be
35、tween absorbance and the thickness of the medium. ii) Concentration of absorbing solute: Beers law: “when a beam of monochromatic light is allowed to pass through a transparent medium, the rate of decrease of intensity with the concentration of absorbing solute is directly proportional to the intens
36、ity of incident radiation”. It gives relationship between absorbance and the concentration of the medium.A = a b c (Fundamental equations of spectroscopy),Deviation from the Beers curve. (Errors in spectrophotometric measurement)Errors may arise from instrumental of from chemical factors. Instrument
37、al errors can arise from several sources. Noise, fluctuation in light source. The ideal absorbance range for most measurement is in the range of 0.2 to 0.8. The calibration curve is relatively linear in this range. Other factor includes Spectral Slit Width (SSW). As slit width is increased, the fine
38、 structure of the absorption band is lost as the incident light is no more monochromatic. Generally, fast scan rates tend to distort spectra, altering the positions of both maxima and minima as well as diminishing peak intensities. This introduces both qualitative and quantitative errors in the meas
39、urement.,A numbers of chemical factors may also produce errors in the analysis. Solute-solute interaction, e.g. aggregation, precipitation, dimerization etc. Ionization or even complexation of the analyte in solution can also lead to apparent deviation from the Beers curve. Fluorescence from absorbi
40、ng species in solution may also contribute to interference.,INSTRUMENTATION,Light source,Slit,Monochromator,Sample Holder,Detector,Display,Light Source: (source of electromagnetic radiation): The tungsten filament lamp is a satisfactory light source for the region 350 to 2000 nm. It consists of a tu
41、ngsten filament contained in a glass envelope. The most convenient light source for UV radiation is discharge lamp. Generally deuterium discharge lamp is used. It is consisting of deuterium-filled silica envelope. It gives radiation from 185 to 380 nm.,2) Slit: (Radiation intensity controlling devic
42、e): Enough light must pass through the sample to elicit a measurable response from the detector.3) Monochromator: (wavelength selecting device). It converts polychromatic light in monochromatic light (light having one wavelength). a) Filters: Glass filters are pieces of colored glass, which transmit
43、 limited wavelength ranges of the spectrum. The color is produced by incorporating oxides of such metals.,b) Prisms: When a beam of light passes through a prism, it is bent or refracted. The amount of deviation is dependent on the wavelength. The prism is made up of quartz for use in the UV light, s
44、ince glass absorbs wavelengths shorter than about 330 nm. Glass prism are preferable for the visible region of the spectrum, as the dispersion is much greater than that obtained with quartz. c) Grating: Most modern UV spectrophotometer uses diffraction grating as a Monochromator. It consisting of a
45、very large number of equispaced lines (200-2000 per mm) ruled on a glass plate. They can be used either as transmission grating or when aluminized, as reflection grating.,4)Sample Holder: The sample holder is known as cuvettes. Cuvettes must be transparent to the light, so the glass cells are used i
46、n the visible region and quartz or silica cells are used in the UV region. The cells used in the UV spectrophotometers are usually 1 cm in path length but cells are available from 0.1 cm to 10 cm or more. 5) Detectors (Radiation measuring device): It is also known as photocell. They convert radiatio
47、n energy in electrical energy. For the determination of substances by spectrophotometric techniques, precise determinations of the light intensities are necessary. Photoelectric detectors are most frequently used for this purpose. They must be employed in such a way that they give a response linearl
48、y proportional to the light input and they must not suffer from drift or fatigue.,a) Barrier-layer photocell:,It one of the simplest detectors, which has the advantage that it requires no power supply but gives a current, which is directly proportional to the light intensity. It is consists of a met
49、allic plate, usually copper or iron, upon which is deposited a layer of selenium. An extremely thin transparent layer of a good conducting metal, e.g. silver, platinum or copper, is formed over the selenium to act as one electrode, the metallic plate acting as the other. Light passes through the semitransparent silver layer causes release of an electron, which migrates, to the collector. The electron accumulating on the collector resulting in a potential difference between the base and collector, which can be measured by a low resistance galvanometer