uvvisiblespectroscopypptuvvisiblespectroscopyppt

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 o

4、f 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 matterclassification of spectroscopy:1) absorption spectroscopy: the

5、 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 gets

6、 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 s

7、tudied, 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 is

8、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 magnetic f

9、orce 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 photon

10、s 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, nm

11、, 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)class

12、ification 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 the

13、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 these

14、 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, the

15、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 e2

16、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. -o

17、n 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.e6* anti-bonding * anti-bonding n non-bonding bonding 6 bonding types of transitions:1) 6 6*: a transitions of electro

18、ns 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 saturat

19、ed 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 unsatu

20、rated 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* and 4*. s

21、o 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 absorpt

22、ion 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 absorption o

23、r 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 bathochromic shift

24、. 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 basic media

25、=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 (a one pe

26、rcent 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 it/i0 a

27、nd % 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 medium. it

28、 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 absorbed by t

29、he molecule.1)factorsaffectingthepositionofthespectrum(-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.g. each alkyl su

30、bstituent 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. the increase in

31、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-isomer is forced in

32、 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 factorsi) ph: e.g. p

33、henolphthalein: in alkaline medium it is pink and in the acidic medium it is colorlessi) temperature: temperature provides more energy to ground state. as a result energy required for excitation will be less, so there is bathochromic shift. 2)factorsaffectingtheintensityofabsorptionofradiation.i) th

34、ickness 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 between absorbance and the

35、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 intensity of incident radiation

36、”. 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. instrumental errors can arise from s

37、everal 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 structure of the absorpti

38、on 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 measurement. a numbers of chem

39、ical 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 absorbing species in solution may

40、 also contribute to interference.instrumentation light sourceslitmonochromatorsample holderdetectordisplay 1)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 tungsten filament contained in a

41、 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 device): enough light must pass thro

42、ugh 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 limited wavelength ranges of t

43、he 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, since glass absorbs wavelengths

44、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 very large number of equispaced

45、 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 in the visible region and quart

46、z 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 radiation energy in electrical energy.

47、 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 linearly proportional to the light in

48、put 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 metallic plate, usually copper or

49、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 cause

50、s 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 circuit. the useful working range of selenium photocell is 380-780 nm. the

51、ir lack of sensitivity compared to phototube and photo multiplier tube, restricts their use to the cheapest colorimeters and flame photometers.it consists of an anode and a cathode sealed in an evacuated glass tube, which may have a quartz or silica window for uv measurement. b) photo emissive tube:

52、 the cathode is coated with a layer of light sensitive material that emits electrons upon absorption of photons.a power supply maintains the anode positive with respect to the cathode so that the photoelectrons are collected at the anode. this current is directly proportional to the light intensity.

53、 phototubes are available for use over the entire uv/visible region of the spectrum, but no single tube covers the entire range satisfactorily. therefore many instruments with phototube detectors employ interchangeable blue and red sensitive phototube in order to provide sufficient sensitivity over

54、the entire spectrum. c) photo multiplier tube: it is very sensitive detectors with very short response times. it contains a photo cathode and a series of dynodes, which are also photosensitive. a higher successive potential is maintained between each dynodes. a photoelectrons released from the photo

55、 cathode is accelerated toward the first dynode by their voltage difference, where it strikes to release several electrons. the secondary electrons are then accelerated toward the second dynode where the process repeats. in this way multiplication of the electrons can be achieved. the current from p

56、hototubes and photo multiplier tubes never falls to zero. a small residual current called dark current is produced, due to long exposure of the light. 6) display (read out meter): the signal from the detector is normally proportional to the intensity of light, and after amplification may be displaye

57、d as % t or after passing through a logarithmic conversion circuit as absorbance (log 1/t)types of instruments:instruments for measuring the absorption of light may be of the single beam or double beam type. in a single beam instrument, light from the sources passes through a filter and then through

58、 the sample and in to the detector. the signal from the detector is proportional to the intensity of the light beam striking it. to make a measurement of absorbance using a manually controlled single-beam instrument, the monochromator is adjusted to the required wavelength and the appropriate lamp a

59、nd photocell are selected by means of switches. the first step is to close a shutter in the path (or adjust dark filter) and adjust 0 %t. the second step is to open the shutter and place the cell containing only the solvent in the light beam and adjust the scale on 100 % t (equivalent to 0 absorbanc

60、e). the third step is to place the sample cell in the light path and measure the intensity it or its equivalent absorbance. in double beam spectrophotometer, the monochromatic light is split by the beam splitter in to two equal intensity light beam, which are directed alternatively in rapid successi