UV-VISIBLE SPECTROSCOPY

INSTRUMENTATION, CHOICE OF SOLVENTS AND SOLVENT EFFECT

1. WELCOME 1

2. ULTRAVIOLET-VISIBLE SPECTROSCopy TOPIC: Instrumentation, choice of solvents and solvent effect. Submitted by BINUJA.S.S First year M.Pharm Department of Pharmaceutical chemistry Ezhuthachan college of pharmaceutical sciences

3. INTRODUCTION INSTRUMENTATION INSTRUMENTS Uv - visible Photometers Uv – visible Spectrophotometers CHOICE OF SOLVENTS AND SOLVENT EFFECT REFERENCE CONTENTS 3

4. INTRODUCTION Oldest physical method for quantitative analysis and structural elucidation. qualitative and quantitative analysis of substances. The spectrophotometer technique is to measures light intensity as a function of wavelength . 4

5. It does this by : diffracting the light beam into a spectrum of wavelengths direct it to an object receiving the light reflected or returned from the object detecting the intensities with a charge-coupled device displaying the results as a graph on the detector. 5

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7. Instrumentation Instruments for measuring the absorption of UV-Visible are made up of following components. Sources Wavelength selectors Sample containers Detectors Readout device 7

8. Sources REQUIREMENTS OF AN IDEAL SOURCE Stable and should not allow fluctuations. Emit light of continuous radiation. Provide sufficient intensity. 8

9. UV Radiation sources Visible sources Deuterium lamp Hydrogen lamp Tungsten halogen lamp Xenon arc lamp Mercury arc lamp Tungsten halogen lamp Mercury vapour lamp Carbonone lamp 9

10. Tungsten Halogen lamp filament of Tungsten fixed in evacuated condition and then filled with inert gas. The filament can be heated up to 3000 k , beyond this tungsten starts sublimating. It is used when polychromatic light is required. To prevent this along with inert gas some amount of halogen is introduced (usually Iodine). 10

11. Sublimated form of tungsten reacts with Iodine to form Tungsten –Iodine complex. Which migrates back to the hot filament where it decomposes and Tungsten get deposited. Wavelength region: 350~2500nm DEMERIT: It emits the major portion of its radiant energy in near IR region of the spectrum. 11

12. Deuterium and Hydrogen lamp By electrical excitation of hydrogen or deuterium at low pressure. A pair of electrodes is enclosed in a glass tube filled with hydrogen gas. Current is passed → discharge of electrons occurs → excitation of hydrogen molecules → emission of UV radiations in near UV region. They are stable. 12

13. Wavelength range: 160-800nm . Quartz windows must be employed. 13

14. XENON ARC LAMP: Two tungsten electrodes separated by some distance. enclosed in a glass tube (for visible) with quartz or fused silica and xenon gas is filled under pressure. An intense arc is formed between electrods . Good source of continuous plus additional intense radiation. Its intensity is higher than the hydrogen discharge lamp. DEMERIT : The lamp since operates at high voltage becomes very hot during operation and hence needs thermal insulation. 14

15. Wavelength range: 200-1000nm Peak intensity at about 500nm. 15

16. MERCURY ARC LAMP Mercury vapour is stored under high pressure and excitation of mercury atoms is done by electric discharge. Not suitable for continuous spectral studies, because of the presence of sharp lines or bands. 16

17. LIGHT EMITTING DIODE p-n junction device. When forward biased, produces radiant energy. Diodes are made from,(mixtures of) Gallium aluminium arsenide Gallium arsenic phosphide Gallium phosphide Gallium nitride Indium gallium nitride. 17

18. Wavelength region: 375-1000nm. MERITS Long life time smaller environmental impact than tungsten filament lamp. 18

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20. WAVELENGTH SELECTORS Filters and Monochromators Filters A device that allows light of the required wavelength to pass but absorbs light of other wavelength wholly or partially. Suitable filter can select a desired wavelength band. A particular filter may be used for a specific analysis. Absorption filters Interference filters 20

21. Absorption filters - works by selective absorption of unwanted radiation and transmits the radiation which is required. Examples- Glass and Gelatin filters. Selection of absorption filter is done according to the following procedure: 21

22. Draw a filter wheel. Write the colour VIBGYOR in clockwise or anticlockwise manner, omitting Indigo. 22

23. If solution to be analyzed is BLUE in colour a filter having a complimentary colour ORANGE is used in the analysis. Similarly, we can select the required filter in colorimeter, based upon the colour of the solution. An Absorption glass filter is made of solid sheet of glass that has been coloured by pigments which is dissolved or dispersed in the glass. The colour in the glass filters are produced by incorporating metal oxides like V, Cr, Mn, Fe, Ni, Co, Cu etc. 23

24. INTERFERENCE FILTERS Works on the interference phenomenon, causes rejection of unwanted wavelength by selective reflection. It is constructed by using two parallel glass plates, which are silvered internally and separated by thin film of dielectric material(CaF2, SiO , MgF2). These filters have a band pass of 10-15nm with peak transmittance of 40-60%. 24

25. Merits - Provide greater transmittance and narrower band pass (10-15nm) as compare to absorption filter. Inexpensive Additional filters can be used to cut off undesired wavelength. 25

26. MONOCHROMATORS A device used to isolate the radiation of the desired wavelength from wavelength of the continuous spectra. PARTS An entrance slit A collimating lens A dispersing device (usually a prism or grating) A focusing lens An exit slit 26

27. The dispersing element in monochromator is 2 types; Prism Grating 27

28. PRISM MONOCHROMATOR Prism is made from glass, Quartz or fused silica. Quartz or fused silica is the choice of material of UV spectrum. Light is passes → dispersion of polychromatic light → rotation of the prism different wavelengths of the spectrum can be made to pass through in exit slit on the sample. 28

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30. Refraction type 30

31. Reflection type 31

32. GRATING MONOCHROMTOR Are most effective one in converting a polychromatic light to monochromatic light. As a resolution of ±0.1nm could be achieved by using gratings, they are commonly used in spectrophotometers. 2 types. 1. Diffraction grating. 2. Transmission gratings. 32

33. DIFFRACTION GRATING More refined dispersion of light is obtained by means of diffraction gratings. consist of large number of parallel lines ( grooves) about 15000-30000/ inch is ruled on highly polished surface of aluminium. 33

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35. TRANSMISSION GRATING Similar to diffraction grating Refraction takes place instead of reflection. Refraction produces reinforcement. Occurs when radiation transmitted through grating reinforces with the partially refracted radiation. 35

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37. SAMPLE CONTAINERS The cells or cuvettes are used for handling liquid samples. The cell may either be rectangular or cylindrical in nature. UV region- quartz or fused silica Visible region- glass 37

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39. DETECTORS Device which converts light energy into electrical signals. The transmitted radiation falls on the detector which determines the intensity of radiation absorbed by sample. 3 TYPES. Barrier layer cell/Photovoltaic cell Phototubes/ Photo emissive tube Photomultiplier tube 39

40. Barrier layer cell/Photovoltaic cell Consists of thin film metallic layer coated with silver or gold and acts as an electrode. metal base plate which acts as another electrode. These two layers are separated by a semiconductor layer of selenium. 40

41. Light radiation falls on selenium layer → electrons become mobile → taken up by transparent metal layer. Creates a potential difference between two electrodes & causes the flow of current. A flow of current observed in galvanometer which is proportional to the intensity and wavelength of light falling on it. 41

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43. PHOTOTUBES/ PHOTOEMISSIVE TUBES Consists of a evacuated glass tube with a photocathode and a collector anode. surface of photocathode is coated with a layer of elements like caesium, silver oxide or mixture of them. radiant energy falls on photosensitive cathode, electrons are emitted which are attracted to anode causing current to flow. More sensitive compared to barrier layer cell. 43

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45. PHOTOMULTIPLIER TUBE Multiplication of photoelectrons by secondary emission of electrons. In a vacuum tube, a primary photo-cathode is fixed which receives radiation from the sample. Eight to ten dynodes are fixed each with increasing potential of 75-100V higher than preceding one. last dynode is fixed an anode or electron collector electrode. extremely sensitive to light and is best suited where weaker or low radiation is received. 45

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48. READOUT DEVICE The data from a detector are displayed by a readout device, such as an analogue meter, a light beam reflected on a scale, or a digital display , or LCD . The output can also be transmitted to a computer or printer. 48

49. INSTRUMENTS PHOTOMETER SPECTOPHOTOMETER 49

50. PHOTOMETER An instrument for measuring the of light or the relative intensity of a pair of lights . Also called an illuminometer. It utilizes filter to isolate a narrow wavelength region. Single beam Double beam 50

51. SPECTOPHOTOMETER An instrument measures the ratio, or a function of the two, of the radiant power of two EM beams over a large wavelength region. It utilizes dispersing element (Prisms/Gratings) instead of filters, to scan large wavelength region. Single beam Double beam 51

52. SINGLE BEAM VISIBLE PHOTOMETERS A direct reading instrument consisting of tungsten filament/LED as a source lens to provide a parallel beam a filter and a photodiode transducer The current produced by the photodiode is processed with electronics or a computer to give a readout in absorbance. Sample is inserted into the light path. The absorbance is calculated as the logarithm of the ratio of these signals. 52

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54. Double beam visible photometer Used to measure the absorbance of sample in a flowing stream. The light beam is split by a 2 branched fibre optic. One beam is passes – sample Other beam- reference. Filters are placed after the cells before the photodiode with nearly identical response. Electrical outputs → voltages and signals are processed by a log ratio amplifier or a computer to give a readout proportional to absorbance. 54

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56. UV absorption photometers Serve as detectors in HPLC. Mercury vapour lamp- source. Wavelength range 254nm. 56

57. Spectrophotometers Visible spectrophotometers Wavelength range: 380-800nm . Simple and inexpensive. Eg ; Spectronic20 Tungsten filament – source Diffraction by a simple reflection grating → radiation passes→ sample/reference 57

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60. UV spectrophotometers Single beam computerised spectrophotometers Light from the source is carried through lens and/or through aperture to pass through a suitable filter. The type of filter to be used is governed by the colour of the solution. The sample solution to be analysed is placed in cuvettes. 60

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62. After passing through the solution, the light strikes the surface of detector (barrier-layer cell or phototube) and produces electrical current. The output of current is measured by the deflection of needle of light-spot galvanometer or micro ammeter. 62

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64. Double beam spectrophotometers two beams are formed in the space by a U shaped mirror called as beam splitter or beam chopper. Chopper is a device consisting of a circular disc. One third of the disc is opaque and one third is transparent, remaining one third is mirrored. It splits the monochromatic beam of light into two beams of equal intensities. 64

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66. Eg ; Varian Cary100 66

67. CHOICE OF SOLVENTS It should not itself absorb radiations in the region under investigations. It should be less polar so that it has minimum interaction with the solute molecule. Most commonly: 95%ethanol Cheap, good dissolving power, does not absorb radiation above 210nm. Typical examples 67

68. Solvent Wavelength(nm) Water 205 Methanol 210 Ethanol 210 Ether 210 Chloroform 245 Carbon tetra chloride 265 Cyclohexane 210 68

69. SOLVENT EFFECT The position and intensity of an absorption band may shift when the spectrum is recorded in different solvents. A dilute sample solution is prefered for analysis. Most commonly used solvent is 95% ethanol. It is best solvent as it is cheap,transparent down to 210nm. Position as well as intensity of absorption maxima get shifted for a particular chromophore by changing the polarity of solvent. By increasing polarity of solvent → dienes , conjugated hydrocarbons→ no shift 69

70. α , β unsaturated carbonyl compound show 2 different shifts. n → Π* transition. Absorption band moves to shorter wavelength(blue shift) by increasing the polarity of the solvent. Ground state is more polar as compared to the excited state. Eg : absorption maximum of acetone is at 279nm in hexane as compared to 264nm in water. 70

71. Π → Π* transition. Absorption band moves to longer wavelength(red shift) by increasing the polarity of the solvent. The dipole-dipole interaction with the solvent molecules lower the energy of excited state more than that of the ground state. 71

72. Effect of solvent polarity on the various types of bands. K-band Due to conjugated enes & enones are affected differently by changing the polarity of the solvent. K bands due to conjugated dienes are not affected by changing the polarity of the solvent, while these bands due to enones shows a red shift by increasing the polarity of solvent. R band The absorption shifts to shorter wavelength (blue shift) with increasing polarity of solvent. 72

73. B band The position as well as the intensity of the band is not shifted by increasing the polarity of the solvent. But the heterocyclic aromatic compound, a marked hyperchromic shift is observed by increasing the polarity of the solvent. 73

74. REFERENCE Principles of instrumental analysis – Doglas A Skoog , James holler, Timothy a nieman , 5 th edition. Instrumental methods of analysis by Willards , 7 th edition. Instrumental methods of chemical analysis by H.Kaur Instrumental methods of chemical analysis by B.K Sharma. Instrumental methods of chemical analysis by Gurdeep R.Chatwal , Sham K.Anand . 74

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