UV-visible spectrophotometry ppt

UV visible spectrophotometry is explained in this presentation

1. UV-Visible Spectrophotometry R Deepthi Assistant Professor Vignan Institute of Pharmaceutical Technology Visakhapatnam

2. Pharmaceutical Analysis Pharmaceutical Industry Bulk - API Formulation – Various Dosage Forms Qualitative analysis Quantitative analysis

3. Spectral methods Light absorption or Emission UV spectroscopy, IR spectroscopy, Flourimetry Chromatographic methods Adsorption/Partition Paper chromatography, TLC and HPLC Electro analytical techniques Electrochemical property Electrophoresis (paper, gel and capillary) Radioactive methods Radio immune assay, ELISA Thermal methods Physical characteristics Differential thermal analysis (DTA), Differential scanning calorimetry (DSC), Thermo gravimetric analysis (TGA) Miscellaneous methods Titrimetric methods/ volumetric methods

4. SPECTROSCOPY It is a branch of Science that deals with the study of interaction of matter with light. Or It is the branch of science that deals with the study of interaction of electromagnetic radiation with matter.

5. Introduction to Spectroscopy Spectroscopy is the measurement and interpretation of Electromagnetic radiation (EMR) absorbed or emitted when the molecules or atoms of a sample move from one energy state to another energy state. This change may be from ground state to excited state or excited state to ground state. At ground state, the energy of a molecule is the sum of total of Rotational, vibrational and or electronic energies. Spectroscopy measures the changes in the Rotational, vibrational and or electronic energies. EMR is made up of discrete particles called Photons . They can also travel in Vacuum. E= hv E= Energy h= Planks constant, v= Frequency of radiation

6. Why we use Spectroscopy? -Detection of Functional groups -Detection of Impurities -Qualitative analysis -Quantitative analysis -Single component without chromophore -Drugs with chromophoric agent -Used to detect conjugation of the compounds

7. UV-Visible Spectroscopy Spectroscopy is concerned with the study of absorption of UV radiation which ranges form 200-400nm. Compounds which are colored absorb radiation from 400-800nm. Compounds which are colorless absorb radiation in UV range. It involves the absorption of UV-Visible light by a molecule, causing the electrons of the molecule to undergo transition from ground state to the excited state.

8. Ɛ = E 1cm 1% X Molecular weight 10 E 1cm 1% means absorbance of 1% w/v solution, using path length of 1cm. E 1cm 1% at a wavelength is a constant value for each drug and can be seen in Pharmacopoeias.

9. Absorption Curve Absorption curve or absorption spectrum of a molecule in the UV-Visible region is a plot between energy absorbed by the molecule or absorbance (on Y-axis) and wavelength (on X-axis). Fig: Absorption spectra

10. From the Absorption curve, the wavelength at which maximum absorption of radiation takes place is called λ max (lambda max) The λ max of every substance is specific and unique, which is useful in identifying the substance. λ max is not affected by concentration of the substance, however absorbance increases with increase in concentration of the absorbing molecules.

11. Quantitative analysis can be carried out by plotting a curve between concentration on X-axis and absorbance on Y-axis which is called as the calibration or standard curve. This curve gives a straight line showing a direct relationship between the absorbance and concentration. Thus, it obeys Beer’s Law. The concentration of a substance in the given sample solution can be determined by extrapolation or intrapolation of the curve.

12. The degree of absorption of the UV-Visible light by the substance and the wavelength at which absorption occurs are measured and recorded by the instrument called UV-Visible spectrophotometer. They operate within the wavelength range of 800-200nm.

13. If the curve between absorbance and concentration is concave upwards, it is termed as positive deviation and if the curve is concave downwards it is a negative deviation. Positive deviation : occurs when a small change in concentration leads to a large change in the absorbance. Negative deviation : occurs when a large change in concentration produces small change in the absorbance.

14. Deviations from Beer’s law are classified as Real Deviations Chemical Deviations Instrumental Deviations Real Deviations : Real deviations are related to the concentration of the absorbing substance. Beers law holds good only for dilute solutions having concentration in the range of 10 -6 to 10 -7 M. At higher concentrations, molecules of the absorbing species undergo collisions and interact with each other. Hence, at higher concentrations, molecules will not absorb radiation in the same manner as in dilute solutions.

15. Chemical Deviations Chemical deviations arise if the absorbing species undergoes chemical changes such as association, complex formation, dissociation, hydrogen bonding, hydrolysis, ionization. Ex: Benzyl alcohol in chloroform exists as a polymer. Upon dilution it dissociates into its monomers. The polymeric form shows positive deviation The monomeric form shows negative deviation

16. b) Presence of impurities in the solution that fluoresence or absorb the radiations at the required wavelength also cause deviation from Beer’s law. c) Reaction of the absorbing species with each other or with the solvent results in the formation of new species. These show different absorbance values which results in deviations. Chemical deviations can be corrected by using appropriate wavelength, buffers and selecting suitable solvents.

17. 3. Instrumental Deviations a) Beer’s law is obeyed only when a monochromatic radiation is used. Polychromatic radiations lead to negative deviations from Beer’s law. b) Any fluctuations in the intensity of the radiation source may also cause deviations from Beer’s law. c) Changes in the sensitivity of the detector employed and defects in the amplication of the radiation also cause deviations. d) Improper slit width also contributes towards deviation from Beer’s law. Improper slit width allows the stray radiations to reach the detector.

18. These stray radiations are absorbed by the impurities present in the sample solution and lead to changes in the absorbance value of the sample. Applications of Beer-Lamberts Law It is used in determining the concentration of unknown solution by comparing with a solution of known concentration (standard solution) using colorimeter or spectrophotometer. Conc. of unknown solution= Absorbance of unknown solution x Conc. of standard solution Absorbance of Standard solution 2. It is used in quantitative analysis of both single and multicomponent analysis. 3. It is used for estimating the concentration of chlorophyll in leaf cells.

19. Limitations of Beer’s Law: It is applicable only at low concentrations. It is not applicable to suspensions . It is not applicable to coagulated particles which cause scattering of radiations, which may either increase or decrease absorbance values Only monochromatic light should be used.

20. In both UV as well as Visible spectroscopy, only the valence electrons absorb the energy, thereby the molecule undergoes transition from Ground state to excited state . This absorption is characteristic and depends on the nature of electrons present.

21. The types of electrons present in any molecule may be conveniently classified as σ, Π and n bonds. ‘ σ’ electrons : These are the ones present in saturated compounds. Such electrons do not absorb near UV, but absorb vacuum UV radiation (<200nm) Π electrons: These electrons are present in unsaturated compounds ex: double or triple bonds ex: c꞊c ‘n’ electrons: These are the non bonded electrons which are not involved in any bonding ex: lone pair of electrons like S, O, N and Halogens.

22. A molecule has either n, Π or σ or a combination of these electrons. These bonding ( Π or σ ) and non-bonding (n) electrons absorb the characteristic radiation and undergoes transition from ground state to excited state. By the characteristic absorption peaks, the nature of the electrons present and hence the molecular structure can be elucidated.

23. Various Electronic transitions are

24. Sigma electron  (σ  electron ) : Sigma (σ) bonds are the single bonds in molecules. Also, recall that a double bond is composed of  1 σ  bond and 1 π bond and a triple bond is composed of  1 σ  bond and 2 π bonds. This means in the given molecule: There are  5 σ  bonds and 1 π bond in CH 2 =CH 2 .

25. Pi electrons

26. Of all the electronic transitions, this type of transition requires highest energy. This is observed with Saturated compounds (especially hydrocarbons). The peaks do not appear in UV region, but occur in vacuum UV region i.e., 125-135nm. Some of the examples with such transition are : Methane -122nm Ethane- 135nm Cyclopropane-190nm

27. Methane CH4

28. Cyclopropane C3H6

29. This type of transition gives rise to B,E and K bands. B bands (Benzenoid bands)-Aromatic and hetero aromatic groups E bands (Ethylenic bands)- Aromatic compounds K bands- Conjugated bonds Extended conjugation (due to addition of more double/triple bonds) and alkyl gps shifts the λ max towards longer wavelength (Bathochromic shift) Trans isomers absorbs at longer wavelength with more intensity than cis isomers (Hyperchromic shift)

30. Compounds containing multiple bonds like alkenes, alkynes, carbonyls, aromatic compounds undergo these type of transitions. Ex: Alkenes generally absorb in the region 170-205nm.

31. This transition occurs in Saturated compounds with hetero atoms like S, O, N or halogens. These compounds usually detected at wavelength 180-250nm. Compound λ max Methylene chloride 173nm Methanol 203nm Ether 215nm Trimethylamine 227nm Chloroform 237nm

32. Of all the transitions this transition requires the lowest energy. The peaks due to this transition are called as R-bands. Compounds containing double bonds involving hetero atoms like S,O,N undergo such type of transitions ex aldehydes and ketones These are detected at 270nm-350 nm These all transitions are used in structural elucidation.

33. Terms used in UV-Visible Spectroscopy Chromophore Chromophore or chromophoric group is a group or part of a molecule responsible for characteristic absorption at a wavelength. Chromophores are covalently unsaturated.

34. Auxochrome These are co- ordinately saturated/ unsaturated groups. They do not have any characteristic absorption on their own but can modify or enhance the absorbing properties of chromophore.

35. Shift in λ max towards longer wavelength is called Bathochromic shift. It is also called as Red shift. It can occur due to increase in conjugation (addition of double or triple bonds), addition of alkyl substituents in the molecule. The effect is due to presence of auxochrome or by change of solvent. This arises due to substitution of Functional gps like: OH, 1 , 2 , 3 amino groups or solvent effect.

36. Shift in λ max towards shorter wavelength is called Hypsochromic shift. Also called as Blue shift It may occur due to removal of double or triple bonds by saturation, dealkylation and also due to change of solvent.

37. Solvent Effect Polarity of the solvent has a great influence on the position of λ max as well as on the intensity of absorption maximum. λ max for polar compounds usually shifts with a change in the polarity of the solvent. However, λ max of non-polar compounds remains same in polar as well as non-polar solvents. For ex: absorption transitions of polar bonds like C=O (but not ethylene) are effected by the solvent polarity.

38. As the polarity of the solvent is increased, π – π * transition bands undergo bathochromic shift. This is because, the π * state is more polar than the π state hence stabilization is greater in excited state than the ground state in polar solvents. The excited state is stable due to the formation of hydrogen bonds with the polar solvents . As the stability is increased, the energy of the excited state decreases which eventually causes Bathochromic shift.

39. In case of n – π * transition , by increasing the polarit y of the solvent, the absorption maximum is shifted towards shorter wavelength because ground state with two n electrons is more stabilized than the excited state with only 1 n electron. In n - π * transition, the ground state is more polar than the excited state and hence is more stabilized because of greater extent of hydrogen bonding with the polar solvents. Similarly with increasing polarity, the n – σ * bands are displaced towards the shorter wavelength i.e., blue shift .

40. Therefore, increase in polarity of the solvent shifts the n – π * and n– σ * absorption bands towards shorter wavelengths ( hypsochromic or blue shift ) and π – π * absorption bands towards longer wavelengths (bathochromic or red shift). Examples of various solvents used in UV-Visible spectroscopy are: Water Ethanol n-hexane Acetone Ether Dioxane Chloroform Benzene etc.

41. Chromogenic agent Chromogen or chromogenic agents are those which is capable of forming a chromophore or color by complexation, chemical reaction, ionisation etc. Ex: Ferric chloride when added to salicylic acid produces a violet colour with λ max at 530 nm. So ferric chloride reagent is called as chromogen.

42. Isobestic point It is a specific wavelength at which the molar absorption coefficient λmax of two or more chemical substances in equilibrium.

43. Instrumentation of UV-Visible spectroscopy The spectrophotometers used in UV-Visible spectroscopy measure the ratio of the intensity of light transmitted (I t ) through a sample and the intensity of incident light (I o ). The components of UV and visible spectrophotometer are identical except that they differ in their radiation sources and material used in Sample holders . The radiation source used in visible region is tungsten lamp whereas in UV region : deuterium lamp, hydrogen discharge lamp, mercury arc and xenon discharge lamp are commonly used.

44. Components of UV-Visible spectrophotometer 1. Source of Light/ Radiation source 2. Filters and Monochromators 3. Sample Holder/container to hold sample/ Cuvvettes 4. Detectors 5. Suitable amplifier and readout device

45. Components of UV-Visible Spectrophotometer Components UV region Visible region Radiation sources Deuterium discharge lamp, Hydrogen discharge lamp, Mercury arc, Xenon discharge lamp Tungsten lamp Monochromators Prism Monochromators Grating Monochromators Prism Monochromators Grating Monochromators Filters Absorption filters Interference filters Absorption filters Interference filters Sample cells Made of Quartz or fused silica Made up of Glass Detectors Photovoltaic cell Phototubes Photomultiplier tubes Photovoltaic cell Phototubes Photomultiplier tubes

46. Source of Light A UV spectrum ranges from 200-400 nm and Visible spectrum ranges from 400-800 nm. Any lamp source which can give adequate intensity of radiation can be used. Ideal characteristics of Light It should provide continuous radiation . It should provide adequate intensity. It should be stable and free from fluctuations . Should not show exhaustion on continuous usage.

47. For visible Radiation 1.Tungsten lamp: is used mostly in colorimeter and spectrophotometers. The lamp consists of a tungsten filament in a vacuum bulb and filled with an inert gas. 2.Carbon arc lamp: For a very high intensity, carbon arc lamp is used. It provides an entire Range of visible spectrum.

48. For UV source of radiation 1.Hydrogen discharge lamp: It is more stable, robust and widely used. It gives radiation from 120-350 nm. The lamp consists of hydrogen gas with high pressure. 2.Deuterium lamp: It is similar to hydrogen discharge lamp, but it is filled with deuterium instead of hydrogen. Offers more intensity than other light sources. This is most widely used, but is little expensive.

49. 3.Xenon discharge lamp: In this lamp, xenon at 10-30 atmospheric pressure is filled in and has two tungsten electrodes. The intensity is greater than hydrogen discharge lamp. 4.Mercury arc lamp: This lamp contains mercury vapour stored under high pressure and offers bands which are sharp. Since the spectrum is not continuous , it is not widely used.

50. Filters and Monochromators The source of light gives radiations from 200nm to 800nm. This is polychromatic in nature (light of several wavelengths) In colorimeter or spectophotometer , we require only monochromatic light (light of single wavelength). Hence a filter or monochromator is used which converts polychromatic light into monochromatic light .

51. Filters are of two kinds: 1. Absorption Filters 2. Interference Filters Monochromators are of two types: Prism type ( Dispersive type or Littow type ) Grating type ( Diffraction grating & Transmission grating )

52. Filters Absorption filters These filters are made up of glass, coated with pigments or they are made up of dyed gelatin . They absorb the unwanted radiation and transmit the rest of the radiation which is required for colorimetry .

53. These filters can be selected according to the procedure given below: Draw a filter wheel (circle with 6 parts). Write the colours (VIBGYOR) in clockwise and anticlockwise manner omitting Indigo. If the colour of the solution is Red, we have to use Green filter and if the colour of the solution is Green, we have to use Red Filter. (The colour of the filter is opposite to the colour of the solution i.e., complimentary in nature). 4. Similarly, we can select the required filter in a colorimeter, based upon the colour of the solution.

54. Merits: Simple in construction Low Cost Selection of filter is easy. Demerits: Less accurate since band pass is more (±30nm) i.e., if we have to measure at 500nm, radiation ranging from 470nm to 530nm falls on the sample. Intensity of radiation becomes less due to absorption by filters.

55. ii) Interference Filters This filter is also known as Fabry -Perot filter. It has dielectric spacer film made up of CaF2, MgF2, or Sio2 between two parallel reflecting silver films. The thickness of dielectric spacer film can be 1/2 λ (1 st order), 2 λ /2 (2 nd order), 3 λ /2 (3 r d order ). Etc. The mechanism is, the radiation reflected by the 2 nd film and the incoming radiation undergoes Constructive interference to give a monochromatic radiation, which is governed by the following equation. λ= 2 դ b/m Where, λ= wavelength of light obtained դ = dielectric constant of layer material b= layer thickness m = order no.

56. Band pass is 10-15nm (i.e., if we select 500nm, the obtained radiation ranges from 490-510nm). Maximum transmission is 40%. Merits Inexpensive Lower band pass when compared to absorption filters and hence more accurate. Use of additional filter cuts off undesired wavelengths. Demerits Peak transmission is low, and becomes so when additional filters are used to cut off undesired wavelength. The band pass is only 10-15nm and hence higher resolution obtained with monochromators or gratings cannot be achieved.

57. b. Monochromators A monochromator is a device which converts a polychromatic beam of light into a monochromatic beam. It consists of the following parts: Entrance slit: It passes the incoming beam of polychromatic light into a narrow beam. Collimator1: It collimates or makes parallel the radiations coming from the entrance slit. Prism / Grating: It disperses the radiations with respect to the component wavelengths. Collimator2: It reforms the images of the entrance slit. Exit slit: It selects a narrow band of dispersed spectrum for observation by the detector.

58. Prism Monochromator These are usually made up of glass, quartz, or fused silica. They disperse the polychromatic light falling on them into its individual rainbow colours according to their wavelengths. They are commonly used in inexpensive instruments. The band pass is lower than that of the filters and hence it has better resolution.

59. Prism monochromators are of 2 types: Refractive type 2. Reflective type Refractive type: Light from the radiation source / source of light falls on a collimator. The parallel radiations from collimator are dispersed into different colours or wavelengths, and by using another collimator, the images of the entrance slit are reformed.

60. The reformed ones will be either Violet, Indigo, Blue, Green, Yellow, Orange or Red. The required radiation on exit slit can be selected by rotating the prism or by keeping the prism stationary and moving the exit slit.

61. 2. Reflective type ( Littrow prism/ Littrow type mounting) Its working is similar to refractive prisms. It consists of a reflective surface on one side so that light does not pass through the prism on the other side. The dispersed radiations are reflected and collected on the same side as the source of light radiation falls.

62. Grating Monochromators Gratings are made up of glass, quartz or alkyl halides like KBr and NaBr . Back surface of the gratings are coated with aluminium to make them reflective. These are highly effective than prisms in converting a polychromatic light into monochromatic light. They consist of densely arranged parallel lines or grooves. The no. of grooves per mm in a grating monochromator vary depending upon the type of spectrophotometer used. They give a resolution of ±1nm. Grating monochromators are of two types: Diffraction gratings Transmission Gratings

63. Diffraction gratings: These are used when polychromatic light is to be separated with high resolution. It works on the Mechanism of reinforcement (strengthening). The incident rays upon the grating gets reinforced with the reflected rays, resulting in a radiation whose wavelength is expressed by the equation: λ = d(Sin i + Sin r) n Where, n = Order no. (0,1,2,3) λ = wavelength of the resultant radiation d = grating space i = Angle of incidence r = Angle of reflection

64. 2. Transmission gratings: Transmission grating is similar to diffraction grating. But, Refraction takes place instead of reflection. Refraction rays produces reinforcement. When the transmitted radiations reinforce with the refracted radiations, a resultant radiation is obtained whose wavelength is given by the equation: λ = d Sin ϴ n Where, λ = Wavelength of the resultant radiation d = Grating spacing ϴ = Angle of Diffraction n = Order no. (0,1,2,3 etc )

65. Thus a light radiation at any angle ϴ or any order can be collected and used in the instrument by either moving the grating and fixing the slit or moving the slit and keeping the grating constant.

66. Sample cells/ Sample Holders: Sample cells or cuvettes are used to hold the sample solutions. Their shape (rectangular or cylindrical) and material of construction varies depending on the instrument and the nature of the sample being analyzed.

67. For ex: cuvvettes made up of quartz are used in UV spectrophotometer, while those of glass are used in visible spectrophotometers. The pathlength of the cell is normally 1cm, however cells with longer pathlengths upto 10cm or shorter pathlengths of 1-2mm are also available.

68. Before taking the measurements, sample cells should be thoroughly cleaned to avoid any contamination. The level of the sample solution must be up to the mark etched on its surface or above the light beam to avoid reflections from the upper surface of the liquid.

69. An ideal characteristics of sample cell The material used in the construction of the sample cell should not react ( chemically inert ) with the solvent it is holding. It must transmit light of the required wavelength. It should have uniform thickness .

70. Detectors Detectors are the devices which convert light energy into electrical signals , that are displayed on the readout device. After passing through the sample cell, a part of the radiation is absorbed by the sample and the remaining is transmitted. The transmitted radiation falls on the detector which determines the intensity of radiation absorbed by the sample. Photomultiplier tubes Barrier layer cell / Photo voltaic cell Photo tubes or P hotoemissive tubes Silicon Photodiode detector

71. Barrier layer cell (Photovoltaic cell) Construction It consists of a photocathode which is a thin metallic layer coated with gold or silver . It also contains a metal base (usually iron) which acts as anode . Between these two electrodes is a semiconductor layer of selenium.

72. Working When light rays falls on the selenium layer , electrons are generated and taken up by the photocathode . Because of the poor electrical conductivity of the selenium , the electrons get accumulated on the cathode leading to the development of potential difference across the two electrodes which results in the generation of electric current.

73. The current flow causes deflection in the galvanometer which gives the measure of the intensity of radiation, i.e., greater the intensity of radiation, greater is the current produced hence greater the deflection in galvanometer. Advantage: It is economical hence commonly used. Disadvantages: For signal amplification, the resistance of the external circuit should be low. It is less responsive towards light except for blue and red.

74. Phototubes ( Photoemissive Tubes) Construction It consists of a hollow glass tube with a photocathode and a collector anode. The surface of the photocathode is coated with a layer of elements like cesium , potassium, silver oxide or a mixture of these . Working When light falls on the photocathode, electrons are produced that travel towards the collector anode and generate current. The amount of current generated is directly proportional to the intensity of light radiation . Compared to barrier layer cell, phototubes are more sensitive and therefore widely used.

75. Photomultiplier Tube This type of detector is the most sensitive of all the detectors, expensive and used in sophisticated instruments. Principle The principle employed in this detector is that, “ multiplication of photoelectrons by secondary emission of electrons”.

76. Construction It consists of a light sensitive cathode (photocathode) and a series of 10 anodes (dynodes) maintained at a potential of 75-100 volts. Photomultiplier tube being sensitive can detect extremely weak signals also, therefore it is used in intricate instruments. PMT can detect very weak signals, even 200 times weaker than that could not be done using Photovoltaic cell. Hence it is useful in flourescence measurements. PMT should be shielded from stray light in order to give accurate results.

77. Silicon photodiode detector Silicon photodiodes have become important recently because 1000 or more can be fabricated side by side on a single small silicon chip. (the width of individual diodes is about 0.02mm). With one or two of the diode-array detectors placed along the length of the focal plane of a monochromator . All the wavelengths can be monitored simultaneously, thus making high-speed spectroscopy possible. Silicon photodiode detectors respond extremely rapidly, usually in nanoseconds.

78. Silicon photodiodes  are solid state semiconductor devices, sensitive to light in the wide spectral range of 200 – 1200nm , which extends from deep ultraviolet through the visible to the near infrared region. Working Photodiodes are semiconductor light sensors that generate a current or voltage when the P-N junction in the semi conductor is illuminated by light. When a photon of sufficient energy strikes the diode, it excites an electron, thereby creating a free electron (and a positively charged electron hole). This mechanism is also known as the inner photoelectric effect. This device can be used in three modes: Photovoltaic as a solar cell, reversed-biased as a photo detector, and forward-biased as an LED.

79. A photodiode is a type of photo detector capable of converting light energy into electrical energy. Photodiodes are similar to regular semiconductor diode except that they may be either exposed (to detect UV or X-rays ) or packaged with a window or optical fibre to allow light to reach the sensitive part of the device. A photodiode is designed to operate in reverse bias.

80. Reverse Biasing In reversed bias the negative region is connected to the positive terminal of the battery and the positive region is connected to the negative terminal. The reverse potential increases the strength of the potential barrier. The potential barrier resists the flow of charge carrier across the junction. It creates a high resistive path in which no current flows through the circuit.

81. Advantages of Silicon Photodiode Excellent linearity with respect to incident light Low noise Wide spectral response Compact and light weight Long life

82. Instrumentation Colorimeters These are usually inexpensive and less accurate. They measure either Absorbance or Transmittance or both. Filters are used for different coloured solutions. The wavelength used is 400-700nm . Spectrophotometers These are little more expensive than colorimeters. They can be used for a wide wavelength range ex 360-900nm. The accuracy of instrument is very high since grating monochromators and photomultiplier tubes are used. They can be supported by amplifiers or recorders. Nowadays microprocessors are used.

83. Different types of Instruments Colorimeters, Spectrocolorimeters and Spectrophotometers Colorimeters: They contain Tungsten lamp Absorption filters and Photovoltaic cell They are designed to read either %Transmittance or absorbance Single beam instruments are non-recording type.

84. Spectrocolorimeters They contain: Tungsten lamp Prisms as monochromators Photovoltaic cell or Phototubes as detector They are also single beam, and non-recording type Designed to measure %Transmittance or Absorbance

85. Spectrophotometers These are expensive Designed to measure %Transmittance or Absorbance Record the absorbance using a plotter or recorder These are of double beam type where we can use both sample and reference solution at a same time. Advantages Storage of Spectrum Comparison of Spectra Rapid wavelength scanning Data Manipulation Derivative spectral mode Software can be used. More accurate and reliable.

86. Single beam UV-Visible Spectrophotometer In a single beam UV-Visible Spectrophotometer, light from the radiation source after passing through a monochromator enters the sample cell containing the sample solution. A part of the incident light I is absorbed by the sample while the remaining gets transmitted It. The transmitted light strikes the detector and produces the electrical signals. The signal produced by the detector is directly proportional to the intensity of the light. The output is measured by a micrometer or galvanometer and displayed on the readout device. The absorbance readings of both the standard and unknown solutions are recorded after adjusting the instrument to 100% transmittance with a blank solution each time the wavelength is changed.

87. Advantages: Simple in construction Easy to operate Economical Disadvantages: Any fluctuations in the intensity of the radiation source affects the absorbance readings. It requires adjustment of transmittance to 0% and 100% whenever the wavelength is changed. Hence, a continuous spectrum is not obtained.

88. Double beam UV-Visible Spectrophotometer Double beam spectrophotometer allows direct measurement of the ratio of intensities of sample and reference beams respectively. The design of double beam spectrophotometer allows direct measurement of the ratio of intensities of sample and reference beams respectively. The design of a double beam spectrophotometer is similar to single beam spectrophotometer except it contains a beam splitter.

89. Monochromator selects the required wavelength of light which is then passed through the exit slit and received by a rapidly rotating beam splitter. Beam splitter is a circular disc one third of which is opaque, one third is transparent and the remaining is mirrored. The beam splitter splits the monochromatic beam of light into two beams of equal intensities. One beam is passed through the sample cell and the other through the reference cell.

90. After passing through the sample and reference cells, the transmitted beams reach the detectors and produce a pulsating current which is proportional to the intensities of the incident light (I ) and transmitted light (I t ). The detectors are connected to an amplifier and readout device which gives the final result in absorbance log I / I t or transmittance log I t / I 0. The ratio between the intensities of incident and transmitted beams gives the direct measure of the absorption or transmittance by the sample and reference solutions. A double beam spectrophotometer can be designed using one or two detectors.

91. Double beam UV-Visible Spectrophotometer

92. Advantages It facilitates rapid scanning over wide wavelength region. Fluctuations due to radiation source are minimized. Disadvantages Construction is complicated. Instrument is expensive

93. Applications Qualitative analysis Detection of Purity/Impurities: Impurities present in the sample can be detected from Absorption spectrum by measuring the absorbance at specific wavelength and can be compared with that of standard. Identification of the compound: Compounds containing lone pair of electrons or conjugated double bonds absorb UV radiations and give characteristic absorption spectrum. The unknown compound can be identified by comparing its absorption spectrum with that of the standard.

94. 2. Quantitative analysis a)A 1% 1cm value(Absorbance factor method) A 1% 1cm values represent the absorbance or extinction value of a 1% solution at a definite wavelength. This method helps in the estimation from raw materials and finished products (formulation). This method is used when reference standard is unavailable. The percent purity can be determined by the formula: %Purity = Observed absorbance X 100 A 1% 1cm X Concentration

95. b) Reference standard method This method involves the measurement of average A 1% 1cm value . The average value can be determined by measuring the absorbance of different standard solutions and calculating their average. The average A 1% 1cm value can be utilized to determine the percentage purity using the formula: %Purity = Observed absorbance X 100 Average A 1% 1cm value X Concentration

96. c) Direct comparison method or single standard method In this method, the absorbance of a standard solution of known concentration and a sample solution is measured. The concentration of can be calculated using the formula A 1 = Ɛ C 1 t A 2 = Ɛ C 2 t Where, A 1 , A 2 = Absorbance of standard and sample C 1, C 2 = Concentration of standard and sample Ɛ = Molar ext. coefficient t = Pathlength (1cm) Dividing 1 st eqn with 2 we get, C 2 = C 1 X A 2 A 1

97. d. Calibration curve Method Calibration curve is a plot of concentration on X-Axis and absorbance of series of standard solution of known concentration on Y-axis. A straight line is drawn through maximum no. of points coinciding. This line is called calibration curve Using this calibration curve- - Concentration of the drug -Amount -% Purity can be determined

98. Overlay spectra or Linearity

99. Difference spectrophotometric method This method is used for quantification of sample drug in the presence of interfering ions or substances especially in biological fluids. It involves the measurement of differential absorption between two different chemical forms of the same drug of equal molarity. The different forms of drug can be obtained by changing the pH with the help of buffers or by chemical reactions such as oxidation, reduction etc. There is a shift in wavelength with different forms of drug.

100. i ) Quantitative Estimation Using UV spectroscopy The following drugs can be estimated by using UV spectroscopy Drugs Solvents λ max (nm) Ampicillin Water 325 Griseofulvin Ethanol 291 Paracetamol 0.1 M NaoH 257 Phenformin Water 520 Verapamil HCl 0.1M HCl 278

101. Estimation of Paracetamol in Tablets using UV- spectroscopy Weigh accurately 20 tablets and powder them. The powdered drug which is equivalent to 150mg of paracetamol is weighed accurately. To the powdered drug, add 50ml of 0.1M sodium hydroxide and dilute it with 100ml of water. Shake the solution for 15minutes. Make the volume with water upto 200ml. Shake gently and filter. 10ml of this filtrate is diluted to 100ml with water. To 10ml of the above solution, add 10ml of 0.1M sodium hydroxide and dilute with water to get 100ml. Shake thoroughly. The absorbance of the resulting solution is measured at 257nm.

102. Concentration of paracetamol is calculated by taking A1%1cm value as 715 at λ max of about 257nm. The amount of drug can be estimated in terms of percentage purity. %Purity = Observed Absorbance X 100 A1%1cm X Concentration ii) Quantitative Estimation Using Visible Spectroscopy: Estimation of Metals and Functional groups Drugs are generally colourless or white in nature. Colourless drugs are rendered coloured by coupling them with chromogenic agents , followed by their estimation using visible spectroscopy.

103. Keto-enol Tautomerism It is a form of Tautomerism in which the keto form (an aldehyde or ketone ) and enol form of a compound are interconvertible and exist in chemical equillibrium . Thus, keto-enol tautomerism can be studied using UV spectroscopy as λ max and molar extinction coefficient € differ among the keto and enol form.

104. Cis and Trans Isomerism UV spectroscopy differentiates cis and trans isomers. Trans-isomer exhibits absorbance at a longer wavelength than cis - isomer. Conversion of cis -isomer to trans-isomer form results in bathochromic shift and hyperchromic effect and viceversa .

105. Conjugation Conjugation can occur between two or more carbons containing double or triple bonds and also in carbon –oxygen double bond. Conjugation helps in determining the presence of an aromatic ring, the number and position of the substituents present on the carbon of conjugated system. Conjugation causes shifting of λ max towards longer wavelength, as the no. of double bonds increases. Compounds λ max H 2 C=CH 2 174nm Ethylene H 2 C=CH-CH=CH 2 217nm Butadiene H 2 C=C=C=CH 2 267nm Butatriene

106. Functional group determination Alkyl substitution Presence or absence of Unsaturation Identification of Unknown compound Structure of Organic and Inorganic compounds Determination of Molecular Weight

107. A solvent for UV spectroscopy should meet the following requirements It should not itself absorb radiation in the region under investigation. It should be less polar, to minimum interaction with solute molecule. Spectroscopic (Analytical grade) solvents should be used. The most commonly used solvent is 95% ethanol. It is cheap Good dissolving power Does not absorb radiation above 210nm

108. 5. Some other solvents which can be used above 210nm are n-hexane, cyclohexane , methanol, water and ether. 6. Hexane and other hydrocarbons are sometimes preferred to polar solvents. Because they have minimum interaction with solute molecule. 7. Benzene, chloroform, carbon tetra chloride cannot be used, because they absorb in the range of about 240-280nm.

109. A solvent is a liquid that dissolves another solid, liquid, or gaseous solute, resulting in a solution. Solvents can be broadly classified into two categories: Polar Non-Polar A drug may show varied spectrum at particular wavelength in one particular condition but shall absorb partially at the same wavelength in another condition. These changes are mainly due to: Nature of the solvent Nature of Absorption Band Nature of the Analyte

110. The position and intensity of an absorption band may shift when the spectrum is recorded in different solvents. A dilute sample solution is preferred 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 Ex dienes , conjugated hydrocarbons no shift

111. Solvent Effects A most suitable solvent is one which does not itself absorb in the region under investigation. A dilute solution of the sample is always prepared for the spectral analysis. Most commonly used solvent is 95% Ethanol. Ethanol is a best solvent as it is cheap and is transparent down to 210 mµ. Commercial ethanol should not be used as it contains benzene which absorbs strongly in the ultraviolet region. Some other solvents which are transparent above 210 mµ are n-hexane, methyl alcohol, cyclohexane , acetonitrile , diethyl ether etc.

112. Hexane and other hydrocarbons can be used as these are less polar and have least interactions with the molecule under investigation. For ultra-violet spectroscopy, ethanol, water and cyclohexane serve the purpose best. The position and the intensity of absorption maximum is shifted for a particular chromophore by changing the polarity of the solvent. By increasing the polarity of the solvent, compounds like dienes and conjugated hydrocarbons do not experience any appreciable shift. Thus, in general, the absorption maximum for the non-polar compounds is the same in alcohol (polar) as well as in hexane (non-polar).The absorption maximum for the polar compounds is usually shifted with the change in polarity of the solvents.

113. α,β -unsaturated carbonyl compounds show two different shifts. n-π* transition (less intense): In such a case, the absorption band moves to shorter wavelength by increasing the polarity of the solvent . In n-π* transition, the ground state is more polar as compared to the excited state. The hydrogen bonding with solvent molecules takes place to lesser extent with the carbonyl group in the excited state. For ex, absorption maximum of acetone is at 279 nm in hexane as compared to 264 nm in water.

114. π-π* transition (intense): For such a case, the absorption band moves to longer wavelength by increasing the polarity of the solvent . The dipole interactions with the solvent molecules lower the energy of the excited state more than that of the ground state. Thus, the value of absorption maximum in ethanol will be greater than that observed in hexane.

115. The absorption spectra of Ethyl-4-hydroxy-1-(4-methoxyphenyl)-2-quinolinone-3-carboxylate (L1) is depicted in attached Figure 1. Two maximum absorption peaks were observed at 231nm and 288nm in ethanol, while in water at 225 and 296nm. according to solvent polarity, water more polar, and increasing polarity of the solvent shifts pi-pi* to higher energy and n-pi* to lower energy. but the situation is reversed, the first peak at 225nm (in water) while 231nm (in Ethanol) and the second peak appears as one peak at 296nm (in water) while it is two peaks at 288-296nm (in Ethanol).  

116. Define Spectroscopy Instrumentation of UV-Visible Spectrophotometer. Beer-lamberts law: Definition, Derivation, Applications and limitations. Types of Deviations and Classify the reasons for deviation and how to prevent them. Explain the working of 4 Detectors along with neat labelled diagram. Explain in detail various applications of UV-Visible spectroscopy. Explain the electrons and different electronic transitions happening in UV spectroscopy. Explain Solvent effect in UV spectroscopy. Define the terms: Bathochromic shift, Hypsochromic shift, Hyperchromc , Hypochromic , Aoxochrome , Chromophore , Chromopgenic agent with suitable examples, λ max, Isobestic point. Explain the working of Double beam UV -visible spectrophotometer.