Instrumental Methods of Analysis

Usually, analysis is not considered an easy subject and it can't be understood on its own if you don't have some proper notes and clear concepts so I am here to help you in analysis for clearing few concepts on UV-Visible spectrophotometer, soon will come up with a new set of notes on new topic depending upon the response.

1. Basics of spectroscopy, Jablonski diagram, Instrumentation of UV-Visible Spectrometer, Beer-Lambert’s law and its deviation Shweta Mishra, M. Pharm (Pharmaceutical Chemistry) [email protected] Sri Aurobindo Institute of Pharmacy, Indore-Ujjain State Highway, Indore , Madhya Pradesh 453555 1 ©

2. ADVANTAGES OF INSTRUMENTAL METHODS OVER CHEMICAL ONES Speed (can be automated, sample throughput is high) Sensitivity (trace or ultra-trace analysis is possible) Selectivity (accurate in the presence of many other components) Reproducibility (reliable, objective) Sample requirement is small (ml or mg or less can be handled) DISADVANTAGES Cost Complexity Maintenance 2 ©

3. Introduction to the electronic structure of the atoms In 1904 Sir JJ Thomson proposed the theory of matter. He pictured the positive charge of an atom d istributed uniformly throughout a sphere of protons and neutrons and the negative charge was surrounding it in a jelly like fashion . Rutherford modified this theory that electrons revolves around the nucleus in such a way that the centrifugal force exactly balances the inward attractive force much like our planets to around the sun . Hence regardless of their speed, they enter the nucleus. Moreover the electron losing energy would omit continuous radiation without any sharp breaks. In 1903, Bohr proposed a radically different view of the atomic a structure based on the optical spectrum of hydrogen. He included the postulates of quantum theory proposed by Max Planck. 3 ©

4. Introduction to the electronic structure of the atoms Bohr proposed that the electron in a hydrogen atom always described a fixed circular path around the nucleus. Search orbits named ‘ stationary states' maybe thought of various circles differing in radius. The angular momentum of each stationary state was an integral multiple of N.H/2П which amounts to angular momentum. The angular momentum mvr is given by the formula : mvr = N (H/2П) w here N is an integer called a quantum number 4 ©

5. He also postulated that as long as the electron remained in a given orbit it neither radiates nor absorbs energy. When the electron moves from one orbit to another it was considered to involve the absorption or emission of definite quantity of energy depending upon whether the electron moved from lower state to higher one or vice versa. This energy manifests as radiation and the frequency of such radiation is manifests as a spectral line which could be related to the energies of electron in the two states E1 and E2: E2 - E1 = hυ 5 ©

6. Line spectra appear in two forms, absorption spectra, showing dark lines on a bright background, and emission spectra with bright lines on a dark or black background. These two types are in fact related and arise due to quantum mechanical interactions between electrons orbiting atoms and photons of light. Photons of light each have a specific frequency. The energy of a photon is a function of its frequency and is determined by: E = h f   where   f  is the frequency of the photon, E is the energy and h is Planck's constant (= 6.626 x 10 -34 J.s) Production of Line Spectra 6 ©

7. An electron orbits around a nucleus in a stable energy level. If a photon of a specific frequency interacts with the electron, it can gain sufficient energy to "jump up" one or more levels. The photon is absorbed by the electron so cannot continue on to be detected by an observer. The electron then "de-excites" and jumps back down to a lower energy orbit, emitting a photon of specific frequency. This photon, however, could be emitted in any direction, not just in the same direction as the original incident photon. The Balmer series of visible lines for atomic hydrogen are caused by transitions from the n = 2 orbit to and from higher orbits. This is shown schematically in the diagram for the Bohr model of the atom. The Lyman Series involves transitions down to the n = 1 orbit and involve higher frequency photons in the UV region whilst the Paschen Series (to n = 3) produces IR spectral lines. 7 ©

8. Spectroscopy It is the branch of science dealing with the interaction of electromagnetic radiations with matter in quantized that is specific energy levels. It refers to the study of how radiated energy and matter interacts e.g. UV-Visible spectroscopy, IR and NMR spectroscopy etc. 8 ©

9. Spectroscopy? Spectrogram? Spectrometry? Spectrograph? T he measurement of a specific spectrum The machine for recording spectra 9 ©

10. SPECTROSCOPY Atomic Spectroscopy Molecular Spectroscopy Absorption Emission Absorption Emission AAS Flame emission/ Flame photometry UV, IR, NMR, Electron spin resonance spectra, Raman spectra Flourimetry , Phosphorimetry 10 ©

11. The absorption and emission spectrum concept 11 ©

12. The origin of UV band If energy is supplied to the sample, for example, by passing electromagnetic radiation through it, the sample atoms or molecules may absorb energy and be promoted into the higher energy level. F or an atom that absorbs in the UV, the absorption spectrum sometimes consist of very sharp lines as would be expected for a quantized process occurring between two discrete energy levels. For molecules, however, the UV absorption usually occurs over a wide range of wavelengths, because molecules (as opposed to atoms) normally have many excited modes of vibration and rotation at room temperature. In fact, the vibration of molecules has many states of vibrational and rotational excitation. 12 ©

13. The energy levels for these states are quite closely spaced, corresponding to energy differences considerably smaller than those of electronic levels. The rotational and vibrational levels are thus superimposed on the electronic levels. A molecule can therefore undergo electronic and vibrational rotational excitation simultaneously. Because there are so many possible transitions, each differing from the others by only a slight amount, each transition consists of a vast number of lines spaced so closely that the spectrophotometer cannot resolve them and usually consider them as a broad band of absorption centered near the wavelength of the major transition in UV spectrum . Deactivation of the thermally excited atoms to lower energy states occurs very rapidly and photons of light are emitted which have energy ( δE ) equal to the difference between the upper and lower energy states. δE = E h - E l = ( E electronic - E vibrational - E rotational ) h - ( E electronic - E vibrational - E rotational ) l 13 ©

14. When a sample containing metallic atoms is heated above 2000 ᵒ C, it undergoes partial or complete partial or complete dissociation into free atoms and then volatilizes to free gaseous atoms. The electrons of the gaseous atoms exist in discrete quantized energy levels, i.e. they are in the orbitals which have specific energy levels that are characteristic of the element. The electrons in the outer orbitals of the atom may absorb thermal energy and be promoted to one or higher energy states. The gain in energy of each electron during a transition is a specific quantity corresponding to the difference between the energy levels after and before excitation. 14 ©

15. δE = E2 - E1 = hυ = hc /λ = hcΰ E α 1/ λ Consequently, a very large number of wavelengths are absorbed, each corresponding to a particular δE value, which are so close together that they appear as a continuous band spectrum. The transition δE 4, given by the highest proportion of molecules, corresponds with the wavelength of maximum absorption ( λ max ) . Light at longer and shorter wavelengths is absorbed less strongly because fewer molecules give related transitions. Longer wavelengths have insufficient energy to induce electronic transitions but they are sufficiently energetic sufficiently energetic to increase the vibrational energy levels, and the associated rotational levels, of many molecular bonds. https://d396qusza40orc.cloudfront.net/physicalchemistry/Spectroscopy_Course/spectroscopy-graphs/visible-spectrum-phenolphthalein.html 15 ©

16. Jablonski diagram 16 ©

17. Transition Time scale Radiative process Internal Conversion 10 -14  - 10 -11  s No Vibrational Relaxation 10 -14  - 10 -11  s No Absorption 10 -15  s Yes Phosphorescence 10 -4  - 10 -1  s Yes Intersystem Crossing 10 -8  - 10 -3  s No Fluorescence 10 -9  - 10 -7  s Yes 17 ©

18. Order of energy:   Energy for absorption > Energy for Fluorescence > Energy for phosphorescence   Order of wavelength:   Wavelength for absorption < Wavelength for Fluorescence < Wavelength for phosphorescence 18 ©

19. EMR 19 ©

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21. Instrumentation of spectrometers: The essential components of spectrometers: A source of EMR A monochromator A sample compartment A detector and associated electronics A recorder or display 21 ©

22. Light source Ideal requirements of light source: It should generate a beam with sufficient power for ready detection and measurement. It should emit continuous radiation in the region being studied. It should be stable. It should emit a measurable signal throughout the region. The distribution of energy through a spectrum is mainly a function of temperature; higher the temperature of the light source the shorter the wavelength of the peak emission. 22 ©

23. Common energy sources for the various regions are indicated below: IR Radiation: Globar and nerst glowers are the common sources of IR radiation. The globar is an electrically heated rod of silicon carbide, and the Nernst glower is a small rod of refractory oxides which, when heated to 1200-1500 degrees, will conduct electricity and thus maintain itself in incandescence. Both these sources operate without a glass envelope, which would absorb IR radiation of wavelength greater than 2 μ m. 23 ©

24. Visible radiation: Tungsten filament lamp is used for the region 350 nm- 2000 nm. Tungsten filament is contained in a glass envelope. IR Radiations which may cause stray light effects can be removed by using suitable filters. The life of the lamp is limited by the evaporation of tungsten, which darkens the inside of the envelope and reduces the energy of the incident light. Tungsten-halogen lamps, are used in more expensive instruments and halogen gas is filled inside a quartz envelope. The halogen prevents evaporation of the tungsten and increases the life of the tungsten. The higher operating temperatures extends the lower wavelength limit to 310 nm and gives greater light intensity. 24 ©

25. UV Radiations: T he most convenient light source for measurement in the UV region is a deuterium discharge lamp. It consists of 2 electrodes contained in a deuterium- filled silica envelope. The passage of high voltage from a special power supply across the electrodes causes emission of deuterium lines which, at low pressure inside the lamp, broaden to give a continuous spectrum in the range 185- 380 nm. Above 380 nm, the emission is not continuous, and the sharp lines at 486 nm and 656.1 nm may be used to calibrate the wavelength scale in this region. 25 ©

26. Below 185 nm the output is reduced by absorption in the silica envelope. Deuterium lamps give approx. five times greater light intensity than hydrogen lamps when operated at same voltage. UV-Visible spectrophotometers normally have both a deuterium lamp and a tungsten (or tungsten-halogen) lamp, and selection of the appropriate lamp is made by moving either the lamp mountings or a mirror to cause the light to fall on the entrance slit of the manochromator . 26 ©

27. Monochromators • Light gives radiation from 400-800nm • This is called polychromatic light which is of several wavelength • Hence a filter or monochromator is used to convert polychromatic light into monochromatic lights used 27 ©

28. Monochromators Ideal requirements of the monochromator : It should be able to isolate a particular wavelength or range of wavelengths. It should adhere to the beer-lambert’s law. It should emit a measurable signal throughout the region. Commonly used monochromators are: Filters Prisms Gratings 28 ©

29. Absorption Filters Glass filters are pieces of coloured glass which transmit limited wavelength ranges of the spectrum. The color is produced by incorporating oxides of such metals as vanadium, chromium, manganese, iron, nickel, and copper in glass. The color absorbed is the complement of the color of the filter. For example, a filter absorbing yellow light will appear blue. The range of wavelengths transmitted (bandwidth) is very high and may exceed 150 nm. 29 ©

30. Wavelength (nm) Color Complement color 400-450 Violet Yellow-green 450-480 Blue Yellow 480-490 Green-blue Orange 490-500 Blue-green Red 500-560 Green Purple 560-575 Yellow-green Violet 575-590 Yellow Blue 590-625 Orange Green-blue 625-750 Red Blue-green Below 400 nm the color gradually becomes invisible as it passes into UV; above 750 nm it passes into IR. 30 ©

31. G elatin filters consisting of a mixture of dyes incorporated in gelatin and sandwitched between glass plates are more selective, with bandwidths about 25 nm. Merits: • Simple, cheaper Demerits : Less acurate , bandpass is more (+/- 30 nm), intensity is less 31 ©

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33. Interferometeric filters They have an even more narrower bandwidth (about 15 nm) and consists of 2 parallel glass plates, silvered internally and separated by a thin film of cryolite or other dielectric material. Such filters make use of interference of light waves rather than absorption to eliminate the undesired radiation, and serve as relatively inexpensive monochromators for a specific purpose. Eg . Isolation of calcium radiation at 626 nm and sodium at 589.3 nm in flame photometer Ordinary filters are incapable for this and cause large errors in measurement of calcium content. 33 ©

34. • Also known as fabry-perot filter • It has dielectric spacer made up of CaF2, MgF2, or SiO . • Thickness of dielectric spacer film can be ½ ʎ (1st order), 2/2 ʎ (2nd order) , 3/2 ʎ (3rd order ). Mechanism is constructive interference followed by this equation . ʎ=2 η b/m ʎ=wavelength of light η= dielectric constant of material B= layer thickness 34 ©

35. Prisms When a beam of monochromatic light passes through a prism, it is bent or refracted. The amount of deviation depends upon the wavelength, eg . Blue light being refracted more than red. If white polychromatic light is substituted for monochromatic light, a separation of different wavelengths lead to the formation of a spectrum from which a required wavelength can be selected. 35 ©

36. Prisms are made up of quartz for use in UV region, since glass absorbs wavelengths shorter than about 330 nm. Glass prisms are preferable for the visible region of the spectrum, as the dispersion is much greater than that obtained with quartz. For the IR region, the transparent substances usually used are NaCl (2– 15 μ m), KBr (12-25 μ m ), LiF (0.2-0.6 μ m ), and CsBr (15-38 μ m ). Prisms produce a non-linear dispersion, with long wavelengths being less efficient separated than short wavelengths. The older spectrophotometers show larger divisions btw each nm in the UV region than in the Visible region. 36 ©

37. Gratings The dispersing element in the monochromator of most modern UV, Vis, IR spectrophotometers is the diffraction grating. It consists of a very large number of equispaced lines (200-2000 per mm) ruled on a glass blank coated with a thin film of aluminium . Parallel lines or grooves are chemically joined on a thin layer of photoresist coated glass which produces interference of light beams. They can be used as transmission gratings or when aluminized can be used as reflection gratings. Rotation of grating permits appropriate wavelengths of the spectrum to emerge from the exit slit of the monochromator . 37 ©

38. The ruling of a grating can be shaped to concentrate the light in certain orders to give greater efficiency which is especially imp in IR. A grating can be plane or concave. Concave gratings are capable of focusing on its own spectra without the use of lenses or mirrors. For convenience plane gratings are used which can be made to cover all spectral regions from 180 nm- 15 μ m. 38 ©

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40. Sample Compartment Ideal requirements: It should hold the sample and should be made of substance which are transparent in the region being studied. It should be reproducible in path length. For example. Quartz cells in UV can hold 3 ml sample with 1 cm pathlength . 40 ©

41. Samples presented for the spectrometric analysis may be in the solid, liquid or gaseous state. For the analysis of liquids and gases in the UV-Visible region above 323 nm, cells (or cuvettes) constructed with optically flat fused glass may be used. Measurements below 325 nm (the UV cut off value of glass), requires the use of more expensive fused silica cells, which are transparent to below 180 nm. The standard path-length of cells for the measurements of molecular absorption or fluorescence in the UV-visible region is 10 mm. The most common sample-containing materials in IR spectroscopy are NaCl (2.5 – 17 μ m) and KBr ( 2.5 – 30 μ m). 41 ©

42. Detector Ideal requirement of the detectors: Respond to the radiant energy over a broad wavelength range. It should be sensitive to low levels of radiant power. It should respond rapidly to the radiation. It should produce an electrical signal that can rapidly be amplified and the signal produced is directly proportional to the power of the striking area. 42 ©

43. Detectors Commonly used detectors are: Barrier-layer cells Phototubes Photomultiplier tubes Photodiodes Thermocouples Bolometers Thermistors Golay detectors 43 ©

44. Barrier-layer cell: One of the simplest detector. Advtg . Requires no power supply but gives a current which, under suitable conditions, is directly proportional to the light intensity. Instrumentation: It consists of a metallic plate, usually copper or iron, upon which is deposited a layer of selenium or sometimes copper oxide. 44 ©

45. An extremely thin layer of a good conducting metal, eg , silver, platinum or copper, is formed over the selenium to act as one electrode, the metallic plate acting as the other. Ordinarily, selenium and metallic oxides and sulphides have extremely small electrical conductivities, the electrons being in energy levels at stationary state that is they are not mobile. Light of suitable frequency, imparts sufficient energy to the electrons so that they leave the selenium and enter the transparent metal layer. If 2 electrodes are now connected to the galvanometer, a current will flow shown by the needle of galvanometer. If the resistance is small, the current produced is directly proportional to the intensity of the light. 45 ©

46. The current output also depends upon the wavelength of the incident light. With higher resistance, there would be loss of linearity. The useful working range of selenium photocells is 380- 780 nm. Demerits: lack of sensitivity Merits: Requires no power supply but gives a current which, under suitable conditions, is directly proportional to the light intensity . Cheapest and still used in colorimeters and flame photometers. 46 ©

47. Phototubes: Electrons are liberated when light falls on a metal surface, and if this is enclosed in an evacuated envelope and kept at a negative voltage, a current which is proportional to the incident light can be drawn from it. The essential feature for a sensitive surface in these cells is that the electrons should be liberated easily from the metal. Elements of high atomic volume, eg . K or Cs, are commonly used and, in order to increase the sensitivity, composite coatings such as Cs/ CsO2/ AgO2 have proved of more value than the metal alone. The sensitive surface is enclosed in a high vacuum and forms the cathode of the cell. 47 ©

48. Application of a sufficiently high potential between cathode and anode ensures that all the electrons liberated by the action of light reach the anode. A saturation photocurrent which exhibits a linear relationship with the intensity of illumination is then obtained. By using different metals, the cells can be made to respond to different regions of the spectrum. In spectrophotometers 2 cells are usually used, one being responsive to the UV and visible radiation of wavelengths upto about 620 nm, and the other to radiation of wavelengths 620-1000nm . 48 ©

49. A modification of the above type of cell involves the inclusion of an inert gas at a low pressure to give gas-filled cells. The underlying principle is that the liberated electrons cause ionization of the gas with consequent increase in the photocurrent but, owing to the lack of linear response, the cells cannot be used in instruments designed for accurate intensity measurement. PHOTOMULTIPLIER tubes (PMTs) In order to obtain greater sensitivity to very weak light intensities, multiplication of the initial photoelectrons by secondary emission is employed. 49 ©

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51. Several anodes at a gradually increasing potential are contained in 1 bulb. Electrons from the photocathode are attracted to anode 1 and liberate more electrons which travel to anode 2 because of its higher potential relative to anode 1. This process then continues to the last anode, and the result is a final photocurrent 10 6 -10 8 times greater than the primary current, which still shows a linear response with increase in the intensity of illumination. PMT are ideal for measuring weak light intensities such as occur in fluorescence and in the determination of trace elements by their emission spectra. 51 ©

52. The current from phototubes and PMTs never falls to zero. A small residual current called dark current is produced, due to spontaneous discharge at the high voltage of the dynodes or to the thermal emission from the cathode. PHOTODIODES In essence, the diode is charged to a pre-set potential by a continuously scanning electron beam. If radiation impinges on the diode, the charge is depleted in proportion to the intensity of the radiation. When the electron beam scans again, a current in proportion to the light intensity flows to recharge the diode to the preset voltage. 52 ©

53. Photodiodes, like vacuum phototubes and PMTs, are single-channel detectors, i.e. they monitor the total intensity of light and cannot distinguish between different wavelengths. A spectrum is obtained with single-channel detectors by varying the monochromatic light passing through the sample. When used in combination with a dispersing system each diode can monitor the light intensity at a different wavelength, and the array provides an almost instantaneous spectrum. The multi-channel detector based upon linear diode arrays is of particular advantage in monitoring fast reaction kinetics and eluate (combination of mp and ana .) in HPLC. 53 ©

54. Thermocouples A thermocouple consists of elements of 2 different metals joined together at one end, the other end being attached to a sensitive galvanometer. When radiant energy impinges on the junction of the metals, a thermoelectromotive force is set up which causes a current to flow. Thermocouples are used in the IR region, and to assist in the complete absorption of the available energy the ‘’hot’’ junction or receiver is usually blackened. 54 ©

55. Bolometers These make use of the increase in resistance of a metal with increase in temperature. Like thermocouples they are used in IR region. If 2 platinum foils are suitably incorporated into a wheatstone bridge, and radiation is allowed to fall on 1 foil, a change in resistance is produced, which results in current which is proportional to the incident radiation. 55 ©

56. Thermistors Its made of metal oxides. It functions by changing resistance when heated. It consists of 2 closely placed thermistor flakes, one of the 10 µm is an active detector, while the other acts the compensating/ reference detector. It is made of semi-conducting material which has a high negative coefficient of resistivity, i.e. the resistance decreases with increase in temp. and this gives the intensity of the IR radiation. 56 ©

57. Golay detector In this detector, the absorption of IR radiation causes expansion of an inert gas in a cell chamber. One wall of the chamber consists of a flexible mirror and the resulting distortion varies the intensity of illumination falling on a photocell from a reflected beam of light. The current from the photocell is proportional to the incident radiation. 57 ©

58. Display system The signal from the detector is normally proportional to the intensity of the light incident on the detector and after amplification may be displayed as % transmittance or absorbance. There are 3 common systems for displaying the %T or absorbance, i.e. Moving coil meter Digital display Strip chart recorder 58 ©

59. Single beam spectrophotometers https:// www.youtube.com/watch?v=XAp-5r3LxQo https://www.youtube.com/watch?v=pxC6F7bK8CU 59 ©

60. Double beam spectrophotometer http:// mas-iiith.vlabs.ac.in/exp1/expt1/instrument_animation.html 60 ©

61. Principle of UV-Visible spectroscopy: BEER-LAMBERT’S LAW: When a beam of light is passed through a transparent cell containing a solution of an absorbing substance, reduction of the intensity of the light may occur. This is due to: Reflections at the inner and outer surfaces of the cell Scatter by particles in the solution Absorption of light by molecules in the solution. 61 ©

62. The reflections at the cell surfaces can be compensated by a reference cell containing the solvent only, and scattering may be eliminated by filtration of the solution. The intensity of the light absorbed is then given by: I absorbed = I - I T Where I is the original intensity on the cell I T   is the reduced intensity transmitted from the cell . 62 ©

63. The transmittance (T) is the ratio I T / I and the % transmittance (%T) is given by : %T = 100 I T / I In 1760, lambert investigated the relationship between I and I T for various thickness of substance and found that the rate of decrease in the intensity of light with the thickness, b, of the medium is proportional to the intensity of incident light. Expressed mathematically, db / dI α I or – dI / db = k’ I w here k’ is a proportionality constant 63 ©

64. - db / dI = 1 / k’ I Integrating, -b = 1/k’ ln I T + C Where I T is the intensity transmitted at thickness b. When b = 0, C = - 1/k’ ln I Therefore, -b = 1/ k’ ln I T – 1/k’ ln I Ln I / I T = k’b 64 ©

65. On conversion to a common logarithm, the expression becomes Log I / I T = k’b / 2.303 The quantity log I / I T is called absorbance (A) and is equal to the reciprocal of the common logarithm of transmittance. A = Log 10 I / I T = log 10 (1/T) = -log T = 2 – log (%T) Older terms for absorbance such as extinction, optical density, and absorbancy are now obsolete. 65 ©

66. Lambert’s law is defined as: The intensity of a beam of parallel monochromatic radiation decreases exponentially as it passes through a medium of homogenous thickness. More simply it is stated that the absorbance is proportional to the thickness ( pathlength ) of the solution. Beer showed in 1852 that a similar relationship exists between the absorbance and the concentration. Log I / I T = k’’c / 2.303 w here k’’ is a proportionality constant and c is the concentration. 66 ©

67. Beer’s law is defined as follows: The intensity of a beam of parallel monochromatic radiation decreases exponentially with the number of absorbing molecules. More simply stated that the absorbance is proportional to the concentration. A combination of the two laws yields the Beer-Lambert Law: A = Log I / I T = abc In which the proportionality constants k’/2.303 and k’’/2.303 are combined as a single constant called absorptivity (a). 67 ©

68. Molar absorptivity When c is in moles per liter, the constant is called molar absorptivity (formerly called as molar extinction coefficient) and has the symbol ϵ . The equation therefore becomes, A = ϵ bc The molar absorptivity at a specified wavelength of a substance in solution is the absorbance at that wavelength of a mol /l solution in a 1 cm cell. The units of ϵ are therefore 1 l/ mol /cm 68 ©

69. Specific absorbance Another form of the beer-lambert proportionality constant is the specific absorbance, which is the absorbance of a specified concentration in a cell of specified pathlength . The most common form is A (1%, 1cm), which is the absorbance of a 1 g/ 100 ml (1% w/v) solution in a 1 cm cell. The beer-lambert equation therefore takes the form, A = A 1cm bc 69 1% ©

70. Deviations from BL Law: Sample conditions Instrumental parameters Chemical effects 70 ©

71. Common organic solvents with their cut-off values 71 ©

72. One factor that influences the absorbance of a sample is the concentration (c). The expectation would be that, as the concentration goes up, more radiation is absorbed and the absorbance goes up. Therefore , the absorbance is directly proportional to the concentration. A second factor is the path length (b). The longer the path length, the more molecules there are in the path of the beam of radiation, therefore the absorbance goes up. Therefore , the path length is directly proportional to the concentration 72 Concentration and pathlength ©

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74. Fundamental requirement under BL-Law is: Every photon of light striking the detector must have equal chances of absorption. Suppose we want to measure some high concentrations, above the usual linear range of calibration curve, without diluting the samples and without shorter path-length cells What will be the result? The absorbance would be non-linear which means the maximum absorbance value will shift from 1 to 2 because of stray light effects. 74 ©

75. In order to reduce that if the wavelength is set to higher values the linearity would be improved that’s bcz the stray light is lessened by the reduced absorbance at the higher wavelength. The intensity of light transmitted from an absorbing solution (I T ) will show small random fluctuations owing to small variations in the light source intensity, detector and amplifier noise etc. Consequently, every absorbance value will have a small random error associated with it. The % relative error is the difference between the true conc. And that calculated from the measured absorbance ( δ c) expressed as a % of the true conc. (c): 75 ©

76. % relative error = 100 δ c/ c The % relative error is dependent on the magnitude of the measured absorbance and the type of detector used to measure the intensities of light. For modern instruments with a phototube detector, it may be shown that the relative error is at minimum when abs is 0.869. The optimum accuracy and precision are therefore obtained when the absorbance is around 0.9. In general practice, the abs values in range 0.3-1.5 are sufficiently reliable, and the combination of cell path-length and conc. of analyte should be adjusted to give an absorbance within this range. 76 ©

77. Instrumental parameters Slit width It is the width (usually in mm) of the entrance and exit slits of the monochromator . The slits are rectangular apertures through which light enters inside and exists from the monochromator . Their purpose is to control the spectral resolution of the monochromator , that is, it has the ability to separate close wavelengths. 77 ©

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79. Light is focused onto the entrance slit B, is focused by concave mirror C onto the grating D, which disperses the light according to wavelength. Concave mirror E then focuses the light on the exist slit F, forming a spectrum across the exit slit. Only the particular wavelength that falls directly on the exit slit passes through it and is detected. Adjusting the rotating angle of the grating changes the wavelength that passes through the exist slit. In a standard monochromator design, the entrance slits and exist slits have equal width. 79 ©

80. The wider the slit widths, the larger the range of wavelengths that passes through the monochromator . Most research-grade instruments have user-controllable slit widths. In general, smaller (narrower) slit widths yield greater spectral resolution but cut down the amount of light that is transmitted through the monochromator . In an Absorption spectrophotometer, a monochromator is used to limit the wavelength range of light passed through the sample to that which can be absorbed by the sample. In the most common arrangement, the light source is focused onto the entrance slit and the absorbing sample is placed immediately after the exist slit, with the photodetector exactly behind it to detect the intensity of transmitted light. 80 ©

81. What is the optimum slit width for absorption spectroscopy? The answer to this question depends on the purpose of the measurement. If the purpose is to record an accurate absorption spectrum, for example, for use as a reference spectrum for future measurements or for identification, then a sufficiently small slit width must be used to avoid the polychromaticity deviation from the BL-law. The requirement is that the spectral bandpass be small compared to the spectral width of the absorber. The monochromaticity or spectral purity of the light incident on the sample cell is defined by the spectral bandwidth, which is the width of the triangle (in nm) at one half of the peak intensity. 81 ©

82. Difference between spectral bandwidth, spectral bandpass and slit width The   spectral bandwidth is  defined as the  width of  the  band of  light at one-half the peak maximum (or full  width  at half maximum [FWHM])  Spectral bandpass  is the FWHM of the wavelength distribution passed by the exit slit. Resolution is related to  bandpass  but determines whether the separation of two peaks can be distinguished. 82 ©

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84. In a dispersive instrument, the spectral bandpass is given by the product of the slitwidth ( sw ) and the linear dispersion (LD). The slit-width is user-variable in many instruments, whereas the LD is fixed by the design of the monochromator . So, the SW is adjustable, setting it to the smallest slit width will insure the smallest spectral bandpass and result in the minimum polychromaticity error. However, the signal-to-noise ratio decreases as the slit width is reduced, so it is not always practical to use the smallest slit width possible. A smallest slit width, even if it is possible on that spectrometer, will not be useful if the random signal-to-noise error exceeds the error caused by non-linearity. 84 ©

85. T he effect that increasing slitwidth has on measured absorbance value depends on the width of the absorption band and on whether the wavelength of measurement corresponds with a maximum or minimum absorbance. The width of the absorption band is defined as natural bandwidth, which is the width of the absorption band at one half the A max. Furthermore, as the absorbance increases due to higher conc. Or longer pathlengths , the % error increases, resulting in a negative deviation from the BL-Law. If the slit width is too narrow, the resultant reduction of the incident energy lowers the signal to noise ratio and decreases the precision of the measurement. The optimum slitwidth is therefore the widest setting that provides spectral fidelity. 85 ©

86. Scanning speed If the scan speed selected for the automatic recording of the spectrum is too fast, electronic and mechanical damping of the spectrophotometer’s signal to the recorder may prevent the recorder pen responding quickly enough to the rapid changes of absorbance. The effects observed in progressively faster replicate scans of an absorption spectrum are that the apparent lambda max is displaced in the direction of the scan, Amax are decreased, Amin are increased and the resolution btw adjacent bands is reduced. The optimum scan speed is normally the fastest speed that maintains the pen fidelity. 86 ©

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88. Stray light Any radiation reaching the photodetector other than the narrow range of wavelengths normally transmitted by the monochromator . It arises from scattering and refraction inside the monochromator , due to mainly imperfections on the optical surface. If stray light comprises wavelengths which the sample does not absorb, it will fall on the photocell during the measurement of the intensity of both the incident light ( I o ) and unabsorbed light (I T ). Thus, the observed absorbances is given by: A obs = log [I o + SL/ I T + SL] 88 ©

89. 89 The effect of stray light is particularly evident at high absorbances , i.e. when IT is low, where deviation from the BL-Law may be observed. This figure shows the effect of increasing levels of stray light on a Beer’s law plot. ©

90. It should be noted that the spectrophotometer cannot measure absorbances greater than that corresponding with the level of stray light. For example, if the unabsorbed stray light is equivalent to 1% of the intensity of light at a particular wavelength, the limiting value of A obs is 2 (approximately). 90 ©

91. The effect of stray light may also be seen at wavelengths near the extremes of the usual wavelength range of light sources, where the available energy is low. For example, the intensity of light from a Deuterium lamp below 220 nm decreases rapidly with decreasing wavelength. Unabsorbed stray light of higher wavelengths may reduced the absorbance sufficiently for a spurious maximum which are common below 220 nm and may be detected by marked deviations in Beer’s law when solutions of different conc. are examined. Alternatively, stray light at a particular wavelength can be detected by placing the solution of a substance that is known to absorb completely at that wavelength. 91 ©

92. Observed absorbances other than infinite absorbance (%T = 0) confirms the presence of stray light. Chemical effects Deviations from BL-Law may occur if the substance undergoes chemical changes (e.g. dissociation, association, polymerization, complex formation) as a result of the variation of conc. For example, a solution of benzoic acid high conc. In a simple unbuffered aqueous solution has a low pH and contains a higher proportion of the unionized form than a solution of low conc. 92 ©

93. The ionized and unionized forms of benzoic acid have different absorption characteristics: λ max = 273 nm λ max = 268 nm ε 273 = 970 ε 268 = 560 Therefore, increasing the conc. o f benzoic acid produces higher absorpitivity values at 273 nm with positive deviation from BL, and lower absorpitivity values at 268 nm with negative deviation from beer’s law. 93 ©

94. References Beckett and Stenlake , 5 th Ed., CBS publishers YR Sharma, Introduction to spectroscopy Pavia, Introduction to spectroscopy https://commons.wikimedia.org/wiki/File:Bohr_atom_animation.gif https://www.priyamstudycentre.com/2019/02/hydrogen-spectrum.html https://www.edinst.com/blog/jablonski-diagram / 94 ©

95. 95 ©