UV Visible SPECTROSCOPY. Points a VVVVVV ° ee wy to be cover Electronic transitions, Chromophores, Auxochromes, Spectral shifts, Solvent effect on absorption spectra, Beer and Lambert's law, Derivation and deviations. Instrumentation Sources of radiation, wavelength selectors, sample cells, detectors-Photo tube, Photomultiplier tube, Photo voltaic cell Silicon Photodiode. Applications Spectrophotometric titrations Single component and multi componentV VISIBLE SPECTROSC © Ultraviolet and visible spectroscopy deals with the recording of the absorption of radiations in the UV and visible regions of the electromagnetic spectrum. © The UV region extends from 10-400nm. It is sub-divided into the near UV (quartz) region (200-400nm) and the far or vacuum UV region (10- 200nm). The visible region extends from 400-800nm. * Absorption of electromagnetic radiations in the UV and visible regions induces the excitation of an electron from a lower to higher molecular orbital (electronic energy level). Since UV and visible spectroscopy involves electronic transitions, it is often called electronic spectroscopy. * Organic chemists use UV and visible spectroscopy for detecting the presence and elucidating the nature of conjugated multiple bonds or aromatic rings. Detector Collimator Prismor Wavelength Sample Grating Selector Solution “ ELECTROMAGNETIC RADIATIONS (EMR) © Electromagnetic radiations (EMR) are the waves (or their photons) of electromagnetic field, propagating or radiating through the space with a specific electromagnetic radiant energy. © EMR or EM radiation consist of electromagnetic waves, which are© synchronized oscillations of electric and magnetic fields. In vacuum electromagnetic waves travel at the speed of light. Speed of light is denoted by 'c' having approx. value 3x 108 m/s. or 3x 1010 cm/s. © Distance of two crest of two trough is termed as length of electromagnetic waves. © Types of Electromagnetic Radiations © Electromagnetic radiations are of following types © Distance of two crest of two trough is termed as length of electromagnetic waves. v Types of Electromagnetic Radiations ° Electromagnetic radiations are of following types Gamma rays <0.00inm X-rays 0.01-10 nm Ultraviolet 200-400 Visible light 400-800 Infrared 0.8-200 (a) NearIR 0.8-2.5 (b) (b) Mid IR 2.5-15 um (Sometimes 2.5-25 um) (©) Far 15-200 um (Sometimes 25-200 um) Microwaves 0.01-1 Radio waves 1-107 © Energy absorbed in UV-visible ranges produces changes in the electronic energy of the molecule resulting from transition of valence electrons in the moleculé, © There are distinct types of electrons are involved in organic molecules i.e. o, mand n electrons.transition of electrons this results in the energy change i.e., AE. *» TYPES OF ELECTRONS v Bonding electrons: o and t y Non-bonding electrons: n v Antibonding electrons: 6 * and n* * o electrons: These electrons are involved in saturated bonds, and known as a bonds. These bonds are excited by the UV-visible radiation. * Eg. CH3-CH; * electrons: These electrons are involved in unsaturated hydrocarbons. * E.g.CH,=CH, * nelectrons: These are the electrons present when hetero atoms are present like nitrogen, oxygen, halogen etc. * Eg. CH3-CH)-OH ° Any molecule has either n, 1, o or combination of these bonding and nonbonding electrons absorbs the characteristic wavelength and undergo transition from ground level to excited state. 0" (anti-bonding) — x" (anti-bonding) 1 (non-bonding) x (bonding) @ (bonding) * o~ o* transition: This is the high energy transition means it require higher energy for the transition of electrons from ground state to the excited state. This trapsition falls in vacuum UV region (150nm). * It (o > o* transitions) is mainly occur in saturated hydrocarbons Eg: methane, ethane etc. | * n~o* -Transition: This is low energy transition than o > o* transitionmeans it require lesser energy than o — o* transitions for the transition of electrons from ground state to the excited state. This transition falls in near UV region (150-250nm), n > o* transitions mainly occur in saturated compounds with heteroatoms (like 0/N/S/Halogen) such as alcohols, water etc. Eg CH30H, CH3l, CH3-S-CH3 etc. 1 > 1* Transition: Less energy is required as compared to n > 1* transitions fall in the range of 200-800nm based on conjugation n > 1* transition occurs in unsaturated compounds having double or triple bonds. Eg Compounds such as aromatic compounds, alkenes, alkynes show this type of transition. n> n* Transition: Lowest energy required for the transition from ‘ground state to excited state. This transition falls nearly 290nm, n > 1* transition occurs in unsaturated compounds with heteroatom such as aldehyde ketone, carbonyl compound ete. The energy required for various transition behaves as following order. o> 0*>n>o0* >To n*>n> T* Based on the energy and intensity transition are classified as * (a) High energy/ Intense transitions: n> o* o> 0* ° Low energy/weak transition: n->1* Q Presentation of spectra ° The ultraviolet-visible spectrum is generally recorded as a plot of absorbance versus wavelength. Absorbance A Mom) Amax is the wavelength where maximum absorption occurred. Solvent in UV spectroscopy: The choice of the solvent to be used in ultraviolet spectroscopy is quite important usually solvents that are notethanol (CH,0H), water etc. but not solvents like toluene as it is the | nonconjugated solvents. CHROMOPHORE It is defined as any isolated covalently bonded group that shows a characteristic absorption of electromagnetic radiation in the UV or visible region. A chromophore is the part of a molecule that absorbs UV or visible light. It isa Greek word (Chroma = Color and Phoros = bearer). re te * Compound containing chromophores known as Chromogen Eg. C=C, C=O Y Chromophores are generally of two types such as (a) Independent Chromophore: If one chromophore is required to impart the color. eg. Azo group (-N-N-), Nitro group (-NO-) etc. (b) Dependent Chromophore: If more than one chromophore is required to impart the color. e.g. acetone having one ketone group that is colorless whereas diacetyl having two ketone groups is having yellow color. “* AUXOCHROME * Itis the functional group of atoms with one or more lone pair of electrons when attached to chromophore, alters both the wavelength and intensity of absorption. ° If these groups are in direct conjugation with pi-system of th chromophore, they may increase the wavelength at which the light i absorbed and as a result intensify the absorption. ° A feature of auxochrome is the presence of at least one lone pair of electron which can be viewed as extending the conjugated system by resonance. ° There are mainly two types of auxochrome, (a) Acidic -COOH, -OH, -SO;H (b) Basic -NH), -NHR, -NR, * ABSORPTION BANDS ° There are four types of absorption bands are classified by the way electronic transition. |(a) K-Bands (b) R-Bands (c) B-Bands (d) E-Bands * Due to which transitions and the compounds in which the bands are observed are explained as a) K-Bands: This type of bands re seen in the conjugated compounds such as dienes, polyenes, enone, aromatic compounds substituted by chromophore like nitrobenzene whereas benzene is the aromatic conjugated system and -NO, is the chromophore group. Intensity of K band is greater than 10*(E,,,, >10*). K bands occurs due to the 1 > 1* transitions. b) R-Bands: R bands occurs due to the n > m* transitions of single chromophore group and having at least one lone pair of electrons on the heteroatom. R bands are also called as forbidden bands. Intensity of R- band is less than 100 (E<100). B-Bands: B bands occurs due to m — m®* transition in aromatic or c heteroaromatic compounds. Benzene shows absorption peaks between 230-270nm, when chromophore group attached to benzene ring, the B- bands are observed at longer wavelength than more intense K-bands. (d) E-Bands: E bands occurs in electronic transitions in the benzenoid system of three ethylenic bonds which are closed cyclic conjugation. These are further characterized as E, and E, bands of benzene at 184nm and 204nm respectively. E, bands appear at lower wavelength (184nm) are more intense than E, band as E, band E,,,, at 184nm is 5000 and E, band E,,,, at 204nm is 7900. * Many compounds absorb ultraviolet (UV) or visible (Vis) light. The diagram below shows a beam of monochromatic radiation of radiant power Iy, directed at a sample solution, some amount of absorption takes place and beam of light leaving the subject is I. * Amount of energy absorbed by the sample this result decrease intransmitted light radiation than that of incidentlight I Transmitted Light Incident Light i © Beer's Law: When a beam of monochromatic radiation is passed through the absorbing medium, then the decrease in the intensity of the radiation is directly proportional to the concentration (c) of the solution. Aac Beer’s Law A= €bc © Lambert's Law. When a beam of monochromatic radiation is passed through the absorbing medium, then the decrease in the intensity of the radiation is directly proportional to the thickness/ pathlength (I) of the solution. Aad Reflection And Interference Losess Absorption Of Light By Particles / Molecules Emergent beam |, with lower intensity Scattering lossesU Beer-Lambert’s law * When a beam of monochromatic radiation is passed through the absorbing medium, then the decrease in the intensity of the radiation is directly | proportional to the thickness/ pathlength (1) as well as concentration of the solution. The Beer-Lambert Law can be expressed in the form of the following ° A=Absorbance 1= Optical path length, i.e. dimension of the cell or cuvette (cm) C = Concentration of solution (mol dm *) E = Molar extinction coefficient, which is constant for a particular substance ata particular wavelength (L Mol ‘ cm ') ° If the absorbance of a series of sample solutions of known concentrations are measured and plotted against their corresponding concentrations, the plot of absorbance versus concentration should be linear. The ratio Ip/ I, is termed as transmittance T, and the ratio log I)/ I, is termed v DERIVATION OF BEER-LAMBERT’S LAW as absorbance A. ® When a beam of monochromatic radiation is passed through the absorbing medium, then the decrease in the intensity of the radiation is directly proportional to the thickness/ pathlength (I) as well as concentration of the solution. ® The Beer-Lambert Law can be expressed in the form of the following equation,4 tec dl Where, d = Small change in the intensity or length I = Intensity of the radiation after passing through |= Thickness of the solution k = Proportionality constant (depends on sol.) c = Conc. of Sol. (in M) I, = Initial intensity of the radiation a4 Lixin da [In /], = -kxC x [In], In] -In, =-kxC x[In/ -0] i In— =-kxCx/ % Int =kxCx/ L) ad jt =logx x When lim its are applied ° Important Units: dk je =Inx Unit of c = Mol/L Unit of /=cm L Unit of ¢ = L Moltcm? 2.303 log > = kxCx/ Unit of A = None In = log, =2.303 log,,The linearity of Beer-Lambert law is limited by chemical and instrumental | factors. Causes of nonlinearity include: (a) The law is not obeyed if the radiation is not monochromatic (b) Deviation in absorptivity coefficients at high concentrations (©0.01M) due to electrostatic interaction between molecule and close proximity. (c) Scattering of light due to particulates of the sample. (d) Fluorescence or Phosphorescence of the sample. (e) Changes in refractive index at high analyte concentration. (f) Shifts in chemical equilibria asa function of concentration. (g) Chemical deviation i.e. dissociation or reaction with the solvent. Problem 1: The solution having maximum absorbance of 275 nm. Molar extinction coefficient is 8400 M? cm“ and the path length is 1 cm. Using a spectrophotometer, you find that absorption at 275 nm is 0.70. What is the concentration of guanosine? Solution: To solve this problem, you must use Beer Lambert's Law. 00 M-1 cm-1) X (1cm n m tins Problem 2: There is a substance in a solution (4 g/liter). The length of cuvette is 2 cm and only 50% of the certain light beam is transmitted. What is the extinction coefficient? Solution: Using Beer-Lambert Law, we can compute the absorption coefficient. Thus,> INSTRUMENTATION » Introduction ¥ The essential parts of a spectrophotometer are: (1) Radiation Source: Both the tungsten and D 2 lamp are present in the UV -visible spectrophotometer. (2) Wavelength Selector: It constitutes three essential parts: (i) Filter: Absorption and interference filters are mainly used. (ii)Monochromator: It gives the desired wavelength in the entire UV or visible region. (iii) Slits: There are two slits, i.e., entrance slit and exit slit. (3) Cells or Cuvettes: For holding the sample solution and the pure solvent (reference). , (4) Detector: The most commonly used detectors are photo -emissive cells or phototubes and photomultiplier tubes. (5) Recording System: Recording is done by recorder pen. (6) Power Supply ¥ Spectrophotometers are of two types: 1) Single-beam spectrophotometers, and 2) Double-beam spectrophotometers. Q SOURCES OF RADIATION * The best source of light is the one which is more stable, more intense and which gives range of spectrum from 180-360nm (up to 400nm). The different sources available are: 1) Hydrogen Discharge Lamp: * In these lamps, hydrogen gas is stored under relatively high | | pressure. When an electric discharge is passed through the ®) lamp, excited hydrogen molecules will be produced which emit | UV radiations. ki | {| The high pressure in the hydrogen lamps causes the hydrogen to TE emit a continuum rather than a simple hydrogen spectrum. i. Hydrogen lamps cover the range of 3500-1200A. These lamps are stable, robust, and widely used. '° If deuterium (D_) is used instead of hydrogen, the emission intensity is increased by as much as a factor of 3 at the short-wavelength end of the UV range. 2) Deuterium Lamp: It is similar to hydrogen discharge lamp, but filled with deuterium in the place of hydrogen. It offers 3-5 times more intensity than other types. Deuterium lamps are more expensive than hydrogen lamps but are used when higher intensity is required. 3) Xenon Discharge Lamp: In this lamp, xenon at 10-30 atmospheric pressure is filled in and it has two tungsten electrodes. The intensity is greater than the hydrogen discharge lamp. 4) Mercury Arc : This contains mercury vapour and offers bands which are sharp. The spectrum is not continuous. Hence, it is not widely used. 5) Tungsten Lamp: This lamp is similar in its functioning to an electric light bulb. It is a tungsten filament heated electrically to white heat. It has two shortcomings. The intensity of radiation at short wavelengths (<350nm) is small. Furthermore, to maintain a constant intensity, the electrical current to the lamp must be carefully controlled. O Wavelength Selectors en Wavelength selectors consist of three parts: 1) Filters, i 2) Monochromators, and 3) Slits. “Filters ° Filters provide high radiation throughout, approximately 50-80% efficiency. The two types of filters are:1) Absorption Filters: * These filters derive their effects from bulk interactions of radiation within the material. Absorption filters are produced in a variety of ° host materials, like gelatin, glass, liquid, and Absorption Fitter plastic. * Glass filters are used in automated chemical analysis equipment and colorimetry. ° The scattering type depends on scattering crystals formed within the glass mass through a reduction and thermal treatment. * Shorter wavelengths are scattered and absorbed, while longer wavelengths are unaffected. Cut-on and cut-off (or sharp-cut) filters are widely used as blocking filters to suppress unwanted spectral orders from interference filters and diffraction gratings. * There are a wide variety of plastic filters, of both sharp cut and intermediate bandwidth types. Plastic filters may be produced either by bulk colourants introduced into the basic batch or through subsequent dye treatments of clear base stock. Cut-on types, unlike their glass counterparts, exhibit no fluorescence in the visible region. 2. Interference Filters: ° These filters, as the name implies, are based | ™* Green Interference Filter, Ftaed”™ on the phenomenon of optical interference. A “ee simple two -interface (Fabry -Perot) filter consists of a dielectric spacer film (CaF, MgF,, or SiO) sandwiched between two parallel, partially reflecting metal films, usually of silver. * The thickness of dielectric film is controlled to be only one, two, or three half -waves thick. These are referred to as first-,second-, or third-order filters, respectively.A portion of the incident ~ “Transparent spacer film sia, (one-half wavelength tick) radiation normal to the filter (beam 1) passes through beam 2, while another portion (beam 3) is reflected back from surface B to surface A. Portion of this reflected r adiation is again reflected from surface A through the dielectric layer and exits as ‘Semi-transparent silver films beam 4 parallel (actually Path of Light Rays through Interference Filter coincident) to beam 2. Thus, the path travelled by beam 4 is longer than that of beam 2 by twice the product of the dielectric spacer thickness and its refractive index. When the layer thickness (b) is half the wavelength of the radiation to be transmitted in the refractive index (n) of the dielectric, beams 2 and 4 are in phase and interfere constructively. The expression for central wavelengths at which full reinforcement occurs is: 2np —— m Where, m = Order number. Since partial reinforcement occurs for other path differences, the filter actually transmits a band of radiant energy. Furthermore, the angle of incidence of the radiation must be 90°. Any phase shifts on reflection are ignored. The bandwidth is 10 -15nm, Full Width at Half Maximum (FWHM) transmission; the maximum transmission is usually 40% with this type of filter. Monochromators (Prisms and Gratings) The monochromator is used to disperse the radiation according to the wavelength. The essential elements of a monochromator are an entrance slit ,a dispersing element, and an exit slit .The entrance slit sharply defines the incoming beam of heterochromatic radiation. The dispersing element disperses the heterochromatic radiation into its component wavelengths whereas exit slit allows the nominal wavelength together with a band of wavelengths on either side of it. Position of the dispersing element is always adjusted by rotating it to vary the nominal wavelength passing through the exit slit. The dispersing element may be a prism or grating. The prisms are generally made of glass, quartz, or fused silica. Glass has the highest resolving power but it is not transparent to radiations having the wavelength between 2000-3000A because glass absorbs strongly in this region. . Quartz and fused silica prisms are transparent throughout the entire UV range and are widely used in UV spectrophotometers. Fused silica prisms are little more transparent in the short wavelength region than quartz prisms, and are used only when very intense radiation is required. The mirrors in the optical system are front surfaced because glass starts to absorb in the UV region. ¥ Characteristics of a Monochromator 1) It allows the largest entrance slit width for the band pass required. 2) It has the highest dispersion. 3) The largest optics is affordable. 4) Longest focal length is affordable. 5) Highest groove density will accommodate the spectral range. 6) Optics and coatings are appropriate for specific spectral range. 7) Entrance optics will optimise endues. 8) If the instrument is to be used at a single wavelength in a non-scanning mode, it must be possible to adjust the exit slit to match the size of the entrance slit image. > types of monochromator 1) Prism Monochromator: * The sample is kept at or near the focus of the beam, just before the entrance slit (A) to the monochromator.© The radiation from the source after passing through the sample and entrance slit, strikes the off-axis parabolic Littrow mirror (B), which renders the radiation parallel and sends it to the prism (C). The dispersed radiation after reflecting from a plane mirror (D) returns through the prism a second time and focuses onto the exit slit of the monochromator, through which it finally passes into the detector section. Exit slit _— Source mirror (B) WA) Single-Pass Monochromator In double-pass monochromator, there occurs a total of four passes of radiations, i.e., (1), (2), (3) and (4) through the prism. The double pass monochromator produces more resolution than the monochromator in the radiation, before it finally passes on to the detector. \_ Exit slit / Entrance slit Plane mirrors Double-Pass Monochromator 2) Grating Monochromator: * Gratings provide an alternative means of producing monochromatic light. A diffraction grating consists of a series of parallel grooves (lines) ona reflecting surface that is produced by taking a replica from a master carefully prepared using a machine, or from one which is holographicallygenerated. The grooves can be considered as separate mirrors from which the reflected light interacts with the light reflected from neighbouring grooves to produce interference, and so to select preferentially the wavelength that is reflected when the angle of the grating to the incident beam is changed. When parallel radiation illuminates a reflecting diffraction grating, the multiple reflections from the mirror grooves will overlap and interfere with each other. If the reflected waves are in phase, interference is said to be constructive and the reflected light is not affected. If the reflected waves are out of phase, there is destructive interference and light of the wavelength at which such interference occurs will not be propagated. expressed by: Grating monochromator possesses the following advantages over prism monochromator: i) Grating can be made with materials which are not attacked by moisture like aluminium. 7* On the other hand, metal salt prisms get subjected to etching from atmospheric moisture. ii) Grating monochromators can be used over considerable wavelength ranges. iii) Grating offers better resolution and energy transfer. iv) Grating has constant bandwidth due to linear dispersion. v) Grating requires less complicated wavelength drive mechanism. vi) Stray light is limited to imperfections at the grating surface. “ SLITS There are two slits, i.e., entrance slit and exit slit. The main function of the entrance slit is to provide a narrow source of light so that there should be no overlapping of monochromatic images. From this, the exit slit selects a narrow band Single slit of dispersed spectrum for observation by the detector. In practical spectrophotometry, the monochromator module is not capable of isolating a single wavelength of radiation entrance slit grating exit slit incoming light From the continuous spectrum emitted by the source. Rather, a definite band of radiation is passed by the monochromator. This finite band arises from the slit distributions. The entrance or aperture of a monochromator is a long, narrow slit whose width is generally adjustable. Inside the monochromator, the rays diverge from the entrance slit and illuminate the collimator mirror, which renders the rays parallelly and focuses them on the dispersing element. Leaving the collimator, the parallel set of rays isa broadened version of the entrance slit.Qa Si samplesalle oniveies Sample containers, which are usually called cells or cuvettes, must have | windows that are transparent in the spectral region of interest. Quartz or fused silica is required for the UV region (wavelengths less than _ 350nm) and may be used in the visible region. Silicate glass is ordinarily | used for 375-2000nm region because of its low cost compared to quartz. Plastic cells are also used in the visible =— ita: Open-top Stoppered Stoppered normal with id normal sem-micro: Tall micro Minimum height sampling micro Commercially Available Cells used in UV-Visible Spectrophotometer The best cells have windows that are perpendicular to the direction of the beam in order to minimize reflection losses. The most common cell path length for studies in the UV and visible regions is of 1cm; matched, calibrated cells of this size are available from several commercial sources. Many other cells with shorter and longer path lengths can be purchased. For reasons of economy, cylindrical cells are sometimes used. Particular care must be taken to duplicate the position of such cells with respect to the | beam; otherwise, variations in path length and reflection losses at the | curved surfaces can cause significant error.Q Detectors © Detectors used in UV-visible spectrophotometers can be called as photometric detectors. The most commonly used detectors are: 1) Photo tubes or photoemissive cells. 2) Photomultiplier Tubes (PMT). 3) Photovoltaic cell or barrier-layer cells. 4) Photomultiplier Silicon photodiode array detector. Barrier Cell Silver Plastic case + terminal - terminal a ‘Supply Motorized Grating ) ds ~< Entrance Deuterium stit Exit Slit Grating Deuterium Lamp+ Photo Tubes or Photo-Emissive Cells | © This detector is composed of an evacuated glass tube, which consists of a | photocathode and a collector anode. The photocathode is coated with elements of high atomic volume (like caesium, potassium, or silver oxide), which can liberate electrons when light radiation falls on it. © This flow of electrons towards anode produces a current proportional to the intensity of light radiation. Composite coatings like caesium/caesium oxide/silver oxide can also be used, which increases the sensitivity and range of wavelength in which the detector can be used (UV region). Anode wire Light rays Cathode Electrons uphifier and it device 90 DC Power supply Photo Tube or Photo-Emissive Cell © The signal from the detector can also be amplified using an amplifier circuit, Phototubes have better sensitivity when compared to photovoltaic cell, and hence are more widely used. +“ Photomultiplier Tubes (PMT) © This type of detector is the most sensitive of all the detectors, is expensive, and is used in sophisticated instruments. The principle employed in this detector is multiplication of photoelectrons by secondary emission of electrons. This is achieved by using a photocathode and a series of up to 10 anodes (dynodes).° Each dynode is maintained at 75-100V higher than the preceding one. At each stage, the electron emission is multiplied by a factor of 4 or 5 due to secondary emission of electrons; and hence an overall factor of 106 is achieved. Schematic Representation of Photomultipier Tube «+ Photovoltaic Cell or Barrier-Layer Cells * This cell is also known as photronic cell and operates without the use of a battery. It consists of a metal base plate (of iron or aluminium), that acts as an electrode. On its surface, a thin layer of asemiconductor metal (like selenium) is deposited. * Then, the surface of selenium is covered by a very thin layer of silver or gold that acts as the second collector electrode. Collecting nng Transparent =e AWA. te A Q Galvanometer layer Metal base plate Figure 1.17: A Typical Photovoltaic Cell ° When the radiation is incident upon the surface of selenium, electrons are generated at the selenium-silver interface. These electrons are collected by the silver. Accumulation of electrons on the silver surface* These cells offer the advantages of being rugged and requiring no external power supply; however, their use is generally limited to the visible region. The wavelength response (450-650nm) is close to that of the human eye. High level of illumination is required to avoid the problems of amplifying the output which arise from the small resistance of the external circuit. + Silicon Photodiode Array Detector © The principle involved in silicon photodiode detector is that upon exposure to light the electrical properties of the detector change (this is the internal photoelectric effect). Solar cells have the similar structure and work on the same principle. Silicon photodiodes are advantageous over photomultipliers because of their low cost, low sensitivity in the light-receiving surface, and no need of special power supply. It obtains photometric data like that of photomultipliers if the light intensity is relatively large QO RECORDING SYSTEM The signal from the photomultiplier tube is finally received by the recording system. The recording is done by a recorder pen. This type of arrangement is only done in recording UV spectrophotometers. OQ POWER SUPPLY © The power supply serves three main functions: 1) It decreases the line voltage to the instruments operating level with a transformer. 2) It converts AC to DC with a rectifier if direct current is required by the instrument. 3) It smoothens out any ripple that may occur in the line voltage to deliver a constant voltage to the source lamp and instruments.Q Single beam UV spectrophotometer * The steps involved in a single beam UV spectrophotometer are: 1) UV radiation is given off by the source. 2) A convex lens gathers the beam of radiation and focuses it on the inlet slit. 3) The inlet slit permits light from the source to pass, but blocks-out stray radiation. 4) The light then reaches the monochromator, which splits it up according to wavelength. 5) The exit slit is positioned to allow light of the required wavelength to pass through. 6) Radiation at all other wavelengths is blocked-out. 7) The selected radiation passes through the sample cell to the detector, which measures the intensity of the radiation reaching it. 8) By comparing the intensity of radiation before and after it passes through the sample, it is possible to measure how much radiation is absorbed by the sample at the particular wavelength used. 9) The output of the detector is usually recorded on graph paper. Slit jochromator Signal processor (Grating) and read-out Tungsten lamp Reference cell or sample cell Single Beam Spectrophotometer 2=[ Light Source Lens Grating Wavelength Sample Detector Digital meter selector Single beam spectrophotometerOne problem with the single beam system is that it measures the total amount of light reaching the detector, rather than the percentage absorbed. Light may be lost at reflecting surfaces or may be absorbed by the solvent used to dissolve the sample. Furthermore, the source intensity may vary with changes in line voltage. For example, when the line voltage decreases, the intensity of the light coming from the source may decrease unless special precautions are taken. Consequently, the intensity of radiation may be constantly changing. Another problem is that the response of the detector varies significantly with the wavelength of the radiation falling on it. Even if the light intensity is constant at all wavelengths, if the wavelength is steadily increased from 200-750nm, the signal from the detector starts at a low value, increases to a value that is steady over a wide range, and then decreases once more. This relationship between the signal from the detector and the wavelength of radiation is called the response curve. Q * A double beam spectrophotometer is also called a UV-visible spectrophotometer andit utilizes two sources: (1) A tungsten lamp (400-800nm), and (2) A D, lamp (200-400nm). Tungsten and D» lamps Double Beam Spectrophotometer* The steps involved in a double beam UV spectrophotometer are: 1) The radiation from the selected source passes through a fixed slit to the surface of reflection grating (monochromator), and from the diffracted radiation, the desired wavelength is selected. 2) This selected beam of light falls on a V-shaped mirror, called a beam splitter that splits the radiations into two beams; one of which passes through the reference cell containing pure solvent and the other simultaneously passes through the sample solution cell. 3) The transmitted light from the two cells go to the photoelectric detector alternatively, and the difference in the absorbance by the solvent and the sample solution is measured electronically with very high accuracy. ° Although the double beam instruments are more complicated and expensive, they do offer the following advantages: 1) It is not necessary to continually replace the blank with the sample or to zero adjust at each wavelength as in the single beam units. 2) The ratio of the powers of the sample and reference beams is constantly obtained and used. Any error due to variation in the intensity of the source and fluctuation in the detector is minimized. 3) Because of the previous two factors, the double beam system lends itself to rapid scanning over a wide wavelength region and to the use of a recorder or digital readout. i | Q APPLICATIONS ° UV-Vis spectroscopy has been mainly applied for the detection of functional groups (chromophore), the extent of conjugation, detection of polynuclear compounds by comparison, etc. Some of the important applications are: | Spe , ic Ti “ In a spectrophotometric titration, the equivalence point is determined with a spectrophotometer. In this technique, the titration vessel is kept directly in the light path of the instrument.© Then, the absorbance of the solution is determined after adding titrant, anda plot of absorbance asa function of volume of titrant is prepared. © If the titration reaction is complete, the titration curve will consist of two straight lines intersecting at the equivalence point, similar to amperometric and conductometric titrations. © On the other hand, if the titration reaction is not complete, there occurs appreciable curvature in the equivalence point region but extrapolation of the two linear segments of the titration curve to their intersection gives the equivalence point volume. 1) Curve (a) is characteristic of a case where only the titrant absorbs. An interesting example of this is the titration of arsenic(III) with bromate- bromide, where the absorbance readings are taken at the wavelength where bromine absorbs. As long as arsenic(III) remains in the solution, the absorbance will not be changed because the product does not absorb in that Absorbance Equivalence region. As soon as arsenic(III) is consumed pa by the titrant, the absorbance will increase due to the colour of the titrant (bromine) alone. 2) Curve (b) is characteristic of a case where only the product of reaction absorbs. An example of this is the titration of Cu(II) with EDTA, carried out at 745nm wavelength. This wavelength is selected because at this Volume of Titrant Absorbance ‘Volume of Titrant position the EDTA-copper complex possesses rs) a much greater absorbance compared to copper solution alone. 3) Curve (c) is characteristic of a case where only the substance being titrated absorbs, while the titrant and the product do not absorb. An example is the titration of p-toluidine in butanol with Volume of Tarant perchloric acid at 290nm wavelength. © Equivalence point© This wavelength is selected because p-toluidine sharply absorbs at this wavelength; whereas the titrant, perchloric acid shows no absorbance in | this region. As soon as the whole quantity of p-toluidine reacts with perchloric acid, the absorbance will become constant after the equivalence point. 4) Curve (d) is obtained when a coloured analyte is converted into a colourless product by a coloured titrant. When | titrant is added, the analyte colour starts fading due to the formation of a colourless product. But after the equivalence point, absorbance again rises due to the colour of the titrant alone. Choice of Wavelength © Anumber of criteria are considered in the choice of wavelength. It may be necessary to balance one against another: 1) The highest absorption peak will yield the maximum sensitivity. 2) A chosen wavelength should yield a minimum interference from other species that may be in the system. 3) Because of the errors that arise from sharply rising or sharply falling portions of the absorption curves, these sections should be avoided. 4) Care should be taken to avoid a sharp band if the slit width exceeds the bandwidth. | 7 Apparatus ein order to carry out spectrophotometric titrations, a special titration cell of 5-100ml capacity is employed that fits in the cell compartment of the spectrophotometer. The cell is made up of perspex sheet. As the material perspex is opaque to UV Absorbance, Equivalence point Volume of Titrant @ Vv ; Figure 1.23: Titration Cell light, two openings are made in the cell :to accommodate cellular quartz windows. * These windows are inserted in such a way that the beam of monochromatic light passes through their centres to the photoelectric cell. The cell has two small openings, one for the tip of a microburette and the other for a micro stirrer. Except the quartz windows, the whole cell is covered with black paper to exclude all extraneous light. > Technique * The experimental technique is simple and involves the following steps: 1) The solution to be titrated is taken in the cell. 2) Then, the cell is kept in the light path of a spectrophotometer. 3) The spectrophotometer is adjusted to the wavelength at which experiment is to be carried out and the instrument is set at zero absorbance (if the reactant is colourless) or at some other starting value (if the reactant is coloured) that lies within the linearity range of the instrument. 4) After this a known volume of titrant is added to the stirred solution in the cell and the absorbance is read again 5) This is repeated at several points before the end point and at several points after the end point. 6) Finally, absorbance is plotted against the volume of titrant added. 7) From this graph, the equivalence point is obtained. > Dilution Correction * Since absorbance depends upon concentration, the effect of dilution should be taken into consideration. This error can be overcome by one of the following methods: 1) Either the titrant is made much more concentrated than the solution to be titrated so that there is negligible change in volume, or 2) A simple correction factor is included in the calculation.° Where, Aand A’ = Corrected and measured absorbance, respectively. V = Original volume of solution. v= Volume of titrant added. * For best results, appropriate dilution corrections must be made and the above equation becomes: Where, A, = Corrected absorbance. V, and V, = Respective initial and added volumes. A,, = Measured absorbance. However, a negligible error will result from ignoring dilution corrections if the titrant concentration is much greater than the concentration of the solution being titrated. > Applications * Some typical methods of analysis by spectrophotometric titrations are: 1) Acid-Base Methods: Phenols can be titrated with NaOH. Absorbance occurs due to the formation of phenolate ion. 2) Oxidation-Reduction Methods: Ce(III) can be titrated with Co(II), forming Ce(IV). 3) Complexometric Titrations: Bi(III) can be titrated with thiourea; copper(II) is added, resulting in the formation of Cu -EDTA complex; or thiourea is added resulting in the disappearance of bismuth -thiourea complex. Fe(III) can be titrated with EDTA, resulting in the disappearance of Fe sulphosalicylic acid complex. Cu(II) can be titrated with EDTA, resulting in the formation of Cu -EDTA complex. Copper and bismuth in a mixture can be estimated by just a single titration with EDTA. Measurements are made at 745nm, where copper -EDTA complex absorbs strongly but bismuth complex does not. 4) Precipitation Titrations: $04?- ions can be titrated with Ba(II) ions, resulting in the appearance of turbidity. F1- ions can be titrated with Th(IV) ions. Reaction of Th(IV) with the indicator SPADNS results in a | precipita>» Advantages © Spectrophotometric titrations have several advantages over direct spectrophotometric analysis: 1) It can be applied to a large number of non-absorbing constituents as only one absorber is to be present among the reactant, the titrant, or the reaction products. 2) Presence of other absorbing species at the analytical wavelength does not cause interference because only the change in absorbance is significant. 3) It can be applied to highly coloured solutions that could not be determined by the visual indicators. 4) It can be applied to such reactions that tend to be appreciably incomplete at the equivalence point. 5) It is quite accurate. One can obtain accuracy and precision of a few tenths per cent with comparative ease by spectrophotometric titrations. 6) In contrast to normal absorbance procedures, it is not necessary that the titration be performed in the region or wavelength of maximum absorbance. Thus, a greater choice of wavelengths is available. 7) Relatively large quantities of other absorbing species at the chosen wavelength are necessary before noticeable interference results. 8) Turbid solutions may be titrated in selected circumstances. 9) More dilute solutions may be employed than in other types of titrations. 10) A variety of equipment may be employed. > Single Component Analysis Methods © The methods involved in the calculation of single component analysis are: 1) Direct Analysis: Essentially all compounds containing conjugated double bond or aromatic rings, and many inorganic species absorb light in the UV-visible regions. In these techniques, the substance to be determined is dissolved in a suitable solvent and diluted to the required concentration by appropriate dilutions, and then absorbance is then measured. 2) Indirect Analysis: This method involves analysis after addition of some reagent.