Course: M.Sc (Chemistry) Analytical Chemistry Unit: III
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1 Course: M.Sc (Chemistry) Analytical Chemistry Unit: III Syllabus: Principle of spectrophotometry Types of spectrophotometer Applications - Dissociation constants of an indicator simultaneous spectrophotometric determinations Determination of Stoichiometry of Complexes Job s method of continuous variation mole ratio and slope ratio analysis Advantages and limitations photometric titrations Colorimetry, standard series method duplication method - balancing method photoelectric colorimeter. SPECTROPHOTOMETRY Dr. K. SIVAKUMAR Department of Chemistry SCSVMV University chemshiva@gmail.com 1 ν Electromagnetic Waves - Terminologies Electromagnetic wave parameters: Wavelength (λ): Wavelength is the distance between the consecutive peaks or crests Wavelength is expressed in nanometers (nm) 1nm = 10-9 meters = 1/ meters 1A = meters = 1/ meters 2 1
2 ν Electromagnetic Waves - Terminologies Electromagnetic wave parameters: Frequency (ν): Frequency is the number of waves passing through any point per second. Frequency is expressed in Hertz (Hz) 3 ν Electromagnetic Waves - Terminologies Electromagnetic wave parameters: Wave number ( ν ): Wave number is the number of waves per cm. Wavelength, Wave number and Frequency are interrelated as, Where, λ is wave length 1 ν = λ = ν c ν is wave number ν is frequency c is velocity of light in vacuum. i.e., 3 x 10 8 m/s 4 2
3 Electromagnetic Spectral regions nm 10-4 to to to to to to to 10 9 EM waves γ-rays X-rays UV Visible IR Microwave Radio 5 Electromagnetic radiation sources EM radiation Spectral method Radiation source Gamma rays Gamma spec. gamma-emitting nuclides X-rays X-ray spec. Synchrotron Radiation Source (SRS), Betatron (cyclotron) Ultraviolet UV spec. Hydrogen discharge lamp Visible Visible spec. tungsten filament lamp Infrared IR spec. rare-earth oxides rod Microwave ESR spec. klystron valve Radio wave NMR spec. magnet of stable field strength 6 3
4 Electromagnetic Spectrum Type of radiation and Energy change involved 7 Electromagnetic Spectrum Type of radiation and Energy change involved 8 4
5 Lambert s law fraction of the monochromatic light absorbed by a homogeneous medium is independent of the intensity of the incident light and each successive unit layer absorbs an equal fraction of the light incident on it Lambert Beer s law fraction of the incident light absorbed is proportional to the number of the absorbing molecules in the light-path and will increase with increasing concentration or sample thickness. Beer 9 Beer Lambert law / Beer Lambert Bouguer law / Lambert Beer law log (I 0 /I) = ε c l = A Where, I 0 - the intensity of incident light I - the intensity of transmitted light ε - molar absorptivity / molar extinction coefficient in cm 2 mol -1 or L mol -1 cm -1. c - concentration in mol L -1 l - path length in cm A - absorbance (unitless) Molar absorptivity 10 5
6 Absorption intensity ε ε max Intensity of absorption is directly proportional to the transition probability A fully allowed transition will have ε max > A low transition probability will have ε max < 1000 λ max wavelength of light corresponding to maximum absorption is designated as λ max and can be read directly from the horizontal axis of the spectrum Absorbance (A) is the vertical axis of the spectrum A = log (I 0 /I) I 0 - intensity of the incident light; I - intensity of transmitted light ε max = Applications of Beer-Lambert s Law In determining the concentration of solutions absorbing in UV or visible region. For Example, if we have a standard solution (i.e., a solution with known concentration) then, let consider that, A absorbance of standard solution s C concentration of standard solution s According to Beer-Lambert s law, A s = C l s or A C s s = l 12 6
7 Applications of Beer-Lambert s Law Now, to find out the concentration of a test solution (i.e., solution with unknown concentration), we can measure the absorbance of test solution and according to Beer-Lambert s law, A C u u = l Comparing equations Where, A A C s s u u absorbance of test solution C concentration of test solution = l & A C u u = l C u Au = C A s s 13 Deviations from Beer s Law & / Limitations of Beer-Lambert s Law deviations in absorptivity coefficients at high concentrations (>0.01M) due to electrostatic interactions between molecules in close proximity often assumed that Beer s Law is always a linear plot describing the relationship between absorbance and concentration Beer s Law successfully describes the behaviour of dilute solutions only. At high concentrations (ie greater than 10-2 M) there is interaction between absorbing particles such that the absorption characteristics of the analyte are affected. 14 7
8 Deviations from Beer s Law & / Limitations of Beer-Lambert s Law scattering of light due to particulates in the sample fluoresecence or phosphorescence of the sample non-monochromatic radiation, deviations can be minimized by using a relatively flat part of the absorption spectrum such as the maximum of an absorption band by shifts in the position of a chemical or physical equilibrium involving the absorbing species. 15 Types of spectrophotometer Schematic of a single-beam spectrophotometer 8
9 Types of spectrophotometer Schematic of a double-beam spectrophotometer Types of spectrophotometer Schematic of a split-beam spectrophotometer 9
10 Types of spectrophotometer UV Spectrophotometer Hydrogen Gas Lamp Mercury Lamp Visible Spectrophotometer Tungsten Lamp Light Sources Dispersion Devices Non-linear dispersion Temperature sensitive Linear Dispersion Different orders 10
11 Photomultiplier Tube Detector High sensitivity at low light levels Cathode material determines spectral sensitivity Good signal/noise Shock sensitive Anode The Photodiode Detector Wide dynamic range Very good signal/noise at high light levels Solid-state device 11
12 Cells UV Spectrophotometer - Quartz (crystalline silica) Visible Spectrophotometer - Glass Cell Types I Open-topped rectangular standard cell (a) and apertured cell (b) for limited sample volume 12
13 Cell Types II Micro cell (a) for very small volumes and flow-through cell (b) for automated applications Beer Lambert law / Beer Lambert Bouguer law / Lambert Beer law Bouguer Actually investigated the range of absorption Vs thickness of medium Lambert Extended the concepts developed by Bouguer Beer Applied Lambert s concept to solutions of different concentrations? Bernard Beer released the results of Lambert s concept just prior to those of Bernard 26 13
14 Spectrometer The instrument used for recording the absorption spectra of a compound is called spectrometer. The different components present in various types of spectrometer are shown here. UV - hydrogen discharge lamp Visible - tungsten filament lamp IR - electrically heated rod of rareearth oxides Microwave - klystron valve NMR - magnet of stable field strength. Variable slit, rheostat, etc Prism, ilter, monochromator, grating Cuvette, test-tube, cell Photographic plate, photocell, photomultiplier, photoconductivity device etc Galvanometer; pen recorder; cathode ray oscillograph 27 Colorimetric analysis Principle: Colorimetry analysis method is useful in determining the concentration of coloured solutions using the visible region (400nm 750nm) of electromagnetic spectrum and Beer Lambert s law. If the test solution is colourless then a suitable complexing agent can be added to test solution to get coloured which will absorb light. Example: For cuprous ions (Cu 2+ ) estimation NH 4 OH can be added to get blue colour. Instrumentation: Tungsten filament lamp is used to generate visible region (400nm 750nm) light
15 Colorimetric analysis The molecules in the cuvette absorb light and the remaining light is transmitted to the photocell. In photocell, Current generated α Amount of light transmitted But the amount of light transmitted depends on the depth of colour of test solution. i.e., concentration of test solution. If high concentration solution is analysed then, more number of molecules will be in the path of light and more amount of light will be absorbed. So, the amount of light transmitted will be very less and generates only less current. 29 Colorimetric analysis: Applications useful in estimating the concentration of coloured solutions Example: Estimation of CuSO 4 by colorimetry Series of CuSO 4 solution with known concentration are prepared and ammonium hydroxide is added to each solution to get blue colour. Absorbance of each standard CuSO 4 solution is measured with same filter and tabulated. Concentration (C) of CuSO 4 Absorbance (A) A A 2 A= C l A 6 test solution A t 30 15
16 UV Visible Spectroscopy Principle: Visible and ultraviolet spectroscopy is a study of electronic spectra of organic molecules which are found in the wavelength region of 100nm-400nm (UV region) and 400nm-750nm (Visible region). UV and visible radiations absorbed by the molecules will bring transition of outer shell electrons(σ, π and n electrons). According to molecular orbital theory when a organic molecule absorbs UV or visible radiations its electrons are promoted from a bonding to an antibonding orbital. 31 The Ultraviolet region [10 800nm] The Ultraviolet region may be divided as follows, 1. Far (or Vacuum) Ultraviolet region [ nm] 2. Near (or Quartz) Ultraviolet region [ nm] 3. Visible region [ nm] 32 16
17 UV - VISIBLE SPECTROSCOPY In UV - Visible Spectroscopy, the sample is irradiated with the broad spectrum of the UV - Visible radiation If a particular electronic transition matches the energy of a certain band of UV - Visible, it will be absorbed The remaining UV - Visible light passes through the sample and is observed From this residual radiation a spectrum is obtained with gaps at these discrete energies this is called an absorption spectrum 33 Instrumentation log(i 0 /I) = A UV-VIS sources monochromator/ beam splitter optics I 0 sample I 0 I 2 reference I 1 detector I λ, nm 34 17
18 Instrumentation Radiation source, monochromator and detector Two sources are required to scan the entire UV-VIS band: Deuterium lamp covers the UV Tungsten lamp covers The lamps illuminate the entire band of UV or visible light; the monochromator (grating or prism) gradually changes the small bands of radiation sent to the beam splitter The beam splitter sends a separate band to a cell containing the sample solution and a reference solution The detector (Photomultiplier, photoelectric cells) measures the difference between the transmitted light through the sample (I) vs. the incident light (I 0 ) and sends this information to the recorder 35 Sample Handling Virtually all UV spectra are recorded solution-phase Only quartz is transparent in the full nm range; plastic and glass are only suitable for visible spectra nm Concentration: 0.1 to 100mg 10-5 to 10-2 molar concentration may safely be used Percentage of light transmitted: 20% to 65% At high concentrations, amount of light transmitted is low, increasing the possibility of error A typical sample cell (commonly called a cuvet): Cells can be made of plastic, glass or quartz (standard cells are typically 1 cm in path length) 36 18
19 SPECTROPHOTOMETRY - Applications Structure identification Useful in the identification of a newly synthesized compound. The spectrum of unknown compound can be compared with the absorption spectrum of several known compounds in the literature. On comparison, If the spectrum of unknown compound correlates with a specifically known compound then the structures of both will also be similar. This method is called finger printing technique. Concentration of impurities During the purification process of a compound, if the absorption spectrum is recorded at a particular interval of time the decrease in the concentration of impurities can be monitored. The purification can be continued till it gives a less value of absorbance. To study the rate of a reaction To study the rate of formation of product in a reaction, the absorbance of product can be measured at definite intervals of time. We know that, the absorbance is directly proportional to concentration and hence the absorbance value will be increasing with respect to time. 37 SPECTROPHOTOMETRY Applications Determination of Dissociation constant Note the several curve cruse to same point is called isobestic point, it is very important because two champers has same absorptivities, since at this point (ph) has not effect. If the absorbance with ph plots at λmax, we get S-shape curve as shown in figure bellow: Example: Absorption spectrum of M methyl red as a function of ph between ph 4.5 and
20 SPECTROPHOTOMETRY Applications Determination of Dissociation constant 39 SPECTROPHOTOMETRY Applications Determination of Dissociation constant 40 20
21 SPECTROPHOTOMETRY Applications Determination of Dissociation constant of Indicator Methyl Red two forms of methyl red absorb strongly in the visible range, the ratio (MR - )/(HMR) may be determined spectrophotometrically 41 SPECTROPHOTOMETRY Applications Determination of Dissociation constant of Indicator. The composition of a mixture of HMR and MR - may be calculated from absorbancies A 1 and A 2 at wavelengths λ 1 and λ 2 using, at unit cell thickness Absorbance of acid and basic form of methyl red at two wavelengths 42 21
22 SPECTROPHOTOMETRY Applications Determination of Dissociation constant of Indicator. From the plots of absorbancy versus wavelength which just obtained, two wavelengths are selected for analyzing mixtures of the acidic and basic forms of methyl red. pk can be calculated by knowing the ratio of (MR - )/(HMR) Absorbancy of HMR and MRas a function of wavelength λ. 43 Simultaneous analysis of mixture The total absorbance of a solution at any given wavelength is equal to the sum of the absorbances of the individual components in the solution. Mixtures:Determining the concentration of mixtures the components of which absorb in the same spectral regions is possible. Strategy of the analysis. Total absorption at some wavelength of a two component mixture: A total,λ1 = A Μ,λ1 + A N,λ1. Each should obey Beer's law at this wavelength as long as concentration is sufficiently low. The contribution from each would then be: A M,λ1 = ε M,λ1 bc M and A N,λ1 = ε N,λ1 bc N. and A total, λ1 = ε M,λ1 bc M + ε N,λ1 bc N. Similarly at some other wavelength we would have, A total, λ2 = ε M,λ2 bc M + ε N,λ2 bc N..εb can be determined for each using standard solutions. Take absorbance readings of mixture at the two λs. Substitute into above so that there are two equations with two unknowns
23 (a) Two cases for analysis of a mixture. Spectra of the pure components have substantial overlap. (b) Regions exist in which each component makes the major contribution. Visible spectrum of MnO 4, Cr 2 O 7 2, and an unknown mixture containing both ions. 23
24 Methods for obtaining the stoichiometry of complex (determination of the composition of complexes) Continuous-variation method (Job s method) M + nl = ML n Methods for obtaining the stoichiometry of complex (determination of the composition of complexes) 1) Continuous-variation method (Job s method) This method is based on the measurement of a series of solutions in which molar concentrations of two reactants vary but their sum remains constant. The absorbance of each solution is measured at a suitable wavelength and plotted versus the mole fraction of one reactant. A maximum in absorbance occurs at the mole ratio corresponding to the combining ratio of the reactants. M + nl = ML n Plot peak 0.5 for 1:1 (MX) complex 0.33 for 1:1 (MX 2 ) complex 0.67 for 2:1 (M 2 X) complex Continuous variations plots for 1:3, 1:2 and 1:1 metal to ligand complexes. 24
25 (Job s method) Plot peak 0.5 for 1:1 (MX) complex 0.33 for 1:1 (MX 2 ) complex 0.67 for 2:1 (M 2 X) complex Continuous variations plot for 1:1 metal to ligand complexe. 2) Mole-ratio method A series of solution is prepared in which the analytical concentration of one reactant is held constant while that of other is varied. A plot of absorbance versus mole ratio of the reactants is then prepared. If the reaction is sufficiently complete, two straight lines of different slopes are obtained. The intersection of the extrapolated lines corresponds to the combining ratio in the complex. Unlike the method of continuous variations, the measured absorbance does not have to be corrected by subtracting the absorbance. Mole-ratio plots for 1:1 and 1:2 metal-toligand complexes. 25
26 3) Slope-ratio method This method, used mainly in studying weak complexes, requires that the formation reaction can be forced to completion with a large excess of either metal or ligand. Two sets of solutions are prepared : The first contains various amounts of metal ion each with the same large excess of ligand, while the second consists of various amounts of ligand each with the same large excess of metal. For the reaction xm + yl = M x L y when L is present in large excess, the concentration of product formed is limited by the concentration of the metal, or [M x L y ] = C M / x If Beer s law obtains, A = εb[m x L y ] = εbc M / x and a plot of A versus C M will yield a straight line with a slope of εb/x. Similarly, for the solutions containing M in large excess, [M x L y ] = C L / y A = εb[m x L y ] = εbc L / y The ratio of the two slopes is the combining ratio for the reaction (εbc M / x )(εbc L / y) = x / y Colorimetry Balancing Method Duboscq colorimeter Unknown and standard solutions are taken in cylinders. The transparent plungers are moved up and down until the colours seen from the top of each cylinder become identical. From the readings of the depth of the samples, the concentration of unknown can be evaluated using the equation given below, Jules Duboscq C u Au = C A s s 52 26
27 Colorimetry Balancing Method Duboscq colorimeter 53 Colorimetry Multiple Standard Method Unknown solution is taken in 50ml or 100ml Nessler tube and made up to the mark The solution is compared with a series of standard solutions with known concentrations The concentration of unknown solution will then be equal to that of the known solution whose colour it matches exactly. If the unknown matches with the solution containing 0.3g and 0.4g, a series of 0.32g, 0.34g, 0.36g. Containing solutions are prepared to get an exact or nearly exact match 54 27
28 Colorimetry Duplication Method Unknown solution is taken in a Nessler tube and appropriate colour developing reagent is added to produce a colour. In another Nessler tube the colour developing reagent is taken and made up little lower than the mark. From a burette, a standard solution is added to this Nessler tube with constant agitation until the colour of unknown solution becomes duplicated in this job. This method is less accurate 55 Photoelectric Colorimeters Single beam (Evelyn) photon-electric colorimeter Gurdeep Chatwal pp
29 Photoelectric Colorimeters Double beam photon-electric colorimeter Gurdeep Chatwal pp Photometric titrations Absorbance of solution is measured after adding titrant Typical photometric titration curves. Molar absorptivities of analyte titrated, product, and the titrant are ε A, ε P, ε T, respectively. 29
30 Photometric titrations (a) Spectrophotometric titration of 30.0 ml of EDTA in acetate buffer with CuSO4 in the same buffer. Upper curve: [EDTA] = [Cu 2+ ] = 5.00 mm. Lower curve: [EDTA] = [Cu 2+ ] = 2.50 mm. (b) Trans formation of data to mole fraction format
31 Good Luck! Dr. K. SIVAKUMAR Department of Chemistry SCSVMV University 61 31
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