1. CHAPTER 15 APPLYING MOLECULAR AND ATOMIC SPECTROSCOPIC METHODS: SHEDDING MORE LIGHT ON THE SUBJECT Introduction to Analytical Chemistry

2. Copyright © 2011 Cengage Learning 15-2 15A-1 Overview Absorption by Organic Compounds. The shared electrons in such single bonds as carbon/carbon or carbon/hydrogen are so firmly held that absorption occurs only with photons more energetic than normal UV photons.

3. Copyright © 2011 Cengage Learning 15-3 15A-1 Overview Unsaturated organic functional groups that absorb in the UV/visible region are known as chromophores. Conjugation between two or more chromophores tends to cause shifts in peak absorbances to longer wavelengths.

4. Copyright © 2011 Cengage Learning 15-4 Table 15-1

5. Copyright © 2011 Cengage Learning 15-5 15A-1 Overview Absorption by Inorganic Species. Ions and complexes of elements in the first two transition series absorb broad bands of visible radiation. With these complexes, absorption involves transitions between filled and unfilled d-orbitals of the metal ion with energies that depend on the bonded ligands.

6. Copyright © 2011 Cengage Learning 15-6 Figure 15-1 Figure 15-1 Ultraviolet spectra for several typical organic compounds.

7. Copyright © 2011 Cengage Learning 15-7 Figure 15-2 Figure 15-2 Absorption spectra of aqueous solutions of several transition metal ions. These ions exist as aquo-complexes in aqueous solutions.

8. Copyright © 2011 Cengage Learning 15-8 15A-1 Overview Charge-Transfer Absorption. Molar absorptivities are unusually large. A charge-transfer complex consists of an electron- donor group bonded to an electron acceptor. The excited state is thus the product of a kind of internal oxidation/reduction process. The red color of the iron(III)/thiocyanate complex is a example of charge-transfer absorption.

9. Copyright © 2011 Cengage Learning 15-9 15A-1 Overview Qualitative Analysis  Qualitative applications of UV/visible spectroscopy are limited because the spectra of most compounds in solution consist of one or, at most, a few broad bands with no fine structure.

10. Copyright © 2011 Cengage Learning 15-10 15A-1 Overview Fundamental Studies  Spectrophotometry in the UV/visible region is one of the major tools for studying chemical equilibria and kinetics.  For equilibrium studies, known concentrations of reactants are mixed, and the absorption spectrum of the reaction mixture is obtained after equilibrium has been established.

11. Copyright © 2011 Cengage Learning 15-11 15A-1 Overview Quantitative Analysis  Ultraviolet /visible spectrophotometry is one of the most powerful and widely used tools for quantitative analysis.  detection limits of 10¯⁴ to 10¯⁷ M.

12. Copyright © 2011 Cengage Learning 15-12 15A-2 Making UV/Visible Spectrophotometry Quantitative Conventional Spectrophotometry  Conventional spectrophotometric methods involve either a direct measurement of the analyte absorbance or an indirect measurement after reacting the analyte with a reagent to form an absorbing product.

13. Copyright © 2011 Cengage Learning 15-13 15A-2 Making UV/Visible Spectrophotometry Quantitative External Standards and the Calibration Curve. In most spectrophotometric methods, calibration is achieved by the method of external standards. Here, a series of standard solutions of the analyte is used to construct a calibration curve of absorbance versus concentration or to produce a linear regression equation.

14. Copyright © 2011 Cengage Learning 15-14 15A-2 Making UV/Visible Spectrophotometry Quantitative External Standards and the Calibration Curve. The ideal standards for the determination would be identical in composition to the samples except that the analyte concentrations would be known. Real standards, however, are usually made from pure chemicals.

15. Copyright © 2011 Cengage Learning 15-15 15A-2 Making UV/Visible Spectrophotometry Quantitative The Standard Addition Method. The difficulties associated with production of standards with an overall composition closely resembling that of the sample can be formidable. Method of standard additions may prove useful. Multiple additions can be made to several aliquots of the sample and a multiple standard addition calibration curve obtained.

16. Copyright © 2011 Cengage Learning 15-16 Example 15-1 The single-point standard addition method was used in the determination of phosphate by the molybdenum blue method. A 2.00-mL urine sample was treated with molybdenum blue reagents to produce a species absorbing at 820 nm, after which the sample was diluted to 100.00 mL. A 25.00-mL aliquot gave an absorbance of 0.428 (solution 1). Addition of 1.00 mL of a solution containing 0.0500 mg of phosphate to a second 25.0-mL aliquot gave an absorbance of 0.517 (solution 2). Use these data to calculate the number of milligrams of phosphate per milliliter of the sample.

17. Copyright © 2011 Cengage Learning 15-17 Example 15-1 The absorbance of the first solution is given by where c x is the unknown concentration of phosphate in the first solution. The absorbance of the second solution is given by

18. Copyright © 2011 Cengage Learning 15-18 Example 15-1 where V x is the volume of the solution of unknown phosphate concentration (25.00 mL), V s is the volume of the standard solution of phosphate added (1.00 mL), V t is the total volume after the addition (26.00 mL), and c s is the concentration of the standard solution (0.500 mg mL1). If we solve the first equation for εb, substitute the result into the second equation, and solve for c x, we obtain

19. Copyright © 2011 Cengage Learning 15-19 Example 15-1 This result is the concentration of the diluted sample. To obtain the concentration of the original urine sample, we need to multiply by 100.00/2.00. Thus,

20. Copyright © 2011 Cengage Learning 15-20 Example 15-2 A multiple standard addition method was used for determining Fe³⁺ in a natural water sample. Ten- milliliter aliquots of the sample were pipetted into 50.00-mL volumetric flasks. Exactly 0.00, 5.00, 10.00, 15.00, and 20.00 mL of a standard solution containing 11.1 ppm of Fe³⁺ were added to each followed by an excess of thiocyanate ion to give the red complex Fe(SCN)²⁺. After dilution to volume, absorbances of the five solutions were measured in a 0.982-cm cell at 480 nm and found to be 0.240, 0.437, 0.621, 0.809, and 1.009, respectively. What is the concentration of Fe³⁺ in the water sample?

21. Copyright © 2011 Cengage Learning 15-21 Example 15-2 For several identical aliquots of a standard solution having a known concentration c s, the absorbance after each addition, A s, is given by where V t is the total volume of each flask, V s is the variable volume of standard added, c x is the unknown concentration, and k is a constant equal to εb/ V t. A plot of A s as a function of V s should yield a straight line of the form

22. Copyright © 2011 Cengage Learning 15-22 Example 15-2 where the slope m and intercept b are given by By combining these equations for the slope and intercept, the concentration of the unknown can be found as

23. Copyright © 2011 Cengage Learning 15-23 15A-2 Making UV/Visible Spectrophotometry Quantitative Analysis of Mixtures. To analyze the mixture, molar absorptivities for M and N are first determined at wavelengths λ 1 and λ 2.

24. Copyright © 2011 Cengage Learning 15-24 15A-2 Making UV/Visible Spectrophotometry Quantitative To complete the analysis, the absorbance of the mixture is determined at the same two wavelengths. From the known molar absorptivities and pathlength, the following equations hold: (15-1) (15-2)

25. Copyright © 2011 Cengage Learning 15-25 15A-2 Making UV/Visible Spectrophotometry Quantitative Following Titrations by Measuring UV/Visible Absorption − The method requires that one or more of the reactants or products absorb radiation or that an absorbing indicator be present. In spectrophotometric titrations, the spectrophotometer serves as the detector

26. Copyright © 2011 Cengage Learning 15-26 15A-2 Making UV/Visible Spectrophotometry Quantitative Titration Curves. The absorbances are corrected for dilution by the titrant by multiplying the measured values by (V T +V A )/V A, where V A and V T are the volumes of the analyte solution and titrant, respectively.

27. Copyright © 2011 Cengage Learning 15-27 Figure 15-7 Figure 15-7 Shapes of spectrophotometric titration curves. In (a), only the titrant absorbs or the excess titrant reacts with an indicator. The absorbance increases sharply after the titrant becomes in excess. In (b), only the product absorbs and the absorbance increases until the analyte has completely reacted. In (c), only the analyte absorbs and its absorbance decreases steadily until the end point. The curves in (d) through (f) are typical of those in which more than one species absorbs. The molar absorptivities of the analyte, product, and titrant are labeled A, P, and T.

28. Copyright © 2011 Cengage Learning 15-28 15A-2 Making UV/Visible Spectrophotometry Quantitative Applications of Spectrophotometric Titrations. Figure 15-8 illustrates the application of this technique to the successive titration of Bi(III) and Cu(II). At 745 nm, the cations, the reagent, and the bismuth complex formed in the first part of the titration do not absorb, but the copper complex does. Thus, the solution exhibits no absorbance until essentially all the Bi(III) has been titrated.

29. Copyright © 2011 Cengage Learning 15-29 15B-1 Molecules That Absorb Infrared Radiation With the exception of homonuclear diatomic molecules, such as O₂, Cl₂, and N₂, all molecules, organic and inorganic, absorb infrared radiation.

30. Copyright © 2011 Cengage Learning 15-30 15B-3 Making Infrared Absorption Spectroscopy Quantitative Quantitative applications of IR spectroscopy are much more limited than are such applications in the UV/visible region. degradation of the transparency of IR cell windows (typically polished NaCl) pathlengths are hard to reproduce because the thickness of IR cells is often less than 1 mm to permit the transmission of measurable intensities.

31. Copyright © 2011 Cengage Learning 15-31 15B-3 Making Infrared Absorption Spectroscopy Quantitative Measuring Absorbance Degradation of the transparency of IR cell windows (typically polished NaCl). Pathlengths are hard to reproduce because the thickness of IR cells is often less than 1 mm to permit the transmission of measurable intensities.

32. Copyright © 2011 Cengage Learning 15-32 15B-3 Making Infrared Absorption Spectroscopy Quantitative Measuring Absorbance The presence of significant solvent and other background absorption can make the measurement of the absorbance of a single component somewhat difficult in the IR region. Infrared absorption remains a good quantitative technique for mixtures of organic compounds.

33. Copyright © 2011 Cengage Learning 15-33 15C Assessing Errors In Spectrophotometry Concentration measurements at absorbances lower than about 0.1 are not very reliable and should be avoided. The reason for the poor precision in this region can be understood by writing Beer’s law in the form

34. Copyright © 2011 Cengage Learning 15-34 15C Assessing Errors In Spectrophotometry Concentration is directly proportional to the difference between the two measured quantities, log P₀ and log P. At low concentrations, log P is nearly as large as log P₀ and A is a small difference between large numbers. Therefore, the relative uncertainties in A and c are large.

35. Copyright © 2011 Cengage Learning 15-35 15D-1 Molecular Fluorescence Spectroscopy One of the most attractive features of molecular fluorescence is its inherent sensitivity, which is often one to three orders of magnitude better than absorption spectroscopy. Another advantage is the large linear concentration range of fluorescence methods.

36. Copyright © 2011 Cengage Learning 15-36 15D-1 Molecular Fluorescence Spectroscopy Relaxation Processes. When the molecules are irradiated with a more energetic band of radiation. Several processes can occur that cause the molecule to lose its excess energy. Two of the most important of these mechanisms, nonradiative relaxation and fluorescence emission. The two most important nonradiative relaxation methods

37. Copyright © 2011 Cengage Learning 15-37 Figure 15-12 Figure 15-12 Energy-level diagram shows some of the processes that occur during (a) absorption of incident radiation, (b) nonradiative relaxation, and (c) fluorescence emission by a molecular species. Absorption typically occurs in 10 -15 s, whereas vibrational relaxation occurs in the 10 -10 to 10 -11 s time scale. Internal conversion between different electronic states is also very rapid (10 - 12 s), whereas the lifetime of fluorescence is typically 10 -5 to 10 -10 s.

38. Copyright © 2011 Cengage Learning 15-38 15D-1 Molecular Fluorescence Spectroscopy Vibrational relaxation takes place during collisions between excited molecules and molecules of the solvent. Nonradiative relaxation between the lower vibrational levels of an excited electronic state and the higher vibrational levels of another electronic state can also occur. This type of relaxation, sometimes called internal conversion.

39. Copyright © 2011 Cengage Learning 15-39 15D-1 Molecular Fluorescence Spectroscopy Relationship between Excitation Spectra and Fluorescence Spectra. Because the energy differences between vibrational states is about the same for both ground and excited states, the absorption, or excitation spectrum, and the fluorescence spectrum for a compound often appear as approximate mirror images of one another.

40. Copyright © 2011 Cengage Learning 15-40 Figure 15-13 Figure 15-13 Fluorescence spectra for 1 ppm anthracene in alcohol: (a) excitation spectrum and (b) emission spectrum. Note that the two spectra are very nearly mirror images.

41. Copyright © 2011 Cengage Learning 15-41 15D-1 Molecular Fluorescence Spectroscopy The radiant power of fluorescence F is proportional to the radiant power of the excitation beam absorbed: where P₀ is the radiant power of the beam incident on the sample and P is the radiant power after it traverses a pathlength b of the medium. (15-11)

42. Copyright © 2011 Cengage Learning 15-42 15D-1 Molecular Fluorescence Spectroscopy At low concentrations where c is the concentration of the fluorescent species. (15-2)

43. Copyright © 2011 Cengage Learning 15-43 15D-1 Molecular Fluorescence Spectroscopy Fluorescence Instrumentation  Sources for fluorescence are usually more powerful than typical absorption sources. In fluorescence, the radiant power emitted is directly proportional to the source intensity (Equation 15-12), whereas absorbance, being related to the ratio of radiant powers, is essentially independent of source intensity.

44. Copyright © 2011 Cengage Learning 15-44 Figure 15-14

45. Copyright © 2011 Cengage Learning 15-45 Figure 15-14(cont.) Figure 15-14 Typical fluorescence instruments. A filter fluorometer is shown in (a). Note that the emission is measured at right angles to the mercury arc lamp source. Fluorescence radiation is emitted in all directions, and the 90 deg geometry avoids the detector viewing the source. The spectrofluorometer (b) uses two grating monochromators and also views the emission at right angles. The two monochromators allow the scanning of excitation spectra (excitation wavelength scanned at a fixed emission wavelength), emission spectra (emission wavelength scanned at a fixed excitation wavelength), or synchronous spectra (both wavelengths scanned with a fixed wavelength or wavenumber offset between the two monochromators).

46. Copyright © 2011 Cengage Learning 15-46 15D-1 Molecular Fluorescence Spectroscopy Applying Molecular Fluorescence Spectroscopy  In many cases where fluorescence monitoring is ordinarily not feasible, fluorescent probes or tags can be bound covalently to specific sites in molecules such as proteins, thus making them detectable via fluorescence.

47. Copyright © 2011 Cengage Learning 15-47 15D-1 Molecular Fluorescence Spectroscopy Applying Molecular Fluorescence Spectroscopy  Inorganic fluorescence methods can be divided into two classes: direct methods that are based on the reaction of the analyte with a complexing agent to form a fluorescent complex and indirect methods that depend on the decrease in fluorescence, also called quenching.

48. Copyright © 2011 Cengage Learning 15-48 15D-1 Molecular Fluorescence Spectroscopy Applying Molecular Fluorescence Spectroscopy  Because of its sensitivity, fluorescence is widely used as a detection technique for liquid chromatographic methods (see Section 17B-1), for flow analysis methods, and for electrophoresis. In addition to methods that are based on measurements of fluorescence intensity, many methods are based on measurements of fluorescence lifetimes.

49. Copyright © 2011 Cengage Learning 15-49 15D-2 Chemiluminescence Methods: Reactions That Produce Light Chemiluminescence is produced when a chemical reaction yields an electronically excited molecule, which emits light as it returns to the ground state. One attractive feature of chemiluminescence for analytical uses, is the very simple instrumentation. Since no external source of radiation is needed for excitation. Chemiluminescence methods are known for their high sensitivities.

50. Copyright © 2011 Cengage Learning 15-50 15E Molecular Scattering Methods Scattering can be divided into two classes: elastic scattering, in which the scattered radiation is of the same energy as the incident radiation, and inelastic scattering, in which the scattered radiation has higher or lower energy than the incident radiation.

51. Copyright © 2011 Cengage Learning 15-51 15E Molecular Scattering Methods Laser light-scattering methods are widely used to determine molecular weights of compounds and to determine particle sizes. Inelastic light-scattering methods have become very important in recent years, particularly those methods based on the Raman effect. Raman spectroscopy involves inelastic scattering of radiation caused by vibrational and rotational transitions.

52. Copyright © 2011 Cengage Learning 15-52 15F-1 Atomic Absorption Spectroscopy Flame Atomic Absorption Spectroscopy Furnace Atomic Absorption Spectroscopy Furnace, or electrothermal, AA uses the same instrumental setup as shown in Figure 15-17 except that a furnace atomizer is substituted for the burner.

53. Copyright © 2011 Cengage Learning 15-53 Figure 15-17 Figure 15-17 Block diagram of a double-beam atomic absorption spectrophotometer. Radiation from the hollow cathode lamp is split into two beams; one goes through the flame and one bypasses the flame as a reference beam. The sample solution is sprayed into the flame as droplets. In the flame, atoms of the element of interest are formed. These atoms can absorb the source radiation and attenuate the beam. The monochromator isolates the desired radiation from background. A photomultiplier tube acts as the radiation transducer.

54. Copyright © 2011 Cengage Learning 15-54 15F-1 Atomic Absorption Spectroscopy Furnace AA gives a transient signal that reaches a peak in a few seconds. Furnace AA is usually one to two orders of magnitude more sensitive than flame AA. Flame and furnace AA are quantitative techniques only, since a specific hollow cathode lamp is required for each element of interest. The method is highly selective because of the very narrow line widths emitted by the hollow cathode lamp (0.002 to 0.005 nm).

55. Copyright © 2011 Cengage Learning 15-55 15F-2 Some Other Types of Atomic Spectroscopy In addition to atomic absorption spectroscopy, atomic emission and atomic fluorescence are also encountered. atomic emission (AE) use flames to produce excited atoms, which then emit characteristic radiation as they return to the ground state. Other AE methods use more energetic atomizers, such as inductively coupled plasmas (ICPs), arcs, and sparks. In contrast to atomic absorption, atomic emission methods can be used for qualitative analysis.

56. Copyright © 2011 Cengage Learning 15-56 Figure 15-18 Figure 15-18 Emission spectrum of a brine sample obtained with an oxyhydrogen flame. [R. Hermann and C. T. J. Alkemade, Chemical Analysis by Flame Photometry, 2nd ed. (New York: Interscience, 1979), p. 484. With permission.]

57. Copyright © 2011 Cengage Learning 15-57 THE END