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

CHAPTER 15APPLYING MOLECULAR AND

ATOMIC SPECTROSCOPIC METHODS: SHEDDING MORE

LIGHT ON THE SUBJECT

Introduction toIntroduction toAnalytical ChemistryAnalytical Chemistry

Copyright © 2011 Cengage Learning15-2

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.


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.


Table 15-1


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.


Figure 15-1

Figure 15-1 Ultraviolet spectra for several typical organic compounds.


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.


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.


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.


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.


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.


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.



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.



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.



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.


Example 15-1The 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.


Example 15-1

The absorbance of the first solution is given by

where cx is the unknown concentration of phosphate in the first solution. The absorbance of the second solution is given by


Example 15-1

where Vx is the volume of the solution of unknown phosphate

concentration (25.00 mL), Vs is the volume of the standard

solution of phosphate added (1.00 mL), Vt is the total volume

after the addition (26.00 mL), and cs 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 cx , we obtain


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,


Example 15-2A 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?


Example 15-2

For several identical aliquots of a standard solution having a known concentration cs , the absorbance after each addition, As, is given by

where Vt is the total volume of each flask, Vs is the variable

volume of standard added, cx is the unknown concentration, and k is a constant equal to εb/ Vt . A plot of As as a function of Vs should yield a straight line of the form


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



Analysis of Mixtures.To analyze the mixture, molar absorptivities for M and

N are first determined at wavelengths λ1 and λ2 .



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)



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



Titration Curves.The absorbances are corrected for dilution by the

titrant by multiplying the measured values by (VT+VA)/VA , where VA and VT are the volumes of the analyte solution and titrant, respectively.


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 .



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.


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.


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.



Quantitative

Measuring AbsorbanceDegradation 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.



Quantitative

Measuring AbsorbanceThe 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.


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


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.


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.



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


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.



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.



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.


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.



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)



At low concentrations

where c is the concentration of the fluorescent species.

(15-2)



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.


Figure 15-14


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).



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.



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.



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.


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.


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.


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.


15F-1 Atomic Absorption Spectroscopy

Flame Atomic Absorption Spectroscopy

Furnace Atomic Absorption SpectroscopyFurnace, or electrothermal, AA uses the same

instrumental setup as shown in Figure 15-17 except that a furnace atomizer is substituted for the burner.


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.


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).


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.


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.]


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