P.415 Spectrophotometry: Instruments and applications Exploring Chemical Analysis Exploring Chemical Analysis Fourth Edition 19 歐亞書局

19-1 The Spectrophotometer

P.415 single-beam spectrophotometer

Double-beam spectrophotometer

Figure 19-1 P.415 Figure 19-1 The double-beam spectrophotometer in Figure 19-1 features a rotating mirror (the beam chopper), which alternately directs light through the sample or reference cell several times per second.

Figure 19-2 P.416 Figure 19-2 shows a double-beam instrument and the layout of its components.

The two lamps in the upper part of Figure 19-2b provide visible or ultraviolet radiation. P.416 Light Source

Figure 19-3 P.417 An ordinary tungsten lamp, whose filament glows at a temperature near 3000 K, produces radiation in the visible and near-infrared regions at wavelengths of 320 to 2500 nm. For ultraviolet spectroscopy, we normally employ a deuterium arc lamp in which a controlled electric discharge dissociates D 2 molecules, which then emit ultraviolet radiation from 200 to 400 nm.

monochromator 單光器  disperses light into its component wavelengths  selects a narrow band of wavelengths to pass through the sample. P.417 A monochromator consists of: entrance slit exit slit grating - to disperse light

Figure 19-4 Figure 19-4 Czerny-Turner grating monochromator. P.417 In the grating monochromator in Figure 19-4, polychromatic radiation from the entrance slit is collimated ( 對準成直線 ) by a concave mirror. Figure 19-5 Principle of a reflection grating.

The closely spaced parallel grooves of the grating have a repeat distance d. When light is reflected from the grating, each groove behaves as a source of radiation.  If adjacent light rays are in phase, they reinforce one another.  If they are out of phase, they cancel one another. P.418 Principle of a reflection grating Constructive interference and destructive interference

Rotating the reflection grating allows different wavelengths to pass through the exit slit. The grating directs a narrow band of wavelengths to the exit slit.

Figure 19-7 P.419 Resolution of closely spaced bands, which requires a narrow slit width, can be achieved at the expense of decreased signal-to- noise ratio Decreasing the exit slit width in monochromator decreases the selected bandwidth and decreases the energy reaching the detector. sample detector

Figure 19-7 P.419 For quantitative analysis, a monochromator bandwidth that is <1/5 of the width of the absorption band is reasonable. monochromator bandwidth ↗ resolution ↘

For a quantitative analysis, use a wavelength of maximum absorbance so that small errors in the wavelength do not change the absorption very much.

Choose a monochromator bandwidth (controlled by exit slit) small enough thtat it does not distoret the band shape, but not so small that the spectrum is too noisy. Widening the bandwidth distorts the band shape. In the lowest trace, a monochromator bandwidth that is 1/5 of the sharp absorption bands (measured at half the peak height) prevents distortion.

Types of monochromators: (a) grating monochromator (b) prism monochromator. Figure 25-6, Skoog diffraction 繞射 : the bending of light rays is done by a grating ( 繞射光柵 ) diffraction 折射 : the bending of light rays is done by a prism ( 分光稜鏡 )

不同頻率會有不同的波速  經過稜鏡之後往不同方向前進 Open the slit to collect a specific range of wavelengths for measurement.

Detector A detector produces an electric signal when it is struck by photons. Figure 19-8 shows that detector response depends on the wavelength of the incident photons. P.419

Figure 19-9 P.420 A photomultiplier tube is a very sensitive detector.

光電倍增管 ( 放大偵測訊號 )

19-2 Analysis of a Mixture

When there is more than one absorbing species in a solution, the absorbance at a particular wavelength is the sum of absorbances from all species at that wavelength: The molar absorptivity of different species at a particular wavelength. How to determine [X], [Y], and [Z]?

For an unknown mixture containing [X] and [Y] We choose wavelengths of maximum absorption for the individual components, X and Y, and measure the absorbances. absorbance at λ’ absorbance at λ’’ NOTE: The molar absorptivities for the same species at different wavelengths are not the same. D = b(ε X ’ε Y ’’-ε Y ’ε X ’’)

Figure Figure Visible spectra of hydrogen peroxide complexes of Ti(IV)(1.32 mM), V(V)(1.89 mM), and an unknown mixture containing both. At λ’ pure Ti complex has a maximal absorbance At λ’’ pure V complex has a maximal absorbance Take the absorbance at in the mixture Take the absorbance at λ’ and λ’’, we can solve the [Ti-complex] and [V-complex] in the mixture

Example ： Example ： Analysis of a Mixture with Equations 19-5 The molar absorptivities of X (the Ti complex) and Y (the V complex) in Figure were measured with pure samples of each: A mixture of X and Y in a 1.00-cm cell had an absorbance of A’= at 406 nm and A’’=0.641 at 457 nm.  Find the concentrations of X and Y in the mixture. P.422

SOLUTION ： P.422

Isosbestic Points Isosbestic Points 等吸收點 We use this reaction to introduce the isosbestic point. When pH > 5.1, there is more In -. When pH < 5.1, there is more HIn.

Isosbestic Points If species X is converted into species Y in the course of a chemical reaction, a spectrum of the mixture has an obvious, characteristic behavior, shown in Figure In - + H + ↔ HIn pK 2 = 5.1 Figure Absorption spectrum of 3.7 ×10 -4 M methyl red as a function of pH between pH 4.5 and 7.1. Almost pure In - at pH 7.1 Almost pure HIn at pH 4.5

An isosbestic point observed during a chemical reaction is good evidence that only two principal species are present. If the spectra of pure X and Y cross each other at some wavelength, then every spectrum recorded during the X If the spectra of pure X and Y cross each other at some wavelength, then every spectrum recorded during the X ↔Y reaction crosses the same point, called an isosbestic point.

An isosbestic point occurs when ε X = ε Y and [X]+[Y] is constant. pure X pure Y At this wavelength, pure X and pure Y have the same absorptivity (ε X = ε Y ). For the X ↔Y reaction, [X]+[Y] stays constant, so the absorbance stays the same at this wavelength.

Figure In - + H + ↔ HIn pK 2 = 5.1 constant

An isosbestic point observed during a chemical reaction is good evidence that only two principal species are present. For X For X ↔Y reaction

19-3 Spectrophotometric Titrations

with a absorbance maximum at 465 nm Fe 3+ apotransferrin

Figure P.424 Figure Each of the two Fe-binding sites of transferrin is located in a cleft in the protein. Each site has one nitrogen ligand from the amino acid histidine and three oxygen ligands from tyrosine and aspartic acid.

1. When the Fe 3+ transferrin complex is formed, the absorbance at 465 nm develops 2. When the protein is saturated with Fe 3+, the curve levels off The end point is the extrapolated intersection of the two straight lines. NOTE: the absorbance rises slowly because Fe 3+ has some absorbance at 465 nm.

Figure shows the titration of mL of apotransferrin with 1.79×10 -3 M ferric nitrilotriacetate.

Figure Figure Spectrophotometric titration of apotransferrin with ferric nitrilotriacetate. Absorbance is corrected for dilution. The initial absorbance of the solution, before iron is added, is due to a colored impurity. P.425

Example: Correcting Absorbance for the Effect of Dilution The absorbance measured after adding 125 μL(= mL) of ferric nitrilotriacetate to mL of apotransferrin was  Calculate the corrected absorbance that should be plotted in Figure SOLUTION:

19-4 What Happens When a Molecule Absorbs Light?

excited state are shown in Figure Electronic States of Formaldehyde Molecular orbitals describe the distribution of electrons in a molecule, just as atomic orbitals describe the distribution of electrons in an atom. In Figure 19-17, four low-lying orbitals of formaldehyde, labeled σ 1 through σ 4, are each occupied by a pair of electrons with opposite spin (spin quantum numbers= +1/2 and -1/2 represented by ↑and↓). P.425

Figure Figure Geometry of formaldehyde in its ground state (S 0 ) and lowest excited singlet state (S 1 ). P.425

Figure P.426 Figure Molecular orbital diagram of formaldehyde, showing energy levels and orbital shapes. The coordinate system was shown in Figure

In a electronic transition, an electron moves from one orbital to another. There are actually two possible transitions, depending on the spin quantum numbers in the excited state. The state in which the spins are opposed in Figure is called a singlet state. If spins are parallel, the excited state is a tiplet state. P.426

Figure P.426 Figure Two possible electronic states arising from an n→π* transition. The terms singlet and triplet are used because a triplet state splits into three slightly different energy levels in a magnetic field, but a singlet state is not split.

Vibration and Rotational States of Formaldehyde The six modes of vibration of formaldehyde are shown in Figure Combined Electronic, Vibrational, and Rotational Transitions Electronic absorption bands are usually very broad (~100 nm in Figure 18-5 and 18-9) because many different vibrational and rotational levels are excited at slightly different energies.

Figure P.427 Figure The six modes of vibration of formaldehyde. The wavenumber of infrared radiation needed to stimulate each kind of motion is given in units of reciprocal centimeters, cm-1.

What Happens to Absorbed Energy? Suppose that absorption of a photon promotes a molecule from the ground electronic state, S 0, to a vibrationally and rotationally excited level of the excited electronic state S 1 (Figure 19-20). P.427

Figure Figure Physical processes that can occur after a molecule absorbs an ultraviolet or visible photon. S 0 is the ground electronic state. S 1 and T 1 are the lowest excited singlet and triplet states, respectively. Straight arrows represent processes involving photons, and wavy arrows are radiationless transitions. R is vibrational relaxation. P.427

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Figure compares absorption and fluorescence spectra of anthracene. Fluorescence comes at lower energy and is roughly the mirror image of absorption. To understand the mirror image relation, consider the energy levels in Figure Photochemistry is a chemical reaction initiated by absorption of light. Some chemical reactions (not initiated by light) release energy in the form of light, which is called chemiluminescence. P.429

Figure P.429 Figure Spectra of anthracene show typical approximate mirror image relationship between absorption and fluorescence. Fluorescence comes at lower energy.

Figure Figure Energy-level diagram showing why structure is seen in the absorption and emission spectra and why the spectra are roughly mirror images. P.429

19-5 Luminescence in Analytical Chemistry

Luminescence is any emission of electromagnetic radiation and includes fluorescence, phosphorescence, and other possible processes. In Figure 19-23, luminescence is measured by exciting a sample at a wavelength that it absorbs (λ excitation ) and observing at the wavelength of maximum emission (λ emission ). P.430

For quantitative analysis, the intensity of luminescence (I) is proportional to the concentration of the emitting species (c) over some limited concentration range:. P.431

Color plate20 P.431

Fluorimetric Assay of Selenium in Brazil Nuts Hydrogen selenate (H 2 SeO 4 ) in the digest is reduced to hydrogen selenite (H 2 SeO 3 ) with hydroxylamine (NH 2 OH). Selenite is then derivatized to form a fluorescent product that is extracted into cyclohexane. P.431

Maximum response of the fluorescent product was observed with an excitation wavelength of 378 nm and an emission wavelength of 518 nm. The fluorescence calibration curve in Figure is linear. Absorption of excitation energy by neighboring molecules of the same substance is called self- absorption. P.431

Immunoassays Figure illustrates the principle of an enzyme-linked immunosorbent assay, abbreviated ELISA in biochemical literature. Figure Enzyme bound to antibody 2 can catalyze reactions that produce (a) colored or (b) fluorescent products. P.432

Figure P.432 Figure Fluorescence calibration curve for the selenium-containing product.

Figure Figure Enzyme-linked immunosorbent assay. P.432

Figure Figure Enzyme bound to antibody 2 can catalyze reactions that produce (a) colored or (b) fluorescent products. P.432