授課教師: Professor 吳逸謨 教授 Warning: Copyrighted by textbook publisher. Do not use outside class. Principles of Instrumental Analysis Section III – Molecular Spectroscopy Chapter 13 Introduction to Ultraviolet-Visible Absorption Spectrometry + Chapter 14 Applications of Ultraviolet-Visible Absorption Spectrometry Reference: p. 423, Visible and UV spectra, in “Organic Chemistry” textbook – Solomons, 3rd Ed] 1 Molecular Spectroscopy Ultraviolet–visible (UV-Vis) absorption vs. fluorescence Spectroscopy Ultraviolet–visible spectroscopy or ultraviolet-visible spectrophotometry (UV-Vis or UV/Vis) refers to absorption spectroscopy or reflectance spectroscopy in the ultraviolet-visible spectral region. This means it uses light in the visible and adjacent (near-UV and near- infrared [NIR]) ranges. The absorption or reflectance in the visible range directly affects the perceived color of the chemicals involved. In this region of the electromagnetic spectrum, molecules undergo electronic transitions. Fluorescence spectroscopy is based on molecular emission: The “UV-Vis” technique is complementary to fluorescence spectroscopy, in that fluorescence deals with transitions from the excited state to the ground state, while UV-Vis absorption measures transitions from the ground state to the excited state.[1] 2 (2007/3) UV-Vis Spec 儀器 -實驗課 3 FIGURE 6-3 Regions of the electromagnetic spectrum. (For UV-Vis, λ = 100~500 nm, 500~1000 nm) 4 Ch6 An Introduction to Spectrometric Methods P.135 Apendix: Chap. 7E - Radiation Transducers p. 191 Read the texts in Chap. 7 7E-2 Photon Transducers for optical spectroscopy – Barrier-Layer Photovoltaic Cells – Vacuum Phototubes – Photomultiplier tubes (PMT), picture on p. 195 – Silicon Photodiodes (Fig. 7-32) [semiconductor type] [A silicon photodiode transducer consists of a reverse- biased p-n junction.] (The latter two types of transducers are more commonly used in UV-Vis.) – We will discuss more details later. 5 Ultraviolet–visible absorption Molecules containing π-electrons or non-bonding electrons (n-electrons) [will be explained next page] can absorb the energy in the form of ultraviolet or visible light to excite these electrons to higher anti-bonding molecular orbitals.[ UV/Vis spectroscopy is routinely used in analytical chemistry for the quantitative determination of different analytes, such as transition metal ions, highly conjugated organic compounds, and biological macromolecules. Spectroscopic analysis is commonly carried out in solutions but solids and gases may also be studied. 1. Solutions of transition metal ions can be colored (i.e., absorb visible light) because d electrons within the metal atoms can be excited from one electronic state to another. 2. Organic compounds, especially those with a high degree of conjugation, also absorb light in the UV or visible regions of the electromagnetic spectrum. The solvents for these determinations are often water for water-soluble compounds, or ethanol for organic-soluble compounds. (Organic solvents may have significant UV absorption; not all solvents are suitable for use in UV spectroscopy.) 3. While charge transfer complexes also give rise to colours, the colours are often6 too intense to be used for quantitative measurement [Note: pi electrons] The Greek letter π in their name refers to p orbitals, since the orbital symmetry of the pi bond is the same as that of the p orbital when seen down the bond axis. P orbitals often engage in this sort of bonding. D-orbitals also engage in pi bonding, and form part of the basis for metal-metal multiple bonding. Electrons in pi bonds are sometimes referred to as pi electrons. -Molecular fragments joined by a pi bond cannot rotate about that bond without breaking the pi bond, because rotation involves destroying the parallel orientation of the constituent p orbitals. A typical double bond consists of one sigma bond and one pi bond; for example, the C=C double bond in ethylene (CH2=CH2). A typical triple bond, for example in acetylene (CHCH), consists of one sigma bond and two pi bonds in two mutually perpendicular planes containing the bond axis. Two pi bonds (π) are the maximum that can exist between a given pair of atoms. 7 Note: non-bonding orbitals/electrons A non-bonding orbital, also known as non-bonding molecular orbital (NBMO), is a molecular orbital whose occupation by electrons neither increases nor decreases the bond order between the involved atoms. A non-bonding orbital with electrons would commonly be a HOMO (highest occupied molecular orbital). Electrons in molecular non-bonding orbitals can undergo electron transitions such as n→σ* or n→π* transitions [where * indicates “anti-bonding”]. For example, n→π* transitions can be seen in ultraviolet-visible spectroscopy of compounds with carbonyl groups (C=O), although absorbance is fairly weak.[2] The following molecular electronic transitions exist: σ → σ* π → π* n → σ* n → π* aromatic π → aromatic π* * : σ → σ Electrons occupying a HOMO of a sigma bond can get excited to the 8 LUMO of that bond. Sigma bonds (σ bond) : - Ref. to your organic chem. Sigma bonds (σ bond) are the strongest type of covalent bonds due to the direct overlap of orbitals, and the electrons in these bonds are sometimes referred to as sigma electrons.[3] Sigma bonds are the strongest type of covalent bonds due to the direct overlap of orbitals, and the electrons in these bonds are sometimes referred to as “sigma electrons”. Typically, a single bond is a sigma bond, while a multiple bond is composed of one sigma bond together with pi or other bonds. A double bond has one sigma plus one pi bond, and a triple bond has one sigma plus two pi bonds. 9 When the molecule is in the ground state, both electrons are paired in the lower-energy bonding orbital – this is the Highest Occupied Molecular Orbital (HOMO). The antibonding σ* orbital, in turn, is the Lowest Unoccupied Molecular Orbital (LUMO). This is referred to as a σ - σ* transition. ΔE for this electronic transition is 258 kcal/mol, corresponding to light with a wavelength of 111 nm (in UV range !). 10 σ σ* transition absorption is usually very strong (but in UV region)! Where UV-vis spectroscopy becomes useful to most organic and biological chemists is in the study of molecules with conjugated pi systems. [Conjugation: double-single-double-single, etc.] In these groups, the energy gap for π -π* transitions is smaller than for isolated double bonds, and thus the wavelength absorbed is longer. (in Visible region) Molecules or parts of molecules that absorb light strongly in the UV-Vis region are called chromophores11 . Introduction p.336 • Molecular absorption spectroscopy is based on the measurement of the transmittance (T), or the absorbance A of solutions contained in transparent cells having a path length of b centimeters. • Ordinarily, the concentration (c) of an absorbing analyte is linearly related to absorbance (A) as given by Beer's law: P A = −log T = log 0 = εbc (13-1) P All of the variables in this equation are defined in Table 13-1 (next page). 12 TABLE 13-1 Important Terms and Symbols for Absorption Measurements 13 Ch13 An Introduction to Ultraviolet-Visible Molecular Absorption Spectrometry P.337 13A. MEASUREMENT OF TRANSMITTANCE AND ABSORBANCE p.336 • Transmittance and absorbance, as defined in Table 13-1, cannot normally be measured in the laboratory because the analyte solution must be held in a transparent container, or cell. • As shown in Figure 13-1 (next page), reflection occurs at the two air-wall interfaces as well as at the two wall-solution interfaces. • The resulting beam attenuation (decay) is substantial, as we demonstrated in Example 6-2 (Chap 6), where it was shown that about 8.5% of a beam of yellow light is lost by reflection in passing through a glass cell containing water. • In addition, attenuation of a beam may occur as a result of scattering by large molecules and sometimes from absorption by the container walls. 14 FIGURE 13-1 Reflection and scattering losses with a solution contained in a typical glass cell. Losses by reflection can occur at all the boundaries that separate the different materials. In this example, the light passes through the air-glass, glass-solution, solution-glass, and glass-air interfaces. 15 Ch13 An Introduction to Ultraviolet-Visible Molecular Absorption Spectrometry P.337 13A. MEASUREMENT OF TRANSMITTANCE AND ABSORBANCE • To compensate for these effects, the power of the beam transmitted by the analyte solution is usually compared with the power of the beam transmitted by an identical cell containing only solvent. • An experimental transmittance and absorbance that closely approximate the true transmittance and absorbance are then obtained with the equations P P (13-2) T = solution ≈ Psolvent P0 P P (13-3) A = log solvent ≈ log 0 Psolution P The terms P0 and P, as used in the remainder of this book, refer to the power of radiation after it has passed through cells containing the solvent and the analyte solutions, respectively. 16 13B BEER’S LAW Equation 13-1 represents Beer's law (A = εbc). This relationship can be rationalized as follows. • Consider the block of absorbing matter (solid, liquid, or gas) shown in Figure 13-2 (below). FIGURE 13-2 Radiation of initial radiant power P0 is attenuated to transmitted power P by a solution containing c moles per liter of absorbing solution with a path length of b centimeters. 17 Cont’d – differential derivation of Beer’s law • A beam of parallel monochromatic radiation with power P0 strikes the block perpendicular to a surface. After passing through a length b of the material, which contains n absorbing atoms, ions, or molecules, its power is decreased to P as a result of absorption. • Consider now a cross section of the block having an area S and an infinitesimal thickness dx. • Within this section there are dn absorbing particles; we can imagine a surface associated with each particle at which photon capture will occur.