Chapter II Experimental
II- 3. Experimental system
The experimental system used in this study is introduced in this section. A block diagram of the measurement system is shown in Figure II-3. A ceramic IR emitter (JASCO) was used as the light source. A pair of elliptical mirrors was used to focus the infrared light onto the sample. An optical chopper (Stanford Research System Inc., SR540) was placed at the co-focus of two elliptical mirrors to generate a modulation of 250 Hz to the light source. The light source and the sample cell were put on each of the other foci. A function generator (IWATSU, FG-330) and a power amplifier (NF corp., NF-4010) were used to generate a 25 kHz sinusoidal AC voltage (~70V0-p), which was applied across the sample cell ~6 µm thick, producing an electric field of ~107 Vm−1 inside the cell.
A dispersive IR spectrometer (JASCO, TRIR-1000, Littrow mounting) was used for the measurement, whose focal length is 200 mm and the F number is 5. Three gratings are available in this spectrometer; their line density is 300, 120 and 60 mm−1, respectively. In the present study, the grating with a line density of 120 mm−1 was used. A liquid nitrogen-cooled HgCdTe (MCT) detector (New England Research Center, MPP12-2-J3) was used for mid-IR signal detection.
The AC coupling scheme is a technique to probe a small AC signal buried in large direct current (DC) background [Figure II-4(a)]. Typically, the AC (intensity change induced by an external field) and DC (intensity of the IR light) voltages generated by the detector in the IR electroabsorption spectrometer are 10−8 V and 10−1 V respectively. In the measurement, the MCT detector is AC-coupled to a low-noise preamplifier, which removes the DC background so that the output of the preamplifier contains only the AC component [Figure II-4(b)]. The AC component is amplified once again [Figure II-4(c)] by the main amplifier (NF Corp., NF-5307, gain=1~1000 variable), and then fed to a lock-in amplifier (Stanford Research System, SR844). Note that the signal we detect is proportional to F2 (second-order Stark effect), and hence it appears at 50 kHz.
The optical chopper and the oscilloscope were used for the measurement of the DC component, i.e., IR absorption spectrum [Figure II-4(d)]. In single electroabsorption experiment, three spectra are measured; (1) the spectrum of the light source (I0), (2) the spectrum of the light intensity that is passed through the sample without external electric field modulation (I), and (3) the spectrum of the intensity change induced by external electric field (∆I). The absorption spectrum of the sample is calculated from (1) and (2) as:
0
log( I )
A= − I (II-9) The intensity difference spectrum ∆I is converted to absorbance change (∆A) by:
and the IR electroabsorption spectrum are derived.
The combination of a dispersive IR spectrometer and the AC coupling method affords detection the absorbance change (∆A) as small as 10−7, which is hard to be reached by the conventional FT-IR method [25, 26].
FT-IR spectra shown in chapter IV were measured with JASCO FT-IR-6100 spectrometer using a sample cell composed of two CaF2 windows and a lead spacer of 100 µm thick. Resolution was 4 cm−1, and 64 runs were averaged for each FT-IR spectrum.
Figure II-1. Home-made sample cell for IR electroabsorption measurements. A, A’: brass cell holders, B, B’: Si windows/electrodes, C: PET film spacer [24].
Figure II-2. RC circuit equivalent to the sample cell. R1 is the resistance between A and B, R2
the resistance between A’ and B’, and Cc the capacitance of the capacitor C [24].
Figure II-3. Experimental setup of infrared electroabsorption spectroscopy [24].
Figure II-4. Schema of the AC coupled amplification method
Chapter III
Theoretical
III-1. Introduction
The theoretical background and the formulations of several molecular responses to an external field observed in infrared electroabsorption (∆A) spectra are introduced in this chapter. Three molecular responses are considered here, namely, the orientational polarization, the electronic polarization, and the equilibrium change [17, 24]. The ∆A signal originates from the changes in height, peak position, and width of an IR absorption band, and those changes appear in the ∆A spectra as the zeroth [Figure III-1(b)], the first [Figure III-1(c)], and the second [Figure III-1(d)] derivative shapes of the absorption band [Figure III-1(a)], respectively. The origins of these contribution seen in the ∆A spectrum are discussed.
III-2. Orientational polarization
When an external electric field is applied, liquid polar molecules having permanent dipole moment µp will orientate themselves so that their permanent dipole moment aligns parallel to the applied electric field [20, 24, 27]. It is known as an orientational polarization.
The amplitude of the orientational polarization is a function of the permanent dipole moment of molecules. The orientational anisotropy induced in the sample gives rise to the absorbance change (∆A).
In the following, the orientational polarization signal is formulated for (a) normally incident non-polarized IR light and (b) p-polarized IR light with tilt incidence, respectively.
(a) Normally incident non-polarized IR light
The orientational polarization signal ∆A can be derived starting from Beer-Lambert law:
2 2 2
where ε is the molar extinction coefficient, c the sample concentration (mol dm-3), l the path length (cm) of the sample, K a constant, the wavenumber (cm-1), and e a unit vector designating the direction of the electric field of the incident light. We set the molecule-fixed
coordinate system such that the z-axis coincides with the direction of the applied electric field and the propagation direction of the IR light as shown in Figures III-2 and III-3. The angles θ, φ, and ϕ determine the orientations of the dipole moment µp and the transition moment µT.
A spatial distribution function f (θ ), and the square of the scalar product of the transition moment and the unit vector (e · µT)2 are two integrands to be obtained explicitly. The distribution function
f (θ ) is proportional to the probability of finding the dipole moment µp in the direction θ with respect to the applied electric field F.
Using the coordinate system in Figures III-2 and III-3, we have
The number of molecules that have energy E is proportional to exp( )
B
E
−k T , and the energy E due to the dipolar interaction is given by
Thus the distribution function f (θ) becomes
p cos
Here C is a normalization factor, T is the temperature, kB is the Boltzmann constant, and F is the internal (not external) field strength. The parameter γ reflects the extent of the electrostatic interaction and in most cases plays a central role in the IREA spectroscopy of ambient polar liquid/solution.
The normalization factor C is determined by the condition
In the presence of the electric field, f (θ ) becomes from Eqs. III-4 and III-6 1 The scalar product of µT and e can be calculated as follows. The electric field vector e, of the incident light lies in the xy-plane, and a projection of µT onto the xy-plane is related to (e · µµµµT)2. µT is thus expressed as
T T
cos cos cos sin sin sin sin cos sin cos sin cos cos sin cos sin sin sin sin cos
sin cos sin cos cos
0≤ ψ ≤2π (1/2, 1/2, and 1, respectively). In the absence of the electric field, the absorbance Aoff for anα = 0° vibrational mode is calculated from Eqs.III-1, III-8, and III-11 as
Similarly, substitution of Eqs. III-7 and III-11 into Eq. III-1 results in the absorbance for the α=0° mode when the electric field is turned on
By expanding the exponential functions and retaining terms up to third-order in γ, we have on 22 2
6 T
A µ
∝γ ⋅
+ (III-14) To confirm that this approximation is valid, suppose 50 V is applied across liquid acetone 5 µm thick. The electric field strength = 1 × 10 V m . For simplicity, we do not consider the local-field correction. Using the dipole moment of acetone, µP = 2.7 D (1 D = 3.33564 × 10-30 C m), we obtain γ = 0.02, for which γ << 1 holds. The absorbance change caused by the Making use of Eqs. III-1, III-7, III-8, and III-17, we end up with the absorbance change ratio of the form
Generalization of Eqs. III-15 and III-18 to an arbitrary angle α is straightforward. The absorbance change for angle α can be decomposed into its parallel (α=0°) and perpendicular (α=90°) components as follows: Substitution of Eqs. III-15 and III-18 into this equation yields the following expression for the orientational polarization signal probed with the normal incidence
Again γ2 is considered exceedingly smaller than 6 in the present study, so the first term in the denominator of Eq.III-20 is safely neglected. Therefore we are left with
non-polarized light on the xy-plane is parallel to the sample cell. In other words, χ is equal to 90°, where χis the angle between the applied electric field F and the electric-field vector e of the incoming IR light (see Fig. III-4). When p-polarized light whose electric field vector e has only x-component is incident upon the sample with angle χ, the absorbance change ratio is
shows the 1-3cos2χ dependence and that the signal disappears if χ = 54.7° or α = 54.7°. Another important point is that the orientational ∆A spectrum is proportional to the absorption spectrum A, indicating that it is manifested as the zeroth derivative shape of the absorption spectrum. Furthermore, Eq. III-22 can be used to determine the dipole moment [14, 17, 19] or angle α experimentally [20].
III-3. Electronic polarization
An absorbance change also arises from electronic polarization, which is the change by an externally applied field in molecule’s electronic properties such as the dipole moment and the polarizability. A general theory of the electronic polarization signal was established by Liptay et al [2, 4]. For a mobile molecule in solution, the electronic polarization signal is given by
Here ∆µµµµ and ∆αααα denote the changes in permanent dipole moment and polarizability tensor between the vibrational ground state (g) and an excited state (e), respectively, i.e., ∆µµµµ = µe – µg and ∆αααα = αe – αg. mˆ is a unit vector in the direction of the transition dipole moment. αgm
and ∆αm are the components of the polarizability change along the direction of the transition dipole moment, i.e., αgm=mˆ ⋅αg⋅mˆ and ∆αm =mˆ ⋅∆α⋅m . A and B are the ˆ transition polarizability and transition hyperpolarizability, respectively, exhibiting the field dependence of the transition moment: m(F) = m + A F + F B F . ⋅ ⋅ ⋅
As described earlier, the zeroth-derivative component represents the intensity change of the absorption spectrum. Note that the first term in the square brackets in Eq. III-24 corresponds to the orientational polarization contribution, which we already derived above.
The first-derivative component depends on both ∆µ and ∆α, and is responsible for the stabilized species. A good instance for such an equilibrium shift is found in the trans/gauche isomerism of 1,2-dichloroethane [14]. The gauche conformer of 1,2-dichloroethane has a permanent dipole moment, and it will get stabilized by an applied electric field. On the other hand, the trans conformer is non-polar due to the presence of a center of symmetry, and it will not benefit stabilization via interaction with the applied electric field. Thus, the equilibrium shifts to the more stabilized gauche conformer. The IR absorption intensity of the gauche conformer is expected to increase, whereas that of the trans conformer is expected to decrease.
Note that because the equilibrium shift signal changes the intensity of the absorption band, a zeroth-derivative shape will be observed in the ∆A spectrum. The spectral shape of the equilibrium change signal is the same as the orientational polarization signal, but the equilibrium shift signal is χ-independent. So, one can differentiate these two signals by studying the χ-dependent ∆A spectra.
Figure III-1. Spectral features observed in ∆A spectrum. (a) IR absorption band. (b) the zeroth derivative shape, (c) the first derivative shape, and (d) the second derivative shape of the IR absorption band.
A
Wavenumber/cm -1
∆ A b so rb an ce
(a)
(b) (c)
(d)
Figure III-2. Coordinate system used in the derivation of the orientational polarization signal.
α is the angle between µµµµp and µµµµT. This figure corresponds to the α = 0° case.
Figure III-3. Coordinate system used in the derivation of the orientational polarization signal.
α is the angle between µµµµp and µµµµT. This figure corresponds to the α = 90° case.
Figure III-4. Angle χ between the applied electric field F and the electric field vector e of the IR probe light [1]. Note that χ can be determined by applying Snell’s law
[
n1⋅sinθ1= ⋅n2 sin(90−χ)]
.Figure III-5. (a) Electric field effect in the vibrational ground state and excited states. (b) An absorption peak shifts to higher wavenumber (purple to green), and the resulting ∆A spectrum (red) exhibits a first derivative shape.
(a)
(b)
∆ A
Wavenumber/cm -1
A b s
Figure III-6. (a) Electric field effect in the vibrational ground state and excited state. (b) An absorption peak shifts to lower wavenumber (purple to green), and the resulting ∆A spectrum (red) shows a first derivative shape.
(a)
(b)
∆ A
Wavenumber/cm -1
A b s
Figure III-7. (a) Electric field effect on the distribution of the transition frequency from the vibrational ground to an excited state. (b) An absorption peak is broadened (purple to green), and the resulting ∆A spectrum (red) shows a second derivative shape.
∆ A
Wavenumber/cm -1
A b s
(a)
(b)
Chapter IV
Infrared electroabsorption spectroscopic
study of 4(3H)-pyrimidinone in p-dioxane
IV-1. Introduction
The structure and the tautomerism of purine and pyrimidine nucleobases have been the focus of numerous experimental and theoretical studies [6, 27, 30-43]. This interest stems from their relevance to the important biological functions. For instance, the fidelity of DNA replication and transcription as well as the stabilization of secondary and tertiary structures of the nucleic acids are governed by specific hydrogen bonds between proper tautomeric forms of the complementary bases [44]. Vibrational spectroscopy is a powerful technique to investigate nucleobases and related compounds [38, 39, 45]. Changes in tautomerism, molecular structure, and hydrogen bonding state of the bases can be revealed by vibrational spectroscopy.
4(3H)-Pyrimidinone (4(3H)-Pyr) (Figure I-1) is a suitable model compound for studying nucleobases [40]. 4(3H)-Pyr exhibits a structure similar to that of the pyrimidine ring of the bases. Its keto and enol forms resemble the six-membered parts of the “normal” amino-oxo and “mutagenic” amino-hydroxy forms of guanine, respectively (see Figure I-1.). Knowledge of the tautomerism and structure of 4(3H)Pyr is therefore of considerable importance and is useful for gaining insight into the actual nucleobase systems. Previous studies suggested that the keto and the enol forms of 4(3H)-Pyr coexist in the gas-phase and low-temperature matrices in comparable amounts [35, 46]. In contrast, the keto form predominates in the condensed phase [46, 47]. A number of combined experimental and theoretical studies have been performed on 4(3H)-Pyr to predict their tautomeric stabilities and to establish vibrational assignments [35, 36]. Most studies of 4(3H)-Pyr have so far been performed in gas-phase or matrix-isolated conditions, in which little intermolecular interactions is present. In fact, these interactions play a central role in the physicochemical properties of the nucleobases. Scientific studies of 4(3H)-Pyr in the solution phase are limited mainly due to the poor solubility of this compound in less polar organic solvent. Aida et al. performed an IR spectroscopic study of 4(3H)-Pyr in chloroform and carbon tetrachloride solutions in combined with ab inito
calculations. They reported that the keto form of 4(3H)-Pyr monomer in the solution phase is dominant with an estimated tautomeric equilibrium constant K = 0.012, which was obtained from the absorbance ratio of the OH and NH stretch bands of the enol and keto forms, respectively. Furthermore, they discuss the molecular structures of keto-keto dimers coexisting with monomer [40].
In this study, we try to shed more light on 4(3H)-Pyr by means of infrared electroabsorption (IREA) spectroscopy. IREA spectroscopy can probe the molecular responses to an applied electric field as changes in IR absorption intensities. It sharply reflects the molecular structure in terms of the dipole moment [15, 17, 24] and the thermodynamic parameters associated with chemical equilibria [14, 18]. To be more specific, if we can extract orientational polarization signal (Eq. III-22) from angle χ dependent IREA spectra (χ is the angle between the direction of the applied electric field and electric field vector of the IR probe light), we will be able to obtain the information about permanent dipole moment µp or angle α (the angle between vibrational transition moment and dipole moment) [20] of the monomer and dimer of 4(3H)-Pyr simultaneously. Another important issue we want to address is the equilibrium change (see Chapter III-4.) between the monomer and the dimer of 4(3H)-Pyr. The monomer has a permanent dipole moment of 2.72 Debye in p-dioxane [48], whereas the dipole moment of the dimer is zero if a planar base pair-like hydrogen bonded structure is assumed (Figure I-1). We expect that the equilibrium between the monomer and the dimer would shift to the monomer because it is polae and can be stabilized via dipolar interaction with an applied electric field.
In this study we have successfully obtained the IREA spectra of 4(3H)-Pyr at various angles. To our knowledge, the responses of a model compound of the pyrimidine base to an electric field have been measured for the first time at room-temperature. The observed IREA spectra show unusually small χ dependence. A further ananlysis using singular value decomposition (SVD) confirms that the χ-dependent component is indeed predominant in
4(3H)-Pyr. As opposed to our expectation, we do not see equilibrium change signal (i.e., a positive ∆A signal for the monomer, and a negative ∆A signal for the dimer). On the basis of results of a least-squares fitting analysis of the decomposed components, we discuss these findings in terms of the angle α and the contribution of electronic polarization.
IV-2. Methodology IV-2-1. Experiment
The experimental apparatus and the sample cell used in this study have been described in Chapter II. To measure angle χ dependent ∆A spectra, a wire-grid IR polarizer (THORLABS, WP25H-C) was used to obtain p-polarized light (with respect to the silicon window).
Fourteen scans were averaged for each ∆A spectrum, which required about two hours. The spectral resolution of IREA measurements was set to be 12 cm-1 to achieve higher signal-to-noise ratio (S/N). Care was taken in the sample flow through the cell in order to prevent heat accumulation and solvent evaporation.
FT-IR spectra were recorded on a JASCO FT-IR-6100 spectrometer using a sample cell composed of two CaF2 windows and a lead spacer (100 µm thick). Resolution of 4 cm-1 was used, and 64 scans were averaged for FT-IR measurements. All experiments were performed at room temperature (293 K).
p-Dioxane (purity > 99.5%) was commercially obtained from J.T. Baker, and 4(3H)-pyrimidinone (purity > 98%) from Sigma-Aldrich. These chemicals were used as received. 4A molecular sieves were used to remove residual water in p-dioxane, followed by filtration (pore size: 0.2 µm) to remove dusts or unwanted particles in the stock solution.
To prepare the sample solution for IREA measurements, 0.0589g of 4(3H)-Pyr was weighted and added to a 10ml volumetric flask, which was then filled up with p-dioxane. An ultrasonicator was then used to dissolve 4(3H)-Pyr in the solvent (p-dioxane). The concentration of the sample solution for the IREA measurement was 60 mM.
IV-2-2. Analysis: Singular value decomposition
Singular value decomposition (SVD) is an important factorization method of a complex matrix. This technique can be employed in principal component analysis; thus it has found many applications in chemometrics and spectral analysis [14, 49, 50].
SVD is a mathematical technique to decompose an arbitrary matrix M (m×n) into a product of three matrices, U, W, and V as
M=UWV (IV-1) T where U (m×n) and V (n×n) are normalized orthogonal matrices, and W (n×n) is a diagonal matrix, whose diagonal matrix elements are called singular values. In the present study, a set of χ dependent infrared electroabsorption spectra can be regarded as the matrix M.
M is decomposed into the product of U (the angle χ-dependence), W (the singular value matrix) and V (the intrinsic spectral components) :
χ-dependence and the ith row of V gives the corresponding spectrum. The contribution of the ith component to the matrix M is expressed by the value of wi, the ith diagonal element of the matrix W. Components of U and V associated with small singular values can be neglected because their contribution to the matrix M is negligibly small. Then the matrix M is reproduced by considering only those components of U and V matrices that are accompanied by large singular values. The number of large singular values gives the number of independent spectral components that show different χ-dependences.
In our experiment, two components are to be considered: one is the χ-independent (constant) component and the other is the χ-dependent component showing the 1−3cos2χ
dependence. Therefore, we confine our attention to the two largest singular values. In this case, Eq. IV-1 can be simplified to
1 2 1 1 mathematical operation and hence there are no physical meaning attached to vectors u and v as they stand. We need to reconstruct these vectors by taking a proper linear combination (represented by a 2 × 2 transformation matrix K) based on the model for the χ-dependence.
Accordingly, the Eq. IV-3 is rewritten to
(
1 2)
1 1( )
and ub correctly represent the two angle χ-dependences of ∆A spectra, namely, constant and 1-3cos2χ. Using the transformation matrix K thus determined, vectors va and vb are obtained which represent physically meaningful spectral components.IV-3. Results and discussion
IV-3-1. Concentration dependent FT-IR spectra
First, to confirm the presence of both monomer and the dimer of 4(3H)-Pyr in our p-dioxane solution, we measured concentration-dependent FT-IR spectra of 4(3H)-Pyr in p-dioxane in the 1740–1640 cm−1 region at 12 different concentrations (5, 10, 15, 20, 25, 31, 35, 40, 45, 48.95, 55, and 62mM) [Figure IV-1(a)].
Two IR bands are observed in this wavenumber region, and the intensities of both bands
Two IR bands are observed in this wavenumber region, and the intensities of both bands