4. Examples

In a typical UV/VIS absorption experiment performed on a solution, one measures the absorbance <sup>A</sup>, which is related to the molar extinction coefficient <sup>ε</sup>ð Þ <sup>e</sup><sup>ν</sup> via Lambert-Beers law, <sup>A</sup> <sup>¼</sup> <sup>ε</sup>ð Þ <sup>e</sup><sup>ν</sup> Cml, where Cm and <sup>l</sup> are the molar concentration and the path length, respectively. From the application of the quantum theory, the molar extinction coefficient is found to be proportional to the spatial average of the

�D E

the excited electronic state and to the electronic ground state, respectively. Lið Þ <sup>e</sup><sup>ν</sup>

cal FWHM width of the order of magnitude as a vibrational quantum. A calculation requires that the electronic transition moment is Taylor expanded in the nuclear coordinates. Due to the superposition of the intensity of the individual transitions in Eq. (11), it follows that the vibrational fine structure in UV/VIS absorption spectra of larger molecules is not well resolved as also experienced from experiments. This is different in the RADIS spectra, where the narrow Raman bands enable the excitation profiles to be well separated experimentally (as illustrated in Figure 2). Since each excitation profile only has contributions from a single Raman-active vibration, the vibrational fine structure in the UV/ VIS absorption can be resolved. Thus, the amount of available information about the excited electronic molecular states is much larger in RADIS than in UV/VIS

It follows that while VRRS is mainly a vibrational spectroscopic technique, RADIS has more in common with electronic spectroscopy. It follows that each Raman-active vibration just plays the role of a "sensor" used to monitor the vibra-

Since the late 1980s, a very large amount of systematic resonance Raman studies on different metal complexes [28, 29] and different metal-porphyrins [12] including heme-proteins have been performed with the goal of determining their structure, their bio-functionality, and the conditions for aggregation. In these studies both the VRRS and RADIS including both excitation profiles and polarization dispersion have been applied. Recently polarization-resolved VRRS has been combined with dynamic light scattering to study among other things the aggregation of Arenicola marina extracellular hemoglobin, which is a macromolecule with 144 heme groups instead of four as in human hemoglobin [30]. As already mentioned one great advantage of applying Raman scattering is that the technique can be performed as reflection measurements without much sample preparation. Besides, the Raman signals can be obtained through glass and other sheets of protection. Thus, Raman studies can be performed as in vivo or in situ studies. We refer to the comprehensive Raman literature on these matters for details (e.g., see [31–36]). A complete RADIS experiment may be time-consuming or in some cases impossible to carry out due to the lack of excitation lasers with the proper wave number. It may also be time-consuming to determine the correct intensity variation when the excitation wave number is changed due to changes in the scattering conditions (laser intensity and focus, laser-induced degradation of the molecule, change of the fluorescence, etc.). The application of internal standards and other means have to be introduced in order to ensure the correct experimental conditions. However, in some cases of practical interest, it is in fact possible to extract valuable information without completing a full RADIS experiment (see Sections

<sup>3</sup>N�6jρgeð Þj <sup>Q</sup> <sup>0</sup>,0,0,…

<sup>3</sup>N�<sup>6</sup> are the vibrational quantum numbers referring to

th transition j i <sup>g</sup><sup>0</sup> ! ev<sup>e</sup>

� � � 2

avLið Þ <sup>e</sup><sup>ν</sup> (11)

with a typi-

i � � �

absolute square of the transition dipole moment:

Modern Spectroscopic Techniques and Applications

ve <sup>1</sup>, v<sup>e</sup> <sup>2</sup>, ve 3…ve

� �

tional fine structure in the UV/VIS absorption spectrum.

<sup>ε</sup>ð Þ <sup>e</sup><sup>ν</sup> <sup>∝</sup> <sup>∑</sup> <sup>v</sup><sup>e</sup> <sup>1</sup>, ve <sup>2</sup>, ve 3…v<sup>e</sup> 3N�6

> 1, v<sup>e</sup> 2, ve 3…ve

is a normalized lineshape function for the i

where ve � <sup>v</sup><sup>e</sup>

absorption.

4.2 and 4.3).

26

As has been shown, vibrational RS is exclusively a vibrational spectroscopic technique like IR and NIR. However, vibrational Raman spectroscopy performed under resonance conditions may be considered as either a vibrational spectroscopic technique or as an electronic spectroscopic technique, which of the two depends on the way the experiments are performed. Three examples are briefly discussed below. For more applications, the interested reader should consult the comprehensive Raman literature [31–36].

### 4.1 Example 1: perturbation of molecular symmetry

As demonstrated in [11], the non-commuting generator approach to molecular symmetry may be applied to calculate the structure of the state tensors. Figure 3 shows an example for two vibrations in point group D4<sup>h</sup>. The figure also demonstrates what happens when the symmetry is lowered so that the configuration is now described in point group D2<sup>h</sup>. Lowering of the symmetry may be a result of a chemical reaction or may be due to a perturbation of the configuration from the planar square to a planar rectangular shape. To the left in the figure, the state tensors in D4<sup>h</sup> for the Raman-active in-plane vibrations a1<sup>g</sup> , a2<sup>g</sup> , and b1<sup>g</sup> are shown, which are written in front of the tensors. The symmetries of the two components of the resonating, degenerate electronic state with Eu symmetry are written in the tensors on the positions, which correspond to the only nonvanishing elements. The plus and minus signs describe the numerical relations between the tensor elements. For the a1<sup>g</sup> vibration, it follows that S <sup>j</sup><sup>e</sup>0¼Eu,x<sup>i</sup> xx <sup>¼</sup> <sup>S</sup> j<sup>e</sup>0¼Eu, <sup>y</sup>i yy , while for the b1<sup>g</sup> vibration, we have that S <sup>j</sup><sup>e</sup>0¼Eu,x<sup>i</sup> xx ¼ �<sup>S</sup> j<sup>e</sup>0¼Eu, <sup>y</sup>i yy . The state tensor for the a2<sup>g</sup> vibration is antisymmetric, i.e., S j<sup>e</sup>0¼Eu, <sup>y</sup>i xy ¼ �S j<sup>e</sup>0¼Eu,xi yx . From the tensor structure, the DPR values can be calculated by using Eq. (5) and the relations given in [11] or [3]. The DPR values are written to the right side of the tensors and are seen to be constants. By correlating the symmetries of the two point groups, the symmetries of the vibrations are changed as follows: a1<sup>g</sup> ! ag , b1<sup>g</sup> ! b2<sup>g</sup> and a2<sup>g</sup> ! bg1. The state tensors for the ag , b1<sup>g</sup> and bg<sup>2</sup> vibrations in D2<sup>h</sup> are also shown in Figure 3. As before,

inside the tensors the symmetries which the intermediate states must have in order

#### Figure 3.

(Left) Changes of the state tensors, induced by perturbation of a square planar (D4<sup>h</sup>) molecular configuration into a rectangular planar (D2<sup>h</sup>) configuration. (Right) Most notably is the change of the constant DPR into DPR dispersion (red curve) for the b1<sup>g</sup>∧bg<sup>2</sup> vibrations (D2<sup>h</sup>).

to give rise to any Raman signal are written. Due to the lowering of the symmetry, there are no longer numerical relations between the tensor components. Considering, e.g., the totally symmetric vibration, we see that a resonating state must have either B3u, B2<sup>u</sup> or B1<sup>u</sup> symmetry and that, e.g., an electronic state with B3<sup>u</sup> symmetry gives rise to a tensor where only <sup>S</sup>je0¼B3u<sup>i</sup> xx 6¼ 0.

similar molecules, i.e., molecules where a number of identical vibrations can be identified in the vibrational signature of the molecules. The method has been applied in combination with polarized resolved fluorescence to study the stability of the Ruthenium-based dye N719 [37] and to study in vitro the stability of N719 and the adsorption and desorption processes of this complex to the TiO<sup>2</sup> substrate in

What Is Vibrational Raman Spectroscopy: A Vibrational or an Electronic Spectroscopic…

Recently the method has also been proposed as a possible noninvasive screening technique for revealing a content of carbon monoxide in fresh tuna fish or meat. Preliminary experimental results were presented at the Raman conference ICORS

The method is based on the presence of dispersive Raman modes combined with a small spectral difference between the visible absorption spectra of similar molecules. The idea is as follows: the resonance condition for a specific molecule in RRS depends, as we have seen above, on the difference in the wave number between the electronic absorption and the excitation laser. Due to the tensor property of resonance Raman scattering, the value of the DPR depends on this difference (polarization dispersion). Thus, a small spectral shift in the absorption will essentially be equivalent to a displacement of the polarization dispersion curve relative to the excitation wave number of the laser, as illustrated in Figure 5. The change of the DPR value at a specific wave number depends on the shape of the dispersion curve, which depends on the nature of the vibration and the Raman tensor. When the molecule has low or no symmetry, most Raman-active vibrations will be dispersive. Although the ideal symmetry of a chromophore is often high, which limits the number of dispersive modes, the real symmetry is frequently lowered due to perturbations of the chromophore, which opens up for dispersion. The heme group with the ideal symmetry D4<sup>h</sup> is an example. This means that in reality the appear-

In the modified atmosphere packaging of fresh fish and meat products, the products are frequently exposed to carbon monoxide. Due to the higher binding affinity of CO in comparison with O2, CO replaces O<sup>2</sup> in myoglobin in the muscle tissue with high affinity, which results in the cherry-red carboxy-myoglobin complex MbCO. Due to the red color and high stability of MbCO, the fish or meat products will appear to be more fresh and attractive for a longer time period than the unexposed products. In [40] a quantitative method for the determination of CO bound to myoglobin based on visible absorption spectroscopy has been developed. Although this method has a high accuracy, it requires taking a sample from the product followed by sample preparation before the absorption spectra can be measured. Figure 4 shows a fresh tuna beef sample together with the polarizationresolved RRS spectra measured on the sample without any sample preparation but measured before and after approximately 10 minutes of exposure to CO and exciting the sample with a solid-state 532 nm laser. The experimental DPR values estimated from six Raman modes are collected in Figure 5. Further experimental details including details on the data processing of the polarization-resolved RRS data are obtainable from the author. Figure 5 also shows, as an example, a simula-

tion of the DPR dispersion curves for the mode at 1450cm<sup>1</sup> present in the

using a fiber-coupled, 180° Raman microscope consisting of a modified

29

polarization-resolved RRS spectra and which has been assigned as a b1<sup>g</sup> mode. The spectral shift (color shift) due to the exposure with CO is ≈9nm equivalent to 275cm1, estimated from measuring the diffuse reflectance of the tuna beef sample before and after exposure with CO with a Lambda 900 spectrophotometer equipped with an integrating sphere. The polarization-resolved RRS data were collected by

OlympusBX60F5, a SpectraPro 2500i spectrograph (Acton) with 1200lines/mm

dye-sensitized solar cells [38]. We refer to this paper for details.

ance of Raman modes exhibiting dispersion is quite common.

2016 in Brazil [39] and are discussed in the following.

DOI: http://dx.doi.org/10.5772/intechopen.86838

For the asymmetric b2<sup>g</sup> vibration, the B3<sup>u</sup> electronic state would result in a tensor where only the component <sup>S</sup>je0¼B3u<sup>i</sup> zx 6¼ 0, whereas one vibrational excitation of this vibration gives a state with the symmetry b2<sup>g</sup>⊗B3<sup>u</sup> ¼ B1u, which is seen to correspond to the transposed state tensor, i.e., only <sup>S</sup>je1¼B1u<sup>i</sup> xz 6¼ 0. From the power expansion of the vibronic state tensor given in Eq. (7), it may be shown that the two tensor elements satisfy the relation: <sup>S</sup>je0<sup>i</sup> zx <sup>¼</sup> <sup>S</sup>je1<sup>i</sup> xz . A closer investigation shows that this relation is valid in general for asymmetric vibrations [11]. The Raman tensor is calculated by inserting the two state tensors in Eq. (3). The DPR is calculated by using Eq. (5) and the relations between the rotational invariants and the Raman tensor derived in [11]. By group theory, it can be shown that an external electronic perturbation with symmetry B1<sup>g</sup> (in D4<sup>h</sup>) would result in the considered shift in the molecular configuration. As shown in Figure 3, the consequence of the symmetrylowering perturbation of the molecular configuration is that the DPR now shows a characteristic symmetric polarization dispersion with maximum half ways between the energy positions of the two states je0i and je1i. The excitation profile is symmetric around the maximum of the DPR curve and has maxima at the positions of these states, i.e., at je0i and je1i. In the point group D4<sup>h</sup>, the state tensor for the a2<sup>g</sup> vibration is purely antisymmetric with a result that the DPR becomes infinity. After the perturbation, where a2<sup>g</sup> ! bg1, the b1<sup>g</sup> state tensor is seen to have the same structure as the tensor for the b2<sup>g</sup> mode, which means that the DPR dispersion curve and excitation profile also become similar. However, the energy of the state je1i will in general be different for different modes.

It follows that through the application of RADIS, it is possible to study small changes of the molecular configuration in excited electronic states and estimate the various molecular parameters influenced by these changes. As shown in numerous RRS papers on biomolecules, these structural changes, which are typically induced by minor changes in the environment of the molecule, can be studied in vivo, which is of course a major advantage [31–36].

#### 4.2 Example 2: noninvasive color detection using polarization dispersion

The color of a molecular species is associated with the properties of the electronic excited states of the molecule, and in large biomolecules, it is due the presence of a chromophore being typically a metal complex. The red color of the hemeproteins, which is due to the presence of the Fe-porphyrin complex, is a well-known example. A change in color may be due to a change of the molecular configuration (distortion, aggregation) or be a result of a chemical reaction. By monitoring the color change before and after a chemical reaction, the substance concentration in solutions can be determined from the absorbance measured by a UV/VIS spectrophotometer or in the case of a solid by applying the spectrophotometer with an integrating sphere. In the literature several color detection methods have been developed for the detection of various substances. A book on color detection is in the process of publication by IntechOpen and will be published later in 2019.

In this section a reflection technique with high spectral resolution is discussed. The technique is suitable for the detection of minor color differences between
