**3. Nonpolar amino acids**

In the discussion, we will separate the discussion according to the polarity of the lateral groups of the amino acids. We begin with the apolar amino acids. This group is composed of the following amino acids: glycine, alanine, valine, leucine, isoleucine, phenylalanine, methionine, tryptophan, and proline.

*Glycine* is the simplest amino acid, having as the radical a hydrogen atom. Although the molecular simplicity, glycine can crystallize in three different forms at atmospheric pressure and room temperature. The α-form shows a *P*21*/n* monoclinic structure where hydrogen bonds appear in double antiparallel layers; the β-form shows a *P*21 monoclinic structure and individual parallel layers are linked by hydrogen bonds in a three-dimensional network; the γ-form shows a *P*31 trigonal structure with zwitterions forming helixes linked in a three-dimensional network [6]. The problem is that the three forms of glycine generally crystallize simultaneously from the same solution. In order to obtain crystals of the γ-form, it is necessary to prepare aqueous solution containing small seeds of the γ-glycine; the metastable β-form can be obtained by a mixture of water and acetic acid having the β-glycine as a seed, among other possibilities [6]. The three forms were studied under high pressure or under temperature variation. From these studies different results were obtained. The α-glycine under pressure is stable up to 23 GPa [7]. The β polymorphic form of glycine

**Figure 1.** Polarized Raman spectra of glycine (α-form) for two scattering geometries in the high wavenumber region of the spectrum.

presents a phase transition at 0.76 GPa [8]; the new phase, β' is noted by the jumps and kinks at the curve of wavenumber vs. pressure as observed in experiment of Raman spectroscopy. Although the γ-form is the most stable among the three atmospheric pressure possibilities, it is possible to observe at ~ 440 K the γ → α phase transition [9]. Also, under pressure, a phase transition from the γ-form to a new δ-form is verified, starting at ~ 2.7 GPa [10]. Additionally, when the δ-form is decompressed, between 0.95 and 0.2 GPa, a new polymorph is obtained, the ζ-glycine [11]. In relation to the Raman spectroscopy, as it is expected, each of the different polymorphs exhibits different spectrum. To give an example, **Figure 1** shows the Raman spectra of the (predominantly) α-form of glycine in the high wavenumber region for two scattering geometries, Z(YY)Z and Z(XX)Z. In this figure, it is possible to observe bands associated with symmetric stretching of CH2, νS(CH2), at 2971 cm −1; antisymmetric stretching of CH2, νA(CH2), at 3006 cm−1, and stretching of NH, ν(NH), at 3145 cm−1. Depending on the kind of polymorph, the stretching of NH, in particular, is observed at different wavenumbers and with different intensities.

**3. Nonpolar amino acids**

204 Raman Spectroscopy and Applications

tryptophan, and proline.

the spectrum.

In the discussion, we will separate the discussion according to the polarity of the lateral groups of the amino acids. We begin with the apolar amino acids. This group is composed of the following amino acids: glycine, alanine, valine, leucine, isoleucine, phenylalanine, methionine,

*Glycine* is the simplest amino acid, having as the radical a hydrogen atom. Although the molecular simplicity, glycine can crystallize in three different forms at atmospheric pressure and room temperature. The α-form shows a *P*21*/n* monoclinic structure where hydrogen bonds appear in double antiparallel layers; the β-form shows a *P*21 monoclinic structure and individual parallel layers are linked by hydrogen bonds in a three-dimensional network; the γ-form shows a *P*31 trigonal structure with zwitterions forming helixes linked in a three-dimensional network [6]. The problem is that the three forms of glycine generally crystallize simultaneously from the same solution. In order to obtain crystals of the γ-form, it is necessary to prepare aqueous solution containing small seeds of the γ-glycine; the metastable β-form can be obtained by a mixture of water and acetic acid having the β-glycine as a seed, among other possibilities [6]. The three forms were studied under high pressure or under temperature variation. From these studies different results were obtained. The α-glycine under pressure is stable up to 23 GPa [7]. The β polymorphic form of glycine

**Figure 1.** Polarized Raman spectra of glycine (α-form) for two scattering geometries in the high wavenumber region of

Although the chirality itself is a theme of great relevance, in the present chapter we discuss only the properties of the l-chiral sister of amino acid crystals (those present in the proteins) and do not furnish further information about the phenomenon. The simplest chiral amino acid is *alanine*. It crystallizes in an orthorhombic *P*212121 space group with four molecules per unit cell. The Raman spectrum of l-alanine was studied in a series of papers throughout the years [12–14]. Two studies have furnished the assignment of the normal modes through the analysis of deuterated analogs [13, 14]. Under temperature variation, l-alanine seems to be stable in the interval of 20–300 K, as indicated by several studies, including both infrared spectroscopy [15] and Raman scattering [16] studies. **Figure 2** shows the Raman spectrum of l-alanine recorded at room temperature in the interval of 300–700 cm−1. This spectral range presents two important vibrations, e.g., the torsion of NH3 + , *τ*(NH3), and the rocking of CO2 − unit, *r*(CO2). The study of the *τ*(NH3) mode as a function of pressure showed that its wavenumber presents *dw*/*dP* < 0, in contrast with other amino acid crystals, such as l-threonine and taurine. Such fact was interpreted as a consequence of the behavior of hydrogen bonds that due to the short dimension should move away the N, H, and O atoms (that participate of a specific hydrogen bond) from a straight line, instead of to approximate the N and O atoms [17]. The discussion about the effect of pressure on l-alanine crystal is also of relevance. A first work indicated that l-alanine crystal undergoes a phase transition at ~ 2.2 GPa [18]. Additionally, the same work showed that the bands appearing at 42 and 48 cm−1 change intensity above the pressure where the supposed phase transition occurs [18] and the variation of intensity of the two bands was also noted by Tumanov et al. [19]. However, two studies point that instead of a phase transition, in fact, at 2.2 GPa, only the inversion of the **a** and **c** crystallographic axes occurs [19, 20]. Because at ~2.2 GPa, the two crystallographic axes had the same dimensions, technically, at this specific pressure, the structure should be tetragonal, but above the critical pressure value, the structure continues with its original orthorhombic structure. Therefore, l-alanine is an interesting example where the union of both Raman spectroscopy and X-ray diffraction furnished a wide picture about the behavior of the system.

**Figure 2.** Raman spectrum of l-alanine in the spectral range from 300 to 700 cm−1. Some important modes related to hydrogen bonds are shown, as rocking of CO2 − , r(CO2 − ), and torsion of NH3 + , *τ*(NH3 + ).

**Figure 3.** FT-Raman spectra of l-valine in the spectral range from 20 to 3500 cm−1. The most intense peaks appear in the high wavenumber region of the spectrum.

**Figure 3** shows the FT-Raman spectrum of *L-valine* in the range of 20–3500 cm−1. This aliphatic amino acid crystallizes in a monoclinic structure with a *P*21 space group. l-valine is characterized by the (CH3)2-(CH) groups as radicals [l-leucine and l-isoleucine, which are discussed below, are characterized by (CH3)2-CH2-CH2]. There are four molecules per unit cell of l-valine, two of them in the *trans* conformation and two of them in the *gauche I* conformation. As usual, the modes in the high wavenumber region are associated with the stretching of CH and CH3; modes related to the bending of CH3, δ(CH3), were assigned for the bands between 1400 and 1460 cm−1; modes assigned as rocking of CH3, r(CH3), were observed between 1125 and 1200 cm−1; bands identified as the stretching of CC, ν(CC), were observed between 900 and 970 cm−1 [21]. The lattice modes were assigned as bands with wavenumber lower than 177 cm−1 and the torsion of CO2, *τ*(CO2), was identified with a band at 185 cm−1. A Raman spectroscopic study showed that the l-valine crystal seems to undergo a phase transition at ~100 K, as indicated by changes in the lattice mode region of the spectrum [21]. In fact, unless the crystal is ferroelastic and presents domains, modification in the lattice mode spectral region means change in the symmetry of the unit cell. This behavior differs from the behavior of the l-alanine crystal, for example, that is stable under cryogenic conditions.

**Figure 2.** Raman spectrum of l-alanine in the spectral range from 300 to 700 cm−1. Some important modes related to

**Figure 3.** FT-Raman spectra of l-valine in the spectral range from 20 to 3500 cm−1. The most intense peaks appear in the

), and torsion of NH3

+ , *τ*(NH3 + ).

− , r(CO2 −

hydrogen bonds are shown, as rocking of CO2

206 Raman Spectroscopy and Applications

high wavenumber region of the spectrum.

**Figure 4.** Raman spectra of l-leucine in the spectral range from 700 to 1280 cm−1 for two scattering geometries.

Another amino acid with nonpolar characteristics is *l-leucine*. This amino acid crystallizes in a monoclinic structure, space group *P*21, and *Z* = 2. The carboxyl and the amino groups are hydrogen bonded in a double layer, in a similar fashion to l-valine and l-isoleucine [22]. **Figure 4** presents the Raman spectra of l-leucine for two scattering geometries in the spectral range from 700 to 1280 cm−1. The band observed at 777 cm−1 is assigned as bending of CO2 − , δ(CO2 − ); at 838 cm−1 as out-of-plane vibration of CO2, γ(CO2); 849 cm−1 as rocking of CH2, r(CH2). The bands between 919 and 1004 cm−1 are assigned as stretching vibration of CC, ν(CC), and the bands at 1032 and 1083 cm−1 are assigned as stretching of CN, ν(CN), while the band at 1131 cm−1 is assigned as rocking of NH3 + , r(NH3 + ) unit [22]. The Raman spectroscopic study showed a series of modifications at about 353 K in both the internal and lattice modes of l-leucine, indicating a possible modification of the structure. This was interpreted as a phase transition from a C2 to a CS structure, even with the appearance of a TO mode at high temperature. lleucine was also investigated under high pressure with the scrutiny of Raman spectroscopy [23]. Anomalous behavior was observed in two ranges, from 0 to 0.46 GPa and from 0.8 to 1.46 GPa. The first anomaly was realized through the observation of the disappearance of a band in the CH and CH3 stretching region of the spectrum. The second anomaly is verified through the disappearance of lattice modes and splitting of modes in the high wavenumber region. Obviously, some of the modifications must involve molecular rearrangements due to changes of hydrogen bonds. Again, Raman spectroscopy appears as a powerful tool in order to study the phase transitions.

**Figure 5.** FT-Raman spectra of l-isoleucine in the spectral range from 20 to 3500 cm−1. The most intense peaks appear in the high wavenumber region of the spectrum.

**Figure 5** presents the FT-Raman spectrum of *l-isoleucine* in the spectral range from 20 to 3500 cm−1. We observe that the most intense bands is located in the high wavenumber region of the spectrum, corresponding to bands associated with stretching of CH, CH2, and CH3; in fact, the spectral range of 2700–3200 cm−1 presents a very complex profile, with at least seven different bands [24]. On the other hand, the region between about 1700 and 2700 cm−1 does not present bands, as occurs with most proteinogenic amino acids (exception to cysteine that presents bands associated with SH stretching vibration at about 2500 cm−1). A series of bands is observed between 500 and 1650 cm−1, including vibrations associated with stretching of CC, ν(CC), from 872 to 1018 cm−1, stretching CN, ν(CN), at 1033 and 1091 cm−1, rocking of CH3, rocking of CO2 − , at 536 cm−1 etc. Several modes associated with bending vibrations are observed in the 300–500 cm−1 spectral range. The vibration associated with torsion of CO2 − , *τ*(CO2 − ), is observed at ~ 177 cm−1; such vibration is common to most amino acid crystals. Finally, below 170 cm−1 bands are observed that are generically associated with the lattice modes of the crystal. In relation to the behavior of the crystal under low temperature conditions, Raman spectroscopy showed that the l-isoleucine crystal does not present any evidence of phase transition, similarly to l-alanine and l-leucine, but differently from l-valine, which presents a modification at about 100 K. Such fact is very curious and future investigations are demanded in order to shed light in this problem.

at 838 cm−1 as out-of-plane vibration of CO2, γ(CO2); 849 cm−1 as rocking of CH2, r(CH2). The bands between 919 and 1004 cm−1 are assigned as stretching vibration of CC, ν(CC), and the bands at 1032 and 1083 cm−1 are assigned as stretching of CN, ν(CN), while the band at 1131

a series of modifications at about 353 K in both the internal and lattice modes of l-leucine, indicating a possible modification of the structure. This was interpreted as a phase transition from a C2 to a CS structure, even with the appearance of a TO mode at high temperature. lleucine was also investigated under high pressure with the scrutiny of Raman spectroscopy [23]. Anomalous behavior was observed in two ranges, from 0 to 0.46 GPa and from 0.8 to 1.46 GPa. The first anomaly was realized through the observation of the disappearance of a band in the CH and CH3 stretching region of the spectrum. The second anomaly is verified through the disappearance of lattice modes and splitting of modes in the high wavenumber region. Obviously, some of the modifications must involve molecular rearrangements due to changes of hydrogen bonds. Again, Raman spectroscopy appears as a powerful tool in order to study

**Figure 5.** FT-Raman spectra of l-isoleucine in the spectral range from 20 to 3500 cm−1. The most intense peaks appear in

**Figure 5** presents the FT-Raman spectrum of *l-isoleucine* in the spectral range from 20 to 3500 cm−1. We observe that the most intense bands is located in the high wavenumber region of the spectrum, corresponding to bands associated with stretching of CH, CH2, and CH3; in fact, the spectral range of 2700–3200 cm−1 presents a very complex profile, with at least seven different bands [24]. On the other hand, the region between about 1700 and 2700 cm−1 does not present bands, as occurs with most proteinogenic amino acids (exception to cysteine that presents bands associated with SH stretching vibration at about 2500 cm−1). A series of bands is observed between 500 and 1650 cm−1, including vibrations associated with stretching of CC, ν(CC), from

) unit [22]. The Raman spectroscopic study showed

+ , r(NH3 +

cm−1 is assigned as rocking of NH3

208 Raman Spectroscopy and Applications

the phase transitions.

the high wavenumber region of the spectrum.

**Figure 6.** FT-Raman spectra of l-phenylalanine, l-methionine, l-proline and l-tryptophan in the spectral range from 20 to 3500 cm−1.

The Raman spectra of *l-phenylalanine*, *l-methionine*, *l-proline*, and *l-tryptophan* are shown in **Figure 6**. It is very difficult to grow crystals of l-phenylalanine and l-tryptophan in their pure forms. l-proline grows mainly in a hydrated form, while it is relatively easy to grow l-methionine. As a consequence there are few studies reporting vibrational properties of l-phenylalanine [25] and l-tryptophan [26], as well l-proline [5], and a little more on l-methionine [27]. In relation to the previous amino acid presented in this chapter, phenylalanine and tryptophan show an additional benzene ring. As a consequence, this unit presents vibrations associated with it: stretching of CH at 2979, 3030, and 3057 cm−1; rocking at ~1260 cm−1; deformation at about 704 cm−1; ring breathing at ~ 1010 cm−1 and wagging at ~ 744 cm−1, for l-tryptophan [25]. The l-phenylalanine shows that vibrations related to the benzene ring are observed at 848, 912, and 949 cm−1 (out-of-plane bending); 1001 cm−1 (breathing of the ring); 1025 and 1076 cm−1 (in-plane bending); 1600 cm−1 (stretching of CC in the ring). Some results on l-proline monohydrated were presented in reference [5], where a high pressure investigation is furnished; the work shows evidence of two phase transitions, between 0.0 and 1.1 GPa and another between 6.5 and 7.8 GPa. In relation to lmethionine, a detailed study of the Raman spectra of the crystal under high pressure was revealed in reference [27]. Although the methionine molecule presents a sulfur atom, differently from the cysteine, in the methionine there is a connection linking two carbon atoms, C–S–C; as a consequence, the very intense band observed at ~ 2500 cm−1 appearing in the Raman spectrum of cysteine due to the S-H stretching is not observed in the Raman spectrum of methionine. However, a very characteristic band associated with the C-S stretching vibration is observed at 659 cm−1 in the spectrum of l-methionine. Following this band, when the l-methionine crystal was subjected to high pressure into a diamond anvil cell, it was observed an impressive modification that was associated with a phase transition undergone by the crystal at about 2.2 GPa [27]. Also interesting is the fact that the phase transition occurs with a hysteresis of ~ 0.8 GPa, suggesting the transition can be classified as a first-order one.
