Heat Capacities of l-Histidine, l-Phenylalanine, l-Proline, l-Tryptophan and l-Tyrosine

In an effort to establish reliable thermodynamic data for proteinogenic amino acids, heat capacities for l-histidine (CAS RN: 71-00-1), l-phenylalanine (CAS RN: 63-91-2), l-proline (CAS RN: 147-85-3), l-tryptophan (CAS RN: 73-22-3), and l-tyrosine (CAS RN: 60-18-4) were measured over a wide temperature range. Prior to heat capacity measurements, thermogravimetric analysis was performed to determine the decomposition temperatures while X-ray powder diffraction (XRPD) and heat-flux differential scanning calorimetry (DSC) were used to identify the initial crystal structures and their possible transformations. Crystal heat capacities of all five amino acids were measured by Tian–Calvet calorimetry in the temperature interval from 262 to 358 K and by power compensation DSC in the temperature interval from 307 to 437 K. Experimental values determined in this work were then combined with the literature data obtained by adiabatic calorimetry. Low temperature heat capacities of l-histidine, for which no literature data were available, were determined in this work using the relaxation (heat pulse) calorimetry from 2 K. As a result, isobaric crystal heat capacities and standard thermodynamic functions up to 430 K for all five crystalline amino acids were developed.


Introduction
This work represents a continuation of our previous project [1][2][3][4], which aimed to establish reliable thermodynamic data along the saturation curve for proteinogenic amino acids. Despite their significance [5,6] and wide use, for example, in pharmaceutical formulations as coformers used to increase the bioavailability of poorly water soluble drugs [7,8], there are still considerable gaps in the availability of fundamental thermodynamic data for basic amino acids [2,9,10]. In addition, the values for properties such as sublimation or crystal-phase enthalpies of formation often show very poor interlaboratory agreement [2,10]. The discrepancies in sublimation thermodynamic properties can be ascribed to the zwitterionic nature of amino acid crystals, which causes their extremely low volatility and renders the experimental determination of their sublimation pressure and enthalpy difficult, prone to systematic errors, or even unfeasible at room temperature using current available experimental techniques. Thermodynamically controlled extrapolation [11] of sublimation pressures measured at higher temperatures therefore seems to be the only method to obtain sublimation thermodynamic data for amino acids at temperatures relevant to their biological activity. For the application of this method, several thermodynamically linked properties are necessary as inputs: (i) sublimation pressure, which was determined at higher temperatures by the Knudsen effusion method for several amino acids including L-proline (from 373 to 416 K) in our previous work [1] (the measurements of other amino acids are in progress); (ii) heat capacity in the ideal gas state, which is typically calculated by the combination of quantum-chemical and statistical thermodynamics methods [3]; and (iii) experimental crystal heat capacities, which were measured in this work for L-histidine, L-phenylalanine, L-proline, L-tryptophan, and L-tyrosine in the temperature range overlapping with that of sublimation pressure measurements by the Knudsen effusion method. Simultaneous correlation of the above-mentioned properties also ensures their thermodynamic consistency.
In order to establish a safe upper temperature limit for the heat capacity measurements, the decomposition temperatures of the amino acids studied were first determined by thermogravimetry. Consequently, after determining the initial crystal structure at 298.15 K by X-ray powder diffraction (XRPD), the phase behavior was studied in the temperature range from 183 K to the decomposition temperature of each amino acid using a heat flux differential scanning calorimeter (DSC) to detect possible phase transitions. Heat capacities in the temperature range from 262 to 358 K were determined with Tian-Calvet calorimetry and extended up to 437 K by using a power compensation DSC. Literature low-temperature heat capacities, which are determined by adiabatic calorimetry from about 10 K to 300 K, were found for all amino acids studied except L-histidine for which new measurements by means of thermal-relaxation calorimetry were performed in this work in the temperature range from 2 to 300 K.

Thermogravimetric Analysis (TGA)
All studied amino acids decompose at/prior to melting using the usual temperature scanning rates (5 to 40) K·min −1 . The samples were studied by TGA previously and the literature temperatures of decomposition along with the results of this work are summarized in Table 1. It should be noted that the decomposition kinetics is influenced by a number of factors (heating rate, sample size, purge gas, etc.). This is reflected by the relatively large range of reported decomposition temperatures. Despite that, thermograms of this work (shown in Figure 1) agree qualitatively with the literature ones for L-histidine [12,13], L-proline [14], L-phenylalanine [15,16], L-tryptophan [16], and L-tyrosine [16].
The decomposition behavior of the amino acids studied exhibits various patterns. The simplest decomposition path can be observed in the case of L-tyrosine (Figure 1e), which shows a simple one-stage decomposition. For L-histidine ( Figure 1a) and L-tryptophan (Figure 1d), two decomposition phases can be clearly distinguished. The situation is more complicated in the case of L-phenylalanine ( Figure 1b) and L-proline (Figure 1c). In both cases, four peaks can be observed on the heat flow curve (red), two of which are, however, quite small. In the case of L-proline, the first small peak at 484 K does not seem to be accompanied by any mass loss, pointing to a possible crystal-crystal transition. Note that the temperatures of fusion reported by Do et al. [17] (see Table 1) are higher than those of decomposition by (20 to 100) K. In order to avoid decomposition, Do et al. [17] used scanning rates up to 20,000 K·s −1 .  The decomposition behavior of the amino acids studied exhibits various patterns. The simplest decomposition path can be observed in the case of L-tyrosine (Figure 1e), which shows a simple one-stage decomposition. For L-histidine ( Figure 1a) and L-tryptophan (Figure 1d), two decomposition phases can be clearly distinguished. The situation is more complicated in the case of L-phenylalanine ( Figure 1b) and L-proline (Figure 1c). In both cases, four peaks can be observed on the heat flow curve (red), two of which are, however, quite small. In the case of L-proline, the first small peak at 484 K does not seem to be accompanied by any mass loss, pointing to a possible crystal-crystal transition. Note that the temperatures of fusion reported by Do et al. [17] (see Table 1) are higher than those of decomposition by (20 to 100) K. In order to avoid decomposition, Do et al. [17] used scanning rates up to 20,000 K·s −1 .   [17] for T fus determination (see text). c "Temperature of fusion" (in fact T decomp ) recommended by the Syracuse Research Corporation and used by many authors. d DTA stands for differential thermal analysis. e Phase transition assumed (mass loss not detected). f Small peak preceding the main decomposition peak. g Main decomposition peak.

Phase Behavior
All amino acids studied are zwitterionic crystals at room temperature. Their initial crystal structures determined at 298.15 K by XRPD are provided in Table 2. Subsequently, the phase behavior was investigated in the temperature range from 183 K to the thermal decomposition temperature using heat-flux DSC for all amino acids studied to confirm/exclude the presence of phase transitions. Except for L-phenylalanine, no phase transitions were detected in the temperature range studied. A subtle endothermic peak on heating was detected for L-phenylalanine at around 440 K ( Figure S1 in the Supplementary Materials), in agreement with Cuppen et al. [23], who, based on a detailed study of this phenomenon by a combination of experimental and computational methods, as-cribed this feature to the conformational phase transition of form I (QQQAUJ05) to its high-temperature form Ih. The upper temperature limit of the heat capacity measurements in this work was 437 K in order to observe the trend of the apparent heat capacities in the region of the phase change. The shape of the heat capacity data agrees well with the DSC thermogram ( Figure S1 in the Supplementary Materials), but the peak is, despite its small area, so broad that all the apparent heat capacity data above 422 K had to be discarded. Thus, all the reported heat capacity data for L-phenylalanine corresponds to the form I. For L-proline for which the TGA measurements suggested a phase transition at around 484 K, no visible thermal event was detected in the temperature range from 183 K to its decomposition temperature using heat-flux DSC. Based on the XRPD and DSC analyses along with heat capacity measurements, which did not show any anomalies in the temperature range studied, it can be concluded that the heat capacity data reported in this work corresponds to the crystal structures listed in Table 2.

Heat Capacities
Experimental heat capacities obtained in this work with the SETARAM µDSC IIIa, PerkinElmer DSC 8500, and Quantum Design PPMS are listed in the Supplementary Materials in Tables S1-S6. Available literature data on crystal heat capacities are summarized in Table 3. The heat capacity data for L-phenylalanine, L-proline, and L-tyrosine reported by Cole et al. [30] and those reported for L-tyrosine by Huffman and Ellis [31] are consistent with our measurements and were therefore correlated together with the data listed in Tables S1-S6 by Equations (1) and (2), for which the parameters are provided in Table 4. The literature heat capacity data for L-proline [32] were excluded from the correlation because of their slight inconsistency in the low-temperature region with more recent measurements reported in [30]. Table 3. Overview of the literature crystal heat capacities of L-histidine, L-phenylalanine, L-proline, L-tryptophan, and L-tyrosine.    (1) and (2) for crystal heat capacities in J K −1 mol −1 .   (1) and (2)) heat capacity, N is the number of data points fitted, and m is the number of independent adjustable parameters.

Reference N a (T min -T max )/K u r (C pm )/% b Calorimeter
The experimental heat capacities for all the amino acids studied are compared with the values calculated using Equations (1) and (2) with parameters from Table 4 in Figure 2. The deviations of the selected experimental data from the calculated values do not exceed 2%. Adiabatic heat capacity data by Huffman and Ellis [31] and Cole et al. [30] agree with our measurements within 1%.  [30]; empty circles: Huffman and Fox [32]; purple circles: Huffman and Ellis [31]. Vertical lines denote the knot temperatures Ti.
The thermodynamic functions derived using Equations (1) and (2) are tabulated in Tables S7 to S11 in the Supplementary Materials and shown in Figure 3.   [30]; empty circles: Huffman and Fox [32]; purple circles: Huffman and Ellis [31]. Vertical lines denote the knot temperatures T i .
The thermodynamic functions derived using Equations (1) and (2) are tabulated in Tables S7-S11 in the Supplementary Materials and shown in Figure 3.

Samples Description
The title amino acids were of commercial origin and were used as received without further purification. The sample purities (as stated in the certificate of analysis provided by the manufacturer) are reported in Table 5.

Thermogravimetry
Prior to heat capacity and phase behavior measurements, thermogravimetric analysis (TGA) was performed. Traditionally, amino acids are reported to melt or decompose at temperatures above 500 K [5,6]. However, the literature data exhibit a significant scatter. Moreover, a recent work by Weiss et al. [12] suggests that all standard amino acids do not melt but rather decompose, which might damage calorimeters. Weiss et al. [12] reported the evolution of CO2, NH3, and H2O for L-histidine at the decomposition temperature of

Samples Description
The title amino acids were of commercial origin and were used as received without further purification. The sample purities (as stated in the certificate of analysis provided by the manufacturer) are reported in Table 5.

Thermogravimetry
Prior to heat capacity and phase behavior measurements, thermogravimetric analysis (TGA) was performed. Traditionally, amino acids are reported to melt or decompose at temperatures above 500 K [5,6]. However, the literature data exhibit a significant scatter. Moreover, a recent work by Weiss et al. [12] suggests that all standard amino acids do not melt but rather decompose, which might damage calorimeters. Weiss et al. [12] reported the evolution of CO 2 , NH 3 , and H 2 O for L-histidine at the decomposition temperature of about 553 K. For the remaining four amino acids of this study, Weiss et al. [12] reported the decomposition without specifying their temperature. Therefore, reinvestigation of the melting/decomposition process for all five amino acids was undertaken to reconcile the literature data. The thermogravimetric analyzer SETARAM Setsys Evolution was used. The samples were placed in an open platinum 100 µL crucible and their thermal stability was investigated in the temperature range from 298 to 573 K with a temperature gradient of 5 K min −1 under an inert Ar atmosphere.

Phase Behavior Measurements
XRPD was used to characterize initial crystal structures of the amino acids studied using a θ-θ powder diffractometer X'Pert 3 Powder from PANalytical in Bragg-Brentano para-focusing geometry using wavelength CuKα radiation (λ = 1.5418Å, U = 40 kV, I = 30 mA). The samples were scanned at 298.15 K in the range of 5 • to 50 • 2θ with a step size of 0.039 • 2θ and 0.7 s for each step. The diffractograms were analyzed with the software HighScore Plus in combination with yearly updated powder diffraction databases PDF4+ and PDF4/Organics.
The heat flux DSC TA Q1000 was used for investigation of the phase behavior of the amino acids studied in the temperature range from 183 K to the decomposition temperature. The combined expanded uncertainty (0.95 level of confidence) in the phase transition temperatures and enthalpies is estimated to be 0.3 K and 3%, respectively.

Heat Capacity Measurements
A Tian-Calvet type calorimeter (SETARAM µDSC IIIa) with operating temperature range 262 K to 358 K was used for the measurement of heat capacities. As the detailed description of the calorimeter and its calibration and operation was reported previously [4], only the most salient information is provided here. The heat capacity measurements were carried out by the continuous heating method [33] using the three-step methodology, i.e., the measurement of the sample is followed by the measurement of the reference material (synthetic sapphire, NIST Standard reference material No. 720) and by performing the blank experiment. The saturated molar heat capacities C sat obtained in this work are identical with the isobaric molar heat capacities C o pm in the temperature range studied given the very low sublimation pressures of amino acids. The combined expanded uncertainty (0.95 level of confidence) of the heat capacity measurements is estimated to be U c C o pm = 0.01 · C o pm . The PerkinElmer power compensation DSC 8500 equipped with autosampler was used for the heat capacity determination in the temperature range from 307 to 437 K. The heat capacity measurements were carried out by the temperature increment method and repeated three times to eliminate systematic errors. The combined expanded uncertainty (0.95 level of confidence) of the heat capacity measurement is estimated to be U c C o pm = 0.03 · C o pm . Due to a lower accuracy, the results obtained by this calorimeter were slightly adjusted to agree with those determined by the more accurate SETARAM µDSC IIIa, following the common practice [34].
For low-temperature heat capacity measurements of L-histidine, a commercially available apparatus Physical Property Measurement System (PPMS) Model 6000 EverCool II (Quantum Design, USA) equipped with a heat capacity module ( 4 He, T min = 1.8 K) was used. The calorimeter uses the thermal-relaxation measurement technique which is an alternative to the time-intensive and labor-intensive adiabatic calorimetry. The specific heat capacity of a sample is determined by measuring the thermal response to a change in heating conditions [35]. Uncertainty in the heat capacity obtained using PPMS was investigated recently in our laboratory [36]. Based on testing with several compounds [36], the combined expanded uncertainty (0.95 level of confidence) of the heat capacity measurements is estimated to be U c C o pm = 0.10 · C o pm below 10 K, U c C o pm = 0.03 · C o pm in the temperature range from 10 to 40 K and U c C o pm = 0.02 · C o pm in the temperature range from 40 to 300 K.
In order to describe the temperature dependence of heat capacity in a wide temperature range (including literature heat capacities obtained using adiabatic calorimetry and data obtained by Quantum Design PPMS), the following equation proposed by Archer [37] was used: is defined by the following equation: where only a single parameter d i per each temperature interval is to be optimized, while the values of the other three are imposed by a constraint of continuity and smoothness of the resulting temperature dependence. Parameter b is estimated from the slope of f (T) at temperatures greater than 70 K prior to the optimization procedure [37].

Conclusions
The decomposition temperatures of the studied amino acids were determined by means of TGA (see Table 2). The thermograms obtained qualitatively agree with those found in the literature. The phase behavior was studied with a heat flux differential scanning calorimetry (calorimeter TA Q1000) in the temperature range from 183 K to the decomposition temperature to determine the presence of possible phase transitions. For L-phenylalanine, a subtle endothermic event corresponding to form I to form Ih transition was detected at around 440 K, in accordance with [23]. New heat capacity values were obtained for L-histidine, L-phenylalanine, L-proline, L-tryptophan, and L-tyrosine (Tables S1-S6 in the Supplementary Materials). Accurate equations in the temperature range from 0 to 437 K for crystal the heat capacity were established. Equations are based on new heat capacity measurements performed in this work as well as on low-temperature heat capacity data from the literature which showed an excellent agreement with our data in the overlapping temperature interval. Standard thermodynamic functions (entropies, enthalpies, and Gibbs energies) of the crystalline phase at p = 0.1 MPa were evaluated in the temperature range from 0 to 430 K (Tables S7-S11 in the Supplementary Materials).

Supplementary Materials:
The following are available online: Figure S1: DSC thermogram and apparent heat capacity for L-phenylalanine, Tables S1-S6: Experimental crystal heat capacities for the studied amino acids, Tables S7-S11: Tabulated standard thermodynamic functions (heat capacity, entropy, enthalpy, and Gibbs energy) for the studied amino acids.
Author Contributions: Investigation, formal analysis, writing-original draft and visualization, V.P.; investigation, data curation and writing-review and editing, V.Š.; investigation, J.H.; supervision, methodology, writing-original draft and writing-review and editing, K.R., conceptualization, supervision, validation, writing-original draft and writing-review and editing, M.F. All authors have read and agreed to the published version of the manuscript.

Conflicts of Interest:
The authors declare no conflict of interest.
Sample Availability: Samples of the compounds are not available from the authors.