Molecular interaction studies of aqueous l-proline using volumetric measurements at various temperatures

A detailed volumetric investigation of L-proline was undertaken in pure water and in aqueous media containing potassium chloride (electrolytic additive) and D-glucose (non-electrolytic additive). Density data were recorded within the molality interval 0.03-0.20 mol·kg⁻¹ over the temperature range 288.15-318.15 K at atmospheric pressure. Using the experimental density values, several thermodynamic quantities such as apparent molar volume(Vϕ ) limiting apparent molar volume (Vϕ0 ), limiting apparent molar transfer volume (ΔtrVϕ0 ) and Hepler’s parameter were determined. These volumetric functions were employed to examine the nature of molecular interactions, particularly solute-solvent and solute-solute associations, and their temperature dependence. The obtained results demonstrate that both KCl and D-glucose markedly modify the hydration and solvation characteristics of L-proline. The study provides useful information regarding intermolecular behaviour in biologically significant aqueous systems and clarifies the effect of co-solutes on hydration phenomena.

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ordering of the solvent network. Owing to these characteristics, D-glucose represents an appropriate model compound for examining nonelectrolyte effects on solvation and hydration behaviour [4,5] . In the present investigation, density measurements of Lproline were carried out in aqueous KCl and aqueous Dglucose media over a broad temperature range. From these measurements, various volumetric and thermodynamic parameters were evaluated. The central aim of this work is to explore the influence of temperature and co-solutes on molecular interactions, particularly solute-solvent and solute-solute interactions, in binary and ternary liquid systems.

Materials and Methods

Materials and Methods

Materials

L-proline, potassium chloride and D-glucose used in this study were procured from Sigma-Aldrich (India). All reagents were of analytical-reagent purity and employed without additional purification. Prior to use, the compounds were dried in a vacuum oven at 343.15 K and subsequently stored in a vacuum desiccator containing fused anhydrous calcium chloride for more than 48 h.

Instrumentation and Experimental Procedure

Deionized distilled water with conductivity lower than 0.1 μS•cm⁻¹ was supplied by an Evoqua purification unit. Solutions were prepared gravimetrically at ambient temperature within the concentration range 0.03-0.20 mol•kg⁻¹ and utilized immediately after preparation. Weighing was performed using a Sartorius Quintix analytical balance (Model 125D-101N) having an accuracy of ±0.01 mg. Density and ultrasonic velocity measurements were obtained using an Anton Paar DSA 5000M digital densimeter-sound analyzer (Austria). The instrument operates on the Pulsed Excitation Method, providing density accuracy up to 0.007 kg.m⁻³. The corresponding sensitivities for density and sound velocity measurements are 1×10 -3 and 1×10 -2 m.s⁻¹, respectively. Estimated standard uncertainties were 0.5 kg.m⁻³ for density and 1.0 m.s⁻¹ for sound velocity. Temperature regulation was maintained by an integrated PT100 thermostat with stability of ±0.01 K. Prior to each measurement sequence, calibration was verified using deionized water throughout the investigated temperature range. The liquid sample was positioned between two piezoelectric transducers; one generated ultrasonic waves of nearly 3 MHz frequency, while the other detected the transmitted signal.

Results and Discussion

Results and Discussion

Density Behaviour

Densities of aqueous L-proline solutions and ternary systems containing 0.01 mol.kg⁻¹ KCl or 0.1 mol.kg⁻¹ Dglucose were measured at temperatures from 288.15 K to 318.15 K over different solute concentrations. The experimental observations indicate that density increases almost linearly with increasing concentration of L-proline in all systems studied. Conversely, at constant concentration, density decreases slightly with rise in temperature due to thermal expansion of the solvent medium. These findings are graphically represented in Figure 1.

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Plot of variation of density () against molality (m) for aqueous solution of L-proline (a) in water (b) in aqueous 0.1 mol.kg-1 KCl and

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Apparent Molar Volume(𝑉 𝜙 )

The apparent molar volume (𝑉 𝜙 ) was evaluated from density data using the expression:

where M denotes molar mass of the solute, m is molality, and ρ and ρ 0 represent densities of solution and solvent, respectively. The calculated values are reported in Table 1 and graphically shown in Figure 2 [6,7] . Positive values of 𝑉 𝜙 were obtained for all concentrations and temperatures, suggesting strong interactions between solute and solvent molecules. Increasing temperature resulted in higher 𝑉 𝜙 values, indicating enhanced solvation effects. In aqueous D-glucose medium, 𝑉 𝜙 increased noticeably, reflecting stronger solute-solvent association, whereas a decreasing trend was observed in KCl solutions [14][15] .

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Plot of 𝑉𝜙 against molality (m) for L-proline in (a) in water (b) in aqueous 0.1 mol.kg-1 KCl and (c) in aqueous 0.1 mol.kg-1 D-glucose

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𝒎/ (mol.kg-1)	106· 𝑽 / (m3 ·mol-1) 𝝓	—	—	—	—	—	—
	T=288.15 K	T=293.15 K	T=298.15 K	T=303.15 K	T=308.15 K	T=313.15 K	T=318.15 K
L-proline + water
0.0300	80.56	81.31	82.07	82.41	82.86	82.95	83.42
0.0505	81.25	81.47	82.29	82.45	82.90	83.23	83.76
0.0700	81.48	81.71	82.23	82.55	83.02	83.39	84.09
0.0905	81.58	81.95	82.33	82.58	83.16	83.60	84.13
0.1004	81.67	81.92	82.34	82.76	83.04	83.75	84.46
0.1204	81.73	82.01	82.52	82.79	83.32	83.73	84.45
0.1404	81.66	81.94	82.52	82.86	83.20	83.72	84.41
0.1603	81.67	82.21	82.60	83.01	83.34	83.73	84.35
0.1801	81.78	82.15	82.71	82.91	83.47	83.72	84.43
0.2023	81.76	82.09	82.67	82.91	83.46	83.67	84.39
L-proline + 0.1 mol.kg-1 KCl
0.0301	85.34	87.60	90.36	92.10	96.05	98.15	103.30
0.0512	85.17	87.24	89.86	91.76	95.71	97.67	102.81
0.0705	84.84	86.81	89.24	91.25	95.29	97.16	102.44
0.0914	84.26	86.21	88.76	90.85	94.70	96.69	101.91
0.0995	83.86	85.76	88.24	90.37	94.30	96.10	101.02
0.1201	83.28	85.33	87.85	89.94	93.81	95.76	100.75
0.1402	83.04	84.83	87.20	89.54	93.33	95.14	100.31
0.1614	82.73	84.05	86.77	89.03	92.95	94.75	99.87
0.1803	82.27	83.71	86.36	88.63	92.57	94.18	99.33
0.2003	81.71	83.21	86.00	88.21	91.04	93.82	98.11
L-proline + 0.1 mol.kg-1 D-glucose
0.0314	91.11	93.82	95.81	98.50	101.03	104.51	107.12
0.0498	91.54	94.21	96.13	99.10	101.49	105.17	107.60
0.0706	92.12	94.84	96.67	99.45	102.03	105.46	108.34
0.0903	92.64	95.43	97.03	99.90	102.60	105.87	108.91
0.0991	93.00	95.92	97.40	100.34	103.08	106.29	109.29
0.1203	93.31	96.21	98.05	100.74	103.48	106.75	109.47
0.1402	93.81	96.71	98.48	101.09	104.06	107.11	109.95
0.1605	94.33	97.27	99.08	101.85	104.57	107.53	110.23
0.1801	94.83	97.61	99.52	102.02	104.89	108.16	110.73
0.1998	95.24	98.01	99.98	102.36	105.40	108.45	110.98

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Values of apparent molar volumes (𝑉𝜙) of L-proline in water, and aqueous 0.1 mol. kg-1 KCl and aqueous 0.1 mol.kg-1 D-glucose at

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Limiting Apparent Molar Volume (𝑉 𝜙 0 )

The limiting apparent molar volume (𝑉 𝜙 0 ) was determined by extrapolating plots of 𝑉 𝜙 versus molality to zero concentration using Masson's relation:

where values observed in some systems imply stronger solutesolute interactions, while negative values suggest comparatively weaker association effects [8,9] .

Limiting Apparent Molar Volume of Transfer (𝛥 𝑡𝑟 𝑉 𝜙 0 )

The limiting apparent molar transfer volume was evaluated from:

The values of 𝛥 𝑡𝑟 𝑉 𝜙 0 for all the studied systems are reported in Table 2. Depending upon the co-sphere overlap model regarding the values of 𝛥 𝑡𝑟 𝑉 𝜙 0 there is negligible contribution from (solute + solute) interactions and hence they provide information regarding (solute + solvent) interactions. The various interactions that occur between amino acid and solvent molecules are graphically represented and can be summarised as follows, 1. (hydrophilic + hydrophilic) interactions (between hydrophilic groups of L-proline and hydrophilic groups of KCl/D-glucose). 2. (ion/hydrophilic + hydrophobic) interactions (between ions of KCl/hydrophilic groups of D-glucose and hydrophobic centres of L-proline.

𝑻/𝑲	𝟏𝟎𝟔. 𝑽𝟎/𝐦𝟑. 𝐦𝐨𝐥−𝟏 𝝓	𝟏𝟎𝟔. 𝑺 /𝐦𝟑. 𝐦𝐨𝐥−𝟐. 𝐤𝐠 𝑽	𝟏𝟎𝟔. 𝜟 𝑽𝟎/𝒎𝟑. 𝒎𝒐𝒍−𝟏 𝒕𝒓 𝝓	𝟏𝟎𝟔. 𝑽𝟎/𝐦𝟑. 𝐦𝐨𝐥−𝟏 (𝒄𝒂𝒍𝒄𝒖𝒍𝒂𝒕𝒆𝒅) 𝝓
L-proline + water
288.15	80.94	5.07		80.94
293.15	80.87	3.07		80.87
298.15	81.69	2.26		81.62
303.15	81.98	2.26		81.98
308.15	82.4	2.36		82.48
313.15	82.69	2.63		82.69
318.15	83.07	3.41		83.07
L-proline + 0.1 mol.kg-1 KCl
288.15	88.19	-13.84	7.25	88.19
293.15	91.02	-16.92	10.15	91.02
298.15	93.56	-16.77	11.87	93.45
303.15	95.04	-14.86	13.06	95.04
308.15	99.54	-16.98	17.14	95.39
313.15	101.36	-16.54	18.67	101.36
318.15	106.94	-18.17	23.87	106.94
L-proline + 0.1 mol.kg-1 D-glucose
288.15	88.09	15.59	7.15	88.09
293.15	90.7	16.18	9.83	90.70
298.15	92.53	16.15	10.84	92.12
303.15	95.7	14.76	13.72	95.70
308.15	97.78	16.74	15.38	98.70
313.15	101.72	14.68	19.03	101.72
318.15	104.49	14.57	21.42	104.49

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Limiting apparent molar properties (𝑉𝜙

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(ion-ion) interactions between zwitterionic centres of

L-proline and ions of KCl. Positive transfer volume values were obtained for both cosolutes, indicating predominance of hydrophilic-hydrophilic interactions according to the co-sphere overlap model proposed by Gurney, Frank, and Evans [10][11][12][13][14][15][16][17] . These interactions arise mainly between polar groups of amino acids and the hydrophilic moieties of KCl or D-glucose. Table 2 reports the 𝑉 𝜙 0 , 𝑆 𝑉 and 𝛥 𝑡𝑟 𝑉 𝜙 0 values for all the systems under study.

Temperature Dependence and Structure-Making/Breaking Effects

The variation of limiting apparent molar volume with temperature is described by:

According to Hepler's criterion,

Table 3. Empirical parameters of Eq. ( 4), limiting apparent molar expansivity (𝐸 𝜙 0 ) of solute, coefficient of thermal expansion (𝛼 * ) and

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Empirical parameters of Eq. (4), limiting apparent molar expansivity (𝐸𝜙

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Hepler's constant (𝜕𝐸 𝜙 0 𝜕𝑇 ⁄ ) for L-proline in aqueous and in aqueous solutions of 0.1 mol.kg -1 KCl and 0.1 mol.kg The sign of this parameter provides information regarding structure-making or structure-breaking tendencies of solutes. Positive values correspond to structure-promoting behaviour, whereas negative values indicate disruption of solvent structure [18][19][20] . The present systems predominantly exhibited positive values, suggesting that both co-solutes act as structure makers in aqueous L-proline solutions. The thermal expansion coefficient was calculated using:

The obtained 𝛼 * values indicate comparatively stronger solute-solvent interactions in aqueous KCl and D-glucose systems than in pure aqueous L-proline solutions [21][22][23][24][25] .

Conclusion

The present investigation offers a comprehensive volumetric examination of L-proline in aqueous as well as mixedsolvent environments containing potassium chloride and Dglucose. The evaluated thermodynamic parameters reveal that solute-solvent interactions become progressively stronger with increasing temperature, as evidenced by the rise in apparent molar volume and limiting apparent molar volume values. Among the investigated co-solutes, D-glucose exerts a more pronounced influence on the interaction behaviour of Lproline compared with KCl. This difference may be attributed to the extensive hydrogen-bonding capability and structural characteristics of glucose molecules in aqueous media. In contrast, the electrolyte KCl primarily affects the system through ion-solvent interactions and electrostatic effects.

The calculated Hepler parameters indicate that L-proline predominantly behaves as a structure breaker in most of the studied solutions. However, in aqueous D-glucose medium, the amino acid exhibits structure-promoting characteristics, suggesting enhanced ordering of the surrounding solvent network. Such behaviour highlights the important role of cosolutes in modifying hydration structure and intermolecular association patterns. Overall, the present findings improve the understanding of amino acid-solvent and amino acid-co-solute interactions in biologically relevant systems. These observations are valuable for interpreting the influence of ions and nonelectrolytes on amino acid solubility, conformational stability, and hydration behaviour. Furthermore, the study provides useful insight into the role of co-solutes in protein denaturation and renaturation phenomena, where amino acids are often employed as simplified model compounds. Knowledge of these thermophysical properties also contributes to a better understanding of protein folding mechanisms, molecular stability, and solvent-mediated biochemical processes.

Acknowledgement

AcknowledgmentsThe author is thankful to Fr. Praveen D'Souza, Principal of St. Francis de Sales College, Nagpur for providing all support and encouragement during this work.

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Plausible interactions between L-proline and KCl/D-glucose

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