Density, viscosity, and refractive index of mono-, di-, and tri-saccharides in aqueous glycine solutions at different temperatures

1 Introduction

Saccharides are the most abundant and diverse classes of organic molecules found in nature and their conjugates with proteins and lipids play key roles in the immune and endocrine systems, fertilisation, brain development, prevention of pathogenesis and blood clotting (Safina, 2012). They serve as energy sources (Safina, 2012; Wang et al., 2012; Knapp et al., 2008) and play a very crucial role in biological recognition phenomena in the process of exchanging information between the cells like cell–cell interactions and cell death signal transduction inflammatory processes, cancer metastasis bacterial and viral infections, fascinating the researchers to design a new category of anticancer drugs (Safina, 2012; Carroll et al., 2006; Kaminski et al., 2012). Recently, Montesarchio and co-workers designed saccharide based synthetic-ion transporters which are of great interest for both technological and biomedical applications (Montesarchio et al., 2012). Interactions of saccharides with proteins play an important role in a wide range of biochemical processes, for example, the application of saccharides as lyoprotectant. It is well-known that there are a lot of protein pharmaceuticals which are prepared by freeze-drying. However, in many cases this process breaks the native structure of protein and leads to its destabilisation. Addition of saccharide minimises the unwanted processes of freeze drying. Due to the great ability of saccharides to form hydrogen bonds with target molecules as well as their weak tendency to recrystallize, aggregation and destabilisation of proteins can be completely avoided (Kaminski et al., 2012). Understanding the mechanism of protein–saccharide interaction and the subsequent stabilisation of proteins by saccharide molecules is still incomplete (Karlsson, 1987; Osawa and Tsuji, 1987). However, due to the complex conformation and configuration of proteins in various solvents, a direct study on proteins is difficult, so we select glycine, the simplest amino acid and constituent of proteins to study the thermodynamic properties of saccharide–amino acid interactions in aqueous solutions. Literature survey indicates that interactions between saccharides and proteins have been investigated using chromatography data, (Karlsson, 1987; Osawa and Tsuji, 1987) X-ray crystallography,(Quiocho and Vyas, 1984; Bundle, 1989) NMR spectra, computer simulations, (Thøgersen et al., 1982; Bock, 1983) and kinetics (Miller et al., 1983; Miller et al., 1980). However, thermodynamic and transport studies of interactions of saccharides with model compounds of proteins (amino acids) are rare. Interactions between glucose and amino acids (glycine, alanine, serine, and valine) (Ali et al., 2006) using volumetric, viscometric, and refractive index techniques, and those between disaccharides (d-maltose and sucrose) and amino acids (glycine, alanine, leucine, and serine) (Parfenyuk et al., 2004) using calorimetric titrations have been reported in dilute aqueous solutions. Such studies can provide better and valuable information towards understanding the behaviour of these biomolecules in aqueous media. Lack of thermodynamic and transport studies on saccharides in aqueous amino acids led us to investigate saccharide–amino acid interactions through the determination of various volumetric and viscometric parameters of glucose, sucrose, and raffinose in aqueous glycine at different temperatures. The derived parameters are used to discuss saccharide–glycine and saccharide/glycine–water interactions.

2 Materials and methods

High purity glycine (E. Merck, Germany, mass fraction 0.99) was used after recrystallisation from ethanol + water mixture and dried over P₂O₅ in a vacuum desiccator. d-glucose (Thomas Baker Chem. Ltd., Mumbai, India, mass fraction 0.99), sucrose (E. Merck, Germany, mass fraction 0.99) and D- (+)- raffinose pentahydrate (raffinose) (Sigma Aldrich, USA, mass fraction 0.99) were used as such, except drying them under vacuum at 40°C for two days. Aqueous solution of 0.015 m (mol kg⁻¹) of glycine was prepared using deionised double distilled degassed water (conductivity 1.3 × 10⁻⁶ S cm⁻¹ at 298.15 K) and was used as solvent for the preparation of 0.02, 0.04, 0.06, 0.08, and 0.10 m solutions of glucose, sucrose, and raffinose. The molalities of the solutions prepared were within an accuracy of ±0.0001 mol kg⁻¹. The solutions were stored in special air tight bottles to avoid the absorption of atmospheric moisture and carbon dioxide, if any. All the solutions were prepared afresh and weighing was done on a Precisa XB-220 (Swiss-make) electronic balance with a precision of ±1 × 10⁻⁴ g.

The densities of the solutions were measured using a pycnometer (Borosil glass, total volume of 8 × 10⁻⁶ m³) having a graduated capillary of narrow bore (internal diameter 1 × 10⁻³ m). The capillary was provided with a well-fitted glass cap in order to avoid changes in composition due to evaporation. The marks on the pycnometer were calibrated at experimental temperatures using known densities of double distilled deionised water and extra pure ethanol (E. Merck, Germany, highly pure). The accuracy of the density measurement was checked by comparing the experimental values of the densities of water and ethanol and good agreement was found with the corresponding literature values (Stokes and Mills, 1965; Ali and Nain, 1997). Details regarding calibration, experimental set up and procedure have been described elsewhere (Parfenyuk et al., 2004; Ali and Nain, 2002; Pal and Kumar, 2005; Behrends et al., 2002). Density measurements were made in triplicate and an average value was used for all the calculations. The uncertainty in density measurement was less than ±5 × 10⁻² kg m⁻³. The viscosity measurements were made by using a thoroughly cleaned Ubbelohde type suspended-level viscometer with a flow time of 300 s for pure water at 298.15 K. Since the flow time was greater than 100 s, kinetic energy corrections were not considered. The time of flow was recorded with a digital stop-watch, accurate to ±0.1 s. An average of four sets of flow times for each reading was taken and used for the calculation of viscosity of the solutions. The accuracy in viscosity measurement was found to be ±3 × 10⁻⁴ N s m⁻². Refractive indices of the solutions were measured with the help of a thermostatic Abbe – refractometer. Before use, the refractometer was calibrated with double distilled deionised water and toluene (E. Merck, India, mass fraction 0.99) at experimental temperatures. The accuracy in refractive index measurement was up to ±0.0002 units. The temperature of the solutions during the measurements of ρ, η, and n_D was maintained (±0.02 K) in an electronically controlled thermostatic water bath (JULABO, Germany).

3 Results and discussion

The density (ρ), viscosity (η), and refractive index (n_D) measured for the solutions of glucose, sucrose, and raffinose in aqueous glycine at 298.15, 303.15, 308.15, and 313.15 K are listed in Table 1.

3.2

3.2 Viscometric study

The data on viscosities, η (Table 1) are also graphically displayed in Fig. 1. It reveals that η tends to increase linearly with an increase in the concentration of saccharides at each investigated temperature. This clearly indicates increased saccharide – glycine/water interaction as the amount of saccharide increases in the solution. Moreover, at a given temperature, the value of η increases from glucose to raffinose in the sequence: glucose < sucrose < raffinose, which, in turn, suggests the increased strength of saccharide – glycine/water interaction. As the temperature of the system increases the value of η decreases for each studied saccharide. Increase in thermal energy of the system, due to rise in temperature, promotes the breaking up of saccharide–glycine/water aggregates. This facilitates the flow of the system, making η decrease with temperature.

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Figure 1

Plots of viscosities, η versus molality, m of (a) glucose, (b) sucrose, and (c) raffinose in aqueous glycine at different temperatures.

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The viscosity data (Table 1) for saccharides in aqueous glycine at different temperatures have been analysed by using the expression (Jones and Dole, 1929):

(8)

$${\eta }_r=\eta /{\eta }_0=1+{\mathit{Ac}}^{1/2}+\mathit{Bc}$$ where η_r is the relative viscosity, η₀ and η are the viscosities of solvent (aqueous glycine) and solution, respectively, and c is the molarity calculated from molality data (Motin, 2004) for saccharides. A, Falkenhagen coefficient reflects the solute–solute interactions, whereas, B, the Jones–Dole coefficient, provides information regarding solute–solvent interactions in the solution. A- and B-coefficients were obtained by the least-squares method as intercept and slope of the linear plot of (η_r − 1/c^1/2) against c^1/2 and are summarised in Table 6.

Table 6 Values of A- and B-coefficients, n_H, $\mathrm{\Delta }{\mu }_1^{0\#}$ , and $\mathrm{\Delta }{\mu }_2^{0\#}$ of glucose, sucrose, and raffinose in aqueous glycine at different temperatures.

Parameter	T/(K)
	298.15	303.15	308.15	313.15
Glucose + aq. Glycine
A (10−2 dm3/2 mol−1/2)	−0.41	−0.27	−0.17	−0.10
B (dm3 mol−1)	0.93	0.63	0.50	0.36
nH	8.65	5.27	3.01	1.84
$\mathrm{\Delta }{\mu }_1^{0\#}$ (kJ mol−1)	60.52	61.27	62.02	62.78
$\mathrm{\Delta }{\mu }_2^{0\#}$ (kJ mol−1)	201.79	162.00	146.33	128.45
Sucrose + aq. Glycine
A (10−2 dm3/2 mol−1/2)	−1.41	−1.26	−0.80	−0.53
B (dm3 mol−1)	1.02	0.94	0.67	0.49
nH	10.64	8.93	5.62	2.46
$\mathrm{\Delta }{\mu }_1^{0\#}$ (kJ mol−1)	60.52	61.27	62.02	62.78
$\mathrm{\Delta }{\mu }_2^{0\#}$ (kJ mol−1)	227.72	219.14	184.88	160.70
Raffinose + aq. Glycine
A (10−2 dm3/2 mol−1/2)	0.14	−2.76	−1.65	−1.90
B (dm3 mol−1)	1.48	1.53	1.15	1.12
nH	14.9	11.17	8.96	5.12
$\mathrm{\Delta }{\mu }_1^{0\#}$ (kJ mol−1)	60.52	61.27	62.02	62.78
$\mathrm{\Delta }{\mu }_2^{0\#}$ (kJ mol−1)	316.18	327.78	278.59	278.84

An inspection of Table 6 shows that B-coefficients are larger than A-coefficients, suggesting the presence of strong solute–solvent interactions as compared to weak solute–solute interactions, hence supporting the behaviours of $V_{\varphi }^0$ and S_v, respectively. In our studied systems, the observed values of B-coefficients are positive for all the saccharides at each investigated temperature, follow the sequence: glucose < sucrose < raffinose. This suggests increased saccharide–glycine/water interaction and that the strength of the interaction follows the above sequence. This is explained by considering that as the number of hydroxyl groups increases from glucose to raffinose so does the number of H-bonds formed between saccharides and water/glycine molecules, (Mishra et al., 1997) resulting in increased B-values in the sequence mentioned above.

The hydration number, n_H of saccharides studied is obtained by the relation (Mishra et al., 1997; Linow and Philipp, 1984):

(9)

$$n_H=\left(\overline{V_{\eta }}-\overline{V_2^0}\right)/\overline{V_1^0}$$ where $\overline{V_{\eta }}$ is the volume of the hydrated solute and can be obtained as the slope of the plot of η_sp/c vs. η_sp (specific viscosity of the solution) (Linow and Philipp, 1984). $\overline{V_2^0}$ and $\overline{V_1^0}$ are the partial molar volumes of solute and solvent, respectively. Hydration numbers vary with the method used to calculate them, (Parke and Birch, 1999) so it is not fair enough to compare the values obtained by different methods. Thus, it is reported that for saccharides n_H may range from 1.8 (NMR) to 21 (NIR), (Allen et al., 1974) for sucrose in aqueous solutions, its values are reported as 5.3 (intrinsic viscosity), (Bressan and Mathlouthi, 1994) 5 (activity measurement), (Akhumov, 1981) and 13.8 (ultrasonic velocity data) (Chen et al., 1981). The value of hydration numbers of the saccharides investigated is given in Table 6. The hydration number mainly comes from the hydrogen bond formation by hydroxyl groups of saccharides with water as these hydroxyl groups have strong hydration ability. The hydration number so obtained follows the order glucose < sucrose < raffinose, but the computed values (Table 6) are not showing a proportional increase in the hydration number with the number of hydroxyl groups in glucose, sucrose, and raffinose which lead us to conclude that all the hydroxyl groups are not freely available for hydration with water. The compatibility of the studied saccharides with the 3-D hydrogen bond structure of water depends not only on the number of hydroxyl groups and oxygen atoms but also on the position (axial or equatorial) of the hydroxyl groups. In our system glucose is a monosaccharide with three equatorial and one axial hydroxyl group (Chen et al., 1981). At 298.15 K the n_H for glucose is approximately 9 which is quite a large number suggesting that the hydroxyl groups of glucose are available for hydrogen bonding with water molecules and hence pack well within the structure of water. The sucrose molecule consists of glucose and fructose monosaccharide units and contains two intramolecular hydrogen bonds (Seuvre and Mathlouthi, 2010). It has a large number of equatorial hydroxyl groups, which might be the reason for its higher hydration (approximately 11 at 298.15 K) than glucose, whereas raffinose is a trisaccharide in which galactose is connected to the glucosyl group of sucrose via α-1, 6 linkage (Cheng and Lin, 2006). It’s n_H at 298.15 K is approximately 15, which is larger than the n_H of glucose and sucrose. This suggests that though the value of n_H increases with the increase in the number of –OH groups as we move from glucose to raffinose, the increase is not in proportion to the number of hydroxyl groups in these saccharides. This is due to the fact that steric hindrance is least for glucose and maximum for raffinose molecules for the formation of H-bonds with water molecules.

The free energy of activation of viscous flow per mole of solvent $\mathrm{\Delta }{\mu }_1^{0\#}$ was calculated using the relation proposed by Eyring and co-workers (Glasstone et al., 1941):

(10)

$${\eta }_0=({\mathit{hN}}_A/{\bar{V}}_1^0)\exp (\mathrm{\Delta }{\mu }_1^{0\#}/\mathit{RT})$$ where h, N_A, and ${\bar{V}}_1^0$ are the Planck’s constant, Avogadro number, and partial molar volume of the solvent, respectively, and R, the universal gas constant. The rearrangement of Eq. (10) yields:

(11)

$$\mathrm{\Delta }{\mu }_1^{0\#}=\mathit{RT}\ln \left({\eta }_0{\bar{V}}_1^0/{\mathit{hN}}_A\right)$$ The free energy of activation of viscous flow per mole of solute $\mathrm{\Delta }{\mu }_2^{0\#}$ can be calculated by using the (Feakins et al., 1974, 1993) extension of Eyring transition-state theory:

(12)

$$B=\left({\bar{V}}_1^0-{\bar{V}}_2^0\right)/1000+{\bar{V}}_1^0\left[(\mathrm{\Delta }{\mu }_2^{0\#}-\mathrm{\Delta }{\mu }_2^{0\#})/1000\mathit{RT}\right]$$ where ${\bar{V}}_2^0(=V_{\varphi }^0)$ is the partial molar volume of the solute. The rearrangement of the above equation gives:

(13)

$$\mathrm{\Delta }{\mu }_2^{0\#}=\mathrm{\Delta }{\mu }_1^{0\#}+\left(\mathit{RT}/{\bar{V}}_1^0\right)\left[1000B-({\bar{V}}_1^0-{\bar{V}}_2^0)\right]$$ The computed values of $\mathrm{\Delta }{\mu }_1^{0\#}$ and $\mathrm{\Delta }{\mu }_2^{0\#}$ at different temperatures are given in Table 6. It reveals that the values of $\mathrm{\Delta }{\mu }_2^{0\#}$ are much larger than $\mathrm{\Delta }{\mu }_1^{0\#}$ values which suggest that the formation of the transition state is less favoured in aqueous glycine and that there are strong glucose/sucrose/raffinose–solvent (glycine + water) interactions in the ground state than in the transition state, which might be because of the breaking and distortion of intermolecular bonds in the latter state. The values of $\mathrm{\Delta }{\mu }_2^{0\#}$ of studied saccharides are increasing in the order glucose < sucrose < raffinose. This implies that more energy is required for the saccharides having more sugar units with complex structure in transferring from ground state to the transition state.

The free energy of activation of viscous flow of solution ΔG^0# was evaluated from the relation:

(14)

$$\mathrm{\Delta }{G}^{0\#}=n_1\mathrm{\Delta }{\mu }_1^{0\#}+n_2\mathrm{\Delta }{\mu }_2^{0\#}$$ where n₁ and n₂ are the respective number of moles of binary solvent (aqueous glycine) and solute (glucose/sucrose/raffinose), respectively. The enthalpy, ΔH^0# and entropy, ΔS^0# of activation of viscous flow were calculated by using the equation:

(15)

$$\mathrm{\Delta }{G}^{0\#}=\mathrm{\Delta }{H}^{0\#}-T\mathrm{\Delta }{S}^{0\#}$$ The values of ΔH^0# and ΔS^0# were obtained as intercept and slope, respectively, from the plots of ΔG^0# vs T. The results are shown in Table 7. ΔH^0# and ΔS^0# are useful in providing the structural information about solute species and solute–solvent interactions. It is clear from the Table 7 that ΔH^0# values are positive and increase with an increase in the concentration of glucose, sucrose and raffinose which indicates that the formation of activated species necessary for viscous flow seems to be difficult as the amount of these saccharides increases in aqueous glycine. The ΔS^0# values are negative and exhibit a pronounced decrease with the increase in concentration of all the three saccharides, pointing out that the systems become more structured as a result of strong interactions between solute and solvent molecules.

Table 7 Enthalpies, ΔH^0# and entropies, ΔS^0# of activation of viscous flow of solutions of glucose, sucrose, and raffinose in aqueous glycine at different temperatures.

m (mol kg−1)	ΔH0# (kJ mol−1)	ΔS0# (10−2 kJ mol−1 K−1)
Glucose + aq. Glycine
0	0.23	0.23
0.02	9.35	−1.55
0.04	18.46	−3.32
0.06	27.57	−5.10
0.08	36.69	−6.87
0.10	45.80	−8.64
Sucrose + aq. Glycine
0	0.23	0.23
0.02	32.97	−9.19
0.04	65.70	−18.60
0.06	98.43	−28.01
0.08	131.16	−37.43
0.10	163.90	−46.84
Raffinose + aq. Glycine
0	0.23	0.23
0.02	25.95	−6.22
0.04	51.67	−12.67
0.06	77.39	−19.12
0.08	103.10	−25.57
0.10	128.82	−32.02