Physicochemical, Spectroscopic and Rheological Studies on Eucalyptus Citriodora (EC) Gum

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Table 1. Physicochemical parameters of EC gum

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Table 1 also presents proximate composition of EC gum. From the results obtained, excessive concentration of carbohydrate (90.11 %) and absence of dietary fibre were observed. This finding is consistent with those reported for most plant gums ^[13]. Generally, plant and seed gums are complex polysaccharides/carbohydrate polymers and are mostly used as thickening agent, foaming agent, film, emulsifier, stabilizer and drug delivery agent because of their high carbohydrate content. Concentrations of nitrogen and protein in the gum were 0.69 and 4.29 % respectively, which is comparable to those reported for Ferula gumosa gum (0.69 % ;^[14]), Albizia gums (0.46 – 1.09 %; ^[3]). However, mean concentration of nitrogen in EC gum was found to be higher than those reported for gum Arabic (0.26 – 0.39 % ; ^[15]) and are also comparable to the range of values reported for some species of Acasia sengal gum (0.21 to 0.35 %; ^{[16, 17, 18]}). The important of nitrogen (hence protein) in gums cannot be overemphasized. The immune responses, which are important in providing evidence for the safety of food additives, are customarily accredited to the proteinaceous component of food (Youssef et al., 2009). According to Pablyana et al. ^[4], the presence of protein in polysaccharide can induce inflammatory response to tissue and the response may have a vital role to play in its pharmacological applications.

Moisture content of EC gum (4 %) was relatively low indicating that the gum has low water absorbing or retaining capacities. Similarly, the lipid content of the gum was as low as 1.3 % indicating that the gum has low potential to turn rancid. The low ash content of the gum (0.30 %) also suggests that it has much organic content than inorganic constituents. Plant gums are polysaccharides containing various metal ions as neutralized cations. Therefore, concentrations of Mn, Fe. Zn. Mg, Ca, Cu, Ni, Na and K in EC gum were analysed using atomic absorption spectrophotometer and the results obtained, indicated high concentrations of essential metal ions (Ca = 8.74 %, Fe = 2.41 % and Zn = 0.75 %). Trace metals were presence in trace concentrations (i.e, Mn = 0.212 %, Mg = 0.085; Cu = 0.039 %; Ni = 0.124 %) while toxic metals were found in concentrations that did not exceed their tolerance limit (i.e Pb = 0.088 and Cd = 0.001 %) ^[19]. Therefore, EC gum is may be a good source of mineral nutrition.

2.2. Spectroscopic Study

The GCMS spectrum of EC gum indicated the presence of 15 peaks (spectrum not shown). Chemical structures of compounds identified from each line on the spectrum are presented in Figure 2 while Table 2 presents the molar mass, retention time and percentage concentrations of the identified compounds. From the results obtained, line 1 to 4 revealed the presence of 1.13 %, 3.70 %, 4.93 % and 3.58 % of pyran-4-one, 1,3-dioxolane, benzofuran and 1,2-ethanediyl acetal while lines 5 to 14 revealed the presence of carboxylic acids at concentrations and retention time presented in Table 2.

Table 2. Analytical parameters deduced from GCMS spectrum of EC gum

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Figure 2. Chemical structures of compounds in EC gum

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Figure 3 presents FTIR spectrum of EC gum while Table 3 presents peaks and frequencies of IR adsorption as well as associated functional groups. The spectrum displayed features, typical of polysaccharides. Thus OH stretch vibrations were found at 3492, 3379 and 3268 cm^-1 respectively. The peak obtained at 2911 cm^-1 is due to stretching modes of C-H bonds of methyl group (CH₃). According to Nep and Conway ^[20], natural gums usually contain fractions of sugar acid units which will usually impart a weakly anionic character to the gum macromolecule. C=O stretches due to acetyl groups were prominent at 1710 and 1245 cm^-1 respectively. Absorption bands at 1615 and 1049 cm^-1 are typical for carboxylate groups of the galacturonic acid residues ^[21]. Other functional groups identified were CH₂ twisting vibration at 1313 cm^-1, OH stretch due to carboxylic acid at 1448 cm^-1, NO asymmetric stretch at 1510 cm^-1 and C-X stretch vibrations due to halides at 837, 749, 572 and 524 cm^-1.

Table 3. Peak, frequency and assignment of FTIR absorption by AO gum

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Figure 3. FTIR spectrum of EC gum

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Figure 4. Scanning electron micrograph of EC gum at 1.00 KX magnification and scale bar of 2 µm

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Scanning electron microscopy (SEM) gives image of a surface of a materials/specimens at a desired position and therefore displayed topographic/morphological picture with better resolution and depth of focus. SEM is often used in studying the morphology of polymer materials ^[22]. The scanning electron micrographs of EC gums at 1.00, 3.00 and 5.00 KX magnifications are shown in Figure 4. At magnification of 1.00 KX, the micrograph revealed a smooth surface with irregularly spaced pores. The system has an irregular network but could likely provide favoured adsorption sites. At 3.00 and 5.00KX, the micrographs indicated the existence of particle aggregations with some internal bridges within the system

Figure 5. Variation of (a) relative viscosity with pH (b) absolute viscosity with concentration for EC gum

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2.3. Rheological Study
2.3.1. Effect of Concentration and pH

Figure 5a shows the variation of viscosity with concentration for EC gum. The absolute viscosity of the gum is found to increase with increase in concentration indicating that there is an increase in internal resistant of the gum to shear or flow. This may be attributed to intermolecular friction exerted when layers of fluids attempt to slide over one another.

According to Newton law of friction, dynamic or absolute viscosity of a polymer can be expressed as follows ^[23],

(1)

where τ is the shearing stress, dC/dy is the concentration gradient. From equation 1, it can be seen that at constant shear rate, the absolute viscosity of a viscous fluid is expected to increase with increase in concentration as shown in Figure 5a. On the other hand, Figure 5b presents plots for the variation of specific viscosity with pH for EC gums. It is evident from the Figure that the specific viscosity of the gum tends to increase with increase in pH. The observed trend may be explained as follows. At low pH, the electrostatic repulsions between gum particles tend to increase indicating that the particles will present a decreasing association as a results of the formation of weak intermolecular forces (including hydrogen bond, Van der Waals and other weak forces). Therefore the viscosity of the gum will increase until a certain critical pH is approached. Above the critical pH (pH = 8), the dissociation of the particles may increase due to decrease in the strength of electrostatic repulsions.

2.3.2. Intrinsic Viscosity

In viscosity study, several terms relating solution and solvent viscosities can be defined as follows,

(2)

(3)

(4)

(5)

where η_sp is the specific viscosity, η_rel is the relative viscosity, η_solutionis the viscosity of the solution, η_solvent is the solvent viscosity and C is the concentration. The intrinsic viscosity [η] is a measure of the inherent ability of a polymer to increase solution viscosity. The most appropriate relationship that relates intrinsic viscosity with the viscosity of a dilute polymer solution is a power series in concentration and can be written as follows;

(6)

The expression, η_sp/C is the reduced viscosity and as C→0, it becomes the [η] hence the Power series expressed in equation 5 becomes truncated to a linear approximation which is known as the Huggins equation ^[24]:

(7)

where η_red is the reduced viscosity and k_H is the intrinsic viscosity. From equation 7, a plot of η_red = (C) should be linear with intercept and slope equal to [η] and k_H[η]² respectively. Figure 6 presents Huggins plot for EC gum. The plot reveals excellent degree of linearity for the plot (R² = 0.9996), which confirm the application of Huggins model. The intrinsic viscosity calculated from the intercept of the plot was found to be 4.31 dL/g while the k_H value was 0.13. Theoretically, the k_H should lies between 0.3 and 0.8 in a theta solvent. According to Ma and Pawlik (2007), a polymer exhibits a higher value of k_H in a poor solvent than in a good solvent because polymer-polymer interaction is favourable. However, calculated value of the k_H for EC gum is relatively lower than the expected range hence water is a theta solvent for EC gum. It has also been found that intrinsic viscosity can also be obtained from the Kraemer equation by extrapolation to zero concentration ^[25]:

(8)

where k_K is the Kraemer constant. Kraemer’s plot for EC gum is also shown in Figure 6. The intrinsic viscosity calculated from the Kraemer’s plot ([η] = 2.72 dL/g) was lower than the value obtained from the Huggins plot. The degree of linearity of the plots (R² = 0.9980) was also excellent and the Kraemer constant (k_K = 0.23) was relatively higher than the k_H. However, the sum of Kraemer and Huggins constants was 0.36 which is below the range (i.e 0.5 ± 10 %) of values expected for gums that are characterized by the absence of molecular association. This suggests the existence of molecular association between the molecules of EC gum.

Figure 6. Huggins and Kraemer plots for EC gum

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2.3.3. Molecular Conformation and Coil Overlap Parameter

The Power law model (equation 8) is useful for studying molecular conformation as well as the level of interactions between molecules in a solution of a polymer ^[26]

(8)

Simplification of equation 8 yielded equation 9 and from equation 9, a plot of ln(η_sp) versus C was found to be linear with slope and intercept equal to b and ln(a) respectively(plot, not shown).

(9)

The results obtained indicated an excellent correlation (R²= 0.9329) while calculated ‘b’ value was 0.812 for EC gum. The value of ‘b’ is significant in rheological studies because it is an index for predicting the conformation of polysaccharides ^[26]. Since ‘b’ value greater than unity is associated with random coil conformation and ‘b’ value less than unity is associated with rod like conformation, the conformation of EC gum is more rod/coil like than random like ^[27].

It has been found that in dilute solution, the individual polymer coils are separated from each other and are free to move independently but with increasing concentrations, the coil starts to overlap and interpenetrate one another. According to Morris et al. ^[28], the transition from dilute solution to concentrated solutions is usually accompanied by pronounced change in concentration dependence of solution viscosity and the corresponding concentration is called critical or coil overlap concentration (C*). The concentration dependence of the specific viscosity (at zero shear rate) on the coil overlap parameter, C[η] for EC gum (plot not shown) was found to displayed two regimes indicating the existence of molecular entanglement. The first and second regimes were observed at slope values of 1.04 and 0.95 respectively. This indicate that when C> C*, η_sp0α C^1.04 while at C < C*, η_sp0α C^0.95. Hence, C*[η]∼ 3.37 and the critical concentration for coil overlap is 1.12 g/dL.

2.3.4. Effect of Temperature

The effect of temperature on the viscosity of EC gum was studied between a temperature range of 303 and 343 K. A decrease of viscosity was observed and there was no degradation or conformational transitions during heating. Since there was no conformational transition, the Arrhenius-Frenkel-Eyring equation was applied to calculate the activation energy of flow using equation 10 ^[29]

(10)

where η is the absolute viscosity of EC gum, B is the pre-exponential constant, E_F is the activation energy of flow, R is the gas constant and T is the absolute temperature. From the logarithm of equation 10, equation 11 was developed and used to plot ln(η_sp) = (1/T) as shown in Figure 7. The plots exhibited good correlation coefficient (R² = 0.8723) while calculated value of E_F was 16.67 kJ/mol. This value is within the range of values reported for some plant gums including Albizia lebbeck gum (15.9 to 17.2 kJ/mol, ^[2]), Arabic gum (15 kJ/mol, ^[30]), A. occidentale gum (16.2 kJ/mol, ^[31]) and A. macrocarpa gum (16.8 kJ/mol, ^[32]). Generally, higher value of E_Findicates that the polymer solution is less sensitive to change in temperature and vice versa. Low E_F also indicates the existence of few inter- and intra interaction between polysaccharide chains in the studied concentration range, which is the case for the studied gum. The E_F values for polymers strongly bonded by inter and intra-molecular interaction has been found to in the range of 27 kJ/mol and above ^[33].

Thermodynamic parameters of viscous flow were also calculated using the Frenkel-Eyring equation ^[23], which can be written as follows:

(11)

where A is the pre-exponential factor, R is the universal gas constant, T is the absolute temperature, and are the entropy and enthalpy changes of viscous flow. From equation 11, a plot of versus 1/T is expected to be linear with slope and intercept equal to and respectively. Figure 7 also presents the Transition state plot for EC gum. Calculated values of ΔS_V and ΔH_V were -55.77 and 14.09 kJ/mol. The negative and positive values obtained for ΔS_V and ΔH_V respectively, suggest that the attainment of transition state of viscous flow is accompanied by bond breaking. The entropy change of activation from the initial to the transition state at a given composition is significant during an activated viscous flow process; indicating that this process may be entropy-controlled ^[34]. Negative values of is associated with uncoiling orientation of the polymer molecules. Thus, the system becomes more ordered in the course of flow. On the other hand, is related to the amount of energy needed by the gum molecules to jump from one equilibrium position to another (i.e the potential energy barrier) ^[35].

Figure 7. Arrhenius and Transition state plots for EC gum

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2.3.5. Effect of Electrolytes

Like all other exudate gums, EC gum is acid polysaccharides containing various metal ions (including, Mn, Fe. Zn. Mg, Ca, Cu, Ni, Na and K) as neutralized cations. Since this gum will behave like polyelectrolyte, then solution viscosity of the gum will be affected by the addition of other electrolytes. In this study, effects of KCl, CaCl₂, AlCl₃ and urea on the viscosity of EC gum were investigated. Addition of these electrolytes was found to reduce the viscosity of the gum as shown in Figure 8. The trend in the decrease corresponded to decreasing charge of the ions (i.e, Al³⁺>Ca²⁺>K⁺). The decrease in viscosity can be attributed to the existence of fewer intermolecular interactions due to the screening of charges and contraction of the macromolecule in the presence of counter ion ^[2]. From literature, it is evident that aluminum has a tendency to establish strong interaction with macromolecules through intermolecular crosslinking effect. On the other hand, the strength of intermolecular crosslinking effect is lower in Ca²⁺ than in aluminum while sodium is not a cross linking agent.. Ahmad et al. ^[35] found that in dilute solution, the turbidity of dilute polyelectrolyte increases with increase in the charge of the ions. Therefore, the variation of the viscosity of EC gum with increasing charge can also be attributed to their effect on turbidity. It is significant to state that the affinity between the gum and counter ions depends on the charge/ionic ratio. Generally, ion with higher charge will have a stronger affinity for the molecular chain of the gum. The charge to ionic radius ratio of K⁺, Ca²⁺ and Al³⁺ are 0.66, 1.75 and 4.41 respectively hence the expected order for the interaction should be Al³⁺> Ca²⁺>K⁺, which is in agreement with our findings. Urea was also seen to decrease the viscosity of EC gum because it has the ability to trap some of the gum’s molecules into its clathrates, which is an organic guest that are held in channels formed by interpenetrating helices of hydrogen-bonded molecules. The withdrawal of some organic molecules by urea guest may lower polymer-polymer interaction and will results in a decrease in viscosity as observed in the study.

Figure 8. Variation of viscosity of EC gum with concentration in the presence of 0.1 M of various electrolytes (KCl, CaCl₂, AlCl₃ and urea)

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Ionic strength is a very important factor in studying the rheology of polymer solutions. Ionic strength affects the thickness of the electrical double layer around the charged interfaces as well as modifying the conformation of the polymer attached to the surface (i.e spatial extension). The effect of ionic strength on the viscosity of EC gum was studied by monitoring the effect of various concentrations of the electrolytes on the apparent viscosity of 1% (w/v). The results obtained (plots not shown) revealed that the apparent viscosity of EC gum decreases with increase in the logarithm of the salt concentration, which may be attributed to shielding and cross linking effects. The cross linking effect seems to prevail more in the presence of Al³⁺, while the contribution of shielding effect led to chain contraction in Ca²⁺^.As stated earlier, shielding and cross linking effect is associated with the ratio of charge to ionic charge of the metal. In Al³⁺, the ratio is highest therefore cross linking effect dominates but K⁺ having the least ratio displayed strong shielding effect. Sanin ^[36], found that an increase in the ionic strength of a polymer solution will lead to a decrease in viscosity because as more ions are added to the system, the viscosity starts to give less respond by changing minimally. Consequently, the electrical double layer is compressed at high ionic strengths. Particles and flocs may assume closer localities with each other and the surface polymer may also assume a coiled conformation rather than a stretch conformation.

2.4. Steady Shear Properties

In studying steady shear properties of a polymer solution, parameters of interest are shear stress, shear rate, speed of rotation and viscosity. Base on the variation of viscosity with shear rate or shear stress with shear rate, polymer solution can be classified as Newtonian or non Newtonian. Newtonian fluids are fluids which have a constant viscosity indicating that a plot of shear stress versus shear rate for Newtonian fluid should be linear [37-38]^[37]. Deviations from Newtonian systems have been attributed to several factors and it varies from one polymer to another polymer systems.

Fig. 9 shows plots for the variation of (a) shear stress with shear rate (b) speed of rotation with viscosity and (c) speed of rotation with torque. The shape of the plot in Figure 9a indicated that EC gum is non Newtonian and displayed characteristics shear thinning property which is typical for a pseudoplastic flow behavior. Figure 9b revealed a linear plot which is typical for non Newtonian behavior ^[39]. From Figure 9a, the yield stress (i.e the amount of force needed to be applied to the gum before it can flow) was estimated through the intercepts.

Figure 9. Variation of (a) shear stress with shear rate (b) speed of rotation with viscosity and (c) speed of rotation with torque for EC gum

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