Hydrogen bonding between KC and gelatin For further understanding the interaction of KC and gelatin, mixing isothermal calorimetry was performed at different temperatures.Fig. 2a shows the heat flow as a function of time after mixing of 0.75% KC/0.75% gelatin. The absolute enthalpy value of mixing at each temperature determined from the area enclosed by the curve and the baseline is shown inFig. 2b. At temperatures higher than 40◦C, the mixing enthalpy is relatively low, which is contributed mainly by electrostatic complex coacervation. The mixing enthalpy becomes increasingly negative (exothermic) when the temperature is below 40◦C. It suggests that a second mechanism comes into play and reinforces the complexation between KC and gelatin. The transition of the mixing enthalpy is in line with the turbidity increase described above.
To confirm the nature of the molecular interaction that underlies the transition, the effects of urea and glycerol on the transition were investigated. Urea is known as a hydrogen bond breaker[15,24]. Glycerol, as a hydrogen donor, can decrease biopolymer collision rate and increase biopolymer aggregation[25], and is thought to promote hydrogen bond formation. As shown inFig. 3,the onset temperature of turbidity increaseTodecreases strongly with increasing urea concentration. Actually, the transition is completely suppressed at 2 M urea and no turbidity increase can be observed (data not shown). On the contrary, the addition of glycerol slightly increasesTo. These results indicate that urea can greatly inhibit the transition while glycerol can promote it. It manifests that the transition around 40◦C for KC/gelatin mixture during cooling is most likely to originate from hydrogen bonding between KC and gelatin. Since KC and gelatin attract each other by electrostatic interaction as mentioned above, the distance between disordered
chains of these two polymers might be shorten by the electrostatic attraction to favor hydrogen bonding formation. Since the transition is well above the conformational ordering temperatures of KC and gelatin, it could be possible that the local structural arrangement of the biopolymers or the change in local solvent environment prior to their conformational transitions enables the formation of intermolecular hydrogen bonds. Indeed, hydrogen bonding is also
the major molecular force that stabilizes gelatin and KC helices in the ordered state[20].
KC and gelatin exhibit conventional complex coacervation due to electrostatic interaction. The electrostatic interaction is less temperature dependent than hydrogen bonding and hydrophobic interaction [15], and therefore the electrostatic complex coacervate shows a nearly constant turbidity in the temperature range of >40◦C(Fig. 1c). When the temperature is below 40◦C, intermolecular hydrogen bonding between KC and gelatin comes into play, which as an attractive interaction could reinforce the existing complex coacervation induced electrostatically. This leads to an extensive turbidity increase, as observed inFig. 1c.