Ethyl Hexanoate Study

3.1 Preliminary optimization studies

3.1.1. Effect of reaction time:

Figure.3 represents the time progression for the enzymatic esterification of ethanol and hexanoic acid with 1:1 substrate ratio by Novozyme 435 (2 %) at 50 ˚C. It was observed that percentage conversion of ethyl hexanoate reached up to 73.6% in the initial 120 min. However, as the reaction proceeds further, a marginal change in conversion was observed after 120 min because of equilibrium of the reaction. This is attributed to the reversible nature of the esterification reaction. The slow rate of reaction after initial hours is due to collection over time of the reaction product on the enzyme this way reducing the surface area available for the reaction. Those moderate rate

The three-level-three-factor (Three levels as +1, 0, -1 and three factors as molar ratio, enzyme, temperature) CCD requiring 20 experiments were employed to optimize the significant parameters. The variables and their levels selected for the study of ethyl hexanoate synthesis were ethanol/hexanoic acid molar ratio (1:4 to 1:2), enzyme dosage (1 to 3 % w/w), reaction temperature (40 to 60°C). Table 1 shows the independent factors, levels and experimental design in terms of coded and uncoded

It was well known that the collisions between enzyme and substrate molecules increased with an increase in temperature that result in an increase in reaction rate and thus the conversion. It was observed that with an increase in enzyme loading from 1-2% (w/w) increase in percentage conversion. Usually, an increase in enzyme loading increases the number of active sites and therefore more substrate molecules converted into products. The highest conversion could be attained when the enzyme loading was taken to be 2% in combination with the temperature 50 ˚C. The extent of reaction was found to decrease with an increase in temperature from 50 to 60ºC. Because at high temperature, the active site of the enzyme got denatured and no more accessible for distinguish substrate 25. However, with an increase in the enzyme amount above 2 %, decreases the percentage conversion. This can be attributed to disruption of enzyme tertiary structure and denaturation at high temperature