2 Boiling tube racks,
Milk,
Sodium Carbonate,
Phenolphthalein,
Spirit Thermometer,
5% Lipase.
Procedure:
Step 1: We mixed 5cm³ of milk with 7cm³ of Sodium Carbonate and added 6 drops of phenolphthalein into each of 5 boiling tubes.
Step 2: We next prepared 1cm³ of Lipase into the remaining 5 boiling tubes.
Step 3: Boiling water from the kettle was then added to the beaker and with cold water the temperature was lowered to 80°C.
Step 4: Into this beaker was then placed a boiling tube containing the milk mixture and a boiling tube containing the lipase, and were left to stand for 5 minutes.
Step 5: After 5 minutes of allowing the two mixtures to heat up the lipase was poured into the milk mixture, gently shaken and then placed back into the beaker.
Step 6: We then time how long it took for the pink colour change to a white mixture indicating the fat had been hydrolysed.
Step 7: The beaker of water was then allowed to cool to 60°C and steps 4 to 6 were repeated.
Step 8: The water was further cooled to 40°C and steps 4 to 6 were repeated.
Step 9: Then the water was cooled to 20°C and steps 4 to 6 were repeated.
Step 10: Finally a control experiment was preformed using tap water at room temperature following steps 4 to 6.
Safety procedures
- Lipase is irritant take care not to get it on your skin, to prevent this wear surgical gloves
- Phenolphthalein is flammable, keep away from naked flames
Below is a table of the times it took for lipase to convert enough fats into fatty acids and glycerol to lower the pH below 8.3 so that the indicator would show this.
If the results are put into a scatter graph (below) and a polynomial trend line added you can see the rate of reactions in comparison.
At 20°C we can see the lowest ROR at 2.9 g/s, next is the control experiment which was carried out at room temperature 21°C and the ROR was slightly higher at 3.2 g/s.
At 40°C the rate of reaction increase noticeably up to 5.2 g/s as the time taken was nearly halved.
At 60°C there was a slight increase in ROR than previously with the time taken only slightly quicker.
When tested at 80°C the reaction surpassed the allocated 30min barrier set out and there was no change in the colour of the mixture.
From the graph we can see the general trend that if you raise the temperature you will get an increased rate of reaction (ROR) until a certain temperature, when the reaction slows and stops.
At 20°C the ROR was slowest due to the kinetic energy being applied, in the form of heat. The chemical reactions in this experiment rely on random collision between the Lipase enzymes and the substrate, which in this case was the fat in the milk. At 20°C the kinetic energy is low so the particles will only be travelling in the mixture at a slow speed. Therefore results in a slower ROR because less enzymes and substrates collide.
For the control experiment, at 21°C the ROR was slightly higher. This is due to the kinetic energy. At a higher temperature there is more kinetic energy so as stated above more collisions therefore faster rate of reaction.
At 40°C we saw a steep increase in the rate of reaction from 2.9g/s at 20°C to 5.2g/s at 40°C. At 40°C there is a large increase in heat and therefore a large increase of kinetic energy. The particles are moving around much faster resulting in many more collisions than previously so there are more enzyme complexes formed, therefore a faster ROR.
At the temperature of 60°C the ROR was only slightly higher than for 40°C. This is due to the lipase beginning to be denatured. As the kinetic energy increases the lipase molecules starts to vibrate more. At a certain point this vibrating becomes too great and the weak hydrogen bonds that hold the proteins into its structure begin to break. These breaks result in the lipase’s structure shape changes, which changes the shape of the active site. With a different shaped active site the substrate will be not fit into it and will no longer be able to form complexes with it. (~~~~~~~~~~) (See fig1)
However the ROR is still higher than for 40°C, which shows us that some of the lipase reacted with the fat before it started denaturing. This is because at the rate of denaturing increases as the temperature increases. Denaturing usually takes place in human enzymes between 40°C and 45°C and this can be seen in the graph as ROR begins to slowdown increasing around 40°C.
At the top temperature of 80°C there was no change detectable due to the large amount of kinetic energy subjected to the lipase and it had become denatured. There would have been a little amount of reactions taking place but not enough to lower the pH so that we could observe a change.
