NOTE This ratio determines how many parts water to amylase. If the concentration is too weak then the experiment will take too long. The ratios of water to amylase must have an equal total; in the case above the totals are 10. The concentrations must all have the same volume and the starch must also have a definite volume.
At the end of the experiment the rate of breakdown will be plotted on a graph against the concentration of amylase. The rate of breakdown can be worked out by the equation on the left. Time is measured in seconds. Rate is measured in arbitrary units of starch/second.
APPARATUS
I will use the following apparatus for this investigation:
- Test tube rack
- Test tubes
- Petite pipette – new one for each concentration.
- Measuring cylinder
- Beaker
- Tile
- pH paper
- Iodine solution
- Amylase
- Water
- Starch
FAIR TEST
- Amount of starch should be kept the same. This can be ensured when using a pipette. (However, when using a pipette, care should be taken to avoid contamination of the solution.) The amount can be decided during the pilot experiment.
- Volume of enzyme must be kept the same. This can be decided during the pilot experiment.
- I must mix the immediately after the enzyme has been added.
- Temperature must be kept the same because if it is not kept the same in all of the test tubes, some of the enzymes will work faster in some tubes than in others. In order to keep the temperature the same, the tubes will be stood in cold water at room temperature, so that it will not fluctuate. Therefore, the experiment will work better because of the increase of kinetic energy and collisions.
- The pH needs to be tested as amylase works best in alkaline conditions, because the pH effects the shape of the enzymes. The pH must be above 8.5 and below 6.5. If the pH is too acidic, then the enzyme will work slower in some test tubes than in others. In order to test the pH of the amylase a drop of the solution should be put on pH paper. This should be done both at the beginning of the experiment and at the end to see if the pH stayed the same throughout.
- In order to keep it a fair test, I must choose a shade of brown that I will stop the stopwatch at. The reason for this is because if I leave the stopwatch on until the iodine turns dark brown and then on one of the other tubes until the iodine changes to light brown, the timing would be unreliable.
PILOT EXPERIMENT
I will use the pilot experiment to determine the volumes of starch and amylase that will be used in the main experiment. If too much is starch is added, the experiment will take too long because there are too many enzymes to active sites. I will use the following method for this experiment:
- I will prepare a tile with drops of iodine solution. I will get a test tube rack and fill it with nine test tubes. The starch for each test tube will be prepared.
- I will choose a concentration of water and amylase from the list of ratios (see table under independent variables).
- Add the concentration of amylase and water to the starch in one of the test tubes. Immediately after this start the stopwatch and stir the solution.
- Every 30 seconds add a drop of the solution to the iodine solution, using a petite pipette.
- Once the iodine solution is no longer black and has changed to the chosen shade of brown, I will record the results.
- If the iodine solution turns brown before the 30 seconds the concentration of amylase is too strong and if it takes over 3 minutes to breakdown the starch then the solution is too weak.
PILOT EXPERIMENT RESULTS
The Pilot experiment was carried out to determine the volumes of starch and amylase that were going to be used in the final experiment. The results above show that the higher the volume of 100% amylase to the volume of 100% starch then the quicker it takes to breakdown. Other volumes of amylase to starch were used, but these took to long to breakdown. This is because there were too many enzymes to active sites.
In the final experiment, I have decided to use a volume of 4cm³ of
amylase to 6cm³ of 100% concentrated starch. I have decided to use
these volumes, as this is the result that worked to a reasonable time. I
have also decided to use the concentrations 20%, 40%, 60%, 80% and 100%. I
will also need to use a 0% concentration of amylase, or 100% water, as a
control experiment, in order to prove that the enzyme is digesting the
starch, not the water.
FINAL METHOD
I will use the following method to determine the effect of the concentration amylase on the rate of breakdown of starch:
- All the apparatus will be set up as in the pilot experiment. A tile will be prepared with a couple of drops of iodine on it. The starch for each test tube will be prepared and the concentrated amylase solution will be added later. A drop of the Amylase and water will be placed on the pH paper.
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The starch will be taken and added together with the amylase. The two volumes are 4cm3 of amylase and 6cm3 of starch, concluding a total volume of 10cm3. NOTE as soon as the two have been mixed together, the stopwatch should be started.
- Every thirty seconds a few drops of the solution would be added to the iodine. A different petite pipette should be used for each concentration so as not to contaminate them.
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Once the iodine solution is no longer black I will record the time. Once I have the results I will record the rate at which the starch broke down. This will help me to come to a conclusion about the effect of the concentration of the amylase on the rate of breakdown of starch.
- Replicates of each test tube and its contents will be made to allow the most accurate results to be gained. Three replicates of each test tube will be created.
- At the end of experiment the solution will be put on the pH paper to determine whether the pH has stayed the same throughout the entire experiment.
FINAL EXPERIMENT RESULTS
CONCLUSION
As I predicted at the beginning of this investigation, the results show that the higher the concentration of amylase, the faster the breakdown of starch. The control test tube shows that starch does not break down when added to water. The table shows that as the concentration was doubled, the rate of breakdown of amylase also doubled. Although the table does not seem to show the anomalous results they can be clearly seen on the graph.
The graph shows the rate of breakdown plotted against the concentration of amylase. In order to see the trend of the results more clearly I used a line of best fit. This made the results that were not on the line more visible. Although the results were not on the line, they were very close to it. This can be justified by the fact that when the experiment took place, inaccuracies may have happened with the higher concentrations because the breakdown took less time than the weaker solutions.
EVALUATION
During the experiment various problems were encountered. Although the graph does not show any anomalous results as such, not all of the results are on the line of best fit. There are several things that could have caused this.
Room temperature may have varied throughout the experiment, which may have effected the efficiency of the enzyme. If the temperature had have decreased, it would have caused the enzymes to become less and less active. If the experiment was performed again, to make the results more accurate, the water bath would be used to make the temperature more controlled. This would make it fair test, because all the tubes could be kept at the same temperature. However, in the final experiment the temperature was the same at the end, as at the start. The pH also remained the same during the experiment meaning it was working in the best conditions throughout.
If we were allowed longer to complete the investigation then more replicates could have been made. This would ensure that the final result was as accurate as possible. Also with more time, more concentrations could have been created. All in all with more time allowed for this investigation, more results would have been gained. In terms of the graph, the more replicates being made and the more concentrations being tested would have meant that it would have been easier to see a pattern, as there have been more results.
Another problem which may have made the results vary would have been the fact that the starch was left out so long. Being left out for a while meant that the starch became viscous and stuck to the sides of the boiling tube. This could have resulted in a variation of the volume of starch being mixed together with the different concentrations of amylase.
In the future a more accurate measuring cylinder could be used because the measuring cylinder used in this investigation was only to the nearest 0.1 cm3. The colour of the iodine solution could have also caused some inaccuracies because different shades of brown could have been chosen. This would have made the time taken for the starch to break down different.
Overall, I think I used my time well, I followed my method and gained fairly accurate results in the time given. My prediction was supported by the results.