Diagram:
Results: It took 5 minutes for the iodine to stop changing to a blue/black colour.
Evaluation: The change of the iodine’s colour to blue/black indicates that there is still starch present. After 5 minutes, when the iodine stopped changing colour, remaining brown/orange, we can tell that the amylase has digested all the starch, as there is no longer any starch present. The starch has been broken down into another substance by the amylase.
Because I am only investigating one factor, pH, I will need to control all other factors that could possibly affect the experiment. This means that I need all the chemicals to be kept at the same temperature and that temperature will need to be constant. I will make sure that all the chemicals are at room temperature at the start of the investigation. I shall also need to make sure that the concentrations of each chemical are constant throughout. This will not be hard to do, as each chemical comes ready-mixed. I also need to ensure that the same amount of each element is used in each experiment. I will use the same quantities as I did in my preliminary work, 5 ml of starch and 1ml of amylase, as I have tried them and found them to be appropriate. I will test a sample of the solution every 30 seconds. This is what I did in my preliminary work and I found that it worked well. Unlike my preliminary work, I have decided that three drops of the solution will be tested with iodine instead of the one I used before. I think that this will make the gradual change in the colour of the iodine, easier to assess because the quantity of iodine and solution will be greater. These are the factors that I will keep constant throughout the experiment. I will measure out the amount of starch using a measuring cylinder and the volumes of amylase and buffer solution using the scale on a pipette. I will also measure the time it takes for the reaction to take place, using a stop clock. I shall change the pH of the solution with the aid of a buffer solution. I will add this instead of the distilled water I used in my preliminary work. I shall use a range of pH3 –pH9. This will give me a range of results including varying strengths of acid and alkali. Because I am using chemicals, I will wear safety glasses to protect my eyes throughout the experiment.
The equipment I shall require for performing the experiment at each different pH level:
- Spotting tile
- Iodine
- Three 2ml pipettes
- Test tube
- Test tube rack
- 2% starch solution
- appropriate pH level buffer solution
- Stop clock
- 0.25 M Amylase
- Thermometer
The procedure that I will go through at each different pH level, will be the same. First, as in my preliminary work, I shall put one drop of iodine in each well of the spotting tile. This does not need to be measured out because it is only being used as an indicator to test whether starch is present. Then I shall measure out 5ml of 2% starch solution, using the measuring cylinder. Then put this into the test tube. Add 1 ml of buffer solution, measured out using a pipette, to the test tube containing the starch. Mix the two chemicals together. Next I will ensure that the stop clock is set to zero. This is just to make sure that I cannot make an error with the timing because it will affect the whole experiment and cause it to be inaccurate. Before adding the amylase, I will use the thermometer to measure the temperature of the solution in the test tube. I will then measure out 1ml of amylase using a pipette and add it to the solution in the test tube. As I do this, I will also start the stop clock. So that the concentration of amylase is constant throughout the solution, I will then gently mix the contents of the test tube, not too violently in case I affect the reaction. The remainder of the experiment is exactly the same process that I followed in my preliminary work. Every 30 seconds, I will use a pipette to remove 3 drops of the solution and add it to a well in the spotting tile. I will do this until the iodine in the spotting tile ceases turning blue/black and remains its original colour. This is when I will stop the stop clock. This process will be repeated for each different pH level. Because I am performing exactly the same experiment for each pH, I can compare the results fairly.
Method:
I carried out the experiment explained above, exactly as planned. Due to my preliminary work, I had already identified any problems and overcome them when it came to collecting the results for my main experiment. The only problem I encountered was when I performed the tests for starch with an extreme acidic or alkaline solution. I found that at these pH levels, the amylase took a very long time to digest the starch. Because of this I have not obtained results for pH 3 and 9. To ensure the accuracy of my results, I performed the whole experiment three times. The results from all of these trials are presented in the results table. If found that the room temperature was constant for every reading, making it fair to compare the results because they were carried out under the same conditions.
Diagram:
Results:
Analysis:
Graph: a graph of my results can be seen on the following page. It shows how the nearer the pH is to pH 6, the quicker the amylase was digested. The pattern of the results depicts a graph in the form of a ‘bucket’ or curve shape. At the base of the curve, the results form a sharp point. This is because there is clearly only one pH level at which you will get the shortest reaction time. Very few of the points fit exactly on the line of best fit marked on the graph. This is because of the three different trials of the experiment, all marked on the graph. The line, in effect shows the average digestion time for each pH. Because there was quite a large variation between the times for each different trial, the average does not match any of the obtained results exactly. It does however follow the same pattern.
The shape of the graph shows that as the pH gets further away from its minimum digestion time, the reaction time increases. It is this relation that gives the graph its ‘U’ shape where the time taken decreases as it approaches pH6 and then increases again as it moves further away from this pH level. Notice that both ‘sides’ of the curve are approximately equal in gradient. This shows the time to decrease/increase at the same rate. It also means that the graph is symmetrical.
This is the rough shape of the graph on the next page:
As you can see from the picture opposite, I have
numbered several areas of the graphs. This is so that I
can clearly refer to them in the text. As I said in my
prediction, the active site of an enzyme can be changed
by very acidic or very alkaline conditions. Each enzyme
has its own pH at which it works best. This happens
because an acid has spare electrons called H+ ions, an
alkali has spare electrons called OH- ions. It is these
spare ions that attract or repel the enzymes. They can
attach onto or break bits away from the enzyme’s active
site and change its shape. This means that the substrates can no longer react with the enzyme because they don’t fit its active site. At stage 1 on the diagram, there are lots of spare H+ ions in the strong acids. These will change the shape of the enzyme so that the substrate will not fit the active site. At stage 2 on the diagram, the pH is approaching neutral, so there are very few H+ ions. This means that they affect the enzyme less. Because it is a pH 6 that the amylase works best, it obviously needs the few H+ ions to change the active site, so that the substrate matches it. At stage 3 on the graph, the same happens as at stage 1, except that it is OH- ions that are changing the enzyme, not the H+ ions.
My results agree with my prediction. On page 1 explained why I expected the amylase to digest the starch the fastest when at a pH of 6. The graph clearly shows that the lowest value of the time taken occurs at pH 6. Although the exact time taken varies according to the three different trials, they all agree that the starch is digested quickest when at pH6.
Evaluation:
As you can see from my graph on the previous page, I had no anomalous results. None of the results I collected were very accurate because there was wide variation between the 3 sets of results, but they still showed a definite trend. There are many possibilities as to why I got such a variation in my different sets of results. One is that the concentration of enzyme varied. Because the chemicals come ready prepared, there is no way I could have overcome this problem in a practical way. I just have to allow for my results and that they might not be as accurate as possible. Another possible reason for the inaccuracy might have also been a factor that was beyond my control. Sometimes the technicians, who keep the enzyme in the fridge, might have only removed it shortly before I used it. This would mean that the enzyme would be at a colder temperature and would therefore work slower. This might have been the case. I measured the temperature of the solution before I added the amylase. This is recorded as ‘temperature’. It does not, however include the temperature of the amylase. If I repeated this experiment that is certainly a factor that I would look at closely. I would measure the amylase’s temperature before using it and if necessary, I would wait until it has returned to room temperature, as the other chemicals were.
The main problem, which I encountered, was the amylase, at pHs of 3 and 9, took too long to digest the starch. I therefore do not have any results for these pHs. This does not affect the fact the amylase works best at pH 6. Any other problems, which I might have encountered, were solved at preliminary work level.
I would have been more accurate if I had measured all the chemicals out using a measuring cylinder, instead of a pipette. The results of this experiment are very hard to interpret because they are purely a matter of opinion. Because I have to judge, for each case, whether the iodine has stopped changing colour or not, they may not be consistent. Because of this chance for human error, there was always a panel of three people to determine the results. This means that it is not just my opinion and is the best way of conducting this experiment, as there is no other way.
As a further line of investigation, I would like to look at one of the other factors listed on page 1. Temperature is a similar factor to the pH levels, in that each enzyme also has a specific temperature at which it works best. I would use principally the same method as I did to investigate the best pH levels. The several things I would alter are; the pH would always be 6 as I now know that it produces the fastest digestion rate and I would also have to perform the entire experiment using a water bath to heat the test tube and its contents. The water bath could be set to heat the chemicals to a certain temperature so it is the best way of changing the temperature. All the factors that I kept constant in the pH investigation, would also be kept constant in this experiment, except that I would now keep the pH constant at 6. This time it would be the temperature that changes instead of the pH. I would aim to test temperatures varying from 10°C to 90°C. After performing a preliminary investigation for the temperature, I would be able to establish whether this temperature range is suitable. I would also know by how much to increase the temperature each time, or how accurate I want the best temperature to be.