Preliminary method:
- Collect the apparatus and set them up (below).
- Place starch into a test tube and amylase into another.
- Put both test tubes into water bath for ten minutes, until amylase and starch have reached correct temperature.
- Into a spotting tile put two drops of iodine into each well, using a pipette.
- When the test tubes have been in the water bath for ten minutes, mix them together and start the stop clock.
- Every fifteen seconds take two drops from the mixture and place into the well with the iodine, record the colour in the results chart.
Preliminary results table:
For my preliminary results I used very low and very high temperatures so that I could find the correct temperature range to use for the actual results, which I will use to further my investigation.
The amylase works too slow at lower temperatures such as 10-20 °C degrees Celsius and denatures at 70- 80 °C degrees Celsius, so I will use values between 30- 70 °C in my experiment, which I will use for real results in my investigation, I will repeat this next experiment three times. I will also use five spotting tiles, because I ran out of spotting tiles on the preliminary experiment and had to put iodine in another spotting tile while putting the mixture of amylase and starch into the one spotting tile I had prepared.
Blank results table
Method:
- Collect apparatus.
- Get two test tubes and put 6ml starch in one and 4ml amylase in another. Put these in the water bath at 30°C.
- While the amylase and starch are in the water bath, fill the five spotting tiles with two drops of iodine in each well.
- When the amylase and starch have been in the water bath for ten minutes, take them out and mix them together in the 100ml-measuring cylinder and start the stop clock immediately.
- Collect some of the mixture in a pipette and every fifteen seconds put two drops of the mixture in a well in the spotting tile.
- When there is a colour change record the time it took for it to happen in the results table.
- Repeat the experiment for 40 °C, 50 °C and 60°C.
Equipment:
Test tube x2: I will place starch and amylase into each test tube so I can heat them up in the water bath and mix them together.
Water bath x1: To place the test tubes in hot water to heat them up to the correct temperature.
Spotting tile x5: To place iodine into and record the colour of the mixture we put into the spotting tile.
Pipette x1: To collect the mixture of amylase and starch and put drops of the mixture of the spotting tile.
Tongs x2: To hold the test tubes (if the tubes are very hot)
Stop clock x1: To measure time intervals of experiment.
10 ml Measuring cylinder x2: To measure the amount of amylase and starch needed. ( Two measuring cylinders are needed to avoid cross-contamination of amylase and starch before the correct temperature has been reached).
Goggles x2: For you and your partner to wear when heating up the water (In case hot water splashes into eyes, they will be protected).
100ml measuring cylinder x1: we will put the amylase and starch into the 100 ml cylinder and mix them when they have reached the right temperature.
Iodine, starch and amylase: The substances needed.
Teacher x1: To provide apparatus and help in the experiment.
Safety:
I will be careful not to spill iodine on my clothes or skin. I will use tongs when taking the test tubes out of the water bath because they could be very hot, up too eighty degrees Celsius and use goggles, goggles are to be used in case water from the water bath splashes and goes into your eyes (goggles to be used at temperatures above fifty degrees Celsius). Tie back long hair so it does not interfere with the water in the water bath, amylase or starch in the test tubes or iodine in the spotting tile.
Fair test:
Always use the same amount of starch (6ml) and amylase (4ml) for each experiment to get accurate and constant results. Put two drops of iodine into each well in the spotting tile. Use the same concentration of amylase and starch for each experiment, for example if you use 1 mole concentration in the first experiment, the same must be used for the second and third experiment. The same PH level of starch, amylase and iodine to be used. To keep the change of temperature constant for each experiment, for example each ten degrees, and starting from twenty degrees each time and ending at sixty degrees. I will also place 1 drop of the mixture into iodine every fifteen seconds, and I will keep the pipette away from the iodine in order not to contaminate the mixture each time I put 1 drop of the mixture into the iodine
Obtaining evidence
Actual results table
The changes I have made to planned method:
The amylase works too slow at lower temperatures such as 10°C degrees Celsius and denatures at 70 and 80 °C degrees Celsius, so I will use values between 20- 60 °C in my experiment, which I will use for real results in my investigation, I will repeat this next experiment two times. I will also use five spotting tiles, because I ran out of spotting tiles on the preliminary experiment and had to put iodine in another spotting tile while putting the mixture of amylase and starch into the one spotting tile I had prepared.
Analysis
Analysis of graph 1: Temperature of enzymes and how long they took to react
My graph shows that the enzymes broke down the starch the fastest at 30°C, it also shows that at 60°C the amylase denatured, this is when the active site becomes damaged and can no longer break down the starch, if this happens to an enzyme it is an irreversible reaction, it can never react again. From the graph I also found out at 30°C the enzyme is working at its optimum temperature, it is reacting fast and breaking down the starch quickly, as it took the shortest time at 30°C, an average of 803 seconds. The rate of reaction increases because of the increasing temperature, this causes the particles to vibrate and collide into each other more and more, this speeds up the break down of starch. As the temperature increases, the collisions per second increase, as the temperature gets too high many of the enzymes start to denature and the active site is damaged and cannot react, this slows down the breakdown of starch and therefore it takes longer for the reaction to finish. At 40°C it took 930 seconds on average, this shows that a few of the enzymes are starting to denature. At 50°C it took 1239 seconds on average, this shows that most of the amylase enzymes have denatured and this slowed down the reaction dramatically. At 60°C the amylase denatured, all of the enzymes had denatured and there was no reaction between the amylase and starch.
My prediction “As the temperature increases, the rate of reaction will increase, because the particles will collide more and speed up the reaction, but if the temperature goes too high then the enzyme will denature. I also predict that the enzymes will not work above 45 °C, they will work best at 37°C (body temperature).” My prediction was correct according to my results. My results showed that the rate of reaction did increase as the temperature increased (from 20°C to 30°C the time to break down starch was shortened). Although the enzymes did work above 45°C (at 50°C), but they worked very slow and took the longest out of all the temperatures, this was because most of the enzymes had become denatured. I did not test 37°C and therefore cannot comment on how well the enzymes worked.
In my scientific knowledge I stated that if the temperature is too low the enzymes will not work and if it is too high they will denature, I was correct, they denatured at 60°C and did not work at 10°C (in my preliminary tests). The graph shows that when the temperature was increased from 20°C to 30°C the rate of reaction will increase, as I stated in my scientific knowledge, this applies for all enzymes. The reason I think the enzymes denatured at 60°C was because the temperature was too high and their active site was damaged and could no longer work, they worked best at 30°C because none of the enzymes were denatured and it was the optimum temperature for them to work at, I heated up the amylase and starch to 30°C and this gave them the energy to work at their best.
At 60°C the enzymes were denatured or working very slow that I could not see a change over a long period of time. On my graph I noted this as infinity (∞), to show that it could go on for a long time or forever.
Analysis of graph 2: Rate of reaction
The graph shows that the rate of reaction was highest at 30°C and from then on a decreasing rate of reaction and no rate of reaction at 60°C at which the enzymes had denatured and the active site was damaged, which means the enzymes could not react and break down the substrate (starch). It shows a pattern of decreasing rate of reaction, because the higher the temperature the more the rate of reaction decreased (only after 30°C) because of the enzymes denaturing and unable to react.
The optimum temperature for enzymes to work is 36-37°C and above this they start to denature. I think that at 40°C a few of the enzymes had denatured and thus not being able to breakdown the starch. At 50°C most of the enzymes had denatured and only a few were left to breakdown the starch and this decreased the rate of reaction dramatically. At 60°C all of the enzymes may of denatured or only very little were left and I could not see a change and therefore I had concluded that they had denatured.
The results do and do not support my prediction of as the temperature increases the rate of reaction will increase. This is because from 20°C to 30°C the prediction was correct, the rate of reaction increased because the enzymes had more energy and hit each other harder, therefore being able to react faster, with no enzymes being denatured. They did not follow the prediction because after 30°C the rate of reaction decreased, this was because the enzymes started to denature and less enzymes were breaking down the starch and therefore less were enzymes were working to break the starch down causing a decrease in the rate of reaction.
Evaluation
Overall the experiment was successful. I used 10°C intervals in which I started from 20°C and ended at 60°C, which gave me enough results to analyse and evaluate.
The experiment was a fair test because I always used the same amount of starch and amylase, two drops of iodine into each well, same concentration and PH of amylase and starch. I used a water bath to heat up the amylase and starch, each time for ten minutes at each temperature, I done this to make sure the temperature for each experiment was constant, for example it was 30°C for trial 1, trial 2 and trial 3, and for all other temperatures.
I made my measurements accurate by using a 10ml-measuring cylinder, this way I could see the 6 and 4ml line (unlike a 100ml measuring cylinder), but to make this more accurate I could use a burette in place of a measuring cylinder. I done this to measure the amount needed accurately. I also used a water bath to heat up the amylase and starch to the correct temperature.
My results did not fit a pattern, because at 30°C it was not decreasing rate of reaction, 30°C was the only one, which increased the rate of reaction.
The experiment worked well but could be improved. I could improve the experiment by doing testing rate of reaction for every 5°C, I could get more results by doing this and analyse the pattern more accurately. Also to improve what I found the optimum temperature to be, I could do the experiment for each degree between 30°C and 40°C. I could also use 1/2ml of iodine in each well to be more accurate, rather than just 2 drops, because this is inaccurate, each drop could be a different value, for example one drop could be 1 ml and another could be 2ml. There was also another problem with the iodine, it was difficult to know when the amylase had turned colourless, there were many different colours and this made the experiment slightly difficult, this could be improved by using a colorimeter, this would give me more reliable results. Another problem that arose was when heating the amylase and starch I did not measure the time that they were exposed to the heat accurately enough, this would therefore mean that if the two substances were exposed to the heat for a longer period of time then they would work better because they would have more energy, or at the higher temperatures, 40°C to 60°C more of the enzymes may have denatured if they were left for a longer period of time. To improve this I could put the substances in a water bath and use a stopwatch, so that I can heat them for an equal time.
My results are reliable because I kept the values of everything constant. The tests I carried out were enough to show the full shape of the graph, so I am sure my conclusion is correct.
Further work can be done. I could test the amylase and starch at different concentrations and PH levels, to see how these variables affect enzymes. I could do the experiment on other enzymes.
Bibliography:
School textbook: Biology for life (second edition)
Internet: Google.co.uk: (images) searched: collision theory enzymes.
Free studentstuff.co.uk: (enzymes coursework).
: GCSE revision bites.
