I am planning to take 7 results over a range of approximately 10-70`C. The temperatures to be measured will be 10’C, 20’C, 30’C, 40’C, 50’C, 60’C, and 70’C. It would be more accurate to take more results but I have a limited amount of time so this amount seems sensible and manageable in the time allowed. I think that below 10`C the reaction may take longer than the time allowed and above 70`C this may also be true because so many enzymes may have been denatured above this temperature. If I do exceed the time allowed this may mean that I will have to carry on the experiment the next day and the next day the starch or amylase may be a slightly different pH, which would make my experiment unfair, this is why it is so important that I do not run over the time limit. I do not have time to repeat all the results, although it would be more accurate, but if I find that the results seem not to be at all what I’d expected or if I had noticed a pattern in the results and then one result seemed to not at all follow that trend I would repeat that particular test.
I will need the following equipment to carry out the investigation:
heat proof mat
tripod
gauze
tongs
Bunsen burner
test tube
water bath
water beaker
thermometer
amylase
indicator paper
starch
iodine
spotting tile
pipette
stop clock
ice
measuring cylinder
I plan to start taking the readings from the lower temperatures first as I predict that these will take the longest and I want to make sure that I will have enough time to do them. Method:
- Measure out 5cm³ of starch using a measuring cylinder and pour into a test tube
- Measure out 2cm³ of amylase and pour into another test tube
- Put a drop of iodine into each dimple of a spotting tile using a pipette
- Put test tube in a beaker of ice and water and use a thermometer to find when the liquid reads 10`C
- When the thermometer reads 10`C and the temperature seems constant, pour the amylase into the starch, start the stop clock and put the first drop of starch and amylase into the first dimple using a pipette at the same time
- Every 2 minutes put one drop of the mixture into a new dimple
- Stop the clock when the iodine stays its original colour
- Make sure the pH is still the same using indicator paper
- Repeat except this time start the clock when the mixture is 20`C
- Repeat except there will probably no need for the ice as the temperature you are aiming for is 30’C, but this time release a drop of the solution at 30 second intervals- this is because the reaction will be faster so if the drops were released every 2 minutes the reaction would not be measured as accurately
- Repeat except use a water bath to heat the starch and amylase to 40`C, if there is no such water bath available put the test tube in a water bath about half full of water and light the Bunsen- use the thermometer to show you when the solution is 40`C
- Repeat except this time the thermometer should read 50`C
- Repeat except this time the thermometer should read 60`C
- Repeat except this time the thermometer should read 70`C
- Record all results
N.B Make sure the temperature is constant when you start taking the readings or it may change too much during the experiment causing that reading to be not as accurate as the others
During the practical I will be taking safety precautions, I will be wearing goggles and an overall in case of any splashes, I will also tie my hair back in case in catches on fire from the Bunsen or gets in the amylase or starch. I will also use tongs to handle the test tube at the higher temperatures to prevent burning. When walking around the lab I will be looking where I am going to prevent any collisions.
I have done a fair amount of work previously, which has helped me plan my practical work. I carried out an “experiment to find the effect of salivary amylase on starch solutions”. We measured 5cm³ of starch and 2cm³ of amylase and mixed them in a test tube, we then put one drop of the mixture into a spotting tile which had a drop of iodine in each dimple every 30 seconds. This experiment showed me that salivary amylase digests starch because after 3 minutes the iodine did not turn black (showing the presence of starch) but stayed the same colour. This helped me as it showed that 2cm³ of amylase is enough to digest 5cm³ starch, it also helped as it showed that starch takes 3 minutes to be digested at room temperature therefore I will have enough time to carry out my experiment in the time allowed. I have also done an “experiment to find the effect of boiling salivary amylase on its activity”. This experiment was the same as the last except this time we also carried out an experiment when we had boiled the amylase. This showed that boiling salivary amylase denatures it, as the starch did not turn black using the boiled amylase. This experiment helped me with my prediction.
Obtaining Evidence
I have collected my results and managed to complete the experiment in the time allowed. During the experiment I made sure I was taking all the safety precautions I stated in “plan and prediction”. Results:
Final results table:
I made a slight change to my method, which can be seen on the large results table; I started releasing drops at 10 second intervals, from 28`C onwards because I found that I could do this to make my results more accurate, as it is to the nearest 10 seconds rather than the nearest 30 seconds. I also changed my interval readings on the ones above to 30 seconds rather than every 2 minutes. These changes have made my experiment more accurate.
Also, I have not taken my results from the exact temperatures that I specified in my method. This is because some of the water baths were not at exactly 60`C, for example, and I wanted my results to be as accurate as possible so I recorded them at their actual temperature. At the 18`C, this was not achieved by using a water bath as there was not a 20`C water bath, it was achieved by using cold water, so it is not exactly 20`C because for the experiment to be fair the temperature has to be constant and the closest and most constant temperature I was able to achieve was 18`C.
I have decided to repeat the experiment to try and make the results more accurate, I have then taken the average of the two. The graphs I construct will account for both of the experiments as I will use the averages to plot the points. I will make graphs because this will allow me to spot any anomalous points and see the general trend of the points easily. When I was doing the second experiment I decided to dry the spotting tile fully or use an unused dry one because when I was doing the first experiment I realised that all of the experiments apart from the first one were damp from washing off the previous experiment. I decided that this may have made the results slightly inaccurate as all of the dimples would have had a different amount of moisture left in each.
Before and after each experiment I checked the pH with indicator paper to make sure that the pH had stayed the same. Another way of finding out the pH would be to us Benedicts solution but I chose to use indicator paper as it is simpler, takes less time and I think they are both as accurate as each other. Below is a piece of the paper with the beginning solution pH and the end solution pH, it shows that the pH stayed the same. On the right is the pH before the experiment and on the left is the pH after. It shows that the solution stayed pH 7.
Analysing evidence
I have made a graph of how temperature effects the time taken to digest (look graph A) so that the trend can be seen easily and any anomalous points spotted. The trend of the points can be seen easily and the trend shows that as the temperature increased to 40’C the rate of reaction got faster and then up to the temperature of 70’C the rate of reaction decreased again. This suggests that the enzyme amylase has an optimum temperature of approximately 40’C, which is as predicted.
My graph suggests that the results are fairly accurate as most of the points are close to the line of best fit and the curve is fairly smooth. There is one point that is a bit further away from the line of best fit than the others, this is the 28’C one but there are no anomalous points. I think the 28’C point may be a bit inaccurate because of a variety of things – maybe the first drop was not taken immediately after the amylase and the starch was mixed and so there was time for the reaction to start slightly before the others, or the measurements of starch and/or amylase were not as accurate as they should have been or my judgement of when the reaction was not as accurate as the judging of the others.
I have decided to construct a second graph (look graph B) as it makes directly shows my results to the coursework title (-shows rate of reaction rather than the last graph which showed how long it took). Also I made it to check to see if the same points were slightly off the best fit line, which they were. This graph backs up my prediction and it also gives a fairly smooth curve indicating the accuracy of my experiment. I think the curves could have been improved if I had taken more readings within that range of temperatures.
From my investigation I have found that my prediction was correct: as temperature increased the rate of reaction increased up to about 40’C, and from this point more and more enzymes are likely to be denatured so the rate of reaction will decrease. This is because as a liquid is heated the molecules gain energy from the heat and so they move around faster, this causes there to be more collisions and so enzymes meet substrates faster, resulting in a quicker reaction. But the denaturing of the enzymes starts at about 40’C and from then on there are less and less active sites for reactions to take place in. My conclusion matches my prediction well because from the 10’C the time taken to react got faster, at 28’C the time was 135 seconds and at 40’C it was 70 seconds and then after this the reaction got slower- at 50’C the time taken to react was 80 seconds. I conclude that my experiment has backed the statement that as temperature increases to about 40’C the enzyme amylase reacts faster but after this the reaction gets slower as the enzymes get denatured.
Evaluating Evidence
I think that my method was a fairly good way of carrying out the investigation because it was fairly simple and possible to carry out in the time allowed for the experiment but, because you had to judge whether the iodine was unchanged or changed there was room for judgmental errors which would be individual to each person. This questions the reliability of the evidence but I think it is enough to back up my prediction. I think I could have made it more accurate if I had put a drop in at more frequent intervals as this would mean that when I did decide that iodine’s colour had not changed the time would be closer to when the reaction was fully complete. Also when measuring out the 5cm³ of starch and the 2cm³ of amylase I could use a burette to make this measurement as exact as I can, using the equipment available to me. I think I could also improve the experiment by taking more result from within the range that I have chosen, as this would make the resulting best fit curve more accurate. I think my results could have been improved on by repeating the experiment once more and taking the average from that but from my graph I think that my results are accurate enough to back up my prediction.
There are no results that do not fit my prediction suggesting that they are fairly accurate.
Extension
To extend and improve the work I have completed so far I could use actual human amylase because I think that the amylase we used for the experiment is not likely to have been actual human amylase but bacterial amylase. This would be good to find if the results are similar and show the same trend, if I was doing this experiment I would use the same method as I did for this one and include the improvements. It would be interesting to use amylase at varying time away from the last time the person’s it was had eaten to find how this effected the results. For example, maybe just before a meal a person may have more amylase present in their saliva. Also I could find the exact optimum temperature by heating the starch and amylase to, for example 37 and doing the experiment, then 39, then 41, then 42 etc. Then when I found the temperature with the shortest reaction time I could do the experiment for the two temperatures above and below it, and find the optimum temperature from these three. I would then repeat this to make the results more accurate.
