I have based these two hypotheses on the facts that I have learnt earlier in my work on this topic, where I found out that the more collisions that occur, the faster the rate of reaction occurs and the quicker it finishes.
Preliminary Work:
I will be carrying out some preliminary work before I carry out my main experiment in order to gain some knowledge on how I should carry out my investigation so that it will be very reliable, accurate and fair.
Preliminary Work Method:
- Put one drop of iodine into each well on a spotting tile.
- Pour 1cm³ of amylase and 5cm³ of starch in a test tube.
- Straight after doing this, start the stopwatch and take 2 drops of the solution in the test tube and add it to the first well of iodine in the spotting tile.
- Continue adding 2 drops of the solution every 30 seconds until the reaction forces the colour of the liquid to turn brown.
- Record the results on a table.
Preliminary Work Apparatus:
- Spotting Tile
- Iodine
-
1cm3 of Enzyme Amylase
-
5cm3 of Starch
- Digital Stopwatch
- Pipette
- Goggles
Preliminary Work Results Table:
The results on the previous page tell me that the enzyme amylase took two minutes to break down the starch.
Preliminary Work Conclusion:
This work has forced me to not trust these results because of a few reasons:
- The timings won’t be accurate i.e. the reaction could have finished in between the 30 second gaps. That is why I will use boiling tubes in the actual investigation to make it accurate.
- The 2 drops of the solution is a very small volume and so I will use a volume of 1ml instead to make sure that my conclusion will be more accurate. The drops from the pipette may also not be reliable, as some drops may not come out or too many drops may be forced out.
- Using test tubes in my method will allow me to clearly see whether the reaction has completed or what stage it is at. It is not possible to clearly see what stage the reaction is at with the use of a spotting tile.
- One variable that I pointed out I will have to control was temperature, which cannot be controlled if I am using the spotting tile method.
- One final alteration that this preliminary work has forced me to make on my method is that the broken down solution will only reach a brown colour, whereas if I were to use a larger volume and more time, it can reach a colourless liquid, which is what I want for my test to be accurate.
Method:
- Measure 10ml of amylase using a measuring cylinder.
- Pour the 10ml of amylase into the boiling tube.
- Put the boiling tube in a hot water bath so it will be a constant temperature of 37°C.
- Then measure 10ml of starch and pour it into a boiling tube and put it into the water bath.
- Add a few drops of iodine to starch (the starch should turn dark blue/black).
- Add amylase into the starch solution with the the test tube still in the hot water beaker.
- Start the stopwatch immediately and measure the time taken until the solution turns colourless.
- Record the results on a simple table as soon as you have taken the reading to make sure you don’t forget.
- Replicate the same procedure for the remaining concentrations.
10) Repeat the experiment twice to make sure results are reliable.
Safety Precautions:
Safety will be an important issue in this experiment, as I am going to be using Iodine, which is an irritant. I will be wearing goggles for this reason, particularly while handling the Iodine, so that it doesn’t have a chance to affect my eyes. I will also make sure that the area around my experiment is clear, so that there can be nothing to affect my experiment and endanger myself and others.
Apparatus:
- Graduated Pipette (this will be vital in allowing me to take accurate and reliable measurements)
- 10ml Measuring Cylinder
- Digital Stopwatch
- Iodine
- 4 Boiling Tubes
- Thermometer
- 0.5% of starch (50ml), 1.5% of starch (50ml), 2.5% of starch (50ml), 3.5% of starch (50ml), 4.5% of starch (50ml), 5% of starch (50ml) [6 different concentrations]
- Amylase (3%) 50ml
- 1 Beaker (250ml)
- Hot Water (from kettle)
- Stirring Rod
- Goggles (safety precautions)
Dilution Table:
Fair Test:
To make my test as fair as possible, I am going to keep certain variables constant throughout. The temperature will be kept the same, as enzymes could denature or not work to their full potential if the temperature is either too low or too high. The concentration of amylase will also be controlled, as it can affect how fast the reaction occurs – the higher the enzyme concentration, the faster the rate of reaction (but to a limit). The volume of starch and amylase will be kept constant, as different volumes take different amounts of time to react with the enzyme. Finally, the pH level will be kept the same, due to the fact that different pH levels affect the work rate of enzymes.
However I will also have an independent variable (something I will keep the same) – the concentration of starch – which I will to a different concentration change every time I carry out the experiment. This will allow me to carry out the aim of my investigation.
Although, there is a variable that I cannot control - time. This is because, time will be dependant on the reaction rate and I cannot control how long it will take for the reaction to finish. I don’t think this will really affect my results, apart from the fact that my graph could be less accurate, as the time scale may be too large.
Accuracy:
I will try to keep my investigation as accurate as possible so that I can be sure that my final conclusion is one that I can go by and trust. I will do this by carrying out the following checks.
To make sure that I am as accurate as possible while measuring how much liquid I have in my measuring cylinder, I will always count the point for the lower meniscus, which will mean that I will be more accurate.
I will also be using a digital stopwatch, therefore increasing the accuracy as it will be very reactive and I will also be able to see the split-second times, and allowing me to round up or down to a whole number.
One final thing I will do to aid my experiment to be as accurate as possible is use a graduated pipette to transfer liquids from one place to another, making sure that it’s accurate to the markings on the pipette.
Reliability:
The main thing I will do to make my experiment reliable, is repeat it three times, so that I am sure that the trend between starch concentration and the enzyme amylase is genuine.
I will use 6 different concentrations of starch, ensuring that I will have at least that much points on results graph, which also means that I will have a reliable graph to analyse. It will also be clear with this number of points on my graph, which ones are anomalous – making it easier for me to know where I have gone wrong.
I will have also carried out some preliminary work before my actual experiment to make a few things clear and so that I will know what to do to make my test reliable. There will also be a pilot run carried out for one concentration of starch with 10ml of amylase to find out whether the amylase will break down the starch too fast or not.
Results:
Results Tables:
Pilot Run Results: I tried using 10ml of the amylase enzyme firstly but it proved to break up the starch concentration way too quickly so I decided to change the amount of amylase used to 5ml, which worked rather better.
From these results, I can figure out a couple of statistics:
- The highest leap in the rate of reaction was between the concentrations 3.5% and 4.5%.
- The fastest average time taken for amylase to break down starch was for the highest concentration tested – 5%.
One thing I changed from my original plan to the actual experiment, was the temperature at which I made the reaction take place. This was changed from 37°C to 30°C. I did this so that the enzyme amylase didn’t react with the starch too quickly, and so that I would have a reasonable range of results to analyse.
Now I will draw two graphs; a time graph and a rate of reaction graph, by using certain columns from the able above on the next few pages.
Time Graph:
The time graph for each substrate concentration shown above shows that as the concentration of starch increased, the time taken for the enzyme amylase to break it down decreased. This is because, there will be more molecules involved in the reaction with an increase in substrate concentration and thus leading to an increase of amylase molecules. The more collisions, the faster the reaction occurs. Therefore, this graph supports my prediction, which states that the time taken for amylase to break down starch would decrease as the starch concentration increases. My graph also shows a sufficient range of points for me to analyse whether it backs up my prediction or not.
Rate of Reaction Graph:
This rate of reaction graph shows me that as the substrate concentration increased, so did the rate of reaction. This also backs up my prediction, for the same reason as the time graph. My prediction also goes on to say that there will be a peak point - the V-max point - for the rate of reaction, which is when the rate of reaction will remain constant, as the enzyme molecules will be saturated with starch molecules and will therefore be unable to break them down any faster. However, this isn’t apparent on my graph but theoretically the same would happen for this graph if I were to continue my investigation to higher concentrations of starch. This graph also has an adequate number of points to allow me to comfortably support my prediction.
Evaluation:
To evaluate, I am going to assess how well I carried out my investigation and whether any improvements are necessary.
My method was fairly accurate; as I had tried my best to always measure the liquid levels in the measuring cylinder and graduated pipette to the lower meniscus, but I may have sometimes unconsciously measured the upper meniscus. My experiment was also fair; as I kept the variables I had stated (temperature, pH level, amylase and starch volume and enzyme amylase concentration) the same and kept the independent variable (starch) constant.
I found a couple of anomalous points, which are circled on my graphs on the previous pages. These may have occurred by a slight glitch in my method, where I may have accidentally put in a bit too much starch concentration or too much amylase, which would have caused the the reaction to take place faster/slower depending on the circumstances.
Other improvements I could make include not rounding up my measurements on the digital stopwatch to a whole number; this would have made it more accurate and reliable. I could also keep my hot water at a constant temperature of 30°C, rather than change the water every time I change the concentration. This would have made sure that the temperature would have strictly remained constant, and it would have no chance at all to affect my experiment. One way to do this is by using an electronic water bath.
Another improvement I could make is using more digital equipment. This would mean using a digital hot water bath and a digital thermometer, which would make my experiment all the more accurate. I could also use data logging equipment to measure my results, as it would mean they would be to their utmost accuracy level. This would also make my test more reliable, due to the fact that the results would be more trustworthy, whereas, I had to use a digital stopwatch, and had to time it myself straight after the reaction is started. I also had to stop it myself and my human reaction time may not have been quick enough or may have been too quick. This means that there would have been a gap between the reaction starting and me starting the stopwatch, even if it may have been a small gap. The data logging equipment would make sure that the time would start as soon as the reaction starts and stop as soon as the reaction stops.
The test was also carried out over a period of 2 days, which may have interfered with the room temperature levels and therefore also affected the experiment. Maybe next time, the test could be carried out over a strict period of time e.g. 3 successive hours. Other errors include liquids being leftover in the boiling tubes, which may also interfere with the reaction and how it occurs. We could maybe use dry boiling tubes every time I start to investigate a different concentration. The water I also used was from the tap and thus would have contained some impurities – causing the investigation to also be flawed. Pure water or filtered water can be used to tackle this problem.
I also suffered from a few problems while analysing the solution colours. I would never be fully sure whether the finished product was completely colourless or was still reacting a little. This could be avoided by using a colorimeter, which measures the density of a colour, and therefore would have allowed us to see whether the solution is actually colourless or not. Other miscalculations could include me adding more drops of iodine than needed e.g. adding 4 drops instead of 3 drops each time. The iodine could have then acted as an inhibitor. To deal with this hitch, we could make a fixed mixture of iodine and starch in a beaker, with 1ml iodine to 50ml of starch, and take the volumes from that.
The method can also be improved by adding something at the end. Instead of finishing with the colourless liquid, I could also add a step where I measure the product of the reaction with Benedict’s solution. This would allow me to see whether maltose is the product of the reaction or not. It would also let me see how much maltose is made. I could also continue experimenting with more concentrations of starch, as in my prediction, I had stated that there will be a point where the rate of reaction will stop increasing and I couldn’t find that out with only the 6 different concentrations that I measured.
Conclusion:
To conclude, I have successfully supported my prediction and found out that as the substrate concentration increases, so does the rate of reaction. I have also found out that the time taken for the enzyme amylase to catalyse starch decreases as the substrate concentration increases. However, I didn’t find out whether the rate of reaction stops increasing to a limit even after the substrate concentration continually increases, which is also what I predicted. My results show that there is a trend between the two variables – substrate (starch) concentration and the rate of reaction. I used a graph to represent this trend, as it made it easier to showcase it. The trend appeared because as more and more enzymes are added there will be more successful collisions as there are more active sites free for the substrate molecules to fit into.
