Therefore the concentration of starch will be kept constant throughout the experiment.
Volumes of amylase concentrations:
The concentration of amylase solution refers to the number of amylase enzyme molecules in a given volume. However two different volumes of the same concentration of amylase enzyme will contain different numbers of enzyme molecules, and therefore most likely react with starch differently. Therefore to ensure this is a fair test it is necessary to keep all concentrations relative to one volume, so that valid and reliable comparisons can be made.
Therefore the volume of amylase solution must be kept constant throughout the experiment.
Outlined method
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When starch is treated with iodine, a dark blue/black colour is formed, the intensity of this colour is dependant on the concentration of starch i.e. the higher the concentration of starch, the darker the colour. Therefore iodine can be used to detect the presence and concentration of starch in a given solution, and this is the technique that I will be using (with the help of a colorimeter to detect the intensity of the colour) to measure the rate at which starch is broken down.
Starch can be separated into two fractions, amylose and amylopectin. Natural starches are mixtures of amylose (10-20%) and amylopectin (80-90%). It is the amylose in starch that is responsible for the formation of a deep blue/black colour in the presence of iodine. Iodine is not very soluble in water therefore the iodine reagent is made by dissolving iodine in water in the presence of potassium iodide. This makes a linear triiodide ion complex, which is soluble. The triiodide ion slips into the coil of the starch causing an intense blue-black colour, and this is the colour that will be tested for.
Risk Assessment
Amylase enzyme
All enzymes are biologically active and therefore needed to be treated with care.
Any spillages on skin should be washed with water, however it is not toxic and is low risk.
If in powdered form avoid inhalation
Iodine solution:
Can be toxic and an irritant, therefore eye protection must be worn at all times.
Any spillages should be washed off with water as soon as possible.
Starch solution
Very low risk
Long hair must be tied back at all times (especially when using a Bunsen burner).
Bunsen burners must be left on yellow flames when not in use, as blue flames are not always clearly visible.
Any hot equipment after the use of Bunsen burner must be handled with extreme care.
The area being worked within must be clean and clutter free to ensure no contamination of chemicals or accidents.
Eye protection must be worn at all times.
All chemicals must be kept in clearly labelled containers to avoid confusion and save time.
Pilot experiment
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Due to time restrictions, it is necessary to ensure all time available is efficiently used. To do this I must firstly ensure I am using the optimum temperature for the amylase enzyme, and secondly ensure a suitable concentration of starch is being used, and ensure, these results are viable in relation to the time available.
I will be looking for the lowest concentration of amylase (2.5%) to take under 240 seconds to turn clear. As this will allow me to take a minimum of three repeats and therefore increases the reliability of my results. Also, to maximise efficiency, it may be useful to carry out more than one experiment at a time. As to would take approximately 15 seconds to take a colorimeter reading during and experiment and note it down, if I were to carry out two experiments at once, I would need to take readings every 30 seconds so I can alternate from each experiment while only using one colorimeter. Taking readings every 30 seconds in a space of 240 seconds will require me to take 9 readings. This is a sufficient amount of readings in order to base a reliable conclusion upon. I will therefore be targeting to take readings every thirty seconds and for each concentration experiment to take approximately 240 seconds.
Also, no precautions are necessary to maintain a specific pH as all solutions used are only very slightly acidic or alkali, and therefore will not cause any noticeable variations in results.
Temperature Pilot Experiment
Using 10% amylase and 2% starch
These results show that 40°C is the optimum temperature for the amylase enzyme and is therefore the temperature I will be using in the next pilot test and the final experiment.
Starch Concentration Pilot Experiment
Using 2.5% amylase at 40°C
From these results, I have found the optimum temperature for the amylase enzyme was 40°C, and the most suitable concentration of starch for the experiment was 5% (closest to my 240 second target). Consequently during my final experiment all solutions will be kept at a constant 40°C and all starch solutions used will stay at a constant 5% to ensure a fair test.
Method
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I measured and placed into a set of clean test tubes, using a pipette and measuring cylinder, 5cm3 of 5% starch solution, then into a second test tube 5cm3 of 2.5% amylase solution and into a third test tube 9cm3 of the iodine reagent (1cm3 per treatment every 30 seconds). I then placed them into a test tube rack.
I then repeated this for 5% amylase. As all of these test tubes were to be placed in the same water bath it was necessary to mark all test tubes involved with the 5% amylase experiment, to ensure no mistakes were made in confusing the two concentrations.
I then prepared and calibrated the colorimeter by using the correct colour filter, and placing a test tube of the final colour I was expecting in the colorimeter and ensuring the counter was on zero.
I then prepared a water bath at 40°C using a Bunsen burner, tripod, gauze and beaker and ensured this temperature was maintained by testing it with a thermometer. I then placed the 2.5% amylase solution in the water bath with its corresponding starch solution, and 15 seconds later placed in the 5% amylase solution with its corresponding starch solution and left them for two minutes (starting from the entry of the first set of solutions), allowing the solutions to reach this temperature. After two minutes, I mixed the 2.5% amylase and its corresponding starch solution together. I then immediately removed 1cm3 of the amylase/starch solution, and 1cm3 of the iodine reagent, mixed these together in to a new test tubes and placed it in the colorimeter to get my first reading at 0 seconds. I then noted these readings in a results table I had prepared earlier. This procedure was then carried out with the 5% amylase concentration.
I then continued to carry out this same procedure every 30 seconds for both concentrations until I reached 240 seconds, continually noting the results.
I then repeated this experiment for the other amylase concentrations (7.5% and 10%) and again noted all results in a table, and repeated the entire experiment a further two times.
Control Experiment
As a control experiment I wanted to ensure that the only substance present during the reaction breaking down starch was the amylase enzyme, if this was found not to be the case alternative methods would have to be implemented due to the a new variable in the experiment. In order to do this, I decided to test the other substances present during the reaction i.e. iodine and water. I carried out this experiment in a similar manner to the final experiment, using 5% starch solution, a water bath and colorimeter. However as I was testing the iodine reagent and also using it to determine changes in starch levels, it was not necessary to treat each 1cm3 starch/iodine portion again with iodine reagent after being removed from the initial solution. Instead each portion was placed directly into the colorimeter. Also it was only necessary to take two readings at 0 and 240 seconds to check if there have been any changes in starch concentration.
. Results of control experiment:
As there was no change in the reading for either water or iodine reagent, there is no evidence starch is being broken down. Also I am able to conclude that there for a 0% amylase concentration experiment there will be no reaction, as the control experiment with water would be exactly the same as my final experiment with 0% amylase.
Final Results Table
*Anomalous results marked in red
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Anomalous results were disregarded, as they were inconsistent and would have disrupted the average results and consequently the graphs.
Average of results from final experiment
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As comparisons for the rate of reaction cannot easily be made from looking at this table it is necessary to express the results in a different format using the average results and plotting them on a graph.
I used the gradient of each line from graph 1 to work out the rate of reaction. The bigger the gradient, the higher the average rate of the reaction and vice versa. To work out the rate of reaction, I drew a line of best fit, and then found the gradient of this line (graph 1). To work out the gradient I divided the arbitrary units by the time taken to reach zero. The results are shown in the table below:
Rate of reaction of starch and amylase at varying amylase concentrations.
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(Graph present)
Conclusion
Main trends and patterns
The original data and graph shows two major patterns.
Firstly, that the larger concentrations react faster than the smaller concentrations. This is evident from the results as the 10% amylase solution took an average of only 90 seconds for the reaction to end, whereas 7.5% amylase solution took an average of 120 seconds, 5% took and average of 180 seconds and 2.5% took 240 seconds. It is clear from these figures that
Secondly, when analysing the data more carefully, in general the rate of reaction seemed to decrease with time. This is evident from graph 1, whereby each curve levels off as it approaches the x-axis, this is particularly apparent from 2.5% and 5% amylase concentrations. This is also illustrated in the table below.
Changes is readings from final experiment
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The table shows the difference between each average reading and clearly shows a trend of decreasing numbers (excluding the anomaly), and so a decrease in the rate of reaction as time progresses. This trend is evident throughout all of the results (excluding anomalies), however is far more so when looking at the 2.5% and 5% results on graph 1. These patterns will be discussed further in the explanation section.
Looking at the average rate of reaction from graph 2, the most prominent trend is that the average rate of reaction increases steadily as amylase concentration increases. Furthermore the rate of reaction is increasing by an average of 19% after every increase of 10% in amylase concentration. This is an area that may be worth further investigation.
Although there is an anomaly (marked red in the table) during the 2.5% amylase at 60 –90 seconds, this is most likely due to experimental errors as no trend of these anomalies is carried through the experiment.
There were also four anomalous results during the final experiment, which were disregarded, as they would have disrupted the final results and graphs and were again most likely down to experimental errors.
Explanation of results
Enzymes are globular proteins that consist of a quaternary structure, which give rise its complex three-dimensional shape and consequently an active site. An active site is an area on the enzymes surface where the substrate molecule attaches to and is broken down. If the concentration of enzymes increases there are subsequently more active sites for the substrate molecules to bond to and so more substrate molecules are broken down. This directly increases the rate of reaction. This information explains the general increase in rate of reaction with increasing amylase concentrations, however does not explain the general decrease in rate of reaction with time.
As I have stated above, the rate of reaction between the enzyme and substrate depends on the formation of enzyme-substrate complexes. The collision theory states that a reaction occurs when a successful collision occurs between reactant molecules. As the reaction progresses, the number of non-reactant molecules (constituent molecules of starch) produced by the reaction between amylase and starch increases. This decreases the number of collisions between the amylase and starch as these reactant molecules are also now colliding with an increasing number of non-reactant molecules.
Also, as you increase the enzyme concentration, you are increasing the number of active sites, but the amount of substrate molecules stays constant. This causes the rate of each concentrations reaction to slowly plateau off. This is because as more substrate is broken down by the enzyme, there is less substrate left in solution and so less enzyme-substrate complexes will be formed. This is also explained by the collision theory that states that reaction occurs when collisions occur between the reactant molecules. So, the rate of reaction is dependant on the number of collisions. Therefore, as there are less substrate molecules left in solution, the number of successful collisions will decrease and so the rate will not increase by as much. However from the results, this pattern of a decrease in rate of a particular concentration was far more visible with lower concentrations i.e. 2.5 and 5% amylase solutions. This is because with significantly higher concentrations of amylase, there are a much greater number of amylase enzymes, therefore in order to disrupt the reactions occurring, a similarly larger number of non-reactant molecules must be present. However this number was kept constant through the experiment as the concentration of and volume of amylase was kept constant.
In summary, even though the rate of reaction increases as the concentration of amylase increases, the rate of a specific concentrations reaction plateaus as time progresses
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Experimental limitations
As mentioned in my analysis, I have small anomalies present in my results. These could be due to experimental limitations. One important experimental limitation would be the accuracy of the equipment used throughout my experiment:
- I used a measuring cylinder to measure out my solutions. The measuring cylinder was accurate to 0.2ml, and so introduced a large error percentage. I measured out 5ml of amylase and starch which introduced a 4% error;
(0.2/5.0 x 100%). I also measured out 1ml of iodine and so this introduces a 20% error; (0.2/1.0 x 100%)
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Due to the unavailability of an electronic water bath, I had to create my own (using a tripod, gauze, beaker, water, thermometer and a Bunsen burner). This was hard to keep at the same temperature (40oC) at all times, and so could also make the results vary, as a change in temperature can also cause a change in the rate of reaction.
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Another experimental limitation would be the fact that I had to read off the colorimeter by eye. Also, the value that needed to be read was not always constant, as the needle kept moving. This could easily cause me to slightly misread the colorimeter.
In order to make my experiment more accurate, I would like to have more accurate equipment and the use of an electronic water bath. Also, I would like to have a digital colorimeter so that no errors could be made when reading.