Fair test:
In a fair test, it is expected that only one variable changes at a time to see what factors affect the rate of reaction and the experiment.
Temperature-controlled
PH level-controlled
Amount of starch used-controlled
Concentration of amylase enzyme and distilled water-changeable
In this investigation the affect of concentration of an enzyme is being investigated. Make sure that all the apparatus used are washed, so that there is no solution or dirt, etc, to give inaccurate results. Start stopwatch, as soon as the two solutions and water reach 37ºC. To make sure that accurate results are gained repeat the experiment two to three times. Finally start the stopwatch as soon as the two substances have been mixed, and stop it straight as the colour has gone. To do this accurately staying eye level to the experiment is necessary.
Safety:
Before doing the experiment, make sure the area where experiment is taking place in is safe. Wear eye protection goggles so the chemicals, etc do not accidentally enter the eyes as this may be harmful. Roll up sleeves to prevent exposure to heat or corrosive substances. Make sure no bags or coats are lying around the floor, as this could cause injuries by tripping over them, and finally make sure that the hot water is kept in a plastic beaker and not glass, just in-case it heats up the glass, and cracks causing risks of injuries. Any spillage of iodine or starch will be washed thoroughly with soap and water.
Variables:
After investigating my sources I have found out that there are some variables which will affect my experiment:
- the temperature of water
- the volume of starch used
- the concentration of the amylase
- the volume of iodine used- iodine is an inhibitor
Inhibitor: An inhibitor is a substance, which prevents an enzyme from catalyzing its reaction. I now know that if there are lots of substrate and only a little inhibitor, it will be more likely that substrate will get in the active site than the inhibitor, so if there is a lot of inhibitor and not much substrate, then the rate of reaction may be greatly reduced. So basically the inhibitor and the substrate are both competing to reach the active site first.
‘This is able to happen because when the iodine is added to the substrate solution (starch) the iodine molecules link into the gaps of the starch molecules shaped in a spiral also known as a helix shape (this is how iodine turns to a blue/black color). As the breakdown of the starch begins the iodine is released due to the splitting up of the helix removing the gaps, resulting for the color to change into a clear looking solution again (this hypothesis is show in diagram below). The amount of iodine used must be constant as the increase in the amount of starch used can result in the slowing down of the reaction between amylase and starch. This change in time occurs as now there is a larger amount of iodine to be changed therefore making the end time this takes, to be longer. This also means there are more iodine molecules to occupy, resulting in all of the ‘gaps’ in the helix’s to be ‘filled’, leaving remaining iodine molecules to enter the active sites of the enzyme molecules, causing it to be inhibited. Inhibitor is the term given to the substance, which causes the stoppage of the substrate molecules from entering the enzyme. If the enzymes are inhibited, this would mean that the starch molecules could not be broken down by the enzyme making it ‘wait’ for another enzyme, which is ‘free’ eventually slowing down the whole time for the reaction to take place or the time taken for the color of the solution to change from blue/black to clear. If the iodine is kept at a constant amount and mixed with the starch for a reasonable time, the iodine molecules will mix more with the starch molecules therefore more iodine molecules can link into the ‘gaps’ between the helix resulting in no remaining iodine molecules therefore no iodine molecules are left to enter the active sites of the enzyme causing it to be inhibited.-Biology text book reference:’ Understanding biology for advanced level’.. Written by’ Glenn and Susan Toole’
Color change from blue/black to clear-
The temperature will be kept at 37°c all the way through the experiments, because if each temperature were different for each experiment it wouldn’t be a fair test. As the warmer the water the more energy gained by the substrate so it would work faster, which means the rate of reaction will also be faster, whereas if the water is cool then the rate of reaction will be slower as the substrate will work slower due to the fact that it wont gain much energy, from the water. Also if I don’t keep the temperature the same throughout the investigation my results wouldn’t be correct which would mean I wouldn’t be able to draw a fair graph and also some enzymes prefer neutral conditions, whereas some prefer highly alkaline conditions. Most enzymes work at an optimum temperature of between 37-40ºC, this is because as the temperature increases the enzyme gains more energy starts to vibrate faster and faster, this then leads to high speeds of vibration, which causes the bonds to break. As the bond break, the enzyme changes its shape and the substrate can no longer fit into the active site. This sometimes happens when enzymes work at the wrong pH level. When an enzyme is no longer working it is known as being ‘denatured’. When an enzyme is denatured by heat, the shape of the active site is changed so that the substrate no longer fits. A change in pH has a similar effect.
The diagram below shows the optimum temperature of an enzyme is 37°c, written by ’David Applin’ published in 1994
It is also vital that we keep the PH level the same. This is because enzymes work best as a PH level of around ‘10’ as the graph shows the highest point on the curve is between 7 and 14, which is just above neutral, meaning that the optimum PH level for amylase is 10, where as the optimum PH level for enzymes in general is 7. If the Ph level was to become more acidic or more alkaline, this would also prevent the enzyme from working at its best. If the PH level was to drop or to increase the concentration of hydrogen ions would change. These hydrogen ions create bonds between the enzyme and the substrate, therefore the change Ph will decrease the amount of bonds connecting the enzyme and substrate, causing an increase of the rate of reaction making the process of breaking down the substrate a slow procedure. This is also know as denaturing the enzyme, but unlike the increase in temperature the increase in PH level would temporarily damage the enzyme, where as the increase in temperature would disable the enzyme forever. The diagram above is from a text book… ‘ key science- biology’ written by…’David Applin’ published in 1994 -shows the optimum PH level of an enzyme…
The volume of starch used also has to be kept the same (60ml) throughout the experiment, because if one contains more starch than the other, the enzymes will have to do more work at a different pace, which will mean that the test will not be a fair test. I will measure the starch accurately by using a measuring cylinder.
As this investigation is about the concentration of amylase, this is one variable, which I will be changing, and my results table will also be based on this. I will dilute the solution of amylase by adding water to it. You can see in the table below how this will be done to achieve the amount I need.
Amylase dilution table:
This table will help me vary the percentage of amylase:
Apparatus for the experiment:
The apparatus that I plan to use for my experiment is:
- Test tube rack
- 30ml amylase solution (5%)
- 20ml measuring cylinder
- 100ml beaker
- 250ml beaker (plastic for water bath)
- Glass stirring rod
- Iodine solution (normal strength two drops mixed with starch)
- 13 test tubes
- thermometers
- Stop watch
- Kettle for Hot/cold water (heated according to temperature wanted)
- 60ml starch solution
- 5ml syringe for accurate measurements
Method:
Step 1- Firstly, before starting to use the chemicals, make sure that the area experiment is taking place in is safe. Remove any coats or bags lying around. Wear safety goggles and gloves to protect eyes and skin. Safety is very important when doing experiments like this.
Step 2- Set out the apparatus as above
Step3- measure out the amylase using a syringe according to the amounts stated in the dilution table, and mix with the appropriate amounts of water also stated in the dilution table. Put the diluted amylase in to a test tube. (Repeat this for all 7 concentrations listed in the dilution table) however fill the 7th test tube with just water (last line of dilution table) - this is known as the control.
Step 4- put 10ml of starch mixed with two drops of iodine using a syringe, into ANOTHER set of 6 test tubes. Total test tubes in use=13
Step 5- put all 13 test tubes into a beaker of warm water at 37°C (it might help if u put labels on each test tube of different concentrations with masking tape)
Step 6- pull out the test with the first concentration on the dilution table along with one with starch when they are at 37°C. Mix the two together and instantly start the stopwatch stir the mixture with a stirring rod but ONLY two full turns. Do this for all 6 concentrations of amylase and record results in a table.
NOTE: between experiments the water could cool down so before u do each 1 check the temperature of the water and keep it at 37°C (add more warm water if needed but ensure the amylase concentrations and the starch solution are both at 37°C before mixing)
Step 7-
Plot a graph to show the results. Analyze all the information to create a conclusion!!!
REVISED METHOD
Due to a shortage of apparatus I had to make a change in the way the experiment was carried out. Instead of using 13 test tubes at the same time I used only two. I did this by going through each concentration of amylase and washed them out before each concentration.
This process caused me to take longer in carrying out the experiment. How ever if I had the equipment and limited time I would use the original method explained above as it is time saving and more accurate in the sense that all the test tubes would be exactly at the same temperature.
Results
ANALYSING AND CONCLUDING
As you can see both my graph and my table show that as the concentration decreases the time it takes for the amylase to break down the starch increases. The patterns on my graphs show that the increase of the concentration between amylase and distilled water has an effect an effect on the rate of reaction. It causes the rate of reaction to increase-the more enzymes there are the quicker the time taken for all the starch molecules to be broken down. This conclusion proves my prediction… ‘the greater the number of enzymes, the increase the in rate of reaction’ this is proven by my results and my graphs, as looking at the readings on my graph, where the concentration of amylase is 100% the time taken for the reaction to take place was 37.85 seconds and where the concentration of amylase is 10% the time taken for the reaction to take place was 152.33 seconds. This clearly shows an increase in the rate of reaction as the amylase concentrations increases.
My graphs and results also show that I managed to control my variables.
- I kept the temperature at 37°C,
- Constant amount of starch used
- Correct amounts of amylase used at different concentrations with water
- Amount of iodine used
- Ph levels-controlled
However my anomalous result found at the concentration at 40%, I think could have been made by an incorrect measurement of iodine as I did not accurately measure the iodine as I had done with the measurements of starch, amylase and the distilled water.
My rate of reaction graph shows the time it had taken for all the starch molecules to be broken down using the formula…
Rate of reaction = Where... t = time taken for complete breakdown,
Looking at my rate of reaction graph I can see that it is accurate as if I compare it to a graph showing ‘the effect of substrate concentration on the rate of an enzyme controlled reaction’ taken from a biology textbook I can see that they are similar.
The diagram below, from a biology text book ‘Understanding biology for advanced level’ written by ‘Glenn and Susan Toole’ shows the rate of reaction using the formula shown above.
The graph shows that as the substrate concentration increases the rate of reaction increases dramatically until there are no longer any active sites available, which then at this point it stops increasing and continues horizontally. This horizontal line is the maximum rate of reaction and is known as the V-max. This no longer increase in the rate of reaction is due to the lack of ‘free’ active sites (all enzymes occupied). Because the substrate concentration is limited, the enzyme active sites are ‘occupied’ leaving the remaining substrate molecules to ‘wait’ until an enzyme is ‘free’.
I have not included a v-max on my graph for a number of reasons:-
- not enough results to reach a visible v-max
- Graph paper not large enough to show results to the most accuracy.
Looking again at my results with in the graphs and my results tables I can see that my results are fair, reliable and most importantly accurate. To prove this I have referred to a set of results I have obtained from a fellow member of the class ‘Anish Amin’ who had done this same experiment taking on the same methods and precautions. His results were as shown below.
Anish’s results clearly show a similar pattern to mine. His results also explain the theory that I have come across and they prove it as his result at an amylase concentration of 100% took 38.405 seconds to break down the starch and at an amylase concentration of 10% it took 153.16 seconds to break down the starch. These figures clearly show along with the trend of my results, the statement in my prediction-‘as the concentration increases the time taken for the amylase to break down the starch decreases’
EVALUATING EVIDENCE
Evaluating my investigation I have found and proved that my prediction was correct. The experiment with 100% amylase broke down the starch in the fastest time. On the whole I think I carried out the experiment very well. Despite this came across an anomalous result. This was found on the graph for the average time taken for the break down of starch at an amylase concentration of 40%. At 40% the time taken was 53.69 seconds, which appeared to be too fast according to my line of best fit. This is also shown by the rate of reaction graph, at 40% the same anomalous result is shown and the rate of reaction was too fast to fit the line of best fit and the trendy curve. This anomalous result could have arisen due to a number of reasons such as:-
- The amount of starch solution may have been less than the others resulting in mess starch molecules to be broken down consequently causing a faster rate of reaction.
- Slightly more than 40% amylase measured enabling more complexes to be formed increasing the rate of reaction.
- Iodine- not proportional to starch may have been less therefore indicating to our eye a quicker reaction time as the solution will go clear quicker than supposed to although the reaction may still be taking place.
To improve the reliability of my results I could have repeated the experiment a few more times. I think that my results are reliable enough to make a convincing conclusion as they
Convey what I explained in my prediction and they fit the scientific theories. From this experiment many errors could have occurred resulting in an eventual difference in the results, which can cause the conclusion to be incorrect.
The variables measured in the experiment were…
- Temperature –(ºc)
- Amount of iodine- drops
- Amount of starch used- (ml)
- Amount of amylase used – (ml)
- PH level
As the iodine was measured in drops you can see that this was not an accurate form of measurement and this could have resulted in errors to occur.
The PH level could not be controlled entirely as at the time of the experiment there was no mean of controlling it.
The temperature of the amylase depends on the temperature of the water. If there were to be a variation in the temperature by only a few degrees, there would only be a small effect on the results. This may have happened due to the water cooling or by misreading the thermometer. However I kept the temperature at 37ºC as best as I could by adding cold water if it was too hot or warm water if it began to cool. It would not be likely to have a great impact on my results as water is able to retain heat well therefore resulting in the error being minor.
The amount of iodine used must have been kept at a constant amount at all times. This is not able to be achieved using the method of droplets. An error here could have been caused from the increase of iodine added as using the measurement of drops is not accurate. This error could have had happened many times during the experiment resulting what I think would be a reasonable impact on the results as the increase in iodine effects the active site of the enzyme, which resists the substrate from entering the enzyme to be broken down. Next time measuring the iodine in measuring cylinders or small syringes is a procedure to improve my results, to be taken into account.
The amount of amylase used should always be kept to the amount the dilution table gives as an increase in the amount, means more substrate molecules can be broken down at one time, increasing the overall time it takes to break down all the substrate molecules. This error again could have a great error in the final results, but as before this is dependent on the size of the error.
The amount of starch used should always be kept to a constant amount as an increase of starch means there would be more starch molecules to be broken down that in those in the other concentrations leading to an unfair test giving in correct results. On the other hand if there was a decrease in the amount of starch used there would be a faster reaction time as there would be less starch molecules to be broken down that in those in the other concentrations again giving in-accurate results.
Another technique that I used to reassure that my measurements were accurate was that I found that in a contained area a liquid forms a meniscus (a curve) at the surface due to the surface tension. Therefore to gain accurate readings I read off from the bottom of the curve. However for further accurate readings I could have used a syringe to measure every thing (although I did use it for some things) as in a syringe a meniscus cannot form as due to the compression of air there is no surface tension to form a dip in the surface of the water as the compressed air is forcing it down.
EVEN MORE ACCURATE MEASUREMENTS, READINGS AND OBSERVATIONS- Looking at my results I can see that they were fairly accurate and reliable but to make my results even more reliable and more convincing if I had the facilities I could…
-Use an electronic device called a colorimeter, which allows you to rely on an electronically developed assumption of when the solution is clear as this device passes light rays through the test tube and will alert you when a chosen level of light rays have shone through (internet research- searched for ‘colorimeter’ in google search engine).
This would be great to notify us exactly when the iodine has cleared and all the starch has been broken down.
- I could have done the experiment at least 4-6 times to group up my results and to find out whether or not similar patterns are found each time.
-carry out the experiment on the same day in the same period of time (e.g. morning, mid-day, evening or night time) in the same room under same conditions so the room temperature is the same and therefore cannot interfere with each of the experiments.
- Again if I had the facilities I would have carried out the procedure according to my original method and not the revised method. I would do this as I explained earlier in my revised method, to save time and for more accurate readings.
- Use shorter intervals between concentrations, as there would be more results to read a conclusion and plot a graph from rather than having a few, makes observations easier and accurate. I could use intervals of 10% of amylase concentration rather than going up in 20% for example… instead of using 20%,40%,60%...etc of amylase I could use 10%, 20%, 30%, 40%, 50%,60%...etc. continuing in intervals of 10%.
BY HEETEN PINDORIA 10P