What must be kept the same?
To ensure that the investigation is reliable there are several steps which have to be taken to ensure this. These are:-
- Keep enzyme concentration the same
- Stir the yeast
- Repeat test to increase reliability
- Use same enzyme concentration in each test
Prediction
The reaction will start very quickly with as enzyme and substrate are mixed. When the reaction starts bubbles of oxygen will be produced. The largest volume of oxygen will be recorded in the first minute than any other minute. This means the initial speed will be faster in the first minute than any other minute. When substrate concentration is increased the speed of the initial reaction will increase. This tells us that substrate concentration is proportional to the initial rate i.e. as substrate concentration increase the initial rate increases and when initial rate increase substrate concentration increases. In the later stages of the reaction the reaction will get slower and less gas will be produced until in stop completely. This will be cause the reaction not to be proportional any more.
This means the results which should be expected from the experiment are
- Very high volumes of oxygen when there is a high substrate concentration
- Then very low volumes of oxygen when there is low substrate concentration
This will mean the curve on the graph will be steep at the beginning where the reaction is at its fastest but as the reaction goes on it will level off
The graph should look like this: -
Scientific knowledge can be used to explain why the above prediction was correct. The initial reaction rate will be higher with a higher substrate concentration and lower with a lower substrate concentration. This is because at a higher substrate concentration there will be more hydrogen peroxide (substrate) molecules available to collide with the enzyme (yeast) active sites. This means at a lower substrate concentration the rate of reaction will be lower because there will be less substrate so it will take them longer to collide with the enzymes active sites and bind with them.
Diagram to show this theory
High Substrate Concentration Low Substrate Concentration
Enzyme
Substrate
You can clearly see in this diagram how in high substrate concentration collisions between enzyme and substrate molecules would happen a lot quicker than in a low substrate concentration. As in a high substrate concentration the substrate molecules out number the enzyme molecules. While in a low substrate concentration enzymes would out number substrate molecules.
The next part of the prediction is to explain why later on in the reaction the rate of reaction will decrease while the substrate concentration is still increasing. This happens because enzyme concentration stays the same each time so there is only a certain amount of active sites available at one time. This causes the enzyme active site to become saturated. This means if there was 10 enzymes active sites and 20 substrate molecules the first 10 substrate molecules which collide will the 10 enzymes will be very quick, but there will be 10 more substrate molecules left. These 10 molecules will have to wait in a hypothetical queue until an active site is free. This means the rate of reaction will be reduced. This will only happen in the high substrate concentrations because it can only happen when substrate molecules outnumber enzyme molecules.
Secondary written evidence to back up explanation
An explanation for the shape of the enzyme kinetics curve
At low substrate concentration the reaction rate increases sharply with increasing substrate concentration because there abundant free enzyme available (E) to bind added substrate. At high substrate concentration, the reaction rate reaches a plateau as the enzyme active sites become saturated with substrate (ES complex), and no free enzyme to bind the added substrate. (BIOLOGY PROJECT )
What should the graph look like for initial rate?
Choices of equipment
In this investigation a number of different types of equipment are going to have to used to implement the experiment. The equipment needed is: -
- Gas syringe
- Delivery with needle for yeast insertion
- Digital stop watch
- Syringe
Gas Syringe – The Gas syringe has been chosen to be used to measure the oxygen given off in the reaction rather than other pieces of equipment which perform the same job. This choice was made because the gas syringe is a lot easily to operate than other option and you can clearly see the amounts of gas given off. This allows you to record the results quickly. Other options like counting the bubbles of gas are less reliable as you cannot be sure of the number of bubbles as they are too quick.
Delivery tube with needle for yeast insertion – This has been chosen as it reduces the chance of gas being lost. The yeast is inserted into the hydrogen peroxide with a needle through the bung. This allows there to be little amount of gas to be lost. This is unlike the other method which involves removing the bung inserting yeast and then putting the bung back on. This would cause there to be a high amount of gas lost. Using a delivery tube with a needle increases reliability and accuracy.
Digital stopwatch – This type of time measurement is used as it is very precise compared with a stopwatch which is analogue. Using digital stopwatch increases accuracy as it measures time more accurately than analogue stopwatches.
Syringe – Syringes are used to measure the quantities of yeast and hydrogen peroxide. This is because they can measure the quantities more accurately than pipettes can. Using a syringe allows you to measure out the different concentrations very accurately compared with using a pipette. This improves the reliability of the investigation if the quantities are measured more accurately.
Method
Apparatus
- Gas syringe
- Clamp stand
- Tap water
- Yeast enzyme
- Hydrogen Peroxide substrate
- Test Tubes
- Beaker
- Delivery tube with needle for yeast insertion
- Digital Stop Watch
- Syringe
Diagram
Method
- Arrange apparatus to how diagram shows
- Check eye protection to ensure it is in place
-
Measure 60cm3 of hydrogen peroxide and measure 60cm3 of water. Mix them together to make 10% concentration hydrogen peroxide
- Measure out the correct concentration which is to be tested (see table3)
- Stir the yeast 5 times, then measure 2cm3 of yeast
- Place the syringe in the holder on the bung on the test tube
- Check gas syringe is fully compressed
- Insert yeast into test tube and start stop clock
- Keep a finger over the end of the syringe to ensure no air leaks out.
- Record results in a table
Preliminary study to discover what quantities of enzyme and substrate to use in my investigation
This preliminary study we help decide what quantities of enzyme and substrate should be used in the investigation. The investigating is to find what the effect of substrate concentration will have on the rate of reaction. This means it should use about 5 different concentrations of Hydrogen peroxide (the substrate). This will it will give a range of results i.e. from a very strong concentration to a very weak concentration. The 5 concentration which will be used in this investigation are: -
The next stage is to decide what the quantities of enzyme and substrate should be. This is very important as if you get the quantities wrong a number of things could happen: -
- reacts too quickly
- reacts too slowly
To ensure the right quantities are used in this investigation, a number of different combinations of substrate/enzymes combinations will have to be investigated to find the best combination. Below is the first test:
Table to show results for test 1 of the preliminary study
Table 1
Results from test 1
The oxygen given off by the reaction filled the air syringe within the 3minutes (180secs). This may have been because the catalase quantity was too high. This was reduced to 2cm3 for the next test and there was a little difference in the results. For next stage volumes of substrate were reduce and catalase volume was kept a 2cm3.
Table to show results for test 2 of the preliminary study
Table 2
Results from test 2
Once again the oxygen given off by the reaction was at such a large volume that the gas syringe did not cope. These quantities were also very difficult to measure, this meant mistakes could have easily been made meaning the reliability would have been reduced. It was also found to be very difficult to measure the substrate volume and water volume to any high accuracy. This means these quantise are ‘no’ good.
This meant a rethink into the concentration of the substrates had to be taken. It was decided that the concentration should be changed to: -
These concentrations were tested in test 3. In test 3 concentrations were changed but catalase volume was kept a 2cm3. The 10% concentration of the hydrogen peroxide (substrate) was made by diluting the 20% hydrogen peroxide by added water to it i.e. adding 50 cm3 hydrogen peroxide to 50 cm3 of water.
Table to show results for test 3 of the preliminary study
Table 3
Results to test 3
This change to quantity’s worked well with the gas syringe taking the oxygen given off in the first 180sec in its measured space.
Bibliography
Web Sites used
An explanation for the shape of the enzyme kinetics curve -
Information on catalase -
Books used
Cambridge Advanced Sciences – Biology 1
Biological Sciences 1&2
Implementing
Safety issues
When working with chemical of any kind care has to be taken to ensure there are no accidents occur. This means you must implement safety procedures. The safety procedures which will be adopted when implementing the experiment: -
- Wear safety glasses to protect eyes from splashes of hydrogen peroxide
- All round care needs to be taken to avoid breakages of glass
- When throwing away chemical ensure they are thrown away correctly i.e. into a basin in a sink not in a bin or down the drain
- When clearly up breakages do not throw broken glass straight into bin. They have to be disposed correctly i.e. wrapped in paper of disposed in a bin dedicated to glass.
Risk Assessment
Precision of apparatus
The results of this investigation can only be good as the apparatus made available. The results can only be measured to the accuracy of the apparatus. The accuracy of the apparatus used in this investigation is: -
Gas Syringe – this can only measure to whole numbers i.e. 5cm3 it cannot measure to decimals. This means the results have to be measured in whole numbers only.
Syringes – these can own be measure one decimal place i.e. 1.1cm3. This tells us that are measurement can only be accurate to one decimal place.
Stop Watch – these can measure in Hrs, Min and Sec so this investigation can only be accurate to this.
Table’s of results
A table to show the results of the volume of oxygen given off as Substrate concentration changes
A table to show the average volume of oxygen given off as Substrate concentration changes
Initial Rate
In this investigation an initial rate graph has to be shown to show the difference in initial rate as concentration increases. It is used to work out the initial rate at the beginning of the reaction so we can compare the initial rates of all the concentrations to see if they change.
Initial rate is worked out by calculating the slope closes to the tangent to the curve, as close to 0 as possible. This means you measure from just before the graph starts to curve.
e.g. The graph starts to curve on about 30 second. This means you take 30seconds. You then look on the graph to see the amount of gas produced in this time. It says 2.7cm3 of oxygen. This mean 2.7 cm3 are produced per 30sec. You then do a calculation of:-
Initial rate = Volume 2.7cm3
Time 30 = 0.09 cm3/sec
A table to show the initial rate of the volume of oxygen given off as substrate concentration changes
The graphs are labeled 1 to 6 for concentrations. See key below
- 10% = No.1
- 8% = No. 2
- 6% = No.3
- 4% = No. 4
- 2% = No. 5
- Initial rate graph = No.6
Analysing Evidence and drawing conclusions
Analysing Evidence
In this section of the investigation it will present the data collected in the appropriate forms. The section will include: -
- Table of results
- Table of averages
- Table for initial rate
- Graphs for each concentration 10%, 8%, 6%, 4% and 2%
- An initial rate graph
Drawing Conclusions
If you look at the results you can see there is a very clear trend. This is that as the concentration of substrate is increase the volume of oxygen given off is increased. This can be shown by looking at these figures. At 10% concentration 91cm3 of oxygen is given off while at 2% concentration only 21cm3 is given off. This proves that at lower concentrations of substrate is less volume of gas given off. While at higher concentrations more oxygen is given off. This tells us that there must be a relationship between substrate concentration and volume of gas given off.
The amount of gas given of in the reaction decreases as it progresses. This is shown on the graphs by the line becoming more level late on in the experiment. The results for these experiments do not show it well but you can see the start of the volume of gas starting to decrease. The graph starts to level off for one main reason. The enzyme active sites are becoming saturated. This means that all the active site are being used and there is a queue of substrate waiting for the active site to come free. This means the rate of reaction is reduced causing the graph to level off.
You can see in the graphs for the higher concentration i.e. 10% and 8%. That there graphs have a steeper gradient at the start of the reactions compared with the lower gradients, which have gentler gradients. This may suggest that these have a faster reaction rate.
We can prove that initial rate is faster in the higher concentrations by looking at the graph for initial rate. This graph shows that as concentration is increased the initial rate will increase. There is some little evidence that the initial rate could be slowing down though at the higher concentration. This is because the differences between each initial rate are decreasing (except the last one which is an anomaly). This is telling us that the initial rate is getting close to its Vmax.
Compare with prediction
What my prediction states in a summary: -
- When very high volumes of oxygen are produce it is a high substrate concentration and when very low volumes of oxygen are produce it is a low substrate concentration
- As reaction progresses the volumes of oxygen will be reduced with the graph levelling off.
-
Initial rate will increase but then level off as it reaches its Vmax.
(1)
You can see clearly on the graph that at high concentration i.e. 10%, 8% and 6% that the volume of is high. The volume of gas given off at 10% concentration after 180secs is 94cm3 if you compare this with the 2% concentration it is only 20 cm3. This happens because at the higher concentration there are more substrate molecules than enzyme molecules this means there can be more reactions = more gas produce. While in the lower concentrations there are less substrate molecules. This means that there can be fewer reactions than in the higher concentrations causing a lower volume of gas to be produced. This backs up the first bit of the prediction.
(2)
There is very little evidence that this has happened in these results. There is evidence though that it is starting too happen and if the timing had been extended for a few more minutes then maybe the graph would show the oxygen levels decreasing and levelling off. The little evidence that the results give is that as time goes on the difference between each volume of gas decreases between each time interval i.e. At 4% concentration
Difference between 90sec and 120sec = 14
“ “ 120sec and 150sec = 10
“ “ 150sec and 180sec = 9
There is a clear trend that as the rime goes on the defence reduces thus suggesting it is slowing down. This then tell us that the line must then level off. The line levels off as all the enzymes active sites become saturated and causing the substrate molecules to queue waiting for a free active site.
The results do support the prediction
(3)
Once again the results do not fully support the prediction as the reaction was taken over a too short period of time. They do support the first part of it; the initial rate does increase as the substrate concentration is increased. This happens because in higher concentration there are more substrate molecules available so the reaction at the beginning very quick. Compared with lower concentrations which have low initial rates as they react slow as there are little substrate molecules available.
The graph does show the beginning of the initial rate reducing and levelling off. The preceding initial rates do show that the reactions are slowing down at higher concentration. This allows us to say that if the test had been continued the graph would have levelled off. The graph would have levelled off as all the active sites are used up so they are saturated causing the rate to slow. The substrates could also reached there vmax so they couldn’t go any faster. This is backed up by the prediction in most parts.
Compare with secondary data for initial rate graph
See graph no. 7
The secondary data does back up the results as the graphs look similar. These secondary results continue up to 20% concentration and you can see that the graph starts to level off unlike the graph for this investigation results.
Evaluation
This section of the investigation will evaluate the investigation. It will highlight areas of the investigation which went well, areas which did not go so well as well stating ways of how the investigation could be improved. I will comment on anomalous results, accuracy, reliability and ways to improve the investigation.
Suitability of techniques
The techniques used in this investigation work reasonably well under the conditions which they were performed in. If the investigation could have taken place in Biology lab then there would have been more accurate tools at disposal which would have made it more accurate. The gas syringe worked well showing good results so there were no major problems with the suitability of the techniques used.
Anomalous results
No experiment is ever perfect; if it was then there results would be false. Every investigation has results which were wrong these are called anomalous results. The anomalous results are shown below in the table: - (The Yellow/blue cells are anomalous results)
The Cells in Grey – these cells are anomalous because they are a lower concentration than the concentration above but they have given off a higher volume of oxygen. The reason for this could have been that the concentration was not correct i.e. too much/little water or substrate. If it was not this it would have been because the yeast was not stirred meaning that there might have been stronger solution yeast than on the higher substrate concentration causing more gas to be produced. They could have been also been caused by the gas syringe sticking.
Main sources of error
Operator error is a main source of error in this investigation because the operator may have misread the measurement on the gas syringe or wrote down results wrong. They could also have mixed up concentrations or may have put wrong concentrations in i.e. too much water etc. These would all lead to the results being wrong and anomalous.
Design error is also a main source of error. This is because errors in the design of the experiment could lead to there being anomalous results. The main design errors of this investigation were that the yeast was not stirred each time like it should have been done. This has caused the yeast to have some areas which were high concentrations of yeast and others which were low concentration yeast. This would have led to there being some anomalous results. Other errors were caused by operator errors and design error. Other design errors were that the gas syringe did not run smoothly because it had no Vaseline on it. This meant it got stuck some times causing anomalous results.
How have these errors affected results?
Errors will lead to anomalous results being caused. Below will explain how the errors will affect the results.
When the yeast is not stirred it will cause there to be anomalous result. The anomalous results will be fairly erratic. This is because the yeast will have areas of it with different concentrations. This means if the operator took a sample of yeast in a low concentration the reaction would be slower while if they took it in an area of high concentration it would have a fast reaction.
The gas syringe did not run smoothly this led to anomalous results. This is because the syringe would stick on a certain measurement. You would then record that measurement and as soon as you wrote it done enough pressure builds up to unstuck it and it jumps to what it really should be. This caused the results to be too low in places.
Improvements
Improvement could be made to the apparatus which would reduce errors are: -
Gas syringe – this would be check before the experiment to ensure it runs smoothly. This will allow it to give more accurate results as it would not stick. This would be a good improvement as it would stop results from being to low.
Yeast – this will be stirred before each experiment 15 times in future experiments. This will ensure the solution is of the same concentration each time and will prevent there from bring difference in the concentration.
Organisation – organisation has to be improved in the future. This would involve labelling test tubes to show there concentration. This would avoid mix-ups between different concentrations. Also labelling of the Hydrogen Peroxide and water would also help as these can be easily mixed up. This will prevent mix up and allow results to be more accurate.
Accuracy
The accuracy in this investigation was poor. This is because the operator did not record the results well as they misread the gas syringe on a number of occasions which led to anomalous results. This has to be improved in any future experiment. This could be done by always double checking the results to ensure they are correct.
The accuracy of the Gas Syringe was not very good as it could only be accurate to whole numbers. It have done with being accurate to one decimal place so that the results could have been more accurate as with no decimal place is up to the operator to decide what the result is. The reliability of the other pieces of the apparatus was good and could not be improved.
Reliability
This investigation could have been more reliable but it was limited to the resources of the school. This meant that only two sets of results could have been taken. This reduced the reliability of the investigation because the averages of two sets of results are not as good as three sets. If the investigation was done again a minimum of 3 set would be taken. This would increase reliability of the results. This would allow the results to be more valued so the analysis could be proven.
Overall this investigation has gone well. The results would have been better if 3 sets had been taken by the schools resources limited it to only two. If the investigation was done again then the improvements which were mentioned above would be implemented. This would improve the overall results and make it the whole investigation more reliable.