Key Variables
The volumes with drawn from the reacting boiling tube must be the same for all 10 of the test tubes.
The same colorimeter has to be used through out the experiment as well as the same blank. Also to check the colorimeter does not vary its starting point, therefore the transmission should be checked that it is at 0 at the start of each reading.
The time intervals at which samples from the test tubes are taken have to be at relative intervals to enable the results to be easily conveyed whilst analyzing.
The temperature of the test tubes which the enzymes lay in has to be kept at a constant temperature, in this case room temperature. This has to be kept the same as a rise or decrease in temperature will affect the temperature greatly.
All measurements are taken at the same part of the meniscus, for example if you have measured from the bottom (the incorrect place) carry on doing so until the end of the experiment.
Keeping these various variables the same will hopefully make the experiment accurate and fair. If these variables are broken it should be highlighted in the results and there fore can be stated as an anomaly.
To ensure that these variables are met there must be a simple case of careful measurement of the variables above. These must all be kept to the specific volumes and concentrations, which will be stated, in the detailed method to follow. It is very important that these variables are kept as the accuracy of the experiment depends on these features also to keep a fair test. I will be making a control for the experiment; this will be in the form of a blank. The blank compensates the colorimeter for the colour which is naturally present in the iodine. It will now register changes in opacity relative to the blank reference solution. The colorimeter shall have a red slide used, as this is the colour at the other end of the spectrum to the colour of iodine.
The measurements in the experiment that have to be kept the same are mainly the amount of the dilute iodine solution added to the timed interval extractions from the reacting mixture. Also the amount of the reacting solution withdrawn from the reacting boiling tube has to be kept the same for each of the 10 test tubes at the various timed intervals.
The colorimeter should give the reading of 100% transmission; this means that it is 0% absorbance. The change in reaction stage will be picked up by the change in light transmission, as the starch broken down by the Amylase the more light transmission will be read of the colorimeter. This will be the opposite at the start of the experiment as in the first few minutes I don’t think there will be must starch broken down. There fore the light transmission will be very low. In comparison with the blank (which has 100% light transmission) it will be very different until the later stages.
From the colorimeter it will show how much starch has been broken down, as the blank tested on had no traces of starch in, only the Amylase and water.
Apparatus
12 Test tubes- The purpose of these are to be a middle container for the reacting substances to be added to the dilute iodine with out mixing the whole reacting solution at once.
1 Boiling tube- To hold the reacting solution through out the experiment, this is where at timed intervals small amount will be taken from.
Colorimeter- This is used to find out how much light is absorbed and the light transmission of the digested starch with the dilute iodine solution.
Timer- To time the intervals for the iodine test as well as the colorimeter.
Pipettes 1ml 5ml 10ml- For fine measuring
Test tube rack- Hold all the test tubes and boiling tubes.
Starch solution
Amylase solution
Method
To be able to make sense of the results once they have been collated I must make a calibration curve so that I can work out the concentration of the substrate of the 10 test tubes. This will tell me how much substrate is left and will give me an indication to how much starch has been broken down by the Amylase. First to make a calibration curve to find out the concentration I must follow a particular plan of how to make the curve.
A colorimeter measures the intensity of colour in a solution, in this case the colour of the 10 different test tubes with the substrate and enzyme which has been tested at various times. When starch is present the iodine solution turns a blue/black colour, there fore a red filter will be used to measure the colour.
To make the calibration curve I must first set up a series of test tubes, these test tubes shall contain different concentration of the starch. The following step will show how the concentration are made:
Before this a blank should be produced, it is vital that the blank does not restrain any starch and only the iodine solution. This blank must then be registered so that we have something to judge against the 7 dilute concentration that will be made. This blanks should read 100% transmission through the colorimeter, which means 0% absorbance.
- Provided with the 1% starch solution, this shall be the source of starch. Use a clean pipette to put 10mls of the starch into test tube 1. Make sure that you shake the starch solution regularly as it can settle into segments. This is the concentration of 1%.
- Take 5mls from test tube 1 and place in test tube 2; now top up test tube 2 with 5mls of distilled water. Mix solution until completely mixed; this has created the concentration of 0.5%.
- Withdraw another 1ml from test tube 1, place in test tube 3. Top up test tube 3 with 9mls of distilled water. Mix till satisfied, now you have diluted the 1% 10 times there fore test tube 3 has the concentration of 0.1%.
- Test tube 4 then needs to be 0.05% starch; this is done by diluting 1ml from test tube 2 with 9mls of distilled water.
- Test tube 5 is then diluted to 0.01% of the original starch solution, this is done by taking 1ml from test tube 3 and then diluting it with 9mls of distilled water.
- Test tube 6 is then made for 0.005% starch, this is simply a follow on from the rest of the trend, withdraw 1ml from test tube 4 and dilute with 9mls of distilled water.
- Test tube 7 has then to 0.001% of starch, this is 1ml from test tube 5 and then diluted by 9mls of distilled water.
From these dilution I will have 7 different concentration of the starch concentration, the next step is to line up the all the variations of concentration (8) and the proceed in putting them into the colorimeter one at a time. First make sure the blank shows 100% light transmission, and then proceed with the rest of the concentrations. The results gained now will be able to convert the light transmission from the core experiment and determine concentrations of the starch.
Plot a calibration cure of transmission Vs known starch concentration. You will find a scale for starch concentration works better if you plot the log of starch concentration or use logarithmic graph paper. Estimate a best-fit curve through the points collected by the results; this will then estimate the starch concentration from the core experiment.
The core experiment method: the main aspect of the experiment is to determine the rate at which the starch is broken down by the Amylase and to test the hypothesis. There fore another step process must be followed to accumulate a dependable accurate set of data.
- First gather all the relative equipment that is necessary, as this experiment needs speed to make it work. Once you have assembled the 10 test tubes in the test tube rack along with the boiling tube for the break down of the starch. Add the Amylase enzyme to the Starch substrate and start the timer. The experiment has started and the breakdown of the starch will have begun.
- Immediately withdraw 1ml from the solution using a clean pipette, transfer into a iodine calibration tube and mix thoroughly. The solution will now have turned blue, an indication that starch is present.
- Now the mixture is thoroughly mixed you need to fill a colorimeter vial ¾ full using a pipette. This is then ready to put into the colorimeter.
- Now record the time that the sample was taken from the reacting mix and then record the transmission% of the blue solution in the vial.
- Record the exact time that the sample was taken, the first reading has to be taken right away to find out the initial rate of the reaction. Proceed to take 1ml from the solution every minute until all 10 test tubes have been used.
- This needs to be repeated 10 times with an interval of 300 secs between every reading.
- Using the calibration curve that has already been made you can now convert the light transmission% to a concentration of starch from the 1% starch solution.
Risk Assessment
Amylase Enzymes
These biological enzymes all have biological activity and are to be treated with care.
Glass pipettes are being used greatly through out the experiment.
These are to be used with care as they are very fragile and can easily splinter.
Starch solution
Very low risk
Keep in clearly labeled container
Results
Calibration curve
Results from Enzyme controlled reaction
Analysing evidence and drawing conclusions
The raw data has been collated by two different groups and collated into a several tables of results, these contain the raw data and averages between the two set that we received. Already there is a pattern forming in the data just by looking at the tables of results.
On the trust that all the groups that carried out the experiment properly and there were no initial errors, which may have affected the results, the result stated have been taken and recorded with a degree of accuracy. From a first assessment of the data it appears to be correct, little error and with no major anomalies.
However a small degree of measurement of the pipette will create a small error percentage, this error comes form the meniscus reading from the pipette, because the surface tension drags the solution up is a small amount inaccurate. The 1st sight of error is the initial withdrawal of the reacting solutions at the various interval. This incurs an error of 1mm, and there fore will make the volume in each of the 10 test tubes slightly less than planned. This totals to around a 10% error for each of the 10 test tubes.
It is necessary to plot two different table of results as the time intervals that each of the groups took was different. Because of this it is not possible to averages results however it is possible to plot both set of data on one graph, this enables us to have various points added to original results gained by the individual group and there fore actually makes it easier to construct a line of best fit.
The calibration graph enables me to calculate the concentration of the starch left in the solutions taken from the reacting boiling tube. This concentration of starch shows that as time goes on the starch concentration becomes less and less. This is because the Amylase enzyme in the solution is breaking down the starch to maltose and therefore not leaving as much starch in to solution.
We know that less starch is present in the later samples of the enzyme and substrate mix as the light transmission becomes greater and the absorption becomes higher. This is because the iodine in the solution, which makes the starch turn blue, is less apparent and is therefore making the transmission greater as there is a greater amount of red light from the filter passing through the solutions.
It is noticed that for the first 300 secs of the experiment that there is very little break down of starch by the enzyme as the light transmission was not any greater the 3%, this converted by the calibration curve means that from the 1% starting starch solution it has only been reduced to 0.9%. For this there could be various reason to do with the enzyme. This may be to do with the fact that the enzyme needs time to get used to the environment before it starts to work, the activation energy needed was high and lowering it took time and the fact that the boiling tube to which the enzyme and substrate were in may not have been mixed regularly to refresh the enzyme with different un-touched molecules.
After 300 secs passed the rate at which the Amylase broke down the Starch increased up until 1200 secs. Until 300 secs the light transmission had gone up to 3%, however from 300 secs onwards the light transmission went up between 300-1200 sec to 34%, that’s a 33% light transmission increase over 900 secs. This had reduced the concentration from the starting 1% to 0.4%, which is a 0.5% deduction form after the initial 300 secs.
However after the 1200 secs mark had passed the light transmission the rate at which the Amylase broke down the starch slowed dramatically, from 1200 secs to 2400 secs the light transmission only increased by 11%, which is a drop in concentration from 1% starch solution to a 0.25% concentration (This means from 1200-2400 secs there was a 0.25 decrease in the concentration) which is a big fall considering the huge increase of 34% from 300-1200 secs.
From the graphs shown in the results it is quite obvious that a positive correlation is present, this means that the enzyme was constantly active with the substrate thought pout the experiment.
From the results it is worth considering that there may be slight variation in data to how fast the experiment was carried out at, for example when you have taken the sample at the timed interval the enzyme is still acting on the substrate and is there fore still breaking down the substrate concentration.
The movement of light transmission thought the experiment changed in a fairly conservative manor, as at the start of the experiment the breakdown of the starch was slow. Then a sudden increase in the amount of starch broken down occurred and finally towards the end the rate at which the concentration of starch was decreased slowed rapidly.
For these different rates to which the enzyme worked can be few reasons, the enzyme may have encountered a sudden decrease in temperature. This will cause the enzyme to work less efficiently as a low temperature will lower the rate they work at. However a sudden decrease in temperature I feel is not a very likely case as the experiment was done inside a room which is nor likely to suffer from temperature irregularity.
It may have been because the enzymes active site was not the right shape for the substrate to slot into to form a substrate complex, however I very much doubt this is the case as Amylase specifically helps the break down of starch so there fore it is very unlikely that this is the cause.
These points stated can be clearly shown on the graphs produced, and it is noticed that there is appositive correlation in the results.
The calibration curve enabled me to convert the relative information from the results to convert the amount of light transmission % into the concentration starch in the solution. The light transmission as seen on the graphs has a positive correlation and it is noticed that as time goes on the more light transmission % is produced and the Absorption gets lower. This is because the red filter in the colorimeter does not pick up on the blue, as there is gradually less starch in the solutions. This on the other end of the scale e means that the colorimeter is receiving more red light on the photosensitive element. Hence absorption % goes up with the less starch present.
From the results there does not appear to be any apparent anomalies that would alter the data greatly. The only odd change is in the difference to which the amount of light transmission% between each 300secs is the fact that from 1200-1500secs it only goes up 2 and does not follow the same pattern, which seems to be occurring around the end of the experiment.
Reasons for why the break down of the starch seemed to slow towards the end is the aspect that there maybe little substrate left in the solution so therefore all the enzymes are not in use and the rate to which the substrate is broke down is therefore reduced. Also if the reactions solution is not constantly mixed there maybe the case that there is not fresh substrate for the enzyme to act upon. If this is the case it could be a worthwhile thought to consider.
Amylase comes in contact with starch whilst in the first stage of digestion, in the first stage of digestion mechanical digestion plays a big part in increasing the surface area. The Amylase in this experiment however only came into contact with the starch with no added bonus of the chewing mechanisms which consequently increases the surface area as well as constantly replacing reacted substrate with fresh molecules. This mechanism will make the rate that the starch is broken down rapidly increase; therefore the amylase may need these conditions to help the digestion of the starch in the first stages of the break down.
In conclusion, it may be said that the enzyme breaks down the substrate in different rates at various points in the experiment. This is proving that the hypothesis is not correct and that there is not a proportional relationship between the Light Transmission/Concentration against time.
Evaluation
Although only two sets of results were used, they are in broad agreement (within the accuracy of the experiment) so the conclusions may be stated with confidence. Even though the two sets of data did not help very much when it came to finding different rate etc. but came in useful when it was necessary to find anomalies in the data as well as constructing best-fit curves.
However, these conclusions rely on a number of assumptions, which although reasonable, were not fully investigated.
-
The boiling tubes that contained the substrate and enzyme where at a constant temperature through out the experiment and for every volume of substrate. This is because during the experiment we noticed that a difference in temperature would drastically affect the rate at which the reaction was carried out. This is because the nature of enzymes and how they act under certain conditions.
-
The size of cross sectional area was the same for every pipette used in the experiment.. The pipettes are most likely mass produced and are not manufactured with amazing amounts precision so it is likely that there could be some variation in the cross sectional area from different manometer tubes. There fore volumes measures could have varied if different pipettes were used during the experiment.
-
The substrate in the reacting solution had continuous fresh molecules being exposed to the enzyme. This could effect the light transmission% as if the enzymes only had molecules that had already been aligned they would there fore slow down the reaction as they cannot get to the fresh substrate.
-
The colorimeter always started on 0% at the start to test each individual vial containing the various samples from the 300 sec intervals. The colorimeters have tendencies to change or be undecided to where they are starting from as well as the decision of a Transmission %. This means that we have to assume it was o 0 every time we used the apparatus.
Readings in the experiment were taken to the nearest whole unit, in this case transmission %. The measurements of the meniscus can vary from the different groups that carried out the experiment; there fore a 1ml error may have occurred when transferring the reacting solution into the iodine solution. This creates little change o the results and is of no relevance to change the raw data to accompany this error.
Anomalies could be counted as the results show that the initial start for the enzyme to start working on the substrate took up to 300 secs and that the end of the experiment had no reason for a sudden decrease in rate. I feel that this is an unexplainable anomaly, which cannot be explained given the condition that the experiment was undertaken in.