Investigating the effect of Enzyme Concentration.

Using my knowledge of Enzymes, I have decided to do my experiment in the following way:

Here is a diagram of the experiment I conducted:

Equipment used

• Boiling Tubes X 5 – to conduct experiments in
• Test Tube rack – to hold up the test tubes
• Large Syringe – to measure 5cm³ of starch
• Small Syringe – to measure different amounts of Amylase
• Pipette – to measure precisely the volume used in each experiment is the same
• Iodine Solution – used as a test for starch
• Benedict Solution  - used for reducing sugars (glucose, maltose etc)
• Spotting Tile – Used to test for starch
• Stop Clock – to time how long it takes for the starch to become negative
• Amylase – 1cm³ of amylase at different concentrations: 0.5%, 0.75%, 1.0%, 1.5% and 2.0%
• Starch – 5cm³ used in each experiment

Method

• Gather all equipment
• Take a large syringe and measure 5cm³ of starch and place in a boiling tube.
• Next, take a small syringe and measure 1cm³ of amylase depending on the following concentration: 0.5%, 0.75%, 1.0%, 1.5% or 2.0%
• Place iodine in a spotting tile. Two drops per spot.
• Add amylase to starch and begin the timer.
• Add an equal mixture to the spotting tile every 30 seconds.
• When the solution turns from black to light brown, record the time it took for the starch to become negative.
• With the remaining mixture, add benedict solution and place in the water bath at 80º for 5 minutes and see if sugar is present.

Fair Test 

        For each experiment, the stop clock needs to begin as soon as the starch mixes with the amylase, to make it a fair and reliable test.  Otherwise if the stop clock is started halfway through the experiment, the results become unreliable and variable.  

        Also, the concentration in each experiment should be measured precisely, measuring the exact concentration of the amylase by using a small syringe and a pipette.  If not, the results will be inaccurate and there will not be a clear pattern showing how the concentration of amylase affects the rate of reaction the enzyme controls. Also, another point to bear in mind is to not measure out the two solutions in the same measuring cylinder before the experiment, otherwise the solution will have already mixed thereby making it very unreliable.

        Another way of making sure that the experiment is fair is to ensure that the temperature surrounding the investigation, is the same for each experiment.

        Also if the amount of amylase was more, it will affect the experiment, because the more amylase in the experiment, the more particles, meaning it will affect the Rate of Reaction the enzyme controls.

Safety Test

• When dealing with acids in a lab, you should always wear goggles in case there is a spillage, which could be very harmful and cause the Acid to burn your skin and eyes,
• Another basic rule when in the lab working with Acids is, never run around.  This floor could be very slippery and if your holding acids you could harm yourself and others working nearby.
• Always tie your hair when doing practical experiments, especially when working near a Bunsen Burner.  You could get distracted for a moment and the situation could be fatal.  
• Amylase is a digestive enzyme. It can be dangerous if it gets on wounds. Therefore, avoid direct contact with it.
• Make sure that when you put iodine it does not get on your clothing as it can leave permanent stains.
•  Do not swallow or drink any substances, as it can be very harmful.
• Do not eat or drink during any part of the experiment.
• When using matches, be sure to handle them with care.
• If a bottle of a chemical is broken on the floor immediately locate and contact the lab technician.
• After the experiment ensure to wash your hands thoroughly before touching any part of your body.
• Do not run around in the laboratory and make sure all bags are underneath the tables.

Observation

         Here are the Results I have collected from my experiment in the form of a table:

Table 1

Table showing my First set of Results using Different Concentrations of Amylase

        From Table 1, it is clear to see that there is a steady decrease in the amount of time taken for the solution to turn Light Brown.  But around 0.5% to 0.75% there is a sudden drop in time by 60 seconds, clearly showing that the reaction speeds up Also, from the table, it is clear to see that when the solution was placed in a water bath at 80º with Benedict solution, it showed us that sugar was present

Table 2

Table showing my Second set of Results using Different Concentrations of Amylase

        From Table 2, it more or less shows that there is a definite drop in time and that the Reaction speeds up when the amount of Amylase concentration is increased.  Also in my second experiment it is clear to see from my Benedicts test that sugar is present.

Table 3

Table showing my Average

Table 4 – Melisha’s results

From the Table above, it is clear to see there is a constant time decrease as the amount of Amylase increases, except in Experiment 2, where there is a time increase for 1.5% of Amylase, 90 seconds.  This may be due to a number of reasons, such as some of the solution may have been mixed before the experiment etc.

Table 5 – Danielle’s Results

In the Table above, the Time Taken for the Amylase to mix with the Starch decreases in both experiments, although in the second experiment, 1.5% and 2.0% of Amylase it took both 90 seconds to react.

Table 6 – Adrienne’s Results

In the Table above there is a steady decrease in the time taken for the Solution to mix in both experiments. Although in the second experiment, it took longer for the 2.0% of amylase to mix with the starch, 70 seconds, where as t only took 60 seconds for the solution to finish with only 1.5% of amylase.

Table 7 – Charmaine’s Results

In the set of results shown above, there is a steady decrease in the Time Taken to complete.

Table 8 – Skevi’s Results

In the Table above, there is also a steady decrease in the Time taken to complete, although in Experiment One, the Time taken is much faster than in any other experiment.

Table 9 - Egle’s Results

In the Table above, there is a slight decrease in the Time Taken for the solution to finish.  Although in the first Experiment, for 0.75% and 1.0% of amylase the Time Taken stays the same, as well as in experiment Two, 0.5% and 0.75% of amylase, the Time Taken is 210 Seconds for both Experiments.

Graphs

        On the next four pages are Graphs showing:

1. My Two sets of Results
2. My Average
3. The Class Average – 6 Students Results.
4. Class Line of Best Fit

Conclusion

        From the Investigation I have conducted, it is clear to see from my results that: there is a steady decrease in the Time taken for the Amylase to react with the starch, when the Amylase concentration is increased.  This is mainly because when the concentration is increases, there is more molecules to react with, meaning they are more likely to collide with each other.  This would mean as the concentration increases the amount of Time Taken for the molecules to react Decreases, speeding up the reaction.

In my Hypothesis, I clearly stated that ‘as the concentration of amylase increases, the time taken for the starch and amylase to React decreases.’  This proves that my prediction is correct.  Another factor that can also affect my results though is the temperature.  If the temperature changes while doing the experiment then I have not kept the variables constant meaning my experiment is not accurate.

        From Background reading, I have also discovered that ‘

Evaluation

        As shown in my Graphs, I do have a few anomalies, which prove that I may have had a few problems when performing my experiment:

        When measuring the amount of Acid in the measuring Cylinder, I always brought the Cylinder to my eye level, thus making the test unreliable and inaccurate.  To make my experiment more accurate, I should put the measuring cylinder on a flat surface and see if it the correct amount.

        Another problem I encountered was with the stop clock, which made the test very unfair as each time I started the experiment I kept pressing the wrong button, and by the time I got it right, the experiment had already turned cloudy.  

To make the experiment more accurate next time, I should let one partner do the timing, and let the other pour the hydrochloric acid into the beaker.

        The test could have also been more accurate if I repeated the experiment a couple more times, thus getting a more accurate result or even by making the temperature go up by 2º, where I would have been able to see where exactly the Rate of Reaction begins to speed up and at what Temperature.

        Also, a burette could have been used to measure the volume more accurately, to give a more precise volume.  This test may have also been unfair due to the lack of accurate equipment.

        Overall, I am pleased with my results and findings, and have a better knowledge of how a Reaction such as Sodium thiosulphate and Hydrochloric Acid can be affected by not just the surface area, a catalyst and the concentration, but also the temperature.