Variables
There are several variables that could affect the rate of reaction
Temperature:
I believe that with a higher temperature the higher rate of reaction up to a certain point. This is because as particles gain heat they move about much more, therefore thus increases the chance of a successful collision taking place. The reacting particles will collide more frequently, also with more energy; this will increase the rate of reaction.
This is the variable that I will be testing; all of the other variables will be kept constant.
pH:
pH is a measure of the concentration of hydrogen ions in a solution. The lower the pH, the higher the hydrogen ion concentration hydrogen ions can interact with the R groups of amino acids, affecting the way in which they bond with each other and therefore affect their 3D arrangement. A pH that is very different from the optimum pH (usually 7) can cause denaturation of an enzyme. Denaturation is when the enzyme molecule begins to its shape and activity.
The pH of the solution will not be altered and will remain at pH7.
Concentration of diastase:
A higher concentration of diastase means that the rate of reaction will be higher. The reason for this is that with more diastase molecules in a sample, the chance and therefore frequency of successful collisions between the particles is increased.
I will therefore keep the volume and concentration of the diastase the same in all cases (25cm3, 1.0%). As this is what I used in my preliminary work and it gave reliable and accurate results.
Concentration of substrate:
The higher the concentration of substrate, the higher the rate of reaction. This is because the more substrate molecules there are around, the more often an enzymes active site can bind with one.
However, if we go on increasing substrate concentration, keeping the enzyme concentration constant, there comes a point where every enzyme active site is working continuously. If more substrate is added, the enzyme simply cannot work faster; the molecules are effectively queuing up for an active site to become vacant.
I will keep the substrate concentration at 25cm3, 1.0%. As this is what I used in my preliminary work and it gave reliable and accurate results.
Measuring methods
The way that I will measure how long it takes for the reaction to cease is to add iodine to a drop of the mixture, it should turn blue if starch is present, if starch isn’t present the iodine will not change colour. I will measure the colour change by sight, as the blue colour will vary depending on the amount of starch present. The darker the colour the more starch there is in the mixture.
I will use diastase solution (25cm3, 1.0%), and starch solution (25cm3, 1.0%). These solutions proved reliable and gave good results in my preliminary experiment.
I could use a colorimeter to be precise, but through my preliminary experiment I found this unnecessary.
Prediction
I predict that the higher the temperature the higher the rate of reaction. This means that the molecules will move faster and have more energy. This means that the molecules will have an increased chance of a successful collision therefore the rate of reaction will be increased. If the temperature is increased to roughly 60oC, I predict that the reaction will slow down, as the enzymes will start to become denatured.
This means that the active site will change its shape, so the substrate will no longer be able to react.
However when I carried out my preliminary experiment, the enzymes didn’t denature until over 60oC, although theoretically they should have denatured at roughly 60oC. As this happened I will test up to 100oC.
This should mean that in my experiment the reaction should slow down just over 60oC, and by 100oC the reaction should cease, as all of the enzymes should be denatured.
Apparatus
- Test tubes x5
- Labels for test tubes and tiles
- Test tube rack x5
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Pipette (5cm3)
- White tile
- Glass rods x6
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Water baths maintained at 25oC, 40oC, 60oC and 100oC.
- Stop clock
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Diastase solution (25cm3, 1.0%)
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Starch solution (25cm3, 1.0%)
- Iodine solution
- Thermometer
Method
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Label 5 test tubes at room temperature, 25oC, 40oC, 60oC and 100oC. To each test tube add 5cm3 of diastase solution, using the pipette.
- Place each tube in the relevant water bath for exactly five minutes, using the stop clock. The first test tube should be kept at room temperature by just leaving it in the room. Measure the temperature of the room with a thermometer and keep it the same temperature by keeping windows shut etc. check the temperature occasionally, to make sure it stays roughly the same temperature.
- At the end of the five minutes remove the tubes from the water baths, and cool them rapidly to room temperature by running the outside of the tubes under a cold tap for about 30 seconds to 1 minute, depending on how high the tube was. To test that they have cooled to room temperature put a thermometer in them and compare it to the temperature you took in step 2.
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Now add to each tube 5cm3 of starch solution and mix with a clean glass rod for each tube.
- At intervals of one minute test each tube for the presence of starch-diastase mixture, place it on a white tile, and add one drop of iodine solution. Use one glass rod for each tube and a separate one for the iodine solution
- Make a complete record of your observations, noting how long it takes in each case before a blue colour ceases to be given when iodine is added to the mixture.
I chose this method, as this is the same method that I used in my preliminary experiment so I know it is reliable.
Risk Assessment
All glassware must be handled with appropriate care, especially when it is immersed in a hot water bath, also take care with the hot water as it is a burn hazard at 100oC.
Results Table
(From my preliminary experiment I found it useful to create a scale for the variation of the colour of the solution when iodine was added. 0 being very dark and 5 being light)
The add results to the table you use the scale to find out what number you think best represents the colour and then add it into the correct time row and the correct temperature column.
Alan Julyam
Biology Coursework