Prediction: Increasing the bead size decreases the rate of reaction i.e. proportional assuming no other limiting factors. Graph above
I aim to keep all of the variables the same. There are many factors, which can increase the rate at which an enzyme works at. These include the following:
- Light (which pigment of light) green, red or blue
- Size of beads and ration it is made at
- Wind, no wind in classroom
- Experiment placed in water-bathes to control the temperature.
- The concentration of enzyme yeast and the amount of hydrogen peroxide
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Use a stopwatch to measure the time to provide accuracy start and stop timer when the beads are first added and when the experiment reaches 80cm3 of O2 produced.
- Use the same equipment and procedures so that if there are errors in the experiment they are carried forward and apply for each experiment so can be ignored.
- Wash all the equipment thoroughly to avoid cross contamination of equipment maintaining accuracy and safety.
- Control the temperature of the reaction by placing the experiment in a water bath and measuring the temperature of that too.
I aim to keep the other factors the same, as they are potential candidates to ruin the accuracy of the experiment. I will keep variables such as enzyme concentration constant by taking the percentage of 5 % enzyme in water so it would be 5 grams of yeast in 100 ml of water. The pH levels should be the same as it was not a varied factor in this experiment and where possible I will use such substances as distilled water to make sure that the ph is not altered. Enzyme concentration is kept the same because the catalase is in immobilised yeast, which contains the same amount of catalase in each bead as in any other bead.
Equipment
3 x 50 cm3 beakers
2 x 100 cm3 beakers
1 x 250 cm3 beakers
4 test tubes
test tube rack
Glass stirring rod
Sieve or tea strainer
5 or 10 cm3 syringe
Clamp stand
20 or 25 cm3 with rubber tubing & screw clip
Distilled water
20 cm3 of sodium alginate solution
1.4 grams of calcium chloride
2 cm3 of catalase solution
50 cm3 of hydrogen peroxide
250 cm side arm flask with stopper
glass gas syringe connected with tube to the side arm flask
Stopwatch
water bath
Justification of equipment
I choose a gas syringe, rather than other such as the u shaped tube-containing water as the products could dissolve in the liquid this is Le Chatelier's Principle, this is where a gas will dissolve into and out of the liquid until there are as many gas molecules going in as out and so the net overall effect is that some of the volume of gas is lost to the liquid. A water bath was used so that the temperature is kept constant throughout out the experiment. I chose to use yeast that contained the enzyme Catalase because it made the experiment far simpler and so saved time, which meant I could do more experiments, and collect more accurate results.
Control
I will repeat the test at each value as many times as possible until I reach minimum of three values within 10% of each other then take an average to make it a fair test. I will do five different sizes of beads
Accuracy I will be accurate by measuring the Hydrogen Peroxide and the water using a measuring cylinder. I have chosen to use a measuring cylinder because they are easy to use and are good for measuring large volumes. To use it to its best I will place it on a flat surface and measure from the bottom of the meniscus. I will measure the time using a stopwatch, which measures to the nearest hundredth of a second. I will use a mercury thermometer to ensure I keep the temperature of the water in the water bath so that it is the right temperature. I will clean the balance of and reset it to zero to ensure the sodium alginate, yeast and calcium chloride is weighed accurately.
Safety
While carrying out this investigation I had to follow these safety rules. Always wear safety goggles to protect the eyes from the Hydrogen peroxide. Keep all apparatus away from the edge of the desk to prevent breakages. Do not run around or rush about. Calcium chloride is an irritant to the eyes, and is also an irritant to the skin and respiratory system. So goggles should be worn and hands should be washed at the end of the experiment. When handling hydrogen peroxide take care because a solution equal to or stronger than 5.9 moles i.e. 20 % or 71 volume is corrosive and causes burns. Solutions stronger or equal to 1.5 moles i.e. 5% or 18 volume but less than 5.9 moles are irritants to the eyes and skin. Wear gloves and eye protection. It’s a dangerous substance if swallowed, as it causes serious internal damage due to the releases of oxygen. Yeast is only classed as a multi celled fungal organism, so there are not really any ethical issues. For safe disposal of the microorganism the equipment will be autoclaved which is a process where they are washed at 100 ºc where all organisms are killed off, and the sink will be treated with bleach.
Method
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Collect 20 cm3 of 2 % sodium alginate solution.
- Stir it with a glass rod to ensure an even consistency.
- Fill the water bath to near full and switch on, turn temperature to 40ºc, and allow water to heat up.
- Dissolve 1.4 grams calcium chloride in 100ml of deionised water.
- Weigh out 5 grams of yeast and pour it into another beaker of 100 ml of deionised water, stir the mixture.
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Add 2 cm3 of yeast solution to the alginate gel, and stir to mix.
- Draw some of the alginate mixture into a syringe. Expel it drop by drop into the calcium chloride solution, from a height of about 10 cm this will allow it to form beads.
- Leave the beads to stand in the calcium chloride solution for 10 minutes, to allow the beads to set completely.
- Drain the beads through a piece of muslin necklace gauze, rinse with tap water.
- Attach a glass syringe to a side arm flask via airtight tubing, and place the side arm flask in the water bath.
- Measure out 50 ml of hydrogen peroxide and pour into side arm flask.
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Measure out a volume of 8 cm3 of beads.
- Empty the beads into the side arm flask put the stopper and simultaneously start the stopwatch.
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Stop the timer when the gas syringe volume reaches 80 cm3.
Preliminary
A preliminary experiment was done to gauge how the results would look and determine around what volume to add drop by drop. Thus reducing the overall time of each experiment and allowing for more experiments to be done in the allocated time. As this was a preliminary experiment the method could be tweaked after finding out what could of affected the results and also provided a test run to implement the plan. From the preliminary I found that the volume of gas expelled should be set at 80 cm3, I found this number was a reasonable figure which all the different sizes reached within a small time. This is also where I perfected my measurements for the percentages of enzyme concentration.
For my preliminary experiment I chose only to do two experiments so that I could get a get a range for which my results would be and to highlight any experimental error procedures, which could be changed for my final experiment.
From these results it shows that both produce the wanted amount of gas increase is relatively quickly. The results could be slightly different to what expected because a lot of the hydrogen peroxide was spilt into the water bath when trying to put it in quickly with the enzymes, however on the whole they seem to follow the prediction, my final results should be similar to the preliminary due to the theory of surface area to volume ratio.
Results 1st experiment anomalous
Second experiment
Analysis
I can see from my first set of results that the rate of reactivity for the immobilised enzymes increased with the decrease in size. This is due to the surface to volume ratio, as there is more surface area more enzymes are exposed to the substrate, this was a prominent factor because I kept the volume for all of the beads the same. Therefore overall the smaller beads had more enzymes that could react with the substrate. When measuring the rates of reaction it would be unfair to have one very large bead compared to one very small bead, the smaller bead might have a good surface to volume ratio of enzymes compared to the bigger immobilised enzyme bead however counting up all he enzymes on the outer surface might show that although the bigger bead has a smaller ratio of surface to volume there could just be more enzymes on the surface. So by taking equal volumes of the enzymes it was a more accurate set of results, thus allowing for the surface area to volume ratio to work.
My first set of results where anomalous because the yeast used was a day old so the enzymes where less reactive causing the bigger enzymes to be more reactive, another factor to consider would be whether if the bead is large enough whether the substrate can diffuse through the encapsulated bead and so the surface area to volume ratio doesn’t become such a factor because the substrate can be broken up by the enzymes inside the bead. I also found it difficult to get accurate measurements of liquids until I found out the best way to take an accurate measurement was to take the volume from the lowest point of the meniscus this happens because of the adhesion of the water molecules in attaching to the sides of the wall’s of the test tube. By taking the bottom of the meniscus the lowest point of the watermark you in fact actually taking the volume, to get an accurate reading of the volume it must be read from eye level to stop the effects of water and glass diffracting the reading of the light.
When looking at my results from experiment two I realised that the percentage increases for time where getting smaller and smaller meaning that the difference between the rates of reactivity where getting smaller, therefore this could be the onset where the surface area to volume ratio effect becomes the less dominant force and the substrate can diffuse through and into the membrane meaning that the surface area to volume ratio of the immobilised bead becomes less important although still a factor which does contribute to the rate of reactivity, all the results should be pretty similar in increasing times because there is a set time for the molecule to diffuse in and out. There will be a point (bead size) where the majority of enzyme/hydrogen peroxide molecules happen inside the bead and only a small proportion happen outside the bead on the surface, once at this point because the diffusion times are the same for whatever molecule the only thing that will affect the difference in the rate of reactivity will be the difference in surface area to volume ratio’s and in the bigger molecules these play a smaller role thus there is a smaller increase in the time taken for the gas to be produced between the larger size of molecules.
Conclusion
From my first set of results it was quite obvious that there was a serious error in the experiment, this can be put down to series of errors. One of these was the fact that I used the yeast enzyme mixture, which I had mixed the preliminary the day before in the real experiment, thus the yeast mixture was exposed to air, causing. Other experimental errors, which lead to, the anomalous set of results in the first experiment is also due to the difficulty in measuring the volumes of beads, as I was using a simple method of pouring the beads into a measuring tube however this could of lead to miss calculations into the total volume of beads due to the air spaces in between the beads. Another factor to consider would be whether in the larger sized beads actually allowed for the hydrogen peroxide molecules to diffuse through and into the bead itself, thus the surface area enzymes with a high surface area to volume ratio which have a larger amount of enzymes around the surface of the bead. However this becomes a less prominent effect if the hydrogen peroxide molecules can diffuse through the membrane of the bead because they can react with the enzymes inside the bead itself.
To further investigate this experiment I could vary the temperature of the reaction, this would show the how stable the immobilised enzymes are and whether the size contributes to their stability. Another factor to consider would be whether to when measuring out the volume of beads or the number of beads, in my experiment I measured the volume this meant that the surface area to volume would definitely work. If I took the number of beads though the result might be different because a small bead might have a larger surface area to volume ratio however the bead might be so big that there might be more enzymes on the outer surface.
Percentage errors
I can work out the percentage errors for my measurements, by using this formula.
Mass of calcium chloride
(0.005 x 100)/1.4 = 0.7%
Mass of sodium alginate
(0.005 x 100)/0.4 = 0.2%
Pipette
(0.05cm3 x 100)/10=5%
(0.05cm3 x 100)/5=2.5%
(0.05cm3 x 100)/15=7.55%
(0.05cm3 x 100)/2=1%
(0.05cm3 x 100)/50=25%
Measuring beaker
(0.2 cm3 x 100)/250 =0.08%
Measuring tube
(0.1 cm3 x 100)/10 =1%
Measuring tube containing hydrogen peroxide
(0.1cm3 x 100)/50 =0.2%
Measuring tube containing water
(0.1cm3 x 100)/100 =0.1%
Measuring tube to calculate volume of beads
(1cm3 x 100)/8 =12.5%