I then used a measuring cylinder to measure out 100cm3 of calcium chloride into a large beaker. I ensured that this beaker was thoroughly cleaned before I used it, using first tap water and then distilled water. This helped ensure there were no impurities in the beaker before I added the calcium chloride. I also ensured that I poured the calcium from the measuring cylinder to the beaker carefully so it would not splash as calcium chloride is an irritant and could cause irritation to the skin but more importantly, the eyes.
Following this, I began to add the mixture in the syringe drop-wise to the calcium chloride solution. Alginate beads containing the immobilized enzyme began to form immediately. I left this mixture to one side in order to give the beads some time to form. I then washed my syringe and the nozzle thoroughly with distilled water in order to ensure that the chemicals did not react and form a sticky substance inside the nozzle, as this would have made it very difficult to use again.
I repeated the above procedure another four times for each different sized syringe. Each time I finished using a different nozzle, I tried to ensure I washed it thoroughly with distilled water. I had to leave the beads in the calcium chloride solution for around 10-20 minutes each time in order for the beads to form, so I had time to ensure that all my syringes were thoroughly cleaned.
After the beads had hardened, I cleaned a small plastic tea strainer with distilled water and then strained the beads. I then washed the beads thoroughly with distilled water. I put each different bead size in labelled evaporating basins. This helped to see the range in sizes as well as make it easier to measure the beads using the micrometer.
I had to take an average for each bead size according to the nozzle they came from. In order to do this, I used a micrometer to measure 10 different beads. I recorded these sizes in a table and later took the average sizes according to which nozzles the beads came from.
Following the recording of the measurements of the beads, I used an empty plastic syringe barrel, which has a thin piece of rubber tubing at the end, with a screw clip holding the tube shut. I put a small piece of nylon gauze at the bottom of the syringe in order to ensure that the beads did not get stuck. I then added the smallest beads to the syringe and placed the syringe in a retort stand. I then closed the screw clip tightly in order to ensure no chemical would drip out and I then added the sucrose to the beads.
I took readings to determine how much glucose there was in the mixture every 30 seconds using Diabur test strips. I started a stop clock and took readings every 30 seconds for 5 minutes. I tired to be as thorough as possible whilst taking my readings. I recorded the amount of glucose there was each time in a table. I then repeated this procedure for each of the different bead sizes and recorded my results.
At the end of the experiment, I washed all of the equipment I had used using first tap water and then distilled water to ensure it was properly cleaned.
Key Variables
- The Independent variable
The only aspect I will vary whilst carrying out my experiment is to change the size of the nozzles. This is the independent variable. I will start with the smallest nozzle size, which will make the smallest beads and work my way up to the largest each time I carry out the experiment.
- The Dependent variables
There will be some dependent variables in my experiment. As a result of changing the nozzles, the sizes of the beads I will be making will change. They will have different dimensions, the most important being the diameter, which is what I will measure and record. The number of beads that will be will also differ according to how large the beads are. This will in turn affect the surface area and therefore the rate of reaction, which means that the amount of glucose in each of the different bead dimensions will be different when the sucrose is added to them.
- The Controlled Variables
I will not vary anything else during my experiment but I will try to maintain a lot of aspects in order to ensure that the experiment is a fair one. I will observe these variables to the best of my ability whilst carrying out the experiment:
- I will maintain the temperature of the chemicals, as they will all be kept at room temperature. I will work away from any water baths, radiators and open windows in order to ensure that the surrounding environment does not affect the temperature of the chemicals I will be using. I will try to work in the same place in the lab I am in each time to maintain fairness of the experiment.
- I will keep the rate at which I make the beads constant throughout the experiment. I will try very hard to push the syringe at the same speed with each different size nozzle in order to ensure that I make as many beads as I can with each different sized nozzle. This means I will be using all of the sodium alginate and invertase mixture productively as much as possible.
- I will always use the same volumes and concentrations of the all the chemicals each time I use a different nozzle. I will use:
2cm3 Invertase solution
8cm3 2% Sodium alginate
2% Sucrose (always measured to the top of the syringe 50cm3)
100 cm3 2 % Calcium chloride solution
- I will always use the same syringe for making the beads to ensure that the test is fair. I will use the same syringe that I intend to put the beads into before adding the sucrose. This is because I want to ensure that the volume of sucrose is always the same but also to make sure that the beads are equally distributed within the syringe each time and cover approximately the same volume of the syringe.
- I will leave the beads to form in the calcium chloride mixture for 17minutes. I will time this using a stop clock. The reason I have chosen to use 17 minutes is that after preliminary trialling, I discovered this was the ideal time to leave the beads to harden and that they would produce the best results after this time.
- I will ensure that I use the same beaker, measuring cylinder and pipette dimensions throughout my experiment. Although I could use many instruments to take measurements, I will always use the same ones to make sure that the experiment is fair.
- I will always be using the same brand of Diabur 5000 test strips to measure how much glucose there is in the bead and sucrose mixture and I will always take down the results in mmol/litre as this will produce the most creditable results.
- I will also make sure that the test strips are used every 30 seconds for 5 minutes with every bead size as this will ensure the results are fair. I will time the reaction using a stop clock, taking readings every 30 seconds without pausing the stop clock for any reason.
- I will always mix the chemicals such that the sodium alginate will always go into the beaker before the invertase and they will always be stirred with a glad rod to ensure equal distribution.
- I will always measure 10 beads of each size, using the micrometer as this will help me to work out a sensible average. It will also make the experiment more fair and plausible because the results will not be based on only one bead coming from each nozzle as even beads from the same nozzle may very considerably in size.
- Finally, I will make sure that all the glassware, measuring cylinders, pipettes, nozzles, syringes, strainer and glass rod are thoroughly washed with tap water and then distilled water each time I use them.
Risk Assessment
Calcium Salts
Calcium Chloride
Calcium chloride is an irritant; wear goggles to protect eyes; avoid inhalation and raising dust. Also irritant to the skin and respirator system. Although calcium chloride is relatively safe to handle, care should be taken that it is not ingested. Calcium chloride reacts with water and can burn the mouth and .
If swallowed- Wash out mouth, give glass or two of water. Seek medical attention as soon as possible.
If solution gets into the eyes- Flood eye thouroughly with gently running water from the tap for 10 minutes. Seek medical attention as soon as possible.
If spilt on skin/clothes- remove contaminated clothing, wash affected area thouroughly.
If spilt in lab- Wear eye protection and rinse area of spill.
2% solutions- Sodium Alginate, Sucrose solution
Low risk. Use eye protection and wash any spillages from skin with water as soon as possible.
Basic safety procedures
- Wearing safety goggles was very important throughout the experiment because I had to ensure I did not get any of the chemicals into my eyes.
- I washed my skin if anything spilt, particulaly the yeast derived invertase which may be more irritating to the skin as it is active.
- I took special precautions with the glasswear to avoid any breakages. I was careful not to place beakers too close to the edge of the bench and always washed up the glasswear I had finished using. This helped to avoid breakages but also keep the bench free of clutter.
- Fortunately, glassware did not pose a problem for me during my experiment but if a breakage had occurred, I would inform a member of staff immeadiately.
Choice of apparatus
Evaluation
The final rate graph I produced did support the hypothesis I predicted before carrying out my experiment. As the beads decreased in size, the rate of reaction increased. This is due to an increased surface area each time the beads became smaller. However, due to my range bars, I can see that some of the results obtained are not as accurate as they primarily appear. The results, particularly for the smallest bead diameter (2.5mm), show the range bar as being spread out over a range of values. This may suggest some variability of the data, which can have an overall effect on the accuracy of the investigation.
Though apparatus and measurements are not central to explaining variability of data, they can, in some cases, play an important role in explaining why a certain aspect of the investigation may not have produced the desired results.
One of the main difficulties with my investigation was the potentially challenging skill of making the sodium alginate beads. My trailing experiment did give me an opportunity to practice this skill. However it seems clear from my results that the bead dimensions may be more difficult to control than I had previously anticipated. Although I used 5 different nozzle sizes, three out of the five were quite difficult to tell apart. It was therefore a problem when I had to decide in which order to place them. In addition to this, making the alginate beads required a steady hand and at times, it was difficult to control the rate at which the beads went into the calcium chloride solution.
I measured the amount of calcium chloride I was using with a measuring cylinder, which may have not been the most appropriate piece of apparatus for accuracy. A biruet or graduated pipette would have been more appropriate for my investigation.
Measuring the glucose concentration was another concern. This was mainly because the colour of the Diabur test strips is at times difficult to determine. Despite having a range of colours to compare the test strip to, it was sometimes hard to see where one colour ended and the next began.
As my beads took approximately 17 minutes to harden, I often made the beads, allowed them to harden, then transferred them to an evaporating basin for use an hour or more later. This may have affected the beads as they were exposed for quite a long time.
I feel that my results were adequate for this particular experiment as I managed to obtain some repeat readings. This was helpful in working out an average for my data. I made two rate graphs using different bead diameters (210mm and 270mm) but I decided that the 270mm rate graph gave the best results, so I chose to use this graph as the basis for my analysis and evaluation. The 210mm is also shown on the next page.
Another important factor is the various limitations involved in drawing a conclusion from a single investigation. The use of 5 different nozzle sizes, though perhaps sufficient for my investigation, is not sufficient for drawing a firm conclusion. This is because a lack of further investigation prevents the more extensive results, which would be required in order to assert a conclusion.
Overall it would appear that my investigation does support my hypothesis that as the beads decrease in size, the rate of reaction will increase.
In regard to further work, I would need far more different nozzles of distinguishably different diameters in order to come to a definite conclusion. In addition, carrying out more repeat tests would help ensure the experiment was more reliable and this may lead to a better end result. I also faced a problem in that the colours shown on the Diabur test strips are of course limited and I had to assume the colour according to the scale I had. However, it might have been a good idea to decide on a value for colours in between the values I was given. This may have made the data more reliable as well as providing a higher degree of accuracy. Finally, I could have used pH buffers in order to ensure the pH of every solution was exactly the same. This would have made the test more fair and perhaps given more accurate results.
For this particular investigation I used yeast-derived invertase but had more time been available, it would have also been profitable to investigate a wider range of invertase enzymes, perhaps derived from different sources. Furthermore, I used the method of immobilizing the enzymes by entrapping them within a gel to form the sodium alginate beads. However, there are a variety of other ways I could have done this:
- Carrier-Binding: the binding of enzymes to water-insoluble carriers
- Cross-Linking: intermolecular cross-linking of enzymes by bi-functional or multi-functional reagents.
- Covalent bonding to a solid support
- Encapsulation behind a selectively permeable membrane
