Iodine
This will be used in the experiments to show the progress of the reaction. Iodine reacts with starch to produce a black/ blue colour. This colour indicates the presence of starch. Thus when there is no positive starch test it shows that it has been broken down by the enzyme.
Conclusions
From the research several points can be made:
- It is important that no inhibitors are present in the reacting mixture between starch and diastase, as this will affect the results on the effect of pH and temperature.
- The pH also needs to be maintained so a buffer will be used in all the experiments.
- As it has been shown that if the pH and temperature is are too extreme then the reaction will not occur. Thus in the trial experiments extreme values will not be used. With pH, not above 13 or below 2. With temperature not above boiling or below freezing. These are counted as being extreme.
- Enzyme concentration must be kept at a constant for all experiments and same volumes used.
- Substrate concentration must be kept at a constant for all experiments and same volumes used.
Key Variables
Before a practical experiment can be carried out, the factors of the reaction which when changed might affect the rate of the reaction, must be identified. These factors are known as the key variables, and deciding which to vary, and which to keep constant during the experiment is important.
The following, are all the factors that should be considered when investigating the rate of this reaction:
- The concentration of diastase
- The concentration of starch
- The pH of the buffer
- The temperature of the reaction mixture
Risk Assessment
Before any laboratory procedures are undertaken potential hazards need to be identified.
- Enzymes are potential allergens and should be handled so as to minimise contact or inhalation. They can be irritants. In case of eye contact, immediately flush eyes with plenty of water for at least 15 minutes. Seek medical attention if irritation develops or persists
- All substances must be in clearly labelled bottles.
- Lab Coat and Goggles to be worn AT ALL TIMES during the experiment
- Sodium Chloride solution causes eye irritation, so avoid contact with eyes and wash hands after handling. In case of eye contact, immediately flush eyes with plenty of water for at least 15 minutes. Seek medical attention if irritation develops or persists.
- As glassware will be used, the up most care to prevent breakage will be taken. This includes never placing cylindrical glassware on the bench where it is able to roll off. If glassware is broken then it should be cleared using appropriate equipment.
-
Iodine solution is toxic and an irritant. Avoid contact with eyes. Avoid direct contact with skin. Only use small quantities at any time. In case of eye or skin contact, immediately rinse with plenty of water for at least 15 minutes. Seek medical attention if irritation develops or persists.
Trial Experiments
These will be conducted in order to identify any possible problems and to find the approximate level of enzyme activity at different pH and temperature values so an appropriate range of temperatures and pH values can be used.
Experiment 1
Aim:
In this initial trial experiment, the aim is firstly, to become acquainted with the method and procedure of the experiment; and secondly, to confirm the fact that the pH affects the rate of the reaction.
Apparatus/Equipment:
In these trial experiments, the following apparatus will be used:
- 0.2M NaCl
- Buffers
- Test tubes
- pipette
- 2% starch solution (fresh
- volumetric flasks
- 0.001M iodine solution
- Graduated pipettes
- Stop clock
- Spotting Tiles
- Thermostatically controlled water bath
Trial Experiment for Enzyme Activity on pH
5cm3 of 1% starch was put into 5 test tubes along with 1cm3 of 0.2M of sodium chloride that was acting as a cofactor. Then 2cm3 of buffer solutions was placed into each test tube. In each test tube a different buffer was used, with pH’s of 4.5, 5.9, 7.0, 8.0, and 8.8. Then using a dropping pipette, 5 drops of 0.001M of iodine solution were placed in each of the hollows in a spotting tile. Using a graduated pipette 1cm3 of the diastase enzyme was added to each test tube and immediately shaken. Then as soon as this had been done 3 drops of the reacting mixture in each test tube was placed in the spotting tile with the iodine. From then on 3 drops was taken out every two minutes, until there was no positive starch test or if there was still a positive starch test after 10 minutes. Table 1 shows the results of the first trial experiment. The, x, indicates a positive starch test while, –, indicates no positive starch test.
Table 1
From these initial results it seems that the enzyme works best at a lower pH. Thus the same experiment was done as above but with buffers of lower pH values. Table 2 shows the results.
Table 2
Graph 1 shows the results of the trial experiment with pH against rate.
Conclusions From Trial Experiment of pH
- In the main experiment pH’s 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0. This range of pHs will give a good set of results to plot a graph.
- In order to be able to get an accurate rate of reaction the sample will be tested every 30 seconds rather than every 2 minutes, as in the trial experiments.
- To ensure accuracy of the pH of the buffer, a pH meter will be used. Graduated pipettes will be used to measure out all the volumes, which should again increase the accuracy and avoid contamination.
- The test tubes in which the reaction is taking place in, will be kept at room temperature to ensure that the only variable being manipulated is that of pH
Trial Experiment For Enzyme Activity on Temperature
5cm3 of 1% starch was put into 5 test tubes along with 1cm3 of 0.2M of sodium chloride that was acting as a cofactor. Then 2cm3 of a buffer solution at pH 7 was placed into each test tube. Then each test tube was placed at different temperatures at degrees Celsius at 10, 20, 30, 40, 50, 60, and 70. This was done using a water bath with the higher temperatures and test tubes were placed in a beaker of ice cubes to be able to reach the lower temperatures. The temperature was monitored by placing thermometers in the test tubes. Then using a dropping pipette, 5 drops of 0.001M of iodine solution were placed in each of the hollows in a spotting tile. Using a graduated pipette 1cm3 of the diastase enzyme was added to each test tube and immediately shaken. Then as soon as this had been done 3 drops of the reacting mixture in each test tube was placed in the spotting tile with the iodine. From then on 3 drops was taken out every two minutes, until there was no positive starch test or if there was still a positive starch test after 10 minutes. Table 3 shows the results of the first trial experiment. The, x, indicates a positive starch test while, –, indicates no positive starch test.
Table 3
Conclusions From Trial Experiment of Temperature
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It can be seen that the rate of reaction between 30-500C is similar. Thus readings will be taken every ten seconds rather than every two minutes as in the trial and the rate will be measured every five degrees at these points due to the sensitivity of temperature on the rate.
- A buffer of pH 7 will be used in the main experiment because if the optimum pH was used the reaction would go too quickly. Thus a higher pH will be used to slow the rate. The buffer ensures that the pH is not changing and the temperature is the only variable that is changed.
Main Experiment For Enzyme Activity on pH
Prediction
From the research it can be predicted that there will be an optimum pH for the enzyme, and this will produce the fastest rate. On either side of this optimum pH, the rate will decrease the further away it is from the optimum.
Method
Buffer solutions of pH 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0 were made up. The accuracy of the buffers was tested using a pH meter and altered if necessary. Then for each pH, three test tubes were arranged and in each one was put the following, using a graduated pipette: 2cm3 of the buffer, 5cm3 of 1% starch solution, and 2cm3 of 0.2M of sodium chloride. Then using a dropping pipette, 5 drops of 0.001M iodine solution were placed in each of the hollows in a spotting tile. Using a graduated pipette 1cm3 of the diastase enzyme was added to each test tube and immediately shaken. Then as soon as this had been done 3 drops of the reacting mixture was placed in the spotting tile with the iodine using a dropping pipette. From then on 3 drops was taken out every 30 seconds, until there was no positive starch test or if there was still no positive test after 15 minutes. The reacting mixture was kept constant at room temperature. At every pH the experiment was repeated three times so that error bars could be plotted on a graph.
The results of the experiments are shown in table 4. It shows the time taken for no positive starch test to be shown. The results are plotted in graph 3.
All of the results gained from all of the experiments conducted will now be presented in a series of tables and graphs. In each case, through conducting a large number of experiments it has been possible to gain Maximum, Minimum, and Average results, displaying the occurrence of any anomalies, or inaccuracies in the results. Where possible this has been shown in the graphs, in the form of error bars.
Table 4
Main Experiment for Enzyme Activity on Temperature
Prediction
From the research it can be predicted that as the temperature increases the iodine will show no positive starch test quicker, because of the sugar being produced. However after a certain temperature the reaction will slow down and eventually stop as the enzyme will become denatured.
Method
The rate was measured at temperatures of 0, 10, 20, 30, 35, 40, 45, 50, 60 and 70 degrees Celsius. Three experiments were taken at each temperature in a test tube, so error bars could be plotted on a graph. In each test tube the following was put in, using a graduated pipette: 5cm3 of 1% starch, 1cm3 of 0.2M of sodium and 2cm3 of a buffer solution at pH 7. the test tubes were then placed at the desired temperature using a water bath for the higher temperatures and test tubes were placed in a beaker of ice cubes to be able to reach the lower temperatures. The temperature was monitored by placing thermometers in the test tubes. The diastase was then placed in a separate test tube so it could reach the same temperature. Then using a dropping pipette, 5 drops of 0.001M of iodine solution were placed in each of the hollows in a spotting tile. Using a graduated pipette 1cm3 of the diastase enzyme was added to the test tube and immediately shaken. Then as soon as this had been done 3 drops of the reacting mixture in each test tube was placed in the spotting tile with the iodine. From then on 3 drops was taken out every two minutes, with the temperature continually being monitored. This was stopped if there was still no positive starch test after 15 minutes of the reaction. Table 5 shows the results of the main experiment. The results are plotted in graph 3.
Table 5
Analysis And Conclusions of Results
Now that all of the experiments have been conducted, the results gained that have been displayed in the form of tables and graphs can now be analysed. It may now be possible to draw certain conclusions from the results about the nature of the reaction that has been investigated.
- The Effect of pH on Enzyme Activity
From graph 3, which shows the rate (min-1) against pH, it seems that the enzyme diastase works best between pH’s 4 – 5.at pH 4.5 the average rate was 0.010. The graph shows that as the rate of reaction increases, the size of the error bars also increases. However the shape of the graph does fit in with that of the predicted graph, as shown in the initial research (Figure 4). There were a couple anomalous results. For pH 5 the rate seemed to be too fast, at 0.011, compared to the other results and pH 5.5 was too low. However, it depends on the curve on the graph that was drawn. The actual process of drawing graphs can provide an error. Drawing an accurate curve freehand is quite difficult requiring a steady and smooth action, and although this skill can be improved with practise, it still provides sources of error. At pH 8, the reaction did not go too completion. Graph 1, which was the trial experiment of pH is fairly similar too that of graph 3. However, at pH 3 in the trial experiment the reaction did not finish, unlike in the main experiment.
In conclusion, it would seem that the optimum pH is quite acidic, between 4 – 5, and the enzyme stops working at pH 8.
- The Effect of pH on Enzyme Activity
From the results it seems that the enzyme works best at around 40 - 45°C. The shape of the graph is similar to the expected shape as shown in Figure 3. However, there were a couple of anomalous results that were missed out from the curve. The rate at 10°C and at 50°C were missed out.
Q 10 Rule
The results gained from investigating how the temperature affects the rate of the reaction can now also be used to test the Q 10 rule.
The table below showing just a selection of results gained gives a mixed response to the generally accepted rule that as there is a 10°C rise in temperature, the reaction time is halved. Although from 0 – 10°C, the rule seems to work from 10 - 20°C it is clear to see the rule has not worked.
Evaluation
Now that the experiment has been conducted and the results have been analysed, the investigation can be evaluated. By assessing methodology and results, and identifying both errors and their sources.
Firstly, the reliability of the results should be assessed; can these results be relied upon to give conclusions that show the true patterns, or trends that actually occur in this reaction? It seems that there is no reason to doubt the methodology behind this investigation, the results gained show what was required to see how pH and temperature affect the rate of reaction. The results in general were consistent to what was expected. Whether the results compiled show exactly what was happening when the experiments were being conducted, is another question because there may be some doubt about the accuracy of the results.
After assessing the methods and procedures used throughout this investigation, areas where errors may have occurred have been identified, and this may explain some of the uncertainty experienced when analysing the results. In the results, some of the error bars were quite large. This shows that the accuracy for these reactions was not particularly high.
Firstly, the various solutions that were being used were not all taken from the same batch of solutions. Due to the allotment of laboratory time, it was impossible to use the same batches of solutions throughout the whole experiment. This was because during the periods when experiments were not being conducted, it was possible that the solutions that were being used may have ‘gone off’, and therefore new batches had to be made up. Therefore, each time a new batch was made up, to say that it was exactly the same concentration as the previous batch would be impossible. And so this is a very real area where errors in the results may have occurred. Modifications that could be made to increase the accuracy would be to only make up one large batch of solutions, and conduct all of the experiments in one go, taking up no more time than perhaps 48 hours. Along with this because many people used the same solutions, due to practical reasons it is possible that they may have become contaminated. If some of the starch had been contaminated with the enzyme for example this would cause problems and account for anomalies in the results.
Secondly, during the preparations of each experiment, the solutions were measured out using graduated pipettes that measured to the nearest 0.1cm3 only. Therefore there is a possibility that volumes were not always measured to the accuracy capable, this may have been due to bad technique, or possibly the fact that there was limited time and a certain amount of pressure to complete all of the practical work. Modifications that could be made to perhaps increase the accuracy of the volumes measured could include using more accurate pipettes; spending more time on both practising the technique, and conducting the actual experiment.
When using the buffers, if left over a long period of time it was found that the pH had actually changed. This could have been due to oxidation of the solutions. However, because of time constraints it was not practical to constantly make new buffers. Thus this can account for errors in the results, as the pH of the buffer may have been different to what was originally measured out. Thus to increase the accuracy, new buffers should have been made up every day, using an accurate pH meter.
Deciphering exactly when the reaction was complete was not always as clear as expected, and this may account for any possible errors. The nature of deciding when the reaction had finished was entirely subjective, and may have changed from day to day, thus brining in error. For a number of the experiments conducted, the colour change that indicates when the reaction has completed, was not always as instantaneous as previously described. On these occasions, the colour change was relatively slow, and this made it difficult to determine when exactly the whole of the solution had changed colour. Therefore there may have been some variation in actually deciding the end point of the reaction, which may have lead to errors and inaccuracies in the results. One possible way to bring in a more objective test would be to use Benedict’s reagent as this would show when sugars were produced. However, this would have been impractical considering the timescale allowed and it does involve a colour change, which again brings in subjectivity
When the ways in which temperature affected the rate of reaction was investigated, it was decided to use a thermostatically controlled water bath. Although this was much more accurate than using a bunsen to heat the water, the accuracy of the water bath to maintain the desired temperature is questionable. The water bath that was being used seemed to be temperamental and not always particularly accurate, this may have lead to errors in the results. Therefore a modification that might be made could be to perhaps use a more sophisticated and reliable water bath. The temperature couldn’t be adjusted so the reaction was occurring at the precise desired temperature; it was always fluctuating.
Thus, as shown above there are numerous possibilities where error may have occurred. These areas of error must therefore be used to explain why the results appeared as they did, making it difficult draw definite conclusions.
Products of the Hydrolysis of Starch
Aim:
To find out the products when starch is hydrolysed by the enzyme diastase.
can be considered to be a of . may be highly branched () or relatively unbranched (amylose). Starch is a polysaccharide. This is made up of a series of monosaccharides, glucose, linked by 1∝-4 linkages. Each pair of glucose units forms a maltose unit, as shown in figure 7.
Fig 7
It is already known from the research that diastase hydrolyses starch. It contains amylases for conversion of starch to maltose and maltase for conversion of maltose to glucose. Thus it can be predicted that the starch hydrolysed by diastase will contain both maltose and glucose.
Iodine solution and Benedict’s solution can both tell the difference between starch and reducing sugars. It cannot however tell what the reducing sugar is or if there are more than one type. Chromatography however can distinguish each compound, by separating chemicals according to their Relative Molecular Mass.
Identification of the products can be achieved in one of two ways:
1. The Rf value of each solute is calculated and compared to published values in the same solvent. (Rf values are less than zero and have no units.)
Rf = Distance moved by the solute
Distance moved by the solvent
2. A number of known substances are run in the same solvent as the unknown substance, and the final positions of each are compared.
Risk Assessment
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Diphenylamine - Toxic. Possible mutagen. Harmful in contact with skin, and if swallowed or inhaled. Irritant.
-
Phenylamine - is toxic and harmful by skin absorption
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Phosphoric acid - Corrosive, causes burns. Harmful if swallowed and in contact with skin. May be harmful through inhalation. Very destructive of mucous membranes, respiratory tract, eyes and skin.
Paper Chromatography Method
A piece of chromatography paper was cut to about 25cm3 in length and placed on a clean surface. To avoid contamination, the paper was held at the top and plastic gloves were worn throughout the whole experiment. A pencil line was drawn three centimetres from the bottom of the paper and then four marks lightly along the line at four centimetre intervals. At each mark a different letter was written. G represented a sample of glucose, M represented a sample of maltose, E represented a sample of starch that had been hydrolysed by the diastase and A represented a sample formed by hydrolysing starch with acid. Using a micropipette, the samples were taken and a small amount was lightly dotted on the corresponding letter on each pencil mark. The chromatography paper was then placed in a glass jar in a solvent containing a mixture of propan-2-ol, ethanoic acid and water in the ratio 3:1:1. A cover was then placed over the top and the chromatogram was left to run, until it had reached ¾ of the way up. A locating agent was then prepared to show up the spots. This consisted of 25cm3 of 2% phenylamine in propanone, 25cm3 of 2% diphenylamine in propanone, and 5cm3 of 85% phosphoric acid. The paper was then drawn through the locating agent in a shallow dish and then put in the oven to dry.
Results
From Fig 8, the following can be said. The highest level reached is that of glucose because it is the smallest molecule. Therefore, G and A contain only glucose. This is acceptable with the research made. M contains only maltose and is the lower than that of glucose because it is a smaller molecule. E, which is the products of the hydrolysis of starch by diastase, has two lines showing it contains both glucose and maltose.
Fig 8 – chromatography paper from experiment
Rf Values
Rf of G = 2.6/4.7 = 0.55 Rf of A = 3.2/5.5 = 0.58
Rf of M = 1.0/4.3 = 0.23 Rf of E = 1.2/4.4 = 0.27
= 3.5/5.0 = 0.70
This shows that G, A and E all contain glucose, even though the Rf of E, being 0.70 is higher than the others, it can still be counted as being glucose. Experimental error can account for this, as the drops of liquids were not all at the same starting point on the chromatography paper.
It also shows that M and E contain maltose.
Conclusions
From the results it can be said that the products of the starch hydrolysis by diastase contain both maltose and glucose, which is in line with the prediction made.
Bibliography
‘Chemistry Students Book’ – Nuffield Advanced Science
‘Chemistry In Context’ – Graham Hill And John Holman
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