Pilot Test
Prior to conducting the full procedure a minor test was conducted to ensure that the chosen dilution of the potato sample would produce suitable readings on the colorimeter. A potato was taken, (does not need to have been stored for a week) and the skin removed, then blended to homogenise it. A sample was prepared as in the method below. A 0.006mol/dm³ sample of glucose solution was prepared as in the below method. Each solution was testes using the Benedict’s test and the colour intensity of the samples compared. It was found that the potato had to be diluted with the same mass of water to facilitate the blending process and produce as smooth sample. Due to unforeseen changes in the thickness of the solution following the Benedict’s test further dilution was found to be necessary to produce appropriate readings on the colorimeter. Other elements of the procedure conducted in the preliminary test which appeared unsuitable or unwieldy were amended for the final Method. In particular the concentration of the potato sample was further reduced than initially planned, to counter the effect of expanded starch molecules on the absorbency of the potato solutions, and a second filtering stage was introduced to help reduce this by reducing the starch in the sample.
Final Method (revised following pilot tests)
Potato Preparation
-Store potatoes at recorded temperatures for a week
-Peel off skin from samples.
-Cut and weigh approximately 20g of potato from each sample piece/potato. Finely chop to speed blending process. Record sample mass and label each tray to avoid confusion.
-A 25ml measuring cylinder will be needed to add an equal mass of water to the mass of each sample during the blending process.
Homogenise Potato Samples
-Place chopped potato sample into blender
-Measure out and pour in an equal mass of distilled water as determined above to dilute the potato and aid the blending process.
-Blend until sample is smooth
-Pour all of blended sample into a relevantly labelled 50ml beaker.
-Repeat for all three samples, rinsing the blender between each sample to prevent contamination.
-At this stage each sample represents a 50% potato solution
Centrifuge Potato Samples
-Once all samples have been homogenised, prepare and label 3 plastic test tubes and ready the centrifuge. Do not use glass tubes inside the centrifuge.
-Draw off enough of each sample using a measuring syringe to nearly fill the relevant plastic test tube. Use a separate 10ml syringe for each sample or thoroughly sluice syringe with water between samples to prevent contamination.
-Place the labelled tubes inside centrifuge, lock the lid and set on a high setting for around 10 minutes although longer is not detrimental so this can be left to run while preparing the glucose solutions.
-If much of the samples remain syringe into similar labelled plastic test tubes and centrifuge with the others to enable the entire sample to be used.
-Rinse the 50ml beakers but retain them and their labels, these will be used in a later stage.
Prepare Standard Glucose Solutions
-Place 6 test tubes in a test tube rack and label each with a concentration of glucose solution from the table below. Ascending order of concentration is advised when preparing the samples.
-Use a 150ml beaker of distilled water and a similar one containing 0.01mol/dm³ glucose solution to measure out using 5ml syringes the correct volume of each liquid for each sample (as shown in the table).
Dilute Centrifuged Potato Samples
-Once all 10ml standard glucose solutions are prepared cease centrifuging and remove the potato samples
-Prepare a labelled test tube for each tube of sample and place in a rack with a prepared funnel and filter paper in the neck of each.
-Pour each sample into the relevant funnel and leave to filter. Most of the solids in the solution will have been pulled to the bottom of the tube during the centrifuging process and the samples should not take long to filter.
-Once each sample has been filtered pour it into the relevant 50ml beaker (those prepared and rinsed previously once potato samples were drawn off)
-Rinse labelled test tubes retaining their labels. Draw off 5ml of each sample from the beaker and place in the relevant labelled test tube. Then draw off 5ml of distilled water to make the quantity in each test tube up to 10ml. Mix thoroughly by swirling/stirring. Concentration of samples at this stage = 25% potato
Prepare for and Conduct Benedict’s test
-Use the dropper in the lid of the bottle of Benedict’s solution to drop around 7 drops (keep number constant) into each test tube of glucose solution and the prepared potato solutions.
-Ensure that the heating block is at a constant temperature of 105C. The thermometer may be required to do this.
-Set the timer for 11 minutes, preferably on alarm. Calmly start the timer and place all the test tubes into the heating block, making a note of the order they were placed in.
-Allow 11 minutes to pass then remove the test tubes in the same order and reinstate them in the test tube rack. (The wait time could be used begin clearing used apparatus)
Filter and Dilute Potato Samples
-Repeat filtering process for each of the potato solutions
-Prepare the 10ml measuring cylinder, a 5ml syringe, 10ml syringe, the test tubes of potato solution and the Beaker of distilled water.
-Using a clean 5ml syringe draw off 3cm³ of the potato solution.
-Using a clean 10ml syringe make the solution up to 10ml using 7cm³ of distilled water.
-Repeat for each potato solution using clean syringes and apparatus.
-This was found to be the necessary level of dilution required to obtain solutions of which the absorbency could be tested. The potato sample at this stage represents a concentration of 7.5% potato
Conduct Absorbency Tests
- Prepare Colorimeter. Only one cuvette will be needed, however this will need to be rinsed between samples. Each solution will be tested in turn in order of concentration (see table). This will be repeated twice to give three results for each sample.
-Turn on the colorimeter, set the wavelength to 470nm using the large wheel on the lower right of the unit. Set the ‘Abs / %T’ Button to Abs.
-Using a dropper pipette siphon enough of the distilled water solution into the cuvette to fill it 2/3 to the top and place the cuvette into position ensuring the arrow points forwards. Press the R button on the colorimeter (used to set the colorimeter to zero).
-Press T to measure absorbency and record in table. Tip out solution and prepare each glucose solution in turn, mixing each before placing into the cuvette and into the device. Press the button marked ‘T’ and record the absorbency of each solution in turn (it is recommended to start with the lowest concentration and work upwards).
-Do the same for each of the three potato solutions.
-Ensure this is repeated each time, including the distilled water test and the results recorded in a suitable table.
Labelled diagram of the Colorimeter which was used
Reliability of Results
A number of measures will be taken throughout the procedure to ensure that the results are reliable and accurate.
-Accurate Measurements
Precise measuring equipment including the syringes, thermometer, timer and measuring scales will be used in the preparation of samples and testing. The same settings will be used for all the measurements taken with the colorimeter throughout. This should ensure a fair and accurately conducted test. The % accuracy of equipment will be recorded to produce a more detailed judgement on the accuracy of result sets.
-Repeats of Results
The tests will be repeated to allow for anomalous results in the first test. Based on the findings of the pilot test this will be done twice to produce three results for each sample.
-Control of Variables
All significant variables in the experiment will be controlled as far as possible. The Temperature of potato storage will be kept at a constant temperature for each of the samples, this will be for the same period of time for all samples (a week) to ensure each has the same length of time to acclimatise its starch/reducing maltose levels. The potato type will be the same for all samples, and samples will be taken from the same potato to ensure that initially their reducing sugar levels are as similar as possible. The constant conditions of tests, care in not contaminating equipment, precise timing and accurate measuring procedures listed in the method should produce a set of reliable and repeatable results.
Results
Storage Temperatures
Room Temperature Sample = 21°C
Fridge Sample = 7°C
Freezer Sample = -9°C
Analysis
Table 3 –Processed mean Absorbencies for all the tested solutions
Table 4 – Estimated Concentrations of Potato Samples derived from the graph of glucose concentrations, showing estimated values for the actual concentration of the samples and scaled up values for a 100% sample.
Graph 1 – Plotted mean absorbency values for the standard glucose solutions with error bars showing minimum/maximum values. Includes estimations for potato sample concentrations.
Conclusions
•The results used to draw conclusions are those which represent 7.5% potato samples. Due to the filtering and blending processes the composition of the samples was significantly changed and so the values for a 100% solution in the table are not appropriately accurate to draw firm conclusions. As such the 7.5% potato values for reducing sugar concentration do not represent the actual concentration within potato cells and are instead used to determine the relationship between reducing sugar levels in the different potato solutions.
•The values for potato sugar concentration in the second column of table 4 will be those used to draw conclusions.
•The graph clearly indicates the expected relationship between volume of precipitate produced during the Benedict’s test and the sugar concentration of the glucose solutions. This trend was used to determine approximate sugar concentrations for the potato solutions by plotting these values on the graph and using the relationship between absorption and concentration to estimate the sugar concentration of the potato sample solutions.
•None of the results appear anomalous as all were repeatable within the region of accuracy found to be obtainable with the colorimeter. The error bars on the graph support this conclusion however the results from the same samples were variable enough to cast doubt on the accuracy of the colorimeter or on the method of its use.
•The results for the estimated sugar concentrations in each of the potato samples do not seem to support the initial hypothesis.
•Assuming that the absorbencies measured correlate directly with the sugar content then it appears that the sample stored at room temperature contained the highest levels of maltose sugar and not as expected the sample stored in the freezer. This is contrary to the hypothesis, the lowest expected concentration appearing to be the highest as determined by this test. Other factors than sugar content however affected the absorbency of the solutions
•It is likely that this caused the room temperature sample to appear to have a higher reducing sugar concentration than the other samples. In particular the attempts to filter the potato sample of dissolved solids were not very effective, affecting the solutions absorbency when the absorbency test was conducted.
•As this was a significantly more noticeable problem with the room temperatures sample and this sample was predicted to have the highest starch concentrations it is probable that the starch expanded when ‘cooked’ during the Benedict’s test and caused the solution to contain more precipitate than that produced from the presence of reducing sugar.
•This would have affected the absorbency of the solution as measured by the colorimeter, the measurements from which were derived the estimated reducing sugar concentrations of the samples. This meant that using the proportional relationship of sugar concentration: absorbency of the solution (saturation of precipitate) was inappropriate in determining an accurate concentration.
•The results for the room temperature sample will be discounted from further conclusions because they were clearly not caused by a temperature related conversion of amylose to maltose and neither are they anomalous and so are not relevant to further conclusions. As the other samples were affected far less by suspended starch I will continue to treat them as suitably reliable results from which to draw a basic conclusion.
•If the Potato samples which were stored in chilled temperatures are taken into account independently from the room temperature sample then the results do appear to support the hypothesis.
•Of the 7.5% potato samples the Fridge sample was estimated to contain 0.005mol/dm³ of reducing sugar and the Freezer sample was estimated to contain 0.007mol/dm³ of reducing sugar. (The room temperature sample contained by comparison a higher concentration of approximately 0.008mol/dm³ reducing sugar)
•This implies that the samples stored at -9°C (freezer sample) converted approximately 40% more of their starch into maltose than those stored at 7°C (fridge sample).
•This supports the hypothesis as it suggests that as the freezer sample was stored in colder conditions its emergency mechanism for converting starch to sugars activated to a greater extent, producing more soluble sugar from the its starch to lower the freezing point of the solutions inside the potato cells. This would in theory have prevented the potato from suffering cell damage until several degrees below freezing. The lower apparent sugar content in the Fridge sample suggests that as the storage temperature was higher, less starch was converted to maltose as the temperature was not dangerously low for the potato. Without a base value for the sugar content in the potato sample (due to the room temperature sample being affected by other factors) it is impossible to tell whether more sugar was present in these samples than there would have been in a sample stored in normal conditions where starch conversion would be unnecessary. There is clear evidence however for the Freezer potato sample having converted starch to maltose when placed in extreme cold conditions, from which a degree of support for the hypothesis can be deduced.
•With this considered I conclude that the results for the chilled potato samples achieved in the experiment imply partial support the hypothesis, however significantly more testing would be necessary to make a supported judgement. I would adapt the hypothesis to take into account a possibility of other factors that could have affected the reducing sugar content of the potato sample but i do accept the original hypothesis based on the loose support of the results for the background theory.
Evaluation
Accuracy of Procedure
-The repeats were sufficient and without significant anomalies.
-There were unexpected factors encountered while conducting the procedure which affected the accuracy and resultantly the reliability of the processed final results derived from the potato samples. The results did not represent the actual sugar concentration of a 7.5% potato sample, and instead were used abstractly to make a judgement on the proportions of sugar content in each sample relative to the others. This was a compromise and did not constitute the planned level of accuracy.
-The methods of measuring the absorbency were not at fault however as the repeated tests for both glucose sugar solutions and potato solutions were concurrent within a significant margin (0.1 arbitrary units) without anomalies. These values represent the absorbencies they were intended to show and their accuracy is not flawed, the error is with the absorbency of the potato sample solutions being affected by factors other than the precipitate from the Benedict’s test which means that the final values for absorbency were not wholly indicative of potato sugar content.
-This is evidenced by the unexpectedly high estimated sugar concentration of the Room Temperature potato sample (0.008mol/dm³) which the theory behind the emergency starch conversion mechanism indicates cannot only be a result of the effect of storage temperature.
-The most significant factor in this was likely caused by difficulties in purifying the samples that necessitated further dilutions to counter the effect of expanded starch molecules in the solutions (on their absorbency readings). This extract further explains the effect of starch on the viscosity and absorbency of a solution:
“When heat is applied to starch granules suspended in a liquid, the starch granules absorb water and swell. Starch molecules have many hydroxyl groups which react with the water molecules, attracting and holding them. The smaller amylose molecules diffuse out of the swollen starch granule and form a 3-D network which traps additional water. The loss of free water and restricted flow of the water due to the enormously swollen granules occupying more space, contribute to the increased viscosity of the dispersion”
taken from http://www.library.ubc.ca/ereserve/hunu201/fdmanual/page46.htm
As the Room Temperature sample was expected to contain more starch (No need for higher than normal maltose conversion by hydrolysis of amylose because of milder conditions so more of the initial starch was intact), it seems probable that this was a far greater problem for this sample than for the others stored in the colder conditions that would have provoked excess starch conversion. This made it necessary for the room temperature sample to be discounted which severely reduced the conclusions which could be drawn from the results in regards to the accuracy of the hypothesis.
-A further possible interpretation of the contrary results is that if the potato that was used had begun germinating there would have been a higher than normal breakdown of starch to simpler sugars. This would have been inhibited by storage in cold conditions whereas the germination process would have continued unabated in the potato sample stored at room temperature. A potential explanation then is that the potato had begun to germinate prior to being cut into samples and used for testing and this continued in the Room Temperature sample and not in the other samples leading to more maltose production in this sample. Although this did not appear to be occurring it remains an unexpected variable which was not controlled and so could have caused the estimated high concentration of sugar in the Room Temperature sample. I am also uncertain that separate pieces of a potato can begin or continue to germinate, if this is indeed the case then this seems a possible explanation for the contrary to hypothesis results.
-In addition the Benedict’s test appeared to occur at a different rate in some of the samples as some took longer to change colour than others. This may have influenced the quantity of precipitate produced and so the absorption of the different samples.
-Other unexpected variables also need to be taken into account. The frozen sample appeared to have suffered massive cell damage - how this would have affected its reducing sugar content, the ability of its cells to convert amylose to glucose and the concentration of precipitates which could potentially influence the absorption reading are all unknowns, none of which were prepared for. This casts further doubt on the use of the final values to determine anything more than which of the samples had the largest concentration. Numerically supported conclusions on the proportion of sugars in each of the samples are very unreliable hence their lack of inclusion in the analysis of the results.
The suitability of the procedure is in question as relevant variables were not controlled. This severely affected the accuracy of the results and limited the conclusions which could be drawn. Were this experiment to be repeated or extended then major changes would have to be made to attempt to minimise or remove the above uncertainties in order to obtain supportively accurate results.
Accuracy of Measurements
-The colorimeter did not volunteer the same reading for identical samples taken from an identical source, even when the same sample was retested the value rarely was the same. This casts some doubt on the accuracy of this piece of equipment although the range in values was small enough to be acceptable within the greater context of this investigation. (Within 0.1 Arbitrary units for all repeats)
-It was also not possible to remove the entirety of potato residue from the blender when creating the homogenised samples. This caused the dilution of the potato in the prepared samples to be an unknown value, greater than the planned value of 50%. which affected the accuracy of the final results.
-Using samples from the same potato limited the repeats possible due to the small volume of the processed homogenised potato solution.
-The other measurements were as accurate as possible and did not have a significant degrading effect on the accuracy of the final results. The % error for the syringes, thermometer and measuring cylinders were all small enough to be insignificant compared to the other errors found when conducting the procedure and as such were not a problem.
Significant Errors
-Uncontrolled Variables were the most significant error, this includes:
The potato solutions at the point of the Benedict’s test were more viscous than the distilled water and glucose solutions and contained suspended starch molecules that the glucose solutions did not making the use of the glucose absorbency curve to estimate the concentration of the potato solutions in appropriate. This is because the starch in the samples expanded when exposed to the high temperatures in the heating block and significantly affected the absorbency of the potato sample solutions. This was not a constant factor across the samples as the very nature of the mechanism being tested meant that some would contain more starch than others. This necessitated a further filtering stage and dilution in addition to that initially planned which failed to totally neutralise the problem as evidenced by the results. This is the most significant error in the method that was used.
Uncertainty over the effect of freezing on the 'freezer' potato sample.
-Errors in the accuracy of the dilution of the potato samples were also very significant. This includes the blending stage and further filtering stages and was a result of an incompletely blended solution due to the limits of the apparatus used and the planned procedure. This meant an uncertain concentration for the final tested samples and should be resolved.
Proposed Improvements and Revisions
Based on inaccuracy and flaws in the procedure and measurements the proposed changes to the method are as follows:
-A whole non-germinating potato from the same batch should be used for each of the samples to allow for the potential of more repeats of or in the experiment on the same sample potato sample and help control the variable of potato germination. This would introduce an uncontrollable variable but so long as the type and source of the potatoes could be accurately traced back to the same place (those grown in the same plot of an allotment would be ideal) it would in general enhance the method and improve accuracy not detract from it.
-A specialised blender for potato homogenisation should be used to ensure a sufficiently blended sample and prevent the some of the potato solid remaining in the equipment after the sample was prepared. If more specialised equipment is not a viable option then the blender jug could be rinsed with a specific volume of water and added to the potato sample taken following the blending process. So long as the volume of water used was not too high then the dilutions prior to the Absorbency testing phase could be adjusted to compensate.
-A more accurate colorimeter should also be used to prevent the medium variability of the results which occurred.
-To counteract the effect of suspended starch on the absorbency readings the potato solutions should be heated at around 50-60°C (to prevent evaporation yet ensure expansion of suspended starch) for 10 minutes and then tested for their absorbencies in relation to a distilled water sample that had undergone the Benedict's test. The value for absorbency prior to the test on the potatoes could be deducted from the final absorbency values after the test to try and eliminate the negative effect of suspended starch on the accuracy of the results. This would work best if the Benedict's test heating stage for the standard glucose solutions occurred at the same time as the heating of the potato samples, and these two sets of samples were tested for their absorbency separately.
-An additional or repeated additional filtrations on the potato samples would also improve accuracy by further reducing the suspended starch in the samples. This could be implemented after the proposed heating phase for the various potato samples prior to the Benedict's Test but before the first absorbency test on the samples. This would work best were a greater volume of potato sample prepared as filtration of small samples using the method used in this experiment is difficult.
-An increase in the volume of potato sample produced would benefit the accuracy of the samples and make filtering samples more efficient. It would also enable more repeats on the same sample. In addition were the blender to be swirled out as suggested above then using more water to do this would increase the effectiveness, increasing the volume of sample produced would allow a greater volume to be used. As such I would propose doubling the mass of potato to be blended and scaling up further stages of the procedure accordingly.
-A change to more specific temperatures that would prevent the unknowns surrounding the sample being frozen.
The proposed tested temperatures and storage locations would be:
Freezer between -1°C and -3°C
Fridge at no higher than 5°C
Room Temperature as in initial method.
Implementing the above improvements would greatly increase the accuracy of the procedure and potentially allow better supported conclusions to be drawn from the results. This would enable far more accurate judgements as to the accuracy of the hypothesis to be made.
(Pilot testing would be necessary to ensure that implementation of these changes into the existing method is a possibility.)
