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Investigation of the effect of adding different concentrations of NaCl to an enzyme-substrate (amylase-starch) solution.

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Introduction

BIOLOGY Coursework - Investigation of the effect of adding different concentrations of NaCl to an enzyme-substrate (amylase-starch) solution. Investigation The objective of the assessed practical is to investigate the effect of different concentrations of sodium chloride on the rate of hydrolysis of starch using the enzyme diastase, or more commonly known as amylase. Therefore, the principal behind the investigation is to look into different factors that affect the rate of enzyme activity. Background Information Starch Starch is made up of combinations of two different polymers of alpha glucose, amylose and amylopectin. Amylose is composed of approximately 300 glucose units connected by alpha 1,4 glycosidic bonds. Amylopectin is a branched chain composed of approximately 1500 glucose subunits, which are connected by the cross linking of alpha 1,4 glycosidic bond chains by alpha 1,6 glycosidic bonds. The chains branching off the CH2OH on alpha glucose molecules cause the polysaccharide to take up a helical structure. The diagrams below represent the points stated: Amylose - alpha glucose molecules connected by alpha 1,4 glycosidic links Amylopectin - branched chain of alpha glucose molecules joined by 1,4 and 1,6 glycosidic cross links From the diagrams above it is apparent that there are very few free ends on a starch molecule as a whole, this would therefore simply mean that there are not many regions on the molecule where hydrolysis by amylase could commence. An easy method of testing whether starch is present is to add 2cm3 of the solution that is to be tested into a test tube. Add 2 drops of the solution into a spotting tile using a pipette. Add a drop of iodine solution on to the same spot on the tile. If starch is present the solution should change in colour to blue-black. The principle behind this experiment is due to the ability of iodine to bind to the centre of the starch helix, which forms a starch-iodine complex. ...read more.

Middle

19. Once the desired colour is reached then stop the timer and record the time. 20. Repeat steps 11-20 for each of the different concentrations of NaCl. 21. Repeat all the steps for each run of the experiment 3 times to obtain a reliable and dependable average set of results. Pilot Experiment For my preliminary work I will plan a pilot experiment first as a trial run. This will help me to confirm whether my planned method and choice of apparatus is appropriate and works effectively to produce reliable results. Also, it will allow me to make any amendments to the planned method if there are any significant inaccuracies, before the actual investigation is carried out. Trial Run Table to show change in time taken for an iodine solution to change the colour from blue-black into orange after single drops of a starch-amylase solution, containing different concentrations of sodium chloride, are added at 60 second intervals. Amount of NaCl in solution (cm�) Number of iodine drops added at 60 second intervals Time taken for solution to change colour from orange to blue/black (seconds) 0.0 6 540.0 1.0 1 60.5 0.8 1 60.0 0.6 2 119.5 0.4 3 360.5 0.2 4 420.0 The errors in the plan are shown in the table by the fact that all the recordings are very close together and there is very little distinguishing between them. This would lead to inaccurate and reliable results, as it would imply that both 1 cm� and 0.8 cm� concentrations of NaCl produce the same time reading. This would also not show how far apart the actual readings were in seconds as both readings are in the first 60 seconds bracket. Justifying Changes to Method It would be appropriate to analyze the results obtained above before justifying the changes made to the plan. The results above are very close together and the reading show that the reactions occurred far too quickly; therefore it would be appropriate to reduce the amount of certain substances used in the solution. ...read more.

Conclusion

Another factor may have been that the tested solution may not have had chanced to mix, therefore the amylase would take longer to break down the starch due to lack of efficient supply of Na+ ion cofactors. However, the inconsistency described above is only slight and do not generally affecting the overall trends as shown by the graphs. Therefore it can be concluded that there was only one anomalous point. Therefore I can conclude that my results are very reliable, due to the fact that all the three different runs of the same experiment produced the same trend of results, as they were all very close together, in terms of values, apart from the value described in the above. This is also shown in Graph 1. I think that through the evaluation of the results that the conclusions, accuracy, reliability and validity of the experiment are very much secure. None of the anomalous points had a major effect on the trends. The results were accurate to one significant figure because it was possible to time the colour change from orange to blue black to the nearest 0.5 seconds. The results were reliable because I repeated the experiment four times and they produced a trend on the graph. The results did support my hypothesis. By looking at my t test results it has statistically proven that the experiments conducted were not due to chance. Apart from, when comparing 0.8mol concentration of NaCl with 0.6mol concentration of NaCl and 0.6mol concentration of NaCl with 0.4 concentration of NaCl the null hypothesis was not rejected this could have been due to a number of reasons. But I believe it was mainly because of some larger drops of water in the spotting tiles for these particular runs, as this would allow the iodine to change colour a lot easier than with the usual amount. This explanation is more feasible than stating that it was caused by inaccurate measuring on syringes as a pipette is not very accurate because there are no specified measurements on it. ...read more.

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