ANALYSIS OF POLYSACCHARIDES

Experimenter

: Volkan Alyama

Experiment Date

: 25/11/2011

Submission Date

: 032/2011

Purpose
The aim of this experiment is to hydolyze starch using enzyme or changing heat and pH; control if the experiment is successful or not by using positive and negative control groups.

Results
In this experiment, our objective was to hydrolyze starch into its monomer, glocose and detect glucose by using a detective reagent, Benedict's solution. Table 1 : Solutions incubated at 37 for 1 hour Tubes A B C D Ingredients 15 ml H2O + 3 ml saliva 3 ml saliva + 15 ml starch 15 ml H2O + 3 ml starch 15 ml starch + amylase Color change No change From blue to greyish yellow No change From blue to greyish yellow

Table 2: Solutions heated in a boling water bath for 15 minutes Tubes E F G H I J K L Ingredients 15 ml H2O 15 ml starch soln. One toothpick broken into tiny pieces + 15 ml H2O 15 ml glucose soln. 15 ml H2O + 10 ml 5N HCl 15 ml starch soln.+ 10 ml 5N HCl One toothpick broken into tiny pieces + 10 ml 5N HCl + 15 ml H2O 15 ml glucose soln.+ 10 ml 5N HCl Color change No change No change No change From blue to yellow No change Became more transparent No change From blue to greyish yellow

Discussion
As we know the monomer of starch is glucose and glucose causes Benedict's solution to turn into yellow or red from blue ( Benedict. 1911 ), we tried to get glucose from starch and observe it using Benedict's solution. In part I, we tried to get glucose from starch via enzymatic reaction. Tube A was a negative control

group, because it did not contain starch, the substrate of this rection. Observing no color change in this tube, we concluded that saliva does not give any color change with Benedict's solution. Tube C was also a negative control group, it did not contained any enzyme. There was not a color change, so starch does not cause Benedict's solution to change color. Tube D was a positive control, it contained both the substrate and the enzyme and it turned Benedict's solution from blue into greyish yellow. As starch does not causes a color change, we can say that reaction occured in the tube and lead glucose to come up and glucose makes Benedict's solution yellow. Tube B, the tested sample, contained the substrate and saliva and caused Benedict's solution to turn into greyish yellow. As we controlled that saliva does not give a color change, it is obvious that reaction took place and glucose became present. So, saliva should contain an enzyme hydrolyzing starch. In Part II, we increased heat upto 100 and observed if increasing heat will cause the hydrolysis of starch to occur or not. Tube E, containing only water, was a negative control and there were no color change. Tube G, containing toothpick so celluluse, was a positive control and there were no color change. If cellulose was hydrolyzed, reaction will produce glucose and it would cause a color change. It can be said that cellulose cannot be hydrolyzed by heating. As a positive control tube H, containing glucose, produced a yellow color with Benedict's solution. So, even heated, glucose, undergoes reaction with Benedict's solution. Our tested sample, Tube F did not cause any color change, so heating is not enough to hydrolyze starch to its monomers. Using Part II as a negative control ( Tube E for Tube I, Tube F for Tube J, Tube G for Tube K and Tube H for Tube L ), the pH of the solutions decreased by adding HCl, in Part III. Tube I was a negative control and we seen that no color change takes place due to HCl. Tube K was a positive control and there were not a color change. So, increasing pH is not enough to break the strong glycosidic bonds between glucose monomers in cellulose. Tube L was also a positive control and we expect to see a complete yellow color after adding Benedict's solution, but the resulting color was greyish yellow. HCl interferes with Benedict's solution and prevents it from reacting with glucose. Probably, we could not neutralize all HCl and instead of yellow we saw greyish yellow. Tube J, containing starch and HCl was tested and there were no color change. What we expected was to see at least a greyish yellow, because high temperatures and low pH makes hydrolysis reactions to take place. Probably, we did not increase temperature and/or decrease pH enough, so the reaction did not take place. We may also failed to neutralize HCl and glucose could not react with Benedict's solution.

Appendices
Questions
1) For Part I, Tube A and C were negative control tubes, Tube D was positive control and Tube B was test tube. For Part II, Tube E was negative control tube, Tube G and H were positive control tubes and Tube F was test tube. In Part III, Tube I was negative control tube, Tube K and L were positive control tubes and Tube J was test tube. 2) It contains sodium citrate, sodium carbonate, copper sulfate. It undergoes a redox reaction with aldehyde groups. Cu ions are reduced to Cu ions by aldehyde groups which are oxidized. 3) Benedict's solution has a very low solubility in water. Tubes are heated after adding Benedict's solution to make Benedict's solution more soluble,so be sure that it didnot precipitate after adding.

4) Benedict's solution contains Cu ions . Glucose causes Cu ions become Cu ions, so reduces them. Therefore, glucose is called a reducing sugar. 5) In the presence of starch and saliva, we observed a greyish yellow color after adding Benedict's solution. The color changes implies that there should be glucose in the tube, so saliva should contain enzyme(s), probably amylase, hydrolyzing starch to glucose. Even cellulose is mainly made up of glucose monomers, it is very stable. Cellulose requries different enzymes than starch to be hydrolyzed and most animals do not have that enzyme(s). So, even we added cellulose to Tube B instead of starch, we would not have any glucose in the tube and would not observe a color change. 6) HCl + Na2CO3 2NaCl + H2O + CO2 CO2 leaves the solution as gas and leads the bubble formation. HCl interacts with Benedict's solution and blocks it. So, sugar and Benedict's solution cannot interact. We use Na2CO3, to neutralize HCl and stabilize pH.

References
Benedict, S.R. 1911. The detection and estimation of glucose in urine. The Journal of the American Medical Association. 1911;LVII(15):1193-1194. http://en.wikipedia.org/wiki/Benedict's_reagent