Analysis of Polysaccharides Experiment

Performer: Aylin NEBOL

Partners' names: Ceylan Nur BULUT & Dila ERDEM & Ceren KURT

INTRODUCTION

A positive control group is the group that is expected to have positive results and tells whether the protocol is functioning as planned or not. Negative control groups are expected to have negative results and according its results, again, it is understood that if the procedure has flaws. Test group is tested through an experiment and its results are compared to the positive and negative groups (M. Anne Helmenstine 2017). For example, when researching about bacteria on lettuce leaves, a negative control group is set up in a way that no bacteria could grow. The lettuce leaf and bacterial growth plate are wiped then some bacteria are put on the plate and observed whether bacterial growth occurs or not. As a negative test group, it is expected not to see any growth but if it does occur then this means there is something wrong about the experiment like contamination. In positive control groups, the plate is not cleared and some lettuce bacteria are put on it. It is expected to see positive results which means bacterial growth (Frequently asked questions about how science works n.d.). Carbohydrates are the molecular compounds which are usually called sugars and consist of C, H, O. They are divided into three main groups: monosaccharides, disaccharides and polysaccharides. Polysaccharides are very large molecules composed of numerous monosaccharides via condensation polymerization. Three types of them can be listed: Starch which is found in plants and storing energy, glycogen is a storage in animals and cellulose which is a part of cell wall in plant cells (Chemistry for Biologists: Carbohydrates n.d.). Polysaccharides can be degraded by using enzymes that can break glycosidic bonds. A substance that can be oxidized and reduces others in a chemical reaction is called reducing substance. Reducing sugars display same features as other reducing substances additionally they have an open chain of aldehyde or ketone group in their molecular structure. Since monosaccharides, including glucose, have these properties they are all called reducing sugars. Digestion enzymes can be used for the hydrolysis like amylase. It digests the polysaccharides into smaller fragments by breaking the linkage. Enzymes only work at specific temperatures, since they can be easily effected by changing temperature and become denatured (Held 2012). Heat and strong acids can be used to hydrolyze substances. Both methods can break the linkage between substances and degrade them into monomers.

Benedict's test is a procedure that helps to find out whether a solution contains reducing sugar. Qualitative test produces colour change depending on the ingredients of the solution. Quantitative test is the precipitation of potassium or copper of the solution (Food Tests - Benedict's Test for Reducing Sugar - Brilliant Biology Student n.d.).

Reducing sugar in solutions reduce Cu²⁺ ions in benedict's solution and precipitate them (Food Tests - Benedict's Test for Reducing Sugar - Brilliant Biology Student n.d.).

AIM

The aim of this experiment was to observe how temperature change, and enzymatic and chemical reactions affects carbohydrate structure; how experiments are conducted considering the results of control groups.

MATERIALS

Chemicals

• 0.5% Starch
• Na₂CO₄
• dH₂O

Solutions

• Benedict' solution
• 6M HCl
• 1M Glucose

Lab equipment

• Test tubes
• 5 & 10 ml serological pipettes and bulb
• Fume hood
• Water bath
• Beaker
• Aluminum folio

Biological Samples

• Saliva
• Amylase
• Toothpicks

METHODS

Set #1:

• The test tubes were labelled as 1A, 1B, 1C and 1D.
• Saliva samples were gathered from each group member in a beaker.
• Into the 1A tube, with the help of serological pipette and bulb 7ml dH₂O and 1ml saliva were put.
• Into the 1B tube, with the help of serological pipette and bulb 7ml of 0.5% starch and 1ml saliva were put. The starch was shaken in case the solution precipitated before addition to the tube.
• Into the 1C tube, with the help of serological pipette and bulb 1ml dH₂O and 7ml of 0.5% starch -already shaken- were put. The starch was shaken.
• Into the 1D tube, with the help of serological pipette and bulb 1ml amylase and 7ml of 0.5% starch -already shaken- were put.
• Top of the tubes were covered with small pieces of aluminum foil and mixtures were shaken one time.
• The tubes were held at 37 ^oC for an hour.
• After the one hour, with the help of serological pipette and bulb 2ml of Benedict's solution was added to each tube.
• Colour changes were observed and noted.
• The test tubes were put into water bath for 5 minutes.
• Colour changes were observed and noted again.

Set #2:

• The test tubes were labelled as 1E, 1F, 1G and 1H.
• Into the 1E tube, with the help of serological pipette and bulb 8ml dH₂O was put.

• Into the 1F tube, with the help of serological pipette and bulb 8ml of 0.5% starch was put. The starch was shaken in case the solution precipitated before addition to the tube.

• Into the 1G tube, with the help of serological pipette and bulb 8ml dH₂O and a few pieces of toothpick were put.
• Into the 1H tube, with the help of serological pipette and bulb 8ml of 1M glucose solution was put.
• Top of the tubes were covered with small pieces of aluminum foil and mixtures were shaken one time.
• The tubes were put into water bath for 15 minutes.
• After 15 minutes, the tubes were waited to cool for couple of minutes.
• After tubes were cooled, with the help of serological pipette and bulb 2ml of Benedict's solution was added to each tube.
• Colour changes were observed and noted.
• The test tubes were put into water bath for 5 minutes.
• Colour changes were observed and noted again.

Set #3:

• The test tubes were labelled as 1I, 1J, 1K and 1L.
• Into the 1I tube, with the help of serological pipette and bulb 5ml dH₂O and 3ml HCl were, respectively, added.
• Into the 1J tube, with the help of serological pipette and bulb 5ml of 0.5% starch and 3ml of 5N HCl was put, respectively.
• Into the 1K tube, with the help of serological pipette and bulb 8ml of 5N HCl and toothpick pieces were put.
• Into the 1L tube, with the help of serological pipette and bulb 3ml 5N HCl and 5ml of 1M glucose were added.
• Top of the tubes were covered with small pieces of aluminum foil and mixtures were shaken one time.
• The tubes were put into water bath for 15 minutes.
• After 15 minutes, the tubes were waited to cool for couple of minutes.
• After tubes were cooled, until the bubble formation ended Na₂CO₃ was added slowly.
• With the help of serological pipette and bulb, 2ml of Benedict's solution was added to each tube.
• Colour changes were observed and noted.
• The test tubes were put into water bath for 5 minutes.
• Colour changes were observed and noted again.

RESULTS

The tubes were labelled to make the experiment easier and not to confuse about the ingredients of tubes. For the first setup saliva was gathered to see if amylase enzyme degrades starch. In the second setup effect of heat on carbohydrate degradation was observed. Finally, in the third setup effect of acidic and basic substances on polysaccharide structure was observed.

In the set #1, negative control tubes were 1A tube and 1C tube; positive control tube was 1D tube, and test tube was 1B tube. In set #2, negative control tube was 1E tube; positive control tube was 1H tube, and test tubes were 1F tube and 1G tube. In set #3, negative control tube was 1I tube; positive control tube was 1L tube, and test tubes were 1J tube and 1K tube.

In set number 1, after one hour at 37 ^oC no colour change was recorded however, after the addition of 2ml Benedict's solution all tubes became blue. After 5 minutes in water bath, colour change was not observed at 1A and 1C tubes they stayed blue, both tubes did not contain glucose; 1B tube had glucose and after 5 minutes in water bath colour change could be easily seen, it turned to dark orange; 1D tube also had glucose and after 5 minutes in water bath colour was seen as relatively light orange.

In set number 2, after the addition of 2ml Benedict's solution, in 1E and 1F tubes colour of the mixtures became blue and after 5 minutes in water bath no colour change was observed but only it was noted final colour of 1F tube was blurrier than of 1E tube and it was getting darker through the top. In 1G tube, contained glucose, after addition of 2ml Benedict's solution the colour became light green and after 5 minutes in water bath the colour was seen as relatively dark. In 1H tube, contained glucose, after addition of 2ml Benedict's solution the colour became yellow and after 5 minutes in water bath colour turned to dark orange.

In set number 3, in 1I, 1Jand 1L tubes addition of Na₂CO₃ did not affect the colours however, after the addition of 2ml Benedict's solution to each tube, colour of the mixtures turned to blue, in L tube the colour was greenish blue. Both 1I and 1J tubes did not contain glucose while 1L tube contained. After 5 minutes in water bath in 1I and 1J tubes no change was observed and in 1L tube orange-red colour was observed. In 1K tube, contained glucose, addition of Na₂CO₃ took very long time, after the addition of Na₂CO₃ and 2ml Benedict's solution the color was turned to green, after 5 minutes in water bath colour of the solution turned to reddish brown.

DISCUSSION

The aim on this experiment was to obtain the information about not only the carbonhydrate structure and also effects of different chemicals and temperature on the structure. Analyzing of carbohydrates was done in three main groups each of those was divided into four groups. First group examined enzymatic effect, second group examined heat effect and the third one examined acidic effect.

The reaction with Benedict's solution needs heat to accelerate the redox reaction of Cu²⁺ and reducing group in the sugar, so colour change can be seen quickly. With the aid of this property of the solution, sugar presence in a mixture was deduced in the experiment.

In 1C tube water could not digest starch, in 1D tube amylase enzyme and in 1B tube saliva could digest starch. So, it can be concluded that saliva contains amylase enzyme. If toothpick, which essentially is a cellulose, had used instead of starch solution it would be expected that amylase enzyme would not break the bonds between cellulose monomers so it cannot digest toothpick.

In the 3^rd experimental set up Na₂CO₃ was added to each test tube to neutralize HCl in the solutions. The reaction was Na₂CO_3(aq) + 2 HCl_(aq) → 2 NaCl_(aq) + H₂O_(l) + CO_2(g)

For each setup, control groups were chosen considering their results. For example, for the 1^st setup it was examined that if starch can be digest with water. Since the result is negative, the tube is chosen as negative control group likewise, positive control groups can be determined in the same way. Test tubes are the ones whose results are not known and want to be examined. Their results are deduced from the results of positive and negative control groups.

Expectations for 1^st setup were satisfied by the observations. It was expected that amylase enzyme in saliva could digest starch and H₂O could not. By the colour change in the B and D tubes it can be seen that observations are compatible with the expectations. In the 1^st setup, after the addition of 2ml Benedict' solution colours of all solutions in the tubes turned to blue. After 5 minutes in water bath in the tubes 1A and 1C no change was observed whereas, in 1B and 1D tubes colour of solutions turned to dark and light orange.

Expectations for 2^nd setup were satisfied by the observations. In this setup, the effect of heat on carbohydrate structure was examined. Colour changes tell that heat achieved to break the glycosidic bonds that bind carbohydrate monomers to form polymer chains. In the 2^nd setup, after the addition of 2ml Benedict' solution in 1E and 1F tubes, colour became blue. Colour of the solution in 1G tube turned to greenish shade of blue and in 1H tube the colour was seen as yellow. After 5 minutes in water bath in 1E and 1F tubes no colour change was observed; in 1G tube colour turned to dark green and the solution in the 1H tube was seen as dark orange.

Expectations for 3^rd setup were not completely satisfied by the observations. Expectations for the J tube was to see colour change to pink-red after the process of addition of Na₂CO₃ and heating in water bath for 5 minutes on the contrary there was no considerable colour change in the tube. This might be caused by adding insufficient Na₂CO₃ to the solution to neutralize HCl so starch could not be degraded and colour change could not be observed. In the 3^rd setup, after the addition of 2ml Benedict' solution 1I and 1J tubes turned to blue, solution in 1K tube turned to green and in 1L tube observed in greenish shade. After 5 minutes in water bath in the tubes 1I and 1J no colour change was observed where the colour of the solution in the J tube should have been turned to pink-red, in 1K tube the colour was change from green to reddish-brown, and in tube 1L orange-red colour was observed.

Colour change is observed when the Benedict's solution gives reaction with glycose. Different concentration of glycose causes different colours to emerge. Degradation of starch and cellulose reveals glycose. Colour changes means starch or cellulose was degraded appropriately. Concerning the Benedict's solution image, it can be concluded that 1A, 1C, 1E, 1F, 1I and 1J tubes did not contain glycose since their colours stayed blue. Also, 1K seemed reddish- brown and 1L seemed orange which means the former has more concentrated glycose. Comparing 1B tube with 1D it can be seen that test group (1B) is more concentrated and has more capacity to digest starch than the positive control group (1D) since the former is has darker shade of orange than the latter. 1G tube was seen green and 1H was yellow-orange, which indicates the H tube has more concentrated glycose. If there was no mistake made in the 2^nd setup, 1J tube would be seen as pinkish. Overall, the 1K tube has the highest concentration among all tubes.

A solution with strong acid and water should be prepared by pouring the water first and then the acid. Acid and water react in an exothermic reaction and such release great amount of heat especially when dealing with strong acids that can boil the water. Moreover, density gradient is also one of the facts. Acids are more likely denser than water and when acid is added to water, the reaction occurs at the bottom of a beaker preventing any splash.

The enzymatic experiment group were put at 37^oC, since it is the body temperature of humans. Because that temperature is the optimum one for the enzymatic activity and because it is wanted to observe in this experiment how the enzymes in human saliva dealt with starch, the group was put at their optimum temperature (Marie Anne Helmenstine 2016).

REFERENCES

Boundless. 2016. "Hydrolysis." https://www.boundless.com/biology/textbooks/boundless-biology-textbook/biological-macromolecules-3/synthesis-of-biological-macromolecules-53/hydrolysis-295-11428/ (March 22, 2017).

Briers, Demarcus. 2012. "Glucose Molecule - DBriers.com." http://www.dbriers.com/tutorials/2012/11/draw-the-structure-of-glucose-molecule/ (March 22, 2017).

"Chemistry for Biologists: Carbohydrates." http://www.rsc.org/Education/Teachers/Resources/cfb/Carbohydrates.htm (March 22, 2017).

"Food Tests - Benedict's Test for Reducing Sugar - Brilliant Biology Student." http://brilliantbiologystudent.weebly.com/benedicts-test-for-reducing-sugars.html (March 22, 2017).

"Frequently Asked Questions about How Science Works." berkeley.edu. http://undsci.berkeley.edu/faqs.php (March 17, 2017).

"Glycosidic Bond | The Biochem Synapse." 2013. https://thebiochemsynapse.wordpress.com/tag/glycosidic-bond/ (March 22, 2017).

Held, Paul. 2012. "Enzymatic Digestion of Polysaccharides." http://www.biotek.com/resources/articles/enzymatic-digestion-of-polysaccharides.html (March 22, 2017).

Helmenstine, M. Anne. 2017. "What Is a Control Group?" https://www.thoughtco.com/what-is-a-control-group-606107 (March 22, 2017).

Helmenstine, Marie Anne. 2016. Acid to Water or Water to Acid? https://www.thoughtco.com/add-sulfuric-acid-to-water-606099 (March 22, 2017).

APPENDIX

1. (b)

Figure 1: Structure of Glucose (a) The cyclic (ring) structure (b) Open chain structure (Briers 2012)

1. (b)

Figure 2: Structure of Glycosidic bonds in (a) Starch and (b) Cellulose (glycosidic bond | The Biochem Synapse 2013)

Figure 3: Structure of the hydrolysis reactions (Boundless 2016)