Organic Molecules Lab Report PDF

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lab report with carbohydrates and lipids...

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Starch

Maltose

Finding Maltose DETECTION OF ORGANIC MOLECULES Hiba Kausar | Biology 2107 | 06/25/2019

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Detection of Organic Molecules Introduction All living matter is made up of carbon and so molecules that contain carbon are known as organic molecules. They form the building blocks of cells and their organelles. The way organic molecules differ is largely dependent on the different atoms present (especially the number of carbons and hydrogen atoms present) as well as the types of bonds and linkages these atoms form. We hypothesize that we will be able to identify three categories of organic molecules; carbohydrates, proteins, and lipids, with their respective tests. We will also design an experiment to test a solution containing starch (a polysaccharide) and investigate its breakdown or hydrolysis, into maltose (a disaccharide) in the presences of a digestive enzyme, Amylase. Materials and Methods Benedict’s Test to identify the presence of monosaccharides: For this test Benedict’s reagent was tested with one monosaccharide, glucose, in a test tube to act as our positive control as well as with water in another test tube to act as our negative control. Two ml’s of Benedict’s reagent were added to two ml’s of both solutions and heated in a boiling water bath for three minutes or until there was a color change (Shanholtzer, 2018). Benedict’s Test to identify the presence of disaccharides and a polysaccharide: The previously stated method was repeated with two ml’s of three disaccharides; maltose, lactose, and sucrose, each in their own respective test tubes. An additional test tube containing two ml’s of starch, a polysaccharide, was also tested with the Benedict’s reagent (Shanholtzer, 2018).

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Lugol’s test to identify the presence of polysaccharides: This test included monosaccharides and disaccharides from the previous sections; glucose, maltose, lactose and sucrose, to show that Lugol’s will not react with the same carbohydrates that Benedict’s reagent did. To conduct this test, one ml of each of the named substance as well as starch (positive control) and water (negative control) were placed in separate test tubes and five drops of the Lugol’s reagent were added (Shanholtzer, 2018). Biuret’s test to identify presence of proteins: For the experiment, two ml’s of water in a test tube with five drops reagent was used as a negative control, while the same values for albumin (a protein) were used in another test tube as the positive control (Shanholtzer, 2018). Brown paper bag test to identify the presence of lipids: Two pieces of brown paper bag were obtained. On one, two drops of water were used, while on the other two drops of oil. Evaporation of the stains was observed. Testing Unknowns: With the results obtained from the above-mentioned tests, we identified an unknown solution A from a selection of solutions that contained any one of the following substances; monosaccharides, disaccharides, lipids, proteins or a combination of these. Each test was conducted on aliquots of this solution (Shanholtzer, 2018). In a similar method, the tests were repeated with a food sample (bread). A small piece of brownbread was torn into smaller pieces and ground in a mortar pestle with a small amount of water to form a sample. Each test was conducted on aliquots of this solution.

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Testing hydrolytic cleavage: In this experimental design, only the formation of maltose was of interest, therefore only one positive control testing for maltose was required. In a test tube, two ml’s of maltose was tested for with Benedict’s reagent in a boiling water-bath where the color change was observed from blue to brick-red. In separate test tubes the following solutions (two ml’s of each) were tested with Lugol’s reagent as negative controls as they indicated the presence of starch; starch and water, and starch and water with five drops of amylase. To test the reaction of hydrolysis of starch to maltose, starch and water with five drops of amylase were added to a test tube along with Benedict’s reagent. This was left for a few minutes at room temperature before being brought to water-bath in order to observe the different phases of color change and to also compare with the same starch solution that was tested with Lugol’s reagent at room temperature. Results Benedict’s Test to identify the presence of carbohydrates: Benedict’s reagent reacted in the presence of reducing sugars like maltose and lactose, changing from a blue color to a variation of brick-red color (table 1). This indicates that both of those disaccharides had free aldehyde groups, like glucose which was our positive control. Unlike these sugars, sucrose and starch did not change color, as they are complex polysaccharides and do not have available aldehyde groups to react with Benedict’s reagent (Holland, 1999). Tube Contents

Color Change

Water Sucrose Starch Glucose Maltose Lactose

N/A (remains light blue) N/A (remains light blue) N/A (remains light blue) Light blue to brick-red Light blue to brick-red Light blue to brick-red

Positive/Negative for the presence of reducing sugars Negative Negative Negative Positive Positive Positive 4

Table 1; results for the Benedict’s reagent test, testing positive in the presences of monosaccharides and negative in the presence of disaccharides as observed by color change from light blue to brick-red.

Lugol’s test to identify the presence of polysaccharides: Starch, a polysaccharide was detected in the presence of Lugol’s reagent as the color change was observed from amber-brown to blue-black. However, monosaccharides and disaccharides, including sucrose, tested negative in the presence of Lugol’s reagent as there was no color change observed (table 2). Tube Contents

Color Change

Water Starch Sucrose Glucose Maltose Lactose

N/A (remains amber-brown) Amber-brown to blue-black N/A (remains amber-brown) N/A (remains amber-brown) N/A (remains amber-brown) N/A (remains amber-brown)

Positive/Negative for the presence of polysaccharides Negative Positive Negative Negative Negative Negative

Table 2; results for Lugol’s test, testing positive only in the presence of complex carbohydrates like polysaccharides, as observed by the color change from amber-brown to blue-black.

Biuret’s test to identify the presence of proteins: Our prediction was true in that Biuret’s reagent tested negative while there was no protein or peptides present as was in the case of water. But it did test positive when tested with albumin, where a color change from blue to violet, indicating the presence of polypeptide chains (table 3) (Holland, 1999). Tube Contents

Color Change

Water Albumin (protein)

N/A (remains light blue) Light blue to violet

Positive/Negative for the presence of polypeptides Negative Positive

Table 3; results for Biuret’s reagent test, testing positive only in the presence of proteins/ polypeptides, as observed by the color change from light blue to violet.

Brown paper bag test to identify the presence of lipids: We allowed the stains an approximate time of thirty minutes to evaporate at room temperature.

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The water stain, as predicted, evaporated completely while the oil stain did not at all. It tested positive for the presence of lipids. Testing Unknowns: With the unknown solution A, we predicted based on its appearance that it was starch. After testing it with the Benedict’s reagent for a reducing sugar, with Lugol’s reagent for polysaccharides, with Biuret’s reagent for proteins and for lipids on the brown paper bag we found our prediction to be accurate. Solution A tested positive only in the presence of Lugol’s reagent, changing color from amber-brown to blue-black, indicating that it was indeed starch (table 4). For our food sample, we applied the same methods to the solution that we had made in the mortar and pestle. We had predicted that it would test positive for starch, and it did change color to blue-black, but it also tested positive for reducing sugars by changing from blue to orange as well as testing positive for protein with the solute at the settled at the bottom of the test tube (table 4). Reagent/test

Results for Sol. A

Results for food, Bread

Reducing sugars

Benedict’s reagent

Polysaccharides

Lugol’s reagent

Lipid Protein

Brown paper bag Biuret’s reagent

Negative/ No color change Positive/ Amberbrown to Blue-Black Negative Negative/ No color change

Positive/ Light blue to orange Positive/ Amberbrown to Blue-Black Negative Positive/ Solute turned deep purple

Table 4; results indicated that Solution A contained a polysaccharide (Starch), and that the bread sample contained starch, reducing sugars as well as traces of protein.

Testing for hydrolytic cleavage: As we had predicted, starch was broken down to maltose in the presence of amylase (enzyme). With our starch, water and amylase sample, the only positive result for maltose was detected in

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the presence of Benedict’s reagent and not Lugol’s, as the color changed from light blue to brickred. All of our other samples with starch were negative as the color remained unchanged for the presence of maltose (table 5). Solution Starch + water Maltose + water

Reagent/Test Lugol’s reagent Benedict’s reagent

Starch + water + amylase Starch + water + amylase

Lugol’s reagent Benedict’s reagent

Color change N/A Light blue to brickred N/A Light blue to brickred

Positive for Maltose Negative Positive Negative Positive

Table 5; results show that the hydrolytic cleavage of Starch to Maltose occurred, in the presence of the digestive enzyme Amylase.

We observed the starch, water and amylase solutions (both with Lugol’s and Benedict’s reagents, respectively) at room temperature and in the boiling water bath. We found, that the solution with Lugol’s reagent did not change its color to indicate anything other than presence of starch, while the solution containing Benedict’s reagent began changing color slightly even at room temperature and then at a faster rate in the water-bath. Discussion: We hypothesized that the reagents used would help identify the different organic molecules; carbohydrates (Benedict’s reagent for reducing sugars and Lugol’s for non-reducing polysaccharides), proteins (Biuret’s for proteins) and lipids. Our results support our hypothesis, even while testing for unknown solutions and complex combinations of organic molecules in the form of bread. For hydrolytic cleavage of starch, our results concluded that the enzyme amylase began the hydrolysis of starch to maltose, a reducing sugar, at room temperature but the reaction rate increased as it was introduced into the water-bath. This occurred because amylase is a naturally occurring enzyme in the human body, and its optimum temperature to work at is 37

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degrees Celsius. Its enzymatic action reduced a complex carbohydrate down to a simple reducing sugar, as predicted. This experiment design could be made more sophisticated to test for the effects of Amylase’s enzymatic activity at different temperature. There were no unexpected results throughout the lab.

Works Cited

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Holland, M. (1999, August 30). Organic Molecules, Laboratory Notes. Retrieved June 25, 2019, from http://faculty.baruch.cuny.edu/jwahlert/bio1003/organic.html Shanholtzer, S. (2018). Principles of Biology Laboratory Exercises (2nd ed.). Plymouth, MI: Hayden-Mcneil.

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