• identify the presence of various macromolecules due to their structural properties.
• determine the identity of unknown food products based on their molecular make-up.
B. Textbook Correlation:
Please review Sections 3.2-3.5 of Chapter 3: Organic Molecules when preparing for the lab.
C. Introduction:
Cells are the basic unit of life. In order to understand the nature of cells, we have to appreciate their chemical make-up. The four most abundant elements of life include oxygen, carbon, hydrogen and nitrogen. These elements make up 96% of your body and can be organized into molecules through chemical bonding. An example of a molecule that is essential in supporting life is H2O, otherwise known as water. Other examples of biologically important molecules include amino acids, fatty acids, monosaccharides, and nucleotides. These four molecules are the basic building blocks of the macromolecules used to construct our cells. There are four types of macromolecules found within all cells: carbohydrates, lipids, proteins, and nucleic acids. These organic (carbon-containing) macromolecules are organized into a functional unit we call cells. In this exercise, we will analyze the properties of the following macronutrients (required in large amounts): water, carbohydrates, lipids, and proteins.
D. Unknown Food Project
Throughout your introductions, you have emphasized the importance of macronutrients on our health and discussed the food products that contain each type. We often classify food groups based on their nutrient content. The goal is to determine the identity of the four unknown food products based on the nutrient make-up of each. While certainly these unknowns may have more than one of the nutrients listed below, each food products is representative of each class of macromolecules.
To accomplish this goal, you will have to devise experiments to detect the presence of these nutrients. Below is a list of materials that will be available:
1. Test Tubes
2. Plastic droppers
3. Hot plate and beaker of water containing boiling chips
4. Distilled H2O
5. Known food products: glucose, starch, egg, and vegetable oil
6. Chemical reagents for testing. In the list of reagents below, I have included the volumetric ratio of test substance/reagent for each and included the order by which materials should be added to their respective tubes:
a. Biuret reagent: 20 drops (1mL) test substance/10 drops (0.5mL) of reagent
b. Lugol's solution: 60 drops (3 mL) test substance/10 drops (0.5 mL) of reagent
c. Benedict's solution: 40 drops (2 mL) test substance/40 drops (2 mL) of reagent
d. Sudan reagent: 60 drops (3 mL) of water/60 drops (3 mL) of test solution/10 drops (0.5 mL) of reagent/40 drops (2 mL) of water
Your job is to research these reagents before lab to determine the appropriate use for each. You also need to determine if there are any special condition necessary (i.e. heat, pH) that are necessary. You also need to specify the negative and positive control for each experiment.
In the space provided below outline the experiment you will use to detect the presence of each nutrient in the four unknown food products. After the experimental section, you will record your data and write a conclusion for each experiment (i.e. which nutrient(s) was present in each unknown).
A. Simple Carbohydrates:
Experimental Design (outline the procedure for identifying simple sugars HERE. Make sure to include both a negative and positive control.
General Info:
Benedict's Solution is used for the testing of simple sugars or simple carbohydrates. It is a blue liquid and contains copper (II) sulfate. This is then reduced to red copper (I) oxide. This happens when carbohydrates are reduced due to an increase in heat. Copper Oxide is not soluble in water and precipitates out of the solution. The substance that is being tested should be heated with the Benedict's solution. The presence of a simple sugar will cause the blue solution to turn green to yellow to brick-red, due to the presence of the aldehyde group in the carbohydrate. Complex carbohydrates like starches don't react positively with the benedicts test unless they are broken down by heat which is why we must heat the Benedict's solution.
Procedure:
We will have to test Benedict’s solution on all four of the unknown substances to find out which of the four is a simple carbohydrate. We must maintain the suggested concentration of unknown substance to reagent. In this case, 40 drops (2 mL) the unknown substance to 40 drops (2 mL) of Benedict’s solution. Thus, for each of the four test tubes containing the four separate unknown substances, we will add 40 drops of the unknown substance and 40 drops of Benedict’s solution. Once the four test tubes, each containing a different unknown food substance, are filled with 40 drops of the given unknown substance, we finally add the 40 drops of Benedict’s solution to each test tube, one at a time. Once this is complete, we will heat the test tubes in a hot water bath, at about 50 to 60 degrees Celsius for 5 minutes, using the hot plate and beaker of water containing boiling chips we are provided. When the four test tubes are done being heated, we will remove them and observe any color changes. The test tube containing the simple carbohydrate will undergo a color change from clear blue to green, yellow or brick-red, depending on the concentration of the sugar.
Positive and Negative Controls:
In an experiment, there are two types of controls: positive and negative. In positive controls, an event is expected to occur. In this experiment, the test tube containing simple carbohydrates is expected to turn from blue to either green, yellow or red. Thus, a color change is a positive control in this experiment because it is expected to occur. A negative control is an event in an experiment that is not expected to occur. For example, we would not expect the substance containing lipids to change color. In this experiment, Benedict's solution having an effect on any other molecules other than simple carbohydrates is a negative control.
B. Complex Carbohydrates:
Experimental Design(outline the procedure for identifying complex sugars HERE. Make sure to include both a negative and positive control.):
General Info:
Lugol's Solution is used for the testing of starch, which is a complex carbohydrate. It is an aqueous solution that is composed of Iodine and Potassium Iodine, in a ratio of 1:2. When starch reacts with this solution, it produces a deep blue-black color. This results from the formation of the polypeptide chains from the reaction between starch and iodine. The amylose, which is the straight chain portion of the starch, produces the dark blue/black color that we see. The formation of a yellow/orange color is because of the amylopectin, the branched portion of the starch. If this yellow/orange color is seen, it indicates that there is no complex carbohydrate in the solution. When this starch is broken down into its building blocks or smaller carbohydrate units, the deep blue/black color will not be seen.
Procedure:
First, we will put the four unknown substances into four separate test tubes. We must maintain the test substance to reagent concentration of 60 drops of unknown substance to 10 drops of Lugol's solution. When the four test tubes are prepared with proper amount of test substance, we will add the Lugol’s solution. Finally, we will gently shake each test tube to mix. The test tube that exhibits a color change from a yellow color to a dark blue/black color has the presence of complex carbohydrates.
Positive and Negative Controls:
If there is the appearance of a dark blue/black color, then this will be a positive control because we expect that a solution with complex carbohydrates to turn a deep blue/black color when reacting to Lugol's solution. If there is the presence of a yellow/orange color in a test tube at the end of the experiment, this is a negative control because there is no change in color with molecules other than proteins. For example, the test tube containing lipids would be a negative control because no change in color occurs.
C. Proteins:
Experimental Design (outline the procedure for identifying proteins HERE. Make sure to include both a negative and positive control.):
General Info:
Biuret reagent is used for testing the presence of proteins. It is made up of sodium hydroxide and copper (II) sulfate. This test for proteins, using this reagent, relies entirely on the reaction that would occur between the copper ions and the peptide bonds in the solution, under alkaline conditions. A solution is alkaline when it has a pH between 7 and 14, exclusively. The biuret reagent is a blue reagent that will turn a violet color in the presence of proteins. The proteins will interact with the biuret reagent because it contains a large number of peptide bonds. It will turn a pink color when it combines with short-chain polypeptides, since they have a smaller number of peptide bonds present in them. The intensity of the color that is produced, after the substance has interacted with the biuret reagent, all depends on the number of peptide bonds that reacted with the copper ions found in the biuret reagent.
Procedure:
The reagent used for proteins is called Biuret reagent. First you add 20 drops of each of the four different test substances into four separate test tubes. Next, to maintain the test substance to reagent concentration, we add 10 drops of the Biuret reagent solution to each of the test tubes with the four different test solutions. Finally, we gently shake the tube to mix the solution. We will know if the solution contains proteins because it will turn from blue to pink or purple.
Positive and Negative Controls:
In this experiment, a positive control would be an event that is expected. The positive control in this case is the substance containing protein would be expected to turn pink or purple. The negative control is when a certain reaction is expected NOT to take place. In this experiment, the negative control would be a substance containing molecules other than protein would not change color in response to the Biuret reagent.
D. Fats:
Experimental Design (outline the procedure for identifying fats HERE. Make sure to include both a negative and positive control.):
General Info:
The sudan reagent is used to test the presence of lipids in a solution. Sudan IV is a red dye that is fat-soluble that is used for the staining of lipids, triglycerides, and lipoproteins. This technique of staining is very important because it allows us to actually visualize the presence of a fatty compound without isolating it. The solution will turn red due to the presence of fatty compounds because it stains the fat cells red.
Procedure:
We will fill four separate test tubes with the four unknown test substances. We will use the Sudan reagent in our procedure to detect which test tube contains fats. The suggested concentration of water to test substance to reagent in this experiment is a total of 100 drops of water to 60 drops of test substance to 10 drops of reagent. Although there is a total of 100 drops of water applied to the final mixture, we apply 60 drops of water first, then add the test substance followed by the reagent, and then another 40 drops of water. Once all four test tubes contain the appropriate concentration of all three substances, we shake gently to mix. We then must play close attention to note any color changes. If fat is present, a red-stained oil layer will float on the water surface.
Positive and Negative Controls:
If the lipid molecules in a given solution interact with the sudan reagent and produces a red-stain oil layer that spreads across the wurface of the water, then this is a positive control because we would expect that to happen when lipid molecules react with the sudan reagent. If this did not occur, then it would be a negative control because we did not expect a solution containing lipid molecules to not interact with the sudan reagent.
E. Results and Conclusions
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