Procedure
- Prepare a hot water bath using a 600 mL beaker half filled with water. Place on a hot plate and heat to boiling.
- Using the following steps, test each of the substances in a table for the presence of reducing sugars.
- Number clean test tube 1 – 10
- To each test tube, add 1 mL of substance to be tested
- To each test tube, add 1 mL of Benedict’s reagent
- Place the test tubes in the hot water bath for 3 – 4 minutes
- Record color.
Data Collection
The Color Change of the Solutions Before and After the Benedict’s Reagent was Added
Data Processing
The Presence of Simple Sugars (Reducing Sugars) Determined by the Benedict’s Reagent
Analysis
Based on the results shown above, it was found that sucrose, glucose, fructose, skim milk and the unknown solution contained simple sugars, based on their color changes. Conversely, starch, albumin, gelatin, water and corn oil did not have colors which would result if there was a presence of simple sugars. Going back to our hypothesis, the unknown solution has a possibility of being sucrose, glucose, fructose, or skim milk.
Evaluation
Our experiment in testing for the presence of simple sugars using the Benedict’s reagent was generally successful; however, there were several weaknesses with this method of testing the presence of simple sugars using the Benedict’s reagent. Some of the colors as a result of the Benedict’s reagent were difficult to tell apart. One suggestion that could be made to improve this experiment would be to conduct several trials in order to ensure the proper colors were recorded.
Testing for the Presence of Starch (Iodine Test)
Background Information
The Iodine test is used to test for the presence of carbohydrates. Iodine solution — iodine dissolved in an aqueous solution of potassium iodide — reacts with starch producing a deep purple color. Iodine tests for the presence of starch in particular. Starch is composed of polymers of glucose. Long linear chains are called amylose. Amylopectin is similar but contains a branch point about every 25th glucose or so. This reaction is the result of the formation of polyiodide chains from the reaction of starch and iodine. The amylose, or straight chain portion of starch, forms helices where iodine molecules assemble, forming a dark blue/black color. The amylopectin, or branched portion of starch, forms much shorter helices and iodine molecules are unable to assemble, leading the color to be of an orange/yellow hue. As starch is broken down or hydrolyzed into smaller carbohydrate units, the blue-black color is not produced. Therefore, this test can determine completion of hydrolysis when a color change does not occur. Iodine solution will also react with glycogen, although the color produced is browner and much less intense.
Problem
Can the effect of the reaction of the Iodine solution to test for the presence of starch with the known solutions be used to determine the unknown solution?
Hypothesis
Based on the background information, if the Benedict’s reagent causes the solution to turn brown, blue or black, we can conclude that there is a presence of simple sugars (reducing sugars); however, if there is another change in color than the ones presented above, there is no presence of starch. Therefore, we can compare to the unknown solution in addition to its color change to determine which substance it is.
Materials
- Solutions – Sucrose, Glucose, Fructose, Starch, Albumin, Gelatin, Skim Milk, Water, Water, Corn Oil, Unknown Solution (1 mL of each; equivalent to 10 eyedropper drops)
- 150 mL Graduated Cylinder (Uncertainty ± 0.005 mL)
- 600 mL Beaker (Uncertainty ± 0.005 mL)
- Iodine solution (10 mL)
- Water (300 mL)
- 10 Test Tubes
- Eyedropper
Variables
Independent Variable
- The different solutions to be combined with the Benedict’s reagent to determine the presence of simple sugars.
Dependant Variable
- The color change that occurs in each of the solutions after the Benedict’s solution is added.
Controlled Variables
- Amount of reagent added
- Amount of the different solutions added
- Room temperature
Controlling of Variables
-
Independent (Manipulated) Variable: The independent variable was varied by using different known solutions with the Iodine solution to create a variety of results.
-
Dependent (Responding) Variable: The dependent variable was monitored by recording the color change of the result between the reactions of the solutions with the Iodine solution.
-
Controlled Variables: The amount of Iodine solution added to the each of the solutions was controlled by limiting the amount added to 10 eyedropper drops (equivalent to 1 mL). The amount of the different solutions added was controlled by also limiting the amount to 10 eyedropper drops, or 1 mL. The room temperature was controlled by keeping the room constant at 27 degrees.
Procedure
- Using the following steps, test each substance for the presence of starch.
- Number clean test tube 1 – 10
- To each add 1 mL of substance to be tested
- To each add 1 mL of Iodine solution
- Record color
Data Collection
The Color Change of the Solutions Before and After the Iodine Solution was Added
Data Processing
The Presence of Starch Determined by the Iodine Solution
Analysis
Based on the results shown above, it was found that only starch and gelatin contained starch, based on their color changes. Conversely, sucrose, glucose, fructose, albumin, water, corn oil and the unknown solution did not have colors which would result if there was a presence of simple sugars. Going back to our hypothesis, the unknown solution has a possibility of being sucrose, glucose, fructose, albumin, water, or corn oil.
Evaluation
Our experiment in testing for the presence of starch using the Iodine solution was generally successful; however, there were several weaknesses with this method of testing the presence of starch using the Iodine solution. Some of the colors as a result of the Iodine solution were difficult to tell apart. One suggestion that could be made to improve this experiment would be to conduct several trials in order to ensure the validity of the colors recorded.
Testing for the Presence of Proteins (Biuret Test)
Background Information
The Biuret solution is a mixture of copper sulfate and potassium hydroxide, which can detect the presence of proteins (specifically, peptide bonds). In order to test solutions with the Biuret solution, a small amount is added to the solution and then is shaken gently to test the amount simple sugars present. The results will show a ring of color within the tested substance. The color of the ring within the tested substance indicates whether or not proteins are present. In the presence of proteins, the Biuret solution changes the solution color from blue to pink-purple, depending on the amount of proteins present. In the presence of a peptide chain, four of the N atoms in the peptide bonds of 2 chains will form a complex with the Cu (II) ion in solution. This complexation causes a change in the electronical properties of the Cu ion, and results in the observed color change. This color change is dependant on the number of formed complexes, so longer chains will have a more pronounced color change.
Problem
Can the effect of the reaction of the Biuret solution to test for the presence of proteins with the known solutions be used to determine the unknown solution?
Hypothesis
Based on the background information, if the Biuret solution causes the solution to turn pink or purple, then we can conclude that there is a presence of proteins; however, if there is another change in color than the ones presented above, there is no presence of proteins. Therefore, we can compare to the unknown solution in addition to its color change to determine which solution it is.
Materials
- Solutions – Sucrose, Glucose, Fructose, Starch, Albumin, Gelatin, Skim Milk, Water, Corn Oil, Unknown Solution (1 mL of each; equivalent to 10 eyedropper drops)
- 150 mL Graduated Cylinder (± 0.005 mL)
- Test Tube Stand
- Biuret Solution (10 mL)
- 10 Test Tubes
- Eyedropper
Variables
Independent (Manipulated) Variable
- The different solutions to be combined with the Biuret solution to determine the presence of proteins.
Dependent (Responding) Variable
- The color change that occurs in each of the solutions after the Biuret solution is added.
Controlled Variables
- Amount of reagent added
- Amount of the different solutions added
- Room temperature
Controlling of Variables
-
Independent (Manipulated) Variable: The independent variable was varied by using different known solutions with the Biuret solution to create a variety of results.
-
Dependent (Responding) Variable: The dependent variable was monitored by recording the color change of the result between the reactions of the solutions with the Biuret solution.
-
Controlled Variables: The amount of Biuret solution added to the each of the solutions was controlled by limiting the amount added to 10 eyedropper drops (equivalent to 1 mL). The amount of the different solutions added was controlled by also limiting the amount to 10 eyedropper drops, or 1 mL. The room temperature was controlled by keeping the room constant at 27 degrees.
Procedure
- Using the following steps, test each of the substances for the presence of peptide bonds.
- Number clean test tubes 1 – 10
- To each test tube, add 1 mL of substance to be tested
- To each test tube, add 10 drops of Biuret solution
- Look for a ring of color at the top of the substance. If no ring appears, shake gently and wait for a minute or two.
- Record color.
Data Collection
The Color of the Solution After the Biuret Solution was Added
Data Processing
The Presence of Proteins Determined by the Biuret Solution
Analysis
Based on the results shown above, it was found that glucose, albumin, gelatin, skim milk and the unknown substance contain proteins, based on their color changes. Conversely, sucrose, fructose, starch, water, and corn oil did not have colors which would result if there was a presence of simple sugars. Going back to our hypothesis, the unknown solution has a possibility of being glucose, albumin, gelatin, or skim milk.
Evaluation
Our experiment in testing for the presence of proteins using the Biuret solution was generally successful; however, there were several weaknesses with this method of testing the presence of proteins using the Biuret solution. The differentiation between the colors blue and purple were difficult to tell apart. Suggestions to improve this experiment would be to use more of the Biuret solution, as that might help with the distinguishing of different colors. Another suggestion would be to conduct several trials in order to ensure the validity of the colors recorded.
Testing for the Presence of Lipids (Translucence Test and Sudan IV Test)
Background Information
There are two tests that can be used to test for the presence of lipids. The first is the translucence test. This is the test that uses unglazed brown paper. If lipids are present, the lipid is absorbed into the paper and causes it to appear translucent which means that light is able to pass through and it looks like a greasy spot. This is because the paper in a brown paper bag or any other paper under microscopic examination is made from thousands of fibres that are separated by gaps of air. When oil or any liquid absorbs into the paper, it acts like a fibre optic cable, it transmits the light from one side to the other. it fills the gaps and makes light shine through the paper by acting like a light pipe. The gaps are filled with oil, so the light can pass through the paper from one side to the other. The second test is the Sudan IV test. Sudan IV is a special biological stain that is soluble in nonpolar solvents such as lipids and turn from a pink to red colour. Polar compounds will not take up the pink or red colour. Like lipids, the chemical Sudan IV is not soluble in water; it is, however, soluble in lipids. Therefore to test for the presence of lipids in a solution you will use a Sudan IV Test. In this test dark red Sudan IV is added to a solution along with ethanol to dissolve any possible lipids. If lipids are present the Sudan IV will stain them reddish-orange, giving a positive test.
Problem
Can the effect degree of translucency when the solution is placed on the brown paper to test for the presence of lipids, as well as the reaction of the Sudan IV solution with the known solutions, be used to determine the unknown solution when the results are compared?
Hypothesis
Based on the background information, if the solutions cause translucent spots on the brown paper, there are lipids present. Lipids will also be determined to be present if the Sudan IV combined with the substance turns reddish–orange. These results are positive, meaning that the presence of lipids was found. Any other result from these tests will show no presence of lipids. Therefore, we can compare the two results to the unknown solution to determine its identity.
Materials
- Solutions – Sucrose, Glucose, Fructose, Starch, Albumin, Gelatin, Skim Milk, Water, Water, Corn Oil, Unknown Solution (1 mL of each; equivalent to 10 eyedropper drops)
- Sudan IV solution (10 mL)
- Eyedropper
- 10 Pieces of Brown Paper (unglazed)
Procedure
- Place a small sample of the substance to be tested on a piece of unglazed paper.
- Spread it as thinly as possible over a small area. Solid foods should be ground on the surface of the paper.
- Discard excess solids.
- After 5 minutes, hold the paper up to the light to look for translucent spots.
- Record results of Sudan IV in a table.
Data Collection
The Degree of Translucency of Each Solution When Placed On a Piece of Unglazed Paper
The Color of Water and Corn Oil When Mixed With the Sudan IV Solution
In the water, a small amount of Sudan IV dissolved, and the water turned yellow. The rest of the Sudan IV can be seen floating on the water as powder. In the oil, Sudan IV dissolves completely in the oil and the oil turns a reddish colour.
Data Processing
The Presence of Lipids As Determined By the Translucence Test and the Sudan IV Test
Analysis
Based on the results shown above, it was found that only gelatin and corn oil contained lipids based on their translucency with the paper and with the Sudan IV test. Conversely, sucrose, glucose, fructose, starch, albumin, skim milk, water and the unknown solution had results that indicate no presence of lipids. Going back to our hypothesis, the unknown solution has a possibility of being sucrose, glucose, fructose, starch, albumin, skim milk, or water.
Evaluation
Our experiment in testing for the presence of lipids was successful. Whether or not the solutions caused translucent spots on the pieces of paper were easy to see. The color change of the water and corn oil with the Sudan IV solution were also clearly noticeable. One suggestion for improvement would be to conduct several trials in order to ensure the validity of the colors recorded.
Data Processing
The Presence Of Simple Sugars, Starch, Proteins, And Lipids As A Result Of All The Tests Made
Analysis
Our hypothesis of whether or not we could figure out the identity of the unknown solution based on the results of the different tests with the known solutions was correct. The results above are a combination of all the data collected from the previous tests done. From the results, we can see that the unknown solution resulted positive for the presence of simple sugars and proteins, while resulting in negative (meaning no presence) for the presence of starch and lipids. From the known solutions, the only solutions that match this are skim milk and glucose. However, if we refer to the Iodine test portion of this lab, we see that the original color of the unknown solution was clear, ruling skim milk out. Therefore we can conclude that the unknown solution is glucose. However, in the Benedict and Biuret solutions, the results were a little different. This is because the glucose has been diluted with water.
Evaluation
Our experiment in generally, to find successful; however, there were several weaknesses with this method of testing for the unknown solution. Some of the colors of the different tests were difficult to tell apart. One suggestion that could be made to improve this experiment would be to conduct several trials in order to ensure the validity of the colors recorded. Another suggestion that could be made to improve this experiment would be to introduce experiments that don’t necessarily deal with starches, proteins, sugars and lipids – for example, conductivity, as well as melting and boiling point tests.
BIO 35 – Identification of Proteins, Carbohydrates and Fats