Iki indicator 1 ml 2.1 in glass vial

Chemical Basis of life Hands-on labs, inc. Version 42-0144-00-01

Review the safety materials and wear goggles when working with chemicals. Read the entire exercise before you begin. Take time to organize the materials you will need and set aside a safe work space in which to complete the exercise.

Experiment Summary:

Students will learn about chemical reactions, qualitative versus quantitative testing, and the four classes of macromolecules and their functions. Students will perform qualitative tests to determine the presence of lipids by using Sudan III, of proteins by using biuret reagent, of simple sugars by using Benedict’s reagent, and of complex carbohydrates by using IKI. Students will also use biochemical tests to identify an unknown.

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ExpErimEnt

ObjEctivEs ● To identify the four classes of macromolecules

● To learn the relationship between reagents and macromolecules

● To understand how to test for carbohydrates, proteins, and lipids

● To use a biochemical test to identify an unknown

Time Allocation: 2–3 hours

Safety: So that you understand the proper procedures to use when handling, pouring, heating, etc. chemicals, if you have not already done so, read the section in the introduction to this Lab Manual titled “Laboratory Equipment and Techniques” before beginning this experiment. Also, always wear goggles when handling chemicals, and use gloves when directed to do so.

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Experiment ChemiCal Basis of life

matErials

MATERiAlS FRoM:

lABEl oR BoX/BAg: QTy iTEM DESCRiPTioN:

Student Provides 1 Water, distilled 1 Cereal, 1 tsp (optional) 1 Egg 1 Plastic wrap or foil, 5 × 5-in square 1 Pencil 1 Sauce pan or Deep skillet 3 Cups 1 Stove

1 1

Oil, vegetable, 1 tsp Zip Lock bag

From LabPaq 1 Benedict’s Reagent – 5 mL in dropper 1 Biuret Reagent – 2 mL in dropper 1 Cylinder graduated, 10 mL 1 Filter paper, 12.5 cm 1 Glucose solution in Dropper Bottle , 1 % solution 1 IKI Indicator, 2.1% - 1mL in glass vial in bubble bag 1 Starch solution, 1% 2 mL in Pipet 1 Sudan III-2mL in Glass Vial 3 Pipet, graduated, jumbo 5 mL 1 Test-tube-clamp-holder 1 Test-tube-cleaning-brush 1 Test tubes(6), 13 x 100mm in Bubble bag 1 Unknown, 0.1 g 1 Test-tube-rack, 6x13-mm

Note: The packaging and/or materials in this LabPaq may differ slightly from that which is listed above. For an exact listing of materials, refer to the Contents List form included in the LabPaq.

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Experiment ChemiCal Basis of life

DiscussiOn anD rEviEw There are four main classes of molecules that are essential to the biology of life: proteins, carbohydrates, lipids, and nucleic acids. These molecules are large, so they are called macromolecules. All living organisms are composed of these four macromolecules, and this lab will expose you to the chemistry and function of these molecules. Most macromolecules are joined together by covalent bonds. A dehydration reaction, in which two molecules are joined and a molecule of water is removed, is responsible for the covalent bonds that join macromolecules together.

Proteins are involved in the growth and repair of organisms, and are present in all basic cell structures. One protein consists of one hundred or more amino acids. Amino acids are the smallest components of a protein. An amino acid is composed of an amino group, a central carbon, an acid group, a hydrogen, and a side chain (R-group). This R-group varies and is what gives each amino acid its unique identity. The amino acids are joined together by a covalent bond called a peptide bond.

Carbohydrates are used in the body as a source of quick energy. Sugars and starches are the components of a carbohydrate, and are composed of carbon, hydrogen, and oxygen often occurring in a 1:2:1 ratio, as a “hydrate of carbon,” CH2O. Monosaccharides contain one molecule of sugar and are the simplest form of a carbohydrate. They form building blocks for more complex carbohydrates. When two monosaccharides join together, a disaccharide is formed. One common disaccharide is sucrose (table sugar). When many monosaccharides join together, the resulting molecule is called a polysaccharide. Polysaccharides are the largest carbohydrate molecules.

The most common type of polysaccharide is starch. Starch is produced in plants, and serves as a storage of chemical energy for plants and as a structural component in cell walls. The cell wall is composed of indigestible sugars commonly called fiber. Polysaccharides also are structural components in the exoskeleton of insects (called chitin).

lipids are large, organic, hydrophobic molecules composed of fatty acids. Fatty acids are long hydrocarbon chains ranging from 10 to 30 carbons in length. Triglycerides, a common form of lipids, include one glycerol and three fatty acids. Types of lipids include fats, waxes, oils, and sterols. Lipids play an important role in cellular membranes, nervous transduction, energy storage, and insulation. The amount of saturation, which is measured by the number of hydrogen atoms attached to central carbons, determines whether a lipid will be a solid or a liquid at ambient temperature.

Nucleic acids are found in the genetic material deoxyribonucleic acid (DNA). They are also present in ribonucleic acid (RNA). The smallest component of a nucleic acid is a nucleotide, which is made of a sugar, a phosphate group, and a nitrogen base. There are five different bases: adenine, guanine, cytosine, thymine, and uracil. Adenine, cytosine, and guanine are found in both DNA and RNA. Thymine is only present in DNA, and uracil is only present in RNA. The arrangement and pairing of these nucleotides are responsible for the genetic coding, which is found in all living organisms.

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Experiment ChemiCal Basis of life

In order to determine which types of macromolecules are present in specific substances, scientists may conduct chemical tests. Tests that show the presence of a substance, but not necessarily a measurement, are called qualitative, and tests that show numeric values are considered quantitative. For example, using litmus paper to simply determine if a substance is acidic or basic is considered qualitative. However, if more sophisticated equipment is used to determine that a substance has a certain pH number value, then the test is considered quantitative. For this experiment, you will be using qualitative tests in which specific numerical values cannot be determined with any of the tests.

When using chemical tests, an observation may occur such as a color change, change in odor, formation of a precipitate (a solid substance that comes out of a solution), change in temperature, or gas production. The changes occur because the substance has gone through a chemical reaction. In the following exercises, you will perform tests using chemicals that indicate whether proteins, amino acids, glucose, starch, and lipids are present. You will then determine the composition of an unknown compound.

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Experiment ChemiCal Basis of life

Exercise 1: Proteins In this exercise, you will test for the presence of a protein in egg white and also in an unknown substance using biuret reagent. The positive control (a substance that should test positive) will be egg white, which contains a protein called albumin. If the positive control does not test positive, the results of any test will not be valid. The main cause of failure is usually a reagent that has lost its effectiveness and thus needs to be replaced. Biuret reagent has a blue color and in the presence of protein it will turn purple. See Figure 1. The degree of color change is indicative of increasing concentrations of protein.

Figure 1: The test tube on the left shows a positive result for protein using biuret reagent. On the right is a negative result.

prOcEDurE 1. Prepare a data table similar to Data Table 1 in Observations.

2. Prepare the unknown solution using the following steps:

a. Pour the contents of the small bag labeled “Unknown powder” into a cup.

b. Label the cup “X”.

c. Use the 10-mL graduated cylinder to measure out 10 mL of distilled water to the unknown powder. Then gently swirl the cup to mix.

d. Use this mixture when the procedure calls for an unknown solution.

3. Prepare an egg albumin solution by cracking an egg and collecting only the clear egg white into a cup.

4. Using a fork or a whisk, beat the egg white.

5. Label four test tubes “albumin”, “A”, “B”, and “X”. These test tubes will have the following contents:

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Experiment ChemiCal Basis of life

Tube # Contents

Albumin 9 mL distilled water + 1 mL egg white A 1 mL solution from albumin tube + 5 drops biuret reagent B 1 mL distilled water + 5 drops biuret reagent X 1 mL unknown solution + 5 drops biuret reagent

6. Measure out 9 mL of distilled water in the graduated cylinder and add it to the albumin test tube.

7. Add 1 mL of distilled water (dH2O) to test tube B.

a. Note the height in the test tube. Because this test is qualitative, accurate measurement is not necessary. When adding 1-mL volumes to the remaining test tubes, use the height in test tube B to estimate the volume needed to add to other tubes.

8. Using a graduated 5-mL jumbo pipet, pull up 1 mL of the egg white and put it into the clean test tube labeled “albumin” for a total volume of 10 mL.

9. Rinse out the pipet:

a. Pour distilled water into another cup.

b. Pull up the distilled water from the cup with the pipet, swirl water around inside the pipet, and discard the water into the sink.

c. Repeat several times.

10. Using the rinsed 5-mL jumbo pipet, add 1 mL of the albumin solution to test tube A.

11. Repeat Step 9.

12. Add 1 mL of the unknown solution to test tube X.

13. Put 5 drops of biuret reagent into each test tube (A, B, and X) and watch for a color change. Hold the test tubes up against a white background to have a better view of the color changes.

14. Record the results in Data Table 1.

15. Wait 5 minutes and then add 5 more drops of biuret reagent to the test tubes.

16. Record any color changes in Data Table 1.

17. Save the remainder of the unknown solution for use in future exercises. If you must take a long break between exercises, place the unknown solution in the refrigerator.

18. Discard all contents (other than the unknown solution) down the sink while running water.

19. Thoroughly clean and dry all equipment for future use.

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Experiment ChemiCal Basis of life

Exercise 2: Simple Sugars In this exercise, you will use Benedict’s reagent to test for the presence of sugars. Depending on the amount of sugars in a solution, adding Benedict’s reagent will cause the solution to change color. A negative result to a Benedict’s reagent test is signified by blue. In the presence of a small amount of glucose (a monosaccharide), the solution will change to green; if more glucose is present in the solution, it will change to yellow, then orange, then red, and then finally brown in the increasing presence of sugars. See Figure 2. Sucrose (also known as table sugar) is a disaccharide (glucose + fructose) that does not react with Benedict’s reagent.

Figure 2: Benedict’s reagent color results

prOcEDurE 1. Prepare a data table similar to Data Table 2 in Observations.

2. Retrieve a small saucepan and fill it with 8 to 10 cm of water.

a. Test the amount of water by placing the test tube rack with a test tube into the pan.

b. The water should reach about halfway up the tube.

c. Remove the test tube, but leave the test tube rack in the pan.

3. Set up a simmering water bath by placing the small saucepan on the stove.

4. Reduce heat until only a few bubbles emanate upward through the water from the bottom of the pan.

5. Label test tubes A,B, and X, which correspond to the following:

Test tube Contents

A 1 mL glucose solution + 3 drops Benedict’s reagent (positive control) B 1 mL dH2O + 3 drops Benedict’s reagent X 1 mL unknown solution + 3 drops Benedict’s reagent

6. Using the jumbo 5-mL pipet, add 1 mL of glucose solution to test tube A.

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Experiment ChemiCal Basis of life

7. Rinse out the pipet:

a. Pour distilled water into a cup.

b. Pull up the distilled water from the cup into the pipet, swirl water around inside the pipet, and discard the water into the sink.

c. Repeat several times.

8. Add 1 mL of distilled water to test tube B.

9. Repeat Step 7.

10. Add 1 mL of unknown solution to test tube X.

11. Repeat Step 7.

12. Add 3 drops of Benedict’s reagent to each test tube. Note the color of the Benedict’s reagent.

13. Use the test tube clamp holder to place the three test tubes (A, B, and X) into the test tube rack in the simmering water bath.

Note: Benedict’s reagent needs to be heated in order to cause a chemical reaction.

14. Heat the test tubes until a color change occurs in test tube A (the positive control). This should take approximately five minutes.

15. After the color change has occurred, turn the stovetop heat off and allow the water to cool.

16. When the water is cool enough, remove the test tube holder from the saucepan.

17. Observe and compare the test tubes to each other.

18. Record the results in Data Table 2.

19. Save the remainder of the unknown solution for use in future exercises. If you must take a long break between exercises, place the unknown solution in the refrigerator.

20. Discard all contents (except the unknown solution) down the sink while running water.

21. Thoroughly clean and dry all equipment for future use.

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Experiment ChemiCal Basis of life

Exercise 3: Complex Carbohydrates In this exercise, you will use iKi (lugol’s iodine, iodine-potassium-iodine) to test for the presence of starch. You will test a known starch solution and then test an unknown substance.

prOcEDurE 1. Prepare a data table similar to Data Table 3 in Observations.

2. A solution of 1% starch in water has been provided in your kit.

3. Label three test tubes “A”, “B”, and “X”, which correspond to the following:

Test tube Contents

A 1 mL starch solution + 1 drop IKI B 1 mL distilled water + 1 drop IKI X 1 mL unknown solution + 1 drop IKI

4. Add 1 mL of starch solution to test tube A.

5. Add 1 mL of distilled water to test tube B.

6. Add 1 mL of the unknown mixture to test tube X.

7. Record the initial color of each test tube in Table 3.

8. Be careful when pipetting iodine as it will stain clothing and skin. Use the empty pipette that is labeled IKI and pull up a small amount of iodine solution from the glass vial of iodine.

9. Carefully add one drop of iodine to each test tube (A, B, and X).

10. Record the immediate results in Table 3.

11. Save the remainder of the unknown solution for use in future exercises. If you must take a long break between exercises, place the unknown solution in the refrigerator.

12. Discard all contents (except the unknown solution) down the sink while running water.

13. Thoroughly clean and dry all equipment for later use.

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Experiment ChemiCal Basis of life

Exercise 4: lipids Sudan iii is the reagent used in this exercise. It will only dissolve in lipids. You will test a positive control, a negative control, and the unknown for lipids.

prOcEDurE 1. Prepare a data table similar to Data Table 4 in Observations.

2. Lay the filter paper flat on a piece of plastic wrap or aluminum foil to keep the area from staining.

3. Draw up a small amount of the Sudan iii with a pipette and place three individual drops in three different spots on the filter paper as illustrated in the drawing.

4. Label each spot “a”, “b”, and “x”, as shown in Figure 4.

Figure 3: Spot labeling

5. Draw a line around these spots after they dry.

6. Add one drop of vegetable oil to spot a.

7. Add one drop of distilled water to spot b.

8. Add one drop of the unknown to spot x.

9. Allow the paper to dry completely.

10. Record the observations in Data Table 4. If red coloration was carried outside of the pencil line drawn, the substance dissolved with the Sudan III, indicating the substance was a lipid.

11. Include a sketch of the filter paper results in your lab report.

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Experiment ChemiCal Basis of life

Exercise 5: Testing Breakfast Cereal (Optional Exercise) In this exercise, you will test a cereal for protein, sugar, starches, and lipids.

prOcEDurE 1. Prepare a data table similar to Data Table 5 in Observations.

2. Record the name of the cereal being tested in your lab report.

3. Grind 1 teaspoon of cereal by placing the cereal into a ziplock bag and crushing it with the back of a spoon or a rolling pin.

4. Mix the ground cereal with 10 mL of distilled water.

5. Prepare a simmering water bath as described in Exercise 2.

6. Label six test tubes “A”, “B”, “C”, “D”, “E”, and “F”, which correspond to the following:

Test Tube Contents

A 2 mL cereal mixture + 5 drops biuret reagent B 2 mL cereal mixture + 5 drops Benedict’s reagent C 2 mL cereal mixture + 1 drop IKI D 2 mL dH2O + 5 drops biuret reagent E 2 mL dH2O + 5 drops Benedict’s reagent F 2 mL dH2O + 1 drop IKI

7. Use the clean 5-mL jumbo pipet to drop 2 mL of the cereal-water mixture into test tubes A, B, and C. Add further contents as shown in the above list in Step 6.

8. Clean the 5-mL jumbo pipet.

9. Use the pipet to add 2 mL of dH2O to test tubes D, E, and F.

10. To test tubes A and D, add 5 drops of the biuret reagent. Observe the color of the resulting solution and record in Data Table 5.

11. To test tubes B and E, add 5 drops of Benedict’s reagent.

12. Create a hot water bath as you did in Exercise 2, and place test tube B and E in the hot water bath.

13. Allow the test tubes to remain in the hot water bath until a color change is present in test tube E.

14. Observe the color of the resulting solutions in test tube B and E and record in Data Table 5.

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Experiment ChemiCal Basis of life

15. To test tubes C and F, add 1 drop of IKI.

16. Observe the color of the resulting solution and record in Data Table 5.

17. Discard all solutions down the drain while flushing with running water.

18. Thoroughly clean and dry all equipment for later use.

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