Ex03 Basic Chemistry-Macromolecules 6E P1 PDF

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Laboratory exercise to determine macromolecules...

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EXERCISE 3

B A S I C C H E M I S T R Y: M A C R O M O L E C U L E S LEARNING OBJECTIVES • Recognize chemical and structural formulas for common molecules. • Provide examples of carbohydrates and proteins that are found in our diet. • Perform tests to detect the presence of carbohydrates and proteins. • Recognize the importance of a control in a biochemical test. • Use biochemical tests to identify an unknown compound.

MATERIALS • Test tubes

• Onion extract

• Test tube holders

• Potato extract

• 250-mL beakers

• Sucrose solution

• Boiling water bath

• Glucose solution

• Disposable 1mL pipettes

• Corn extract

• Wax pencil

• Amino acid solution

• Benedict’s reagent

• Milk

• Iodine-potassium iodide, I2KI

• Egg albumin

• Biuret reagent

• Chicken broth

• Distilled water

© b l u ed o o r, L LC ( SF 0 0 3 - N M )

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INTRODUCTION Biomolecules are macromolecules (large molecules) found in living systems that contain five or more carbon atoms in either ring or chain formation. Organisms are composed of four key biomolecules essential to life – carbohydrates, lipids, proteins, and nucleic acids. These substances are called organic compounds because they all contain carbon, hydrogen, and oxygen. We obtain macromolecules from the food we eat. Various chemical tests can be used to detect the presence of each type of biological molecule. Indicators are special chemicals that change color in the presence of a specific organic substance. Most of the tests involve a color change that is visible to the naked eye. A change color indicates a positive test result. If a color change is not observed, the test is negative, indicating that a particular molecule is not present. A control will also be used in all of the chemical tests we will be performing. Your control will be distilled water (dH2O), which does not contain any macromolecules. If your control gives you a positive result, your test is invalid.

CARBOHYDRATES Carbohydrates provide substrates for cellular respiration and therefore lead to energy production. Some organisms, like plants, also use carbohydrates as part of their structure. The basic building blocks for carbohydrates are sugars, also called monosaccharides. Glucose and fructose are monosaccharides. Two monosaccharides joined together by a dehydration synthesis reaction form a disaccharide; sucrose and maltose are examples of common disaccharides. Monosaccharide molecules are often linked together to form a medium-length chain called a polysaccharide (many-sugar); examples include starch, cellulose, and glycogen. See Figure 3.1 for examples of a monosaccharide, disaccharide, and polysaccharide. The chemical and physical properties of monosaccharides and polysaccharides are different so the two types of molecules can be detected and differentiated by using specific chemical tests. Glucose

Maltose

CH2OH

CH2OH

CH2OH O

H

H

O

H

H

O

H

H

OH

OH

O

OH

H

H

H

H

OH

H

OH H

OH

C6H12O6

OH

C12H22O11

O O

O

O O

O CH 2

O

O

O

O O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O O

O

O O

O

O

O O

O O

O

O O

Starch

O

O

O

O

CH 2

O

O O O

Figure 3.1: Examples of carbohydrates.

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Basic Chemist r y: Macr omolecules | Exer cise 3

PROTEINS The basic unit of protein is the amino acid. The bond between two amino acids is called a peptide bond and, for this reason, short protein chains are referred to as dipeptides and polypeptides. Proteins perform a variety of functions in cells and are probably the most diverse macromolecule in living systems. They can also be enzymes or antibodies that fight disease. Proteins are found throughout our cell membranes, helping cells communicate and function properly. For example, one very important protein in your body is hemoglobin. Hemoglobin is found in red blood cells and carries oxygen to all cells of the body. Figure 3.2 illustrates a polypeptide chain. Peptide bonds

H

O

C

C

H

O

N

C

C

H

ile

H

O

N

C

C

H

val

H

O

N

C

C

H

glu

H +H

N

O–

H gly

Figure 3.2: A short polypeptide chain.

Exer cise 3 | Basic Chemist r y: Macr omolecules

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PRE-LAB QUESTIONS 1.

What is happening to a compound undergoing oxidation? (refer to the electrons)

2.

What are the four types of biomolecules?

3.

What are the two types of carbohydrates we will be testing for? Which indicator will be used for which type?

4.

Which test solution in this lab changes from a blue color to a violet color due to the amine groups in the amino acids reacting with the copper ions in the solution?

5.

What compound will be used as a control for all tests?

6.

Which reagent is used to identify the presence of starch? What is a positive test using this reagent?

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Basic Chemist r y: Macr omolecules | Exer cise 3

7.

What is the repeating unit that is used to create a protein?

8.

What reagent is used to detect a peptide bond?

9.

According to the information provided in the introduction, generally, what type of carbohydrate is not a reducing sugar?

10. What tests (or reagents) in this lab require hot water to complete the reaction properly?

Exer cise 3 | Basic Chemist r y: Macr omolecules

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ACTIVITY #1 – BENEDICT’S TEST FOR REDUCING SUGARS The Benedict’s test detects the presence of reducing sugars. Reducing sugars are usually either monosaccharides or disaccharides, which have a free aldehyde group (-CHO) that can be oxidized. Oxidation occurs when a compound loses an electron. In a positive reaction, the blue Cu+2 ion is reduced to red Cu+1 ion with the monosaccharide donating the electron. Generally large polysaccharides are non-reducing sugars. When heated to boiling in the presence of Benedict’s reagent, a positive test for reducing sugars will produce a colored precipitate (a solid that will separate from the solution and sink to the bottom of your test tube). The precipitate can be green (indicating low concentration), yellow, orange, or red-orange (indicating high concentrations), depending on the concentration of reducing sugars present. If no reducing sugar is present, the Benedict’s reagent stays blue or may be a shade of blue or purple.

Procedure 1.

Notice the location(s) of the hot plate(s) and the boiling water as indicated by your instructor that you will use to heat the test tubes.

2.

Obtain ten clean test tubes and number them 1-10 using a wax pencil. Be sure the number is written high on the test tube to prevent the label from coming off in the hot water. Use a plastic pipette, add 2 mL of each of the following solutions in the corresponding test tubes: 1 = dH2O; 2 = glucose; 3 = sucrose; 4 = corn extract; 5 = onion extract; 6 = potato extract; 7 = amino acid solution; 8 = egg albumin; 9 = chicken broth; 10 = milk

3.

Record your hypothesis regarding your predictions of the solutions listed reacting with the Benedict’s solution.

4.

Add ten drops of Benedict’s reagent to each tube. Record initial color of the solution after the Benedict’s has been added. Mix well so that there are no layers in the two liquids.

5.

Heat all ten tubes for 1-2 minutes in the boiling water bath. Note that sometimes the wax pencil labels will be obscured or lost while heating. Place your test tubes in the water bath so that you can identify them even if the labels come off. Caution: Use a test tube holder and not bare hands when handling hot test tubes.

6.

Record all color changes in the Table 3.1 and answer the following questions.

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Basic Chemist r y: Macr omolecules | Exer cise 3

Table 3.1: Using the Benedict’s Reagent in Various Substrates to Detect Reducing Sugars. Test Tube

Substance Tested

1

Distilled water

2

Glucose

3

Sucrose

4

Corn extract

5

Onion extract

6

Potato extract

7

Amino acid solution

8

Egg albumin

9

Chicken broth

10

Milk

Initial Color Before Heating

Final Color After Heating

Reducing Sugars Present?

QUESTIONS 1.

What was the purpose of test tube #1?

2.

Were your hypotheses supported or not? Were you surprised by any of your results? Explain.

3.

How are sucrose and glucose different from one another?

4.

Which contains more reducing sugars: potato extract or onion extract? How do you know?

Exer cise 3 | Basic Chemist r y: Macr omolecules

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5.

Based on your answer to question 4, what would you conclude about the difference between the storage of sugars in onions and potatoes?

6.

Would you expect to find reducing sugars in fruit juice? Why or why not? What other foods would you expect to test positive for reducing sugars?

ACTIVITY #2 – IODINE TEST FOR STARCHES The iodine test (I2KI or Lugol’s reagent) is a carbohydrate test that identifies the presence of starch. Starch molecules are polymers of glucose commonly found in plants. In the presence of starch, the color of the iodine reagent changes from amber to a very dark blue-black color. If the test is negative, the solution stays amber. CAUTION: Iodine will stain your clothes and your skin. Wear gloves when handling. It is not safe for internal use.

Procedure 1.

Number ten test tubes 1-10 with a wax pencil.

2.

Add 2 mL of each of the following solutions to the appropriate numbered test tube: 1 = dH2O; 2 = glucose; 3 = sucrose; 4 = corn extract; 5 = onion extract; 6 = potato extract; 7 = amino acid solution; 8 = egg albumin; 9 = chicken broth; 10 = milk

3.

Record your hypothesis here about which substances you predict will test positive for starch.

4.

Record the initial color of each solution before the Iodine is added in Table 3.2.

5.

Add 10 drops of iodine solution to each test tube. Mix well by shaking the test tube from side to side. Do NOT heat these tubes.

6.

Record all color changes in Table 3.2 and answer the following questions.

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Table 3.2: Using Iodine or Lugol’s Reagent to Detect Starch in Various Common Foods. Test Tube

Substance Tested

1

Distilled water

2

Glucose

3

Sucrose

4

Corn extract

5

Onion extract

6

Potato extract

7

Amino acid solution

8

Egg albumin

9

Chicken broth

10

Milk

Initial Color Before Adding Iodine

Final Color After Adding Iodine

Starch Present?

QUESTIONS 1.

What was the purpose of water in this experiment?

2.

Were your hypotheses supported or not? Were you surprised by any of the results? Explain.

3.

What can you infer about the storage of carbohydrates in onions and potatoes?

4.

Why did glucose (a carbohydrate) give a negative result for starch?

5.

Would you expect to find starch in beans? Why or why not? What other foods would you expect to test positive for starch?

Exer cise 3 | Basic Chemist r y: Macr omolecules

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ACTIVITY #3 – BIURET TEST FOR PROTEIN The Biuret test detects the presence of proteins and short peptides (short chains of amino acids). Biuret reagent contains a strong solution of sodium hydroxide (NaOH) and a very small amount of dilute copper sulfate (CuSO4) solution. In the presence of a protein, the reagent changes to a violet color (from blue) due to the amine groups in the amino acids reacting with the copper ions. A blue color indicates a negative result. CAUTION: Biruet solution contains a strong base. It may burn your clothes and skin. Wear goggles and gloves while handling. In case of contact, inform your instructor. Rinse the area with cold water. Do not rub.

Procedure 1.

Obtain ten clean test tubes and number them 1-10 using a wax pencil.

2.

Place 2 mL of the following solutions into each tube: 1 = dH2O; 2 = glucose; 3 = sucrose; 4 = corn extract; 5 = onion extract; 6 = potato extract; 7 = amino acid solution; 8 = egg albumin; 9 = chicken broth; 10 = milk

3.

Record your hypothesis here as to whether or not you think each solution contains proteins.

4.

Record the initial color of each solution before the Biuret’s solution has been added in Table 3.3.

5.

Shake the reagent carefully and add 20 drops of Buiret reagent to each tube and gently shake to mix. Do NOT heat this solution.

6.

Record your results in the Table 3.3.

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Basic Chemist r y: Macr omolecules | Exer cise 3

Table 3.3: Common Foods Tested for Protein Using Biuret’s Reagent. Test Tube

Substance Tested

1

Distilled water

2

Glucose

3

Sucrose

4

Corn extract

5

Onion extract

6

Potato extract

7

Amino acid solution

8

Egg albumin

9

Chicken broth

10

Milk

Initial Color Before Biuret’s

Final Color After Biuret’s

Protein Present?

QUESTIONS 1.

What are the monomers of proteins? What type of bond holds these monomers together?

2.

Were your hypotheses supported or not? Were you surprised by any of the results? Explain.

ACTIVITY #4 – TESTING UNKNOWN SOLUTIONS Procedure You will perform three tests on each of the unknowns. Set up the test tube rack as shown in Table 3.4. Using this pattern, you can add Benedict’s reagent to column 1, mix, and place the four tubes in the hot water. You can then add Iodine to column 2, and Biuret’s reagent to the four tubes in column 3. You will follow the procedure for each test as you did for the known solutions. See the individual procedures to remind you of how much solution is needed, etc.

Exer cise 3 | Basic Chemist r y: Macr omolecules

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Record your results in Table 3.5. Table 3.4: Set Up for Test Tube Rack with Tubes to Test Unknown Solutions. Add Benedict’s Reagent to tubes in this column

Add Iodine (Lugol’s) Reagent to tubes in this column

Add Biuret’s Reagent to tubes in this column

A

A

A

B

B

B

C

C

C

H2O

H2O

H2O

Table 3.5: Chemical Testing to Identify an Unknown. Write whether the results were positive or negative for the unknowns for each test. Unknown

Benedict’s Test

Iodine Test

A B C

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Basic Chemist r y: Macr omolecules | Exer cise 3

Biuret Test

Unknown Identity

EXERCISE 3

POST-LAB QUESTIONS NAME:

D AT E :

INST RUCTOR:

SECTION:

1.

What was the control group in all activities of this lab? Why was it necessary? Reducing sugar activity

Starch activity

Protein activity

2.

Complete Table 3.6 below to demonstrate your understanding of each of the assays performed. Table 3.6: Summary of Biochemical Tests. Test

Molecule Tested For

Color of Positive

Color of Negative

Ex er c ise 3 | Ba sic C h em ist r y: Ma c r om olec u les: Post - L a b

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