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LABORATORY

Chemistry of life and nutrition: Are you what you eat?

In the very first lab we looked at cells using our microscope and did an experiment to determine the presence of starch in onions versus potatoes. In this lab we will use similar techniques to identify which of the three major nutrient macromolecules we find in food. In this lab we will: • learn how to test for the molecules that makeup 99% of the human body • determine the chemical content of a solution of unknown composition • understand the role that biological molecules play in our nutrition • analyze and interpret data • learn the importance of controls in experiments Safety reminders: Chemicals used in this lab that should be handled with care are the following: Iodine (Stains clothing and skin; some individuals may have an allergic reaction) Benedict's and Buiret's reagent contain copper, which can be toxic in high concentration. Please minimize contact with skin. Be sure to thoroughly wash your hands if any reagent spills on your skin. Recall from the chapter on chemistry in your textbook that biological molecules come in four varieties: proteins, carbohydrates, lipids, and nucleic acids. Living organisms such as humans are composed of a variety of these biological molecules that come directly or indirectly from our food. However, different foods contain different amounts of the various biological molecules. For example, grains and potatoes are rich in starch, while meats are typically rich in protein and fat. It is often necessary to determine the types and relative amounts of organic molecules present in particular foods since each one plays an important role in the body. These various roles explain the need for a balanced diet - the intake of appropriate types and amounts of foods and drink to supply nutrition and energy for the maintenance of the body. Table 1 shows the recommended distribution in your diet of three biological molecules. Table 1 - Macronutrient Distribution Carbohydrates: Fat: Protein: 4-1

45 to 65% of total calories 20 t0 35% of total calories 10 to 35% of total calories

What are the unique properties of each class of biological molecules that can be used to distinguish one from the other? Protein: _____________________________________________________________ Carbohydrate: ________________________________________________________ Fat: ________________________________________________________________ To help use make an informed decision about our diet and the choices that contribute to a healthy diet, the food nutrition label is displayed on many of the containers of the foods we consume. The label lists the amount of fat, carbohydrate, and protein contained within the food product as well as other nutrients. By examining the label below, we can gather vital information about the composition of food products. The information in the main top section (see #1-4 and #6 on the sample nutrition label below), can vary with each food product. This section contains product-specific information such as serving size, calories, and nutrient information. The bottom section (see #5 on the sample label below) contains a footnote with Daily Values (DVs) for 2,000 and 2,500 calorie diets. This footnote provides recommended dietary information for the intake of important nutrients, including fats, sodium

source: www.fda.gov

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and fiber. You will find this footnote only on larger packages and it is not product specific. The DVs are based on recommendation for key nutrients for a 2,000 calorie diet. Those values are used as a reference point to help you determine if you are receiving the needed amounts of those nutrients in your diet. Notice that the DVs percentages do not add up to 100%. Each nutrient value is based on 100% of the daily requirements for that nutrient. Use the food label to estimate the amount of fiber recommended for your daily consumption. To do this you must set up a ratio. If 3g of fiber equals 12% of your daily requirements, 100% can be calculated as how many grams? Complete the ratio. ___________grams of fiber needed per day. Besides using grams, we also calculate nutritional information in calories. Calories are determined as the amount of energy needed to raise 1g of water 1oC. Dietary calorie (written as Cal) is equal to 1000 calories (kcal). According to the Association of Official Analytical Chemists (AOAC) one gram of protein yields 4 Cal, one gram of carbohydrate yields 4 Cal and one gram of fat yields 9 Cal. Using the caloric content of the three biological molecules verify the estimate of the total calories per serving of the food product. Use the label to calculate the following:

____ grams of Total Fat in product x ___ calories per gram = ______ calories from Fat ____ grams of Total Carbs in product x ___ calories per gram = ______ calories from Carbs ____ grams of Protein in product x ___ calories per gram = _______ calories from Protein

Total Calories ___________ Using Table 1, does this product meet the recommended distribution of calories?_________ Notice that the number of calories calculated above does not match up precisely with the number of calories reported on the food nutrition label. The Food and Drug Administration (FDA) is the federal agency that regulates the look and content of the food nutrition labels. The FDA requires manufacturers to round calories to the nearest 4-3

5-calorie if the food products has 50 or less calories and the nearest 10-calorie if the food has more than 50 calories. If you consume 2000 Calories per day, how many grams of the following do you require? Carbohydrates: ___________________

Fat: ___________________________

Protein is used for building and repairing body tissues, red blood cells, hair and other body structures as well as for synthesizing some hormones. To estimate how much protein (grams) is required for an individual each day, multiply your body weight (lbs) by the factor below: Sedentary adult Active adult Growing athlete Adult building muscle mass

0.4 x _____ lbs = ________ grams of 0.6 x _____ lbs = ________ grams of 0.7 x _____ lbs = ________ grams of 0.9 x _____ lbs = ________ grams of

protein protein protein protein

Laboratory identification of Biological Molecules The purpose of today's lab exercise is to introduce you to tests used to detect biological molecules. It is possible to identify these molecules because of their unique properties. Typically, one can detect a particular class of biological molecules by adding a chemical called a reagent to a solution. If the class of biological molecules is present, the reagent will react with it to form a colored product, whereas if it is not present there will be no color change. Such a test is called a colorimetric test because the results can be visualize as a change in the color of the solution. Besides the known substances we will test, you will notice that each test includes water. Water should not react with any of the reagents because we know that it does not contain any biological molecules. In each of the test you will notice that pure water is tested along with the other samples. Water will be used as the control in our test. Why would these tests not be valid without a water control?___________________ _________________________________________________________________ Note: Although good scientists always keep their work area clean, in this lab it is important to keep your test tubes with your results from each part of experiment. Once your unknown is completed, be sure to clean your area (including thoroughly washing all test tubes that you use) before you leave this lab.

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Identification of Biological Molecules - Techniques Benedict's Test for Reducing Sugars (Carbohydrates) 1. 2.

Get six tubes and label each with the numbers 1 – 6 In each test tube place approximately 2 ml of the following substances, the first bend of your index finger can be used to approximte 2 ml

Tube #

Substance

4

Maltose Solution

1 2 3

Sucrose Solution Distilled Water

5 6

3. 4. 5. 6.

Banana Juice Glucose Solution Fructose Solution

Add 2 ml of Benedict’s solution to each of the six test tubes Place the six test tubes in a hot-water bath for 10 minutes Allow the tubes to cool and then examine the tubes for changes in color Record your results on the data sheets at the end of the exercise

If a reducing sugar is present the solution will change from an aqua blue color to a green, brown, or rusty orange color with a precipitate at the bottom of the tube.

Iodine Test for Starch (Carbohydrates) 1. 2.

Get five test tubes and label each with the numbers 1 – 5 In each test tube place approximately 2 ml of the following substances, the first bend of your index finger can be used to approximte 2 ml

Tube #

Substance

4

Sucrose solution

1 2 3 5 3.

4.

Cornstarch solution Glucose solution Salad Oil Distilled water

Add 2 drops of Lugol’s iodine solution to each test tube and shake gently Record your results on the data sheets at the end of the exercise

If there is a starch present in the solution, it will change to a deep blue/blue –black color.. Variation in the intensity of the blue/black color is related to the concentration of starch presence in the solution. 4-5

Test for Lipids (Fats and Oils) 1.

Get one paper square, draw six dime-sized circles on the paper square and label each circle with the numbers 1 – 6

Circle #

Substance

2

Glucose solution

1 3 4

Cornstarch solution Margarine

5 6

2. 3. 4.

Salad oil

Bacon grease Distilled water

On each circle place a drop of the following substances Leave paper to dry Record your results on the data sheets at the end of the exercise

If a lipid is present, the paper will have a saturated greasy appearance

Biuret Test for Proteins 1. 2.

Get five test tubes and label each with the number 1 – 5 In each test tube place approximately 2 ml of the following substances, the first bend of your index finger can be used to approximte 2 ml

Tube #

Substance

4 5

Salad Oil Distilled Water

1 2 3

3. 4. 5.

5% Fructose Egg albumin Cornstarch

Add 1 ml (20 drops) of Biuret solution to each test tube and mix Let stand for 5 minutes and examine for a change in color Record your results on the data sheets at the end of the exercise

If a protein is present, the solution color will change to a light purple/lavender color

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Data Collection Sheets Benedict’s test for reducing sugars Tube #

Substance

Results

Conclusion

Substance

Results

Conclusion

Substance

Results

Conclusion

Lugol’s test for starch Tube #

Spot test for lipids Paper #

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Biuret’s test for protein Tube #

Substance

Results

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Conclusion

Student Name: __________________________________________ Unknown Tube #: ___________________ What substance(s) are found in your unknown solution? ________________________________________________________ Now that you have familiarized yourself with different techniques for identifying biological molecules, each of you will obtain a solution from your instructor and perform the four tests on that solution to determine ALL the substances in the solution. Your solution may have up to three substances found in the lab. It may be only water or a combination of all three biological molecules. To get it right you must indicate which substances specifically are in the solution. So, to get started write down what you will need to complete this task. Note: you may need to add more than one drop of the Lugol’s solution when testing the unknown solution for starch. How many test tubes will you require? ______________ How many paper squares will you require? ___________

Tube #

Test Performed

Results

Conclusion

Paper

When you have completed the testing of your unknown solution, you should report to your instructor the specific substances that tested positive from the following list. Remember, you may only have water or one substance or two substances, or as many as three substanes. Do not report that you have protein, fats, or carboydrate (BE SPECIFIC). Circle the substances you found in your unknown Banana Juice

Glucose Salad Oil

Egg Albumin

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Cornstarch

Margarine