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AP BIOLOGY (DOC download) by zhouwenjuan

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									AP BIOLOGY                                                 NAME______________________
                                                               Sept. 28/29/30, 2010
LAB 2: ORGANIC MOLECULES




PURPOSE:
To learn how to identify several major types of organic molecules found in living systems.

OVERVIEW
A cell is a living chemistry laboratory in which most functions take the form of interactions
between organic (carbon-containing) molecules. Most organic molecules found in living systems
can be classified as carbohydrates, fats, proteins, or nucleotides. Each of these classes of
molecules has specific properties that can be identified by simple chemical tests.

In this lab you will be learning how to identify:
         • reducing sugars using the Benedict’s test
            (one major group of monosaccharides and disaccharides)
         • polysaccharides using Lugol’s iodine
         • lipids using the Sudan IV test
         • proteins using the Biuret reagent
         • amino acids using Ninhydrin

EXERCISE 1: TESTING FOR CARBOHYDRATES
The basic structural unit of carbohydrates is the monosaccharide (or single sugar).
Monosachharides are classified by the number of carbons they contain: for example, trioses have
three carbons, pentoses have five carbons, and hexoses have six carbons.
            Glucose                                            Fructose

Figure 1. Glucose, an aldose sugar, and fructose, a ketose sugar. Both are hexoses.

Monosaccharides are also characterized by the presence of a carbonyl group, either a terminal
aldehyde group or an internal ketone group. Both of these groups contain a double bonded
oxygen atom that will react with Benedict’s reagent to form a colored precipitate.
When two monosaccharides are joined together (via dehydration synthesis), they form a
disaccharide. If the reactive aldehyde or ketone groups are involved in the bond between the
monosaccharide units, the disaccharide will not react with Benedict’s reagent. Sucrose is an
example of such a disaccharide. If only one carbonyl group is involved in the bond, the other is
free to react with the reagent. Maltose is such a disaccharide. Sugars with free aldehyde or
ketone groups, whether monosaccharides or disaccharides, are called reducing sugars. These
sugars are oxidized (lose electrons) by the Cu 2+ in the Benedict’s reagent and the Benedict’s
reagent is reduced (gains the electrons lost by the sugar). Hence the name reducing sugar!




Figure 2. Two disaccharides: maltose and sucrose.

Monosaccharides may join together via dehydration synthesis to form long chains, known as
polysaccharides. These chains may be either straight or branched. Starch is an example of a
polysaccharide formed entirely of glucose units. Starch does not show a reaction with Benedict’s
reagent because the number of free aldehyde groups (found only at the end of each chain) is
small in proportion to the rest of the molecule. Many polysaccharides will react with Lugol’s
reagent (iodine/potassium iodide, I2KI). Lugol’s reagent changes from a brownish or yellowish
color to blue-black when starch is present, but there is no color change in the presence of
monosaccharides or disaccharides

In this section the lab, you will be using Benedict’s reagent to test for the presence of reducing
sugars and Lugol’s iodine to test for the presence of starch.

PROCEDURE:
PART A: BENEDICT’S TEST FOR REDUCING SUGARS
1. Set up a row of nine test tubes. Use a marker to number the tubes 1 through 9.
2. Add 2 ml of the solutions listed in Table 1, matching the number of the solution to the
   number on the tube.
3. Add one dropperful (approximately 2 ml) of Benedict’s reagent to each tube.
4. Mix the reagent and the sample by agitating the solution in each tube from side to side.
   Record the original color of each tube’s contents in Table 1 in the appropriate column.
5. Heat the test tubes in boiling water for 3 minutes. Record the color of each tube at the end of
   three minutes in Table 1.

PART B: LUGOL’S TEST FOR STARCH
6. Place five microscope slides on top of a white piece of paper. Divide each down the middle
    with a line drawn with a marker. Number each half, from 1 to 9.
7. Place a drop or two of each of the solutions listed in Table 1 on the slides, matching the
    number of the solution with the number on the space on the slide.
8. Record the original color of each solution in Table 1 in the appropriate column.
9. Add one or two drops of Lugol’s reagent to sample of solution. Mix using toothpicks. Use a
    clean toothpick for each numbered sample.
10. Record the color of each sample after mixing in Table 1.

Table 1. Results for Benedict’s test for reducing sugars and Lugol’s test for starch

 Tube # and         Benedict’s Test                          Lugol’s Test
 Contents           Original color       Final color after   Original color       Final color after
                    before boiling       boiling             before adding        adding I2KI
                                                             I2KI
 1. Water
 2. Starch
 3. Glucose
 4. Maltose
 5. Sucrose
 6. Onion Juice
 7. Potato Slice
 8. Milk
 9. Corn Syrup
EXERCISE 2: TESTING FOR LIPIDS

The word lipid refers to any member of a rather heterogeneous group of organic molecules that
are soluble in nonpolar solvents such as chloroform (CHCl3) but insoluble in water. Although
lipids include fats, steroids, and phospholipids, this lab will focus primarily on fats.
Triglycerides, a popular topic in the discussion of diet and nutrition, are the most common form
of fat. They consist of three fatty acid molecules attached to a molecule of glycerol.
Triglycerides are found predominantly in adipose tissue and store more energy per gram than any
other type of compound found in living tissue. Triglycerides store approximately 9 calories per
gram, carbohydrates and proteins both store approximately 4 calories per gram.

At room temperature, some lipids are solid (generally those that are found in animals) and are
referred to as fats, or more properly as saturated fats. Other triglycerides are liquid at room
temperature (generally those found in plants) and are referred to as oils, or more properly as
unsaturated fats. Vegetable oil, a liquid fat, is a mixture of triglycerides.
Since both liquid and solid fats are nonpolar, you will test for their presence by using Sudan IV,
a nonpolar dye that dissolves in nonpolar substances such as fats and oils, but not in polar
substances such as water.




Figure 3. A tryglyceride, an example of a lipid.

Procedure
1. The familiar “grease spot” is the basis of a very simple test for fats. On a piece of unglazed
   paper, such as brown paper lunch or grocery store bags, place one drop of oil and one drop of
   water. Allow the drops to dry and observe the difference between the spots where the drops
   were applied.
2. Label six test tubes in sequence, 1 through 6. Add 1 ml of each substance listed in table 2 to
   the appropriate tube.
3. Add 3 drops of Sudan IV to each tube and mix.
4. Finally add 2 ml of water to eachg tube.
5. If fats or oils are present in the sample, these will appear as floating red droplets or a floating
   red layer colored by the Sudan IV.
Record your observations in Table 2.
Table 2. Results for Sudan IV test for lipids.

 Tube number and              Sudan IV Solubility Reaction
 Substance

 1. Distilled Water

 2. Vegetable Oil

 3. Onion Juice

 4. Hamburger Juice

 5. Heavy Cream

 6. 1% Milk



EXERCISE 3: TESTING FOR PROTEINS AND AMINO ACIDS

Proteins are made up of one or more polypeptides, which are linear polymers of smaller
molecules called amino acids. Amino acids derive their name from the amino group and the
carboxyl group (acidic) that each possesses. Polypeptides are formed when amino acids are
joined together by peptide bonds between the amino group of one amino acid and the carboxyl
group of another amino acid.




Figure 4. The amino acids leucine and proline    Figure 5. A peptide bond between 2 amino acids

The Biuret reagent reacts with the peptide bonds and therefore reacts with proteins, such as egg
albumin, but not with free amino acids, such as glycine and alanine. The Biuret reagent is light
blue, but in the presence of polypeptides and proteins it turns violet. Other types of molecules
may cause other color changes, but only the violet color indicates the presence of polypeptides.

On the other hand, the reagent ninhydrin reacts with the amino group of free amino acids, but not
with polypeptides. Ninhydrin turns purple or violet in the presence of the free amino groups in
amino acids. In the presence of the amino acid proline, however, it turns yellow. Proline reacts
differently because its amino group is not free but is, instead, part of the ring structure of the
molecule.


PROCEDURE
PART A: TESTING FOR PROTEINS WITH THE BIURET REAGENT
1. Obtain seven clean test tubes and number them 1 through 7.
2. Add 2 ml of the solutions listed in Table 3 to the test tubes, matching the number of the
   substance to the number on the tube.
3. Add 2 ml (one dropperful) of Biuret reagent to each tube.
4. After an incubation period of 2 minutes, record your results in Table 3 and determine which
   of the samples contain protein. Base you conclusions only on the presence or absence of the
   violet color.


Table 3. Results for Biuret test for the presence of proteins


 Tube Number and             Color with Biuret reagent after 2 minutes      Protein Present (+)
 Contents                                                                   or Absent (- )

 1. Distilled Water

 2. Egg Albumin

 3. Potato Starch

 4. Glucose

 5. Amino Acid

 6. Hamburger Juice

 7. Chicken Broth



PART B: TESTING FOR AMINO ACIDS WITH NINHYDRIN
1. Write the numbers 1 through 6 around the perimeter of a circular piece of filter paper. Put
   your name and your lab partner’s name on the filter paper.
2. Apply one drop of each of the substances listed in Table 4 in the appropriate spot on the filter
   paper.
3. Let the filter paper dry. You may speed up the drying by using a hairdryer.
4. Give your filter paper to your instructor, who will spray the paper with ninhydrin under the
   fume hood.
5. Leave overnight in the fume hood to dry.
6. When you come to class tomorrow, observe your filter paper and record your observations in
   Table 4. If the substance reacts with ninhydrin, a pink or purple spot will be seen on the filter
   paper.

Table 4. Results for Ninhydrin test for amino acids.


  Substance                             Reaction with ninhydrin (+ or – and color)


  1. Distilled Water

  2. Egg Albumin


  3. Potato Starch


  4. Glucose


  5. Alanine


  6. Glycine




EXERCISE 4: TESTING UNKNOWN FOOD SUBSTANCES

In this exercise you will be determining the presence of carbohydrates, lipids and proteins in in
several unidentified food substance. Once you have determined which types of organic
molecules are present in each you will hypothesize as to the true identity of the substance!

PROCEDURE:

1. Obtain a sample of each of the unknown substances.
2. Perform the following tests on each sample: Benedict’s, Lugol’s, Sudan IV, and Biuret. Use
   the procedures given earlier in this handout for each of the tests.
3. Record the results of the tests in Table 5.
4. Based on your results, hypothesize as to the true identity of each food substance. Write your
   analysis in the space provided under Table 5.


Table 5. Analysis of several unknown food substances.

                      Substance # 1            Substance # 2           Substance # 3
 Test                 Results                  Results                 Results

 Benedict’s

 Lugol’s

 Sudan IV

 Biuret



Unknown # 1:

Contains the following types of organic molecules:

______________________________________________________

Possible identity of Unknown #1:

______________________________________________________


Unknown # 2:

Contains the following types of organic molecules:

______________________________________________________

Possible identity of Unknown #1:

______________________________________________________


Unknown # 3:

Contains the following types of organic molecules:

______________________________________________________
Possible identity of Unknown #1:

DISCUSSION QUESTIONS

1. Explain the limitations of the Benedict’s test in determining whether or not sugar is present
    in a certain food product. Why do all monosaccharides, but only some disaccharides, react
    with Benedict’s reagent?
2. What did you learn about the specificity of the Biuret reagent? When would you use
    ninhydrin instead of the biuret reagent? Give an example
3. The leaves of many plants are coated with a waxy substance that causes them to shed water.
    Would you expect this substance to “react” with Sudan IV? Explain.
4. Ninhydrin reacts with a mixture of amino acids and turns purple. Could proline be one of the
    amino acids? Describe a way that you could conclusively determine if proline was present in
    the mixture?
5. A sample of food product X has a positive test for both Benedict’s and Lugol’s reagents. For
    what group of nutrients would this food be a good source? Why is this specific group of
    nutrients needed by living organisms?
6. Nutritionists recommend that some fats be present in a person’s daily diet. Why? What
    nutrients (biological molecules) does a person obtain from eating fish? Why are these
    nutrients an important part of a healthy daily diet?
7. Why are vitamins a necessary part of a healthy diet? Give the names of two vitamins and
    describe what their function is in living systems.
8. Vitamins fall into two general categories: water soluble and fat soluble. Which one of these
    groups should not be taken in excess? Give an example of one of these vitamins and the
    dangers associated with taking it in excess.
9. Why are some amino acids called essential amino acids? Give the names of these amino
    acids.
10. Fatty acids with more than one double bond are considered essential fatty acids. Are these
    fatty acids considered saturated or unsaturated? Explain why. What are the sources of
    essential fatty acids?
11. In winter, plants exchange the saturated lipids in their cell membranes for unsaturated lipids.
    Unsaturated lipids are bent and keep the membrane more fluid because they cannot be
    stacked closely together. What advantage would this exchange have for herbaceous plants
    that live through the winter? (Sort of a hint: what happens to bacon grease or the layer of fat
    on top of soup when you put it in the refrigerator?)
12. Do some online research into what nutritionists recommend for daily calorie and food group
    intake for a person your age. Create a one-day menu for three meals (breakfast, lunch and
    dinner) that would meet these requirements. You may also use the food pyramid article given
    to you in class.

								
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