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Classification of Macromolecules

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The purpose of this lab was to determine if various substances contained macromolecules, specifically; carbohydrates, proteins, or vitamin C. The tests used were the Biuret test, the xanthoproteic test, the Benedict’s test, the starch test, and the indophenol test. Many of the substances were positive for that which they were being tested, proving the hypothesis partially correct.

INTRODUCTIONThis lab was conducted to determine if various substances contained various macromolecules. The macromolecules tested for were carbohydrates, proteins, and vitamin C. The substances tested were glucose, fructose, sucrose, maltose, milk, corn syrup, starch, glycogen, gelatin, egg albumin, ascorbic acid, apple juice, orange juice, and fruit punch. It was hypothesized that all the substances tested for carbohydrates would test positive, that all the substances tested for proteins would test positive, and that ascorbic acid and orange juice would test positive for vitamin C. To test for carbohydrates, a Biuret test and a starch test were conducted on each substance. To test for proteins, a Benedict’s test and a Xanthoproteic test were conducted on each substance. Finally, to test for vitamin C, each substance was titrated with indophenol.

Carbohydrates are macromolecules consisting of carbon, hydrogen, and oxygen atoms. Most carbohydrates have a hydrogen to oxygen ratio of two to one. When photosynthesis takes place, the result is carbohydrates and oxygen gas. These macromolecules are the main source of energy for both plants and animals. In plants, the carbohydrate cellulose is used in the physical structure of the organism. Carbohydrates are referred to as saccharides, or simply as sugars, and are vital to life. They can be divided into monosaccharides, disaccharides, and polysaccharides, depending on the number of sugar molecules involved. A single sugar molecule is a monosaccharide and includes things such as glucose and fructose, and two sugar molecules are a disaccharide. When many sugars (sometimes thousands) are joined together, they form a polysaccharide. (Campbell, 2004) (Malyk, 2007a) (Malyk 2007b) (Brown, 2007)The other main distinction between different sugars is whether they are an aldehyde or a ketone.

This difference affects what sort of structure the sugar will display. An aldehyde’s hydrocarbon chain ends in a carboxyl group, whereas a ketone group is found in the middle of the chain. The reducing sugar test checks to see if the carbohydrate has carboxyl groups with double bonded oxygen atoms, thus a positive result indicates an aldehyde. The test for reducing sugars functions by creating a colored precipitate when the oxygen is bonded to the Biuret solution. Starches, a polysaccharide, are a form of storage sugar made up of thousands of sugar molecules. The test functions through the iodine, which reacts with the starch to create a blue-black color. The substances tested for reducing sugars and starch were glucose, fructose, sucrose, maltose, milk, corn syrup, starch, and glycogen. (Campbell, 2004) (Malyk, 2007a) (Malyk, 2007b) (Reusch, 2007a)Proteins are another of the macromolecules tested in this lab. Structurally, protein is made up of amino acids, joined together by peptide bonds.

Amino acids consist of an amino group and a carboxyl group linked to a side chain, which determines the nature of the amino acid. They form much of the structure of animals, including skin and muscles. Twenty amino acids allow for an almost unlimited variety of proteins. Structurally, proteins are made up of four levels. The primary level is formed based on the order of amino acids. The secondary structure is the folds and coils in the protein, caused by the hydrogen bonds in the polar sections of the amino acids. The third level is formed by a variety of causes, including hydrogen bonds, the tendency of hydrophobic group to go together, ionic bonds, and disulfide bridges, bonds between two sulfur atoms. The last level of structure isn’t always present but is caused by the bonding of two polypeptide chains together. Proteins are vital to many processes in the human body, such as movement and oxygen transport (in the form of hemoglobin). Elementally, proteins are made up of hydrogen, oxygen, carbon, and nitrogen. Smaller chains, of only a few amino acids, are called peptides rather than proteins. Gelatin, egg albumin, and milk were the substances tested for proteins.

The Benedict’s test tested for the presence of peptide bonds, whereas the xanthoproteic test tested for a benzene ring. Peptide bonds are found in all proteins, while benzene rings are not. (Campbell, 2004) (Malyk, 2007c) (Reusch, 2007b)The last macromolecule tested for was vitamin C. Vitamin C is an acid, which is produced from glucose or gathered from nature. Being a vitamin, it is essential to animals but cannot be synthesized by the animal. It plays a crucial role in the production or collagen, helps wound healing, and helps boost the immune system. It is found naturally in foods like citrus fruits, cabbage and tomatoes. Vitamin C is made up entirely of hydrogen, carbon and oxygen, and has the chemical formula of C6H8O6. Vitamin C is best taken from raw foods due to the fact that the chemical structure can easily be changed by heat or air exposure. To test for vitamin C, it will be titrated with a known volume of indophenol. When equilibrium is achieved, indicated by a color change to pink or colorless, the test is considered positive.

Ascorbic acid, orange juice, apple juice, and fruit punch were tested for vitamin C. (Malyk, 2007d) (Higdon, 2007)MATERIALSBenedict’s solutions1% glucosetest tube rack1% fructosetest tube tongs1% maltosetest tubes (35 total)1% sucrosehot plate1% glycogen400 mL beaker1% starchdisposable pipettes10% corn syrupdrop plateskim milkLugol’s reagent (iodine solution)potato extractdistilled water0.02M CuSO410% egg albumin6M NaOH1% gelatinconcentrated HNO3milk0.125 % ascorbic acid solutionapple juiceorange juicefruit punchMETHODS OR PROCEDURESThis lab was broken down into three smaller experiments; one for each of carbohydrates, proteins, and vitamin C. Each set of tests was done separately.

The first set of substances tested were glucose, fructose, maltose, sucrose, glycogen, starch, corn syrup, skim milk, and potato extract. These were all put through the Benedict’s test, for reducing sugars, and through a starch test.

For the Benedict’s test, 4 mL of each solution were placed into labeled test tubes. 4 mL of each solution were then placed in a second set of test tubes, which served as a control. Next, 5 drops of Benedict’s solution was added to each of the labeled test tubes. All the test tubes were then placed in a hot water bath using the hot plate. After 2 to 3 minutes, any color changes were observed and recorded. A yellow color indicated a positive result.

The starch test was conducted next and consisted of putting five drops of each solution in a well on a drop plate. 5 drops of water was also added to a well, functioning as a control. To each well, 1 drop of iodine solution was added. Any color changes were observed and recorded. A color changed indicated a positive result.

The results from the first two tests were then recorded in a table. (Malyk, 2007b)Tests for protein were conducted next, and the substances used were egg albumin, gelatin, and milk. These substances were tested using the Biuret test and the xanthoproteic test.

The Biuret test was conducted by added 2 mL of each solution to a test tube, along with a distilled water control. 2 mL of 6 M NaOH was then added to each test tube followed by 4 drops of 0.02 M CuSO¬4. Each test tube was then gently shaken to encourage mixing. Any color change was observed and recorded in a table. A violet or pink color indicated a positive result.

Next, the xanthoproteic test was conducted on the same substances. 1 mL of each substance was placed into a labeled test tube and 5 to 10 drops of concentrated HNO3 was added. Any color change was noted and recorded in a table. A yellow color indicated a positive result. (Malyk, 2007c)The last test was conducted on ascorbic acid, apple juice, orange juice, and fruit punch to test for the presence of vitamin C. 10 drops of indophenol was placed in a test tube and 0.125% ascorbic acid was added drop by drop until a color change in the indophenol was noted. The number of drops required was noted and recorded. This procedure was repeated for the other substances, as well as a distilled water control. These results were also recorded in a table. (Malyk, 2007d)


Carbohydrates can be divided into three different classes: monosaccharides, disaccharides, and polysaccharides. In this experiment, the Biuret test was used to test for reducing sugars, that is carbohydrates with double bonded oxygens that can be reduced into to saccharides. A colored precipitate formed from the double bonded oxygens indicated this result. Glucose, fructose, maltose, milk and corn syrup all tested positive for reducing sugars. The starch test, however, resulted in a positive from starch. The starch test worked by added iodine to the samples, relying on the fact that when iodine comes into contact with starch, it reacts by turning a blue or black color. Starch is a polysaccharide and is primarily used as a storage molecule, and thus is an excellent source of energy for living beings. Since reducing sugars and starch are different kinds of carbohydrates, the test results for each differed substantially. If testing an unknown substance for carbohydrates, it would be best to perform both tests, due to their relative simplicity. (Malyk, 2007b) (Reusch, 2007a)

Proteins are made up various amino acids joined together by peptide bonds, which form between the carboxyl group of one amino acid and the amino group of another utilizing dehydration synthesis. Proteins are used in various roles in the human body, including motion, enzymes, and structure. Milk, gelatin, and egg albumin all tested positive in the Benedict’s test, indicating the presence of peptide bonds, and thus of proteins. The xanthoproteic test revealed that milk’s proteins contain benzene rings. Each test functioned by denaturing the protein, and then causing a reaction to reveal the peptide bonds and benzene rings respectively. In the event that an unknown sample must be tested for proteins, it would be best to test using the Benedict’s test, since it revealed peptides bonds, which are required for all proteins, rather than benzene rings, which are not in all proteins. (Malyk 2007c) (Reusch, 2007b)Vitamin C is an acid, also known as ascorbic acid, which is required for many processes in the human body. It is crucial to gum health, collagen production, and the immune system.

Orange juice, and ascorbic acid, tested positive for vitamin C. This was determined by using a test in which indophenol was placed in a test tube, functioning as an indicator, and ascorbic acid was added until it changed color to pink or colorless. This test is excellent for using with unknown substances as it not only reveals the presence of ascorbic acid, but also reveals how much, through comparison to a known concentration, 0.125% in this case. (Harrison, 2007a) (Higdon, 2007)CONCLUSIONSIn conclusion, the hypothesis that all the substances would test positive for carbohydrates was incorrect, in that glycogen and sucrose both tested negative for starch and reducing sugars. It was, however, correctly hypothesized that all the substances would test positive for protein and also that ascorbic acid and orange juice would test positive for vitamin C.


The limiting factor in this lab was the difficulty in correctly reading any color change that took place in the various tests. This was impossible to fix due to the fact that the tests required human interpretation. The only way to improve the procedure would be to photograph the resultant color change, allowing for further study and interpretation.


1.AP Biology. (2007). AP Biology Formal Lab Results of September 12. Ridley College. St. Catharines, Ontario, Canada.

2.Brown, W P. “Naming and Structure of Aldehydes and Ketones”. (2007) http://www.wpbschoolhouse.btinternet.co.uk/page06/AldehydesKetones.htm3.Campbell, Neil A. and Reece, Jane B. Biology Sixth Edition Benjamin Cummings: San Francisco: 20044.Harrison, Karl. “Vitamin C or Ascorbic Acid”. (2007a) http://www.3dchem.com/molecules.asp?ID=695. “Sucrose”. (2007b) http://www.3dchem.com/molecules.asp?ID=596.Higdon, Jane. “Vitamin C”. Oregon
State University. (2007) http://lpi.oregonstate.edu/infocenter/vitamins/vitaminC/7.Malyk, R.J. (2007a). AP Biology Class Notes of Macromolecules. Ridley College. St. Catharines, Ontario, Canada.

8. (2007b). AP Biology Identification of Carbohydrates Lab Instructions of September 12. Ridley College. St. Catharines, Ontario, Canada.

9. (2007c). AP Biology Identification of Proteins Lab Instructions of September 12. Ridley College St. Catharines, Ontario, Canada.

10. (2007d). AP Biology Identification of Vitamin C Lab Instructions of September 12. Ridley College St. Catharines, Ontario, Canada11.Reusch, William. “Carbohydrates”. Michigan State University. (2007a) http://www.cem.msu.edu/~reusch/VirtualText/carbhyd.htm12. “Proteins”. Michigan State University. (2007b) http://www.cem.msu.edu/~reusch/VirtualText/proteins.htm

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