Macromolecules are organic compounds that are present in both plants and animals. They are made up of simple structures called monomers. When the monomers combine, they form a more complex structure that is commonly referred to as polymers. Scientists have developed reagents that can be used to test for the presence of the various macromolecules. These macromolecules include lipids, proteins, carbohydrates and nucleic acids.
Carbohydrates
Carbohydrates are macromolecules mainly contain carbon, hydrogen, and oxygen that are covalently linked together. Carbohydrates are important since they form important structural part of the plant cell. In addition, they are broken down during respiration process to provide energy that is used to drive the various biological processes within living things. Carbohydrates have been categorized into three main groups namely, disaccharides, monosaccharides, and polysaccharides. Monosaccharides are often referred to as reducing sugars. They include glucose, fructose, and galactose. When two monosaccharides combine, they form a complex structure called a disaccharide. Examples of this category of carbohydrate include sucrose, lactose, and maltose. If three or more monosaccharides are linked together, a more complex structure called a polysaccharide if formed. An example of a polysaccharide includes cellulose and glycogen. Usually, the Benedict reagent is used to test for the presence or absence of reducing sugars (Perry, Morton & Perry, 2007).
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Proteins
Like carbohydrates, proteins are also organic compounds. The structure of proteins comprises of carbon, hydrogen, oxygen, nitrogen and sulfur in some cases. The building blocks of proteins are called amino acids. Specific gene sequences code for amino acids. For instance, the genetic code for the amino acid called phenylalanine is AAA. A, in this case, represents one of the four bases in the DNA stand called Adenine. Notably, when different amino acids are covalently joined, they form a polymer that is usually referred to as proteins (Bowman & Friedman, 2013). The bond type, in this case, is known as peptide bonds. In order to test for the presence of both protein and amino acids, Ninhydrin reagent is usually performed. However, when the test is specific for the protein, the Biuret test is the most appropriate (Perry, Morton & Perry, 2007).
Lipids
Lipids are organic compounds that are a hydrocarbon in nature. This implies that their structures are made up of carbon and hydrogen and oxygen in some instances. The structure of lipids comprises of carbon chains that can be either closed or open. The term triglycerides are also used when referring to lipids. There are two types of lipids namely oils and fats. Oils are unsaturated while fats are saturated. This means that oils can either contain a double or triple (C=C or C≡C) bond while fats have a single bond (C-C). At room temperature, oils are liquid while fats are solid. Notably, lipids are found in cell membranes, and they also provide energy as well as metabolic water for some biochemical processes. The test for lipids is performed using Sudan III reagent (Perry, Morton & Perry, 2007).
Method
Testing for Carbohydrates
Materials : In this first experiment, the following materials that were used: Heating source, 4 test tubes, test tube rack, razor blade, solutions of glucose, sucrose and starch, potato, Benedict and Iodine solutions, test tube holder, onion, potato, microscope, and distilled water.
Test 1
Procedure : First the test tubes were cleaned and labeled from 1 to 4. Test tube 1 was loaded with 10 cm3 of distilled water. Test tube 2, 3, and 4 were loaded with 10 cm3 of glucose, sucrose and starch solutions respectively. In each tube, 5 cm3 of Benedict reagent was added. The tubes were transferred to a hot water bath, and the heating was continued for about 2 minutes. The tubes were then removed from the water bath. The researcher observed and recorded changes in color that were noted in Table 1.
Test 2
Procedure : In the second test for carbohydrate, the four test tubes were washed thoroughly and labeled from 1 to 4. Test tube 1 was loaded with 10 cm3 of distilled water. Test tube 2, 3, and 4 were loaded with 10 cm3 of glucose solution, onion juice, and potato juice respectively. In each tube, 5 cm3 of Benedict reagent was added. The tubes were then transferred into a hot water bath for 2mins. The tubes were then removed from the water bath. The researcher noted the changes in color and recorded the results in Table 2.
Test 3
Procedure : In the third test for carbohydrate, the four test tubes were washed thoroughly and labeled from 1 to 4. Test tube 1 was loaded with 10 cm3 of distilled water. Test tube 2, 3, and 4 were loaded with 10 cm3 of glucose, sucrose and starch solutions respectively. In each tube, 3 cm3 of Iodine reagent was added. The tubes were then swirled. The researcher noted the changes in color and recorded the results in Table 3.
Test 4
Procedure : The fourth carbohydrate test involved slicing a piece of onion and observing it under a microscope. 1 cm3 of iodine reagent was added on the slice and then studied under a microscope. The same procedure was repeated for a thin piece of potato. Changes in color were noted and recorded in Table 4.
Testing for Amino Acids and Proteins
Materials : The materials used were 4 Test tubes and Hot water bath. The samples involved Albumin Solution, Amino Acid solution, Starch solution, and distilled water. The reagents for this experiment were 1% CuSO4 solution, Sodium Hydroxide and 0.1% Ninhydrin solution.
Test 1
Procedure : The four test tubes were washed to remove contaminants. They were then labeled 1 through 4. Test tube 1 was loaded with 10 cm3 of distilled water. Test tube 2, 3, and 4 were loaded with 10 cm3 of Amino Acid, albumin and starch solutions respectively. In each tube, 5 cm3 of Ninhydrin solution was added. The researcher then noted the changes in color and recorded the results in Table 5.
Test 2
Procedure : The four test tubes were cleaned and then labeled 1 through 4. Test tube 1 was loaded with 10 cm3 of distilled water. Test tube 2, 3, and 4 were loaded with 10 cm3 of Amino Acid, albumin and starch solutions respectively. In each tube, 10 cm3 of sodium hydroxide was added. Afterward, 5 cm3 of 1% CuSO4 solution was added in each test tube and the mixture swirled. The researcher then noted if a violet color appeared and recorded the results in Table 6
Testing for Lipids
Materials : The materials for this test included Test tubes, test tube holder, and test tube rack. The samples for this test were a honey solution, distilled water, Corn Oil, egg yolk, egg white, glucose solution, Cornstarch suspension, salad oil, honey solution, lard, and margarine. The reagent was Sudan III dye.
Test 1
Procedure : The four test tubes were cleaned and then labeled 1 through 4. Test tube 1 was loaded with 10 cm3 of distilled water. Test tube 2, 3, and 4 were loaded with 10 cm3 of egg white, corn oil and honey solutions respectively. In each tube, 10 cm3 of Sudan III was added, and the mixture swirled. The results for color change was recorded in Table 7
Test 2
Procedure : In this second test, the experiment was conducted to test for fats and oils. An unglazed paper was used for this test. Notably, 6 circles were drawn on the paper. Notably, the circles were 3 cm apart. The circles were allocated numbers from 1 to 6 (Pratt, 2011). Glucose solution, cornstarch, lard, distilled water, salad oil, and margarine were rubbed on each circle separately. The observations made were recorded in Table 8.
Results
Table 1: Testing for reducing sugar using Benedict’s solution
| Samples | Observation | Inferences |
| Water | No color change | Reducing sugar absent |
| Glucose | Orange solution was formed | Contains large amount of reducing sugar |
| Sucrose | The solution turned to Green | Contains little amount of reducing sugar |
| Starch | No color change | Reducing sugar absent |
Table 2: Testing for reducing sugar using Benedict’s solution
| Samples | Observation | Inferences |
| Onion Juice | The solution turned to Green | Contains little amount of reducing sugar |
| Potato Juice | Yellow solution was formed | Contains large amount of reducing sugar |
Table 3: Testing for starch using Iodine solution
| Samples | Observation | Inferences |
| Water | No color change | Starch Absent |
| Glucose | No color change | Starch Absent |
| Sucrose | No color change | Starch Absent |
| Starch | Blue Black solution formed | Starch present in large amounts |
Table 4: Testing for starch using Iodine solution
| Samples | Observation | Inferences |
| Onion | No color change | Starch absent |
| Potato | Changed to dark black | Starch present in moderate amount |
Table 5: Testing for Proteins or Amino Acids (AA) using Ninhydrin reagent
| Samples | Observation | Inferences |
| Water | No color change | AA or protein absent |
| Amino Acid | Purple solution was formed | AA or protein present |
| Albumin | Purple solution was formed | AA or protein present |
| Starch | No color change | AA or protein absent |
Table 6: Testing for Proteins using Biuret reagent
| Samples | Observation | Inferences |
| Water | No color change | Protein absent |
| Amino Acid | No color change | Protein absent |
| Albumin | Purple solution was formed | Protein present |
| Starch | No color change | Protein absent |
Table 7: Testing for Lipids using Sudan III Reagent
| Samples | Observation | Inferences |
| Water | No color change | Lipid absent |
| Honey | No color change | Lipid absent |
| Corn Oil | Changed to red | Lipid present |
| Egg White | No color change | Lipid absent |
Table 8: Testing for Lipids using unglazed paper
| Samples | Observation | Inferences |
| Water | No Translucent spot | Lipid Absent |
| Lard | Translucent spot formed | Lipid present |
| Salad oil | Translucent spot formed | Lipid present |
| Margarine | Translucent spot formed | Lipid present |
| Cornstarch | No Translucent spot | Lipid Absent |
| Glucose solution | No Translucent spot | Lipid Absent |
Discussions
Carbohydrate Tests
In the first test, that was carried to test for the presence of reducing sugar; the results went as per the expectation of the researcher. Notably, reducing sugars have a functional group called an aldehyde in their structure. The presence of an aldehyde group in simple sugars explains why they reacted with the Benedict solution when the samples were heated in a hot water bath. The simple sugar reduces the copper sulfate solution by accepting am oxygen atom from the Benedict solution. On the other hand, showed that sucrose solution contained little amount of reducing sugar. As aforementioned, sucrose is a disaccharide that is formed when glucose and galactose molecules combine covalently.
A process in which two monosaccharide units combine to form a disaccharide is called condensation. In that respect, a disaccharide molecule is not expected to reduce Benedict solution unless it is first broken down into its constituent parts using dilute hydrochloric acid. The carbohydrate experiment that was carried to test for starch obtained results that could easily be predicted. Notably, glucose being a monosaccharide and sucrose being a disaccharide do not contain starch. Starch itself is a polysaccharide. The iodine reagent reacts with the starch molecule to form a blue-black solution to show that starch was present in the sample. It was apparent that onions do not contain starch. However, potatoes have starch though in moderate amounts.
Protein tests
This experiment used the Ninhydrin reagent to test for both AA and proteins. The Biuret’s reagent was used to test for the presence of proteins specifically. As aforementioned, proteins are formed when the genetic code first codes for an amino acid. When various amino acids are joined covalently via peptide bonds they form a long polypeptide called a protein (Cozzone, 2010). The Ninhydrin reagent in this reaction oxidatively decarboxylates AA to CO2NH3 and the resulting aldehyde group will have one carbon less than the original AA. The reduced Ninhydrin, therefore, reacts with the freed ammonia. Proteins also can react with Biuret reagent. However, this type of reaction is particular for compounds that have more than two peptide bonds. Buret solution is contains NaOH or KOH and a small amount of CUSO4. Furthermore, Biuret solution gives positive results when reacted with Albumin.
Lipid Test
Compared to other tests, the test for lipids was easier to accomplish. However, the changes in color when using Sudan III reagent was the most challenging part of the experiment. Lipids are not polarized therefore they are not soluble in polar liquids. However, they dissolve in non-polar liquids like chloroform.
Conclusion
Macromolecules are important compounds in all living things. There are various types of macromolecules such as proteins, lipids, carbohydrates and nucleic acids. It is imerative for researchers to understand how these compounds can be distinguished chemically. This is important when it comes to understanding certain processes within living things. For instance, the understanding of such components has led to the development of genetically engineered organisms that have been helpful in various fields such as medicine. Nevertheless, scientists have developed reagents that have been instrumental when distinguishing between the different macromolecules. As this paper has shown, observing a change in color is essential when establishing the macromolecules that are present in samples.
References
Bowman, K., & Friedman, D. (2013). Glycoscience: Integrating a Key Macromolecule More Fully into the Curriculum. CBE Life Sciences Education , 12 (1), 5–8. doi: http://doi.org/10.1187/cbe.12-12-0217.
Cozzone, A. J. (2010). Proteins: Fundamental chemical properties . eLS . Retrieved from http://www-sop.inria.fr/axis/cost282/kelsi04/Brito/Brito3.pdf.
Perry, J. W., Morton, D., & Perry, J. B. (2007). Laboratory Manual for General Biology for Starr's Biology Texts . Belmont, CA: Thomson Brooks/Cole. ISBN-13: 978-0534380250
Pratt, C. W. (2011). A Biology Laboratory Exercise Using Macromolecule Assays to Distinguish Four Types of Milk. Journal of Microbiology & Biology Education : JMBE , 12 (1), 44–45. doi: http://doi.org/10.1128/jmbe.v12i1.242.
