Exp 2 Organic Compounds PDF

Experiment 2: Organic Compounds Biologically important molecules: carbohydrates, proteins and lipids  Objectives: By the end of this exercise you should be able to: Perform tests to detect the presence of carbohydrates, proteins and lipids Explain the importance of a contr...

Experiment 2: Organic Compounds Biologically important molecules: carbohydrates, proteins and lipids  Objectives: By the end of this exercise you should be able to: Perform tests to detect the presence of carbohydrates, proteins and lipids Explain the importance of a control in biochemical tests  Organic compounds All living things are made up of four classes of large biological molecules Carbohydrates Lipids Proteins Nucleic Acids Macromolecules are large molecules composed of thousands of covalently bonded atoms  Organic compounds Macromolecules are polymers, built from monomers A polymer is a long molecule consisting of many similar building blocks These small building-block molecules are called monomers A dehydration reaction occurs when two monomers bond together through the loss of a water molecule Polymers are broken down to monomers by hydrolysis, a reaction that is essentially the reverse of the dehydration reaction  Controlled experiments Biochemical tests are used to identify the major types of organic compounds in living organisms. Each of these tests involves two or more treatments 1. An unknown solution to be identified (the unknown may or may not be or contain the substance that the investigator is trying to detect) 2. Controls to provide standards for comparison (controls are known solutions)  Controls are used to validate that our procedure is detecting what we expect it to detect and nothing more. Positive control: contains the variable for which you are testing. It reacts positively and demonstrates the test’s ability to detect what you expect. It also shows what a positive test looks like Negative control: It does not contain the variable for which we are searching. It contains only the solvent (often distilled water with no solutes). It shows what a negative test looks like  Example: Iodine test for starch Distilled water+ Potato juice + Sucrose solution Starch + iodine iodine iodine + iodine Compare with Compare with blue yellow positive and positive and negative controls negative controls Positive control Negative control unknown unknown  Carbohydrates Carbohydrates are the most abundant organic compounds in the plant world Carbohydrates are molecules made of C, H, and O in a ratio of 1:2:1(ex: C6H12O6)  Monosaccharide The simplest carbohydrates are monosaccharide, or single sugars Glucose (C6H12O6) is the most common monosaccharide Fructose and galactose are also examples of monosaccharide  Disaccharides A disaccharide is formed when a dehydration reaction joins two monosaccharide This covalent bond is called a glycosidic linkage Maltose, sucrose and Lactose are examples of disaccharides  Polysaccharides Linking three or more monosaccharide forms a polysaccharide such as starch, glycogen, or cellulose The structure and function of a polysaccharide are determined by its sugar monomers and the positions of glycosidic linkages  Types of polysaccharides Starch, a storage polysaccharide of plants, consists entirely of glucose monomers  Types of polysaccharides Glycogen is a storage polysaccharide in animals Humans and other vertebrates store glycogen mainly in liver and muscle cells  Types of polysaccharides The polysaccharide cellulose is a major component of the tough wall of plant cells Like starch, cellulose is a polymer of glucose, but the glycosidic linkages differ  Carbohydrates Benedict’s test for reducing sugars Tests for Iodine test for carbohydrates starch  Benedict’s test for reducing sugars: Many monosaccharide such as glucose and fructose are reducing sugars, meaning that they possess free aldehyde (-CHO) or ketone (-C=O) groups that reduce weak oxidizing agents such as copper in Benedict’s reagent. Reducing sugars reduce the cupric (Cu2+) ions to cuprous oxide Cuprous oxide is green to reddish orange. Benedict’s test for reducing sugars:  Benedict’s test: to detect the presence of reducing sugars Procedure: Obtain seven tubes and number them 1-7. Add to each tube the materials to be tested (table 1). Add 2 ml of Benedict’s solution to each tube. Place all of the tubes in gently boiling water – bath for 2 min After 2 min, remove the tubes from the water – bath and let them cool to room temperature. Record the color of their contents.  Results, Analysis and Conclusion Table 1 Solution Benedict’s color reaction 1 ml onion juice brick red precipitate 1 ml potato juice blue color 1 ml sucrose juice blue color 1 ml glucose solution brick red precipitate 1 ml distilled water blue color 1 ml reducing sugar solution brick red precipitate 1 ml starch solution blue color  Iodine test for starch: Staining by iodine distinguishes starch from monosaccharide, disaccharides, and other polysaccharides. The basis for this test is that starch is a coiled polymer of glucose; iodine interacts with this coiled polymer and becomes bluish black Iodine test for starch:  Iodine test: to detect the presence of starch Procedure: Obtain seven test tubes and number the 1-7 Add to each tube the materials to be tested (table 2). Add 3 drops of iodine to each tube. Record the color of their contents Results, Analysis and Conclusion Table 2 Solution Iodine color reaction 1 ml onion juice Yellow color 1 ml potato juice Blue to black color 1 ml sucrose juice Yellow color 1 ml glucose solution Yellow color 1 ml distilled water Yellow color 1 ml reducing sugar solution Yellow color 1 ml starch solution Blue to black color  Proteins Proteins are made up of smaller units called amino acids, which are building blocks of proteins. They are attached to one another by peptide bonds forming a long chain of proteins. Each amino acid has an amino group (-NH2), a carboxyl (acid) group (-COOH) and a variable side chain (R) There are 20 amino acids. Based on the nature of their ‘R’ group  Proteins Amino acids are linked together by ‘amide groups’ called peptide bonds. During protein synthesis, the carboxyl group of amino acid at the end of the growing polypeptide chain reacts with the amino group of an incoming amino acid, releasing a molecule of water. The resulting bond between the amino acids is a peptide bond. Peptide bond  Biuret Test: Peptide bond (C-N bonds) in proteins complex with Cu+2 in Biuret reagent and produce a violet color. A Cu+2 must complex with four to six peptide bonds to produce a color Individual amino acids do not react positively  Biuret Test: to detect the presence of protein Procedure: Obtain 4 clean tubes and number them. Add 2 ml of the solutions listed in table 3 in each tube. Add 2 ml of Biuret reagent (already prepared) and mix Record the color of the tubes’ content in table 3 Results, Analysis and Conclusion Table 3 Tube Solution Color 1 Egg albumin solution violet color 2 Distilled Water blue color 3 Amino acid solution blue color 4 Protein solution violet color  Lipids Lipids are a diverse group of hydrophobic molecules The unifying feature of lipids is having little or no affinity for water (water fearing) Lipids are hydrophobic The most biologically important lipids are fats, phospholipids, and steroids  Fats Fats are stored in the adipose tissues in the form of triglycerides. That is why fats can be dissolved in non-polar solvents instead In a fat, three fatty acids are joined to glycerol by an ester linkage, creating a triacylglycerol, or triglyceride  Sudan IV test It is used to detect the presence of lipids Sudan IV is a fat-soluble (hydrophobic) dye. Sudan IV is soluble in non-polar solvents. Sudan IV is dissolved in a non-polar solvent such as diethyl ether or chloroform producing a dark red colored solution. When the above solution is added to a lipid-containing solutions homogeneous mixture But when added to aqueous solutions heterogeneous mixture Remark: lipids are non-polar molecules and thus they are soluble in nonpolar solvents and insoluble in polar solvents.  Sudan IV test: to detect the presence of lipids Procedure 4: Obtain 4 clean tubes and number them. Add the material listed in table 4 and mix Record the color of the tubes’ content in table 4 Results, Analysis and Conclusion Table 4 Tube Solution Number of Analysis layers 1 1 ml salad oil + 1ml Sudan IV one layer presence of lipids 2 1 ml egg albumin + 1ml Sudan IV two layers absence of lipids 3 1 ml distilled water + 1ml Sudan IV two layers absence of lipids 4 1 ml known lipid solution + 1ml one layer presence of Sudan IV lipids  Recommended videos https://amrita.olabs.edu.in/?sub=79&brch=17 &sim=205&cnt=694 ( To open the above link press right click, then press open hyperlink) (You will be responsible only for the procedures in the ppt)