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QualificationHigher National Diploma in Biomedical ScienceLevel5Module Name NumberBiochemistry (BIOM 502/1)Name of CandidateFathmath DheemaStudent Number516031822Title of PracticalChemical Tests for BiomoleculesDate of Practical25th April 2018Submission Date9th May 2018 MARKS SCHEME LABORATORY REPORTIntroduction and Objectives/ 20Materials and Methodology/ 20Results/ 20Discussion and Conclusion/ 20Academic Writing/ 10Log Book / 10TOTAL / 100 Contents Introduction Biomolecules are those primarily made up of hydrogen, carbon, and oxygen and are synthesized by living organisms. The most common group of biomolecules known as macromolecules, which are massive in size, include carbohydrate, protein and lipids. Each of these macromolecules are made up of small monomers. ( Donohue, 2018). Carbohydrates, also known as saccharides or sugars, are the most plentiful molecules found in nature. They are made up of monomers called monosaccharides. Examples of monosaccharides include, dextrose, levulose etc. (Introduction to Biomolecules, 2012). Monosaccharides are used to build cell arrangement and they also function as an energy source during cellular rrespiration. When two monosaccharides join with each other they form a dissaccharide. For exmaple Galactose ( figure1.) is formed from lactose joined with glucose. And polysaccharides are formed when a long chain of monosaccharides bond with each other, like starch which is composed of many alpha glucose molecules joined together. They act a source of energy in human body in the form of glycogen and in plants, in the form of starch. And also they componenets of other molecules such as DNA, and RNA. Lipids are biomolecules that consists of glycerol and fatty acid tails. These fatty acid tail are composed of long chain of carbon and hydrogens and are hydrophobic, which can either be saturated or non saturated. They play an important role in energy storing, as they store more amount of energy than carbohydrates. Lipid together with a phosphate group forms a phospholipid which is an important componenet of the cell memebrane. All organelles from mitochondria to nuclues, are surrounded by lipids. Many amino acids join together to form the complex structure of a protein. The 20 amino acid that forms a protein, are similar in structure except for the R group in each amino acid, as in figure 2a which shows Alanine with the R group CH3 and figure 2b. shows Cysteine with CH2 SH. (VirtualChembook,2003) Proteins play many vital roles in our body, from providing support to be regulatory proteins such as enzymes which helps in cell processes. Our immune system contains antibodies which are made up of proteins. (staff, 2013). This lab report is about the chemical tests that was done to identify the biomolecules in unknown samples of carbohydrates, amino acids, and lipids. The specific functional groups of biomolecules are distinguished with the help of these of these tests. First of all, for identification of carbohydrates, there are 6 tests iodine test used to test for the presence of starch. When starch react with iodine solution a bluish color is obtained. (Arshad, 2015) 2. Molischs test When concentrated sulphuric acid is treated with a carbohydrate, it is dehydrated to form furfural derivatives, when Alpha naphthol is added they condense to form a colored compound. 3. Benedict test this test is mainly used to identify reducing sugars and it can also detect carbohydrates with free aldehyde and ketone groups, which has the ability to reduce certain metallic ions. (Chabra, 2014) 4. Tollens test used to distinguish between aldehyde and ketones. Tollens solution readily oxidizes aldehydes but doesnt oxidize ketone. (Nichols, 2017) 5. Barfoed test this test is to tell apart monosaccharaides and disaccharides. Monosaccharaides reacts faster than disaccharides. 6. Seliwanoffs Reaction (for ketose sugars) when HCL keto hexoses is treated with HCL form 5-hydroxy methyl furfural, and then condenses to give a red colored complex. (Chabra, 2014). Secondly, the tests for amino acids include 1. Ninhydrin test this test is common for all amino acids. Ninhydrin reacts with the amino group to form a blue substance. But since proline has an imino group, it forms a yellow substance and arginine reacts with Ninhydrin to form a brown colored product. 2. Xanthroproteic test aromatic R groups containing amino acids, such as tyrosine, tryptophan and phenylalanine can be detected through this test. The aromatic benzene ring undergoes nitration to from a yellow colored compound. Since phenylalanine is difficult to titrate under normal condition it gives a negative or slightly positive result. 4. Millions test this test is specific to detect the presence of tyrosine and protein containing this amino acid as it is the only amino acid that contains a hydro benzene ring in it. When tyrosine and millions reagent is reacted together it gives a yellow precipitate, upon heating it changes into red colored precipitate. (Karki, 2018) 5. Paulys test It is a test which is usually used to test the presence of histidine or tyrosine. (Shresta, 2014) Lastly, for lipids the first test is, ethanol emulsion which forms a cloudy white suspension if lipid is present. 2. Grease spot test where an unglazed paper is used to identify whether water is more translucent or oil is. 3. Sudan 3 test, if lipid is present, upon shaking the mixture a red color is seen. Objectives To identify unknown samples of carbohydrates To discriminate reducing sugar from non-reducing sugars To identify certain amino acids To identify which is more translucent among water and oil To identify ketoses Materials Test tube rack Test tube holder Bunsen burner Test tubes Beaker Pipette Gloves For carbohydrate tests 3 unknown carbohydrate samples ( A, B, C) Molischs reagent Concentrated H2SO4 Iodine solution Benedicts solution AgNO3 Dilute Sodium hydroxide Dilute NH4OH Bafoed reagent Seliwanoffs reagent For Amino acid tests Ninhydrin solution Concentrated HNO3 Millions reagent Sulphanilic acid Ice bath Dilute HCL 2 NaOH For lipid tests Cooking oil Ethanol Distilled water Unglazed brown paper Cotton swab Water Sudan 3 red solution Methodology For the carbohydrates test, the first test that was done was Iodine test Using a pipette, 3 drops of iodine solution was added to the test tube containing 1 ml of unknown carbohydrate sample A. This was repeated for the rest 2 samples. (B and C). Molischs test 3 test tubes containing 1 ml of unknown samples were taken and to each few drops of Molischs reagent was added. Then to each tube, 1 ml of concentrated sulfuric acid was added down the sides of it. Benedicts test I ml of each unknown sample of carbohydrate was added into 3 test tubes. To this 5 ml of Benedicts solution was added using a pipette and was heated above a Bunsen flame for 2 minutes. The test tube was held by a test tube holder. Tollens test To three test tubes, 1 ml of silver nitrate was added, and dilute sodium hydroxide was also added. When a slight precipitate was formed, dilute NH4OH was added until the precipitate dissolved. To each test tube 1 ml of unknown samples of carbohydrate was added and each test tube was heated above the Bunsen flame for 5 minutes. Barfoed test To three test tubes, 1 ml of unknown samples of carbohydrate was added, then to it 5ml of Bafoed reagent was added separately. Each test tube was heated for 3 minutes. Seliwanoffs Reaction 5 ml of seliwanoffs solution was added to three test tubes, to each test tube 1 ml of an unknown sample of carbohydrate was added. Each test tube was heated for 30 seconds. Test for Amino Acids Ninhydrin test 0.5 ml of an unknown sample of amino acid was added into a test tube, to this 1 ml of ninhydrin solution was added and was heated for 3 minutes. The same procedure was repeated for the remaining 2 unknown samples as well. Xanthoproteic test 2 ml of unknown samples of amino acids was taken into 3 test tubes, to each test tube 2 ml of concentrated HNO3 was added. Each test tube was heated for 2 minutes. Millions test 2 ml of three unknown samples was added to test tubes, to each test tube 3 drops of Millions reagent was added and was heated. Paulys test 3 test tubes were taken, and to each test tube 1 ml of unknown samples of amino acid was added and to each test tube 1 ml of sulphanilic acid was added. 1 ml of HCl was added into three different test tubes. All 6 test tubes were kept in an ice bath for 3 minutes. After that the one HCL test tube and amino acid test tube was added. Same was done for the other two test tubes as well. Test for lipids Ethanol emulsion test 5 drops of oil sample was added to a test tube. Then 2 ml of ethanol was added and it was shaken thoroughly. After this 2 ml of distilled water was added to the test tube. Grease spot test Unglazed brown paper was taken, and two boxes were drawn on it with a pencil. To one box an oil drop was put and it was spread using a cotton a swab. And on the second box, a drop of water was put, it was spread using another cotton swab. The paper was held against a light source. Sudan 3 red test 2 ml of water and 2 ml of oil were added into a test tube. To this 3 drops of sudan 3 red was added and the test tube was shaken well. Results For carbohydrates TestsObservations of samplesInferenceABCIodine testYellow color changes to brownYellow color changes to brownYellow to blueA and B are branched but C is not branchedMolischs testA purple ring formedNo purple ring formedPurple ring formedA and C contains carbohydrates but B does notBenedicts testNo color changeBlue to greenNo changeB is a reducing sugar. A and C are not.Tollens testNo observationA silver mirror formedNo observation.B is an aldehyde and A and C is notBafoeds testNo changeRed precipitate formedNo changeB is a mono saccharideSeliwanoffs testRed color formedNo change in colorNo changeA is a keto sugarTable SEQ Table ARABIC 1 Protein tests TestsObservationInferenceABCNinhydrin testDark purple precipitate formedDark purple precipitate formedDark purple precipitate formedAll the samples contain an amino acidXanthoproteic testNo changeNo changeYellow colored complex formedC has an aromatic groupMillions testNo changeNo changeYellow complex formedC has a benzene ring in it.Paulys testNo changeNo changeNo changeTable SEQ Table ARABIC 2 Lipid tests TestsObservation ( Oil sample)inferenceEthanol emulsionCloudy white suspension formedLipids are presentGrease spot testOil allowed light to pass through it more than it did for waterOil is more translucent than waterSudan 3 red testRed color observed Oil layer on top Oil stained red and oil is more denser than water Table SEQ Table ARABIC 3 Discussion Sample B, among carbohydrate unknown samples, is a reducing sugar which is a monosaccharide and has an aldehyde group. B is also branched. Sample A is a ketose sugar which is branched. C is a carbohydrate and is not branched. Sample C of among the unknown samples of amino acids, is an aromatic amino acid. A and B are also amino acids. Oil sample contains lipids and is more translucent than water. It is also denser. According to table1 Carbohydrate sample B showed all positive results for the tests Benedicts test and silver mirror test. Thats why it is a reducing monosaccharide. The results for the seliwanoffs test confirmed that A is ketose sugar, and C is not branched because it didnt show a positive result for the iodine test, the color changed to blue instead of brown. Sample C of amino acids is tryptophan since it formed a yellow complex in the Xanthoproteic test. Paulys test didnt show any results. This was due to not Oil is denser than water according to sudan red 3 test, red colored oil layer was on top of the water layer. Oil let light pass through it so it is more translucent that water. 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