 As-salamu alaikum. Dear students, carbohydrates, also known as saccharides or sugars, are the most abundant biomolecules and are made up of carbon, hydrogen and oxygen. These are essential for proper body functioning and provide energy to cells. Based upon their structure, carbohydrates may be classified as monosecarides, disaccharides, oligosaccharides and polysaccharides. In the biochemistry laboratory, there are certain tests that we can use to identify whether a sample contains carbohydrate or not and if it does contain a carbohydrate, what type of carbohydrate is present in the sample. These tests can be used for the detection of carbohydrates in various body fluids, thus helping in disease detection. Today, we will be performing three tests for the identification of carbohydrates. The first test that we will perform is known as the MOLISH test. The MOLISH test is basically a general test used for the identification of carbohydrates and will give a positive result for all carbohydrates regardless of the fact whether a monosecaride is present or a disaccharide is present. The principle of this test is based upon the dehydration of the carbohydrate in the presence of concentrated sulfuric acid to yield an aldehyde which will condense with two molecules of a phenol, usually alpha nephthol to give a purple or violet colored product. The aldehyde formed is perforal if the sugar present is pentose and hydroxy methylperforal if the sugar present is a hexose. To proceed with this test, we will first take one ml of the sample in a test tube. We will add two to three drops of alpha nephthol. Fix the contents of the tube to ensure that alpha nephthol mixes well with the sample. We will add one to two ml of concentrated sulfuric acid to this tube. This step needs to be done carefully since sulfuric acid is corrosive. The formation of a purple or violet colored ring will indicate that a carbohydrate is present in this sample. The main precaution for this test is ensuring proper mixing once alpha nephthol is added since alpha nephthol is dense and tends to settle at the base of the test tube. The second test that we will perform is the Benedict test which is used for the detection of reducing sugars which are carbohydrates in which the carbonyl carbon is free for reaction. The Benedict test makes use of the Benedict reagent which contains copper tucil fate, sodium citrate and sodium carbonyl. Copper tucil fate provides copper ions for the reaction, sodium carbonate provides alkaline conditions for this reaction and sodium citrate prevents the reduction of copper ions during storage of the reagent. The principle of this test is that when a carbohydrate is heated in the presence of an alkali it yields to form an n-diol which reduces copper 2 ions to copper 1 ion. These copper 1 ions tend to form the brick red precipitate of copper 1 oxide which is visible at the end of the reaction. To proceed with this test we will first take one ml of the sample in a test tube. This sample we will add 2 ml of Benedict's reagent. This is then heated for approximately 2-3 minutes and checked for color change. Ideally this heating should be done on a water bath but if a water bath is not available direct heating can be done carefully. If the sample contains a carbohydrate you will notice that the colors of the solution changes from blue to green to yellow to orange and eventually red. The formation of this red color indicates that this sample contains a reducing sugar. The last test that we will perform is the iodine test which is used to detect the presence of a polysaccharide namely starch in the sample. The principle of this test is that iodide ions present in the iodine solution get absorbed in the amylose present in the starch molecule and produce a blue black color. To proceed with this test we will take one ml of the sample in a test tube. To this sample we will add 2-3 drops of iodine solution. Iodine solution is originally brown in color to 3 drops of iodine solution to this. Iodine solution is originally brown in color. The presence of blue black color indicates that this sample contains starch. It is worth noticing that all three tests done today are qualitative tests that is they will give us an idea about the identity of the carbohydrate present but not the amount of carbohydrate that was present in the sample. These are the building blocks of proteins and are named so because they contain an amino group that is NH2 and a carboxylic acid group that is COOH. There are certain tests that can be done in the biochemistry laboratory to identify whether a sample contains an amino acid or a protein. Today we will be performing three such tests namely the NIN-hydrin test, the biure test and the xanthoprotic test. NIN-hydrin test is the first test that we will be performing today and is a general test that indicates the presence of any amino acid or protein in the sample. To proceed with the test we will first take one ml of the sample in a test tube. We will add to this sample a few drops of 1% NIN-hydrin reagent. It will be mixed and heated using a spirit lamp or a Bunsen burner. The principle of this test is that NIN-hydrin carries out oxidative deamination of the amino acid leading to the formation of purple or violet colored complex and amino acids will give a positive color that is purple color for this reaction except for proline which will give us a yellow colored product. Proline contains a secondary amino group that is NH rather than a primary amino group that is present in all other amino acids hence the difference in the color of the product form. It is the biure test which will only give positive result if the sample contains at least two peptide bonds or any protein. The sample if it contains any amino acid will not give a positive result with this test. To proceed with the biure test we will first transfer one ml of the sample to a test tube. One ml of 10% sodium hydroxide or NaOH to this sample. Next step we will use 1% copper sulfate solution and add it drop by drop to this sample that already contains 10% sodium hydroxide solution. We will observe the formation of violet color if the sample contains at least two peptide bonds or protein. The principle of this reaction is the incorporation of copper ions between the peptide bonds. Third test is the xanthoprotec test. This test is specific for amino acid that have an aromatic ring in them that is tyrosine or cryptophan. The principle of this test is when the amino acid is heated with concentrated nitric acid there is nitration of the aromatic ring giving us a yellow colored compound. Addition of sodium hydroxide will convert this into a salt of this sodium hydroxide resulting in the formation of a bright orange or red precipitate. To proceed with this test we will first take one ml of the sample in a test tube. Next we will add approximately two ml of concentrated nitric acid to this sample. This step again needs to be done very carefully since nitric acid is concentrated and corrosive. We observe that yellow color of the solution if the amino acid present is an aromatic amino acid. Heat the tube slightly you will observe that the intensity of the color increases. Next very carefully add a few drops of 40% sodium hydroxide to this solution. This is the formation of orange color when sodium hydroxide is added to this solution. Keep orange color formed at the end of the reaction indicates that the sample contained an aromatic amino acid such as tyrosine or cryptophan. Similar to carbohydrates these three tests can only give us an idea about the presence of a certain amino acid or protein in the sample but they do not tell us about the amount or quantity of the sample present. Hence these three tests can be qualified as qualitative tests not quantitative tests. Dear students lipids are a class of heterogeneous compounds that contain hydrocarbons and include baths, oils, steroids, waxes and their derivatives. To identify whether a sample contains a lipid or not there are certain tests that we use in the biochemistry laboratory. Today we will perform three such tests namely the grease spot test, the solubility test and the sponification test. To proceed with the grease spot test we will require a small piece of filter paper. On this piece of filter paper we will place one drop of our sample. Once the drop of sample is placed you will notice that it spreads over the filter paper and becomes translucent. Light can now pass through this piece of filter paper. The principle of this grease spot test the second test that we will perform is the solubility test where we will compare the solubility of the sample in distilled water and chloroform. The principle of this test is that fats are generally not soluble in water but are soluble in organic solvents such as chloroform and ether. To proceed with this test we will take a small volume of the sample in a test tube. One test tube we will add 2 ml of distilled water. We will add 2 ml of chloroform. It's the contents of both test tubes. Two will observe that the test tube in which chloroform was added gives a clear solution indicating that our fat sample that was present is soluble in chloroform. Whereas the test tube in which water was added contains two distinct layers. One layer contains oil while the other layer contains water. This further confirms that our sample contained a lipid. The third test is the spawnification test. The principle of this test is that when a lipid is heated with an alkali in the presence of an alcohol it will hydrolyze to yield glycerol and a salt of that fatty acid. The salt of the fatty acid formed in this case gives a soap. To proceed with this test we will take one ml of sample in the test tube. To this sample we will transfer 3 ml of 0.5 normal alcoholic sodium hydroxide or NaOa. It's of the test tube heated using a water bath. If a water bath is not available the test tube can be heated directly for approximately 5 to 10 minutes. After heating you will observe that there are two distinct layers in the test tube. The denser layer contains soap or the salt of the fatty acid formed as a result of this reaction whereas the liquid layer contains glycerol and any leftover alkali. This test confirms that the sample contained a lipid.