1. Isolation of Casein From Milk Aim: To Isolate Casein

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Bioorganic chemistry
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  1.ISOLATION OF CASEIN FROM MILK Aim: To isolate casein from milk, i.e., milk protein from milk. Principle:Precipitation: It is widely used in down stream process of biological products such as proteins. This unitoperation serves to concentrate and fractionate the target product from various contaminants.Protein precipitate formation occurs in a stepwise process. The addition of a precipitating agentand steady mixing destabilizes the protein solution. Mixing causes the precipitant and the target product to collide. The mechanism of precipitation is to alter the salvation potential of thesolvent and thus lower the solubility of the solute by addition of a reagent. Casein: It is a predominant phospho protein that accounts for nearly 80% of protein in milk and cheese.When co-agulated with rennet, casein is sometimes called a paracasein. It consists of a fairlyhigh number of proline peptides which do not interact. There are also no disulphide bridges. As aresult it has relatively little secondary and tertiary structures and it cannot denature. It ishydrophobic making it poorly soluble in water. The isoelectric pH of casein is 4.6. The purified protein is water insoluble; insoluble in neutral salt solution. It is readily dispersible in dilutealkali and salt solution such as sodium acetate, sodium oxalate. Apparatus required:  Beaker   Test tube  Watch glass  Electronic heater   Tripod stand Chemicals required:  Ethanol  Ether   Acetic acid   NaOH  Conc. Hydrochloric acid  Benzene. 1BIOORGANIC CHEMISTRY LAB MANUAL  Procedure: Dilute 70ml of milk in 250ml of water in a 600ml beaker and warm it to 40 . C . Then add 10%acetic acid with constant stirring to obtain all the casein precipitate. Allow the beaker to standundisturbed for 5minutes. Filter the precipitate in the bukner funnel with help of watmann filter  paper and wash successively thrice in 5ml of water, 20ml of ethanol, 10ml of ether to remove allthe fat. Dry the wet solid in a vacuum desicator and weigh the dry powder. Test the solubility of casein with water, benzene, 5% hydrochloric acid and 5%NaOH. Result: 2BIOORGANIC CHEMISTRY LAB MANUAL  2.PREPARATION OF OLEIC ACID FROM OLIVE OILAim: To prepare oleic acid from commercially available olive oil. Principle:Extraction: Extraction with solvents is used as a method for separation of dissolved substances fromsolution. It can also be used for the separation of one constituent from a solid mixture as well asfor the removal of undesired soluble impurities form mixtures.The common solvents used for extraction in the lab are diethyl ether, benzene, petroleum ether,chloroform and carbon tetrachloride. A good solvent for extraction should satisfy two importantconditions,1). the substance extracted should be highly soluble in the solvent.2). after extraction the solvent should be easily separable from the solute.In the extraction of organic substance generally diethyl ether is used because organic substancesare generally soluble in ether. Oleic acid: Oleic acid is mono unsaturated fatty acid found in various animal and vegetable oils. It has theformula C 18 H 34 O 2 [CH 3 (CH 2 ) CH=CH (CH 2 ) 7 COOH]. Oleic acid also called as an octadecanoicacid. Its molecular weight is 231.38. The unsaturated form of this acid is stearic acid. Oleic acidmakes up to 55 to 80% of olive oils, though there may be only 0.5 to 2.5% are actual free acidand 15 to 20% of grape seed oil. It is tasteless, colorless oily fluid at a temperature of 44 . C. it isinsoluble in water but soluble in alcohol, ethanol and ether. Apparatus required:  Beaker   Separating funnel  China dish  Stirrer   Stand Chemicals required:  Hydrochloric acid 3BIOORGANIC CHEMISTRY LAB MANUAL   Diethyl ether   KOH  Lead acetate  Olive oil Procedure: To isolate oleic acid add 30ml of 10% KOH to the given olive oil and mix well. To the soapformation add 30ml of lead acetate. Insoluble lead salts of free fatty acids are obtained. It isseparated using separating funnel. The net salts are treated with ether, in which only lead oleatedissolves leaving behind insoluble salts. The solution of lead oleate is treated with dil.HCl toliberate oleic acid as an oily layer. This is separated, dehydrated with anhydrous calcium chlorideand cooled to -7.2 . C. the pure crystals of oleic cid formed are tested for solubility. Result: 4BIOORGANIC CHEMISTRY LAB MANUAL
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