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

1. ISOLA ISOLATI TION ON OF OF CASE CASEIN IN FRO FROM M MILK 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 unit operation serves to concentrate and fractionate the target product from various contaminants. Protein precipitate formation occurs in a stepwise process. The addition o f a precipitating agent and 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 the solvent and thus lower the solubility of the solute by add ition of a reagent. Casein:

It is a predominant phospho protein that accounts for nearly 80% of protein in milk and an d cheese. When co-agulated with rennet, casein is sometimes called a paracasein. It consists of a fairly high number of proline peptides which do not interact. There are also no disulphide bridges. As a result it has relatively little secondary and tertiary structures and it cannot denature. It is hydrophobic 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 dilute alkali 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.

1 BIOORG BIOORGANI ANIC C CHEM CHEMIST ISTRY RY LA LAB B MAN MANUAL UAL

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 stand undisturbed 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 all the 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:


2. PREPAR PREPARATIO ATION N OF OLEIC OLEIC ACID ACID FROM FROM OLIVE OLIVE OIL OIL Aim:

To prepare oleic acid from commercially available olive oil. Principle: Extraction:

Extraction with solvents is used as a method for separation of dissolved substances from solution. It can also be used for the separation of one constituent from a solid mixture as well as for 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 important conditions, 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 substances are generally soluble in ether. Oleic acid:

Oleic acid is mono unsaturated fatty acid found in various animal and vegetable oils. It has the formula C18H34O2 [CH3 (CH2) CH=CH (CH2)7COOH]. Oleic acid also called as an octadecanoic acid. Its molecular weight is 231.38. The unsaturated form of this acid is stearic acid. Oleic acid makes up to 55 to 80% of olive oils, though there may be only 0.5 to 2.5% are actual free acid and 15 to 20% of grape seed oil. It is tasteless, colorless oily fluid at a temperature of 4 4.C. it is insoluble in water but soluble in alcohol, ethanol and ether. Apparatus required: 



Beaker Separating funnel China dish Stirrer Stand



Hydrochloric acid

  

Chemicals required:




  

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 soap formation add 30ml of lead acetate. Insoluble lead salts of free fatty acids are obtained. It is separated using separating funnel. The net n et salts are treated with ether, in which only lead oleate dissolves leaving behind insoluble salts. The solution of lead oleate is treated with dil.HCl to liberate oleic acid as an oily layer. This is separated, dehydrated with anhydrous calcium chloride and cooled to -7.2.C. the pure crystals of oleic cid formed are tested for solubility.

Result:


3. SYNT SYNTHE HESI SIS S OF ASP ASPIR IRIN IN Aim:

To synthesis aspirin from salicylic acid by acetylation method. Principle: Acetylation:

Acetylation (or in IUPAC nomenclature ethanoylation) describes a reaction that introduces an acetyl functional group into an organic compound. It is that, process of introducing a n acetyl group (resulting in an acetyl group) in to a compound specifically, the substitution of an acetyl group for an active hydrogen atom. A reaction involving the replacement of the hydrogen atom of a hydroxyl group with an acetyl group g roup (CH3CO) yields a specific ester, the acetate. COOH

COOH

OH

Salicylic acid

+ CH3-COOCO-CH3 ------→ Acetic anhydride

OCOCH3

+ CH3 COOH

Aspirin

Aspirin:

Aspirin or acetyl salicylic acid is a salicylate drug, often used as an analgesic to relive minor aches and pains, as an antipyretic to reduce fever and as an anti inflammatory medication. It also has an antiplatelet or anti clotting effect and is used in long term, low doses to prevent heart attacks, strokes and blood clot formation in peop le at high risk for developing blood clots. The main undesirable side effects of aspirin are gastrointestinal ulcers and stomach bleeding and tinnitus, especially in higher doses. Aspirin was the first discovered member of class of drugs known as non-steroidal anti inflammatory drugs (NSAIDS). (NSAIDS). The synthesis of aspirin is classified classified as esterification reaction, where the alcohol group from the salicylic acid reacts acetyl anhydride to form an ester. Aspirin is commercially synthesized using a 2 step process. 1. Phenol treated with sodium base which generates sodium phenolate, which is then reacted with CO2 under high temperature and pressure to yield salicylate, which is acidified, yielding salicylic acid. This process is known as Kolbe- schmitt reaction.


2. Salicylic Salicylic acid acid is then acetylated acetylated using using acetic anhydrid anhydride, e, yielding yielding aspirin aspirin and acetic acid acid as a by product. Apparatus required:        

Conical flask Beaker Stirrer Funnel Spatula Watch glass Filter paper Hot water bath.

Chemicals required:    

Acetic anhydride Salicylic acid Con. H2SO4. FeCl3

Procedure:

Dissolve 7.5gm of salicylic acid in 11.5gm of freshly distilled acetic anhydride in a conical flask. Add 4-5 drops of Conc.H2SO4 and stir the contents thoroughly. Heat the flask between 50 – 60.C for 30 minutes. Transfer the contents of the conical flask to a beaker containing co ntaining ice cold water. Stir the contents in the beaker. Filter the solids, dry it and weigh.

Result:


4. HYDRO HYDROLY LYSIS SIS OF SUCR SUCROS OSE E Aim:

The objective of the experiment is to determine the condition suitable for hydrolysis of disaccharide to list the functional group and to prepare its derivatives. Principle:

Hydrolysis of sucrose is catalyzed by H+ ions. C12H22O11+H2O

-----→ C6H12O6+C6H12O6.

H+ ions act as a catalyst. The reaction is therefore b imolecular, but it is found to be of o f ist order, which may be explained in the following way,

Water is in excess, its molar concentration concentration remains unchanged. Thus, the rate of reaction is determined only by sucrose concentration. Hence the reaction is of Ist order. Such reactions are referred as pseudo u nimolecular reaction. Procedure:

Prepare 100ml of 0.1M sucrose solution. Take 4 test tubes and name it 1,2,3,4 respectively. Fill the sample to 3/4th of each test tube. Add 5 drops of Conc.HCl to the test tube 2, 3 and 4. Let the test tube 2 to be at room temperature and heat the other test tubes in hot water bath for the following period of time. Test tube 1 = 15 minutes. Test tube 3 = 2.5 minutes Test tube 4 = 5 minutes As each test tube is removed from water bath it is cooled to room temperature. It is tested for functional groups. Test for functional groups: 1. Molisch Molisch’s ’s test: test: ( genera generall test for for carboh carbohydr ydrate) ate) 7 BIOORG BIOORGANI ANIC C CHEM CHEMIST ISTRY RY LA LAB B MAN MANUAL UAL

Take 1 ml of sugar solution; add 3-4 drops of α-napthol in ethanol and 1.5ml of Con.H2SO4 along the sides of test tube violet v iolet and red color is formed at the interface of 2 liquids. The solution on shaking attains a dark violet color.

2. Be Bene nedi dict ct’s ’s test test::

It is the most convenient test. Thus it is done for identification of reducing sugar and nonno nreducing sugar. Procedure and observation :

To 2 ml of Benedict’s reagent add 0.2ml of sugar solution and boil. The mixture is allowed to cool. A red precipitate indicates the presence of reducing sugar and red solution indicates presence of non-reducing sugar.

3. Seliwan Seliwanoff’ off’ss test: test: ( Test for Fructos Fructose) e)

It is used to distinguish aldose and ketose in which ketose undergoes dehydration to give further derivatives which condense with resorcinol to form red color. One way to differentiate ketoses and aldoses is to look for the functional group attached to it. When a ketone is attached to it. When a ketone is attached to it, it is a ketose. However if an aldehyde is attached to it, it is an aldose. The dehydrated ketose then reacts with resorcinol to produce a deep cherry red color. Aldoses may react slightly to produce faint pink colour. Procedure and observation:


Take 2 ml of aqueous solution of a sugar add 2ml of seliwanoff’s reagent and warm it in boiling water for one minute. A deep red color appears followed by a precipitate. 4. Barfoed’s test:

It is a chemical test used for detecting the presence of a monosaccharide. It is based b ased on the reduction of copper (II) oxide to copper copp er (I) oxide to form brick red color. Procedure and observation:

Take 2ml of barfoed’s reagent add 1ml of sugar solution in it. Heat the test tube in a briskly boiling water bath.

Result:



5. ISOLATI ISOLATION ON OF OF LYCOPE LYCOPENE NE FROM FROM TOMA TOMATO TO PASTE PASTE Aim:

To isolate lycopene from tomato paste.

Principle: Solvent extraction:

Extraction with solvents is used as a method for separation of dissolved substances from solution. It can also be used for the separation of one constituent from a solid mixture as well as for 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 important conditions, 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 substances are generally soluble in ether. Lycopene:

Lycopene is a bright red carotenoid pigment (of phytochemical) found in tomatoes and other red fruits, lycopene is the most common carotenoid in hu man body and it is one of the most potent ant-oxidant. The color of lycopene is due to its many conjugated carbon bonds. Each double bond may reduce the energy ene rgy required for electrons transition to higher energy states allowing the molecule to absorb visible light of progressively longer wavelength. Lycopene absorbs most of the visible spectrum. So it appears red. 11 BIOORG BIOORGANI ANIC C CHEM CHEMIST ISTRY RY LA LAB B MAN MANUAL UAL

Apparatus required: 

Conical flask



Beaker



Thermometer



Heating mantle



Stirrer



Shaker



Water bath.



Sodium chloride



Sodium sulphate



Ethanol



Diethyl ether

Chemicals required:

Procedure:

1. 5gm of tomato tomato paste paste was was weighed weighed and placed placed in a 100ml round round bottom bottom flask. flask. 2. 10ml of ethanol ethanol was added added to it and heated heated on water water bath for for 5 minutes. minutes. 3. This hot mixtur mixturee was filtered filtered using using small small funnel funnel and flask flask was allowed allowed to drain drain thoroughly. 4. The liquid liquid out out of the solid solid residu residuee was squeezed squeezed with with spatula. spatula. 5. The solid solid residue residue was returned returned to to flask and and 10ml of diethyl diethyl ether ether was was added and reflex reflex for for 3-4 minutes. 6. The yellow yellow extract extract was filtere filtered d and the extractio extraction n was repeated repeated two to to four times times with with 10ml of diethyl ether. 7. The combined combined extract extract was was taken and and poured poured into the the separating separating funnel funnel 10% 10% NaCl solution was added for layer separation. 12 BIOORG BIOORGANI ANIC C CHEM CHEMIST ISTRY RY LA LAB B MAN MANUAL UAL

8. On adding NaCl solution yellow colour layer is obtained. On passing the color solution over sodium sulphate lycopene obtained.

Result:


6. PREPARATION OF α-D-GLUCOPYRANOSE PENTA ACETATE

Aim:

To prepare α-D-Glucopyranose penta acetate (or) 1,2,3,4,6- penta-o-acetyl α-D-Glucopyranose in the lab.

Background:

Pyranose is a collective term for carbohydrates which have a chemical structure that includes a six membered ring consisting of five carbons and one oxygen. The pyranose ring is formed by the reaction of C-5 alcohol group of a sugar with its C-1 aldehyde forming an intramolecular hemiacetal.

Materials required: 

Conical flask



Measuring cylinder



Water bath



Spatula



Anhydrous zinc chloride



Acetic anhydride



Powdered glucose

Reagents required:


Procedure:

0.5gm of anhydrous zinc chloride is taken in a 250ml conical flask along with 11.5ml acetic anhydride and shaked well and placed in a water bath for 10 minutes. About 2.5gm of α-DGlucose is added very slowly and the mixture is shak en slowly; during the addition to control vigorous reaction. Finally the flask is heated for one hour in a boiling water bath. The contents are poured into 70-100ml of ice cold water and stirred vigorously. The product obtained is filtered.

Result:

7. PREPARATION OF 5,10,15,20- TETRA PHENYL PORPHYRIN

Aim:

To prepare 5, 10, 15, 1 5, 20-tetraphenyl porphyrin using pyrrol and benzaldehyde.

Principle:


Porphyrins bind metals to form complexes. The metal ion usually with a charge of 2+ or 3+, resides in central N4 cavity formed by the loss of 2 protons. Most metals can be inserted. A porphyrin in which no metal is inserted in its cavity is sometimes called a free base. Some iron containing porphrins are called hemes and heme containing proteins or hemoproteins is found extensively in nature.

Chemicals required: 

Propionic acid



Pyrrol



Benzaldehyde



Ethanol



Round bottom flask



Condenser.

Apparatus required:

Procedure:

Take 10ml of propionic acid in 250ml round bottomed flask. Add some boiling stones. Fit it the reflex condenser and reflux the acid. Add 1ml of pyrrol and 2 ml of benzaldehyde to the propionic acid through the condenser. Continue to heat under refluxing condition for 30 minutes. Cool to room temperature and collect co llect the deeply colored product by suction filtration. Wash the product thoroughly with ethanol until ethanol washings are colorless. Dry the product under suction for few minutes.

Result:


8. PREPARATION OF 5,10,15,20- TETRA PHENYL PORPHYRIN COPPER(II) ACETATE

Aim:

To prepare 5,10,15,20- tetraphenyl porphrin copper (II)acetate complex.

Principle:

Porphyrins bind metals to form complexes. The metal ion usually with a charge of 2+ or 3+, resides in central N4 cavity formed by the loss of 2 protons. Most metals can be inserted. A porphyrin in which no metal is inserted in its cavity is sometimes called a free base. Some iron containing porphrins are called hemes and heme containing proteins or hemoproteins are found extensively in nature.


Dimethyl fluoramide



TPP



Copper (II) acetate



Round bottom flask



Condenser.

Apparatus required:


Procedure:

Take 10ml of dimethyl fluoramide (DMF) in a 100ml beak er and few boiling stones and heat it on a hot plate. When the solvent begins to boil add 100mg of TPP and allows it to dissolves. Add 40mg Copper (II) acetate and continue co ntinue to heat till 5 minutes after the boiling bo iling point of the solid. Cool the beaker in an ice bar for about 15 minutes and an d dilute the mixture with 100ml of distilled water. Collect the solid product by suction filtration. Wash the product well with water and dry by suction.

Result:

9. PREPARATION PREPARATION OF 1,2,5,6 1,2,5,6 DI-O-CYCL DI-O-CYCLO O HEXYLIDIN HEXYLIDINE E α-D-GLUCO α-D-GLUCOPYRANO PYRANOSE SE

Aim:

To prepare crystals of 1,2,5,6 di-O-cyclo hexylidine α-D-Glucopyranose

Chemicals Required: 

Cyclo hexanose



Conc. H2SO4




Dry α-D-Glucose



Round bottomed flask



Cooling bath with an intimate mixture of ice and salt.



Shaker

Apparatus required:

Procedure:

Take a round bottomed flask. Immerse it in a large plastic or metal container filled with freezing mixture. Add 10.6 ml of cyclohexanose to the flask and cool it at 0.C. Add 1 ml of Conc.H2SO4 using separating funnel into the vigorously stirred cyclo hexanone. Final solution should be light straw color. Add slowly with vigorously stirring 4.5 gm of finely powdered dry -D-Glucose. Remove the cooling bath and allow the reaction mixture to reach ambient temperature continuously stirring it. Put the flask in the shaker for 8 hours so that there is a continuous mechanical stirring. The reaction mixture becomes progressively more viscous and finally sets into the solid between crystalline mass on cooling. Now keep it in room temperature for sometime. Keep the conical flask in a slant position so that the solvent separates from the solid mass. Remove the solvent; take the solid mass in a filter paper and dry it well to get crystals of 1,2,5,6 di-o-cyclo hexylidine α-D-Glucopyranose.

Result:


10. PREPARATION PREPARATION OF PYRUVIC PYRUVIC ACID FROM TARTARIC ACID Aim:

To prepare pyruvic acid using tartaric acid Principle:

Tartaric acid is heated with potassium hydrogen sulphate at 210 to220.C to produced pyruvic acid


Tartaric acid



Potassium hydrogen sulphate (freshly fused)



Glass mortar



Round bottomed flask



Leibeig condenser

Apparatus required:

Procedure:

Take 20gm of powdered tartaric acid and 30gm of freshly fused potassium hydrogen sulphate in a glass mortar and grind well to form an intimate mixture. Place the mixture in a 250ml round bottomed flask and connect the leibeig condenser; heat the flask in an oil bath maintained at 210.C to 220.C until no liquid distills over. Fractionate the distills under reduced pressure and collect the pyruvic acid at 78.85.C 20 BIOORG BIOORGANI ANIC C CHEM CHEMIST ISTRY RY LA LAB B MAN MANUAL UAL

Result:


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

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