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 unit
operation serves to concentrate and fractionate the target product from various contaminants.
Protein precipitate formation occurs in a stepwise process. The addition of 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 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 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 BIOORGANIC 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 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 BIOORGANIC CHEMISTRY LAB MANUAL

 2. PREPARATION OF OLEIC ACID FROM OLIVE 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 44.C. it is
insoluble in water but soluble in alcohol, ethanol and ether.

Apparatus required:

 Beaker
 Separating funnel
 China dish
 Stirrer
 Stand
Chemicals required:

 Hydrochloric acid

3 BIOORGANIC 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 soap
formation add 30ml of lead acetate. Insoluble lead salts of free fatty acids are obtained. It is
separated using separating funnel. The net 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:

4 BIOORGANIC CHEMISTRY LAB MANUAL

 3. SYNTHESIS OF ASPIRIN

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 an 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 (CH3CO) yields a specific ester, the acetate.

COOH COOH

OH + CH3-COOCO-CH3 ------→ OCOCH3 + CH3 COOH

Salicylic acid Acetic anhydride 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 people 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). The synthesis of aspirin is 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.

5 BIOORGANIC CHEMISTRY LAB MANUAL

 2. Salicylic acid is then acetylated using acetic anhydride, yielding aspirin and acetic 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 ice cold water.
Stir the contents in the beaker. Filter the solids, dry it and weigh.

Result:

6 BIOORGANIC CHEMISTRY LAB MANUAL

 4. HYDROLYSIS OF SUCROSE

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 bimolecular, but it is found to be of ist order,
which may be explained in the following way,

Water is in excess, its molar 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 unimolecular 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’s test: ( general test for carbohydrate)

7 BIOORGANIC CHEMISTRY LAB MANUAL

 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 and red color is formed at the interface of 2
liquids. The solution on shaking attains a dark violet color.

2. Benedict’s test:

It is the most convenient test. Thus it is done for identification of reducing sugar and non-
reducing 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. Seliwanoff’s test: ( Test for Fructose)

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:

8 BIOORGANIC CHEMISTRY LAB MANUAL

 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 on the
reduction of copper (II) oxide to copper (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:

9 BIOORGANIC CHEMISTRY LAB MANUAL

10 BIOORGANIC CHEMISTRY LAB MANUAL
 5. ISOLATION OF LYCOPENE FROM TOMATO 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 human 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 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 BIOORGANIC CHEMISTRY LAB MANUAL

Apparatus required:

 Conical flask

 Beaker

 Thermometer

 Heating mantle

 Stirrer

 Shaker

 Water bath.

Chemicals required:

 Sodium chloride

 Sodium sulphate

 Ethanol

 Diethyl ether

Procedure:

1. 5gm of tomato paste was weighed and placed in a 100ml round bottom flask.

2. 10ml of ethanol was added to it and heated on water bath for 5 minutes.

3. This hot mixture was filtered using small funnel and flask was allowed to drain
thoroughly.

4. The liquid out of the solid residue was squeezed with spatula.

5. The solid residue was returned to flask and 10ml of diethyl ether was added and reflex for
3-4 minutes.

6. The yellow extract was filtered and the extraction was repeated two to four times with
10ml of diethyl ether.

7. The combined extract was taken and poured into the separating funnel 10% NaCl
solution was added for layer separation.

12 BIOORGANIC CHEMISTRY LAB MANUAL

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

Result:

13 BIOORGANIC CHEMISTRY LAB MANUAL

 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

Reagents required:

 Anhydrous zinc chloride

 Acetic anhydride

 Powdered glucose

14 BIOORGANIC CHEMISTRY LAB MANUAL

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-D-
Glucose is added very slowly and the mixture is shaken 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, 20-tetraphenyl porphyrin using pyrrol and benzaldehyde.

Principle:

15 BIOORGANIC CHEMISTRY LAB MANUAL

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

Apparatus required:

 Round bottom flask

 Condenser.

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 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:

16 BIOORGANIC CHEMISTRY LAB MANUAL

 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.

Chemicals required:

 Dimethyl fluoramide

 TPP

 Copper (II) acetate

Apparatus required:

 Round bottom flask

 Condenser.

17 BIOORGANIC CHEMISTRY LAB MANUAL

Procedure:

Take 10ml of dimethyl fluoramide (DMF) in a 100ml beaker 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 to heat till 5 minutes after the boiling point of the solid.
Cool the beaker in an ice bar for about 15 minutes and 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 OF 1,2,5,6 DI-O-CYCLO HEXYLIDINE α-D-GLUCOPYRANOSE

Aim:

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

Chemicals Required:

 Cyclo hexanose

 Conc. H2SO4

18 BIOORGANIC CHEMISTRY LAB MANUAL

  Dry α-D-Glucose

Apparatus required:

 Round bottomed flask

 Cooling bath with an intimate mixture of ice and salt.

 Shaker

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:

19 BIOORGANIC CHEMISTRY LAB MANUAL

 10. PREPARATION OF 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

Chemicals required:

 Tartaric acid

 Potassium hydrogen sulphate (freshly fused)

Apparatus required:

 Glass mortar

 Round bottomed flask

 Leibeig condenser

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 BIOORGANIC CHEMISTRY LAB MANUAL

Result:

21 BIOORGANIC CHEMISTRY LAB MANUAL