EXTRATION OF CAFFEINE FROM TEA LEAVES

SYNONYM                           :Camellia thea

BIOLOGICAL SOURCE     : It consists of prepared leaves and leaf buds of ‘Thea sinensis belonging

                                              to family ‘Theaceae’.

CHEMICAL CONSTITUENTS:

*Tea leaves contains rich source of cfaffine (1-3%),Thea-1bromine and theophylline in major

   quantites.

*The colour of leaves is due to gallatanic acid.

*The aggreable colour is due to presence of yellow volatile oils.

*Tea leaves also contain enzymatic mixture ‘Thease’.

STRUCTURE:

CHEMICAL NAME:

          1,3,7 Trimethyl xanthine

USES:

          *Act as CNS Stimulant

          *Act as diuretic

IDENTIFICATION TESTS:

         

S.NO	EXPERIMENT	OBSERVATION	INFERENCE
1.	MUREXIDE TEST:To few mg of caffine,3-4 drops of potassium nitrate was added in a small porcelin dish.it was then evaporated to dryness.To the obtained residue,2 drops of Ammonium hydroxide.	Purple colour was observed.	Presence of caffine was confirmed.
2.	MAYER’S TEST: To few mg of caffeine mayer’s reagent was added.	No precipitate was observed.	Presence of caffeine was observed.
3.	To the test sample containing 0.2% of caffeine in chloroform was taken in procelein dish.It  was then evapourated to dryness to the residue,5 drops of 30% w/v hydrogen peroxide aqueoussolution and 5 drops of 1M hcl was added.Heated over dryness.To the residue 1 drop of ammonia solution was added.	No colour change from yellow-red to red violet.	Presence of caffeine was confirmed.

REQURIMENTS:

          *Beakers

          *PH PAPER

          *Glass rod

          *Tripod stand

          *Standard flask

          *Funnel

CHEMICALS:

          *Teadust

          *10% sulphuric acid

          *Chloroform or Dichloromethane

          *Activated charcoal

PROCEDURE:

          50gms of tea dust was taken and mixed with a pintch of sodium bicarbonate.

          To it 800ml of water was added and mixture was heated on water for 15 minutes.

          The solution was filtered and to the filtrate,lead acetate solution was added drop by drop till precipitate ceases and again the solution was filtered.

          To the filtrate dilute sulphuric acid was added till precipitation has occurred.

          To obtained filtrate 1gm of activated charcoal was added.

          The clear filtrate and then evaporated.To obtain filtrate crystalline needles of caffeine.

          To the product was air dried and percentage yield was calculated on dry weight basis.

REFERENCE:

          The approximate yield of silky needles of caffeine is 1gm and should be 1%.

REPORT:

          1.Caffiene was extracted from tea dust and yield was found to be 0.025 gms=25mg.

          2.Various chemical tests of caffeine were performed and reported.

INTRODUCTION FOR EXTRATION

DEFINITION:

                        Extration may be defined as the process in which animal or plant tissue are treated with specific solvents by using extraction process medically active constituents are dissolved out.

METHOD OF EXTRATION:

                   For extraction process there are various method which include the following.

Extraction with organic solvents:

*Percolation

*Maceration

*Extraction using soxhlet apparatus

Extraction with water

          *Infusion

          *Decoction

          *Digestion

          *Steam distillation

Other methods include

          *Super critical fluid extraction and phytonocs

          *Spouted extraction

          *Vertical or turbo extraction

          *Ultra sound extraction

          *Counter current extraction

          *Successive solvent extraction

EXTRATION WITH ORGANIC SOLVENTS:

PERCOLATION:

          Exhaustive extraction or percolation is defined as complete removal of desired extractive substances from the drug substances (material)

GENERAL METHOD FOR EXTRACTING BOTANICALS:

          Moisten 1000gms of powdered botanical with sufficient quantity of prescribed menstrum to render it evenly and distinctly damp

Macerate for 6hrs in light covered containers(this will enable the plant cells to absorb the menstrum)

                                                       

Then pack in cylindrical percolator

Add enough menstrum to saturate powder and leave stratum above it.

When the liquid begins to drop from percolator close. The lower surface cover the percolator and macerate for prescribed time(48hrs)

Then open (hofmann clamp) valve and allow the percolation to proceed slowly.

Collect the preserve first 850ml

Continue percolation by gradually adding more menstrum over the herb until botanical is exhausted.

Recover the alchol from the reminder of percolate and concentrate to soft extract in a vaccum apparatus at a temperature exceed 450 c

Dissolve this residue in the reserved portion of percolate,mix thoroughly add a sufficient quantity of menstrum to make fluid extract measure 1000ml (k)and filter.

MACERATION:

          Maceration is defined as soaking in botanical in suitable solvent for a specified period of time any where from several hours to 3 weeks until solute portions are dissolved in menstrum or maceration is a process of extraction of drug with a solvent with several daily shaking or stirrings at room temperature.

PROCEDURE:

          Solid ingredients+750ml of perscrised solvent.

Allow to stand for a period of atleast one week in warm place with free quent agitation for proper dissolution.

          Filter the mixture.

         

          Residue wash with sufficient quantity of solvent.

          Combine filtrates make upto 1000ml.

ADVANTAGES:

*Small samples are prepared in exactly the same way as technical and production holders.

*highly versatile.

*Drugs passing through smelling(strong)properly or high mucous content can be processed.

HOT CONTINOUS EXTRATION SOXELATION:

          Convinent way to thimble in central plant extract eith pure solvent.

+

PROCEDURE:

          Material is placed in thimble in central compartment with sulphoning device and side arm connected to lower compartment.

          Solvent placed in central compartment

          Solvent is heated to boiling

          Vapour passing through side arm into reflux condenser

          Vapour gets condensed and liquefied and drops into thimble containing the material to be extracted

          Warm solvent percolates through the material and wall of thimble

          The extract is gradually collected in central compartment

          On reaching the top of siphon the entire liquid in central compartment flows through this and back into lower solvent container

          Process in thus repeated.

APPLICATIONS:

          Useful for exhaustive extraction of plant with a particular solvent

                   Eg: defalting or where 100% yield of a particular component is desired.

          Also useful for exhaustive sequential extraction with series of solvents of increased

polarity is desired.

Eg:Hexane,chloroform,and water.

DISADVANTAGES:

Due to continous recycling the extract that collects in lower container is continously being heated that may suffer thermal degradation reaction.

If the operation is carried out on a large scale it may not be suitable for use with solvents with relatively high boiling points such as methanol or water,since the whole apparatus below the condenser needs to be at this temperature for effective movement of solvent vapour.this method is limited for pure solvents or azeotropic mixture.

EXTRATION WITH WATER

INFUSION:

          It is a dilute solution of readily soluble constituents of crude drugs.

PROCEDURE:

          Moisten 60gms of herb cut coarsely ground in 1lit distilled water.

         

          Allow to stand for 15min and then add the balance of litre of boiling water.

          Cover the vessel tightly and allow standing approximately for 30minutes.

          Strain the mixture and pass water to make infusion mixture 1 litre.

          Filter refrigerate or preserve.

DISADVANTAGES:

          Difficult to standardize and are unstable and particularly susceptible to attack by various drugs and bacteria.

2.DECOCTION:

          This process extracts vegetable substances or substances that contain water soluble constituents from crude drugs by boiling in water for 15min.coating straining and passing sufficient cold water through drug to produce required volume.

PROCEDURE:

          Cut the powdered herb(60gms) and place in suitable vessel.

          1lit of cold water preferably distilled is placed over it.

          Cover the vessel well and boil the mixture for 15min.

          Allow to cool and then strain through muslin cloth and pass enough cold water through strainer to make the product measure it.

          Filter,refrigerate or preserve.

3.DIGESTION:

          This is form of maceratin in which gentle heat is used during process of extraction.

ADVANTAGE:

          It is used when moderately elevated temperature is not objectionable and the solvent efficiency of menstrum increased there by.

4.STEAM DISTILLATION,EXPRESSION.EXTRATION AND ENZYMATIC HYDROLYSIS:

METHOD FOR VOLATILE OILS:

METHOD FOR STEAM DISTILLATION AS FOLLOWS:

          *Water distillation

          *water and steam distillation

          *Direct steam distillation

WATER DISTILLATION:

          It is mostly applicable to such material which is dried initially in air and the constituents are not degraded by boiling upto 100Oc 

                   Eg:Turpentine oil

WATER AND STEAM DISTILLATION:

          It is often suitable for such plant material,whether fresh or dried the constituents of which degradation by direct boiling.

          Eg:clove oil,cinnamon oil

DIRECT STEAM DISTILLATION:

          It is invariably applicable to fresh drug that is loaded with sufficient natural moisture and hence no maceration is required.

          Eg:pipperment oil,spearmint oil.

ADVANTAGES:

*Relatively simple equipment.

*No separate filtration step is required to separate,extrated oil from plant material.

DISADVANTAGES:

          Cannot be used where the oil contains hydrolysable substances such as Esters (or) those that are easily oxidized (or) decomposed by heat.

EXPRESSION:

          Expression may be accomplished by four following methods.They are as follows.

                   1. Spooge method.

                   2. Scarification method.

                   3. Rasping process.

                   4. Mechanical process.

EXTRACTION OF VOLATILE OIL: It involves

          *Extraction with volatile solvents.

          *Extration with non-volatile solvents.

EXTRATION WITH VOLATILE SOLVENTS:

          Eg:Hexane,benzene

ADVANTAGES:

          Maintaining an uniform temperature during extraction process ensures retention of more intenses and natural fragnance.

          Flueal concretes-represent an admixture of natural odoriferous components of flowers ,plant waxes,colour pigments and certain albuminous material.

EXTRATION WITH NON-VOLATILE SOLVENTS:

          Three methods adopted for extraction they are as follows

          *Enfleuraga method

          *Pnematic method

          *Maceration method

OTHER METHODS

1)SUPER CRITICAL FLUID EXTRACTION:

          It is process of separating one component from another using super critical fluids as extracting solvent.

ADVANTAGES:

          *Environmental improvement and reduce product contamination.

          *Selectively

          *Speed

DISADVANTAGES:

          *Use of high sophisticated equipment

          *High pressure and high temperature is required.

2)SPOUTED BED EXTRATION:

          This is a process meant for physical removal of pigment layer which gives a better product than thah obtained by solvent extraction.

          Eg:production of annatto powder from seeds of Bixaorellana

3.VORTICO OR TURBO EXTRACTOR:

          A method adopted to reduce the length of time involved is simple maceratin process.

          Eg: cinchona bark.

DISADVANTAGES:

          *Temperature may rise due to high speed stirring.

          *Equilibrium separation of drug residues from miscelles is difficult.

4.ULTRA SOUND EXTRACTION:

          It is defined as frequencies above 20,000 HZ waves used to accelerate extraction.

          Eg:play a major role in decomposition of alkaloid’s in Jaborandi observed after 30s ultra sound treatment on laboratory scale at 20 HZ.

EXTRATION BY ELECTRICAL ENERGY:

          Eg:Extration of scopolamine from seeds and throns of thorn apple.

5.COUNTER-CURRENT METHOD(EXTRACTION):

          It is a process in which plant material is in a contact with charged solvent at same time as fresh solvent is being brought contact in with pre extracted drug.

APPLICATION:

          Alkaloids,Amino acids,Antibiotics,Phenolic including anthroquinone derivates,cardiac glycosides.PGS.Steriods,F.A,essential oils and vitamins.

6.SUCCESSIVE SOLVENT EXTRACTION:

          *This is meant for air dried material.

          *Based on increasing order of polarity.

Limit test for Arsenic

Official compounds

It constitute an important section of pharmacopoeia which gives all necessary information of drugs and compounds that are official. The information includes the title, subtitle, molecular formula, molecular weight , test for identification , tests for purity , description and solubility , method of assay , category and dose it applicable

Assay

The quantitative estimation of the amount of drug present in a given sample constitutes as assay. The percentage purity of the sample is determine by caring out its assay

Limit test

These are qualitative or semi- quantitative test designed to detect and limit small quantities of impurities that are commonly present in pharmaceutical substance. They are based on the comparison or opalescence, turbid ty or colour produced due to impurity in asample with that produced in a standard under t he same test conditions

Ex

Limi t test for CCl, SO4, Fe2 , Heavy metals, lead and arsenic 

Limit test for arsenic

The limit test for arsenic is provided to demonstrate that the content of arsenic does not exceed the limit given in the individual monograph in terms of micrograms of arsenic per gram of the test substance.

To carry out the limit test for arsenic a solution is prepared from the test substance by a procedure specified in the monograph.

This procedure assures that the solution in every case contains the whole of the arsenic (if any) present in the substance.

The standard stain against which the comparison is made contains 10 μg of As.

The procedure described may also be used to determine the amount of arsenic in the substance by matching the depth of color of the stain with a series of standard stains.

In the statements of arsenic limits, the permitted amount of arsenic is expressed as As.

Limit test for Arsenic

Principle

The principle is based on converting any arsenic impurity present in the sample to arsine gas by a series of reaction. The arsine gas is made to come in contact with mercuric chloride test paper when by it produces a yellow or brown s train due to the formation of mercuric arsenic

The stain produce by the sample is compare to a standard stain produced by standard strain

The sample stain should not be darker than standard stain

Reactions

1. The arsenic impurity is converted in acidic medium into arsenious acid or arsenic acid depending upon the valency state of arsenic

As 3+  --------------------------   As (OH)3  or H3 AsO3

Trivalent                                    Arsenious acid

As 5+  --------------------------   O  = As (OH)3  or H3 AsO4

Pentavalent                                    Arsenic acid

2. Any arsenic acid formed is converted int arsenious acid by reduction with stannous chloride and hydro chloric acid

                           Stanous chloride

H3 ASO4    ------------------------------------  H3ASO3

3. The arsenic acid is further reduced to arsine gas with the help of nacent hydrogen obtained in the reaction between zinc and hydrochloride acid

                                 Reduction

H3ASO3 +6H  ---------------------------------------- ASH3  +3H2O

4. Arsenic gas react with mercuric choride test paper to produce yellow to brown stain due to formation  of mercuric arsenide

2ASH3 + HGCl2   -----------------------------------   Hg (As H2)2   + 2HCl 

Apparatus

It consist of  which mouthed bottle of 120ml capacity fitted with a rubber bung.

Through the rubber bung insert a glass tube as a internal diameter of 6.0 mm and external diameter of 8.0 mm

It has a total length of 200 mm , is drawn out at one end to a diameter of about 1 mm, and has a hole not less than 2 mm in diameter blown in the side of the tube, near the constricted part.

The mercuric chloride test paper is placed between the bund and clipped together with a clip

Procedure

The glass tube is packed with lead acetate cotton

Sample Stain

The solution of the sample is placed in the wide mouthed bottle.

10 ml stagnated HCl ,  and 10 grms of zinc is added to the sample solution.

The reaction is allowed to proceed for 40 minutes at the temperature of 40c

Standard stains

Solutions are prepared by adding to 50 ml of water, 10 ml of stannated hydrochloride acid AsT and quantities of dilute arsenics solution AsT varying 0.2 ml to 1 ml. The resulting solutions , when treated as described in the general test yield stains on the mercuric chloride paper referred to as the standard stains

Stannated hydrochloride acid AsT

Stannous chloride solution AsT     -      1ml

Hydrochloric acid AsT                  -       100 ml

Comparison and results

The intensity of sample stain is immediately compared to the that of the standard stain. For the sample to pass the limit test for arsenic , the intensity of sample stain should not be greater than of the standard stain 

Basis of test

Both in the sample and the the standard is done by the combined action of Zinc, acid , stannous chloride and potassium iodide.

The pharmacopoeial test  of arsenic which is a toxic component in medicinal substances is based on the fact that arsenic in the arsenious state can be readily reduced to arsine (As H3) gas which passing over mercuric chloride paper develops a yellow to brown stain

The intensity and length of which are proportional to the amount of arsenic

The solution is reacted with a reducing agent like stannous chloride or sulphurous acid to convert the penta valent arsenic acid into the trivalent arsenious acid which is converted into gaseous arseniou  hydride (arsine gas) with the help of nascent hydrogen produced by the action of zinc with hydrochloric acid

HCl

Arsenic  to arsenious or arsenic acid in presence of acidic media

Stannous chloride

This solution is reacted with a reducing agent like stannous chloride to convert the pentavalent arsenic acid into the trivalent arsenious acid

Zinc with hydrochloric acid

Nascent hydrogen produced by the action of zinc with hydrochloric acid

Trivalent arsenious acid which is converted into gaseous arsenious hydride (arsine gas) with the help of nascent hydrogen produced by the action of zinc with hydrochloric acid

                         Sn Cl2                           Zn + HCl   

H3 AsO4   -------------------  H3 AsO3 -----------------  AsH3   +  3H2O

Arsenic acid                     Arsenious acid                     Arsine gas

Arsine gas is carried out through the tube with the help of hydrogen to the mercuric chloride paper

The reaction of arsine with mercuric chloride produces a yellow colored stain

The intensity of the color is dependent on the quantity of arsenic

2AsH3  + HgCl2 ------------------------  Hg (AsH2)2   +2HCl

                                                             Yellow stain 

The most suitable temperature for carrying out the test is generally abou 40c

The tube must be washed with HCl , rinsed with water and dried between successive tests

The stains may be preserved by dipping in hot melted paraffin or placing over phosphorus pentoxide protected from light

Stannous chloride is required for the complete evolution fo arsine

The acion between pure zinc and hydrochloric acid is slow and he presence of other metals accelerates the formation of hydrogen . Stannous salts reduce arsenic to arsenious state

Hydrogen sulphide

Metallic zinc may contain traces of sulphiide which on  reaction with the acid yields hydrogen sulphide

The lead acetate papers are used to trap any hydrogen sulphide evolved together with arsine.

The hydrogen sulphide reacts with mercuric chloride paper developing a dark stain , thus interfering with the formation of the required mercuric arsenide stain

Apparatus  

A suitable type of apparatus is described below, though other acceptable constructions are available.

A wide-mouthed bottle of about 120 ml capacity, is fitted with a rubber bung through which passes a glass tube.

The tube is passed through the bung fitting the bottle so that, when inserted in the bottle containing 70 ml of liquid, the constricted end of the tube is above the surface of the liquid and the hole in the side is below the bottom of the bung.

The upper end of the tube is cut off square, and is either slightly rounded off or ground smooth.

Two rubber bungs (about 25 mm × 25 mm), each with a hole bored centrally and true and exactly 6.5 mm in diameter, are fitted with a rubber band or spring clip for holding them tightly together.

Alternatively, the two bungs may be replaced by any suitable construction satisfying the conditions of the test, as described below.

Recommended procedure

Pack the glass tube lightly with cotton-wool, previously moistened with lead acetate (80 g/l) TS and dried, so that the upper surface of the cotton-wool is not less than 25 mm below the top of the tube.

Insert the upper end of the tube into the narrow end of one of the pair of rubber bungs, either (1) to a depth of about 10 mm in the case of the tube with the rounded-off end or (2) so that the ground end of the tube is flush with the larger end of the bung. Place a piece of mercuric bromide paper AsR flat on the top of the bung, and place the other bung over it. Secure the assembly by means of a rubber band or spring clip, in such a manner that the borings of the two bungs (or the boring of the upper bung and the glass tube) meet to form a true tube 6.5 mm in diameter interrupted by a diaphragm of mercuric bromide paper AsR.

Instead of this method of attaching the mercuric bromide paper AsR, any other method may be used provided (1) that the whole of the evolved gas passes through the paper, (2) that the portion of the paper in contact with the gas is a circle 6.5 mm in diameter, and (3) that the paper is protected from sunlight during the test.

Place the solution, prepared as specified in the monograph, in the wide-mouthed bottle, add 1 g of potassium iodide AsR and 10 g of granulated zinc AsR, and place the prepared glass tube assembly quickly into position. Allow the reaction to proceed for 40 minutes. Compare any yellow stain that is produced on the mercuric bromide paper AsR, with a standard stain, produced in a similar manner with a known quantity of dilute arsenic AsTS. Make the comparison in daylight and immediately after simultaneous preparation of the test and standard stains; the stains fade on keeping.

The most suitable temperature for carrying out the test is generally about 40°C The temperature may be adjusted to obtain a regular

The reaction may be accelerated by placing the apparatus on a warm surface, care being taken to ensure that the mercuric bromide paper AsR remains quite dry throughout the test.

Between successive tests, the tube must be washed with hydrochloric acid (~250 g/l) AsTS, rinsed with water, and dried.