EXTRACTION PROTOCOLS

Mrs. Pooja H. Khanpara

APIP, Jmnagar

Extraction

Extraction may be defined as the treatment of the plant or

animal tissues with solvent, whereby the medicinally active

constituents are dissolved, and most of the inert matter remains

undissolved.

The solvent used for extraction is known as Menstruum and the

inert insoluble material that remains after extraction is called

Marc

The extract thus obtained, after standardization, may be used as medicinal agent •as such in the form of tinctures •fluid extracts •further processed to be incorporated in any dosage form such as tablets and capsules

Choice of solvents

• Successful determination of biologically active compounds depends on the type of solvent used in the extraction procedure.

• The choice of solvent is influenced by what is intended with the extract.

Properties of a good solvent in plant extractions

low toxicity, ease of evaporation at low heat,promotion of rapid physiologic absorption of

the extract, preservative action, inability to cause the extract to complex or

dissociate.

Solvents used for active component extraction

Steps Involved in the Extraction of

Medicinal Plants

1. Size reduction2. Extraction3. Filtration4. Concentration5. Drying

1. Size ReductionObjective:•To rupture plant organ, tissue & cell structures so that its medicinal ingredients are exposed to the extraction solvent.•Size reduction maximizes the surface area, which in turn enhances the mass transfer of active principle from plant material to the solvent.The 30-40 mesh size is optimal.Hammer mill or a disc pulverizer which has built in sieves controlled by varying the speed of the rotor clearance b/w the hammers & the lining of the grinder.

parameters influencing the quality of an extract

• Plant part used as starting material • Solvent used for extraction • Extraction procedure

Selection of plant

• Plant based natural constituents can be derived from any part of the plant like bark, leaves, flowers, roots, fruits, seeds, etc.

• Plants are usually air dried to a constant weight before extraction.

• oven drying: every part were cut into pieces dried in an oven @ 60°C for 9 hrs. & pulverized.

• Other method for drying the plants is the oven drying at about 40°C for 72 h.

Filtration• The extract so obtained is separated out from the

marc (exhausted plant material) by allowing it to trickle into a holding tank through the built-in false bottom of the extractor, which is covered with a filter cloth.

• The marc is retained at the false bottom, and the extract is received in the holding tank.

• From the holding tank, the extract is pumped into a sparkler filter to remove fine or colloidal particles from the extract.

Concentration• The enriched extract from percolators or extractors, known as

miscella, is fed into a wiped film evaporator where it is concentrated under vacuum to produce a thick concentrated extract.

• The concentrated extract is further fed into a vacuum chamber dryer to produce a solid mass free from solvent.

• The solvent recovered from the wiped film evaporator and vacuum chamber dryer is recycled back to the percolator or extractor for the next batch of plant material.

• The solid mass thus obtained is pulverized and used directly for the

desired pharmaceutical formulations or further processed for isolation of its phytoconstituents.

Drying• The filtered extract is subjected to spray drying

with a high pressure pump at a controlled feed rate and temperature to get dry powder.

• The desired particle size of the product is obtained by controlling the inside temperature of the chamber and by varying the pressure of the pump.

• The dry powder is mixed with suitable diluents or excipients and blended in a double cone mixer to obtain a homogeneous powder that can be straight away used (for example, for filling in capsules or making tablets).

Variation in extraction methods

• Length of the extraction period, • Solvent used, • pH of the solvent, • Temperature, • Particle size of the plant tissues,• The solvent-to-sample ratio.

The general techniques of medicinal plant extraction

maceration, infusion, percolation, digestion, decoction, hot continuous extraction (Soxhlet), aqueous-alcoholic extraction by fermentation, counter-current extraction, microwave-assisted extraction, ultrasound extraction (sonication), supercritical fluid extraction, phytonic extraction (with hydrofluorocarbon

solvents).

Extraction techniques For aromatic plants

Hydrodistillation techniques (water distillation, steam distillation, water and steam distillation),

Hydrolytic maceration followed by distillation, expression and enfleurage (cold fat extraction)

Headspace trapping, Solid phase micro-extraction, Protoplast extraction, Microdistillation, Thermomicrodistillation, Molecular distillation.

Maceration• The whole / coarsely powdered crude drug is placed in a

stoppered container with the solvent.• Allow to stand @ room temperature for a period of at least 3

days (& NMT 7 days) with frequent agitation until the soluble matter gets dissolved.

• The mixture then is strained, the marc (the damp solid material) is pressed,

• The combined liquids are clarified by filtration or decantation after standing.

• This method is best suitable for use in case of the thermo labile drugs.

• This process is modified to multiple • stage extraction to increase yield • of the active ingredient.

Infusion

• Fresh infusions are prepared by macerating the crude drug for a short period of time with cold or boiling water.

• These are dilute solutions of the readily soluble constituents of crude drugs.

Digestion

• This is a form of maceration in which gentle heat is used during the process of extraction.

• It is used when moderately elevated temperature is not objectionable.

• The solvent efficiency of the menstruum is thereby increased.

Image=microwave Digestion system

Decoction• In this process, the crude drug is boiled in a

specified volume of water (1;4) for a defined time,• Volume is reduced to 1/4th the original,• It is then cooled and strained / filtered.• This procedure is suitable for extracting water-

soluble, heat-stable constituents.• Typically used in preparation of Ayurvedic extracts

= “quath” / “kawath”

Percolation• It is continuous flow of solvent through the bed of crude drug to

get extract.• Used most frequently to extract active ingredients in the

preparation of tinctures and fluid extracts.• The solid ingredients are moistened with an appropriate amount

of the specified menstruum,• Allowed to stand for approximately 4 hours (minimum15 min) in

a well closed container, After stand time, the mass is packed & the top of the percolator is closed.

• the mixture is allowed to macerate in the closed V shaped percolator for 24 h.

• It yields greater conc. then maceration process.

,• Additional menstruum is added as required, until the

percolate measures about three-quarters of the required volume of the finished product.

• The marc is then pressed and the expressed liquid is added to the percolate.

• Sufficient menstruum is added to produce the required volume.

• The mixed liquid is clarified by filtration or by standing followed by decanting.

Hot Continuous Extraction (Soxhlet)

• The finely ground crude drug is placed in a porous bag or “ thimble” made of strong filter paper, which is placed in chamber of the Soxhlet apparatus.

• The extracting solvent in flask is heated, and its vapors condense in condenser.

• The condensed extractant drips into the thimble containing the crude drug & extracts it by contact.

Soxhlet apparatus• When the level of liquid in chamber rises to the

top of siphon tube, the liquid contents of chamber siphon into flask

• This process is continuous and is carried out until a drop of solvent from the siphon tube does not leave residue when evaporated.

Aqueous Alcoholic Extraction by Fermentation

• Some medicinal preparations of Ayurveda (asava & arista) adopt the technique of fermentation for extracting the active principles.

• The extraction procedure involves soaking the crude drug, [powder / a decoction (kasaya)], for a specified period of time

• Undergoes fermentation & generates alcohol in situ.• This facilitates the extraction of the active

constituents contained in the plant material.• The alcohol thus generated also serves as a preservative.

,• Some examples of Ayurvedic preparations:karpurasava , kanakasava , dasmularista .

• If the fermentation is to be carried out in an earthen vessel, it should not be new: water should first be boiled in the vessel.

• In large-scale manufacture, wooden vats, porcelain jars or metal vessels are used in place of earthen

vessels.

Counter-current Extraction• Wet raw material is pulverized using toothed disc disintegrators to

produce a fine slurry.• Material to be extracted is moved in one direction generally in the

form of a fine slurry within a cylindrical extractor where it comes in contact with extraction solvent.

• The further the starting material moves, the more concentrated the extract becomes.

• Complete extraction is thus possible when the quantities of solvent & material. Their flow rates should be optimized.

• sufficiently concentrated extract comes out at one end of the extractor while the marc, practically free of visible

solvent falls out from the other end.

Ultrasound Extraction (Sonication)

• The procedure involves the use of ultrasound with frequencies ranging from 20 kHz to 2000 kHz.

• This increases the permeability of cell walls & produces cavitation.Eg: extraction of rauwolfia root.• Deleterious effect: Ultrasound energy (>20 kHz) on

the active constituents of medicinal plants through formation of free radicals and consequently undesirable changes in the drug molecules.

The critical point represents the highest temperature and pressure at which the substance can exist as a vapour and liquid in equilibrium. The phenomenon can be easily explained with reference to the phase diagram for pure carbon dioxide

Supercritical Fluid Extraction

Supercritical Fluid Extraction• Cylindrical extraction vessels are used.• Certain gases behave like free flowing liquids at critical point of

temp. & pressure, which have high penetration power & extraction efficiency.

• This is first used in food packing industry for deodorization of packed food. It is sterile and bacteriostatic, nonexplosive, harmless to env. No waste product found.

• In this CO2 as the extracting fluid.(73.83 bar & 31.06°C)

• The component recovery rates generally increase with increasing pressure/temperature.

• The highest recovery rates in case of argon,@ 500 atm & 150° C.

Advantage & disadvantage of SFE• Advantage:

1. Higher diffusion rate, Lower viscosities, Higher vapour pressure than liquid solvents.

2. Higher densities compared to gases, higher solvating power.

3. Solubility & selectivity can be modified.

4. Low polarity of CO2 can modified cosolvents.5. Suitable for heat sensitive compound.Disadvantage:

1. Low polarity, so cant extract polar compound

2. Presence of water may cause problems

3. Equipment are so expensive.

SEPARATION AND ISOLATION OF CONSTITUENTS

The instrumentation for the structure for the structure elucidation of organic compounds becomes effective and allows the use of increasingly.

The most difficult operation in phytopharmacetical research is the isolation and purification of plant constituents.

The physical methods used are chromatographic techniques and methods such as fractional crystallisation, fractional distillation, fractional liberation.

Chemical method is based on groups or moieties present in the compound and chemical reactions.

FRACTIONAL CRYSTALLISATION

It is an important method for the purification of compounds from mixture.

It depends upon the compound which form crystals at the point of super saturation in the solvent in which it is soluble

Many natural products are crystaline nature even in mixture, process such as concentration, slow evaporation, refrigeration are using for crystalisation

FRACTIONAL DISTILLATION

This method is used for the separation of the components from volatile mixturesLargely using in the separation of hydrocarbons from oxygenated volatile oil eg citral, eucalyptol

FRACTIONAL LIBERATION

In this process the groups of compounds having the tendency of precipitation from the solution.Incertain cases the compounds may modified by converting to its salt form.This proces is often used in separation of cinchona alkaloids, morphine etc.

SUBLIMATION

Here the compound is heated the solid state changes to gaseous state without passing via liquid state. Such compounds get deposited in form of crystals or cake.

This method is traditionally used for the separation of camphor from chips of cinnamomum camphora.

CHROMATOGRAPY

Chromatography is widely used for the separation & identification of components of a mixture.

Separation of chemical compounds is carried out by mobile phase and stationary phase.

Chromatography can be classified according to mechanism of separation as: adsorption chromatography, partition chromatography, ion exchange chromatography, size exclusion chromatography and affinity chromatography.

PAPER CHROMATOGRAPHY

The principle is partition Mainly the stationary phase is moisture present in the cellulose fibers and mobile vary as we using. The components separated based on their solubility

The ratio between the distance travelled on the paper by a component of the test solution & the distance travelled by the solvent is termed the RF value. Under standard conditions, this is a constant for the particular compound.

In practise, however, variations of the RF value often occur & it is best to run a reference compound alongside the unknown mixtures.

ADVANTAGES

i. Simple & inexpensiveii. Sensitive – gives good separation of very

small amounts, of especially water-soluble compounds, e.g. sugars.

DISADVANTAGES

i. Fragile – chromatogram may be destroyed by chemicals used for visualization

ii. May be time-consuming.

THIN LAYER CHROMATOGRAPHY (TLC)

TLC is an e.g. of adsorption chromatography, the stationary phase being a thin layer adsorbent held on a suitable backing. Separation of the compounds present in the plant extract depends on the differences in their adsorptive/desorptive behaviour in respect of the stationary phase.

TLC involves a thin layer of adsorbent, mixed with a binder such as silica gel, which is spread on a glass plate & allowed to dry.The plant mixture to be separated is applied as a spot near the base of the plate, which is then placed in a closed glass tank containing a layer of developing solvent.

ADVANTAGES OF TLC OVER PAPER CHROMATOGRAPHY

Separation of compounds can be achieved more rapidly & with less plant material.-The separated spots are more compact & clearly demarcated from one another-Reagents such as concentrated H2SO4 would destroy a paper chromatogram, but ma be used to locate the separated substances on a TLC plate.

COLUMN CHROMATOGRAPHY

It is a method used to purify individual chemical compounds from mixtures of compounds the principle of separation is adsorption.

The classical preparative chromatography column, is a glass tube with a diameter from 5 mm to 50 mm and a height of 5 cm to 1 m with a tap and some kind of a filter (a glass frit or glass wool plug – to prevent the loss of the stationary phase) at the bottom.

GAS CHROMATOGRAPHY (GC)It is an analytical technique for separating compounds based primarily on their volatilities. (nitrogen, helium or argon) GC provides both qualitative and quantitative information for individual compounds present in a sample. Compounds move through a GC column as gases, either because the compounds are normally gases or they can be heated and vaporized into a gaseous state. The differential partitioning into the stationary phase allows the compounds to be separated in time and space.

High-performance liquid chromatography (HPLC)

High performance liquid chromatography is a powerful tool in analysis. This page looks at how it is carried out and shows how it uses the same principles as in thin layer chromatography and column chromatography.

Application of HPLC

1- Isolation and purification of biologically active natural products2- Control of synthetic reactions Identification of intermediates and target compound.3- Biosynthesis study Detection of biogenetic intermediates and enzymes involved.4-Control the microbiological processUsed for separation of antibiotic from broth mixture5- Pharmacokinetics study Pharmacokinetic study comprises the measurement of drug metabolites concentration in body fluids, absorption, bioavailability and elimination of drugsHPLC determines the drug and its metabolites in one step.6- Stability test Rapid method of analysis in stability test.7- Quality control HPLC is used to know the identity, purity and content of the ingredients (drugs, raw and pharmaceutical products, 8- Drugs metabolisms

9- Purification refers to the process of separation or extraction the target compound from other compounds or contaminants10- Quantification of compounds by HPLC Quantitative (assay) and qualitative determination of natural products11- Is the process of determination of the unknown concentration of a compound in a known solution.12- Identification of compound by HPLC through :- Comparison of retention time with authentic- Comparison of UV spectrum of the compound with that of the authentic.- Comparison of the Mass spectrum with that of the authentic.

Qualitative Reactions For The Detection Of Plant Constituents

Test for alkaloids

1. Dragendorff’s test1 ml of extract, add 1 ml of Dragendroff’s reagent (potassium bismuth iodide solution). An orange-red precipitate indicates the presence of alkaloids.

2.Mayer’s test1 ml of extract, add 1 ml of Mayer’s reagent (potassium mercuric iodide solution). Whitish or cream colored precipitate indicates the presence of alkaloids.

3.Hager’s test1 ml of extract, add 3 ml of Hager’s reagent (saturated aqueous solution of picric acid). Yellow colored precipitate indicates the presence of alkaloids

4. Wagner’s test1 ml of extract, add 2 ml of Wagner’s reagent (iodine in potassium iodide). Reddish brown colored precipitate indicates the presence of alkaloids

Test for glycosides

Bontrager's test In this test boil test sample with 1ml of sluphuric acid in a test tube for 5min,filter while hot. Cool the filterate and shake with equal volume of dichloromethane or chloroform then seperate the lower layer of chloroform and shake it with half volume of dilute ammonia. A rose pink to red colour is produced in the ammonical layer.

Modified Borntragor’s Test:

To 1 gm of drug add 5 ml dilute HCl followed by 5 ml ferricChloride (5% w/v). Boil for 10 minutes on water bath, cool and filter, filtrate was extracted withcarbon tetrachloride or benzene and add equal volume of ammonia solution, formation of pink tored colour due to presence of anthraquinone moiety. This is used C-type of anthraquinoneglycosides

Chemical tests for steroid and triterpenoid glycosidesLibermann Bruchard test:Alcoholic extract of drug was evaporated to dryness and extracted with CHCl 3 , add few drops of acetic anhydride followed by conc. H2SO4 from sidewall of test tube to the CHCl3extract. Formation of violet to blue coloured ring at the junction of two liquid, indicate the presence of steroid moiety.

Salkovaski test:Alcoholic extract of drug was evaporated to dryness and extracted withCHCl3, add conc. H2SO4 from sidewall of test tube to the CHCl3 extract. Formation of yellow colored ring at the junction of two liquid, which turns red after 2 minutes, indicate the presence of steroid moiety

Chemical tests for cardiac glycosides Keller Killiani test:To the extract of drug equal volume of water and 0.5 ml of strong lead acetate solution was added, shaked and filtered. Filtrate was extracted with equal volume of chloroform. Chloroform extract was evaporated to dryness and residue was dissolved in 3 ml of glacial acetic acid followed by addition of few drops of FeCl3 solution. The resultant solution was transferred to a testube containing 2 ml of conc. H2SO4. Reddish brown layer is formed, which turns bluish green after standing due to presence of digitoxose.

Legal test:Treat the test solution with 2ml of pyridine and sodium nitropruside 2 ml was added followed by addition of NaOH solution to make alkaline. Formation of pink colour in presence of glycosides or aglycon moiety.

Baljet test:Treat the test solution with picric acid or sodium picrate solution, it forms yellow to orange colour in presence of aglycones or glycosides

Goldbeater’s skin test: Goldbeater’s skin is a membrane produced from the intestine of Ox. It behaves just like untanned animal hide. A piece of goldbeaters skin previously soaked in 2% hydrochloric acid and washed with distilled water is placed in a solution of tannin for 5 minutes. It is then washed with distilled water and transferred to 1 % ferrous sulphate solution. A change of the color of the goldbeater’s skin to brown or black indicates the presence of tannin. Hydrolysable and condensed tannins both give the positive goldbeater’s test while pseudo tannins show very little color or negative test.

Tests for tannins

Phenazone Test: To 5 ml of aqueous solution of tannin containing drug, add 0.5 g of sodium acid phosphate. Warm the solution, cool and filter. Add 2 % phenazone solution to the filtrate. All tannins are precipitated as bulky, colored precipitate. Gelatin Test: To a 1 % gelatin solution, add little 10 % sodium chloride. If a 1 % solution of tannin is added to the gelatin solution, tannins cause precipitation of gelatin from solution.

Tests for flavonoids:

Shinoda Test: To the test solution, and few drops of conc. HCl. To this solution 0.5 g of magnesium turnings were added. Observance of pink coloration indicated the presence of flavonoids.

With Lead Acetate: To the small quantity of test solution lead acetate solution was added. Formation of yellow precipitate showed the presence of flavonoid.

With Sodium Hydroxide: On addition of an increasing amount of sodium hydroxide, the ethanolic extract showed yellow coloration, this decolorized after addition of acid.

Tests of proteinBiuret test

On adding 1% copper sulphite to alkaline solution (4% NaOH solution) of protein, a violet colour is developed. This test is due to the presence of peptide linkage.

Xanthoproteic Test:To 5ml test solution add 1ml of Con.HNO3 and boil, yellow ppt is formed. On addition of NH4OH, yellow ppt. turned orange.

Nihydrin test When protein is boiled with a dilute solution of ninhydrin, a violet colour is produced.

Aromatic Plant Extracts

• essential oils,• concretes,• absolutes, • pomades• resinoids.

Essential oils

• Used in a wide variety of consumer goods viz. , detergents, soaps, toilet products, cosmetics, pharmaceuticals, perfumes, confectionery food products, soft drinks, distilled alcoholic beverages (hard drinks) and insecticides.

• Production technology is an essential element to improve the overall yield & quality of essential oi l.

Presence of Essential Oils in plant parts.

Hetreogenous chemical group present in essential oils

The traditional methods for essential oil extraction

• Water distillation,• Water and steam distillation,• Steam distillation, • Cohobation, • Maceration,• Enfleurage.

methods

1. Distillation: Originally introduced by Von Rechenberg.•water distillation.•water and steam distillation.•direct steam distillation.2. Hydrolytic maceration distillation. 3. Expression.4. Cold fat extraction / Enfleurage.

• Distillation methods are good for powdered almonds, rose petals and rose blossoms.

• Maceration is adaptable when oil yield from distillation is poor.

• Solvent extraction is suitable for expensive, delicate & thermally unstable materials like jasmine, tuberose, and hyacinth.

• Water distillation is the most favored method of production of citronella oil from plant material.

1. Headspace trapping techniques- Static headspace technique- Vacuum headspace technique-Dynamic headspace technique2. Solid phase micro-extraction (SPME)3. Supercritical fluid extraction (SFE)4. Phytosol (phytol) extraction5. Protoplast technique6. Simultaneous distillation extraction (SDE)7. Microwave distillation8. Controlled instantaneous decomposition (CID)9. Thermomicrodistillation10. Microdistillation11. Molecular spinning band distillation12. Membrane extraction

Modern (Non-traditional) Methods of Extraction

Concrete• This is an extract of fresh flowers, herbs, leaves and the

flowering tops of plants obtained by the use of a hydrocarbon solvent such as butane, pentane, hexane and petroleum ether.

• Concrete is rich in hydrocarbon soluble material & devoid of water-soluble components.

• It is generally a waxy, semisolid, dark-colored material free from the original solvent.

• concretes are produced in static extractors.• It is a normal practice to circulate fresh solvent through a battery of extractors. • The enriched solvent from the extractor is pumped into an

evaporator for solvent recovery & the solvent content is reduced to about 1/10th the original volume.

Absolutes• To make an absolute, the concrete is mixed with absolute alcohol

& agitated thoroughly in a vessel with an agitator.• During agitation, the temperature is kept at 40°-60° C and the

concrete is immersed in the solution.• The solution is cooled down to -5° to -10° C to precipitate out the

wax, since waxes are normally insoluble in alcohol below -1° C. • The precipitated wax is removed by passing the solution through a

rotary filter.• The filtrate from the rotary filter is pumped into a primary

evaporator, where it is concentrated to about 10% alcohol content. • Finally, the concentrated extract is pumped into an agitating-type evaporator, where the alcohol is carefully removed under high vacuum.

Resinoids

• Resinoid is an extract of naturally resinous material, made with a hydrocarbon solvent.

• Resinoids are usually obtained from dry materials.• The extraction process is same as that of concrete

production, except that perforated discs are not used for stacking the material;

• instead powder from dry plant material is fed into the extractor.

Pomades• Pomades are obtained by a process known as enfleurage, which is a cold fat extraction method.• The fat is spread out on glass plates contained in wooden

frames, leaving a clear margin near the edges. • The absorptive surface of the fat is increased by surface

grooves made with a wooden spatula.• Fresh flowers are spread out on the surface of the fat and the

frames are stacked in piles.• After the perfume oils have been absorbed from the flowers,

the spent flowers are removed by hand.• Fresh flowers are again spread on the fat surface.• This is repeated until the fat surface is completely enriched

with perfume oils.• The pomade so obtained is ready for cold alcoholic extraction

major constraints in sustainable industrial exploitation

• Poor agricultural practices for MAPs,• Unscientific and indiscriminate gathering practices from the wild, poor

postharvest & post-gathering practices leading to poor quality raw material,

• Lack of research for the development of high-yielding varieties of medicinal and aromatic plants-MAP’s.

• Poor propagation methods.• Inefficient processing techniques.• Poor quality control procedures.• Lack of research on process & product development.• Difficulty in marketing.• Non-availability of trained personnel.• Lack of facilities & tools to fabricate equipment locally.• Finally lack of access to the latest technologies & market information.

Conclusion • The process of extracting MAP’s determines how

efficiently we add value to MAP bio-resources.

• In the case of essential oils, the extraction process affects the physical as well as internal composition.

• Variations in the chemical constituents of the extracts of medicinal plants may result by using non-standardized procedures of extraction.

• Efforts should be made to produce batches with quality as consistent as possible.

References

S. S. Handa. 2008. An Overview of Extraction Techniques for Medicinal and Aromatic Plants.(© United Nations Industrial Development Organization and the International Centre for Science and High Technology, 2008).

Prashant Tiwari, Bimlesh Kumar, Mandeep Kaur, Gurpreet Kaur, Harleen Kaur. 2011. Phytochemical screening and Extraction: A Review. INTERNATIONALE PHARMACEUTICA SCIENCIA.

Google images.

Atropine (tropane alkaloid)• Present in Atropa belladona, Datura stramonium,

Hyosymus niger, Hyoscymus muticus (Egyptian)

• Family: Solanaceae• M.P.: 115 – 116° C

• PROPERTIES: • Atropine is optically inactive levorotatory isomer of

Hyoscyamine.• It consist of colorless needle like crystals or white crystalline,

colorless, odorless powder.• It is a strong poison with a sharp bitter taste.• It is Sublimes in high vaccum at 93°C to 110°C.• Sparingly soluble in water but freely dissolve in alcohol,

benzene & dil. acids.• USE:• Due to its dilating action on pupils of the eye it is used in

Ophthalmology.• Atropine: water (1:1,30,000) ratio use to cause dilation of the

pupils of cat’s eye.• When taken internally, 1st stimulate & than depresses the CNS.

• ISOLATION:• It is isolated from juice or powdered drug of Hyoscymus muticus

(Egyptian) Powder drug

Moistened with ↓ aq. Sol. Of Na2CO3

extracted with ↓ ether or benzene Free bases are found

extracted with ↓ water & acidified with acetic acid

shaken with ↓ ether to remove coloring matter Alkaloids are precipitated with Na2CO3, filtered off, washed & dried.

Dissolve in ether or acetone & ↓ dehydrated with anhydrous sulphate

filtration ↓ Concentration & cooling Crude crystals of hyoscymine & atropine

dissolved in ↓ alcohol & mixed with NaOH Allow to stand till hyoscymine is completely racemised to atropine crude atropine purified by crystallization from acetone.

• Identification:• Vitali-Morin Test:

• Dilute solution of atropine treated with conc. HNO3 this mixture evaporated to dryness on steam bath produce pale yellow residue.

• This residue gives violate color when a drop of freshly prepared KOH is added.

• TLC of Atropine:• 1 % sol. Of atropine prepared in 2N acetic Acid is spotted

on plate & eluted in solvent system of strong ammonia solution: methanol (1.5:100).

• Than plate spraying with iodoplatinate solution shows Rf value 0.18.

• Atropine sulphate showes Rf value 0.70, in the solvent system acetone: 0.5 M NaCl spraying with dragendorff’s reagent.