WELCOME

GENERAL METHODS OF

ISOLATION AND SEPERATION

OF PLANT CONSTITUENTS

RAHUL B SM PHARM PART 1

CONTENTS

INTRODUCTION

EXTRACTION PROCESS * Types of extraction * Solvents used * Process of extraction * Types of extracts SEPERATION AND IDENTIFICATION OF PLANT CONTISTITUENTS * Fractional crystallization * Fractional distillation * TLC * Fraction liberation * Sublimation * Column chromatography * Counter - current extraction * Paper chromatography QUALITATIVE REACTIONS FOR THE DETECTION OF PLANT CONSTITUENTS APPLICATION OF GLCAPPLICATION OF HPLCCONCLUSIONREFERENCE

INDRODUCTION

• A natural product is a chemical compound or substance

produced by a living organism. They may be extracted from

tissues of terrestrial plants, marine organism or micro - organism

fermentation.

• In that respect any biological molecule is a natural product, but

in general the term is reserved for secondary metabolites

(carotinoids, phytosterines, saponines, phenolic compounds,

alkaloids, glycosinates, terpenes etc).

• The extracts from plant tissue are a rich source of lead

compounds for nutraceutical or pharmaceutical applications

Extraction

SteamDistillation

Pressing

Distillation PLANT MATERIAL

Methods for recovery of secondary metabolites.

EXTRACTION PROCESS

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 various process used for extraction are 1. Maceration

In this process, the whole or coarsely powdered crude drug is

placed in a stoppered container with the solvent and allowed

to stand at room temperature for a period of at least 3 days

with frequent agitation until the soluble matter has dissolved.

The mixture then is strained, the marc (the damp solid

material) is pressed, and the combined liquids are clarified by

filtration or decantation after standing.

2. 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.

3. 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.

4. Decoction

In this process, the crude drug is boiled in a specified volume of

water for a defined time; it is then cooled and strained or filtered.

This procedure is suitable for extracting water-soluble,

heatstable constituents. The starting ratio of crude drug to water

is fixed, e.g. 1:4 or 1:16; the volume is then brought down to

one-fourth its original volume by boiling during the extraction

procedure. Then, the concentrated extract is filtered and used as

such or processed further.

5. Percolation

Percolation is a continuous flow of the solvent through the bed

of the crude drug material to get the extract.

In this process, the powdered drug is moistened with an

appropriate amount of the specified menstruum and allowed to

stand for approximately 4 h in a wellclosed container, after

which the mass is packed and the top of the percolator is

closed.

Additional menstruum is added to form a shallow layer above

the mass, and the mixture is allowed to macerate in the closed

percolator for 24 h.

The outlet of the percolator then is opened and the liquid

contained therein is allowed to drip slowly.

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,

and the mixed liquid is clarified by filtration or by standing

followed by decanting.

MODIFIED PERCOLATIONThe conventional percolation process is modified to include evaporation for the production of more concentrated products, especially when the solvent is dilute alcohol In simple percolation Drug imbibition maceration percolation and collect the percolateIn conventional percolation Drug imbibition maceration percolation and collect the

1000 ml of percolate maceration percolation and collect the

1000 ml of percolatemaceration percolation and collect the

1000 ml of percolateThe process is continued in case the drug is not completely exhausted.

Hot Continuous Extraction (Soxhlet)In this method, the finely ground crude drug is placed in a porous bag or “thimble” made of strong filter paper, 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, and extracts it by contact.

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.

The advantage of this method, compared to previously described methods, is that large amounts of drug can be extracted with a much smaller quantity of solvent.

SOXHLET APPARATUS

Aqueous Alcoholic Extraction by Fermentation

It involves soaking the crude drug, in the form of either a

powder or a decoction for a specified period of time, during

which it undergoes fermentation and 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 such preparations are karpurasava,

kanakasava, dasmularista..

COUNTER-CURRENT EXTRACTION

In counter-current extraction (CCE), wet raw material is pulverized and produce a fine slurry.

Here the material to be extracted is moved in one direction 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 and material and their flow rates are optimized.

Finally, sufficiently concentrated extract comes out at one end of the extractor while the marc falls out from the other end.

Advantages

Smaller volume of solvent as compared to other methods like maceration, decoction, percolation.

CCE is commonly done at room temperature, which spares the thermolabile constituents from exposure to heat which is employed in most other techniques.

As the pulverization of the drug is done under wet conditions, the heat generated during comminution is neutralized by water. This again spares the thermo labile constituents from exposure to heat.

The extraction procedure has been rated to be more efficient and effective than continuous hot extraction.

Ultrasound Extraction (Sonication)

The procedure involves the use of ultrasound with requencies ranging from 20 kHz to 2000 kHz;this increases the permeability of cell walls and produces cavitation.

The process is useful in some cases, like extraction of rauwolfia root, its large-scale application is limited due to the higher costs.

Disadvantage

The deleterious effect of ultrasound energy (more than 20 kHz) on the active constituents of medicinal plants through formation of free radicals and consequently undesirable changes in the drug molecules.

Supercritical Fluid ExtractionThe 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

A super-critical fluid is a substance, mixture, or element, which under certain operative conditions of pressure and temperature, and mechanical operations, is above its critical point but below the pressure needed to condense it into a solid.

Extraction via super-critical fluids is better for the environment than conventional methods of extraction, because it uses gases such as CO 2 at high pressure, in a liquid or super-critical state, instead of chlorinated solvents which produce toxic waste.

carbon dioxide is the preferred fluid for SFE, They are powerful solvents and have a great capacity of penetration in solids, which allows a rapid and almost complete exhaustion of extractable solids.

They can easily be completely separated from extracts, simply by modifying pressure or temperature, up to the point

advantages:i) The extraction of constituents at low temperature, which strictly avoids damage from heat and some organic solvents.ii) No solvent residues.iii) Environmentally friendly extraction procedure.

The main drawback is the time of extraction, which is usually long. In fact, in some cases, it can take as much as 24 hours. With normal fluids, extraction can be speeded up by mechanical shaking, but this presents problems when using super-critical fluids, which limits industrial use.

Types of solventThe solvents used in extraction are capable of penetrating the tissues of the drug and dissolve the active principles contained in its cell.

The various solvents used are water, propene, butane, ethylacetate, ethanol, methanol, CO 2 , N 2 O, acetone etc.WaterIt is the cheap, non toxic, non inflammable and has wide solvent action. Eg; proteins, glycosides, enzymes, sugar etc.Disadvantages•Water may promote growth of mould and bacteria, hence requires a preservative.•It may leads to hydrolysis.•Large amount of heat is required to concentrate the aqueous preparations.•It promote fermentation or decomposition of the preparation

AlcoholIt is the important solvent for dissolves alkaloids, alkaloidal salts, glycosides etc. it also dissolves many colouring matter, tannins, etc. it doesn’t dissolve gums waxes, fats etc.

MeritsIt doesn’t allow the growth of mould and bacteria in above 20% of alcoholIt is nontoxic in the concentration mostly present in the preparations. Small amount of heat is requiered for concentration

DemeritsCostInflammable, volatile etcSolvents such as ether, chloroform, light petroleum are rarely used.

factors considered when selecting a solvent

• Solvent power (selectivity). Only the active, desired constituents should be extracted from the plant material, which means that a high selectivity is required.• Boiling temperature. The boiling point of the solvent is aslow as possible in order to facilitate removal of the solventfrom the product.• Reactivity. The solvent should not react chemically with theextract, nor should it readily decompose.• Viscosity. A low viscosity of the solvent leads to low pressuredrop and good heat and mass transfer.• Safety. The solvent should be non-fl ammable and non-corrosive, and should not present a toxic hazard; its disposal should not imperil the environment.• Cost. The solvent should be readily available at low cost.• Vapor pressure. To prevent loss of solvent by evaporation, alow vapor pressure at operating temperature is required.• Recovery. The solvent has to be separated easily from the

extract to produce a solvent-free extract.

Types of extract

Aqueous extractsThe medicinal preparations intended to be used immediately after preparation or to be preserved for use, solvent used is water. The methods used for their preparation are decoction, infusion, and digestion.

Hydro alcoholic or Alcoholic These are prepared by the methods of maceration and percolation eg tintures, here the solvent using is alcohol

Soft extractsThey are extracts with semisolid or syrup consistency Can be used in a variety of dosage form like ointments and suppositories Eg; glycerriza extracts

Dry extractsThey powdered extracts or dry powder Extract obtained from suitable process is filtered and get concentrated under vacuum, dried completely by spray or tray drying. Eg; belladona used in dossage forms such as capsules, tablets etc.

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 proces 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

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

iv. 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 CaSo4, 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. 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.

APPLICATIONS

Quality controlcontamination of plant and plant based products with pesticides, herbicides and many other materials that are considered a health risk, all such products on sale today must be carefully assayed

Identification of Source /OriginThe source of many plants (herbs and spices) can often be identified from the peak pattern of the chromatograms obtained.Technique of fingerprint could really identify the false herbal products.The fundamental reason of quality control of herbal medicines is based on the concept of phytoequivalence of herbs, and then to use this conception to identify the real herbal medicine and the false one, and further to do quality control.

Qualification and Quantification of PhytoconstituentsAlkaloids Capillary gas chromatography (GC), often coupled with a mass spectrometer as a detector (GC-MS), is a well established technique for analyzing complex mixtures of alkaloids.

Terpenes

A qualitative comparative study was performed for terpenes from volatile oils by GC and GC-MS technique

Flavanoids and Flavones

Flavonoids receive considerable attention in the literature, specifically because of their biological and hysiological importance. Gas Chromatography Coupled to Mass Spectrometry GC-MS is established as a routine technique for the analysis of flavonoid aglycones.

Essential Oils /Volatile oils

Many pharmacologically active components in herbal medicines are volatile chemical compounds. Thus, the analysis of volatile compounds by gas chromatography is very important in the analysis of herbal medicines

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.

CONCLUSION

Extraction, as the term is used pharmaceutically, involves theseparation of medicinally active portions of plant or animal tissues from the inactive or inert components by using selective solvents in standard extraction procedures.

The products so obtained from plants are relatively impure liquids, semisolids or powders intended only for oral or external use.

There are several techniques for the separation and identification of natural products. Selection of method is important in result.

Reference