General Methods for Extraction, Isolation and Identification of Herbal Drugs

Chapter 24

General Methods for Extraction, Isolation and Identification of Herbal Drugs

INTRODUCTION

• The crude drug contains the active constituents, which can be isolated from these drugs by various methods of extraction and separation. Extraction is defined as the process of isolation of soluble material from an insoluble residue, which may be liquid or solid, by treatment with a solvent on the basis of the physical nature of crude drug to be extracted, i.e., liquid or solid, the extraction process may be liquid—liquid or solid—liquid extraction.

• The process of extraction is controlled by mass transfer. Mass transfer is a unit operation, which involves the transfer of mass of soluble material from a solid to a fluid. If a crude drug panicle is immersed in a solvent to be used for extraction, the particle is first surrounded by a boundary layer of the solute; the solvent starts penetrating inside the particle and subsequently forms solution of the constituents within the cells. Escape of these dissolved constituents through the cell wall and through the boundary layer takes place. The process is continued till equilibrium is set up between the solution in the cells and the free solution. Few important factors, which affect the mass transfer are agitation and temperature that increase the concentration gradient to bring about an efficient extraction. Size reduction of the crude drug increases the area over which diffusion can occur. Overall extraction is also dependent on the selection of the method of extraction and the solvent selected for extraction.

EXTRACTION METHODS

• Majority of the small-scale extraction processes of maceration and percolation are generally slow and time consuming and also give the inefficient extraction of the crude drugs. These processes are generally modified for more efficient and faster extraction at the laboratory scale. Large-scale industrial batch operations demand some more modifications of extraction process where the small-scale directions are inappropriate.

Maceration

• Maceration process involves the separation of medicinally active portions of the crude drugs. It is based on the immersion of the crude drugs in a bulk of the solvent or menstruum. Solid drug material is taken in stoppered container as shown, with about 750 ml. of the menstruum and allowed to stand for at least three to seven days in a warm place with frequent shaking. The mixture of crude drug containing solvent is filtered until most of the liquid drains off. The filtrate and the washing are combined to produce 1,000 ml of the solution.

• Maceration method is modified to multiple stage extraction to increase the yield of the active ingredients in the extracts. The crude drug material is charged in the extractor, which is connected with a circulatory pump and spray distributor, along with number of connected tanks to receive the extraction solution. This is known as multiple stage extraction because the solvent added and circulated in the extractor containing drug is removed as extracted solution and is stored in the receiver tanks. This operation is repeated thrice. When the crude drug material is charged in the extractions, the stored solution is once again circulated through fresh drug and then removed as an extract. Likewise, after three extractions, the drug is removed from the extractor, again recharged with fresh drug and the whole cycle is repeated. 

Percolation

Modified percolation 

• The conventional percolation process is modified especially when the solvent is dilute alcohol. In cases when the strength of alcohol needs to be unaffected by concentration of the extract, percolation is continued, and the first quantity of the percolate is collected and set aside. 

• The subsequent quantities of the percolates are collected, concentrated and lastly, the first volume of the percolate is added in the final product. In this way it maintains the required alcohol strength and also produces the higher concentration of the products. The process is known as reserve percolate method.

• In modified process of percolation techniques, continuous or semicontinuous extraction devices are used in some industries for handling the batches of varying size. The extraction batteries which consist of a number of vessels in series are inter-connected through pipelines and are arranged in such a way that the solvent can be added, and the product removed from any vessel. Such type of extraction battery gives maximum efficiency of extraction with minimum use of solvent. The product obtained is more concentrated and less losses of solvent take place due to evaporation.

 Continuous Extraction

Soxhlet extraction 

• Soxhlet extraction is the process of continuous extraction in which the same solvent can be circulated through the extractor for several times. This process involves extraction followed by evaporation of the solvent. The vapours of the solvent are taken to a condenser and the condensed liquid is returned to the drug for continuous extraction.

• Soxhlet apparatus, designed for such continuous extraction, consists of a body of extractor attached with a side tube and siphon tube as shown. The extractor from the lower side can be attached to distillation flask and the mouth of the extractor is fixed to a condenser by the standard joints. The crude drug powder is packed in the Soxhlet apparatus directly or in a thimble of filter paper or fine muslin. The diameter of the thimble corresponds to the internal diameter of the Soxhlet extractor. Extraction assembly is set up by fixing condenser and a distillation flask. Initially for the setting of the powder, solvent is allowed to siphon once before heating. Fresh activated porcelain pieces are added to the flask to avoid bumping of the solvent. The vapours pass through the side tube and the condensed liquid gradually increases the level of liquid in the extractor and in the siphon tube. A siphon is set up as the liquid reaches the point of return and the contents of the extraction chamber are transferred to the flask. The cycle of solvent evaporation and siphoning back can be continued as many times as possible without changing the solvent so as to get efficient extraction. This method, although a continuous extraction process, is nothing but a series of short macerations.

• Similar methodology can be adopted in large-scale production in which the operation principles may resemble the laboratory equipment. Soxhlet extraction is advantageous in a way that less solvent is needed for yielding more concentrated products. The extraction can be continued until complete exhaustion of the drug. The main disadvantage is that this process is restricted to pure boiling solvents or to azeotropes. 

Large-scale extraction 

• As the large-scale extraction is meant for the extra large batches of drug material, the various assemblies which are generally in attachment with the body of soxhlet extractor are modified. The pilot plant extractor generally has a separate extractor and condenser unit. Separate inlet for loading the drug and an outlet for drug discharge are provided. The extractor body is divided into two parts: the upper one for drug material and the lower one as a distillation chamber. The distillation chamber is electrically heated. The vapours of the solvent are passed to condenser and the condensed liquid is sprayed on the bed of crude drug with the help of solvent distribution nozzle. Solution returns to the distillation chamber via solution return pipe. Such large-scale extractors are provided with the outlet from the lower side of the extractor, for removing the extract.

Supercritical fluid extraction 

• The supercritical fluid extraction is a comparatively recent method of extraction of crude drugs. Certain gases behave like a free flowing liquids or supercritical fluids at the critical point of temperature and pressure. Such supercritical fluids have a very high penetration powers and extraction efficiency. This principle was first used in the food packing industries for the deodorization of the packed food products. The gases like carbon dioxide are held as a supercritical fluid at the critical point of 73.83 bar pressure and 31.06°C temperature. At this critical point CO2 behaves as a liquefied gas or free-flowing liquid and assists the extraction of the phytochemical constituents from the crude drugs. The phase diagram of CO2 indicates the characteristic areas for the deodorization, extraction and fractionation.

• The advantages of CO2 in supercritical fluid extraction are that it is sterile and bacteriostatic. It is noncombustible and nonexplosive. CO2 is harmless to environment and no waste products are generated during the process, and it is available in large amount under favourable condition.

• The mixture to be fractionated is passed in the extraction column along the length of which the heater is located. CO2 is purged through the column. Once the extraction column is pressurized, drug material gets saturated in the supercritical fluid which moves along the length of the column. The operating conditions, i.e. pressure and temperature, are selected. In the pressure controlled type of extraction, the solution is just expanded in the separation stage to precipitate the extract and then again the gas is recompressed for recycle. In temperature control type operation, the extract is precipitated by heating the solution which lowers the solvent density. The density is then increased by isobaric cooling for recycling. Operation of supercritical fluid extraction system is controlled from a PC. PC is used to set the operating conditions like pressure, temperature and flow rate. PC is programmed to safely shut down the unit in case of overpressure or over temperature situations.

• Supercritical fluid extraction has many applications in pharmacognosy especially in the extraction and isolation of the active constituents. The process is successfully used in the decaffeination of coffee, extraction of pyrethrins and for the production of terpeneless oils. It has been successful in selectively extracting larger proportions of active ingredients than conventional methods of hydrodistillation or extraction, i.e. in cases of acorone from Acorus calamus, matricin and bisabolol from chamomile flowers, heat labile sesquiterpene hydrocarbons of Valerian and Nardostachys species. It is useful for removing the off odours or ‘still notes’ from the freshly distilled essential oil. The temperature pressure conditions for the extraction of certain constituents from crude drugs.

Advantages of SFE:

•  Higher diffusion rates than liquid solvents.

• Lower viscosities than liquid solvents.

• Higher vapour pressure than liquid solvents 

• Higher densities compared to gases, higher solvating power.

• Solubility and (to some extent) selectivity can be controlled by modification of parameters 

• Low polarity of carbon dioxide can be modified with cosolvents.

• Suitable for heat-sensitive compounds.

Disadvantages of SFE:

• Carbon dioxide, which is the most commonly used solvent, has low polarity and hence cannot extract polar compounds 

• Presence of water may cause problems

•  Unpredictability of matrix effect 

• Need for specialized/expensive equipment

TYPES OF EXTRACTS

• Numbers of different types of methods are used for the extraction of herbal drugs, and the extracts are used for different purposes ranging from internal administration, external use, for further purification of phytopharmaceuticals or for it semisynthetic conversion to some therapeutically more active compounds. The extracts are therefore prepared likewise to achieve the objectives for which it is prepared. Extracts can be in the form of aqueous, hydroalcoholic types in the form of infusion, decoction, tinctures, etc., or they can be more concentrated which may further be transformed into soft, dry or liquid extracts.

Aqueous Extracts

• These are the extracts which are medicinal preparations intended to be used immediately after preparation or to be preserved for use. The following methods are generally more in utility for their preparation.

Decoction: 

• This is the ancient and more popular process of extracting water soluble and heat stable constituents from crude drugs by boiling in water for about 15 min. The boiled crude drug—water mixture is then cooled; filtered and sufficient volume of cold water is passed through the drug to produce the required volume

Infusion: 

• An infusion is generally a dilute solution of the readily soluble constituents of crude drugs. It is nothing but a type of periodic maceration of the drug with either cold or boiling water. The infusion is filtered to remove the crude vegetable material and then produced in a required volume by addition of water.

Digestion: 

• Digestion is also a type of maceration in which moderate heating is preferred during extraction. Heating causes the digestion of drug material and increases the solvent efficiency. It is preferred for the drugs in which the use of moderately elevated temperature does not cause the degradation of constituents

Tinctures: 

• Tinctures are the alcoholic or hydroalcoholic solutions prepared from crude drugs or from the pure organic or inorganic substances. Tinctures of crude drugs may contain 10–20 g of drug per 100 ml of tincture. The methods used for the preparation of tinctures are: maceration and percolation. Iodine tincture is an example of inorganic pharmaceuticals, belladonna tincture is prepared by percolation while compound benzoin tincture, sweet orange peel tincture is prepared by maceration.

Liquid Extracts: 

• The liquid extracts are also termed as fluid extracts in some official books like USP. It is a liquid preparation of crude drugs which contain ethyl alcohol as a solvent and preservative. It may contain active constituents to the extent of 1 g of drug per ml. Pharmacopoeia liquid extracts are prepared by the percolation or modified percolation techniques.

Soft Extract: 

• The extracts which are produced as semisolid, or liquids of syrupy consistency are termed as soft extracts. These extracts are used in the variety of dosage forms ranging from ointments, suppositories or can be used in the preparation of some other pharmaceuticals. Glycyrrhiza extract USP comes in the form of soft extract.

Dry Extract: 

• Dry extracts are also known as the powdered extracts or dry powders. The total extracts obtained by using suitable process of extraction, are filtered, concentrated preferably under vacuum and dried completely.

• The tray drying or spray drying is used for making dry extracts. Just like soft extracts, these powdered extracts can be used for the manufacture of some medicinal preparations. Powdered extracts are preferably used into a solid, dry dosage forms like capsules, powders or tablets. The Belladonna extract, Hyoscyamus extract are the official dry extracts.

ISOLATION AND IDENTIFICATION OF NATURAL PRODUCTS

• The progress in the techniques for isolation and analysis has led to the identification of many unknown compounds. Various processes are involved in the isolation of the particular compound from its plant material. The isolation process possibly will depend on the nature of the active constituent present in the crude drug. For example, trapping of the components is done for the volatile chemicals while extraction of nonvolatile compounds using organic solvents is also done. The isolation of components is done for both known constituents and also for the components which are unknown, and the process of the separation, purification and identification of compounds coupled with biological screening is a demanding task. After the extraction of the required crude extract from the plant, the need of the marker component to be isolated and identified is also equally important for its study with respect to chemical nature or even for the development of newer formulations. The advances in the field of chromatographic techniques have enabled the separation and purification of compounds.

Fractional Crystallization

• Crystallization is an old but a very important method for the purification of the compounds from the mixture. Crystallization mostly depends upon the inherent character of the compound which forms crystals at the point of supersaturation in the solvent in which it is soluble. Many phytopharmaceuticals and natural products are crystalline compounds which tend to crystallize even in the mixtures. Compounds, such as sugars, glycosides, alkaloids, steroids, triterpenoids, flavonoids, etc., show the crystalline nature with certain exceptions. The processes, such as concentration, slow evaporation, refrigeration is used for crystallizing the products. In case of sugars, osazone formation leads to the crystallization of the derivatives in the form of various types of crystals enabling the analysis of the sugars.

Fractional Distillation

• For the distillation the component should have volatile nature. Therefore, fractional distillation is mostly used for the separation of essential oil components. Most of the volatile components are steam volatile and if the process of fractional distillation is skillfully used, various low-boiling and high-boiling components can be separated from the total oil. This process is largely used for the separation of hydrocarbons from the oxygenated volatile oil components—the product referred to as terpenes essential oils. The components like citral, citronellal and eucalyptol are even now separated by fractional distillation. It is used in the separation of hydrocyanic acid from plant material.

Fractional Liberation

• In the process of fractional liberation, groups of the compounds having the tendency of precipitation come out of the solution. In certain cases the nature of the compound such as alkaloids is modified by converting to their salt form or free bases. If such alkaloidal compounds are more in number with variation in basic nature, with such conversions base liberation, these may be brought about from a weaker base to relatively stronger base. The process is often used even at the industrial level for the separation of cinchona alkaloid quinine, isolation of morphine and many other alkaloids. By using the similar processes phenols, organic acids, like compounds are liberated from the solution. 

Sublimation

• As a matter of fact there are very few natural products which have sublimating nature. In this process the compound if subjected to heating, changes from solid state to gaseous state directly without passing through a phase of liquid. Such compounds from the gaseous state get deposited on the cooler surface in the form of crystals or cake. The process is traditionally used for the separation of camphor from the chips of wood of Cinnamomum camphora to obtain solid sublimate of camphor. Sublimation can also be used for the isolation of caffeine from tea or for the purification of material present in a crude extract. In the inorganic compounds, sublimation is the well-known process for the isolation and purification of sulphur.

Chromatography

• Chromatography is a family of analytical chemistry techniques for the separation of mixtures. It was the Russian botanist, Mikhail Tswett, who invented the first chromatography technique in 1901, which was based on adsorption principle. In 1952, Archer John Porter Martin and Richard Laurence Millington Synge were the two scientists who identified partition chromatography. Chromatographic techniques create a basis for analysis and separation of a wide range of physical methods used in complex mixtures. In chromatography, it has two phases: stationary phase and a mobile phase in which the components are distributed. When components pass through the system at different rates they become separated in time, like runners in a 

• mass-start fool race. Each component has a characteristic time of passage through the system, called a retention time. Chromatographic separation is achieved when the retention time of the analyte differs from that of other components in the sample. This difference in rate of travel results in the separation of the individual component. The smaller the affinity a molecule has for the stationary phase, the shorter the time spent in the column. A chromatograph takes a chemical mixture carried by liquid or gas and separates it into its component parts as a result of differential distributions of the solutes as they flow around or over the stationary liquid or solid phase. If the right adsorbent material, mobile fluid and operating conditions are employed, any soluble or volatile component can be separated using chromatography. Even structurally very similar components can be separated with chromatography. The principle of chromatography differs according to the stationary and mobile phase used. According to this the types are:

• Adsorption Chromatography: Adsorption chromatography is probably one of the oldest types of chromatography around. It utilizes a mobile liquid or gaseous phase that is adsorbed onto the surface of a stationary solid phase. The equilibration between the mobile and stationary phase accounts for the separation of different solutes.

• Partition Chromatography: This form of chromatography is based on a thin film formed on the surface of a solid support by a liquid stationary phase. Solute equilibrates between the mobile phase and the stationary liquid.

• Ion Exchange Chromatography: In this type of chromatography, the use of a resin (the stationary solid phase) is used to covalently attach anions or cations on to it. Solute ions of the opposite charge in the mobile liquid phase are attracted to the resin by electrostatic forces.

• Molecular Exclusion Chromatography: Also known as gel permeation or gel filtration, this type of chromatography lacks an attractive interaction between the stationary phase and solute. The liquid or gaseous phase passes through a porous gel which separates the molecules according to its size. The pores are normally small and exclude the larger solute molecules, but allow smaller molecules to enter the gel, causing them to flow through a larger volume. This causes the larger molecules to pass through the column at a faster rate than the smaller ones.

• Affinity Chromatography: This is the most selective type of chromatography employed. It utilizes the specific interaction between one kind of solute molecule and a second molecule that is immobilized on a stationary phase. For example, the immobilized molecule may be an antibody to some specific protein. When solute containing a mixture of proteins is passed by this molecule, only the specific protein is reacted to this antibody binding it to the stationary phase. This protein is later extracted by changing the ionic strength or pH. There are different types of chromatographic methods like, paper chromatography (PC), thin layer chromatography (TLC), column chromatography, high-performance liquid chromatography, gas chromatography and high-performance TLC. All these methods were used in the analysis, separation and isolation of the components in natural products.

Retention

• The retention is nothing but a measure of the speed at which a compound moves in a chromatographic system. In continuous development systems like HPLC or GC, where the compounds are eluted with the eluent, the retention is usually measured as the retention time Rt , the time between injection and detection. In interrupted development systems like TLC, PC the retention is measured as the retention factor Rf , the run length of the compound divided by the run length of the eluent front.

Paper Chromatography (PC)

• The main advantage of the PC is the convenience of carrying out separations simply on sheets of filter paper that serve both as the medium for separation and as support. The technique was extended to maximum all classes of natural products. The solution of components to be separated is applied as a spot near one end of a prepared filter paper strip. Usually several sample and standard spots are placed along the edge. Then the chromatogram is developed by immersing that edge of the paper in a solvent that migrates through the paper as the mobile phase. The solvent often has two, three or four components—one of which is usually water. Development is normally done in a chamber that is saturated with solvent vapour. The water from the solvent, in particular, is adsorbed and tightly held on the paper fibres, so the sample components partition between the migrating mobile phase and the tightly held water. After the separation, any strongly coloured spots are visible on the chromatogram. Colourless materials can be visualized by heating the paper in an oven so that substances (but not the paper) char and leave black spots. Sometimes the paper is first sprayed with a solution of sulphuric acid for better charring. Fluorescent materials can be visualized with ultraviolet light. Reagents specific for certain components may be sprayed on to make coloured spots. Radioactive spots can be located with a detector, or the chromatogram can be pressed against X-ray film for minutes or hours to expose the film. Sample spots can be tentatively identified if they have the same Rf values as known standard spots. 

• Sometimes spots, once located, are cut out so that the material in the spot can be recovered. There are also instruments that (more or less accurately) quantitative the material in the spot by measuring light absorbance.

Thin Layer Chromatography

• The thin layer chromatography is a widely used, fast technique for the qualitative analysis of a mixture of compounds. The stationary phase consists of a thin layer of adsorbent like silica gel, alumina, or cellulose on a flat carrier like a glass plate, a thick aluminium foil, or a plastic sheet. TLC has certain advantages over PC. Fractionations can be affected more rapidly with smaller quantities of the mixture. The separated spots are usually more compact and more clearly demarcated from one another, and the nature of the film is often such that drastic reagents, such as concentrated sulphuric acid, which would destroy a paper chromatogram, can be used for the location of separated substances. TLC plates are made by mixing the adsorbent with a small amount of inert binder like calcium sulfate (gypsum) and water, spreading the thick slurry on the carrier, drying the plate, and activation of the adsorbent by heating in an oven. The thickness of the adsorbent layer is typically around 0.1–0.25 mm for analytical purposes and around 1–2 mm for preparative TLC.

• Several methods exist to make colourless spots visible. Often a small amount of a fluorescent dye is added to the adsorbent that allows the visualization of UV absorbing spots under a black light (UV254). Even UV light without fluorescent dye could scan the compounds, both in long (365 nm) and short (254 nm) wavelength ultraviolet light. Iodine vapors are a general unspecific colour reagent.

• Specific colour reagents exist into which the TLC plate is dipped, or which are sprayed onto the plate. Dragen Dorff ’s reagents are used in the form of sprays for the general detection of alkaloids. Antimony trichloride in chloroform is used as a spray reagent for steroidal compounds and other terpenoids. Ammonia vapour can be used for free anthraquinone compounds and Fast Blue Salt B ‘Merck’ for cannabinoids and phloroglucides.

• It is effective, comparatively cheap as relatively small amounts of analyte, adsorbent and solvents are required. The use of appropriate developing agents can help understand the compound properties and can be quantified by careful standardization of procedures.

High-Performance Thin Layer Chromatography

• The high-performance thin layer chromatography is a sophisticated and automated form of TLC. It is useful in qualitative and quantitative analysis of natural products. The principle of separation is adsorption (same as that of TLC). In HPTLC, the precoated plates are used and the particle size of stationary phase is less than 1μ in diameter. There is a wide choice of stationary phases like silica gel for normal phase and C18, C8, etc., for reverse phase mode.

• HPTLC provides a higher efficiency than TLC because adsorbents used are small and uniform in size. A very less amount of sample is spotted on the plate so the sample prepared should be highly concentrated. The size of the sample spot should not be more than 1 mm in diameter. The samples are spotted by various techniques and commonly used method is by semiautomatic linomet V apparatus.

• New types of development chambers are used in HPTLC that require a smaller number of solvents for developing. A linear development technique is commonly used. The plate is placed vertically in development chambers containing solvent, and chromatogram can be developed from the sides. In HPTLC, UV/VIS/fluorescence scanner is used; therefore, it scans the entire chromatogram qualitatively and quantitatively. The scanner is an advanced type of densitometer.

• HPTLC is used for the standardization of herbal extracts and other formulations. By using this technique, the analytical profiles of alkaloids, cardenolides, anthracene glycosides, flavonoids, lipids, steroidal compounds, etc., have been developed.

Column Chromatography

• Column chromatography utilizes a vertical glass column filled with some form of solid support with the sample to be separated placed on top of this support. The rest of the column is filled with a solvent which, under the influence of gravity, moves the sample through the column. Similarly to other forms of chromatography, differences in rates of movement through the solid medium are translated to different exit times from the bottom of the column for the various elements of the original sample.

Flash Column Chromatography

• This is a fast, simple, widely used preparative separation technique, where the stationary bed is packed in a long, narrow glass lube. The flow rate of the mobile phase of the system can be accelerated either by applying pressure on the top of the column or by applying suction from the lower end of the column to decrease the time that the compounds spend in the column or to increase the flow rate of the mobile phase. Typically, silica is used as the stationary phase but other stationary beds such as reverse phase silica or cellulose are also used depending on the nature of the compounds to be separated. The particles size should be smaller than that of the column chromatography.

High-Performance Liquid Chromatography

• This is a versatile natural product isolation technique which is similar to flash chromatography; however, high pressure (up to 4,000–5,000 psi) is applied to the system to move the mobile phase through the smaller particle sized (2–10 μm) stationary phase bed. The column is stainless steel to withstand the high pressure. It employs relatively narrow columns about 5 mm diameter for analytical work, operating at ambient temperature or up to about 200°C. The apparatus is suitable for all types of liquid chromatography columns (adsorption, partition by the use of bonded liquid phases, reversed phase, gel filtration, ion exchange and affinity). Many stationary phases are available and the most widely used is silica based, silanol groups (SiOH).

• Reversed phase packing material is produced by the bonding of octadecylsilyl groups to silica gel. In the commercial material there appears to be a considerable proportion of residual silanol OH groups, and this would lead to both adsorption and partition effects during separation. Unlike the other two methods (TLC and flash chromatography), the eluting compounds can be detected using their different physical and structural properties by connecting a detector (UV/visible UV/VIS, refractive index RI). Furthermore, the eluting compounds can be connected to a spectrophotometer (NMR, MS) to study the spectral characteristics of compounds eluting through the HPLC system. The stationary phase bed could be either silica or reverse phase Cn bonded silica, depending on the nature (polarity) of the compounds that are to be separated. The possibilities of working at ambient temperature and recovering the sample after analysis and purification associated with HPLC presents more advantages compared to gas chromatography. 

Gas Chromatography

• Gas chromatography is the most widely used chromatographic technique to analyse volatile compounds where those compounds are carried by an inert gas like nitrogen, helium or argon through a heated (50–350°C) stationary bed (silica supported with bonded polar or nonpolar phase). The choice of stationary phase is governed by the temperature at which the column is to operate and the nature of the material to be fractionated; it should be nonvolatile at the operating temperature and should not react with either the stationary and mobile phases or the solutes. Some commonly used, stationary phase materials are nonpolar compound like silicone oils, apeiron oils and greases, high-boiling point paraffins, such as mineral oil, squalene, moderately polar compounds high-boiling point alcohols and their esters and strongly polar compounds polypropylene glycols and their esters.

• There are two general types of columns, packed and capillary (also known as open tubular). Packed columns contain a finely divided, inert, solid support material (commonly based on diatomaceous earth) coated with liquid stationary phase. Most packed columns are 1.5–10 m in length and have an internal diameter of 2–4 mm.

• Capillary columns have an internal diameter of a few tenths of a millimeter. They can be one of two types; wall-coated open tubular (WCOT) or support-coated open tubular (SCOT). Wall-coated columns consist of a capillary tube whose walls are coated with liquid stationary phase. In support-coated columns, the inner wall of the capillary is lined with a thin layer of support material such as diatomaceous earth, onto which the stationary phase has been adsorbed. SCOT columns are generally less efficient than WCOT columns. Both types of capillary column are more efficient than packed columns. Either a flame ionization detector (FID) or electron capture detector (BCD) detects the compounds eluting from the column producing a signal which can transform into a peak or a chromatogram. The technique is very sensitive, and low concentrations of sample (less than nanograms) can be analysed. Preparative GC also can be carried out using a thermal conductivity detector or splitting outlet to connector and FID. But the difficulties in recovering the compound after analysis are the main disadvantage over HPLC and flash chromatography. GC can be connected to a mass spectrometer to analyse the spectral characteristics of separated compounds. Once a compound is isolated, it needs to be identified to determine the chemical structure of the compound. Complete identification of a compound could be afforded using its spectral characteristics. A known compound from a new plant source can be identified using chromatographic, nonchromatographic and a spectral comparison with the authentic material. With a new compound, the relationship of the chromatographic and spectral data of known compounds in the same series together with the chemical conversions, determination of the chemical formula or derivatization could help to investigate the chemical structure. The available modern advanced spectroscopic techniques and chromatography coupled spectroscopy such as gas chromatography coupled mass spectrometry (GC-MS), high-performance liquid chromatography coupled mass spectroscopy (LC-MS) and high-performance liquid chromatography coupled nuclear magnetic resonance (NMR) spectroscopy (LC-NMR) has led to the identification of new molecules more quickly than before.

Spectroscopy

• The isolated and purified plant constituents should be identified, and its chemical nature should be determined. The plant compounds could be identified by their spectral characteristics. Spectroscopy is the use of absorption, emission or scattering of electromagnetic radiation by atoms or molecules (or atomic or molecular ions) to qualitatively or quantitatively study the atoms or molecules or to study the physical process of a compound. The theory behind the spectroscopy is the interaction of radiation with matter. It can cause redirection of the radiation and/or transitions between energy levels of atoms or molecules. This transition could be absorption, emission or scattering. Among the number of spectroscopic methods ultraviolet visible absorption (UV-VIS), infrared (IR), NMR and mass (MS) plays a crucial role in identifying the plant compounds.

Ultraviolet-Visible Absorption Spectroscopy

• Different organic molecules with certain functional groups (chromophores) that contain valence electrons of low energy can absorb ultraviolet (UV) or visible (VIS) radiation at different wavelengths. Hence the absorption spectrum of a certain molecule will show a number of absorption bands corresponding to structural groups within the molecule. Most commonly used solvent is 95% ethanol, because it solubilizes most classes of compounds. Other solvents used are water, petroleum, hexane, ether and methanol. The absorption is recorded using a detector, such as photo diode array (PDA). This phenomenon could be used to identify the functional groups of a certain molecule or when a PDA detector is connected to a HPLC system; it could be used to monitor the separation or purity of a certain mixture or a compound/s that contained different chromophores. Selection of the detection wavelength of a compound determines the nature of the chromophore within the molecule. While the compounds which possess chromophores are detected between 200–700 nm (visible), others which do not possess chromophores are detected between 200–400 nm (UV). Also, the study of the functional group or chromophore could be extended by observing the shifting (red shift or blue shift) of the maximum absorbance (λmax) in the absorption spectrum of the compounds by changing the solvent in which the compound is dissolved. The value of UV and visible spectra in identifying unknown constituents is obviously related to the relative complexity of the spectrum and to the general position of the wavelength maxima.

Infrared Spectroscopy

• This is done by IR spectrophotometer and the plant compounds used is either in liquid, e.g., chloroform, as a mull with unroll oil or in the solid state, mixed with potassium bromide to form a thin disc. The term ‘infra-red’ covers the range of the electromagnetic spectrum between 0.78 and 1,000 μm. In the context of infra-red spectroscopy, wavelength is measured in wavenumbers, which have the unit cm 1.

• It is useful to divide the infra-red region into three sections: near, mid and far infra-red.

• The most useful IR region lies between 4,000 and 670 cm 1. IR radiation does not have enough energy to induce electronic transitions seen with UV. Absorption of IR is restricted to compounds with small energy differences in the possible vibrational and rotational states.

• For a molecule to absorb IR, the vibrations or rotations within a molecule must cause a net change in the dipole moment of the molecule. The alternating electrical field of the radiation interacts with fluctuations in the dipole moment of the molecule. If the frequency of the radiation matches the vibrational frequency of the molecule, then radiation will be absorbed causing a change in the amplitude of molecular vibration.

• IR spectrum is the simplest and most reliable tool because many functional groups can be identified by their characteristic vibration frequencies. It has a role in structural elucidation when new compounds are identified in plants.

Nuclear Magnetic Resonance Spectroscopy

• The nuclear magnetic resonance is a theoretically complex but powerful tool for providing information about the structure of a molecule in a solution. Proton NMR spectroscopy provides a means of determining the structure of an organic compound by measuring the magnetic moments of its hydrogen atom. Theoretically, subatomic particles (electrons, protons and neutrons) can be imaged as spinning on their axis. In many atoms like, I2C, these spins are paired against each other. Those nuclei of atoms have no overall spin. However, in some atoms like, 1 H, I3C, the nucleus does possess an overall spin. The sample of the substance is placed in solution, in an inert solvent between the poles of a powerful magnet and the protons undergo different chemical shifts according to their molecular environments within the molecule. It requires 5–10 mg of sample and this sample could be recovered as it is. The spin of the hydrogen atom can be promoted from the lower to higher level by interacting with and absorbing energy from electromagnetic radiation in the radio frequency region of the spectrum. The separation between the two spin energy levels is a sensitive function of the molecular environment of the hydrogen atom in the molecule. Thus, hydrogen atoms in different environments absorb photons of different energies. The NMR spectrum of the protons in a molecule is obtained by plotting the amount of energy absorbed by the spinning nuclei versus the frequency of the RF radiation applied to the molecule. This spectrum provides information about the chemical environment of the spinning proton and can be used to deduce the atomic bonding patterns in the molecule. The spin between two groups of protons result in small interactions with each other and is called spin—spin coupling where the spacebetween each coupling is called the coupling constant. The multiplication of the coupling and the magnitude of the coupling constant in a 1 H NMR gives information about the number of H atoms on the nearest carbon atom of the relevant peak or proton/s and their chemical nature. But the proton NMR cannot give information on the nature of the carbon skeleton of a molecule but 13C NMR could help to solve it. Even in 13C NMR, the theory is the same as in 1 H NMR and both decoupled and coupled spectra are recorded. But the coupled spectrum of 13C NMR is slightly different from that of 1 H NMR and is called off resonance proton decoupling. I3C NMR helps to identify the number of unique carbon atoms in a molecule, the number of hydrogen atoms attached to that carbon atom, the environment of the carbon atom and C–C skeleton of the molecule. The modern NMR techniques like, heteronuclear multiple bond correlation (HMBC), correlated spectroscopy (COSY), heteronuclear single quantum coherence (HSQC), nuclear overs Hauser enhancement spectroscopy (NOESY), total correlated spectroscopy (TOCSY) have made the characterization of natural products easier.

Mass Spectroscopy 

• In mass spectrometry, the sample in gas or liquid or solid state is introduced to the spectrometer followed by ionization, mass analysis, and ion detection/data analysis. We could get the exact molecular weights of the compounds in microgram amounts of sample. Volatilization of the sample (liquid or solid state) is done either prior to ionization or along with the ionization. The various ionization techniques commonly used are chemical ionization (CI), electron impact ionization (El) and desorption ionization techniques. Charged molecules in the gas phase are produced by fast atom bombardment (FAB), plasma desorption (PD) and thermospray and particle beam. Then, the ion and its fragments are accelerated by electrical and magnetic fields and the ions are separated in the basis of their mass/ charge (m/z) ratio and detected. The data produced by the molecular mass measurements, or the fragmentation data enable us to elucidate the possible chemical structure of the molecule. The use of GC-MS, LC-MS, capillary electrophoresis-mass spectroscopy has made the identification of natural product easier