Introduction

Secondary metabolites, through several exceptions, take place in amounts that are less than 0.01 % of the dry weight of the plant. Extraction of 1 kg of dryplant substance is probable to yield less than 100 mg of a natural product. Such compounds might be unstable and present as part of a complex mixture. The isolation, division and purification of such natural products need considerable skill. The source of a secondary metabolite needs proper identification and a voucher specimen requires to be preserved. Within the equivalent species there are sometimes chemotypes, each through a meticulous composition. Several compounds are established in the roots, several are components of the bark, and others might be established in the leaves, the flowers or the fruit. Various compounds play a seasonal role in the plant, for instance as insect antifeedants. Therefore, the part of the plant and the place and date on that the plant was collected should all be recorded. 

Chemistry of natural products engages the study of such chemicals produced in plants, and other organisms describing their: 

General Methods of Extraction

Aqueous and organic-solvent extraction methods

Extraction process to be chosen should be depends on comprehension of numerous physicochemical properties of the compound of interest. Such comprise partition coefficients in water or organic solvents, relative polarity of the molecule, stability of the molecule in light or dark, in addition to the temperature employed during the extraction procedure. So, if the compound of interest is extremely soluble in water, we utilize hot or cold water to attain an aqueous extract. If, on the other hand, the compound is highly soluble in a particular organic solvent, we employ that solvent to obtain an organic-solvent extract.

Aqueous extraction of compounds

Traditional methods of aqueous extraction 

The preparation of herbal remedies depend on traditional methods of water extraction utilises two different approaches: if extracting herbaceous tissues of leaves, roots, and flowers, or soft-textured fruits with a relatively high water content (in the range of 60 to 95% water) with hot water or cold water, relatively mild physical conditions are used to obtain what is said an infusion. However, for woody, highly lignified tissues through comparatively low water content (in the range of 5 to 50% water), these as roots, barks, twigs, and several dry fruits, we need to employ more vigorous physical extraction process, using longer extraction times and boiling water, to attain what is said a decoction.

There is an approximately endless list of techniques for preparing infusions and decoctions, but all are depend on the similar principles.

Laboratory process of organic-solvent extraction of compounds

Maceration

Powdered crude material is placed in a stoppered container with the solvent and allowed to stand for a period of 24 to 48 hours with frequent agitation until soluble matter is dissolved. The mixture is then stained; the marc (the damp solid material) pressed and may be macerated again. The combined liquid is clarified by filtration or decantation after standing.

Percolation

Performed in a cone shaped vessel, (called percolator) the narrow side of which is fitted with a suitable filter and a stopcock, plant material allowed macerating with the specified menstrum for a specified period in the percolator after which the stopcock is opened so that the solvent drips slowly from the bottom. The collected solvent is evaporated in a rotary evaporator and recovered solvent is put back to percolator. 

Hot continuous extraction (Soxhlet extraction) 

If the compounds of interest are not soluble in water because of their non-polar  nature, select an organic solvent (e.g., acetone, methanol, ethanol, chloroform, diethyl ether, methylene chloride, or a combination of more than one organic solvent) to carry out the extraction. The temperature of this extraction depends on the boiling point of the solvent chosen and must be carefully watched due to the special equipment that is used. One can use a  Soxhlet extractor, which is basically a specialised glass refluxing unit that is used for such organic-solvent extractions.  The method involves continuous extraction by boiling organic solvents. Natural products may be obtained from the crushed biological material by extraction with a solvent such as petroleum ether, chloroform (trichloromethane), ethyl acetate (ethyl ethanoate) or methanol. Several solvents of increasing polarity may be used. Thus lipid material (waxes, fatty acids, sterols, carotenoids and simple terpenoids) can be extracted with non-polar solvents such as petroleum ether, but more polar substances such as the alkaloids and glycosides are extracted with methanol, aqueous methanol or even hot water.

In the following example (using a 1:1 mixture of methylene chloride/methanol), the temperature must be maintained at 40°C for 8hrs in order to obtain complete extraction of each sample. If the temperature falls below this, extraction will be slow. If the temperature goes above this, the risk of degrading the compounds of interest becomes great.

With the presence of higher temperatures, there is always the risk of degrading some of the active compounds; thus, low-boiling-point solvents, such as dichloromethane or diethyl ether, are usually the best choice. The basic procedure is outlined below. Preliminary cleanup procedures are usually necessary before samples are analysed by HPLC or by any of the other chromatographic techniques.

The example we use here employs methylene chloride. It is highly toxic, and therefore, needs to be used in a fume hood to avoid breathing the toxic fumes. It should also be handled carefully, using latex gloves so that none of this solvent comes in contact with the skin. The extraction methods are as follows:

1.  Weigh out a standard quantity of plant tissue sample that was previously stored in deep freeze at -80°C.  

2.  Grind frozen plant tissues to a fine powder using small amounts of liquid nitrogen in a ceramic mortar and grinding with a ceramic pestle.

3.  Weigh out a known quantity of powdered tissue sample in a cellulose thimble in a Soxhlet extractor containing enough solvent or solvent mixture such as a 1:1 mixture of methylene chloride/methanol.

4.  Reflux the sample for 8hrs at 40°C using a condenser (with running cold water) attached to the top of the Soxhlet. This condenser drops the temperature quickly, allowing the solvent to condense on the sides of the glass and drop back into the cellulose thimble.

5.  Allow the solvent to cool to room temperature, and filter with a 40 μm filter to remove any particulate matter.  

6.  Blow the collected liquid to dryness with a stream of N₂ gas, and store the sample in the refrigerator at 40oC until it is used to separate and identify the kinds and amounts of compounds present by means of HPLC/MS or other appropriate procedures.

Newer methods include:

Supercritical fluid extraction (SFE)

A substance is in its supercritical fluid state when both the temperature and pressure equal or exceed the critical point (31°C and 73 atm for carbon dioxide). In its supercritical state, CO₂ is neither a gas nor a liquid and is best described as intermediate to the two extremes. This dual characteristic of supercritical fluids provides the ideal conditions for extracting compounds with a high degree of recovery in a short period of time. Supercritical fluid extractions (SFEs) are currently being carried out with carbon dioxide on a large scale for the decaffeination of green coffee beans (Coffea arabica) and the extraction of hops (Humulus lupulus) for beer production. Carbon dioxide is the most adopted supercritical fluid due to its low cost, environmentally benign nature, lack of flammability, and reactivity. There are several advantages of SFE compared to traditional solvent extractions. Supercritical fluids have lower viscosities and higher diffusivities than conventional solvent systems; therefore,  extraction rates are  enhanced and less degradation of solutes occur. 

SFE with carbon dioxide is carried out at a relatively low temperature,

ca.-40°C, avoiding the decomposition of thermally labile components. In addition, extractions can be selective to some extent, by controlling the temperature or pressure of the medium. Because of the high pressures required to perform CO₂ extractions, the equipment is expensive and must be handled with care. However, commercial SFE instruments are available.

SFEs with CO₂ were used as alternatives to hydro distillation for isolating essential oils.

Because of the non-polar nature of carbon dioxide, supercritical fluid extraction is an attractive technique  for the isolation  of relatively non-polar natural products, such as artemisinin from  Artemisia annua, azadirachtin from  neem (Azadirachta indica), phloroglucinols  from St. John's wort (Hypericum perforatum), or triterpenoids and steroids from cork (Quercus suber). In order to efficiently extract more polar substituents, the addition of a small amount of a liquid modifier can significantly enhance the extraction of these materials. Typical modifiers include methanol, ethanol, dichloromethane, and acetonitrile. For example, vindoline was efficiently extracted from rosy periwinkle (Catharanthus roseus)  using SFE with 3% methanol as  a co-solvent; flavanones and xanthones were extracted more efficiently from Osage orange (Maclura pomifera) using 20% methanol rather than CO2 alone; and the polyphenolics catechin and epicatechin were isolated from grape seeds using 40% methanol. 

Fractionation

The extracts are then subjected to fractionation by partition among solvents of different polarities or can be directly put to chromatographic separation. A typical fractionation is set out in figure

1. All separation processes involve the division of a mixture into a number of discrete fractions. These fractions may be obvious, physically discrete divisions, such as the two phases of a liquid-liquid extraction, or they may be the contiguous eluate from a chromatography column that is artificially divided by the extractor into fractions. The type of fractionation depends on the individual sample and the aims of the separation. Typically, a column is run and the eluate divided into a manageable number of even-sized fractions, followed by analysis of the fractions to determine which contain the desired compounds. Obviously, collecting the eluate as a large number of very small fractions means that each fraction is more likely to contain a pure compound, but it requires more work in analyzing every fraction, This also runs the risk of spreading the target compound over so many fractions that, if originally present in only low concentrations, it may evade detection in any one of the fractions. If the separation process is relatively crude, it is probably more sensible to collect only a few large, relatively crude fractions and quickly home in on those containing the target.

Alternatively, one may monitor "online" and fractionate the eluate accordingly.

This is generally used at the later stages of separation for separations of less complex mixtures, typically on high-performance liquid hromatography (HPLC) divisions monitored via UV, where one can identify and isolate substance analogous to individual peaks.

Fractionation by partitioning between solvents

A solution of the extract in an organic solvent (these as ethyl acetate) is shaken with an inorganic base (such as aqueous sodium hydrogen carbonate) to remove the carboxylic acids as their water-soluble sodium salts. The more weakly acidic phenols may only be extracted with a sodium hydroxide solution. Extraction of the original solution with an acid such as dilute hydrochloric acid will remove the bases such as the alkaloids as their salts. The neutral compounds remain behind in the organic phase. The acids and the phenols may be recovered from the aqueous solution of their sodium salts by treatment with dilute hydrochloric acid and re-extraction with an organic solvent, and the bases may be recovered by treatment of their salts with ammonia and re-extraction.

Fig: A Typical Fractionation of Plant Extract

Fractionation by chromatography

Chromatography is one of the most helpful means of separating mixtures of compounds, as a technique to both purify the components and identify them. In chromatography, the mixture is divided via differential distribution of the components between a stationary phase and a mobile phase. Primary techniques of chromatography in isolation and analysis of natural products comprise the subsequent: thin layer chromatography (TLC), liquid column chromatography (LC), and gas chromatography (GC), high-performance liquid chromatography (HPLC), and fast protein liquid chromatography (FPLC). There are probably 5 major division mechanisms of chromatography. They comprise adsorption chromatography, gas chromatography, liquid-liquid partition chromatography, ion-exchange chromatography, and size-exclusion chromatography. 

Gas chromatography is a chromatographic technique that can be employed to separate volatile organic compounds. A gas chromatograph consists of a gaseous mobile phase, an injection port, a division column enclosing the stationary phase, and a detector. The organic compounds are divided due to differences in their partitioning behavior between the mobile gas phase and the stationary liquid phase in the column. The volatilities of the compounds that powerfully correlate to their boiling points are frequently responsible for the partitioning between the liquid phase and the gas phase.

Liquid-liquid separation chromatography is depends on the dissimilar solubilities of compounds in 2 liquid phases. Here, the stationary phase and the mobile phases are liquids. The oldest recognized technique of liquid-liquid partition chromatography is paper chromatography. More recent implementations are droplet countercurrent chromatography (DCCC), centrifugal droplet countercurrent chromatography (CPC), high-speed countercurrent chromatography (HSCCC), and elution extrusion countercurrent chromatography (EECCC). While paper chromatography is extensively employed in analytical divisions, the other methods are frequently utilized for preparative purposes.

In ion-exchange chromatography, separation is based on ionic interactions of the individual components of a mixture through a stationary phase, which is an ionically charged surface of opposite charge to the example ions. The mobile phase is an aqueous buffer, where both pH and ionic strength are employed to control elution time. The stronger the charge on the example, the stronger it will be attracted to the ionic surface and therefore the longer it will take to elute it from the column. Typical applications in natural products chemistry are in the separation of fruit acids.

In size-exclusion chromatography, the division of mixtures is depends harshly upon size. The column is filled by material that has accurately controlled pore sizes, and the example is simply screened or filtered according to its solvated molecular size. Because larger molecules can't obtain into the small pores of the packing substance, they are speedily washed through the column. A smaller molecule, though, penetrates inside the porous packing particles that lead to a longer path through the column, and as a consequence, makes them elute later. Typical applications for size-exclusion chromatography are divisions of biomacromolecules, these as proteins.

The 1^st kind of chromatography, adsorption chromatography, can be further subdivided into normal-phase chromatography and reversed-phase chromatography.

In normal-phase chromatography, the stationary phase is of a polar nature (hydrophilic), typically silica, and the solvent is of a non-polar nature (hydrophobic). In order to attain division, the polarity of the solvent is adjusted to the polarity of the mixture. It ranges from non-polar hexanes to extremely polar methanol or even water. Polar samples are retained on the polar surface of the column packing longer than less-polar materials. On the other hand, reversed-phase chromatography consists of modified silica surfaces, such that the nature of the stationary phase becomes unpolar. The mobile phase is a polar solvent, such as water-methanol mixtures or water-acetonitrile mixtures. The division procedure in adsorption chromatography is classically completed via probing the diverse functional groups in the molecules to be divided. An exemplary case could be an extremely polar and hydrophilic carboxylic acid function connected to a long alkane chain that is hydrophobic. Attempts to separate molecules of various chain lengths and complexity should then focal point on the hydrophobic part.

For the division procedure, these dissimilar kinds of performance compete. To resolve fluctuations in dissimilar molecules, a delicate balance between those different effects, hydrophobic versus hydrophilic interaction, is required. Since we can fluctuate only the mobile phase during a division, after choosing a stationary phase, we have to complete minute variations via choosing proper solvent systems. As a consequence, we frequently end up by fairly complex mobile phases that address different parts of our molecules. In many cases, however, we have no prior knowledge of such different groups, either since we don't know precise structures or because we don't know exact composition of the mixture, and we are left by easy trial-and-error attempts depend on our experience through a precise group of plants or plant constituents.

Adsorption chromatography

In adsorption chromatography, finely divided inert adsorbent materials (for example, silica gel or alumina) serve as the stationary phase, and organic solvents serve as the mobile phase. Separation of the mixture, then, is achieved by differences in polarity of the individual components. Depending on the pore size of the material, different techniques can be used to achieve separations.

(a)   Thin-layer chromatography (TLC)

In thin-layer chromatography (TLC), the adsorbent is coated on one side of a plate of glass or a strip of plastic or aluminum. Common adsorbents are silica gel and alumina. A few microliters of a solution of the sample to be analysed are spotted onto the plate as a single small dot near one end of the plate using a microcapillary tube. The plate is developed by placing it in a jar or developing chamber that contains a small amount of solvent. The solvent rises up to the plate by capillary action, carrying the components of the sample with it. The different compounds are separated based upon their interaction with the adsorbent coating.

Commercially available TLC plates, 60 Å silica gel, 250 μm layer thickness, on either polyester or aluminum backing, either with or without a fluorescent indicator (for example, Whatman flexible-backed TLC plates), are suitable for the rapid analysis of crude plant extracts and for following the progress of  preparative separations.  These commercial plates can be cut with scissors or a paper cutter to the desired size. Samples are commonly applied with fine capillaries prepared from melting-point capillary tubes. Development of analytical TLC plates can be carried out either in wide-mouth round jars or rectangular TLC developing chambers. The detection of spots is generally achieved using an ultraviolet (UV) lamp (if the TLC plates have the fluorescent indicator) or iodine vapor. The detection of components of TLC plates may also be accomplished via spraying the plates with a appropriate reagent (for example, chromic acid solution or 2, 4-dinitrophenylhydrazine reagent). Preparative TLC plates, as well commercially available (for example, Sorbent Technologies, 20 cm × 20 cm, glass-backed, 60 Å silica gel, 1000 μm layer thickness, through fluorescent indicator), were employed for preparative separations. The sample is applied as a line rather than a spot onto the bottom of the plate with a capillary, and the plates are developed in a solvent chamber. Visualisation of the components is carried out using a UV lamp. The desired components are scraped off of the glass by a razor blade. The compound is divided from the adsorbent via dissolving through an appropriate solvent and filtering from the silica gel.

Analytical in addition to preparative TLC is executed via 1^st spotting the mixture under study onto a dry TLC plate. This task is best performed via adding small amounts of sample at a time, these that only a small area of the TLC plate is covered through the mixture. In the case of comparative studies of several mixtures, or mixtures with reference compounds, care has to be taken that all mixture spots are lined up parallel to the edge of the plate. The mobile phase should be placed into the developing chamber prior to the separation. The chamber should have a lid that closes strongly enough to maintain a steady gas pressure in the chamber.

Necessary for good and reproducible separations is that the solvent system be allowed to equilibrate with its vapor phase. Chamber walls are often covered with filter-type paper to ensure the evaporation of the solvent and a fast equilibrium. The depth of the solvent should be less than the distance of the example spots from the bottom of the plates.

Lastly, the plate is situated in the chamber and permuted to build up. Capillary forces will move the mobile phase into the TLC plate. On their way through the TLC plate, they shift the different molecules at different distances through the plate. Expansion should be stopped before the solvent front achieves the other end of the plate. The exact end-point of the solvent front should be marked.   

(b)   Preparative column chromatography

Column chromatography is most often used for the preparative scale separation of components from a crude plant extract. In practice, a vertical column, usually glass, is packed by a suspension of adsorbent in a nonpolar solvent (for example, hexane). The crude extract to be separated is added to the top of the column, and a suitable solvent or solvent mixture that should be as nonpolar as poss, is allowed to flow down through the column, eluting the various components from the bottom of the column. Common adsorbents for column chromatography include aluminum oxide, Florisil (a magnesium silicate), and silica gel. More recently, Sephadex LH-20 (a hydroxypropylated, cross-linked dextran) and bondedphase silica gel derivatives such as C8 or C18 (for reversed-phase separations) have become popular. In common, the ratio of mass of adsorbent/mass of extract should be around 40:1, and the ratio of column height/column diameter of the packed column of adsorbent should be around 15:1. For gross  preparative separations of multigram quantities of crude plant extracts, we found that columns of silica gel (240 to 400 mesh), 85 cm length × 5 cm diameter, either gravity elution or with slight nitrogen pressure (flash chromatography), eluting by a solvent gradient (for instance, hexane/ethyl acetate or chloroform/ methanol) usually turn out good consequences.

(c)   Affinity chromatography

This is a powerful means of purifying proteins. The principle of this technique is based on the fact that some proteins have a very high affinity for specific chemical groups (ligands) covalently attached to a chromatographic bed material (the matrix). After loading and running the protein mixture sample through the column, only those proteins with a high affinity to the ligand can bind to the column matrix. Other proteins, in contrast, run through the column, because they are unable to bind to the ligand. The bound proteins can then be eluted from the column by a solution containing a high concentration of the soluble form of the ligand or, in several cases, via changes in the buffer conditions that cause the bound proteins to change conformation. A number of affinity columns are commercially available, depending on the particular protein purification desired. One example is ConA-Sepharose affinity chromatography that is exact for glycoprotein purification. Another illustration uses columns through immobilised metal ion covalently linked to the column matrix. In this type of method, the proteins with affinity to the metal ion (including the proteins that have a HIS-Tag or a series of six to ten histidine residues) will bind to the column, while other proteins will be washed away.

(d)   High-performance liquid chromatography (HPLC)

HPLC typically utilizes small particle sizes for the stationary phase, which effects in a fairly huge backpressure when the mobile phase is passed through this bed. As a consequence, the only way to achieve flow of the mobile phase is to employ pump systems. The pressure of HPLC typically reaches 150 to 200 bars. In certain cases, larger pressures are unavoidable. Modern instrumentation is able to handle up to 400 bars.  

Typically, an HPLC system consists of the following components: solvent reservoir, injection system, column, HPLC pump, detector, sample collector (optional), and a computer serving as a data station for the detector information as well as a way to control and automate the HPLC pump.

More sophisticated instruments consist of 2 or 3 pumps in addition to a number of detectors to characterize the example. The benefit of having more than one pump is that solvent gradients can be programmed, meaning that the solvent composition can be transformed constantly throughout the chromatography. This permits for the division of a much larger range of compounds since the mobile phase can be adjusted to the changing polarity of the mixture. As a further advantage, chromatographic peaks get sharper - they elute in a smaller volume from the column - and the division can be done in a much shorter time period. Almost every HPLC system is equipped through a UV detector in order to detect compounds of interest. Traditionally, such UV detectors were single-wavelength detectors; though, through cheaper hardware photodiode-array (PDA or DAD) detectors that allow the scanning of the full UV-visible range (210to 650 nm) are becoming more popular. For the division of natural products, this is of huge benefit since depending on the compounds included; there can be extremely huge dissimilarities in their UV maxima.

In most cases, HPLC is implemented as an analytical technique. With either reversed-phase or normal  phase  substances, column sizes are typically 25 cm in length and 4.6 mm in diameter. The distinctive load for an analytical column is well below 1 mg for the overall mixture. Though, up to 5 mg of example are possible in positive cases. General flow rates for analytical separations are 1 ml·min^-l. Semi preparative divisions can be attained using larger columns; here the diameter of the column is amplified to 10 mm or more. For a 10 mm column, a maximum load of about 50 mg is possible. Preparative columns go up to 50 mm in diameter that permits for the division of a few grams of substance. In the case of preparative divisions, the larger column diameters need raised flow rates (up to 150 ml·min-1), and thus, HPLC pumps through larger pumping capabilities are needed.

For normal-phase HPLC, there are a variety of dissimilar surface chemistries commercially available. Those most usually employed are silica columns; though, utilize of alumina packing or modified silica columns, these as diol, cyano, or amino phases, is possible. The benefit of the modified silica phases is their ability to equilibrate rapidly, contrary to silica or alumina phases. 

Partition Chromatography

Partition chromatography, frequently said liquid-liquid partition chromatography, involves 2 liquid phases. The stationary phase is an adsorbed solvent detained on the surface or inside the grains or fibers of an inert solid supporting matrix. Instances of inert supports comprise sheets of paper (cellulose) as utilized in paper chromatography. The division of mixtures is depends on the partitioning of the individual components between 2 immiscible liquid phases - the solubility differences of each component in the mobile phase and the stationary phase. In paper chromatography, a thin film of water on the paper constitutes the stationary phase. Early liquid chromatography (LC) stationary phases were coated onto the inert support and packed into a column. The mobile phase was then passed through the column.

(a)   Paper chromatography

Paper chromatography is generally carried out in a huge glass tank or cabinet and includes either ascending or descending flow of the mobile-phase solvents. Descending paper chromatography is faster due to gravity facilitating the flow of solvents. Large sheets of Whatman #1 or #2 filter paper (the latter is thicker) are cut into long strips (for example, 22 × 56 cm long) for utilize in descending paper chromatography, or a broad strip of paper (for example, 25 cm wide) of variable height is utilized for ascending paper chromatography. For descending liquid-paper chromatography, substances to be separated are applied as spots (for example, 25 mm apart) along a horizontal pencil line placed down from the V-trough folded top of the paper. 

The V-trough folded paper is placed in a glass trough, held down by a glass rod, and when the tank has been equilibrated (vapor-saturated) through "running solvents" (mobile phase), the comparable solvent is added to the trough via a hole in the lid covering the chromatography tank. The lid is sealed onto the chamber through stopcock grease in order to make the chamber airtight. After the mobile-phase trails to the base of and off the paper sheet, the paper is hung to dry in a fume hood, where it can then be sprayed with reagents (for example, ninhydrin reagent for amino acids) that provide colour to the divided compounds of interest in white or UV light.

Several compounds of interest have their own distinctive colours (for example, chlorophylls), and hence, can be purified using this technique. In other cases, the dyes utilized to stain the location of the compound or protein cause irreversible covalent changes to the compound. In these cases, purification isn't possible. In ascending paper chromatography, the similar essential setup and principles apply, by the exception that the mobile phase is placed at the bottom of the tank. Division is attained when the mobile phase travels up the paper via capillary action. 

Gas chromatography

Gas chromatography was the first commercially available separation technique available. A gas chromatograph consists of an injector system, a column that is placed in a programmable oven, and a detection system. Currently, the columns most widely used are fused silica columns approximately 30 m in length. They provide excellent stability over a wide temperature range, so that compounds up to molecular weights of approximately 450 amu can be analysed by GC. The most common detector for GC is the flame ionisation detector, which provides excellent sensitivity for a wide range of compounds. A more sophisticated detector is a mass selective detector. In natural products, GC instruments are typically utilized for the evaluation of extremely volatile essential oils. Because, in many cases, retention time is the only parameter to differentiate individual compounds, and retention time extremely depends on the column utilized in addition to the exact temperature program incorporated, techniques were expanded to compare dissimilar analyses. In order to attain a scaled retention time, a standard example of a recognized mixture of hydrocarbons is employed to calculate the amalgamation of temperature program and column traits under the exact situations as the actual plant extract.

Using this standard mixture, retention indices (RIs) can be computed, and a comparison of the definite instance through the work done via others is possible depend on these RIs.

It is clear that the individual components are better separated through the slower temperature gradient. Additionally, when inspecting the hydrocarbon mixture, it is apparent that the spacing between the individual components transforms, indicating that it is extremely significant to correlate divisions under different situations through standard mixtures.

Characterization of Natural Products

The bottleneck in natural products chemistry usually is separation/purification of compounds of interest from compound mixtures and structure elucidation of those compounds. The most significant tools for structure elucidation of natural products are nuclear magnetic resonance (NMR) and mass spectroscopic (MS) techniques. In addition, infrared (IR) and ultraviolet-visible spectrophotometric (UV-Vis) techniques are of importance. We as well comprise hyphenated techniques these as gas chromatography (GC)-MS, liquid chromatography (LC) - MS, and LC-NMR. Such techniques provide separation methods coupled by structural (spectroscopic) information. An extremely powerful modern analytical technique is x-ray crystallography.

The stages in structure elucidation in examining the strategies for natural product structure determination, it is probable to discern four overlapping stages. First there is the preliminary characterization in that the physical steadies are determined, the molecular formula is founded and the functional groups are identified. At this stage it is frequently possible to distinguish the class of natural product to which the compound belongs. It may be apparent that the compound is known from another source or is just an easy derivative of another compound of known structure. An easy chemical interrelationship might then be sufficient to establish the structure of the unknown compound.

The 2^nd stage is one of structural simplification in order to identify the underlying carbon skeleton. Chemically, this work engages the selective elimination of the diverse functional groups and the dissection of the carbon skeleton into identifiable fragments. Spectroscopically, it involves identifying adjacent groups of atoms via, for instance, their NMR traits. Evidence may accumulate concerning the position of the functional groups on the carbon skeleton. The willpower of the position and relative stereochemistry of the functional groups shapes the 3^rd phase. There is an interesting distinguish in utilize of spectroscopic and chemical process between the first and third stages of this strategy. In the 1^st stage, the techniques are employed to attain information about the divide functional groups, but in the third phase the methods are utilized to reveal interactions between groups. The 4^th stage engages establishing the absolute stereochemistry of the molecule. Even though this is sometimes assumed, it is significant to verify it in order to understand the biological activity of a natural product. A few natural products are found in both enantiomeric forms.

When a natural product or a simple derivative crystallises particularly well, X-ray crystallography can provide an unambiguous way of establishing the structure. Using a modern diffractometer, structures can be obtained in a matter of hours rather than days. However, unless a heavy atom such as bromine is present or a derivative is made with an optically active compound of known absolute stereochemistry, the structure determined this way will contain only relative stereochemical information. When a structure has been proposed, it is helpful to rationalize this in biogenetic terms. An unambiguous partial synthesis from a compound of identified structure, or a total synthesis, will in conclusion give the ultimate proof of structure. 

Fig: The Stages Involved in Isolation and Characterizations Natural Products

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