Separation and Isolation Techniques + Chromatography (PDF)

PHARMACOGONOSY-III SEPARATION AND ISOLATION OF PLANT CONSTITUENTS The phytochemical investigation of a plant involves the extraction of a plant material, separation and isolation of the constituents of interest; characterisation of the isolated compounds; investigation of biosynthetic pat...

PHARMACOGONOSY-III SEPARATION AND ISOLATION OF PLANT CONSTITUENTS The phytochemical investigation of a plant involves the extraction of a plant material, separation and isolation of the constituents of interest; characterisation of the isolated compounds; investigation of biosynthetic pathways to particular compounds, quantitative evaluations and pharmacological activities. The plant material used should be properly authenticated. The choice of extraction procedure depends on the nature of the plant material and the components to be isolated. Dried materials are usually powdered before extraction, whereas fresh plants (e.g leaves) can be homogenized or macerated with alcohol. Alcohol is a fixed solvent for many plant constituents, except fixed oils. Light petroleum (essential and fixed oils, steroids), ether and chloroform (alkaloids), are used widely for extraction. SELECTION COLLECTION AND IDENTIFICATION OF PLANT MATERIAL Selection of plant material for the study in drug discovery programs may be performed by different approaches such as, random selection, selective selection using ethnopharmacological reports, or by restricting the plant of interest to groups based on chemotaxonomic or geographical preferences. Plant selection should normally involve a literature survey of the floristic diversity of the area of interest and shoul include the plant medicinal uses in the region where collection will take place. During collection of a plant it should be kept in mind that the specimens to be studied should be healthy, since microbial and other infections may change the metabolites produced by the specimen. Variations in the collection sit, altitude, plant age, climate, and soil type can all influence the concentration levels of secondary metabolites and even the kinds of compounds biosynthesized in some cases. Different organs of the plant are known to produce different profiles of secondary metabolites, e.g. flavonoids are found in the flowers and leaves of a particular species, whereas tropane alkaloids occur in the roots and sesquiterpene lactones and essential oils are restricted to glandular hairs and other glands. Once the plant part is collected, at least three herbarium samples should be prepared, and each specimen should be correctly identified by a taxonomist. One of these samples should be deposited in a local herbarium, and the others should be deposited in a specialist museum or herbarium. All voucher specimens should be kept in an appropriate place for future reference. A card with details of the place, altitude, environment and characteristics of each collection should be attached to the herbarium sample which is very important in case a recollection of the plant material is necessary.  PRESERVATION OF PLANT MATERIAL The plant material must first be preserved so that the active compounds will remain unchanged during transport and storage. The cells of living plants contain not only low molecular-weight compounds and enzymes, but they also have many kinds of barriers that keep these constituents apart. When the plant dies, the barriers are quickly broken down and the enzymes then get the opportunity to promote various chemical changes in the other cell constituents, e.g. by oxidation or hydrolysis. Preservation aims A. Drying The most common method for preserving plant material is drying. Enzymic processes take place in aqueous solution. Rapid removal of the water from the cell will, therefore, largely prevent degradation of the cell constituents. Drying also decreases the risk of external attack, e.g. by moulds. Living plant material has a high water content: leaves may contain 60-90% water, roots and rhizomes 70-85%, and wood 40-50%. The lowest percentage, often no more than 5-10%, is found in seeds. To stop the enzymic processes, the water content must be brought down to about 10 %. Drying must be done quickly, in other words at raised temperatures and with rapid and efficient removal of the water vapor. The most efficient drying is achieved in large driers of the tunnel type. The plant material is spread out on shallow trays, which are placed on mobile racks and passed into a tunnel where they meet a stream of warm air. The air temperature is kept at 20-40 °C for thin materials such as leaves, but is often raised to 60-70 °C for plant parts that are harder to dry, e.g. roots and barks. When the crude drug has been collected under primitive conditions, without access to a drier, it must be dried in the open. Even then, the material should be spread out in shallow layers with good ventilation to facilitate the drying. The choice of sunshine or shade is determined by the sensitivity to light of the constituents. In a dried drug the enzymes are not destroyed but only rendered inactive due to the low water content. As soon as water is added, they become active again. Hence, dried drugs must be protected from moisture during storage B. Freeze-drying Freeze-drying (lyophilization) is a very mild method. Frozen material is placed in an evacuated apparatus which has a cold surface maintained at -60 to -80 °C. Water vapor from the frozen material then passes rapidly to the cold surface. The method requires a relatively complicated apparatus and is much more expensive than hot-air drying. For this reason, it is not used as a routine method, but it is very important for drying heat-sensitive substances, e.g. antibiotics and proteins. Fermentation Enzymic transformation of the original plant constituents is sometimes desirable. The fresh material is then placed in thick layers, sometimes covered and often exposed to raised temperatures (30-40 °C) and humidity, so as to accelerate the enzymic processes. (This treatment is usually called fermentation). The fermented product must, of course, be dried afterwards to prevent attack by microorganisms, e.g. moulds. Fermentation is mostly used to remove bitter or unpleasant- tasting substances or to promote the formation of aromatic compounds with a pleasant smell or taste. It is mainly applied to drugs used as spices or stimulants, e.g. vanilla, tea and cacao.  GRINDING Regardless of whether the crude drug is to be used for isolation of a pure compound or for manufacture of a simple preparation, the first operation that must be performed is grinding of the plant material to a powder of suitable particle size. It is important that the particles are of as uniform a size as possible. Large particles take a longer time for complete extraction than small ones and large differences in particle size thus slow down the extraction process. Several types of machines are available for grinding crude drugs: 1. Hammer mill; a common type for grinding crude drugs. 2. Knife mill; is useful for production of low-dust powders of leaves, barks and roots for subsequent percolation or maceration. 3. Tooth mill; is used for production of very fine powders. 4. Blenders Grinding produces a certain amount of heat which must be observed when grinding crude drugs containing heat-sensitive compounds. Mills cooled with liquid nitrogen are available for such purposes. Cold grinding is also preferable for crude drugs containing volatile oils.  EXTRACTS Extracts can be defined as preparations of crude drugs which contain all the constituents which are soluble in the solvent used in making the extract. In dry extracts all solvent has been removed. Soft extracts and fluid extracts are prepared with mixtures of water and ethanol as solvent. Tinctures are prepared by extraction of the crude drug with five to ten parts of ethanol of varying concentration, without concentration of the final product. For both extracts and tinctures the ratio drug/solvent should always be stated. Several factors influence the extraction process. Plant constituents are usually contained inside the cells. Therefore, The solvent used for extraction must diffuse into the cell to dissolve the desired compounds whereupon the solution must pass the cell wall in the opposite direction and mix with the surrounding liquid. An equilibrium is established between the solute inside the cells and the solvent surrounding the fragmented plant tissues. The speed with which this equilibrium is established depends on: 1. Temperature 2. pH 3. Particle size 4. The movement of the solvent  CHOICE OF SOLVENT The ideal solvent for a certain pharmacologically active constituent should: 1. Be highly selective for the compound to be extracted. 2. Not react with the extracted compound or with other compounds in the plant material. 3. Have a low price. 4. Be harmless to man and to the environment. 5. Be completely volatile. 6. According to the pharmacopoeias, ethyl alcohol is the solvent of choice for obtaining classic extracts such as tinctures and fluid, soft and dry extracts The ethanol is usually mixed with water to induce swelling of the plant particles and to increase the porosity of the cell walls which facilitates the diffusion of extracted substances from inside the cells to the surrounding solvent. For extraction of barks, roots, woody parts and seeds the ideal alcohol/water ratio is about 7:3 or 8:2. For leaves or aerial green parts the ratio 1:1 is usually preferred in order to avoid extraction of chlorophyll.  PROCESSES FOR THE EXTRACTION There are many procedures for obtaining extracts such as: 1. Infusion 2. Maceration 3. Percolation 4. Digestion 5. Decoction 6. Continuous hot extraction 7. Solvent-solvent precipitation 8. Liquid-liquid extraction 1. INFUSION In this method the plant material (herbal tea) is placed in a pot and wetted with cold water. Immediately afterwards boiling water is poured over it, then left to stand covered with a lid, for about fifteen minutes after which the tea is poured off. 2. MECERATION This method is used frequently for water soluble active constituents. It consists of mecerating the plant material in cold water for several hours. 3. PERCOLATION In this method the ground plant material is subjected to a slow flow of fresh solvent. 4. DIGESTION This method is suitable for hard barks or woods which are difficult for water to penetrate. Digestion is also considered as meceration but at a relatively elevated temperature. As a general rule the temperature of the extracting medium should be in the range from 35-400 but not exceeding 500. 5. DECOCTION: If the plant material is boiled for ten minutes or if boiling water is poured over it and allowed to stand for thirty minutes the result is called decoction. 6. CONTINUOUS HOT EXTRACTION METHOD This procedure is considered as the most common method used for extraction of organic constituents from dried plant tissue. It can be used both on laboratory andd industrial scales. In lab the powdered material is continuously extracted in a Soxhlet apparatus with a range of solvents of increasingly polarity. 7. SOLVENT-SOLVENT PRECIPITATION 1. The extract dissolved in a suitable solvent is mixed with a less polar but miscible solvent causing the selective precipitation of the less soluble plant constituent, e.g. precipitation of triterpenoid saponins from methanol extract of Phytolacca dodecandra by the addition of acetone and the precipitation of gum from aqueous extracts of Olibnum by the addition of alcohol. 2. By the addition of the extract to a solvent in which the constituent is insoluble or very sparingly soluble, e.g. precipitation of resins from the alcoholic extracts by the 8. LIQUID-LIQUID EXTRACTION This method is also known as Solvent extraction and Partitioning, and is used to separate compounds based on their relative solubilities in two different immiscible liquids, usually water and organic solvent. It is an extraction of a substance from one liquid phase into another liquid phase. This is a basic technique in phytochemical laboratories, where it iss performed using a separating funnel.  AN INTRODUCTION TO CHROMATOGRAPHY AND CHROMATOGRAPHIC TECHNIQUES The study of chromatography has become prominent as a means of separating and analyzing organic and inorganic materials. Large amounts as well as micro quantities may be employed, and the analyses may be conducted either qualitatively or quantitatively. Chromatographic methods are often more effective than other means of separation of drug principles. Such techniques are of wide application in research in pharmacognosy involving the determination and purity of drugs and derivatives of natural origin as well as in pharmaceutical research and manufacturing processes. Chromatography is a combination of two words;* Chromo – Meaning color* Graphy – representation of something on paper DEFINITION“ It is a physical separation method in which the components of a mixture are separated by differences in their distribution between two phases, one of which is stationary (stationary phase) while the other (mobile phase) moves through it in a definite direction. The substances must interact with the stationary phase to be retained and separated by it. Chromatography is also defined as a method of analysis in which the flow of solvent or gas promotes the separation of substances by differential migration from a narrow initial zone in a porous medium.  CHROMATOGRAPHY TERMS Chromatogram: It is the visual output of the chromatograph. Chromatograph: It is equipment that enables a sophisticated Separation. Stationary phase (bounded phase): It is a phase that is covalently bonded to the support particles or to the inside wall of the column tubing. Mobile phase: It is the phase which moves in a definite direction. Analyte (Sample): It is the substance to be separated during chromatography. Eluate: It is the mobile phase leaving the column.  Retention time: It is the characteristic time it takes for a particular analyte to pass through the system(from the column inlet to the detector) under set conditions. Eluent: It is the solvent that will carry the analyte. Retardation factor ( R ): Fraction of an analyte in the mobile phase of a chromatographic system. R = Quantity of substance in mobile phase Total quantity of the substance in the system  Types of Chromatography According to mechanism of separation chromatography is of four types 1) Ion-exchange chromatography 2) Affinity chromatography 3) Size-exclusion chromatography 4) Adsorption chromatography ION-EXCHANGE CHROMATOGRAPHY It is a process that allows the separation of ions and polar molecules based on their charge. DEFINITION: In this type of chromatography, a resin (the stationary solid phase) is used to covalently attach anions or cations onto it. Solute ions of the opposite charge in the mobile liquid phase are attracted to the resin by electrostatic forces. Ion exchange chromatography is performed in columns but can also be useful in planar mode. PRINCIPLE: Ion - exchange chromatography retains sample molecules on the column based on ionic interactions. The surface of stationary phase displays ionic functional groups (R-X) that interact with analyte ions of opposite charge. MECHANISM: Ion exchange chromatography uses a charged stationary phase to separate charged compounds including anions, cations, amino acids, peptides, and proteins. In conventional methods the stationary phase is an ion exchange resin that carries charged functional groups which interact with oppositely charged groups of the compound to be retained. Ion exchange chromatography is commonly used to purify proteins. Cation exchange chromatography retains positively charged cations because the stationary phase displays a negatively charged functional group. Anion exchange chromatography retains anions using positively charged functional group. APPLICATIONS: It can be used for almost any kind of charged molecule including large proteins, small nucleotides and amino acids. Protein purification Water analysis Quality control AFFINITY CHROMATOGRAPHY This is the most selective type of control chromatography. It is a method of separating biochemical mixtures and based on a highly specific biological interaction such as that between antigen and antibody, enzyme and substrate, or receptor and ligand. DEFINITION: “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 a solute containing a mixture of proteins are 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. PRINCIPLE: The stationary phase is typically a gel matrix (often agarose). The molecule of interest has a known and defined property. The process is an entrapment in which the target molecule becomes trapped on stationary phase. The Stationary phase can then be removed from the Chromatography mixture, washed and then target molecule is released from the entrapment. APPLICATIONS: Purify and concentrate an enzyme solution Purification of recombinant proteins Purification of antibodies SIZE-EXCLUSION CHROMATOGRAPHY The technique was invented of antibodies by Grant Henry Lathe and Colin R Ruthven. They later received the John Scott Award for this invention. It is a chromatographic method in which molecules in solution are separated by their size, and in some cases molecular weight. It is usually applied to large molecules or macromolecular complexes such as proteins and industrial polymers. DEFINITION: It is also known as gel permeation or gel filtration chromatography. 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 allows 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.  PRINCIPLE: Smaller molecules are able to enter the pores of the media and, therefore, molecules are trapped and removed from the flow of the mobile phase. The average residence time in the pores depends upon the effective size of the analyte molecules. However, molecules that are larger than the average pore size of the packing are excluded. APPLICATIONS: Purification and analysis of synthetic and biological polymers, such as; Proteins, Polysaccharides, Nucleic acids. It is also useful for determining the tertiary structure and quaternary structure of purified proteins. It is generally a low-resolution chromatography technique and thus it is often reserved for the final “polishing” step of purification. ADSORPTION CHROMATOGRAPHY DEFINITION: “It is a type of chromatography in which a mobile liquid or gaseous phase 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.”  PRINCIPLE: Principle involves competition of components of sample mixture for active site on adsorbent. These active sites are formed in molecule due to *Cracks and *Edges. The electrostatic forces present in the molecule, which hold together the crystal lattice, are directed outward. These forces and electrostatic forces of solute molecule cause separation. Separation occurs because of the fact that an equilibrium is established between molecules adsorbed on stationary phase and those which are flowing freely in mobile phase. The more the affinity of the molecule of particular component, less will be its movement.  TYPES: Adsorption chromatography is of three types 1. Column chromatography 2. Thin layer chromatograph 3. Gas chromatography ADSORBENTS: “An adsorbent is a substance, usually porous in nature and with a high surface area that can adsorb substances onto its surface by intermolecular forces.” AN IDEAL ADSORBENT: The Ideal adsorbent must fulfill the following requirements: Insoluble in mobile phase Inert to solutes (adsorptive) Colorless especially when work with colored mixtures Suitable particle size enough to give good separation and reasonable flow rate Column chromatography Column chromatography has been defined as uniform percolation of fluid through a column of more or less finely divided substance which selectively retards certain components of the fluid. In this method many adsorbents have been used such as sucrose, talc, calcium or sodium carbonate, silicic acid and activated alumina. Solvents include petroleum ether, carbon tetrachloride, carbon disulphide, ether acetone, alcohol, water, and mixtures of acids and bases in water, alcohol and pyridine. The adsorbent is packed uniformly into a suitable glass tube and the solution of the drug or substance in a small amount of solvent is passed through a column. COMMON ADSORBENTS: Hydrated silica gel Silica gel G Silica gel S Silica gel GF 254 Silica gel H Silica gel N Silica gel HF 254 Silica gel PF 254 Cellulose microcrystalline In pharmacognosy column chromatography is used where large quantities of the material to be tested are available. Thin layer and paper chromatography are preferred for purposes of identification because of their selectivity, convenience and adaptability, to small quantities of material. A modification of the column method is known as a Flowing chromatogram. In this method the liquid is allowed to flow continuously through the column until the separated substance appears in the discharged solution. It is then identified either as the solution or as the evaporated material. THIN LAYER CHROMATOGRAPHY Thin Layer chromatography is a modification of the paper chromatography and this was originated by a German pharmacognosist Professor Egon Stahl in 1956. It is characterised by the application of dry finely powdered adsorbent in a thin uniform layer to a glass plate. This coated plate is comparable to an open chromatographic column and the separation of components in the test material is based on adsorption, partition or a combination of both, depending on the adsorbents and the solvents used. For Two dimensional thin layer chromatography the glass plate is turned at a right angle and again the entire process is repeated using the second solvent. PARTITION CHROMATOGRAPHY In this process two immiscible liquids are used one representing an immobile phase the other a mobile phase. It is a process for the separation of mixtures in columns or on filter paper based on partition of a solute between two solvents one of which is immobilized by the substance in the column or by the paper. Separation of components of a sample mixture occurs because of partition. Stationary phase is coated with a liquid which is immiscible in mobile phase. Partition of component of sample between sample and liquid/ gas stationary phase retard some components of sample more as compared to others. This gives basis for separation. The stationary phase immobilizes the liquid surface layer, which becomes stationary phase. Mobile phase passes over the coated adsorbent and depending upon relative solubility in the coated liquid, separation occurs. The components of sample mixture appear separated because of differences in their partition coefficient.  Gas Chromatography Gas chromatography is a specific method in which the moving phase is gas. It is of two types: Gas-liquid chromatography and Gas-solid chromatography, the difference being the type of stationary phase over which the gas flows. In gas-liquid chromatography, the immobile liquid phase consists of a thin film adhering to a finely divided solid support that does not retard the drug mixture from passing through. Crushed fire brick may be employed as the solid support. In gas-solid chromatography the immobile solid phase is represented by an active adsorbent, such as aluminia, silica gel, or carbon which adheres to the support. PAPER CHROMATOGRAPHY Paper chromatography is more popular than column chromatography. Two techniques may be employed: the descending and the Ascending depending upon whether the mobile phase moves downward or upward on the chromatographic paper. In paper chromatography, the sample mixture is applied to a piece of filter paper, the edge of the paper is immersed in a solvent, and the solvent moves up the paper by capillary action. Components of the mixture are carried along with the solvent up the paper to varying degrees. The paper is composed of cellulose to which polar water molecules are adsorbed, while the solvent is less polar, usually consisting of a mixture of water and an organic liquid. The paper is called the stationary phase while the solvent is referred to as the mobile phase. Performing a chromatographic experiment is basically a three-step process: 1.application of the sample, 2. “developing" the chromatogram by allowing the mobile phase to move up the paper 3. calculating Rf values and making conclusions. The ratio of the distance travelled on the paper sheets by the test substance to the distance travelled by the front of the mobile phase from the point of application of the test substance is termed the Rf value of the substance. The resulting spots or pattern of distribution on the paper is called the chromatogram. A modification of the paper chromatographic method is the two dimensional separation of a sample by using another solvent on the same chromatogram. Another chamber is prepared the chromatogram is turned at a right angle and the entire process is repeated using the second solvent. When the substance to be analysed is vaporized and introduced into the moving gas phase it is carried into the column and is distributed between the gas and the stationary phase (liquid or solid) on which it is dissolved or adsorbed. The efficiency of this method depends on the number of factors: the specific solute, the type of liquid solvent or solid adsorbent, the temperature at which the procedure is conducted and the rate of gas flow.