Separation Techniques and Extraction methods of Phytochemicals

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1 Separation Techniques and Extraction methods of Phytochemicals

2 Introduction herbal medicines and the products derived from them are traditionally used for their benefits in prevention and treatment of diseases. In Pharmaceutical terms several factors should be considered: aspects of production (agricultural production or wild-crafting, extraction, fractionation, formulation, quality assurance). Legal framework of their use. Clinical aspects (safety, pharmacovigilance).

3 Plant-derived medicine forms Pure compounds: which are often isolated from botanical drugs. Traditionally used medicinal plants: loose or in teabags to form infusions, including instant teas, and tinctures, essential oils, fatty acids and dried extracts. Crude drugs: Powdered or cut (unprocessed). Non-standardised extracts, with varying information about quality and, consequently, sometimes uncertain information about clinical efficacy and pharmacological effects. Standardised extracts, generally with relatively wellestablished clinical and pharmacological profiles.

4 EXTRACTS According to the European Pharmacopeia, an extract is a concentrated preparation of liquid (water-extract or tinctures) intermediate (semiliquid) solid (dry extract) consistency normally produced from dried botanical or zoological material by a technique involving the use of adequate solvents for obtaining a mixture of compounds. For some preparations, the material to be extracted may undergo a preliminary treatment prior to extraction. (Examples: defatting, inactivation of enzymes or grinding). Extracts are prepared by maceration, percolation or other suitable, validated methods using ethanol or another suitable solvent. After extraction, unwanted material may be removed if this is deemed appropriate.

5 Drug:extract ratio (DER): The ratio of a botanical drug to the amount of extract obtained, for example, 4 to 1 (4:1) four units of a dried starting material (the botanical drug) yield one unit of extract (e.g. kg) Drug:solvent ratio (DSR): The ratio of a botanical drug to the amount of solvent used in the extraction, for example, 1 to 8 (1:8) eight units are used to extract one unit of a botanical drug. In general w/v (weight/ volume) or w/w (weight/weight) are used as units and very often a range is given (e.g. 1 to 6 10). In addition, the solvent and the type of extraction must also be stated.

6 Analysis and standardisation of medicinal plants Extraction is first step to extract the desired chemical components for further separation and characterization. The basic operation included pre-washing, Drying or freeze drying, grinding, to obtain a homogenous sample, improving the kinetics of analytic extraction, and increasing the contact of sample surface with the solvent system.

7 Solvent selection for extraction The selection of solvent system largely depends on the specific nature of the bioactive compound being targeted. Different solvent systems are available to extract the bioactive compound from natural products. The extraction of hydrophilic compounds uses polar solvents such as ethanol or ethyl acetate. For extraction of more lipophilic compounds, dichloromethane or a mixture of dichloromethane/methanol in ratio of 1:1 are used. In some instances, extraction with hexane is used to remove chlorophyll

8 METHODS OF EXTRACTION 1. Plant Tissue Homogenization Plant tissue homogenization in solvent has been widely used by researchers. Dried or wet, fresh plant parts are grinded in a blender to fine particles, put in a certain quantity of solvent and shaken vigorously for 5-10 min or left for 24 h after which the extract is filtered. The filtrate then may be dried under reduced pressure (freeze drying) and re-dissolved in the solvent to determine the concentration. Some time centrifuging the filtrate is used for clarification of the extract

9 2. Serial Exhaustive Extraction This method involves successive extraction with solvents of increasing polarity from a non polar (hexane) to a more polar solvent (methanol) to ensure that a wide polarity range of compounds could be extracted.

11 3. Soxhlet Extraction Soxhlet extraction is only required where the desired compound has a limited solubility in a solvent, and the impurity is insoluble in that solvent. If the desired compound has a high solubility in a solvent then a simple filtration can be used to separate the compound from the insoluble substance. Advantage: instead of many portions of warm solvent being passed through the sample, just one batch of solvent is recycled. This method cannot be used for thermolabile compounds as prolonged heating may lead to degradation of compounds

13 4. Maceration In maceration (for fluid extract), whole or coarsely powdered plant-drug is kept in contact with the solvent in a stoppered container for a defined period with frequent agitation until soluble matter is dissolved. This method is best suitable for use in case of the thermolabile drugs

14 5. Decoction This method is used for the extraction of the water soluble and heat stable constituents from crude drug by boiling it in water for 15 minutes, cooling, straining and passing sufficient cold water through the drug to produce the required volume.

15 6. Infusion It is a dilute solution of the readily soluble components of the crude drugs. Fresh infusions are prepared by macerating the solids for a short period of time with either cold or boiling water

16 7. Digestion It is a type of maceration in which gentle heat is applied during the maceration extraction process. It is used when moderately elevated temperature is not objectionable and the solvent efficiency of the menstrum is increased thereby.

17 8. Percolation It is the procedure used most frequently to extract active ingredients in the preparation of tinctures and fluid extracts. A percolator (a narrow, cone-shaped vessel open at both ends) is generally used. The solid ingredients are moistened with an appropriate amount of the specified solvent and allowed to stand for approximately 4 h in a well closed container, after which the mass is packed and the top of the percolator is closed. Additional solvent is added to form a shallow layer above the mass, and the mixture is allowed to macerate in the closed percolator for 24 h.

18 The outlet of the percolator then is opened and the liquid contained therein is allowed to drip slowly. Additional solvent is added as required, until the percolate measures about three- quarters of the required volume of the finished product. The marc is then pressed and the expressed liquid is added to the percolate. Sufficient solvent is added to produce the required volume, and the mixed liquid is clarified by filtration or by standing followed by decanting

19 9. Sonication The procedure involves the use of ultrasound with frequencies ranging from 20 khz to 2000 khz; this increases the permeability of cell walls and produces cavitation. Although the process is useful in some cases, like extraction of rauwolfi a root, its large-scale application is limited due to the higher costs. Disadvantage: is the occasional but known deleterious effect of ultrasound energy (more than 20 khz) on the active constituents of medicinal plants through formation of free radicals and consequently undesirable changes in the drug molecules

20 Identification of phytochemicals Plant extracts contains various type of bioactive compounds having different polarities their separation still remains a big challenge for the process of identification and characterization of bioactive compounds. In isolation of bioactive compounds different separation techniques are used: such as TLC, HPTLC, paper chromatography, column chromatography, Gas chromatography, and HPLC, should be used to obtain pure compounds. The pure compounds are then used for the determination of structure and biological activity.

21 Chromatography techniques Chromatography is a technique where the molecules are separated based on their size, shape and charge. During chromatography analyte in solvent move through solid phase that acts as a sieving material. As molecule proceeds further through molecular sieve it gets separated. Paper and thin layer chromatography (TLC) are the chromatographic techniques which readily provides qualitative information and through which it become possible to obtain quantitative data.

24 Examples of stationary phases (TLC) Silica gel: Amino acids, alkaloid, sugars, fatty acids, lipid. Aluminium: Alkaloids, phenols, steroids, vitamins and carotenes. Cellulose powder: Amino acids, food dyes, alkaloids

26 Principle of separation of different components Differential affinities (strength of adhesion) of the various components of the analyte towards the stationary and mobile phase results in the differential separation of the components. Affinity, in turn, is dictated by two properties of the molecule: Adsorption and Solubility. We can define adsorption as the property of how well a component of the mixture sticks to the stationary phase, while solubility is the property of how well a component of the mixture dissolves in the mobile phase. Higher the adsorption to the stationary phase, the slower the molecule will move through the column. Higher the solubility in the mobile phase, the faster the molecule will move through the column.

27 Attractive forces

28 1. Adsorption chromatography It is also termed as displacement or liquid/solid chromatography and is based on interactions between the solute and fixed active sites on the stationary phase. The active sites of the stationary phase interact with the functional groups of compounds to be separated by non- covalent bonds, non-polar interactions, van der Waals forces and hydrophobic interactions. The compounds which are loosely bound will be eluted out firstly by the mobile phase at and classes of compounds can be separated.

29 2. Partition chromatography In partition chromatography the molecules to be separated will interact between two immiscible liquid phases according to their relative solubility. This process is also referred as liquid/liquid chromatography.

30 3. Ion-exchange chromatography This allows the separation of ions and polar molecules on the basis of electrical properties of the molecules.

31 4. Affinity chromatography In affinity chromatography, separations are based on the specific interactions between interacting pairs of substances such as macromolecules and it s substrates, cofactor, allosteric effector or inhibitor. During this chromatography, a mixture of substances applied to the columns. Substances that have no affinity with the ligand are washed through with the buffer and desired compound is bind to ligand. Buffer having different ph or an increased ionic strength is used to elutes the analyte out.

32 5. Size exclusion chromatography It also termed as gel filtration, gel permeation chromatography and molecular sieve chromatography. In this chromatography, no chemical attraction or interaction occurs between the solutes and stationary phase and the molecules are separated according to their size.

33 Different types of chromatography; depending on polarity of the stationary phase 1. normal-phase chromatography: the stationary phase is polar (hydrophilic) in nature and the mobile phase is non-polar (hydrophobic) in nature. 2. reverse-phase chromatographic techniques are used for special applications, the scenario is reversed i.e. the stationary phase is non-polar while the mobile phase is polar. There are several types of chromatography, each differing in the kind of stationary and mobile phase they use. The underlying principle though remains the same: differential affinities of the various components of the analyte towards the stationary and mobile phases results in the differential separation of the components

34 normal phase thin layer chromatography is performed on a piece of glass plate that is coated with a thin layer of silica. Here, silica acts as the stationary phase and the solvent in which the plate is dipped and that runs up the plate by capillary action is the mobile phase. The stationary phase i.e. silica is very polar in nature, while the solvent is less polar compared to silica. The polar components of the analyte will adhere to the silica tightly and thus travel slowly up the plate, while the less polar or non-polar components will not adhere that strongly to the silica and travel up the plate relatively fast with the solvent.

35 Classification based on Mobile Phase Mobile Phase Gas Chromatography Liquid Chromatography Gas-solid Gas-liquid Column (gravity flow) Thin layer High Performance (pressure flow) (adsorption)

36 Gas chromatography (GC) Gas chromatography is a method for the separation of volatile compounds. In this method, species distribute between gas and a liquid phase. The gas phase is flowing and the liquid phase is stationary. Species that distribute themselves partly in both phases will migrate at an intermediate rate. Gas chromatography involves a sample being vaporized and injected onto the head of the chromatographic column. The sample is then transported through the column by the flow of inert, gaseous mobile phase. The column itself contains a liquid stationary phase which is adsorbed onto the surface of an inert solid.

37 Paper chromatography In paper chromatography a sheet of paper is used for the inert phase. One of the advantages of paper chromatography is that separations are carried out simply on sheets of filter paper, which acts as both support as well as medium for separation. Another advantage is the considerable reproducibility of Rf (retention factor) values determine on paper. In paper chromatography, filter paper used as solid phase, which is inert phase. A sample is placed near the bottom of the filter paper. Then this filter paper is placed in chromatographic chamber with solvent. The solvent move forwards by capillary action carrying soluble molecules along with it. Low porosity paper will produce a slow rate of movement of the solvent and thick papers have increased sample

38 Rf Value (Retention Factor) In paper and thin layer chromatography the results are represented by Rf value, which represent the movement or migration of solute relative to the solvent front. Rf = Distance travelled by the solute Distance travelled by the solvent front

39 Thin layer chromatography (TLC) Compared to paper chromatography, the special advantage of TLC is the versatility, speedy and sensitive. TLC is an adsorption chromatography where samples are separated based on the interaction between a thin layers of adsorbent attached on the plate. The technique mostly employed for the separation of low molecular weight compounds.

40 As shown above, the three components A, B and C of the reaction mixture travelled different distances, as the solvent moved up the TLC plate. Measured from the origin (where we spotted the reaction mixture): component C travelled 1 cm, component A travelled 2 cms and component B travelled 3 cms. The solvent travelled 5 cms (distance from origin to solvent front).

44 Analytical TLC plates usually have ~ 250 μm thick adsorbent layers. The plates used for preparatory separation can be up to 5 mm thick. A binding agent such as calcium sulfate or gypsum is usually incorporated to ensure the binding of the adsorbent to the solid substrate. Visualization: If the compounds under analysis are colored, they can easily be visualized without any assistance. Most organic compounds, however, are not colored. In most cases, the spots can be detected using ultraviolet light. TLC plates wherein silica gel is impregnated with fluorescent compounds are commercially available. The plates have compounds that fluoresce when excited with the UV light of wavelength 254 nm. The plate, after the developing step, is illuminated with the UV light. The analytes that absorb UV quench the fluorescence and appear as dark spots in the glowing green background. The spots are outlined using a pencil and the plate is taken out of the UV light. In case this method does not work, the bands may be observed by placing the plates with iodine vapors; a large number of organic compounds form a dark-colored complex with iodine. A large number of TLC stains are available for detecting specific classes of molecules

45 Two Dimensional Development In this method the paper or TLC is developed in one direction and after that, the plate is developed in second direction, in same or different solvent system, allowing more compounds to be separated into individual spots.

48 Column chromatography (CC) Column chromatography involves ion exchange, molecular sieves, and adsorption phenomenon. The flushing in conventional chromatography greatly dilutes the material, and the fractions usually require another step for concentration.

Eluent Eluate Elution Analyte fluid entering the column fluid exiting the column (that is collected in flasks) the process

49 Principles of chromatography Several terms that are commonly used in the of chromatography Term Mobile phase or Carrier Definition solvent moving through the column Stationary phase or substance that stays fixed inside the adsorbent column Eluent Eluate Elution Analyte fluid entering the column fluid exiting the column (that is collected in flasks) the process of washing out a compound through a column using a suitable solvent mixture whose individual components have to be separated and analysed

51 Originally referred to as High-Pressure Liquid Chromatography. Now, more commonly called High Performance Liquid Chromatography. It is a form of liquid chromatography used to separate compounds that are dissolved in a solution. Mobile phase is liquid Stationary phase is solid (inside a narrow pressure resistant column).

52 High Pressure Liquid Chromatography (HPLC) Instrumentation 1. Reservoir: One or two reservoir for mobile phase (buffer). 2. Pump: One or two pump to flow the buffer from reservoir. A pump is chosen as per the pressure required to run the mobile phase. (Pressure limit more than bar). 3. Mixer: A mixer is required to mix the buffer received from both pumps to form a linear or step gradient. 4. Column: A column made up of glass or steel. 5. Detector: The elution coming out from column goes to the online monitoring system to test the presence of the analyte based on different properties. There are different types of detectors are known in chromatography such as UV-Visible detector etc. 6. Fraction Collection- The eluent can be collected in different fractions by a fraction collector. 7. Recorder: The profile of eluent with respect to the measured property in a detector can be plotted in the recorder.

54 The solvent is pumped from the solvent reservoir through the pump passing through the filters and the sample is injected through the sample port and gets mixed with the solvent and is passed into the HPLC column and the sample runs on the silica gel. The eluted sample at the end of the column is detected by the detector and the signal is passed to a computer that is converted to a chromatogram and displayed.

58 Technique *Paper chromatography *Thin layer chromatography (TLC) *Liquid column chromatography Size exclusion chromatography Ion-exchange chromatography Stationary phase solid (cellulose) solid (silica or alumina) solid (silica or alumina) Mobile phase Basis of separation Notes liquid polarity of molecules compound spotted directly on a cellulose paper liquid solid (microporous liquid beads of silica) solid (cationic or anionic resin) polarity of molecules glass is coated with thin layer of silica on which is spotted the compound liquid polarity of molecules glass column is packed with slurry of silica liquid size of molecules ionic charge of the molecules small molecules get trapped in the pores of the stationary phase, while large molecules flow through the gaps between the beads and have very small retention times. So larger molecules come out first. In this type of chromatography there isn t any interaction, physical or chemical, between the analyte and the stationary phase. molecules possessing the opposite charge as the resin will bind tightly to the resin, and molecules having the same charge as the resin will flow through the column and elute out first. Affinity chromatography solid (agarose or porous glass beads on to which are liquid immobilized molecules like enzymes and antibodies) binding affinity of the analyte molecule to the molecule immobilized on the stationary phase if the molecule is a substrate for the enzyme, it will bind tightly to the enzyme and the unbound analytes will pass through in the mobile phase, and elute out of the column, leaving the substrate bound to the enzyme, which can then be detached from the stationary phase and eluted out of the column with an appropriate solvent. Gas chromatography liquid or solid support gas (inert gas like argon or helium) boiling point of the molecules samples are volatilized and the molecule with lowest boiling point comes out of the column first. The molecule with the highest boiling point comes out of the column last. *Fall under the category of Liquid Chromatography

Chromatography. writing in color

Chromatography. writing in color Chromatography writing in color Outlines of Lecture Chromatographic analysis» Principles and theory.» Definition.» Mechanism.» Types of chromatography.» Uses of Chromatography. In 1906 Mikhail Tswett used