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Separation and Isolation of Plant Constituents

Separation and Isolation of Plant Constituents. Anna Drew with grateful acknowledgement for inspirational teaching received at The School of Pharmacy, University of London. Plants -> chemicals. Secondary metabolites (primary metabolites sugars, amino acids etc

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Separation and Isolation of Plant Constituents

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  1. Separation and Isolation of Plant Constituents Anna Drew with grateful acknowledgement for inspirational teaching received at The School of Pharmacy, University of London

  2. Plants -> chemicals • Secondary metabolites • (primary metabolites • sugars, amino acids etc • essential functions eg absorbing water) • Many functions • (until 1990s thought to be waste products) • growth • sensory devices – proteins in light-sensitive compounds • roots can detect nitrates and ammonium salts in soil • reproduction • produce chemicals to attract pollinators • protection • bioactive compounds that affect living cells • eg caterpillar eating leaf produce chemical to attract wasp

  3. “Crude drugs” • dried plant parts used in medicinal preparations • complex mixtures of cells and chemicals • previously many used in form of alcoholic extracts (tinctures) • today pure isolated active principles used • not always possible: • difficult to separate – more economic to use extracts • unstable when isolated • active principles not known – activity thought from mixture • pharmacist needs basic knowledge of the ways in which drug plants can be extracted and tested for presence of active principles • quality assurance

  4. Isolation • dried powdered plant material • extracted with solvent • by maceration or percolation • unwanted or insoluble material filtered off • extract concentrated • to low volume under reduced pressure • (minimum decomposition of thermolabile substances) • further purification • to remove unwanted chemicals • chlorophylls, pigments, fats, waxes, oils, resins, proteins, carbohydrates • using one or more: • partition between immiscible solvents (to separate un/wanted) • selective precipitation by adding selected reagents • chromatographic techniques or physical processes (crystallisation, distillation)

  5. Purity • … of isolated active principle via specific tests: • melting point • boiling point • optical rotation • chemical tests* • chromatographic data (Rf, Rt values) • spectral data (UV, IR, MS) • biological evaluation

  6. Natural products • Majority used medicinally are of following types: • alkaloids • glycosides • volatile oils • fixed oils • resins • tannins • polysaccharides

  7. CHROMATOGRAPHY “The uniform percolation of a fluid through a column of finely divided substance, which selectively retards certain components of a mixture” (Martin) F1 = impelling force (hydrodynamic) F2 = retarding force (molecular frictional forces) - Mobile phase - Stationary phase

  8. More definitions • Stationary phase: • solid or liquid • facilitates separation by selectively retarding the solute by: • Adsorption (adsorption chromatography) • Partition (partition chromatography) • Mobile phase: • moving solvent flowing over stationary phase that takes solutes with it. Gas or liquid.

  9. Solid support: • in partition chromatography stationary liquid must be held in position on an inert support material. This is solid support and is evenly coated with stationary liquid. • Elution: • when the separation of solutes is complete they are recovered from the stationary phase (solid or liquid) by washing with suitable solvent.

  10. Classification • (1) Closed column chromatography • stationary phase is packed inside a column • mobile phase + solute flows through the column -> separation • two forms according to mobile phase type • Liquid chromatography • Gas chromatography • (2) Open column chromatography (a) Paper chromatography • sheet of paper is used to support the stationary phase (b) Thin-layer chromatography • adsorbent is spread evenly over the surface of a flat sheet of glass

  11. Mechanisms of separation • depends on distribution of solutes between mobile and stationary phase • Adsorption: between liquid and solid phases • Partition: between two liquids or gas/liquid phase • distribution ratio: • ratio of amount of solute retained in one phase to the amount in the other • Adsorption coefficient (a) • Partition coefficient (α)

  12. ADSORPTION • in a solid/liquid two phase system higher concentration of solute molecules will be found at the surface of the solid than in liquid phase • arises because of attraction between surface molecules of solid and molecules in liquid phase (1) Chemisorption • irreversible - chemical interaction between solute and solid surface (2) Physical adsorption • reversible – electrostatic forces, dipole interactions, Van de Waal’s forces

  13. in a dilute solution adsorption of a solute may be described by the empirical Freudlich equation: x/m = kcn x/m = amount adsorbed per unit weight of adsorbent k & n = constants c = concentration • if x/m is plotted against concentration an isotherm is obtained:

  14. equation holds • at constant temperature • over limited concentration range • assumptions • no chemisorption occurs • only a mono-layer is formed • the number of active sites is constant and propertional to adsorbent weight

  15. However a solution is a binary system and • preferential adsorption depends on • solute-solvent interactions • solute-solvent affinities for the adsorbent surface • In fact a composite isotherm is produced • both molecular species at solid surface • If more than one solute present • competition for active sites on adsorbent surface • chromatographic separation not always predictable • Freudlich equation only holds true for • dilute solutions - concentration dependent adsorption

  16. At higher concentrations • plateau obtained when all active sites are full • adsorption is concentration independent • AVOID in chromatography

  17. Chromatography • only dilute solutions used • on relatively weak adsorbents • separation by physical adsorption • Factors affecting adsorption • govern migration of solute • depend on relative strengths of following molecular interactions: • solute – solute • solute – solvent • solvent – solvent • solute and solvent affinities for active sites • effect of molecules in adsorbed state

  18. PARTITION • if a solute in introduced into a system of two liquid phases and is soluble in both it will distribute itself between the phases according to its relative solubility in each • function of the nature of solvent and solute • ratio in which it distributes itself is the partition coefficient (α) • constant at • constant temperature • over a limited range of concentration α = cA / cB cA and cB are solute concentrations in solvents A and B

  19. equation describes a partition isotherm • linear over a greater range of concentrations • if more than one solute present • (always the case in chromatography) • distribution of each solute is independent of others

  20. Ion exchange • … consists of an insoluble matrix with chemically bound charged groups and mobile counter ions • the counter ion reversibly exchanges with other ions of the same charge without any changes to the insoluble matrix: • separation of a mixed solute consists of binding all solute to matrix then recovering one bound species at a time • conditions (pH, ionic strength) required to liberate species are determined by electrical properties

  21. Diffusion methods • molecular diffusion can be used to separate a mixed solute • in absence of specific binding factors, the rate of diffusion of solute in a stabilising medium (semi-permeable membrane, gel) depends on • radius of solute molecule • viscosity of medium • temperature • can be considered to contain pores • allows certain size molecules to diffuse through • when washed through a column or along a thin film of gel with solvent larger molecules will move further

  22. Electrophoretic mobilities • consider a zone of solute in a stabilising gel – will diffuse slowly to equilibrium • in the absence of specific binding effects, movement can be directed by applying an electric potential across the gel • molecules acquire charges in aqueous solution and move according to: • charge on the species • electric retarding force due to counter-ion atmosphere • viscous resistance of medium (giving different mobility) • constants of the apparatus

  23. Chromatography isotherms • mechanism of separation is never completely one of the following: • adsorption • partition • ion-exchange • diffusion • mixture of all –> “sorption” isotherms • describes conditions encountered not process

  24. Factors affecting migration: [1] The adsorbent • Classified into polar and non-polar types [->] • Non-polar • weak adsorbent forces – Van de Waal’s forces • Polar • stronger - dipole forces, hydrogen bonding between active site on solid surface and solute • Strength of adsorbent modified by • Particle size • surface area – more active sites if smaller • Moisture content • higher with polar adsorbents (free moisture held by H-bonding) • heating will drive off moisture

  25. [A] Strong polar adsorbents • low water content alumina • Fullers Earth • charcoal • silicic acid [B] Medium polar adsorbents • high water content alumina • silica gel • magnesium hydroxide • calcium carbonate [C] Weak adsorbents • Polar: • sugar • cellulose • starch • Non-polar: • talc • Kieselguhr and celite

  26. [2] Nature of solvent • Graded by powers of elution [->] • more polar the solvent greater eluting power • in open-column chromatography pushed further • adsorption strongest from non-polar solvents in which solute is sparingly soluble • solvent-solute affinity weak • solute-adsorbent affinity strong • moderate or non-polar base solvent is chosen • other solvents are added to increase or decrease Rf value according to nature of solutes to be separated

  27. Light petroleum Cyclohexane Toluene Benzene Dichloromethane Chloroform Ether Ethyl acetate Acetone N-propanol Ethanol Water Pyridine Acetic acid [Trapps, 1940] ↓ eluting power increasing ↓

  28. [3] Structure of solute [A] Molecular weight • Non-polar adsorbents: • adsorption increases (Rf value ↓) with increased molecular weight [Traube’s Rule] • Polar adsorbents: • adsorption decreases with increased molecular weight [Reverse Traube’s Rule] • polar groupings between solute-adsorbent important • side chain dilutes this [B] Nature of constituent groups • functional groups which H-bond • dipole interactions • ionised forms • play major roles in determining solute migration

  29. Alkaloids - pKa of nitrogen group important • bases of varying strengths • ionise at different pH’s • ionised form more strongly adsorbed than un-ionised form • pH of solvents and stationary phase has to be controlled • some have more than one ionised form due to more than one basic group • - > multi-spot formation • Substituents groups modify effects of pKa and molecular weight on migration: • R-COOH • R-OH • R-NH2 • R-COOCH3 • R-N(CH3)2 • R-NO2 • R-OCH3 • R-H • Unsaturation in a molecule -> lower Rf • eg aromatic rings – due to greater electron density associate with π orbital electrons in the ring Polar – strong adsorbent affinity, low Rf ↓ active site affinities [Brookmann] Non-polar – weak adsorbent, high Rf

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