S3 L1 Separation and Isolation of Plant Constituents
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Transcript of S3 L1 Separation and Isolation of Plant Constituents
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Separation and Isolation of Plant Constituents
Anna Drew
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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
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“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
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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)
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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
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Natural products
• Majority used medicinally are of following types:
• Alkaloids• Glycosides• Volatile oils• Fixed oils• Resins• Tannins• Polysaccharides
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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
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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.
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• 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.
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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
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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)
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• 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
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• 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:
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• 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
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• 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
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• At higher concentrations• plateau obtained when all active sites are full• adsorption is concentration independent• AVOID in chromatography
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• 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
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• 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
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• 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
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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
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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
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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
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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
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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
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[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
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• [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
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Light petroleum
Cyclohexane
Toluene
Benzene
Dichloromethane
Chloroform
Ether
Ethyl acetate
Acetone
N-propanol
Ethanol
Water
Pyridine
Acetic acid
[Trapps, 1940]
↓
eluting power increasing
↓
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[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
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• 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
↓ active site affinities [Brookmann]
Polar – strong adsorbent affinity, low Rf
Non-polar – weak adsorbent, high Rf