Download Separation and Isolation of Plant Constituents

Separation and Isolation of
Plant Constituents
Anna Drew
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
“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
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)
Purity
• … of isolated active principle via specific
tests:
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melting point
boiling point
optical rotation
chemical tests*
chromatographic data (Rf, Rt values)
spectral data (UV, IR, MS)
biological evaluation
Natural products
• Majority used medicinally are of following
types:
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Alkaloids
Glycosides
Volatile oils
Fixed oils
Resins
Tannins
Polysaccharides
CHROMATOGRAPHY
“The uniform percolation of a fluid through a
column of finely divided substance, which
selectively retards certain components of a
mixture” (Martin)
- Mobile phase
- Stationary phase
F1 = impelling force (hydrodynamic)
F2 = retarding force (molecular frictional
forces)
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.
• 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.
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
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)
• 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
• 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:
• 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
• 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
• At higher concentrations
• plateau obtained when all active sites are full
• adsorption is concentration independent
• AVOID in chromatography
• 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:
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solute – solute
solute – solvent
solvent – solvent
solute and solvent affinities for active sites
effect of molecules in adsorbed state
• 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 = c A / cB
cA and cB are solute concentrations in solvents A and B
• 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
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
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 (semipermeable 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
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:
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charge on the species
electric retarding force due to counter-ion atmosphere
viscous resistance of medium (giving different mobility)
constants of the apparatus
Chromatography isotherms
• Mechanism of separation is never
completely one of the following:
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Adsorption
Partition
Ion-exchange
Diffusion
• Mixture of all –> “sorption” isotherms
• describes conditions encountered not process
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 Hbonding)
» heating will drive off moisture
[A] Strong polar adsorbents
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low water content alumina
Fullers Earth
charcoal
silicic acid
[B] Medium polar adsorbents
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high water content alumina
silica gel
magnesium hydroxide
calcium carbonate
[C] Weak adsorbents
– Polar:
» sugar
» cellulose
» starch
– Non-polar:
» talc
» Kieselguhr and celite
• [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 Rfvalue according to nature of solutes to be separated
Light petroleum
Cyclohexane
Toluene
Benzene
Dichloromethane
Chloroform
Ether
Ethyl acetate
Acetone
N-propanol
Ethanol
Water
Pyridine
Acetic acid
[Trapps, 1940]
↓
eluting
power
increasing
↓
[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
• 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:
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R-COOH
R-OH
R-NH2
R-COOCH3
R-N(CH3)2
R-NO2
R-OCH3
R-H
Polar – strong adsorbent affinity, low Rf
↓ active site affinities [Brookmann]
Non-polar – weak adsorbent, high Rf
• Unsaturation in a molecule -> lower Rf
• Eg aromatic rings – due to greater electron density associate with π
orbital electrons in the ring