Transcript 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
– 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 Rf
value 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