p3 l1 separation and isolation of plant constituents
TRANSCRIPT
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Separation and Isolation of
Plant Constituents
Anna Drewwith grateful acknowledgement for inspirational teaching received at
The School of Pharmacy, University of London
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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 whichdrug plants can be extracted and tested for presence ofactive 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 followingtypes:
alkaloids glycosides
volatile oils
fixed oils
resins
tannins
polysaccharides
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CHROMATOGRAPHY
The uniform percolation of a fluid through acolumn of finely divided substance, whichselectively 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 supportmaterial. This is solid support and is evenlycoated with stationary liquid.
Elution: when the separation of solutes is complete
they are recovered from the stationary phase(solid or liquid) by washing with suitablesolvent.
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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 betweenmobile 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 tothe amount in the other
Adsorption coefficient (a)
Partition coefficient ()
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ADSORPTION in a solid/liquid two phase system higher
concentration of solute molecules will befound at the surface of the solid than in liquidphase
arises because of attraction between surfacemolecules of solid and molecules in liquidphase
(1) Chemisorption irreversible - chemical interaction between solute and
solid surface(2) Physical adsorption
reversible electrostatic forces, dipole interactions, Vande Waals forces
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in a dilute solution adsorption of a solutemay be described by the empirical
Freudlich equation:
x/m = kcn
x/m = amount adsorbed per unit weight of adsorbentk & n = constants
c = concentration
if x/m is plotted against concentration anisotherm 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 toadsorbent 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 followingmolecular 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 willdistribute itself between the phases accordingto its relative solubility in each
function of the nature of solvent and solute
ratio in which it distributes itself is the partitioncoefficient ()
constant at constant temperature
over a limited range of concentration
= cA / cBcA 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 boundcharged groups and mobile counter ions
the counter ion reversibly exchanges with other ions ofthe same charge without any changes to the insoluble
matrix:
separation of a mixed solute consists of binding all soluteto matrix then recovering one bound species at a time
conditions (pH, ionic strength) required to liberatespecies are determined by electrical properties
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Diffusion methods
molecular diffusion can be used to separate amixed 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 gelwith solvent larger molecules will move further
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Electrophoretic mobilities
consider a zone of solute in a stabilising gelwill diffuse slowly to equilibrium
in the absence of specific binding effects,
movement can be directed by applying anelectric potential across the gel
molecules acquire charges in aqueous solutionand 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 nevercompletely 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 forcesVan de Waals 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 inwhich 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
BenzeneDichloromethane
Chloroform
Ether
Ethyl acetate
Acetone
N-propanol
Ethanol
Water
PyridineAcetic acid
[Trapps, 1940]
elutingpower
increasing
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[3] Structure of solute
[A] Molecular weight
Non-polar adsorbents: adsorption increases (Rf value ) with increased
molecular weight [Traubes Rule]
Polar adsorbents: adsorption decreases with increased molecular weight
[Reverse Traubes 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 pHs
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 onebasic 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 bit l l t i th i
active site affinities [Brookmann]
Polar strong adsorbent affinity, low Rf
Non-polar weak adsorbent, high Rf