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:
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:
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 ()

 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 Waals 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:
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 ()
constant at
constant temperature over a limited range of concentration

= 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:
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:
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 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 Hbonding) heating will drive off moisture

[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

 [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 [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

 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 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

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 orbital electrons in the ring