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:

• 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)

F1 = impelling force (hydrodynamic)F2 = retarding force (molecular frictional

forces)

- Mobile phase

- Stationary phase

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:– 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 = cA / 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 (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

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 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

[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 [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:

• 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