Anna Drew

• 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

• 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

• 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)

• … of isolated active principle via specific tests:
• melting point
• boiling point
• optical rotation
• chemical tests*
• chromatographic data (R f
, R t
• spectral data (UV, IR, MS) values)
• biological evaluation

• Majority used medicinally are of following types:
• Alkaloids
• Glycosides
• Volatile oils
• Fixed oils
• Resins
• Tannins
• Polysaccharides

“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)

• 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.

• (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

• 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:
n 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 = c
A
/ c
B c
A and c
B 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

• … 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

• 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

• 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

• 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

[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
– 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 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:
• 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