1. Separation and Isolation of Plant Constituents
Anna Drew

2. 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

3. “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

4. 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)

5. 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

6. Natural products Majority used medicinally are of following types:
Alkaloids
Glycosides
Volatile oils
Fixed oils
Resins
Tannins
Polysaccharides

7. 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

8. More definitions Stationary phase: Mobile 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.

9. Solid support: Elution:
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.

10. 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

11. 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)

12. 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

13. If x/m is plotted against concentration an isotherm is obtained:
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:

14. Equation holds Assumptions 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

15. 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

16. At higher concentrations
plateau obtained when all active sites are full
adsorption is concentration independent
AVOID in chromatography

17. Factors affecting adsorption
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

18. 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

19. 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

20. 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

21. 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

22. 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

23. 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

24. 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

25. [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

26. [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

27. Light petroleum Cyclohexane Toluene Benzene Dichloromethane Chloroform
Ether
Ethyl acetate
Acetone
N-propanol
Ethanol
Water
Pyridine
Acetic acid
[Trapps, 1940] ↓
eluting power increasing

28. [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

29. 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
Polar – strong adsorbent affinity, low Rf
↓ active site affinities [Brookmann]
Non-polar – weak adsorbent, high Rf