P3 L1 Separation and Isolation of Plant Constituents

8/3/2019 P3 L1 Separation and Isolation of Plant Constituents

1/29

Separation and Isolation of

Plant Constituents

Anna Drewwith grateful acknowledgement for inspirational teaching received at

The School of Pharmacy, University of London


2/29

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


3/29

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


4/29

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)


5/29

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


6/29

Natural products

Majority used medicinally are of followingtypes:

alkaloids glycosides

volatile oils

fixed oils

resins

tannins

polysaccharides


7/29

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


8/29

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.


9/29

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.


10/29

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


11/29

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 ()


12/29

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


13/29

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:


14/29

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


15/29

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


16/29

At higher concentrations plateau obtained when all active sites are full

adsorption is concentration independent AVOID in chromatography


17/29

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


18/29

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


19/29

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


20/29

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


21/29

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


22/29

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


23/29

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


24/29

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


25/29

[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


26/29

[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


27/29

Light petroleum

Cyclohexane

Toluene

BenzeneDichloromethane

Chloroform

Ether

Ethyl acetate

Acetone

N-propanol

Ethanol

Water

PyridineAcetic acid

[Trapps, 1940]

elutingpower

increasing


28/29

[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


29/29

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

Documents

P3 L1 Separation and Isolation of Plant Constituents