separation and isolation of plant constituents l.
Skip this Video
Loading SlideShow in 5 Seconds..
Separation and Isolation of Plant Constituents PowerPoint Presentation
Download Presentation
Separation and Isolation of Plant Constituents

Loading in 2 Seconds...

play fullscreen
1 / 29

Separation and Isolation of Plant Constituents - PowerPoint PPT Presentation

  • Uploaded on

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

I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
Download Presentation

PowerPoint Slideshow about 'Separation and Isolation of Plant Constituents' - tavorian

An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.

- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
separation and isolation of plant constituents

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
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
“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
  • 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 (Rf, Rt values)
      • spectral data (UV, IR, MS)
      • biological evaluation
natural products
Natural products
  • 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)

F1 = impelling force (hydrodynamic)

F2 = retarding force (molecular frictional forces)

- Mobile phase

- Stationary phase

more definitions
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.
  • (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
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)
    • 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
    • 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
    • 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
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
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
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
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
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







Ethyl acetate






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

Polar – strong adsorbent affinity, low Rf

↓ active site affinities [Brookmann]

Non-polar – weak adsorbent, high Rf