Extraction, Fractionation and Isolation

of Natural Products

Dr. A. K. Yadav

Assistant Professor-Chemistry

Maharana Pratap Govt. P.G. College, Hardoi

Cast aside for years, natural products drug discovery appears to be

reclaiming attention and on the verge of a comeback

According to a recent survey by David J. Newman, Gordon M. Cragg, and

Kenneth M. Snader of the National Cancer Institute, 61% of the 877 small-

molecule new chemical entities introduced as drugs worldwide during 1981–

2002 can be traced to or were inspired by natural products. These include

Natural products (6%),

Natural product derivatives (27%),

Synthetic compounds with pharmacophores derived from natural-product-

(5%),

Natural product mimic; a synthetic compounds designed on the basis of

knowledge gained from a natural product (23%).

Rediscovering Natural Products

Journal of Natural Products 2003, 66, 1022 Natural Product Report 2005, 22, 162.

Drug Likeness of Natural Products

J. Chem. Inf. Comput. Sci., 2003, 43, 218.

The plots show greater similarity in the

distributions of drugs and natural products

than in the distributions of combinatorial

compounds and natural products. It is

suggest therefore, that combinatorial

libraries that mimic the distribution

properties of natural products might be more

biologically relevant.

In chemical diversity space, combinatorial

compounds densely populate a small area,

whereas natural products are more spread

out.

An extract can be prepared by following various extraction techniques

depending upon the requirement and type of compounds needed.

Following are the general methods to perform an extraction;

Maceration; Extraction with shaking at room temperature.

Percolation; Most commonly used continuous process where saturated

solvent is replaced by fresh solvent until exhaustive extraction achieved.

Counter current extraction; Moving extractive material is extracted by

a liquid phase flowing against it.

Hot continuous extraction (Soxheletion); Performed in Soxhlet

apparatus with refluxing solvent. It can be a good choice when

exhaustive extraction is required. Major drawback is use of higher

temperature.

Infusion and Decoction; Extraction with cold / boiling water.

Steam Distillation; Distillation of volatile oils with steam. Extensively

used for essential oils and aroma chemicals extractions.

Electrodischarge and Ultrasonic extraction methods are of very limited

scope.

Extraction / Fractionation Techniques

Microwave Assisted Extraction (MAE)

Microwaves interact selectively with the molecules present in glands,

trichomes or vascular tissues. Localised heating leads to the expansion and rupture of

cell walls and is followed by the liberation of essential oils into the solvent.

Accelerated Solvent Extraction (ASE)

Accelerated solvent extraction (ASE) is a SLE technique which is an

alternative to current extraction methods such as Soxhlet, maceration, percolation or

reflux. ASE uses organic solvents at elevated pressure and temperature in order to

increase the efficiency of the extraction process.

Recent Extraction techniques Supercritical Fluid Extraction (SFE)

At temperatures and pressures above the critical point there exist a fluid

called supercritical fluid, which can dissolve wide variety of organic compounds and

their solvent power can be varied near their critical point by small pressure and

temperature changes. They have very superior mass transfer properties by virtue of

their low viscosities, high diffusivity and ability to penetrate microporous materials.

SCO2 with critical temp. 304 K and critical pressure 73 atm with added

benefits like nontoxicity, nonflammability, noncorrosiveness, chemical inertness, cost

effectiveness and environmental acceptability is preferred solvent for many

supercritical fluid extractions.

Sepbox concept is based on a combination of High

Performance Liquid Chromatography (HPLC) together

with Solid Phase Extraction (SPE) to enhance the

throughput in natural product reserach.

With this technology a large number of samples can be

efficiently processed.

Up to 600 samples for HTS can be obtained with a

maximum of purity and more than 90% recovery rate

using 2-dimensional separation, both for polar and non-

polar substances.

For highly polar substances an optional polar setup can

be integrated.

SEPBOX

Typical SEPBOX setup

Extraction Procedure Followed at CDRI

Plant Material

Extraction with EtOH

Ethanolic extract

Macerated with Hexane

ChloroformFraction

n-ButanolFraction

AqueousFraction

Residue in Water

Partitioned between water and n-butanol

HexaneFraction

Residue

Partitioned between Chloroform and water

Isolation/Chromatographic techniques

Nobel Prize in Chemistry 1952, was awarded to Archer J.P. Martin and

Richard L.M. Synge for developing partition chromatography.

Michael Tswett (1872-1920) is credited with developing and publishing

the first concept and technique of chromatography (column chromatography)

while trying to analyze vegetable pigments chlorophyll, xanthophyll, and

carotene . Chemistry from its beginning is concentrated to the large extent upon

the study of natural products, which are obtained from plants , animals or

microorganisms. The primary objective of a natural product chemist is to isolate

the substance of interest in pure form and of a synthetic chemist is to obtain

desired product from the mixture in pure form.

From the very beginning of chemistry methods of separating

substances have occupied a key position in the development of science. Even

today, in Holland , chemistry is called “Scheikunde” or “the art of separation”

Chromatography is a physical method of separating mixtures of organic

compounds, biomolecules, organic and inorganic salts, etc., by distribution or

partition between two phases. One of the phases is stationary phase and other is

the mobile phase which moves through the stationary phase. The basic principle

of separation is that, substance to be separated should have different relative

affinities for the stationary and mobile phase. Thus a substance with relatively

higher affinity for the stationary phase moves with a lower velocity, through the

chromatographic system.

For compound “X” the characteristic way in which it gets distributed between

two phases can be expressed in terms of its distribution coefficient.

[X]stationary phase

KD=

[X]Mobile phase

Introduction

Chromatographic Methods

Chromatographic methods can be classified on two basis:

1. According to nature of stationary & mobile phase.

2. According to the mechanism of separation.

1. According to the nature of stationary and mobile phase:

A Solid stationary phase with fluid mobile phase:

Column chromatography.

Thin layer chromatography.

High performance liquid chromatography.

Paper chromatography.

Gas chromatography.

B Fluid stationary phase with a fluid mobile phase.

Liquid chromatography.

Gas liquid chromatography.

Adsorption Chromatography

(Solid stationary phase)

Liquid mobile phase: CC, TLC,

Ion-Exchange etc.

Gas mobile phase: GC.

Partition Chromatography

(Liquid stationary phase)

Liquid mobile phase: Paper

Chromatography, HPLC.

Gas mobile phase: GLC.

It can also be categorized in terms of the mobile phase selected i.e Liquid

Chromatography (CC, TLC, HPLC, IEC etc.) and Gas chromatography (GC,

GLC).

2. According to the mechanism of separation.

Ion Exchange Chromatography

In this type of chromatography, the use of a resin (the stationary solid

phase) is used to covalently attach anions or cations onto it. Solute ions of the

opposite charge in the mobile liquid phase are attracted to the resin by

electrostatic forces.

Molecular Exclusion Chromatography Also known as gel permeation or gel filtration, this type of

chromatography lacks an attractive interaction between the stationary phase and

solute. The solute passes through a porous gel which separates the molecules

according to its size.

Affinity Chromatography

It utilizes the specific interaction between one kind of solute molecule

and a second molecule that is immobilized on a stationary phase. For example,

the immobilized molecule may be an antibody to some specific protein. When

solute containing a mixture of proteins are passed by this molecule, only the

specific protein is reacted to this antibody, binding it to the stationary phase. This

protein is later extracted by changing the ionic strength or pH.

Adsorption Chromatography

Adsorbents Nature

Silica Gel Acidic

Alumina Acidic, basic and neutral

Magnesium Silicate Acidic

Charcoal Neutral and acidic

Sucrose Neutral

Starch Neutral

Adsorbant:

Adsorbents are finely divided , porous particles with

large surface area for adsorption.

Silica gel and alumina are the two most common adsorbents. They

are cheap and readily available commercially.

Silica gel (SiO2xH2O): Surface of the silica gel particles are covered by

hydroxyl groups.

Alumina, acidic (Al2O3): On heating hydrated alumina Al2O3xH2O to 300-

4000C, most of the adsorbed water is drawn off and remaining water reacts with the

surface to form hydroxyl group. Like silica gel it is also highly polar, acidic (pH-4)

solid stationery phase.

Alumina, basic (Al2O3): On heating hydrated alumina Al2O3xH2O to 800-

10000C, total water is drawn off and oxide ions now give the basic property to

alumina. It is also polar, solid stationery phase.

Si

O

O

Si-O Si OH

O

O

O

O

Si

O

Si-O Si OH

O O

Bulk (SiO2)xSurface

Nature of Adsorption forces

Adsorption forced are purely physical in nature and are sum of the van der

Walls’ forces, inductive effects and hydrogen bonding. Polar compound bind

strongly to the polar stationary phase and less polar or non polar compounds

bind weakly to a polar stationary phase.

van der Walls’ forces: Intermolecular forces which held molecules together

in the liquid or solid state.

Inductive effects: Compounds with permanent dipole moments bind to the

stationery phase through their dipolar interactions.ex. C=O, C-O, C-X, C-N

etc.

Hydrogen bonding: Compounds with polar functional groups such as OH,

NH2, COOH, etc. bind with stationary phase surfaces using hydrogen

bonding.

Solvent System

Compounds in the mixture have different adsorption

strengths towards the stationary phase. Their actual

separation can be done only when a mobile phase moves

through the stationary phase. The mobile phase is a pure

organic solvent or solvent mixture and is called eluent.

The increasing order of the polarity of the common organic

solvents are as follows:

Hexane< Cyclohexane < Benzene < DCM < Chloroform <

Ethyl acetate < Acetone < Ethanol < Methanol < Water <

Acetic acid

Less polar compounds are displaced or desorbed by the less

polar eluent from their adsorption and for more polar

compounds which adsorb strongly on polar stationary phase a

polar mobile phase is required.

TLC and CC

Extensively used of all the chromatographic methods for separation of organic

compounds. Theoretical basis for separation in both is adsorption

chromatography. While CC is used to effect large scale quantitative separations

to give pure compounds, TLC needs an extremely small quantity of sample (less

than 1 mg) and an extremely short time for qualitative separation of the

compounds in the sample.

Rf (retardation factor) value: TLC profile of a compound is expressed in terms of

its Rf value in the given conditions of adsorbent and solvent system.

Rf =Distance moved by compound / Distance moved by solvent front.

Applications of TLC

Determination of purity of the sample.

Select the starting eluent for CC.

Monitoring the progress of CC or organic reaction.

Identification of known compounds by comparison with standards.

Effects small scale (10-100 mg) quantitative separation of mixtures through

preparative chromatography.

TLC - Retention Factor (Rf)

The retention factor, or Rf, is defined as the

distance traveled by the compound divided by the

distance traveled by the solvent.

For example, if a compound travels 2.1 cm and the

solvent front travels 2.8 cm, the Rf is 0.75:

Distance travelled by compound

Rf =

Distance travelled by solvent

Schematic representation of column chromatographic separation

Partition Chromatography

Partition chromatography is based on the ability of

the solute molecules to distribute between two

liquid phases. Generally one of the liquid phases is

immobilized on the packing material. The mobile

phase of different composition to the stationary

liquid phase, is then passed through the column.

According to the mobile phase selected they are of

two types

Liquid mobile phase: ex. HPLC, MPLC etc.

Gas mobile phase: GLC.

Stationary phases (Adsorbents)

Packing adsorbents are available in a vide variety of materials ex. silica gel,

polyacrylamide,carbohydrates etc.

Physical state descriptors of the packing adsorbents consist of particle size,

shape, porosity, and surface area.

Main adsorbent parameters for HPLC are:

Particle size: 3 to 10 µm (10-200 µm for CC)

Particle size distribution: as narrow as possible, usually within 10% of the

mean;

Pore size: 70 to 300 Å;

Surface area: 50 to 250 m2/g

Bonding phase density (number of adsorption sites per surface unit): 1 to 5

per 1 nm2

Depending on the type of the ligand attached to the surface, the adsorbent

could be

normal phase (-OH, -NH2).

reversed-phase (C8, C18, Phenyl).

anion (NH4+), or cation (-COO-) exchangers.

Common adsorbants

Bonded silica gel: Silica can be chemically modified in a number of ways

that alter both its chromatographic and physical properties. As shown below

the reactive silanol groups can be blocked with a variety of silylchlorides to

produce a non polar (Reverse phase) or polar (Normal phase) chromatography

support

Si

O

O

Si-O Si OH

O

O

O

O

Si

O

Si-O Si OH

O O

Bulk (SiO2)x Surface

Si

O

O

Si-O Si O

O

O

O

O

Si

O

Si-O Si O

O O

Bulk (SiO2)x Surface

Reverse phase

R = C18H37 (octadecylsilyl deriv. silica “C18”),

R = C8H17 (octylsilyl deriv. silica or “C8”)

Normal phase

R = Functionalized silanes (amino, diol-, cyano-, etc.)

R Si Cl

CH3

CH3

SiR

H3C

CH3

Si

H3C

CH3

R

The different preparative pressure liquid chromatographic

methods are usually classified in four categories according to

the pressure employed for the separation, namely

LIQUID CHROMATOGRAPHY

Flash chromatography (pressure approx. 2 bar),

Low pressure liquid chromatography (< 5 bar),

Medium-pressure liquid chromatography (5 – 20 bar), and

High-pressure (high-performance) liquid chromatography (> 20 bar).

High Performance Liquid Chromatography (HPLC)

HPLC is characterized by the use of high pressure to push a mobile

phase solution through a column of stationary phase allowing

separation of complex mixtures with high resolution.

TLC vs. HPLC

Type of Analysis Qualitative only Qualitative & quantitative

Stationary Phase 2-dimensional

thin layer plate

3-dimensional column

Instrumentation minimal! much! with many adjustable parameters

Sample

Application

spotting

(capillary)

Injection

Mobile Phase

Movement

capillary action

(during development)

high pressure (solvent delivery)

Visualization of

Results

UV lightbox “on-line” detection (variable UV/Vis, RI,

Electrochemical etc.)

Form of Results spots, Rf’s (retention

factors)

peaks, Rt’s (retention times)

HPLC System

HPLC Solvent

Reservoirs

Pump (flow capacity of at

least 10-100 ml/min).

Variable

UV/Vis Detector

Injector

HPLC

Column

Detector Computer

Workstation

HPLC Chromatograms

Rt = 3.0 min.

faster moving

less retained

Rt = 5.2 min.

slower moving

more retained

0 1 2 3 4 5 6 7

Time (minutes)

Ab

sorb

ance

Peak A Peak B

Normal Phase Reversed

Phase

Stationary

phase

Polar (silica

gel, alumina)

Non-polar

(C18, C8)

Mobile phase

Non-polar

(organic

solvents)

Polar

(aqueous/organ

ic)

Sample

movement

Non-polar

fastest

Polar fastest

Separation

based on

Adsorption

partition

Normal vs. Reversed Phase Chromatography

Gas Chromatography

Factors which affect GC separations

Efficient separation of compounds in GC is dependent on the

compounds traveling through the column at different rates. The rate at

which a compound travels through a particular GC system depends on

the factors listed below:

•Volatility of compound: Low boiling (volatile) components will

travel faster through the column than will high boiling components

•Polarity of compounds / Column : Polar compounds will move

more slowly, especially if the column is polar and vice versa.

•temperature: Raising the column temperature speeds up all the

compounds in a mixture.

•Flow rate of the gas through the column: Speeding up the carrier

gas flow increases the speed with which all compounds move

through the column.

•Length of the column: The longer the column, the longer it will

take all compounds to elute. Longer columns are employed to

obtain better separation.

Ion Exchange Chromatography

This technique is used for separation of the charged molecules such as cations (K+, Na+, Ca++, Cu++, Lanthanides etc.), anions (Cl-, Br-, I-, etc.), amino acids, proteins or neutral molecules that can develop a charge in acidic or basic media such as carboxylic acids and amines.

The basis of the ion-exchange separation is the reversible binding of charged molecules, Charged functional groups (G) attached to some support matrix (R) (polystyrene resin, carbohydrate polymers and silica gel), can interact with oppositely charged solute molecules (S), resulting in retention on the column, and displacement of the counter-ion (C).

Depending on the attached charged group IEC can be divided in to two categories.

Cation-exchange chromatography.

RG- C+ + S+ = RG- S+ + C+

Anion-exchange chromatography.

RG+ C- + S- = RG+ S- + C-

Ion exchange on solute molecules can often be manipulated by adjusting the pH.

Theory of ion-exchange

The success and failure of an ion-exchange experiment clearly

depends on the relative affinity of functional group towards

various ions and their relative concentrations.

Polyvalent ions have, in general greater affinity for a charged

stationary phase than monovalent ions.

Ions that are more polarizable will have higher affinity.

Affinity for some common anions towards anion-exchanger is

generally as follows:

OH-< CH3COO-< HCOO-< Cl-< PO4-

Affinity for some common cations towards cation-exchanger is

generally as follows:

Li+< H+< Na+< NH4+< Ca++

These relative affinities constitute one of the major factors

involved in designing an ion-exchange experiment.

Polyacrylamide:

Gel based on copolymerization of N,N’-methylene-bis-acrylamide and

acrylamide.

They are fairly inert, free of charge hence suitable for labile compounds.

These gels are extremely hydrophilic.

They swell in water and are used with water as mobile phase often.

chromatographic interactions revolve around hydrogen bonding.they are suitable

for chromatography of carbohydrates, peptides, tannins etc.

Molecular Exclusion Chromatography

Size exclusion chromatography, also known as gel permeation or filtration

chromatography does not involve any adsorption and is extremely fast. The

packing is a porous gel, and is capable of separating large molecules from

smaller ones. The larger molecules elute first since they cannot penetrate the

pores. This method is common in protein separation and purification.

NH2

O

HN

O O+

H2N HN

HN

H2N

O

O

O

OO

H2NO

H2N

HN

HN

O

OO

H2N

Polyacrylamide gels

Fractionation range of Bio-Gel gels

Gel type Fractionation range (Mol.

Wt.)

Swollen volume in water,

ml/g dry beads

P-2 100-2000 3

P-4 800-4000 4

P-6 1000-6000 6.5

P-10 1500-20,000 7.5

Carbohydrates:

Some of the most useful methods for the chromatography of labile natural

products are the fairly inert polymers of carbohydrates. Polysaccharides can

be crosslinked to produce three-dimensional networks and hence can be

formed in to beads.

Sephadex is such a gel formed by crosslinking of the water soluble dextran

with epichlorohydrin. The resulting water soluble polymer has glycerin-ether

bonds as the crosslinker which swells in water and degree of swelling

determines the chromatographic properties (range of molecular sizes

separable) of the gel.

Fractionation range of Sephadex G gels

Sephadex type

Fractionation range (Mol. Wt.)

Approx Swollen volume in water, ml/g dry beads

G-10 0-700 2.5

G-15 0-1500 3

G-25 100-5000 5

G-50 500-10,000 10

G-100 1000-100,000 15

Sephadex LH-20 is hydroxypropylated Sephadex G-25. The derivatization

adds lipophilicity to the gel, at the same time retaining its hydrophilicity. The

gel swells in polar solvents, such as water, methanol and THF, to about four

times its dry volume.

Separation mode is gel filtration only when single solvent is used.

When solvent mixture is used, the more polar of the solvent will be taken up by

the gel preferentially. This results in two phase system with stationary and

mobile phase of different composition and chromatography now takes place by

partition mechanism.

Chromatography coupled with mass

spectrophotometers/NMR

GC-MS

LC-MS

LC-MS-MS

LC-NMR

LC-MS-NMR

LC-SPE-NMR

Principle of operation of the HPLC-SPE-NMR instrument used in this

work. Separation of sample constituents is performed by reversed-

phase HPLC. Detection of chromatographic peaks in a photodiode

array detector results in automated analyte trapping by solid-phase

extraction after dilution of the HPLC eluate with water. SPE

cartridges with trapped analytes are dried and the analytes transferred

to an NMR spectrometer using acetonitrile-d3.

J. Nat. Prod. 2005, 68, 1500-1509

LC-SPE-NMR

STRUCTURE ELUCIDATION OF COMPOUNDS BY

SPECTROCHEMICAL TECHNIQUES

Ultraviolet Absorption Spectra: pattern of unsaturation

Infrared Spectra: Functional groups

Nuclear Magnetic Resonance Spectra: nature of 1H and 13C,

their connectivities and relative stereochemistry.

Mass Spectra: Molecular weight, Fragmentation pattern or MS/MS

profile.

Circular Dichroism Spectra: Optical behaviour

X-Ray Crystallographic Method: Cryatal structure

Degradative Reactions

Chemical Transformations: Functional group/Certain

structural framwork detection

Synthesis: Confirmation of proposed structure, Absolute

stereochemistry.

Tetrahedron Letters 48 (2007) 7194–7198

New cassane butenolide hemiketal diterpenes from the marine

creeper Caesalpinia bonduc and their antiproliferative activity

Structure elucidation through NMR Sepectroscopy