extraction, fractionation and isolation of natural products natural product extraction.pdf ·...
TRANSCRIPT
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