Extraction and
Phytochemical studies

Introduction
• Currently, there is an increasing interest in the study of natural
products, especially as part of drug discovery programs.
• Plants and microorganisms produce complex mixtures of natural
products.
• Selection of the best protocol for an efficient extraction of these
substances are necessary
• “Classic” solvent based procedures, e.g., maceration, percolation are still
used despite many disadvantages.
pp:27-28

Solvent extraction methods
• Maceration:
• Put pulverised plant material in a closed glass container along with suitable solvent
• After sometime decant the solvent and filter them.
• Store in a fresh container.
Use: Suitable for both initial and bulk extraction.
• Ultrasound Assisted Extraction:
• Place the powdered materials in a glass container with solvent
• Put the container in ultrasonic bath
• Set a suitable temperature and time
• Filter the extract after the process pp:32
Use: Extraction of intracellular metabolites from plant cell cultures

• Percolation:
• In a percolator, place the powdered plant material to soak in contact with the extraction
solvent.
• Pour some additional solvent on top of the plant material and allow the extract to
percolate slowly (drop-wise) out of the bottom of the percolator.
• Perform successive percolations to extract the plant material exhaustively by refilling the
percolator with fresh solvent.
• Pool all extracts together.
Disadv: Large volumes of solvents are required and the process can be time-consuming.
• pp 33

• Soxhlet Extraction:
• Put the powdered plant material in a cellulose thimble and cover with cotton wool.
• Place the thimble inside the Soxhlet extraction chamber.
• Assemble the Soxhlet extraction chamber on top of a collecting round-bottom flask
containing some anti bumping granules.
• Add a suitable solvent to the Soxhlet chamber. When a certain level of solvent has
accumulated in the thimble, it is siphoned into the flask beneath.
• Connect a reflux condenser to the Soxhlet chamber.
• Place the collecting flask in a heating mantle and heat the setup under reflux.
• Soxtec extraction unit is a fully automated system, based on soxhlet aparatus for fast
extraction of soluble matter from a wide range of matrices
Adv: The material is extracted continuously, i.e., the solvent saturated in solubilized
metabolites empties into the flask, fresh recondensed solvent then re-extracts the
material in the thimble.

• Accelerated Solvent Extration:
• This method uses high pressure to maintain the solvent in a liquid state at high temperature
• Mix the powdered material with some sea-sand in a in a 4:1 ratio.
• Load the plant-sand mixture (ca. 40 g) into a 100 mL ASE ® extraction cartridge.
• Place the extraction cartridge in the ASE ® 100 extractor.
• Fill in the reservoir bottles with a suitable extraction solvent.
• Program the ASE ® 100 system to extract at a pressure of1,500 psi and temperature of 100°C in four static
cycles (static time of 8 min/cycle) with a flush volume of 60% and a purge time with nitrogen gas of 150 s.
• 6. Collect the extract, which is automatically filtered, in the receiving flask.
Adv: more economical and environment-friendly alternative to conventional approaches
pp34

Extraction under reflux:
• In a round-bottomed flask, immerse the plant material in a suitable solvent,
and connect the fl ask directly to a refl ux condenser.
• Place the set-up in a heating mantle. When the solvent reaches its boiling
point, the vapor is condensed and the solvent is recycled to the flask
Disadv: Thermolabile components risk being degraded

Modern Methods :
Microwave Assisted Extraction (MAE): It is an efficient method which involves
deriving natural compounds from raw plants.
Microwave extraction allows organic compounds to be extracted more rapidly,
with similar or better yield as compared to conventional extraction methods.
Advantage of MAE over classic procedures :
• Reduction in extraction time
• Improved yield
• Better accuracy
• Suitable for thermolabile substances
pp 91

• The MAE process flow is as follows:
Microwave radiation
Moisture get heated up
Moisture evaporates
Generation of tremendous pressure on cell wall
Swelling of plant cell
Rupture of the cell
Leaching out of phyto-constituents
pp98

Classification of MAE
Two types of system for MAE:
a. Closed System or pressurized
system
b. Open System or focussed
system

Factors affecting MAE
• Extraction time An increase in extraction time increases the extraction yield.
However, it also increases the risk of degradation of thermolabile
components. Therefore, a balance between the extraction yield and the
stability of the components must be achieved to ensure a meaningful MAE.
• Microwave power To optimize an MAE protocol, a combination of low or
moderate microwave power with longer extraction time is a generally
desirable.
• Matrix characteristics The matrix characteristics, e.g., particle size and the
nature of the material, may affect the recoveries of the compounds. Finer the
particle size of the sample larger is the surface area and better is the
penetration of microwaves. pp 98

• Advantage of Closed system:
• Decrease in extraction time.
• Loss of volatile substances is avoided.
• Less solvent is required because no evaporation occurs.
• No hazardous fumes during acid microwave since it is a closed vessel.
• Disadvantages of closed systems:
• High pressure used pose safety risks
• The usual constituent material of the vessel does not allow high solution temperatures
• Addition of reagents is impossible since it is a single step procedure
• Vessel must be cooled down before it can be opened to prevent loss of volatile constituents.
Mandal V and Hemlatha S ( 2007)

Advantages of Open system:
• Increased safety
• Addition of reagent is possible
• Vessels made of various material can be used
• Excess solvent can be removed easily
• Ability to process large samples
• No requirement for cooling down or depressurisation
• Low cost of equipment
• Suitable for thermolabile products
Disadvantages:
• The ensuing method are less precise than in close-vessel system
• The sample throughput is lower as open system cannot process many samples simultaneously
• Require longer time to achieve same results as for closed system
Mandal V and Hemlatha S ( 2007)

Supercritical Fluid Extraction: Separation of chemicals, flavors from the products such as coffee, tea,
hops, herbs, and spices which are mixed with supercritical fluid to form a mobile phase
The process begins with CO2 in vapor form. It is then compressed into a liquid before becoming supercritical.
While supercritical, the extraction takes place.
Critical conditions (For CO2):
• Temperature (tc)= 30.9 0C
• Pressure (pc)=73.8 bar
• Density (dc)=0.467gm/ml
Features:
• Cagniard de la Tour discovered the critical point in 1822.
• At temperatures and pressures above this point, a single homogeneous fluid is formed, which is known as
supercritical fluid (SCF).
• SCFs are produced by heating a gas above its critical temperature or compressing a liquid above its critical
pressure
• The principle of SFE, where an SCF is used as a solvent for extraction, derives from the fact that the solubility
of a solute in a solvent is dependent on its temperature and pressure pp 44

Properties of supercritical fluid
• Supercritical fluids have highly compressed gases, which combine properties of gases and liquids.
• Supercritical fluids have solvent power similar to light hydrocarbons for most of the solutes.
However, fluorinated compounds are often more soluble in supercritical CO2 than in hydrocarbons;
this increased solubility is important for polymerization.
• Solubility increases with increasing density (that is with increasing pressure). Rapid expansion of
supercritical solutions leads to precipitation of a finely divided solid. This is a key feature of flow
reactors.
Hitchen and Dean (1990)

Factors involving in extraction:
(a) The solubility of the target compound(s) in SC-CO2 or other SCF has to be determined . It is necessary to
performsolubility tests to determine the effect of temperature andpressure (which in turn control the
density) on the solubility ofthe target compound(s) in the SCF.
(b) The effect of co-solvents on the solubility of the target compound(s) needs to be determined.
(c) The effect of matrix, either has the analyte lying on its surface (adsorbed), or the analyte is entrained in
the matrix (absorbed),has to be considered carefully.
(d) The solvating power of SCF is proportional to its density which can be affected by any temperature
change for any given pressure. Therefore, strict temperature control has to be in place.
(e) The partition coefficient of the analyte between CO2 and the matrix, which is often affected by the flow
rate, has to be considered. Higher fl ow rates and longer extraction time may benecessary to sweep all the
analyte out of the extraction chamber. Lower flow rates may be applied if the kinetics of the system is slow.
(f) Careful consideration has to be given in choosing appropriate modifiers.
pp 55

SFE Instrument

• Advantages of SFE:
• Lower operating temperatures
• Easier regeneration of the sc solvent
• It is a fast process and completed in 10 to 60 minutes
• Environmental improvement and reduced product contamination
• A supercritical fluid can be separated from analyte by simply releasing pressure.
• Recovery of analytes becomes simple.
• Improved yield
• Solvent power comparable to liquid solvents
• Solvent power adjustable by pressure and temperature changes
• Very hígh volatility compared to the dissolved substances
• Complete separation of solvent from extract and raffinate
• high diffusivity, low viscosity

• Disadvantages:
• Elevated pressures required
• Relative high costs of investment
• Complicated phase behaviour
http://web.ist.utl.pt/ist11061/fidel/flaves/sec5/sec5431.html

• Accelerated Solvent Extraction:
In the ASE system, the extraction process is carried out at temperatures exceeding
the boiling point of a solvent what implies that the pressure inside the extraction
cell must be kept high in order to maintain the solvent in a liquid state.
Three steps can be distinguished during the extraction:
• Desorption from a solid particle,
• Diffusion through the solvent located inside a particle pore, and
• Transfer to the bulk of the flowing fluid.
pp 77

• Parameters of extraction:
Solvents:
i The polarity of extraction solvent should closely match that of the target compounds.
ii Mixing solvents of differing polarities can be used for wide classes of compounds.
iii While many ASE ® methods recommend solvent or solvent mixtures for specific analyte
classes,there may be alternatives that better fi t the needs of a particular laboratory.
Temperature:
i Temperature is critically important in ASE ® extraction.
ii With increasing temperature, the viscosity of the solvent decreases significantly, and it results in
an increased ability of the solvent to wet the matrix and solubilize the target analytes.
iii The added thermal energy also assists in breaking analytes–matrix bonds and encourages
analytes diffusion from the matrix surface.
iv majority of the ASE ® applications is performed in the temperature range of 75–125°C
pp 77-78

• Pressure :
i Commonly used organic solvents like DCM, acetone and MeOH boil at relatively low temperature
between 40 and 65°C at atmospheric pressure.
ii The effect of pressure is to maintain the solvents in liquid state while above their atmospheric boiling
points.
iii Changing of pressure will have very little impact of analyte recovery and it is not usually considered
as critical from experimental view point.
• Static Time
i Certain sample matrices can retain analytes within pores or other structures.
ii Increasing static time at elevated temperature can allow these compounds to diffuse into extraction
solvent.
iii The effect of static time should also be explored with static cycles, in order to produce a complete
extraction in the most effi cient way possible.
pp 78

Instrumental schematic and outline of ASE

Preparative HPLC
• Powerful technique for the isolation and purification of variety of
chemicals, pharmaceutical compounds, natural products and
biological molecules.
• To increase throughput and separation power, the first preparative
HPLC system was developed in the 1970’s.
• Types of HPLC: Based on the scale of operation.
a. Analytical HPLC
b. Preparative HPLC
pp 256

Analytical HPLC.
• Sample goes from detector into waste.
• Use quantification and/or identification of
compounds.
• Column has internal diameter-1-5mm
• Column particles are 5µm or smaller.
• HPLC pump provide up to 10mL/min.
• Solubility of sample in mobile phase usually
not important.
• Mobile phase is not recover.
Preparative HPLC
• Sample goes from detector into fraction
collector
• Use for isolation and/purification of
compounds.
• Column has internal diameter-1-10cm
• Column particles are 7µm or larger
• HPLC pump provide >>10mL/min
• Solubility of sample usually very important
• Mobile phase recovery is possible

Phases of HPLC:
Normal Phase:
• Normal phase chromatography uses a polar stationary phase(usually silica) and less polar
(nonaqueous) eluting solvents
• The more polar the compound, the more likely it is to be adsorbed onto the stationary phase,
and less polar compounds will be eluted first from the column.
• Normal phase prep-HPLC is best suited to the separation and isolation of lipophilic
compounds, long-chain alkane derivatives or where the mixture of interest is sparingly soluble
in aqueous conditions.
• The eluants used in normal phase prep-HPLC are usually mixtures of aliphatic hydrocarbons ,
halogenated hydrocarbons, more polar oxygenated hydrocarbons , or hydroxylated solvents
such as isopropanol and methanol pp 258

• Reversed phase:
• This technique is the reverse on normal phase prep-HPLC whereby the stationary phase
is more nonpolar than the eluting solvent.
• Silica-based reversed-phase sorbents are also called “bonded-phase” materials whereby
the silica particles are derivatized with alkylsilyl reagents
• The eluant used in reversed-phase prep-HPLC usually comprises a mixture of water and
miscible organic solvents, usually acetonitrile , methanol, or tetrahydrofuran.
• Buffers, acids, or bases may be added to suppress compound ionization or to control the
degree of ionization of free unreacted silanol groups to reduce peak tailing and improve
chromatography. pp 259

Instrumentation of Preparative HPLC

• Flash Chromatography:
• Flash chromatography, also known as medium pressure chromatography.
• “An air pressure driven hybrid of medium and short column
chromatography optimized for rapid separation"
• It was popularized in 1970s by Clark Still of Columbia University.
• An alternative to slow and often inefficient gravity-fed chromatography
https://yvesrubin.files.wordpress.com/2011/03/flash_chromatography.pdf

Column Chromatography
• Glass columns with silica gel
• Separation is very slow
• End of the run, silica gel must be
removed
• Both time consuming and
• hazardous
Flash Chromatography
• Pre-packed plastic cartridges
• Solvent is pumped through the
cartridge and separation is rapid
• Much quicker and more reproducible
• Remaining solvent flushed out of the
column using pressurized gas

• Principle:
• The principle is that the eluent is, under gas pressure (normally nitrogen or compressed
air) rapidly pushed through a short glass column.
• The glass column is packed with an adsorbent of defined particle size with large inner
diameter.
• The most used stationary phase is silica gel 40 –63 μm, but obviously packing with other
particle sizes can be used as well.
• Particles smaller than 25 μm should only be used with very low viscosity mobile phases,
because otherwise the flow rate would be very low.
• Normally gel beds are about 15 cm high with working pressures of 1.5 – 2.0 bars.
• In the meantime, reversed phase materials are used more frequently in flash
chromatography.
Roje B et al., (2011)

Instrumentation

Advantages:
• Fast and economic methods for the synthesis laboratory.
• Ideal for the separation of compounds up to gram quantities.
• Automated changes between normal phase and reversed phase
chromatography.

• Application:
• Isolation and Purification of Natural Products/Nutraceuticals
• Purification of Carbohydrate
• Isolation of Pharmaceutical/Small Molecules Application
• Purification of Lipids

Counter-current chromatography Extraction
• Counter-current chromatography has its origins in liquid–liquid extraction
• The distribution of the solute molecules between the two phases is governed
by the partition constant ( K ) which is a constant at any given temperature
• Separation occurs in an analogous manner to solid–liquid chromatography or
gas–liquid chromatography, where solute molecules are separated on the
basis of an equilibrium established between the two phases involved.
K=CA/CB
where, C A and C B represents the concentration of the solute in the two
solvents. pp: 400

Counter-current distribution
• Craig recognized that while liquid–liquid extraction, for example in a
separatory funnel, allowed only limited resolving power that the
method could be extended to multiple equilibrium
• Multiple solutes could be resolved based on their respective partition
constants
• It was also demonstrated that when the ratio of partition constants
between two solutes was greater than four, separation was easily
achieved pp: 400-401

Droplet Counter-current chromatography
• DCCC apparatus contain as many as 600 columns connected by
capillaries through which a mobile phase is pumped.
• The mobile phase flow can be discretionally reversed to account for
the relative density of the stationary phase solvent and the solute
molecules can be added in either solvent.
• A multitude of natural products have been isolated and solvent
systems suitable for a wide variety of structural classes have been
established thereby lessening the ordeal of finding a suitable solvent
system. pp: 401-402

High speed Counter-current chromatography
• High speed counter-current chromatography (HSCCC) represents the
most advanced technique in the evolution of counter-current
chromatography
• It has overcome many of the pitfalls associated with earlier methods
and allows the rapid separation of solute molecules from often
complex mixtures, including extracts of microbial origins.
pp: 403

Selection of solvents as per polarity