EXTRACTION PROCESSES

FIRST SEMESTER LECTURES (5 HOURS)

DEPARTMENT OF PHARMACOGNOSY AND DRUG DEVELOPMENT

FACULTY OF PHARMACEUTICAL SCIENCES AHMADU BELLO
UNIVERSITY ZARIA-NIGERIA

U. H. DANMALAM Ph. D
[email protected]; [email protected] +234 803 616 2831

February, 2020

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COURSE CONTENTS

1.0 Introduction
2.0 Pre-extraction Preparation
3.0 Methods of Extraction
4.0 Steps Involved in Extraction
5.0 Solvents Used in Extraction
6.0 Factors in Selecting Solvents
7.0 Types of Extracts

1.0 INTRODUCTION
Extraction is defined as the separation of active constituents of plant or animal tissues from
the inert components using solvent in standard procedures whereby active constituents
dissolved, while others remain insoluble.

It is usually the first step in the separation of the desired bioactive molecules from their raw
materials. It is the separation of medicinally active portions of natural product using selective
solvents (called menstruum) through standard procedures. The purpose of extraction is to
separate the soluble metabolites, leaving behind the insoluble cellular marc (residue). Some
of the extracts initially obtain may be ready for use as medicinal agents as tinctures or fluid
extracts but majority need further processing. According to the extraction principle, the
commonly encountered methods of extraction include the following:

(i) Solvent extraction

(ii) Distillation method
(iii) Pressing
(iv) Sublimation

Solvent extraction is the most widely used method and the process passes through the
following stages (Fig. 1):

(i) Solvent penetrates into the solid matrix;

(ii) Solute dissolves in the solvents;
(iii) Solute is diffused out of the solid matrix;

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 (iv) Extracted solutes are collected.

Any factor that can enhance the diffusion of solvent and the solubility of solutes in the above
steps will facilitate the extraction. For example, the following factors will affect the
extraction efficiency:

(i) Properties of the extraction solvent;

(ii) Particle size of the raw materials;
(iii) Extraction temperature;
(iv) Duration of the extraction;
(v) Solvent-to-solid ration.

Fig. 1: Representation of Solvent Extraction Method

The selection of the solvent is crucial for solvent extraction. Selectivity, solubility, cost and
safety should be considered in selection of solvents. Based on the law of similarity and inter-
miscibility (like dissolves like), solvents with polarity value near the polarity of the solutes
are likely to perform better and vice versa. Alcohols (EtOH and MeOH) are universal
solvents in solvent extraction for phytochemical investigation.

Generally, the finer the particle size, the better the extraction. The extraction efficiency will
be enhanced by the small particle size due to the enhanced penetration of solvents and
diffusion of solutes. Too fine particle size, however, will cause the excessive absorption of
solute in solid and difficulty in subsequent filtration.

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High temperatures increase the solubility and diffusion. Temperatures that are too high,
however, may cause solvents to be lost, leading to extracts of undesirable impurities and the
decomposition of thermo labile components.

The extraction efficiency increases with the increase in extraction duration in a certain time
range. Increasing time will not have any beneficial effect on the extraction after the
equilibrium of the solute is reached inside and outside the solid material.

The greater the solvent-to-solid ratio is, the higher the extraction yield is; however, a
solvent-to-solid ratio that is too high will cause excessive usage of the extraction solvent and
requires a long time for concentration.

Conventional extraction methods, including maceration, percolation and reflux extraction,

usually use organic solvents and require a large volume of solvents and long extraction time.
Some modern or greener extraction methods such as super critical fluid extraction (SFC),
pressurized liquid extraction (PLE) and microwave assisted extraction (MAE) have also been
applied in natural products extraction, and they offer some advantages such as lower organic
solvent consumption, shorter extraction time and higher selectivity. Some extraction
methods, however, such as sublimation, expeller pressing and enfleurage are rarely used in
current phytochemical investigation. A brief summary of the various extraction methods used
for natural products is shown in Table 1 below.

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Table 1: A Brief Summary of Various Extraction Methods for Natural Products

Volume of organic
Method Solvent Temperature Pressure Time
solvent consumed
Water, aqueous and non-
Maceration Room temperature Atmospheric Long Large
aqueous solvents
Water, aqueous and non- Room temperature,
Percolation Atmospheric Long Large
aqueous solvents occasionally under heat
Decoction Water Under heat Atmospheric Moderate None
Aqueous and non-aqueous
Reflux extraction Under heat Atmospheric Moderate Moderate
solvents
Soxhlet extraction Organic solvents Under heat Atmospheric Long Moderate
Pressurized liquid Water, aqueous and non-
Under heat High Short Small
extraction aqueous solvents
Supercritical fluid Supercritical fluid (usually S-
Near room temperature High Short None or small
extraction CO2), sometimes with modifier
Ultrasound assisted Water, aqueous and non- Room temperature, or
Atmospheric Short Moderate
extraction aqueous solvents under heat
Microwave assisted Water, aqueous and non-
Room temperature Atmospheric Short None or moderate
extraction aqueous solvents
Pulsed electric field Water, aqueous and non- Room temperature, or
Atmospheric Short Moderate
extraction aqueous solvents under heat
Room temperature, or
Enzyme assisted Water, aqueous and non-
heated after enzyme Atmospheric Moderate Moderate
extraction aqueous solvents
treatment
Hydro and steam
Water Under heat Atmospheric Long None
distillations

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2.0 PRE-EXTRACTION PREPARATION
These are the initial preparation steps that are taken before extraction in order to preserve the
biomolecules in the plants. Samples such as leaves, barks, roots, fruits and flowers can be
extracted from fresh or dried plants material. Other factors such as drying and grinding also
influence the preservation of phytochemicals in the final extracts.

2.1 Fresh vs. dried samples:

• Both fresh and dried samples can be used in medicinal plants studies.
• Dried sample in many cases is preferred due to time needed for the preparation of the
experimental design.
2.2 Grinded vs. powdered samples:
Grinding lower the particle sizes of materials and increases contact surface between the
samples and solvents. It usually resulted in coarse and smaller samples thereby giving a more
homogenized and smaller particle. Particle sizes smaller than 0.5 mm are ideal for efficient
extraction. Conventional mortar/pestle, electric blenders and mills are common used in
making powders.

2.3 Air-, microwave-, oven- and freeze-drying (lyophilisation) of plants samples:

Air-drying of plant material may takes from 3 – 7 days and sometimes up to months (e.g.
leaves or seed). No high temperature, as such heat-labile compounds are preserved. However,
it takes longer time in comparison which may lead to contamination at unstable temp.

Microwave-drying uses electromagnetic radiation that causes simultaneous heating by

dipolar rotation and ionic induction. Also oscillation causes collisions between molecules
resulting in faster heating. This shortens the drying time but sometimes causes degradation of
phytochemicals.

Oven-drying uses thermal energy to remove moisture from the samples. This is considered as
one of the easiest and rapid thermal processing that can preserved phytochemicals.

Freeze-drying is base on the principle of sublimation. Sample is frozen at -80°C to -20°C

prior to lyophilisation to solidify any liquid (e.g. solvent, moisture) in the samples. After an
overnight (12 h) freezing, sample is immediately lyophilized to avoid the frozen liquid in the
sample from melting. Mouth of container is wrapped with needle-poked-paraffin to avoid

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loss. Sample may be lost by splattering out into the freeze-flask. The method is complex and
expensive.

3.0 METHODS OF EXTRACTION

3.1 Maceration, Infusion, Percolation and Decoction
In Maceration process, the material (coarse or powdered) is soaked in a closed container
with a solvent and allowed to stand at room temperature for a minimum period of three days
with frequent agitation. The processed intended to soften and break the cell wall to release the
soluble phytochemicals. After three days, the mixture is pressed or strained by filtration. In
this conventional method, heat is transferred through convection and conduction and the
choice of solvents will determine the type of compound extracted from the samples.

• Circulatory maceration: solvent is made to circulate continuously through sample

by pumping from the bottom of container to the inlet and distributed as spray.
Movement of solvent and uniform distribution reduces duration of extraction (Fig. 3).

Digestion: a form of maceration in which gentle heat is used during the process of extraction.
Most common temperatures are 35 ° to 40 ° C., although can rise to no higher than 50 ° C.
Used for thermo-stable active constituents. Efficiency of the menstruum is increased. Sample
is introduced into a container with pre-heated solvent at required temperature. It is maintained
for a period of half an hour to 24 hours with regular shaking.

Infusion uses the same principle as maceration; infusion results from soaking the material in
cold or boiled water. However, the maceration period for infusion is shorter and the sample is
boiled in specified volume of water (e.g. 1:4 or 1:16) for a defined time.

Decoction is obtained by heating or boiling the material and is only suitable for extracting
heat-stable compounds, hard plants materials (e.g. roots and barks) and usually resulted in
more oil-soluble compounds compared to maceration and infusion.

Unique equipment called percolator (Fig. 4) is used in percolation, another method that
shares similar fundamental principle. Dried powdered samples are packed in the percolator,
added with boiling water and macerated for 2 hours. The percolation process is usually done
at moderate rate (e.g. 6 drops /min) until the extraction is completed before evaporation to get
a concentrated extracts.

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Fig. 2: Representation of the Process of Maceration

Fig. 3: Representation of the Circulatory Maceration

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Fig. 4: A Percolator

3.1.1 Strength and limitation

These techniques are the easiest and simplest methods. However, organic waste come into an
issue as large volume of solvents are used and proper management of the waste is needed.
Alteration in temperature and choice of solvents enhance the extraction process, reduce the
volume needed for extraction and can be introduced in the maceration technique, when such
alteration is not objectionable.

3.2 Soxhlet or Hot Continuous Extraction:

In this method, finely ground sample is placed in a porous bag or “thimble” made from a
strong filter paper or cellulose, which is place, is in thimble chamber of the Soxhlet apparatus
(Fig. 4). Extraction solvents is heated in the bottom flask, vaporizes into the sample thimble,
condenses in the condenser and drip back. When the liquid content reaches the siphon arm,
the liquid contents emptied into the bottom flask again and the process is continued.

3.2.1 Strength and limitation

This method requires smaller quantity of solvent compared to maceration. However, it comes
with the disadvantage of exposing the sample to hazardous and flammable liquid organic
solvents, with potential toxic emissions during extraction. Solvents used in the extraction

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system need to be of high-purity that might add to cost. This procedure is considered not
environmental friendly and may contribute to pollution problem compared to advance
extraction method such as supercritical fluid extraction (SFE). The ideal sample for Soxhlet
extraction is also limited to a dry and finely divided solid and many factors such as
temperature, solvent-sample ratio and agitation speed need to be considered for this method.

Fig. 5: Soxhlet Apparatus: A Parts of the apparatus: 1 – anti-bumping chips, 2 – round

bottom flask, 3 – soxhlet flask, 4 – thimble, 5 – drug, 6 – siphon tube, 7 – siphon opening, 8 –
adaptor, 9 – condenser, 10 – water inlet, 11 – water outlet. B – Apparatus set up for
extraction.

3.3 Microwave Assisted Extraction (MAE)

MAE utilizes microwave energy to facilitate partition of analytes from the sample matrix into
the solvent. Microwave radiation interacts with dipoles of polar and polarizable materials
(e.g. solvents and sample) to cause heating near the surface of the materials and the heat is
transferred by conduction. Dipole rotation of the molecules induced by microwave
electromagnetic radiations disrupts hydrogen bonding; enhancing the migration of dissolved

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ions and promotes solvent penetration into the matrix. In non-polar solvents, poor heating
occurs as the energy is transferred by dielectric absorption only. MAE can be considered as
selective methods that favour polar molecules and solvents with high dielectric constant
(Table 2).

3.3.1 Strength and limitation

This technique reduced extraction time and solvent volume as compared to conventional
methods (maceration and Soxhlet extraction). Improved recoveries of analytes and
reproducibility were observed in MAE method but with caution of using proper conditions to
avoid thermal degradation.

Table 2: Dielectric Constant of some Commonly Used Extraction Solvents

Solvent Dielectric constant (20°C)

Hexane 1.89
Toluene 2.4
Dichloromethane 8.9
Acetone 20.7
Ethanol 24.3
Methanol 32.6
Water 78.5

3.4. Ultrasound-Assisted (UAE) or Sonication Extraction

UAE involves the use of ultrasound ranging from 20 kHz to 2000 kHz. The mechanic effect
of acoustic cavitation from the ultrasound increases the surface contact between solvents and
samples and permeability of cell walls. Physical and chemical properties of the materials
subjected to ultrasound are altered and disrupt the cell wall of the material; facilitating release
of compounds and enhancing mass transport of the solvents into the cells. The procedure is
simple and relatively low cost technology (Fig. 5) that can be used in both small and large
scale of phytochemical extraction.
3.4.1 Strength and limitation:
The benefits of UAE is mainly due reduction in extraction time and solvent consumption.
However, use of ultrasound energy more than 20 kHz may have an effect on the active
phytochemicals through the formation of free radicals.

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Fig. 5: Sonicator and Schematic Representation of Bench and Industrial Scale Ultrasounic
Liquid Processor

3.5. Accelerated Solvent Extraction (ASE)

ASE is an automated, efficient form of liquid extraction compared to maceration and Soxhlet
extraction as it uses minimal amount of solvent. Sample is packed with inert material such as
sand in the stainless steel extraction cell to prevent it from aggregating and block the system
tubing. Packed ASE cell includes layers of sand-sample mixture in between cellulose filter
paper and sand layers. The technology is able to control temperature and pressure for each
individual samples and requires less than an hour for extraction. Similar to other solvent
technique, it also depends on the solvent types.

3.6 Supercritical Fluid Extraction (SFE)

Supercritical fluid (SF) (also called as dense-gas) is a substance that shares the physical
properties of both gas and liquid at its critical point. Factors such as temperature and pressure
are the determinants that push a substance into its critical region. SF behaves more like a gas
but have the solvating characteristic of a liquid. An example of SF is CO2 that become SF at
above 31.1°C and 7380 kPa. Interest in Supercritical-CO2 (SC-CO2) extraction due to its
excellent solvent for non-polar analytes and CO2 is readily available at low cost and has low
toxicity. Even though SC-CO2 has poor solubility for polar compounds, modification such as
adding small amount of ethanol and methanol enable it to extracts polar compounds. SC-CO2
also produces analytes as concentrate form as CO2 vaporizes at ambient temperature. SC-

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 solvents strength can be easily altered by changing the temperature and/or pressure or by
adding modifiers that lead to reduce extraction time.

3.6.1 Process of extraction

· Carbon dioxide becomes liquid under pressure while remaining in a gaseous state, which
means it is now "supercritical." In this state, it is pumped into a chamber filled with plant
matter (Fig.

Fig. 6: Representation of Supercritical Fluid Extraction

· Because of the liquid properties of the gas, the CO2 functions as a solvent on the natural
plant matter, pulling the oils and other substances such as pigment and resin from the
plant matter. The essential oil content then dissolves into the liquid CO2.
· The CO2 is brought back to natural pressure and evaporates back into its gaseous state,
while what is left is the resulting oil.

3.7 Enzyme-Assisted Extraction (EAE)

Some bioactive compounds in natural products are dispersed in cell cytoplasm, and the
metabolites are retained in the polysaccharide-lignin network by hydrogen or hydrophobic
bonding and are not accessible with a solvent extraction process. Enzymatic pre-treatment

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has been found as an effective way to release the bounded compounds and increase overall
yield. Specific enzymes such as cellulase, α-amylase, and pectinase added during extraction
enhance recovery by breaking the cell wall and hydrolyzing the structural polysaccharides
and lipid bodies. Two approaches are commonly encountered namely enzyme-assisted
aqueous extraction (EAAE) and enzyme-assisted cold pressing (EACP). EAAE methods have
been employed mainly for the extraction of oils from various seeds. In EACP method,
enzymes are used to hydrolyze the seed cell wall of a plant.

3.8 Pulsed Electric Field (PEF) Extraction

Pulsed electric field extraction significantly increases the extraction yield and decreased the
extraction time because it can increase mass transfer during extraction by destroying
membrane structures. The effectiveness of PEF treatment depends on several parameters
including field strength, specific energy input, pulse number and treatment temperature. PEF
extraction is a non-thermal method and minimizes the degradation of the thermolabile
compounds.

3.9 Distillation Methods

Distillation is the separation process whereby components of a mixture of two or more liquids
are separated due to the difference in their vapour pressures. Volatile plant components such
as essential oils are still obtained mainly by distillation techniques, although working at
elevated temperatures can lead to chemical changes. Hydro-distillation (HD), steam
distillation (SD), simultaneous distillation solvent extraction (SDE), microwave-assisted
hydro-distillation (MWHD), supercritical fluid (CO2) extraction (SFE), purge and trap, and
solid phase micro-extraction (SPME) techniques are employed to extract volatile organic
compounds from fresh plant parts. Among these techniques, HD, SD, and SDE are classical
and conventional methods for extracting bioactive volatile organic compounds.

The limitations of the conventional methods include long extraction times, large amounts of
solvents needed and multiple steps. Additionally, many unstable volatile organic compounds
may be thermally decomposed and degraded during the thermal extraction or distillation.
Because of their simplicity, these methods are still in use to extract fragrance-and-aroma oils
from plants. Purge and trap, SFE, and SPME have aroused much attention from analysts as
they are environment friendly sampling techniques.

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3.9.1. Steam Distillation

Steam Distillation is the most popular method used to extract and isolate essential oils from
plants for use in natural products. This happens when the steam vaporizes the plant material’s
volatile compounds, which eventually go through a condensation and collection process.

Steam Distillation Process

· A large container called a Still, which is usually made of stainless steel, containing
the plant material has steam added to it.
· Through an inlet, steam is injected through the plant material containing the desired
oils, releasing the plant’s aromatic molecules and turning them into vapour.
· The vaporized plant compounds travel to the condensation flask or the Condenser.
Here, two separate pipes make it possible for hot water to exit and for cold water to
enter the Condenser. This makes the vapour cool back into liquid form.
· The aromatic liquid by-product drops from the Condenser and collects inside a
receptacle underneath it, which is called a Separator. Because water and oil do not
mix, the essential oil floats on top of the water. From here, it is siphoned off. (Some
essential oils are heavier than water, such as clove essential oil, so they are found
at the bottom of the Separator.)

Fig. 7: Representation of the Steam Distillation Extraction

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3.9.2 Water Distillation

Delicate flowers such as roses and orange blossoms would clump together when introduced
to steam in the distillation process, so the most effective method of extraction in this situation
is to submerge fragile plant material in pure boiling water instead. The water protects the
extracted oil from overheating. The condensed liquids cool down and separate from each
other. The remaining water, which can sometimes be fragrant, is referred to by several names
including hydrolate, hydrosol, herbal water, essential water, floral water, or herbal distillate.

Fig. 8: Representation of the Water Distillation Extraction

4.0 STEPS INVOLVED IN EXTRACTION

4.1 Size Reduction: dried material is milled or pulverized to 30 – 40 mesh size, this rupture
the tissues and expose the constituents to solvent and maximizes surface area for optimum
reaction.
4.2 Extraction: this is the stage where the actual extraction takes place.
4.3 Filtration: the menstruum is separated out from the marc using filter paper or other
media.
4.4 Concentration: the extract is concentrated under vacuum (e.g. using Rotavapour) to
produce a thick extract.
4.5 Drying: drying of the syrup can be carried out using vacuum chamber dryer, spray dryer
or air (cold or hot) chambers.

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5.0 SOLVENTS USE IN EXTRACTION
Solvents to be used in extraction shall be capable of penetrating tissues of drug to dissolve
active principles in the cells. Various solvents are used including:

o Polar: such as water, ethanol, methanol, acetone, dilute acids, dilute bases etc.
o Intermediate Polar: like ethyl acetate, chloroform.
o Non polar: including n-hexane, petroleum spirit, propane, butane, CO2, N2O, diethyl
ether, benzene etc.
5.1 Water: is cheap, non toxic, non inflammable and has wide solvent action (use extracting
polar and none-lipid components e.g. proteins, glycosides, enzymes, sugar, tannins etc).
5.1.1 Disadvantages of water
o Promote growth of mould and bacteria, hence requires a preservative.
o It may leads to hydrolysis.
o Large amount of heat is required to concentrate.
o It promotes fermentation or decomposition of the preparation.
5.2 Alcohol: solvent for dissolving alkaloids, alkaloidal salts, glycosides etc. it also dissolves
many colouring matter, tannins, etc. it doesn’t dissolve gums waxes, fats etc.
Merits: Doesn’t allow growth of mould and bacteria at concentrations above 20%. It is
non-toxic in the concentration for most preparations. Small amount of heat is required for
concentration.
Demerits: it is costly, flammable and volatile etc
Solvents such as ether, chloroform and light petroleum are rarely used.

6.0 FACTORS IN SOLVENT SELECTION

6.1 Solvent power (selectivity): the solvent should be able to select only the active and
desired constituents from the material.
6.2 Boiling temperature: b. p. should low in order to facilitate removal of solvent from
product after the extraction.
6.3 Reactivity: solvent should not react chemically with the extract, nor readily decompose.
6.4 Viscosity: low viscosity of solvent leads to low pressure drop and good heat and mass
transfer which enhances the efficiency of the process.
6.5 Cost: solvent should be readily available and at low cost.
6.6 Safety: solvent should be non-flammable, non-corrosive, and non-toxic.

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6.7 Vapour pressure: To prevent loss of solvent by evaporation, a low vapour pressure for
the solvent at operating temperature is required.
6.8 Recovery: The solvent has to be separated easily from extract to produce a solvent-free
extract.
7.0 TYPE OF EXTRACTS
7.1 Aqueous extracts: medicinal preparations intended to be used immediately or to be
preserved for use, solvent used is water. The methods commonly used for preparation are
decoction, infusion and digestion.
7.2 Hydro alcohol or Alcohol extract: are prepared by the methods of maceration and
percolation e.g. tinctures, here solvent used is alcohol and mixture of water and alcohol.
7.3 Soft extracts: are extracts with semisolid or syrup consistency, can be used in a variety of
dosage form like ointments and suppositories e.g. glycyrrhiza extract.
7.4 Dry extracts: are powdered extracts or dry powder extract obtained from suitable
process, filtered and concentrated under vacuum, dried completely by spray or tray
drying. e.g. belladonna used in dosage forms such as capsules, tablets etc.

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