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Applied Organic Chemistry

Extraction of Natural Products
Prof Priyani Paranagama
Chair of Chemistry The extraction of terpenes from natural sources is
Department of Chemistry a crucial step in obtaining these valuable
compounds for various applications, including
University of Kelaniya
aromatherapy, perfumery, and pharmaceuticals.

Each extraction method has its advantages and

limitations, and the choice of method often
depends on the specific requirements of the
desired end product, including purity, yield, and
preservation of volatile compounds

The typical extraction process, especially for raw materials Simple cold extraction
incorporates the following steps: non-volatiles

1. Drying and grinding of natural source or homogenizing or

maceration

Choice of solvents
a. Polar extraction
b. Medium polarity extraction
Air drying
microwave vacuum drying spray drying c. Nonpolar
freeze drying Atomizes a liquid feed into a
Involves exposing the material Uses microwave energy to Involves freezing the material hot drying medium, typically a) Polar solvents have a high dielectric constant and can
to ambient air, often with the heat the material under and then reducing the air, causing rapid evaporation dissolve polar compounds effectively due to their ability to
aid of fans to increase air vacuum conditions, causing surrounding pressure to allow of moisture and producing dry form hydrogen bonds. They are suitable for extracting
circulation. rapid evaporation of the frozen water in the material powder particles. hydrophilic (water-soluble) compounds.
moisture. to sublimate directly from solid
Advantages: Fast drying to vapor phase. Advantages: Produces fine, b) Medium polarity solvents bridge the gap between polar
preserves heat-sensitive Advantages: Fast drying uniform powder, continuous and non-polar solvents. They can extract a wider range of
compounds, uniform heating. preserves heat-sensitive Advantages: Preserves the process, suitable for compounds.
compounds, uniform heating. structure, flavor, and bioactive heat-sensitive materials.
Disadvantages: High energy compounds of the material, c) Non-polar solvents have low dielectric constants and
consumption, potential for Disadvantages: High energy minimal heat exposure. Disadvantages: Requires are effective for extracting non-polar compounds. They do
uneven heating and damage to consumption, potential for
liquid feed, potential for not interact well with polar compounds
materials. uneven heating and damage Disadvantages: Expensive, thermal degradation of
to materials time-consuming, requires compounds.
specialized equipment.
There are four common methods of Expression
extractions of volatile compounds

a) Expression method Advantage

- Low cost
b) Steam distillation method - simple

c) Extraction by means of volatile solvents Disadvantage

- High energy
d) Adsorption in purified fats - Long time

This technique involves mechanically pressing plant materials to release

essential oils and other volatile compounds. It is commonly used for citrus
fruits, where the rind is cold-pressed to extract oils without the application of
heat.

Widely used in the production of citrus essential oils (like lemon and orange)
and some other fruits. The method preserves the natural aroma and flavor of
the oils.

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Laboratory extraction of the essential oil

• The essential oil for analysis can be obtained by distillation of the
plant parts.

• Three methods can be employed to extract the essential oil in the

laboratory
Steam distillation method
Hydrodistillation
Clavenger arm distillation

Steam distillation method

Hydrodistillation
• Traditional method of extraction

Advantage
-Easy separation

Disadvantage
- Combustion of the
sample
- Degradation of
Advantage Disadvantage volatile compound

- Easy separation - Degradation of some

- Easy operation analytes
Laboratory setup for steam distillation: plant parts are
- Sample impurities
shade-dried for 2 days and subjected to steam distillation for
3 h at a rate of 150 ml/h. -Expensive equipment

The plant material is submerged directly in water in the distillation

Plant material is placed in a distillation flask and steam is passed through it. flask and heated to produce steam.
The steam vaporizes the volatile compounds, which are then condensed and collected as The steam vaporizes the essential oil compounds which are then
a liquid. condensed and collected.
The condensed liquid contains both the essential oil and water, which are separated Hydrodistillation is a simpler method compared to steam distillation
since they are immiscible. but the plant material may be in direct contact with hot water which
Steam distillation is the most common method for extracting essential oils and produces can degrade some compounds
high-quality, pure oils

Clavenger arm distillation apparatus

• A dried leaves are
chopped (100 g) and
mixed with water (500
ml). Steam distillation is
carried out for 4 hours
using a laboratory
Clavenger light oil arm

Clevenger light oil arm The Clevenger apparatus that

Clevenger heavy oil arm collects both heavy and light oil

A variation of hydrodistillation using a special glass apparatus designed by Clevenger.

The plant material is placed in a round bottom flask with water and heated.
The vapors pass through a graduated collection tube where the condensed essential oil is collected and its volume measured.
The Clevenger apparatus allows for easier quantification of the extracted oil compared to other methods
The Clevenger apparatus is designed to collect both heavy and light essential oils from plant materials through distillation.
It is particularly effective for oils that are denser than water, allowing for accurate measurement and separation.

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Modified Likens and Nickerson apparatus

Commercial extraction of the essential oil
The Modified Likens-Nickerson apparatus is an
advanced laboratory tool designed for the
simultaneous steam distillation and extraction of • Traditional boilers or a simple village type distillation apparatus
volatile compounds from various sources. could be used to extract the essential oil in commercial scale

The apparatus is typically made of borosilicate glass

and consists of two boiling flasks. One flask holds an
aqueous solution containing the desired compounds,
while the other contains an immiscible organic solvent.

The design allows for the return of either the aqueous

or organic layer back to their respective flasks,
enabling continuous operation and recovery of valuable
materials.

suitable for extraction of highly volatile compound like in mint.

It is connected to another condenser with dry ice with acetone.
It helps to higher efficiency of extraction and prevention of evaporation.

disadvantage of this apparatus is hardness of handling.

A traditional boilers

A simple village type extractor

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Supercritical fluid extraction (SFE) Apparatus

High-Pressure Pump:
- Dense gases
This pump is responsible for delivering the supercritical
fluid, typically carbon dioxide (CO), to the extraction
- Critical temperature chamber at the required pressure and flow rate.
& pressure
Extraction Chamber:
The chamber where the raw material is placed. It is
- Resemble designed to withstand high pressures and allows the
properties between supercritical CO to flow through the material, dissolving the
extractable compounds.
both liquids & Heating System:
gases A heating element is used to maintain the temperature of
the CO above its critical point (31°C) to ensure it remains in
a supercritical state. This is crucial for optimizing the
extraction process.
Separator:
After the extraction process, the CO containing the dissolved compounds is directed to a separator
where the pressure is reduced. This causes the solute to precipitate out of the supercritical fluid,
allowing for the collection of the extracted compounds.
Collection Vessel:
A vessel where the extracted compounds are collected after separation from the CO. This may
involve further processing or purification steps depending on the desired end product.
Back Pressure Regulator:
This component maintains the pressure in the system, ensuring that the supercritical fluid remains in
the desired state throughout the extraction process.
The high-pressure pump introduces supercritical CO into
the extraction chamber, where it permeates the plant
material. The supercritical CO dissolves the target
compounds due to its unique solvent properties.
Factor affecting on quality of extract by SFE
SOLUBILITY UNDER LIQUID CO2 Increasing the extraction temperature
generally enhances the vapor pressure Pressure significantly impacts the
1. MW < 500 daltons of the target compounds, which can density of the supercritical fluid.
2. Low - medium MW of halocarbon, aldehyde, keton, improve their solubility in the ➢ Temperature Higher pressures increase the
ester, alcohol, ether supercritical fluid. However, higher density, which enhances the
3. Low MW , non polar, aliphatic hydrocarbons -- 20 carbons temperatures can also decrease the solvent's ability to dissolve more of
4. Polar organic -- low solubility density of the supercritical fluid, the target compounds, leading to
5. Polar groups -- OH, COOH, N decrease solubility potentially reducing its solvating power. ➢ Pressure higher extraction yields.
6. Chorophyll, carotenoids, amino acid, fruit acid
--->insoluble
7. Alkaloids, phenols, aniline compounds The size of the plant material ➢Particle size
----> poor solubility particles affects the extraction
efficiency. Smaller particle sizes
increase the surface area ➢Time
available for the supercritical fluid
The time allowed for
to interact with the material,
extraction affects the overall
facilitating better mass transfer
yield and quality of the
and extraction rates.
extracted compounds. Longer
extraction times can lead to
higher yields, but they may
also result in the extraction of
unwanted compounds or
degradation of sensitive
materials.

Critical properties of various solvents

ADVANTAGES; Super Critical CO2 Extraction
Molecular Critical Critical Critical
Solvent weight temperature pressure density
g/mol ˚C Bar g/cm3 •Easy to remove,
Carbon dioxide (CO 2 ) 44.01 30.95 73.77 0.469
•Not harmful,
Water (H2O) 18.015 373.946 220.65 0.322

Methane (CH4) 16.04 -82.75 46 0.162 •odorless,

Ethane (C2 H6) 30.07 32.15 48.7 0.203
Propane (C3H8) 44.09 96.65 42.5 0.217 •tasteless,
Ethylene (C2H4) 28.05 9.25 50.4 0.215 •non flammable,
Propylene (C3H6) 42.08 91.75 46.0 0.232
• high purity, moderate condition = 310C 74 bar ,
Methanol (CH3OH) 32.04 239.45 80.9 0.272

Ethanol (C2H5OH) 46.07 240.75 61.4 0.276 •no degradation

Acetone (C3H6O) 58.08 234.95 47.0 0.278

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• ---->labile flavor compounds selectivity,

aroma & flavor-->resemblance to original
material

• Disadvantage;
Although the technology is simple, the material cost of the
extraction of essential oil is very high. Therefore this
technology is not practiced in developing countries to
extract essential oils

Soxhlet Extraction Process

Sample Preparation: The plant material is dried, ground, and placed in a thimble-shaped filter paper holder within the Soxhlet extractor.

Solvent Heating: The extraction solvent is heated in a round-bottom flask below the Soxhlet extractor. As the solvent evaporates, it travels up the distillation arm and condenses in the cooler
upper part of the apparatus.

Extraction: The condensed solvent drips into the thimble containing the plant material, extracting the target compounds. When the solvent level reaches the siphon tube, the solution is
siphoned back into the round-bottom flask, carrying the extracted compounds.

Continuous Extraction: The process repeats continuously, with fresh solvent repeatedly coming into contact with the plant material, ensuring efficient extraction of the non-volatile compounds.

Solvent Evaporation: After the desired extraction time, the solvent is evaporated, leaving behind the extracted compounds for further analysis or use.

Soxhelet extraction
(to extract non-volatile
compounds)

Solvents:
Hexane
Chloroform
Ethanol
Water
Hexane: A non-polar solvent commonly used for extracting lipophilic compounds, such as fats, oils, and
waxes, from plant materials.

Chloroform: A medium-polarity solvent that can extract a wide range of compounds, including both polar Advantages and Limitations
and non-polar substances. It is often used for extracting alkaloids and other nitrogen-containing Advantages: Soxhlet extraction is a simple, inexpensive, and efficient
compounds. method for extracting non-volatile compounds. It allows for continuous
extraction with minimal solvent use and is suitable for a wide range of plant
Ethanol: A polar solvent that can extract a variety of compounds, including polar and some non-polar materials.
substances. Ethanol is commonly used for extracting phenolic compounds, flavonoids, and other polar
phytochemicals. Limitations: The technique is time-consuming, with extraction times typically
ranging from several hours to days. It may not be suitable for thermolabile
Water: compounds due to the prolonged exposure to heat. The use of large
The most polar solvent, suitable for extracting highly polar compounds like glycosides, polysaccharides, volumes of organic solvents can also be a concern in terms of environmental
and water-soluble vitamins. Aqueous extractions are often used for preparing herbal teas and decoctions impact and safety
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Extraction of
oleoresins
Oleoresins can be extracted using
various methods, including solvent
extraction, steam distillation, and cold
pressing. The choice of extraction
method can influence the quality and
composition of the final product.

Oleoresins are natural mixtures of essential oils and resins obtained from
various plants, particularly from trees and shrubs. These compounds are
typically extracted from plant materials such as bark, leaves, and fruits
and are known for their aromatic properties and potential therapeutic
benefits.

Oleoresins consist of a combination of volatile essential oils and

non-volatile resinous compounds. The essential oils contribute to the
aroma, while the resins provide texture and additional flavor.

Modern strategies
Natural products can contribute to the search for new drugs in three
different ways:
a. Bioassay-guided isolation and identification of active ‘‘lead’’
compounds from natural sources.
1. by acting as new drugs that can be used in an unmodified state
(e.g., vincristine from Catharanthus roseus).
b. Production of natural products libraries.

c. Production of active compounds in cell or tissue culture 2. by providing chemical ‘‘building blocks’’ used to synthesize more
complex molecules (e.g., diosgenin from Dioscorea floribunda for the
synthesis of oral contraceptives).
d. More focused on bioactivity.
3. by indicating new modes of pharmacological action that allow
e. Introduction of the concepts of chemical fingerprinting, and complete synthesis of novel analogs (e.g., synthetic analogs of
metabolomics. penicillin from Penicillium notatum).

f. Selection of organisms based on ethnopharmacological

information, folkloric reputations, or traditional uses, and also
those randomly selected.

Classical or older chromatographic techniques include: THIN LAYER CHROMATOGRAPHY

Thin layer chromatography (TLC) is an important
1. Thin-layer chromatography (TLC). technique for identification and separation of
2. Preparative thin-layer chromatography (PTLC). mixtures of organic compounds.
3. Open-column chromatography (CC).
4. Flash chromatography (FC). It is useful in:

•Identification of components of a mixture (using

appropriate standards)
•following the course of a reaction,
•analyzing fractions collected during purification,
•analyzing the purity of a compound.

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THIN LAYER CHROMATOGRAPHY

filter paper

A B U C D

Solvent Properties and Elution Strengths

o
Solvent MF Bp ( C) Hazards* Dipole Elution
MW Density (g/mL) Stength
()
Hexane C6H14 68.7 Flammable 0.08 0.01
CH3(CH2)4CH3 86.17 0.659 Toxic

Toluene C7H8 110.6 Flammable 0.31 0.22

C6H5CH3 92.13 0.867 Toxic

Diethyl ether C4H10O 34.6 Flammable 1.15 0.29

CH3CH2OCH2CH3 74.12 0.713 Toxic, CNS
Depressant

Dichloromethane CH2Cl2 39.8 Toxic, Irritant 1.14 0.32

CH2Cl2 84.94 1.326 Cancer suspect

Ethyl Acetate C4H8O2 77.1 Flammable 1.88 0.45

CH3CO2CH2CH3 88.10 0.901 Irritant

Acetone C3H6O 56.3 Flammable 2.69 0.43

CH3COCH3 58.08 0.790 Irritant

Butanone C4H8O 80.1 Flammable 2.76 0.39

CH3CH2COCH3 72.10 0.805 Irritant

1-Butanol C4H10O 117.7 Flammable 1.75 0.47

CH3CH2CH2CH2OH 74.12 0.810 Irritant

Propanol C3H8O 82.3 Flammable 1.66 0.63

CH3CH2CH2OH 60.09 0.785 Irritant

Ethanol C2H6O 78.5 Flammable 1.70 0.68

CH3CH2OH 46.07 0.789 Irritant

Methanol CH4O 64.7 Flammable 1.7 0.73

CH3OH 32.04 0.791 Toxic

Water H2O 100.0 1.87 >1

HOH 18.02 0.998

THIN LAYER CHROMATOGRAPHY

Calculation of Rf’s
Visualization of components on TLC plate Rf (A) =
2.0 cm
5.0 cm
= 0.40

Solvent Front

• Observe under the UV lamp Rf (B) = 3.0 cm

5.0 cm
= 0.60
Distance solvent
• Use of vanillin reagent migrated = 5.0 cm
4.0 cm
Distance A
Rf (C) = 0.8 cm = 0.16
• Use of Iodine bath migrated = 3.0 cm
5.0 cm

Distance B
migrated = 2.0 cm 3.0 cm Rf (D) = 4.0 cm = 0.80
5.0 cm
Distance C
migrated = 0.8 cm
0.8 cm
Rf (U1) = 3.0 cm = 0.60
Origen
x x x x x 5.0 cm
A B U C D
0.8 cm
Rf (U2) = = 0.16
5.0 cm
The Rf is defined as the distance the center of the spot moved divided by the
distance the solvent front moved (both measured from the origin)

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COLUMN CHROMATOGRAPHY
Column chromatography (CC) is an extremely valuable
technique for purification of synthetic or natural
products.

Compounds are separated by CC through the same

mechanism as TLC;

A variety of adsorbents can be used as the stationary

phase;

COLUMN CHROMATOGRAPHY
Monitoring the column:

Preparative TLC

Preparative TLC operates on the same principles as standard TLC, where a stationary
phase (usually silica gel) is coated onto a glass or plastic plate. The sample mixture is
applied to the plate, and a solvent (mobile phase) is used to separate the components
based on their affinities to the stationary phase.

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HPCL

Components of HPLC:
HPLC operates on the principle of liquid chromatography, where a liquid sample is Pump: Delivers the mobile phase (solvent) at a constant flow rate under high pressure.
passed through a column packed with a stationary phase. The different components of Injector: Introduces the sample into the mobile phase stream.
the sample interact with the stationary phase to varying degrees, leading to their
separation as they travel through the column Column: The heart of the HPLC system, containing the stationary phase that separates the
components based on their interactions with it.

Detector: Monitors the eluent coming from the column and provides a signal proportional to the
concentration of the components. Common detectors include UV-Vis, fluorescence, and mass
spectrometry.

Data System: Records and analyzes the detector signals, generating chromatograms that
represent the separation of components.

Bioactivity-Guided Separation
Preparative HPLC
Extraction
Plant
Extraction Crude Extract

Bioassay

Chromatography

Repeat

Pure
Compound

Bioassay
Bioactivity-guided separation is a powerful approach used to isolate and purify bioactive
compounds from complex mixtures, such as plant extracts. The process involves several
steps:
Purpose: Bioassay: The initial extract or fraction is tested for a specific bioactivity using a relevant
Prep HPLC is used to separate and purify compounds in larger amounts than analytical HPLC, bioassay. This helps identify the fraction(s) containing the active compound(s).
making it suitable for applications in pharmaceuticals, natural product isolation, and chemical
research. Separation: The active fraction(s) are then subjected to various separation techniques, such
as column chromatography, HPLC, or HSCCC, to isolate the individual compounds.
Principle:
Similar to analytical HPLC, Prep HPLC relies on the differential interactions of compounds with a Bioassay-Guided Fractionation: Each subfraction obtained from the separation is tested again
stationary phase (typically silica or polymer-based) and a mobile phase (liquid solvent) to achieve for the target bioactivity. This iterative process helps track the active compound(s) and guide
separation. The main difference lies in the scale of the operation and the focus on obtaining pure further separation steps.
compounds.
Purification: The active compound(s) are further purified using preparative techniques like
prep-HPLC to obtain the pure bioactive compound(s).

Identification: The purified compound(s) are then identified using spectroscopic techniques
such as NMR and mass spectrometry.

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