ENVIRONMENTAL ANALYSIS LAB

ETM 521 - 2

PRACTICAL NO - 01
EXTRACTION TECHNIQUES ɪ

P. G.C.N.T.PILAPITIYA

UWU/PGU/MSC/ETM/22/019

MSc IN ENVIRONMENTAL TECHNOLOGY AND MANAGEMENT

1ST YEAR 2ND SEMESTER
Date - 01/22/23
Practical number - 01
Title - Extraction technique 01, simple solvent extraction

Introduction
Solvent extraction is one of the most common and important methods for
separation and purification of many elements. This method has proved itself very
helpful as a recovery methods for many compounds. It is a method of separation
compounds on the basis of their solubility in two different immiscible liquids like
water and organic compounds. Most of the time this technique use to discover drugs,
cosmetics, insect/pest repellents from various biological materials such as plant,
fungi bacterial.

Objective of the practical - Familiarization with different extraction methods.

Materials and methodology 01 - Extraction of metabolites from a plant material

Step 01
Materials
Ethyl acetate, Rotary evaporator,Funnel, Dried plant material, Filter papers, Conical
flask, Hexane, Motor and pestle, Round bottom flasks, Small vials, Methodology
Sonicators, Al foil

Methodology
(i) 10g of air dried curry leaves were measured and crushed.
(ii) 5g of crushed curry leaves were separated and mixed with ethyl acetate and
hexane in 2 250ml conical flasks.
(iii) The mixture was placed in a sonicator for 5 min.
(iv) The extraction was filtered out using a filter paper
(v) The crude was transferred to a weight known empty vial.
(vi) The dry weight of the crude material was measured.

Step 02
(i)The curry leaves dissolved in a small amount of methanol.
(ii) The sample spots was placed in a TLC plate using a capillary tube.
(iii) The TLC test was done in two solvents Methanol: Ethyl acetate and Ethyl acetate:
Hexane.
(iv)The solvent front was marked and visualized it under UV light.
(v)The spotted marked was calculated for Rf value.

Calculation
Step 01

Weight of the empty vials- (01) EA vial - 49.40g (02)Hexane vial - 50.04g
Weight with extraction - (01) EA vial - 49.68g (02)Hexane vial - 56.27g

Weight of the extraction - (01) EA vial - 0.28g (02)Hexane vial - 6.23g

Step 02

Table 01 - TLC plate readings

TLC plate 01 Methanol : Ethyl acetate (5:95) L= 5.9 cm
Stationary phase Distance traveled by spot
A = 2.70cm
M B1 = 4.00 cm
C1 = 4.50 cm
H B2 = 4.20 cm
C2= 4.60 cm
EA B3 = 4.10 cm
C3= 4.70 cm

TLC plate 02 Ethyl acetate: Hexane (25:75) L= 5.98 cm

Stationary phase Distance traveled by spot
M A1 = 0.80 cm
B1 =1.20 cm
C1=2.60 cm
D1 =3.50 cm
E1 = 5.00cm
H A2 = 0.50 cm
B2 =0.80 cm
C2=2.50 cm
D2 =3.70 cm
E2 = 5.50cm
EA A3 = 0.60 cm
B3 =1.00 cm
C3=2.50 cm
D3 =3.50 cm
E3 = 5.20cm

Rf value = Distance traveled by spot / L

Table 02 - Rf values for spots

Methanol : Ethyl acetate Ethyl acetate: Hexane (25:75)
(5:95) L= 5.98 cm
L= 5.9 cm
R f A(M) = 0.457 R f A(M) = 0.137
R f B(M) = 0.678 R f B(M) = 0.207
R f C(M) = 0.763 R f C(M) = 0.448
R f B(H) = 0.712 R f D(M) = 0.603
R f C(H) = 0..780 R f E(M) = 0.862
R f B(EA) = 0.695
R f C(EA) = 0..800 R f A(H) = 0.086
R f B(H) = 0.138
R f C(H) = 0.431
R f D(H) = 0.638
R f E(H) = 0.948

R f A(EA) = 0.103
R f B(EA) = 0.172
R f C(EA) = 0.431
R f D(EA) = 0.604
R f E(EA) = 0.897

Discussion
Plants are an inexhaustible source of bio active compounds that have been utilized
by man since antiquity.Plants bioactive substances are called metabolites.
Metabolites are the intermediate products produce during metabolism, catalyzed by
various enzymes that occurs naturally within plant cells. These metabolites control
the biological functions as survival, protection and communication. There are two
type of metabolites primary and secondary.

Primary metabolites - chemical compounds produced during growth and

development process (carbohydrate, vitamins, ethanol, lactic acid)

Secondary metabolites - Produced by the organisms and have ecological importance

(phenolic, steroids, antibiotic, pigments)

Mwtabolites such as alkaliods, flavonoid, terpenes, saponins, steroids and glycosides

are extract using an appropriate solvent and standard extraction procedure.
The choice of solvent dependent on the type of plant part part to be extracted
nature of bio activate compounds and the availability of compounds. Most of the
time, polar solvent such as water, methanol and ethanol are used to extract polar
compounds, whereas non-polar solvent such as hexane and dichloromethane are
used to extract non-polar compounds. A good extraction solvent was 3 major
properties as immiscibility, dissolves the compound to be extracted and does not
dissolve impurities. Polarity of the substances effects the solubility of the compound
on the solvent. Polar solvents dissolve polar molecules and non solvent dissolve non
polar molecules.

In metabolite extraction fro curry leaves (Murraya koenigii) has use two solvents (i)
Ethyl Acetate (ii) Hexane. M.koeniggi leaves are reported to have rich source of
carbohydrates, proteins, amino acids, alkaloids, mineral, vitamin A and B. They also a
source of calcium but due to the presence of oxalic acid in high concentrations (total
oxalate 1.35%, soluble oxalate 1.15%). It’s nutrition availability is affected. The
leaves also contain a crystalline glucocide, koenigiin and a resin. The most important
constituent of M.koenigii are β-xaryophyllene, β-gurjunene, β-elemence, β-
phellendrene and β-thujene.

Many herbs and spices can not be extracted from distillation method. So solvent
extraction is the safest method for extracting high quality oil. Usually 2 types of
solvents use for extraction process as organic and inorganic solvents.

Organic solvents are commonly used in dry cleaning (tetrachloroethylene) as pain

thinners (toluene, turpentine) and glue solvents (acetone, methyl acetate, ethyl
acetate) in spot removers (hexane, petrol ether). inorganic is limited to research
chemistry.

Ethyl acetate is a organic compound with the formula CH3CO2CH2CH2

C4H8O2. It is an ester of ethanol and acetic acid, solvent on large scale and
moderately polar clear colorless liquid solvent. Ethyl acetate has chosen as a
extraction solvent because of high volatility, low solubility in water and it’s efficacy
as a solvent for organic compounds.

Due to chemical and biological characteristics of ethyl acetate including medium

polarity and minimum toxicity on test strains. These properties help to extract many
biological compounds from their raw materials. Hexane is an organic compound with
a straight -chain alkane with six carbon atoms and C6H14 chemical formula. It is a
colorless liquid and odorless when pure and with boiling point approximately 69 0C.
hexane is used as a non-polar solvent as well as a neurotoxin. It’s a popular solvent
for extraction as it unique properties make it efficient and effective to use. The main
reason behind hexane being the perfect chemical for extractions is because of it’s
high evaporation rate and low boiling point. It’s low boiling point allows hexane to
retain its liquid state in cold temperatures, the high evaporation rate of hexane
allows this solvent to extract the largest amount of oil in the quickest amount of
time.
Table 03 - Relevent properties of hexane and ethyl acetate

Properties Hexane Ethyl acetate

Molecular weight (g/mol) 86.18 88.11
Density 250C (g/ml) 0.655 0.897
Boiling point (0C) 68.5 77.1
Flash point (0C) -26.0 -4.0
Viscosity 25 0C 0.29 0.43
Enthalpy of vaporization 28.85 31.94
(dyne/cm)
Dielecyric constant 1.88 6.02

Due to density and viscosity the of ethyl acetate extraction weight can be slightly
higher than hexane. To test the separation of compounds in mixture can use TLC
method. Like other chromatography techniques , TLC depend one separation and the
separation relies on the relative affinity of compounds towards both the phase. The
compounds in mobile phase move over the surface of the stationary phase. Therefor
it has a high affinity for compounds with high polarity. As a result the polar
compounds will interact with the silica gel. On the other hand low polar compounds
are less likely to interact with silica gel. They are less affected by silica gel.

The other important thing in TLC is mobile phase. TLC first spot a solution on the TLC
plate. This is then immerse in the mobile phase(developed solvent). Then the
solution rises, the compound moves up. But the different compounds move in
different rates, so compounds separate.
As the mobile phase always less polar than stationary phase, polar compounds will
tend to have a lesser affinity for the mobile phase than non-polar compounds.
Therefor high polarity compounds tend to spend less of the elution time in mobile
than the non-polar compound, so it travel slower up to the plate.

The more popular mobile phase combination is Hexane: Ethyl acetate mobile phase.
In this type of mobile phase the polarity of the solvent depend on the ration
between two compounds. In this practical the first mobile is in 3:1 ratio. The solvent
is more non-polar due to high % of hexane, but it is more polar than straight hexane
due to presence of ethyl acetate. The second mobile phase is Methanol: Ethyl
acetate to 5:95 ration. This is a high polar solution than the first mixture because of
the presence of methanol.

The Rf value is the ratio of the solute’s distance travel to the solvents distance
traveled. It act as a physical constant for a organic molecules that can be used to
verify a molecules identity.

-A strong IMF possible with the stationary phase(often more polar function
groups on the compound) the more time the compound will be stationary
-lower Rf value

-The more polar functions groups present on a compound the less it tend to
be attracted to the less polar elutant, and less time spent in the mobile phase.
-low Rf value

Conclution
According to the results , the final weights of the extractions are like ethyl acetate -
0.28g and exane extraction- 6.23g. As the result, the hexane extraction is higher than
ethyl acetate extraction. But in ideal situations the low polar solvent extract low
amount of compounds. This kind of result may be occur due to availability of non-
polar impurities.

In TLC tests, the mobile phase polarity;

Meth:EA (1:19) > Hex: EA (3:1)

Th the TLC test the compound separation with methanol and ethyl acetate is lower
than hexane: ethyl acetate mobile phase and the Rf values are moderately high
(range from 0.457 to 0.800. that means compounds spend less amount of time in
stationary phase and tend to travel with mobile phase.

The Rf values in second TLC with ethyl acetate and hexane mobile phase is range
from 0.086 to 0.948. Among 15 readings 9 readings are more intend to stationary
phase. But in this TLC the component separation is high compare to first TLC test.

The results may conclude as the final extraction may have involved some non-polar
impurities and the extractions are more polar.

References

(i) Bele, A.A., and Khole, A., an overview on thin layer chromatography ‘International
Journal of Pharmaceutical science and research’ viewed om 30th January 2023

(ii) Ghasemzodeg, A. et al (2014) ‘ Evaluation of bioactive compounds,

pharmaceutical quantity and anticancer activity of curry leaves and alternative
medicine, 2014 pp 1-8. viewed 30th January 2023.5.30

(iii)Sablania, V., Bosco, S.J.D., and Bashir, M., (2019) ‘Extraction process optimization
of Murroga koenigii leaf extraction and antibiotic properties’ Journal of food science
and technology 56(12) pp5500-5508. viewed on 3rd February 2023.

(iv)Chemis, Rn 2022, Solvent extraction principle easy process, applications, viewed

on 9th February 2023,< https://www.chemistnite.com/analytical-chemistry-solvent-
extraction-principle>

(v) Nicholes, L 2020, Separation theoty, viewed on 9th February 2023; <
https://www.chem.libretexts.org/cause/SUNY-Oneonta/chem-221%3A-organic-
chemistry-I- (Bennett) 25 3ALAB-Text book.