Bookchapter Fkkksa07

First Edition 2007
ZAINUDDIN ABDUL MANAN, MOHAMED MAHMOUD NASEF

& SITI HAMIDAH MOHD. SETAPAR 2007
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information storage and retrieval system, without permission in writing from Universiti
Teknologi Malaysia, 81310 Skudai, Johor Darul Tazim, Malaysia.
Perpustakaan Negara Malaysia
Cataloguing-in-Publication Data
Advances in separation process / [editors Zainuddin Abdul Manan,

Mohamed Mahmoud Nasef, Siti Hamidah Mohd. Setapar].
ISBN 978-983-52-0595-8
1. Membrance separation. 2. Chemical processes. I. Zainuddin Abdul
Manan, 1966-. II. Mohamed Mahmoud Nasef. III. Siti Hamidah Mohd. Setapar,
1961-.
660.2842
Editor: Zainuddin Abdul Manan dan Rakan-rakan
Pereka Kulit: Mohd Nazir Md. Basri & Mohd Asmawidin Bidin
Diatur huruf oleh / Typeset by
Fakulti Kejuruteraan Kimia & Kejuruteraan Sumber Asli
Diterbitkan di Malaysia oleh / Published in Malaysia by

PENERBIT
UNIVERSITI TEKNOLOGI MALAYSIA
34 38, Jln. Kebudayaan 1,Taman Universiti

81300 Skudai,
Johor Darul Tazim, MALAYSIA.
(PENERBIT UTM anggota PERSATUAN PENERBIT BUKU MALAYSIA/
MALAYSIAN BOOK PUBLISHERS ASSOCIATION dengan no. keahlian 9101)
Dicetak di Malaysia oleh / Printed in Malaysia by
UNIVISION PRESS SDN. BHD
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Jalan Serdang Raya, Taman Serdang Raya,
43300 Seri Kembangan,
Selangor Darul Ehsan, MALAYSIA.
CONTENTS
Preface
vii
Chapter 1 Introduction to Separation Processes

Noorhalieza Ali
Chapter 2 Separation Methods in the Pharmaceutical 15

Industry
Siti Hamidah Mohd. Setapar
Chapter 3 Application of Supercritical Fluid in the
Extraction of Active Compounds From
Plant Material
Mohd Azizi Che Yunus, Wahyu Bahari
Setianto and Zainuddin Manan
31
Chapter 4 Regeneration Technique for the Spent

Bleaching Clay: Oil Extraction and
Activation
Abu Hanipah N., Wahyu Bahari Setianto,
Nur Madehah O., Nik Norulaini Nik A. R,
Mohd Omar A. K and Mohd Azizi Che
Yunus
62
vi
Content
Chapter 5 Ion Exchange Technology: Principles and 75

Progress in Materials Development
Mohamed Mahmoud Nasef, Hamdani
Saidi and Zaini Ujang
Chapter 6 Potential of Bio-Products Adsorption by
Immobilized Metal Ion Affinity
Mesoporous Adsorbents
Azizul Azri Mustaffa
125
Chapter 7 Reverse Micelle Extraction

137
Chapter 8 Liquid-Liquid Separation of Antibiotic

with Reverse Micelles
157
Chapter 9 Extraction And Separation Of Djenkolic 175

Acid From Pithecellobium Jiringan Seeds
Using Supercritical Carbon Dioxide
Extraction
C. Y. Mohd Azizi, N. A. Nik Norulaini,
B. S. Wahyu, A. K. Mohd Omar
Index
197
PREFACE
Separation and purification technologies play a very vital role in a

variety of manufacturing industries and account for a significant
portion of plant energy and overall product cost. The major
industrial separation technologies including distillation,
evaporation, drying, extraction, absorption, adsorption, membrane
and crystallization have been practiced for separation in chemical
plants for many years. Developing efficient separation processes
that can reduce the energy consumption and minimize the
environmental pollution have been the subject of R & D in many
research institutes. This can be achieved through multiple efforts
including optimization of system design and/or developing new
separation materials that promote low energy consumption
separation technologies such as membranes and extraction.
The purpose of this edited book Advances in Separation
Processes is to report on the advances in R & D in some
separation and purification technologies. The book contains 10
chapters ranging from state-of-the-art reviews to research articles
addressed to all chemical engineers, scientists at universities and in
industry who wish to keep abreast of advances in the topics
covered.
The book also provides background information necessary
to understand the need to develop realistic R & D directions
towards acquiring new and advanced system designs and
sustainable separating materials to increase the efficiency of some
viii
Preface
viii
separation processes. The information reported by the contributors

are merely representing their own research interest and findings on
particular separation technologies giving this book a flavor of
broad range of views. Some valid separation techniques or
processes may have not been covered in this book due to
contributors judgments in prioritizing technologies currently
under investigations in our research groups in Chemical
Engineering Department.
Chapters of this book were obtained in a camera ready
form without any content modification from the editor, from a
group of experienced lecturers in Chemical Engineering
Department involved in teaching and practicing separation and
purification technologies at both academic and industrial levels for
many years. The contributors were motivated by the appealing
findings of their research and consultancy projects together with
their desire to promote the ambitious publication policy adopted by
University Teknologi Malaysia to contribute intensively to the
body of knowledge of research niche areas.
I would like to take this opportunity to thank my fellow
contributors for their intensive efforts to prepare the manuscripts of
their chapters according to the required format and abiding by the
given time frame despite being very busy at the beginning of the
semester. The inputs from Dr. Ramli Mat, Prof. Nor Aisha Saidna
Amin and Prof. Arrifin Samsuri were instrumental in completing
this book. Without the dedication and support of all involved
parties, this book would not be possible in this short time.
Zainuddin Abdul Manan

Mohamed Mahmoud Nasef
Universiti Teknologi Malaysia
2007
INTRODUCTION TO SEPARATION
PROCESSES
Noorhalieza Ali
1.1 DEFINITION OF SEPARATION PROCESSES

King (1980) define separation processes as those operations which
transform a mixture of substances into two or more products which
differ from each other in composition. These may involve either
removing a single component from a mixture or separating a solution
into its almost pure components. This is achieved by exploiting
molecular, thermodynamic, and transport properties differences
between the species in the different phases present such as those
shown in Table 1.1.
Table 1.1 Molecular, thermodynamic and transport properties
Molecular properties
Molecular weight
van der Waals volume
van der Waals area
Molecular shape (acentric
factor)
Dipole moment
Thermodynamic and transport

properties
Vapour pressure
Solubility
Adsorptivity
Diffusivity
According to the second law of thermodynamics, which states

that all natural processes tends to increase the randomness (entropy) or
homogeneity of the universe, all naturally occurring processes are
Advances in Separation Processes
inherently mixing processes which occur spontaneously. The reverse

processes, demixing or separation, cannot occur spontaneously. To
separate mixtures into two or more products of different composition,
energy in the form of heat or mechanical work need to be supplied.
For two substances A and B will mix spontaneously when the free
enthalpy of the mixture/product is smaller than the sum of the free
enthalpies of the pure substances. The minimum amount of energy
needed for complete separation is that equivalent to or larger than the
entropy (energy) of mixing.
In practice, the energy requirement for separation will be many
times greater than this minimum value. Most separations use about 50
times the minimum energy requirement based on the ideal
thermodynamic requirements. For example, in the desalination of
water, the theoretical energy requirement for demixing 1000 l of
seawater is only about 4 MJ but 280 MJ is needed to produce
desalinated water depending on the process used. The situation is
similar in many other separation processes.
Separation processes in the industries involve removal of
impurities from raw materials, isolation and purification of products
and by-products, separation of recycle streams and waste treatment
such as air and water effluents prior to discharge to minimize the
environmental impact.
1.2 HOMOGENEOUS SYSTEMS
The feedstocks to separation processes may be either homogeneous,

consisting of a single phase solids, liquids, gases or supercritical
fluids, or heterogeneous mixtures containing any combination of these
phases. Examples of heterogeneous mixtures are slurries, emulsions
and gases or vapours carrying entrained solids or liquids.
Introduction to Separation Processes
Homogeneous systems usually require more effort to separate.

To separate a homogeneous mixture, the mixture is usually converted
into a heterogeneous system by either chemical or physical means. In
physical separations, a liquid solution is made to form a different
phase either by changing the temperature, adding a solvent which is
immiscible with the solution or by selective adsorption on a suitable
solid which will leads to either a partial or complete separation of the
components. Separation of a homogeneous mixture by chemical mean
is by using chemical reactions. One or more of the components are
converted into a more suitable physical state such as converting
substances which are soluble in water into insoluble, easily separable
precipitates such as the precipitation of metal ions as sulfides.
1.3 EQUILIBRIUM PROCESSES
Homogeneous systems can be based on either equilibrium processes or

rate-controlled (diffusional) processes. If mass transfer is rapid
resulting in quick equilibrium, then the separation is equilibrium
processes. On the other hand if the mass transfer is slow such that
equilibrium is not reached quickly, then the separation is ratecontrolled processes.
In an equilibrium process, two phases are brought into contact
with each other, mixed thoroughly, and then separated with a
redistribution of the components between the phases. Stages or
cascades of individual units are operated with two streams flowing
countercurrent to each other so as to effectively bring the two phases
together. The driving force for separation is the desire of the two
phases to try and re-establish equilibrium at each stage and the degree
of separation in each stage is governed by a thermodynamic
equilibrium relationship between the phases. An example is
distillation, in which a new vapor-phase is generated by the addition of
heat to the liquid feed resulting in a vapour phase which is in

equilibrium with the residual liquid. The end result is the separation
of two liquids with different boiling temperatures. Other equilibrium
based processes include extraction (removal of a liquid by means of a
liquid for which it has stronger affinity) and leaching, or solid
extraction (removal of a solid by means of a liquid for which it has
stronger affinity).
1.4 RATE-CONTROLLED PROCESSES
Rate-controlled (diffusional) processes involve differential rates of

mass transfer to achieve a separation. The mass transfer of one
component of a feed stream from the feed phase into a second phase is
due to a gradient in a physical property such as concentration,
temperature, pressure or external force field differences. They achieve
a change in composition of the product from differences in diffusion
rate of feed components through a barrier across which the gradient is
applied. Membrane processes such as ultrafiltration, microfiltration,
reverse osmosis and pervaporation are based upon size and chemically
selective permeability differences between components of a feed
stream. Another example of rate-controlled processes is the processes
that depend on slow chemical reactions to make the separation.
Absorption, a process by which a vapor is removed from its mixture
with an inert gas by means of a liquid in which it is soluble, is one
example of mass transfer based process. Desorption, or stripping, on
the other hand, is the removal of a volatile gas from a liquid by means
of a gas in which it is soluble.
1.5 HETEROGENEOUS SYSTEMS
Heterogeneous systems can be easily separated by mechanical

methods because of differences in the physical properties of their
homogeneous components. Examples are settling, centrifugation,
flotation, filtration, magnetic separation and electrostatic precipitation.
The heterogeneous system can further be classified into two major
groups of unit operations: the solid/fluid (either gas or liquid)
separation, and solid/solid separation. The separation of solid particles
from fluids can be done using a wide variety of methods depending on
the type of solids, the proportion of solid to fluid in the mixture,
viscosity of the solution and other factors. In filtration a pressure
difference is set up across a filtration medium which blocks the
passage of the solid particles and allows the fluid to pass through.
Heterogeneous particles, suspended in fluid can be separated into
individual groups of the constituent homogeneous particles in terms of
the differences in the physical or physical-chemical properties of the
particles such as differences in size or density of the particles. Solidsolid separation processes are generally used to separate minerals
based on differences in intrinsic properties such as specific gravity,
magnetic susceptibility or conductivity. An example is a centrifugal
separator which uses density differences.
1.6 MECHANISM OF SEPARATION
Seader and Henley attributed separation to be caused by any five

general separation techniques or combination thereof, each being
driven by energy and/or the addition of mass to alter properties
important to separation.
The most common industrial technique is the creation of a

second phase by a separating agent which takes the form of an energy
(ESA) transfer or by pressure reduction. In evaporation, heat (ESA) is
supplied to the evaporators so as to cause the more volatile
components of a liquid feed to vapourise into a gas phase. In a second
technique, a second phase is introduced in the form of a solvent/mass
(MSA). This technique is applied in liquid-liquid extraction where one
or two liquid solvents is added to the original feed . When only one
solvent is supplied, the solvent selectively dissolves only one or a
fraction of the components in the feed mixture. For a two-solvent
extraction, each solvent has its own specific selectivity for dissolving
the components in the feed mixture. Some separation processes utilize
more than one separating agent, for example, in extractive distillation
where both solvent (MSA) and heat (ESA) is supplied. The solvent
(MSA) is added so as to increase volatility differences between
selected species of the feed so as to facilitate separation which is
initially not easily accomplished by just adding heat as the volatility
differences between species are so small.
A less common technique but is gaining in popularity, utilizes
the use of a barrier which restricts and/or enhances the movement of
certain chemical species with respect to other species. Membrane
processes using microporous and nonporous membranes as
semipermeable barriers is used resulting in separation as different
components in the feed mixture permeable at different rate due to size
and chemical selectivity for the membrane material. The fourth
tseparation technique uses solid mass-separating agent (MSA) which
act directly or as inert carriers for other substances so as to cause
separation. Adsorption consists of the removal of a species (solute)
from a fluid feed mixture by means of a solid adsorbent with which it
has a higher affinity until it eventually becomes saturated and must be
regenerated or replaced.
Lastly, separation by an external force field or gradient
technique, takes advantage of differing degrees of response of
molecules and ions to forces and gradients. In centrifugation, a
centrifugal force field is applied. An example of a centrifugation is the
use of cyclone separators for the removal of suspended dust particles

from gases where coarse solids are thrown outwards against the walls
of the separators due to the action of the centrifugal force and fall to
the bottom under the
action of gravity. Small particles are removed by means of an exhaust
fan at the top of the separators.
For all the general techniques, the degree to which a separation
can be achieved depends on differing rates of mass transfer of the
individual components of the mixture, with limits dictated by
thermodynamic equilibrium. Often, the degree of separation can be
greatly improved by using multiple contacting stages, with each stage
approaching equilibrium, and/or using a sequence of two or more
different types of separation methods in a hybrid system. Table 1.2
shows some examples of separation processes and their separation
techniques.
Table 1.2 A few examples of separation processes and their separation

techniques.
Separation Operation
Separating Agent(s)
Examples
Distillation(1)
Heat (ESA)
Petroleum refining
Extractive
Distillation(2)
Solvent (MSA) and

heat (ESA)
Separation of
aliphatics from
aromatics
Absorption(3)
Liquid absorbent
(MSA)
Removal of CO2 from

natural gas
Stripping(4)
Stripping vapour
(MSA)
Removal of benzene
from wastewaters
Liquid-liquid
Extraction(5)
Liquid solvent
(MSA)
Extraction of soybean
oil from soybeans
Supercritical Fluid
Extraction(6)
Liquid solvent
(MSA)
Recovery of
carotenoid from palm
oil
Drying(7)
Gas (MSA) and/or

heat (ESA)
Recovery of solvent
from polymer
Evaporation(8)
Heat (ESA)
Concentration of
sucralos
Crystallization(9)
Heat (ESA)
Fat fractionation in
vegetable oil refining
Leaching(10)
Liquid solvent
(MSA)
Extraction of sugar
from sugar beet
Reverse Osmosis(11)
Nonporous
membrane
Porous membrane
Desalination of
seawater
Dialysis(12)
Microporous
membrane
Separation of H2SO2
from nickel and
copper sulfates
Microfiltration(13)
Microporous
membrane
Protein concentrates
from skim milk
Ultrafiltration(14)
Nonporous
membrane
Separation of whey
from cheese
Pervaporation(15)
Nonporous
membrane
Dehydration of Ethyl
Alcohol
Gas permeation(16)
Liquid membrane
Nitrogen from air
Liquid
Membrane(17)
Solid adsorbent
Extraction of organic
acids from wastewater
Adsorption(18)
Solid or liquid
adsorbent
Removal of Arsenic
from water
Chromatography(19)
Resin
Separation of salt
from glycerol.
Ion Exchange(20)
Centrifugal force
field
Recovery of heavy
metals
Centrifugation(21)
Thermal gradient
Separation of palm
kernel shell from palm
kernel
Thermal diffusion(22)
Electrical force field
Separation of chlorine
isotopes
Electrolysis(23)
Concentration of
heavy water
Electrodialysis(24)
Desalination of sea
water
Electrophoresis(25)
Separation of
biochemicals
(1) - (10): separation by phase addition or creation, (11) (17): separation by

barrier, (18) (20): separation by solid agent, (21) (25): separation by force
field or gradient.
10
1.7
SELECTION
PROCESSES
OF
FEASIBLE
SEPARATION
The selection of the proper process depends on several factors, for

example, the nature of the constituents in a mixture, the volume of the
solution to be handled, the degree of separation required and the cost
of the process. Two general criteria apply to all separation processes:
the separation must be feasible technically and economically. The
separation process must be able to achieve the desired separation and
also achieve a quality product. Sometimes a combination of two or
more separation processes is necessary to meet the requirements.
However, economical feasibility depends strongly on the value of the
products isolated. The separation cost is often related directly to the
degree of dilution for the component of interest in the initial mixture.
Seader and Henley listed four important factors in the selection
of feasible separation operation as shown in Table 1.3. When the
species are dilute in the feed, separation operations such as those based
on the use of barriers or solid agents are the preferred operations.
Large differences in certain properties must exist or large number of
stages must be provided when very pure products are required.
Table 1.3 gives a list of the more common separation
operations ranked according to ease of scale-up where the top
operations are frequently designed without any laboratory data or
pilot-plant tests. The middle operations usually need laboratory data
whereas the bottom operations require pilot plant test on actual feed
mixtures. Also included in the table is the ease of providing multiple
stages and to what extent parallel units may be required to handle high
capacities.
11
Table 1.3 Factors that influence the selection of feasible separation

operations.
Feed
Conditions
Product
Conditions
Composition
Flow rate
Temperature
Pressure
Phase state
Required
purities
Temperatures
Pressures
Phase state
Property
differences that
may be exploited
Molecular
Thermodynamic
Transport
Characteristics of
separation operation
Ease of scale-up
Ease of staging
Temperature, pressure
and
phase-state
requirement
Physical
size
limitations
Energy requirements
An optimum separation process involves a balance between energy

costs and the costs for equipment to minimize energy. Ultimately, the
process having the lowest operating, maintenance and capital costs is
selected.
Table 1.4 Ease of scale-up of the most common separation operations.

Operation in decreasing
ease of scale-up
Distillation
Absorption
Extractive and azeotropic
distillation
Liquid-liquid extraction
Membranes
Adsorption
Crystallization
Drying
Ease of staging
Easy
Easy
Easy
Easy
Repressurization
required between
stages
Easy
Not easy
Not convenient
Need for parallel

units
No need
No need
No need
Sometimes
Almost always
For regeneration cycle

Sometimes
Sometimes
12
1.8 IMPORTANCE OF SEPARATION PROCESSES
Separation processes play an important role in petroleum, chemical,

petrochemical, pharmaceutical, mineral and other industries. Often,
separation itself can be the main function of an entire process as is the
case of desalination of seawater. Overall, separation processes
account for 40 - 70% of both capital and operating cost in the industry
(Humprey and Keller, 1997). In the chemical and petroleum refining
industries, 45% of its annual energy consumption is simply in
separating products from its main process streams. In biotechnology,
separation and purification is done in the downstream processing
where 50 90% of the production cost of a typical biological product
is accounted for. Hence, proper application of separation processes can
greatly enhance productivity, profitability and global competiveness.
The energy requirements increase rapidly with the required
degree of purity. As more and more attention is placed on energy
consumption and as industries begin involving themselves in how to
resolve these issues, the role of the separation process is changing.
With a growing emphasis on global warming, diminishing natural
resources, and greenhouse gas emissions, companies are seeking new
ways to slash energy consumption which pose challenges that will
require new processing approaches.
Separation processes are used to reduce the quantity of
potentially toxic or hazardous substances discharged to the
environment. Pollutants are frequently present in only trace quantities.
The problem of removing pollutants from extremely dilute solutions is
very crucial as the allowable levels for pollutants are lowered. The
costs of waste disposal in the manufacturing sector are very high and
diverse as shown in Figure 1.1. Hence, the development of separation
processes with increased cost-competiveness, boost energy efficiency,
increase productivity, and minimal environmental impact is very
crucial for sustainable operation.
The importance of an effective separation processes has never
been greater as the world seeks to reduce its reliance on fossil fuels
13
and primary ores. Previously, petroleum is used to produce a large

number of industrial products but at present, nearly all of these goods
can be produced with biobased materials. Separation processes that
enable valuable materials to be recovered from waste stream are also
likely to be in increasing demand. What was previously considered
waste now can be separated and processed into a variety of products.
In the future, green separation processes will be designed for
optimal energy efficiency with minimal environmental impact. All
materials will exceed environmental standards to reduce or eliminate
the quantity of potentially toxic or hazardous materials in the
manufacturing process as well as in the final product. It is clear that
this is an important technology area and will continue to grow.
Inefficient use of raw

materials
Health & Safety

Pressures from authority &
neighborhood
Costs of Waste
Legislation
Hazard Evaluation
Waste Disposal
Figure 1.1 The costs of waste.
14
REFERENCES
Afonso, C. A. M. and Crespo, J. G., 2005, Green Separation

Processes: Fundamentals and Applications, WILEY-VCH
Verlag GmbH & Co., Weinheim.
Humphrey, J. L. and Keller II, G. E., 1997, Separation Process
Technology, McGraw-Hill, New York.
King, C. J., 1980, Separation Processes, 2nd Ed., McGraw-Hill, New
York
Noble, R. D. and Terry, P. A., 2004, Principles of Chemical
Separations with Environmental Applications, Cambridge
University Press, Cambridge, UK.
Long, R. B., 1995, Separation Processes in Waste Minimization, CRC
Press.
Lu, S., R. J. Pugh and Forssberg, K. S. E., 2005, Interfacial Separation
of Particles, Elsevier,
Seader, J. D. and Henley, E. J., 2006, Separation Process Principles,
2nd Ed., Wiley, New York.
Schweitzer, P. A., 1997, Handbook of Separation Techniques for
Chemical Engineers, 3rd Ed., McGraw-Hill, New York.
Wankat, P. C., 2007, Separation Process Engineering, 2nd Ed., Pearson
Education, Inc.
Wiberg, E., N. Wiberg and Holleman, A. F., 2001, Inorganic
Chemistry, Academic Press.
2
SEPARATION METHODS IN THE
PHARMACEUTICAL
INDUSTRY
2.1
INTRODUCTION
It has been discussed in many papers that the development of efficient

methods for the separation of bioproducts from fermentation broths is
crucial to advancements in biotechnology.
Various forms of
chromatography and electrophoresis are usefully applied in the
purification of high value pharmaceuticals but are expensive and
difficult to scale-up beyond laboratory sizes (Cascaval et al., 2001).
Therefore, there is a significant need for efficient methods that can
separate and concentrate pharmaceutical products continuously, with
new cost-effective and simple separation techniques that can be
scaled-up easily.
For a wide range of chemical and biochemical products, the
cost of separation and purification processes represents a significant
proportion of the unit cost of production (Tessier et al., 2005, Soto et
al., 2005), as well as the contribution of the process to the final cost
being of 20.6% to 90% (Cascaval et al., 2001). It has been reported
that fermentation based products are typically capital intensive
because large and complex fermentors and extensive equipment for
multi-step downstream processing are required to handle large volume
fermentation broths with a low product concentration (Liu et al.,
2004). As a consequence, liquid-liquid extraction has been widely
used. Liquid-liquid extraction is the transfer of certain components
from one phase to another when immiscible or partially soluble liquid
16
phases are brought into contact with each other (Kilikian et al., 2000),
according to their partition coefficient (Chimuka et al., 2004). The
cost of liquid-liquid extraction is much lower than other more
sophisticated bioseparations method such as liquid chromatography
(Gu, 2000). The process design and its scale-up are easier than those
for other separation techniques, and liquid-liquid extractions can
minimise irreversible absorption losses and surface denaturation losses
when compared to absorption methods (Gerson, 1988).
Unfortunately, since liquid-liquid extractions are infrequently
used in the laboratory, they are often overlooked in process
development and scale-up (Gerson, 1988). However, some attempts
have been made to investigate the separation and purification
processes that are based on liquid-liquid extraction techniques,
especially the solvent extraction method as discussed thoroughly by
Soto et al. (2005). Hatton (1985) asserted that an important step in the
production of antibiotics is the recovery and concentration of the
product from the fermentation broth in which it is produced. He
further pointed out that the physicochemical properties which
distinguish the desired solute from other fermentation products must
be exploited in the selection of an appropriate separation technique for
this step; liquid-liquid extraction has been successfully employed in
the isolation of different bioproducts. The successful implementation
of liquid-liquid extraction in the separation and concentration of
bioproducts depends both on thermodynamic equilibrium constraints
and on process considerations such as relative flow rates and the
contacting patterns of the two phases. The partitioning behaviour of
bioproducts can be influenced strongly by solution properties like pH,
ionic strength, antibiotic concentration, and can also be modified
through the introduction of additives which interact with either the
solute and/or the solvent to cause effective changes in the solute
distribution between the phases.
The effectiveness of the liquid-liquid extraction process also depends
on the solvent selected for recovery of the solute from the
fermentation broth (Liu et al., 2004). Popular solvents employed in
Separation Methods in the Pharmaceutical Industry
17
the extraction of bioproducts are alcohols, ketones and acetates. These

are all inexpensive and readily available, immiscible with aqueous
solutions and exhibit the desirable physical properties of low viscosity
and density which are significantly different from those of water. The
selectivity of solvent for the product can usually be influenced by
adjustment of the broth pH, or by the introduction of other additives to
the aqueous phase. Ordinary solvent extraction can be applied to large
scale processes for separation of pharmaceutical products, e.g.
antibiotics; however, its application is not always suitable because of
denaturation in organic solvents (Yang et al., 1994; Liu et al., 2004;
Tessier et al., 2005). Hu and Gulari (1996) argued that conventional
organic solvent extraction for aminoglycoside molecules was
inefficient because of their hydrophilic nature. They suggested that
ion exchange and reverse micelle extraction are the appropriate
alternative methods for the recovery of these products in industrial
operations.
Alternative novel purification techniques such as reverse
micelle systems have been developed, which may be used as a first
step of the purification process, leading to higher recovery yields as
reported by Aires-Barros and Cabral (1991). In the past decade,
reverse micelle have attracted a lot of attention as a novel method for
separating and purifying many biological products (Wolbert et al.,
1989; Leser and Luisi, 1990; Ono et al., 1996), because reverse
micelle provide a special microenvironment in an organic medium that
make it possible to retain the structure of biomolecules that dominates
their specific functions (Ono et al., 1996). A liquid-liquid extraction
with a reversed micellar phase has the capacity to solubilise specific
biomolecules from dilute aqueous solutions such as fermentation and
cell culture media (Kilikian et al., 2000). Reverse micelle is found to
be a versatile tool for separating biomolecules and show a close
similarity with liquid-liquid extraction since both are diphasic
processes which consist of partitioning a targeted solute between an
aqueous feed phase and an organic phase and then operating the back
transfer to a second aqueous stripping phase (Rodrigues et al., 1999;;
Kilikian et al., 2000). Consequently, a potential biotechnology
18
application of reverse micelle as an alternative for solvent extraction

methods is one of the first steps in downstream processing (Regalado
et al., 1996).
2.2
CONVENTIONAL SOLVENT EXTRACTION
Conventional solvent extraction is one of the most important

techniques for recovering pharmaceutical products, e.g. antibiotics,
from fermentation broths. The method has been used for over 100
years as an industrial unit operation (Grinbaum, 2003). The successful
implementation of a solvent extraction process depends on the solvent
selected for recovery of the solute from its fermentation broth (Hatton,
1985). The selection of solvents is crucial and must take account of
their selectivity and capacity, price, availability, toxicity and process
compatibility (Mat and Stuckey, 1994). Popular solvents employed in
antibiotic extractions are butanol, methyl isobutyl ketone and pentyl or
butyl acetate.
Though the application of organic solvents to whole broth
extraction of penicillin and other antibiotics is successful, there are a
lot of potential disadvantages in using this extraction method which
include:
1) The need to use a higher concentration of surfactants to achieve a
reasonable phase separation. Also, for biochemical products such
as penicillin, extracting solvents need to be added to get optimum
product recovery (Verral, 1988).
2) A stable emulsion could be formed when denaturing of the
antibiotics occurs during the solvent extraction process, such that a
very efficient deemulsifier is required.
The addition of
demulsifiers increases the production price and can cause
19
environmental problems as emulsifiers cannot be recycled and

discarded along with the aqueous solution (Yang et al., 1994;
Nabais and Cardoso 1999; Yu et al., 2003; Tessier et al., 2005;
Mathew and Juang, 2007).
3) For an unstable product such as penicillin G, a low temperature
and expensive centrifugal extractor are required; however, there is
still 10 to 15 % loss of products (Yang and Cussler, 2000).
4) Solvents are very volatile compounds and can be evaporated
easily, which leads to wastage of solvents (Yang et al., 1994,
Nabais and Cardoso, 1999).
5) Some antibiotics such as cephalosporin are difficult to isolate by
solvent extraction because of their amphoteric nature and high
solubility in water (Ghosh et al., 1997).
6) The loss of solvents could occur because of only partial miscibility
with water as a result of their fairly high dielectric permittivity
values. Their ionic nature at high pH and instability at low pH
increases the difficulties associated with solvent extraction of
antibiotics (Ghosh et al., 1997).
2.3
LIQUID MEMBRANE EXTRACTION
Extraction using liquid membranes has been studied since the 1980s
and is one of the most advantageous separation techniques present
(Cascaval et al., 2001; Goto, 2006). The method entails transferring a
solute between two aqueous phases at different pH values, which are
separated by a solvent and a carrier layer.
20
A liquid membrane consists of two immiscible phases namely oil

membrane bulk phase and internal phase droplets. The later is also
known as the continuous phase which contained emulsion globules as
the continuous phase (Thien et al., 1986). Commonly, liquid
membranes are obtained either by emulsification (emulsion liquid
membrane, ELM) when their stability is poor, or by including the
solvent in a hydrophobic porous polymer matrix (supported liquid
membrane, SLM). Cascaval et al. (2001) carried out a comparison
between extraction using liquid membranes and conventional liquidliquid extraction. The claimed advantages are as follows:
1) The quantity of solvent used is small because of its continuous
regeneration.
2) The loss of solvent is small during extraction process.
3) Provided the pH gradient between the two aqueous phases is
maintained, there is a possibility of solute transport through liquid
membranes that have been used for the separation of some
biosynthetic products, namely carboxylic acids, amino acids and
antibiotics.
Since its discovery two decades ago, the ELM has been studied for the
separation of metal ions, amino acids, and carboxylic acids from dilute
aqueous (e.g., Ho and Li, 1992). ELM extraction does not encounter
the equilibrium limitations of conventional solvent extraction
processes and can offer high selectivity of separation of solutes from
low concentration feeds (Ghosh et al., 1997).
However, ELM also suffers from some major drawbacks which make
the system difficult to operate commercially (Pellegrino and Noble,
1990):
21
1) The toxicity of the carrier and solvent should be carefully

considered when designing an ELM system for the separation of
antibiotics.
2) Stability of the membrane is needed to obtain an efficient initial
mass transfer and this requirement always causes an increase in
process costs.
A liquid membrane involving a microporous polymeric support
membrane, the socalled SLM, can be more feasible for scale-up and
adaptability for continuous operation (Ghosh et al., 1997). However,
the system suffers from the drawbacks of high mass transfer resistance
across the liquid membrane and low membrane stability.
2.4
AQUEOUS TWO-PHASE SYSTEM
Liquid-liquid extraction processes found in the chemical processing

industry employ immiscible aqueous and organic phases to separate
components, including those used successfully in the purification of
antibiotics. However, many biologically active compounds are prone
to damage by exposure to organic solvents. An alternative to organicaqueous extraction processes is to use aqueous solutions of
hydrophilic polymers, which can be induced to form two aqueous
phases under certain conditions (Cascaval et al., 2001; Mathew and
Juang, 2007). Aqueous two-phase systems (ATPS) are formed by two
different polymers, the most widely used combination being dextran
and polyethylene glycol, or polymer and a salt (Ghosh et al., 1997).
Aqueous two-phase systems have been used for the recovery of a
variety of intracellular enzymes from disrupted cell broths,
purification of interferon from mammalian cell cultures, affinity
purification of enzymes and proteins and extractive bioconversion
22
(Lee and Sandler, 1990). In an ATPS, a given molecule partitions

between the two phases depending on its partition coefficient, defined
as the ratio of the molecule concentration in the one phase to that in
the other phase. The advantages of high water content,
biocompatibility, low cost and space-time yield support the extended
use of this technique (Rito-Palomares et al., 1996). On the other hand,
ATPS has disadvantages as follows:
1) The cost of a polymer/polymer system, which typically consists of
fractioned dextran as the bottom phase polymer, is too expensive
to apply in large-scale applications (Ghosh et al., 1997).
2) The ionic strength of the salt used is high which severely
influences the affinity partitioning of the biomolecules to be
extracted (Ghosh et al., 1997).
3) The polluting effect of the phase components used. The cost of
disposal is increased due to the presence of phosphate , which is
one of the most difficult nutrients to be removed efficiently in
waste treatment plants (Verral, 1988).
4) A high centrifugal force is generally required to obtain an efficient
separation of bio-products (Yang et al., 1994). The consumption
of chemicals gets higher and increase the operational costs.
2.5
CHROMATOGRAPHY
In chromatography, components of a solution are separated due to the

partitioning of the components between a solid (stationary) phase and
a fluid (mobile) phase. Partitioning occurs on the basis of charge,
polarity, hydrophobicity, size or affinity. There are a number of
23
different types of chromatography based on the interactions which

occur between the solutes and the stationary phase (Verral, 1988). In
spite of being relatively costly, chromatographic methods continue to
be dominantly practiced on the preparative scale. Thin layer
chromatography (TLC) and paper chromatography (PC), which offer
good possibilities for physical separation, are applicable for analytical
purposes only. High performance liquid chromatography (HPLC)
offers the advantages of superior resolution and speed of operation and
nearly all-new antibiotics are studied from development to clinical
application by using HPLC. However, the use of HPLC is still limited
to laboratory analysis and difficult to scale up for industrial purposes
(Cascaval et al., 2001). Whilst chromatographic methods show
promising results there are potential drawbacks.
One of the
disadvantages is that the use of these methods is considered as the high
end of separation and purification.
2.6
REVERSE MICELLE
Reverse micelle is an alternative method to conventional separation

and purification procedures due to their potential for large scale use
(Ono et al., 1996; Naoe et al., 2004; Shen and Yu, 2007; Moore and
Palepu, 2007), their potential application to continuous separation of
biological substances (Zhang et al., 1999; Hong and Kuboi, 1999;
Naoe et al., 2002; Noh and Imm, 2005; Juang et al., 2006; Majumdar
and Mahapatra, 2007), the process similarities to liquid-liquid
extraction (Naoe et al., 2004; Hasmann et al., 2007), and easy scale-up
(Chang et al., 1997; Rodrigues et al., 1999; Kilikian et al., 2000;
Mathew and Juang, 2007). Nishiki et al. (1998) showed that reverse
micelle extraction is able to recover proteins with little denaturation
because proteins can be hosted in a solubilised aqueous phase which is
shielded from the organic solvent by surfactant molecules;
24
solubilisation inside reverse micelle does not damage the native

protein structure (Yu et al., 2003; Hetch and Peled, 2006). Therefore,
in an ordinary liquid-liquid extraction, although it has been difficult to
extract large bioactive molecules such as proteins, the limitation can
be overcome by utilising a nanostructural molecular assembly like
reverse micelle (Aires-Barros and Cabral, 1991; Ono et al., 1996).
2.7
CONCLUSIONS
Generally, most conventional extraction methods (as detailed in

Section 2.2) have the potential to be utilised in the downstream
processing of antibiotics and further research is being conducted to
improve these methods for large-scale application. However, one
method that has shown great potential is a system that utilises reverse
micelle in the extraction of the required bioproduct. Carlson and
Nagarajan (1992) discussed the applications in which reverse micelle
systems may offer a promising alternative to chromatography for the
large scale purification of proteins. In theory, this system is much
easier to scale up than chromatographic separation processes and can
allow for continuous separation processes for biomolecules (see Table
2.1).
25
Table 2.1 Features of separation methods in the pharmaceutical industry.
Liquid
membrane
(Pellegrino
and Noble,
1990; Ghosh
et al., 1997;
Cascaval et
al., 2000)
Aqueous
two phase
system
(Verral,
1988; Yang
et al.,
1994)
Chromatography
(Kinugasa et al.,
1991; Pires et al.,
1996; Ghosh et al.,
1997; Kyung and
Jee, 2004)
Efficiency
Yield
/cycle
Very good
Can be poor
Low
Can be
high
Very good
Can be poor
Selectivity
Very good
Time
consumed
Toxicity of
Solvent
No
information
High
Can be
good
No
information
High
Denaturing
of product
Wastage of
solvent
Cost
Scale-up
potential
No
information
Very low
Low
possibility
Very low
Low
Very high
Moderate
(low stability)
Very high
Can be
good
Very high
Poor (limited to
laboratory)
Can be high
Very good
No
information
Can be low
Very good
No
No
No
Yes
No
Method
established
Conventional
solvent
extraction
(Kinugasa et
al., 1991;
Pires et al.,
1996; Ghosh
et al., 1997;
Nabais and
Cardoso,
1999; Liu et
al., 2001;
Naoe et al.,
2004
Good
Very good,
can be low
Reverse
micelle
extraction
(Pires et
al., 1996;
Ghosh et
al., 1997;
Naoe et al.,
2004;
Kyung and
Jee, 2004)
Very good
Moderate
Good
Long
Long
Short
Information not
available
High (because
of
demulsifies)
High
possibility
High
No
information
Low
Very good
Very good
Very low
26
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30
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3
APPLICATION OF SUPERCRITICAL
FLUID IN THE EXTRACTION OF ACTIVE
COMPOUNDS FROM PLANT MATERIAL
Mohd Azizi Che Yunus
Wahyu B. Setianto
Zainuddin Abdul Manan
3.1
INTRODUCTION
In conjunction with the rapid demands natural medicinal product, the

processing technologies used in its production have to make major
technical and scientific improvements in their efficiencies and product
quality. This is important in order to meet the basic requirement of
hygiene and safety standards as well as to survive in this increasingly
competitive and dynamic market product. The current processing
methods are inefficient and the extract produced is low of quality.
Majority of the local herbs medicinal products are still prepared in the
traditional manners involving mainly hot or cold-water extraction,
grinding to powder and conventional drying. By using the traditional
processes of water extraction and conventional drying, the finished
product is not guaranteed to be safe. Both the desired as well as
undesired compounds are extracted and it is difficult to separate
specific compounds of interest. Thus the finished products contain
some undesired compounds such as polar toxic substances too. Most
of factories that produce traditional medicine are aware of these
limitations, but lack of resources and new technologies and expertise
forced them to still use these unconventional techniques.
32
In view of the above statement, the supercritical carbon dioxide

extraction process has a promising field in the industrial application
especially herbs industries where high quality product can be obtained.
There are many reasons which make the use of supercritical carbon
dioxide extraction as a very great potential method for extracting
valuable compounds and at the same time removing of undesired
compounds from plant materials and promising alternative in
extraction process.
The first advantage of this technique is that the final product
fulfils the expectations of health conscious users because it is free of
solvent, does not contain toxic residues as well as undesired
microorganisms. Supercritical fluid extraction using carbon dioxide as
a solvent provides an excellent alternative to the use of chemical
solvents. Since the carbon dioxide is non-toxic, non flammable and
inert in nature, there is no risk of thermal degradation of processed
product. In fact, the use of carbon dioxide has been accepted by most
European Food and Drugs acts as an extraction medium for the
isolation of food-related compounds (Starmans and Nijhuis, 1996).
Over the past three decades, supercritical carbon dioxide extraction
has been used for the extraction and isolation of valuable compounds
from natural products. Supercritical carbon dioxide extraction is also
capable to function as a sterilizer as studies by Banana (2005) showed
that bacteria can be eliminated at pressure as low as 20.68 MPa and
temperature 50o C. These results will contribute to the motives of
choosing supercritical carbon dioxide as the extraction medium and to
improve the extracts quality and compliance customers awareness
elsewhere.
Secondly, most of the medicinal plants extracts are usually in
low concentration and exist in complex mixtures. Therefore the
process selected to obtain the maximum valuable compounds as well
as desired compounds must be effective, selective, and satisfy both
economical requirement and environmental regulations. Bisunadan
(1993) reported that SC-CO2 is more efficient in extracting of trace
amount of compounds such as carotenes and tocopherols from oil
palm fruits at high pressure and temperature of 500 bars and 80o C
Application of Supercritical Fluid in theExtraction of

ActiveCompounds From Plant Material
33
compared with hexane extraction. On the other hand, the extraction of

triglycerides it was sufficient to operate at pressure above 300 bars and
temperature more than 40o C. This scenario indicates the ability of SCCO2 technique on the selectivity of target compounds from the
heterogeneous mixture in nature, by adjusting the temperature and
pressure of the extraction process. Gast et al. (2005) has studied on the
purification of tocochromanols from the crude palm oil and soy oil
deodorizer distillate. The results suggest that a tocochromanols
enrichment starting from crude edible oil to very high concentration is
possible by means of supercritical extraction. Kallio and Kerrola
(1992) have studied the application of supercritical carbon dioxide
extraction on the sample of coriander fruit. Their results show that at
extraction temperature between 40 and 50 o C and extraction pressure
lower than 100 bars, higher molecular weight compounds are not
extracted. Another important aspect on comparing the different
extraction methods is the quality of the resultant extracts where both
the desired and undesired compounds could be extracted. For example,
in the water extraction, the number of chemical constituents that could
be extracted was more than supercritical fluid extraction. This is due to
solubility of polar compounds in water is greater than in carbon
dioxide. Therefore not all the compounds extracted using water could
be considered because most of toxic compounds are present in polar
compounds.
Thirdly, supercritical fluid has relatively lower viscosity and
higher diffusivity therefore, it can penetrate into porous solid materials
more efficiency than liquid solvents and consequently, it may render
much faster mass transfer resulting a more rapid and efficient
extraction. Norhuda (2005) used Transmission Electron Micrograph
(TEM) to study the effect of pressure and temperature on the structure
of the palm kernel after extraction with SC-CO2. The comparison was
made on the transmission electron micrograph of the cross section of a
single palm kernel before extraction and after extraction at optimum
conditions over a 30 minutes extraction. It was found, that the pore of
the solid matrix filled with oil (before extraction) was voids after the
34
completion of extraction. This argument was a good rationale for the

use of supercritical carbon dioxide extraction in the separation of
essential oils from the plants herb. Essential oil compounds are
normally located within the cellular structure in vacuoles matrix.
Therefore by adjusting the operating temperature and pressure; the
penetration depth of solvent carbon dioxide into internal matrix
structure could be achieved, and hence results in exploration of
trapped essential oil compounds.
The fourth reason is to fulfil requirement of the end product
quality. For Malaysian herbs, in order to meet the international herbal
product standards and compete successfully in the international
market, it is essential for the local manufacturers comply with the
highest standards of manufacturing including the WHO-GMP (World
Health Organization-Good Manufacturing Practices), which is
globally accepted. Since the 7th Malaysian Plan, the government made
major demands on the industry through implementation of new
legislative requirement, such as more stringent approval criteria for
new products and good manufacturing practice, GMP (Teik and Azmi,
1997). Since 1st January 1999, all herbal products registered with the
Malaysian Government must comply with Malaysia GMP rulings,
whereby the Malaysian GMP complies with the WHO-GMP
guidelines. One of the aspects emphasized in GMP guidelines is
processing method, referring to high quality and established product.
In fact, to make the Malaysian medicinal product viable, an extraction
process plant has to be developed. Shortcomings of using crude
products such as storage, standardizations, stability and a short shelf
life can be overcome by doing the extraction of natural compounds.
The manufacturers should adopt modern concepts and technologies in
processing and developing their products. Supercritical carbon dioxide
extraction process is one of alternative method and also among the
most recent technologies used in extraction and separation of valuable
compounds from natural sources that have medicinal values.

3.2
35
WHAT ARE SUPERCRITICAL FLUIDS
A supercritical fluid (SCF) is defined as any substance at a

temperature and pressure above the critical values. The critical
temperature of a substance is defined as the temperature above which
a pure, gaseous component cannot be liquefied regardless of the
applied pressure. The critical pressure is then defined as the vapour
pressure of the gas at the critical temperature. Another popular
description of supercritical fluid is the substance in the intermediate
state between gas and liquid; therefore it does inhibit the
characteristics of high diffusivity comparable with a gas and high
solubility as a liquid. Therefore, supercritical fluid extraction provides
the extraction feasible, superior quality products and more efficient.
The most influence factors influencing solvent selection are
critical temperature and pressure. An ideal solvent should have a low
or moderate critical pressure to minimize compression cost (Rizvi et
al., 1986). Basically, high critical temperature may destroy heatsensitive solutes. The temperature range commonly employed in
supercritical extraction falls between 30 150oC. Therefore, in order
to avoid a thermal degradation or deterioration for heat-sensitive
solutes, the solvent must have critical temperatures lying in this range
(Taylor, 1996). Table 3.1 shows the critical properties of various
fluids commonly used in SFE technology.
Table 3.1 Various Solvent as SFs with Critical Properties

Solvent
Sulfur Hexafluoride
Tc (oC)
45.5
Pc (bar)
37.1
36
Chlorotrifluoromethane
29.0
38.7
1,1,1,2-tetrafluoroethane
101.2
40.6
Propane
96.8
42.4
Trichlorofluoromethane
198.0
43.5
Trifluoromethane
25.9
46.9
Chlorodifluoromethane
96.4
48.5
Xenon
16.6
57.6
Dinitrogen Monoxide
36.5
71.7
Carbon Dioxide
31.0
72.9
Ammonia
132.4
111.3
Water
374.0
217.7
3.3
PROPERTIES OF SUPERCRITICAL FLUID
A supercritical fluid (SCF) is a substance that is gas like, which has

been heated above its critical temperature (Tc) and simultaneously
compressed above its critical pressure (Pc). Definition of critical
temperature is the highest temperature at which a gas can be converted
to a liquid by increase in pressure and the critical pressure is the
highest pressure at which a liquid can be converted to a traditional gas
by an increase in the liquid temperature. In other word, supercritical
fluid can also be defined as a heavy gas with a controllable dissolving
power or as form of matter in which the liquid and gaseous state are
indistinguishable. This principle can be explained more clearly by
using pressure-temperature phase diagram as shown in Figure 3.1. In
P-T diagram, there are lines describing the sublimation, melting and

37
vaporization equilibrium, respectively. The three curves intersect at

the so-called triple point, TP, where the solid, liquid and gaseous
phases coexist in equilibrium and ends at the critical point, CP. Above
critical point, and no liquefaction will take place on raising the
pressure and no gas will form on increasing temperature. This region
of pressure and temperature above Pc and Tc is called supercritical
region, which is denoted by the yellowed area in P-T diagram.
Figure 1 Pressure-temperature phase diagram of carbon dioxide.

(Dean, 1993)
In the case of carbon dioxide gas, the critical temperature is 31o

C and critical pressure is 7.38 MPa. If the CO2 gas is heated up to any
temperature over 31o C, which would be over `super the critical point,
then it will not be turned into liquid no matter what ever pressure is
applied. This region of supercritical fluid is unique because it is a
38
phase that possesses some of the properties of both a gas and a liquid.
At supercritical region, the characteristics of solvent is a strong
function of temperature and pressure, can change the property sharply
with very little change in pressure and temperature, able to control
density, polarity, viscosity and other properties of the fluids
continuously over a wide range. According to Mc Hugh and Krukonis
(1986) these unique characteristics make it possible for extraction to
take place rapidly. In supercritical fluid extraction, it is essential to
discuss the properties of supercritical fluids in details such as solvent
power; diffusivity/viscosity and competing factors effect hence it
influence the solubility of desired compounds particularly those
located remotely within solid matrices.
3.3.1 SOLVENT POWER CONSIDERATION
Basically at the beginning of supercritical fluid extraction, the process

is controlled by the solubility followed by mass transfer controlled at
the end of extraction. The solubility is influenced by solvent density
and solute vapour pressure meanwhile mass transfer is influence by
diffusivity and viscosity of supercritical fluids. In order to obtain
maximum extracted mass, the solubility and mass transfer should be in
higher magnitude. For utilization of supercritical fluids as an
extraction solvent, the fluids must have characteristics of solvating
power. Supercritical fluids begin to exhibit significant solvating power
when they are compressed to liquid densities.
Table 3.2 shows the comparison of properties of supercritical
CO2 and typical liquid and gas properties. The density of CO2 is 100 to
1000 times greater than gas and solvating characteristics approaching
those of a liquid. The diffusivity of CO2 is higher than that of a liquid
by a factor of several hundreds. This means that mass transfer in a
supercritical fluid is faster than in a liquid by the same factor. The

39
viscosity also shows a significant difference from that of a liquid, one

hundred times lower. In addition, the Reynolds number becomes 30 to
100 times higher than that of a liquid for a given flow velocity. This
facilitates to develop turbulent flow in tubing, reducing the laminar
flow dispersion.
Table 3.2 Properties of supercritical carbon dioxide and ordinary gases and
liquid.
Region
Density
(g/ml)
Viscosity
(g/cm.s)
Gases
Supercritical
CO2
T c, P c
Tc, 6Pc
Liquids
(0.10.2) X 10-3
(13) X10-4
Diffusion
Coefficient
(cm2/s)
0.1 0.4
0.47
1.0
3 X 10-4
1 X 10-3
7 X 10-4
2 X 10-4
0.6 1.6
(0.23.0) X 10-2
(0.2 2.0) X10-5
This solvating power that is referring to solvent density is

directly related to the thermodynamic state of the solvent that is highly
dependent on its temperature and pressure as shown in Figure 3.2.
Indeed, adjusting the range of temperature and pressure interest can
easily control the solvent strength.
At constant temperature, the solvent power or solvent density
of a supercritical fluid increases as pressure increases. On the other
hand, at constant pressure, as temperature increases, the solvent
density will decrease. Meanwhile, increasing temperature will also
lead to an exponential increase in the vapor pressure of the solute
(solute volatility). The solubility of solute is increased as the solvent
density and solute volatility is increased, hence the oil yield will also
increase. As reported by Murga et al. (2005) the solubility of caffeic
acid in supercritical carbon dioxide is increased with pressure and
temperatures studied. As shown in Figure 3.3, the solubility of caffeic
40
acid is high at higher operating pressure and temperature, 50 MPa and

333 K respectively. In other words, solubility depends on the pressure
and temperature which is represented as solvent density and solute
volatility respectively.
Figure 3.2 Density of carbon dioxide as function of pressure at different

temperature (Fattori et al., 1988).
Solubility of caffeic acid

(mole fraction), y x10E7

41
0.5
313 K
323 K
333 K
0.4
0.3
0.2
0.1
0
15
20
25
30
35
40
45
50
Pressure, MPa
Figure 3.3 Effect of pressure on solubility of caffeic acid in supercritical
carbon dioxide at different temperature (Murga et al., 2005).
3.3.2 CROSS OVER PRESSURE
Cross over pressure or retrograde solubility is a point (graph solubility

versus pressure at different temperature) where the behaviour of
solubility regarded due to the competing effects between the solvent
density (referring to pressure) and solute volatility (referring to
temperature). At pressure above cross over pressure, a CO2 density is
less sensitive to temperature and solute volatility effects dominate, so
the solubility of compounds increases with a higher temperature. On
the other hand, at pressure below the cross over pressure, the solubility
decreases with increasing temperature due to the rapid decrease of
CO2 density. At the point of cross over pressure, the competing effects
42
balance each other and the solubility remains relatively constant with
increasing temperature. As reported by Murga et al. (2003), the
behaviour of cross over pressure occurs in the extraction of ferulic
acid. At cross over pressure, the solubility of ferulic acid relatively
constant even though temperature was increased up to 50o C. The
phenomenon of cross over pressure was shown in Figure 3.4. Lee
(1994) study on the extraction of oil from evening primrose seed using
SC-CO2 determined that the pressure of 200 bar is a pressure at cross
over pressure.
Figure 3.4 Solubility of ferulic acid as function of pressure at various

temperatures. The cross over pressure located at pressure of 13 MPa (Murga
et al., 2005).
Many researchers who have discussed the cross over pressure

phenomenon include Taylor (1996), Kiriamiti et al. (2001), Perretti et
al. (2003), Daood et al. (2002 and Gordillo et al. (2001). The

43
significance for determination cross over pressure is related to the

selection of pressure of interest. If the pressure range selected is above
cross over pressure, that means maximum extracted mass (yield) could
be obtained at the higher interaction condition. Meanwhile at below
cross over pressure, lower temperature will produce the maximum
yield.
3.3.3 DIFFUSIVITY AND VISCOSITY CONSIDERATION
The extraction process is controlled by mass transfer properties, which

are represented by diffusivity and viscosity of fluid solvent. The
values of diffusivity and viscosity are function of temperature and
pressure. Referring to Figure 3.5, the relationship of diffusivity
function with temperature and pressure can be written as follows:
x
x
x
x
at constant temperature, the diffusivity in a supercritical fluid

decreases with increase in the pressure
at constant pressure, the diffusivity increases with increasing
temperature particularly, in the vicinity region of the critical point
the slopes of diffusivity function of temperature isobar increase
with decreasing temperature and pressure
the diffusivity of a solute in a supercritical fluid always exceeds
that in an ordinary liquid solvent
44
Figure 3.5 Diffusivity of carbon dioxide in the supercritical fluid and near
critical region (Weatherly, 2005).
Figure 3.6 shows the variation of the CO2 viscosity as a function of

pressure at different temperature studied. Similarly, the relationship of
viscosity with respect to pressure and temperature were:
x at constant temperature, viscosity increases with given pressure
x at constant pressure, viscosity decreases with increasing
temperature particularly above critical pressure
As shown in Table 3.2 the properties of supercritical carbon
dioxide with high diffusivity and low viscosity have quite significant
properties for the purpose of extraction because their mass transfer
properties are much more favourable. Another word, high diffusivity
is a significant property as the rates of extraction are limited by the
speed because solute molecules are transported by diffusion process

45
from the matrix sample into the bulk fluid of CO2. Meanwhile low
viscosity will allow supercritical fluids to penetrate matrices of desired
compounds with low permeability more rapidly than conventional
solvents because it does not exhibit surface tension limitation (Dean,
1993).
Figure 3.6 Variation on viscosity of carbon dioxide function of pressure at

temperature studied (Mc Hugh and Krukonis, 1986).
46
3.4
OPERATIONAL PARAMETERS IN SUPERCRITICAL
FLUID
For a successful application of SFE technology, several critical

factors or parameters must be taken into consideration in conducting
SFE experiments. These factors include the type of sample, method of
sample, type of fluid, and extraction conditions; pressure, temperature,
solvent flow rate and extraction time (Lang and Wai, 2001).
According to Reverchon (1997), those parameters could significantly
affect the extraction rate. However, the main factor that affects the
extraction performance is the operating conditions, namely pressure
and temperature. In addition, the extraction performance is also
indirectly affected by solvent flow rate, sample and particle sizes as
well as water content in a sample matrix. The efficiency of
supercritical fluid extraction was determined by number of factors as
shown in Table 3.3 (Mohd Azizi, 2007).
Table 3.3 Experimental variables in supercritical fluid extraction.

Sample preparation
Sample size
Particle size
Homogeneity of
sample
Moisture content
Drying agent
Vessel volume
Packing density
Extraction parameters
Pressure
Temperature
Flow rate
Trapping
Collection technique
Trap size
Sorbent type
Modifier
Static or dynamic
Extraction mode
Extraction time
Trap elution solvent

Trap elution volume
Restrictor temperature
Pressurized/depressurized
Collection
temperature

47
3.5
PRINCIPLES OF SUPERCRITICAL EXTRACTION
PROCESS
The principles of extraction from solids consist of two processes,
which can be represented as two-stage process namely the extraction,
and separation of the extract from solvent, (Figure 3.7). During the
extraction step, the solvent is first compressed to above critical
pressure before it flows through a fixed bed of solid particles in the
extractor and dissolves the extractable components of the solid. The
loaded solvent is removed from the extractor and fed to a precipitator.
In a large scale SFE process, the solvent is commonly recycled to
separation stage and solvent losses are compensated by make-up
stream (Brunner, 1994).
3UHVVXUH
LQGLFDWRU
&RROHU
3UHVVXUH
UHJXODWRU
0HWHULQJ
YDOYH
([WUDFWRU
2YHQ
3XPS
&2
F\OLQGHU
3UHVVXUH
LQGLFDWRU
:HWJDV
PHWHU
$QDO\WH
UHFHLYHU
Figure 3.7 Schematic diagram of supercritical fluid extraction using carbon

dioxide
48
Collection of the extract yield is an important step in any separation

application using SFE method. In order to maximise sample
collection efficiency, techniques such as cooled collectors and liquid
traps should be included. Sample collection can be accomplished by
simply allowing the solvent/extract mixture to depressurise completely
to atmospheric pressure whereby the solvent is vaporised and
dissipated, leaving only the extracted material in a collection vial.
Otherwise, pressure could be reduced to the level which is enough to
decrease the solubility of the extract in the solvent. This can be done
without complete depressurisation in a separation vessel held above
atmospheric pressure. In this way, the solvent can be recycled and the
energy costs associated with pressuring the solvent can be saved.
3.6. APPLICATION OF SUPERCRITICAL FLUIDS
Application of SC-CO2 have been studied by numerous researchers on

the different views such as the applications of SC-CO2 in the
extraction of medicinal components from plants, the comparison with
conventional method, determination of SC-CO2 properties behaviors
such as oil solubility, mass transfer coefficient, extraction with
reaction in supercritical fluids and thermodynamic modeling of phase
equilibrium of solvent-solute mixture. The application of SC-CO2
related to this work starting from 1991 was summarized in Table 3.4.
In general, it was noticed that some of the conditions applied in the
previous research contributed brilliant ideas for the selection of
specific extraction conditions will be used in this study.

49
Table 3.4 Summary of application of supercritical carbon dioxide in the

extraction of active compounds from plants.
N
o
Application
of SC-CO2
on Plant
Matrices
SC-CO2
Conditions
SC-CO2 outcomes
Ref.
Extraction of
evening
primrose oil
from the seed
of Oenothera
biennis L.
1.Temperature
ranges=40 to
60o C
2.Pressure
ranges = 20 to
70 MPa
3. CO2 flow
rate is 18 g/min
Favati
et al.
(1991)
Extraction of
oil from Oil
Palm fruits
1. Operating
temperatures
are 40o, 60o and
80o C.
2. Operating
pressures are
300, 400 and
500 bar
3. F = 2.4 kg/hr
Extraction of
oil from
evening
primrose seeds
1.100 P 300
bar, P = 100
bar
2. 20 T 50 o
C, T = 15 o C
3. Sample =
1) Over the temperature range,

only a limited amount of oil
could be removed at the lowest
pressure of 20 MPa.
2) Recoveries above 90% were
obtained when the pressure
increased to 30 MPa and even
higher yields were recorded
when operating at 50 and 70
MPa
1) The optimum condition for
extraction of oil was at highest
pressure and temperature, 500
bar and 80o C.
2) The most suitable solvent
flow rate was determined to
be at 2.4 kg/hr.
3) The optimum condition for
the extraction of -tocopherol
was at 500 bar and 80 o C with
the maximum amount is 160
ppm
4) The efficient condition in
extracting triglycerides at
pressure above 300 bar and
temperature above 40 o C.
1) Under the range covered, the
highest yield was 21 wt% at 300
bar and 50 o C.
2) The mass transfer value of
the oil from the seeds to the CO2
phase was found to increase
Bisuna
dan
(1993)
Lee et
al.
(1994)
50

ground drier
with particle
size of 25 40
mesh
Extraction of
tocopherols
from soyn
flakes, ricebran and
wheat germ
Extraction of
carotenoids
and lipids
from buriti
fruit (Mauritia
flexuosa)
1. 250 bar from

40o to 80o C
2. 700 bar at
80o C
Extraction of
Vit. E and
squalene from
Pandanus
odorus
Ridl./leaves
1.40 T 80,
T= 10 C,
2. P = 80, 100,
200 kg/cm2 ,
3. T= 180 min;
4. Sample = 4
g of ground
freeze dried
leaves
Extraction of
flavonoids of
Scutellaria
1. Temperature
was set at 40,
50, 60 and 70o
1. P = 20MPa
and 30 MPa
2. T = 313K
and 328 K
linearly with the superficial

velocity.
3) The oil extracted with SCCO2 was found to have
comparable extraction yield and
significantly less phosphorus
content compared with nhexane
1) The soy fakes showed the
better yields of vitamin E
2) The optimal condition is at
250 bar and 40o C with vitamin
E in enriched is 0.6 wt/wt %
1) Extraction with SC-CO2 was
capable of removing about 80 %
of the initial carotene content.
2) The extraction curves have
the three distinct region with the
largest amount of carotene was
extracted in the diffusion
controlled region
3) The constant extraction rate
period was longer at 20 MPa
than 30 MPa, while the mas
transfer rate were about seven
times larger at 30 MPa
1) The yield of vitamin E
increases with an increase in
pressure but decrease with
increasing temperature
King et
al.
(1996)
De
Franca
et. al
(1999)
Hassan
et al.
(1999)
2) Maximum yield of Vit. E and

squalene was obtained were 300
ppm and 1200 ppm, respectively
at 200 kg/cm2 and 40 o C during
3 hour extraction
1) At 50o C, with modifier 10 %
v/v, when the pressure increased
from 200, 300 and 400 bar, the
Lin et
al.
(1999)

baicalensis
from
Scutellariae
Radix
C
2. Extraction
pressures was
set at 200, 300
and 400 bar
3. Methanol
was used as
modifier with
concentration is
5 and 10 %
yield decrease 14.3, 15.2 and

18.6 %, respectively.
2) When 5% of modifier was
used, as the pressure was
increased, the decreased
amounts were 48.8, 45.9 and
52.3 %.
Extraction of
lipids from
Pythirm
irregulare
1. Extraction
temperatures
are 40. 50 and
60o C
2. Extraction
pressure are
13.8, 20.6 and
27.6
3. Flow rate are
20, 50, 60, 100
and 400
mL/min
4. Moisture
content are 10,
20 and 30%
Extraction of
oleic
sunflower
seeds
P = 250 bar; T
= 40 and 60 o
C.
10
Extraction of
nimbin from
Azadirachta
indica A.
Juss/seeds
10 P 26
MPa, 308 T
333 K
0.24 F 1.24
ml/min
Sample = 2 g
1) Extraction of oil showed

success for moisture contents as
high as 30% wet basis
2) The extraction rates
decreased substantially after 40
to 50% of the lipids were
removed, which indicated the
transition to a diffusion
controlled regime.
3) The extraction rate that lasted
about 90 min for the 30%
moisture and only 60 min for
the 20% moisture
4) Higher molecular weight
components appeared in greater
amounts toward the end of
extraction process
At 40 o C and flow rate of 3.02
kg/hr, the rate of extraction is
greater than 60 o C. The
experimental operational
pressure is below crossover
pressure.
Best extraction condition
occurred at 23 MPa, 308 K and
flow rate of 1.24 ml/min for
producing 0.175 mg nimbin/ g
neem seeds
51
Walker
et al.
(1999)
Kiriami
ti
et al.
(2001)
Tonthu
bthimth
ong
et al.
(2001)
52

ground neem
powder
11
Extraction of
essential oil
from Piper
nigrum
L./seeds
P = 160 and
200 bar; T = 40
o
C
The solubility of essential oil at

200 bar was found to be 56%
higher than that a 160 bar
Harchar
an S
(2002)
12
Extraction of
tocopherol
from olive tree
leaves
1. Extraction
pressures range
from 25 to 45
MPa
2. Extraction
temperature
range from 313
to 333K
3. Particle size
range from 0.25
to 1.50 mm
4. Solvent flow
rate from 0.5 to
1.5 L/min)
Lucas
de et
al.
(2002)
13
Extraction of
oleoresin from
pungent spice
paprika
powder
1. Extraction
pressure from
100 to 400 bar
2. Extraction
temperature
from 35o to 55o
C
3. Flow rate of
1.0 to 1.5 l/min
1) At constant temperature, the

extraction yield strongly
increased with pressure
2) Oil yield increased with
decreasing particle size
3) Increasing CO2 flow rate
from 0.5 to 1.0 L/min increased
oil yield, while at 1.5 L/min a
slight increased is observed
4) Maximum amount of vitamin
E was obtained at 25 MPa and
further increase of pressure up
to 35 and 45 MPa decrease
vitamin E recovery
5) Effect of flow rate on the
extraction rate of vitamin E is so
small, which is can be detected
when a wider range of fluid
flow is analysed
1) At a given temperature,
increasing the extraction
pressure increased the oil
solubility in SC-CO2.
2) At 55o C, and a pressure
lower than 300 bars, the yield of
oleoresin was markedly low buit
at 400 bars a maximum yield
could be achieved
3) A ratio of solvent/solid is
8:10 was required for the
complete extraction of paprika
oleoresin
Daood
et al.
(2002)

53
14
Extraction of
Anacardic acid
and cardol
from Cashew
shell
40 T 60,
T= 10o C,
P = 30 MPa, F
= 5 liter/min
It proposed a pressure swing

method to extract Anacardic
acid in its natural characteristic.
Wahyu
et al.
(2003)
15
Extraction of
oil from
ground black
pepper
1. Extraction
pressures are
90, 100 and 150
bar
2. Extraction
temperatures
are 40 and 50o
C
3. Solvent flow
rate are 1.1, 2.0
and 3.0 kg/hr
1) Extraction rate increases with

increasing pressure, at 150 bar
the pepper oil was extracted
faster than 100 bar but the total
amount of extract almost the
same
2) Lower final extraction yield
observed at 90 bar
3) At 100 bar, as temperature
increase resulted in decrease of
the extraction rate. Final
extraction yield was envchanced
by 45% as temperature
decreased
Perakis
et al.
(2005)
4) The increase in flow rate

benefited the extraction rate
16
Decaffeination of
raw green
coffee beans
1. Extraction
pressure from
16 MPa to 22
MPa
2. Extraction
temperature
from 70 o C to
90 o C
3. Extraction
time is 10 hour
The caffeine content is reduced

from typical range of 1-3% to
less than 0.04%
Zosel.
K
(1981)
17
Deacidification
1. Pressure
range from 20
1) CO2 selectivity for fatty acids

was higher at 20 MPa and 60o
Brunett
i
54

of olive oils
MPa and 30
MPa
2. Temperature
range from 40 o
C to 60 o C.
C.
2) CO2 selectivity also increased
significantly as the free fatty
acid concentration in oil
decreased.
et al.
(1989)
18
Separation of
triglycerides,
diglycerides,
free fatty
acids,
carotenes,
tocopherol and
tocotrienols
from crude
palm oil
The separation
of components
has been
achieved by
supercritical
fluid
chromatograph
y (SFC) by
using the
programmed
extraction
elution method
Choo
et al.
(1996)
19
Decaffeination of
guarana seeds
1. Extraction
temperatures
were 40o and
70o C
2. Extraction
pressures at
100, 200 and
400 bar
3. CO2 flow
rate is 5.7 and
9.4 g/min 4.
The weight of
sample is 3 g
with water
content of 40%
1) Fraction 1 eluted after 66 min

while using pure CO2 at 18 MPa
pressure as eluent consists of
free fatty acids and a small
amount of tocopherolss and
tocotrienols
2) Eluted during the next 20
min with a mixture of CO2 and
ethanol (5.0/2.0 mL/min)
consists of tocopherols,
tocotrienols and diglycerides 3)
Solutes eluted in the next 54
min by increasing ethanol flow
rate to 0.5 mL/min consisted of
isomer triglycerides
1) Extraction at 400 bar and 70o
C at flow rate of 5.7 g/ min
allowed the removal 98% of the
initial caffeine content in wet
ground guarana seeds
2) When extraction was
performed at lower pressures or
temperatures, additional time
and larger amount of CO2 were
necessary to achieved the same
yield
20
Concentration
of tocopherols
1. Pressure
ranging from
Saldana
et al.
(2002)
3) A retrograde behavior for the

extraction of caffeine from
guarana seeds was observed at
100 bar for the 40 and 70o C
isotherms
1) The tocopherols were

concentrated up to 40%,
Lee et
al.

from the
soybean
sludge
200 bar to 400

bar
2. Temperature
ranging from
35 to 70o C
21
Isolation of
peppermint
oil/leaves
22
Improving the
value of rice
by product
1. 5000 P
10000 Psi,
P = 2500 Psi
2. 40 T 80o
C, T = 20 o C
23
Enhancement
for separation
of lauric acid
and oleic acid
in Palm kernel
oil
Solubility of
Antibiotic
penicillin G
27.6 P 48.3,
P = 6.9MPa
T= 80 o C; 30
t 60 min, t
= 10 min
24
Extraction
condition is:
1. 90 bar and
50o C and 120
bar and 40o C
1. Extraction
pressures from
100 to 350 bar
2.
Temperatures
from 313.5 to
333.15 K
indicating that a countercurrent

multistage separation column
will be necessary to obtain when
pure CO2 is used without
entrainer
2) The tocopherol concentration
in the extract is increase with
increasing pressure
1) On increasing the CO2
density, oxygenated
monoterpenes in the extracts
decreased from 84.4% to 72.2
%, whereas sesquiterpenes
increased from 4.6% to 13.2%.
2) The coextraction of waxes
increased from about 10 % to 30
% by weight compared to the
yield of peppermint essential oil
The extraction conducted at
10000 Psi and 80o C gave the
highest extraction yield 4.93 g
of rice bran with contains of
18.0 mg/g of rice bran of oryzanol
At 48.3 MPa and 80o C lauric
acid was reduced from 53.65%
to 31. 86% and oleic acid was
increased from 9.63% to
23.56%.
1) Pressure above 150 bar, the
solubility of penicillin G
increases with increasing
temperature and pressure.
2) Pressure lower than 150 bar,
the solubility increase with
increasing pressure and decrease
with temperature
3) The predicted solubility data
with the empirical model are in
55
(1991)
Reverc
hon
(1994)
Perretti
et al.
(2003)
Nik
Norulai
ni
et al.
(2004)
Gordill
o
et al.
(2001)
56

good agreement with the
experimental data
25
Extraction
kinetics of
Pre-pelletized
Jalapeno
Peppers
26
Sterilization of
Oil Palm Fruit
Fiber
1. Average
sample particle
size from 0.28
to 3.19 mm
2. Superficial
solvent velocity
from 0.14 to
2.62 mm/s
3. Extraction
condition:
40o C and 120
bar
40o C and 320
bar
1. extraction
temperature are
40, 50 and 70o
C
2. Extraction
pressure are
13.7 and 20.7
MPa
3. Extraction
time is 60 min
1) External mass transfer

coefficient increased with
superficial velocity
2) Values of external mass
transfer coefficient increased as
particle size decreased
3) Internal mass transfer
coefficient increase as increase
in pressure
De
Valle
et al.
(2003)
1) The bacteria present was

completely killed under pressure
as low as 20.7 MPa and 50o C.
2) The temperature plays a
fundamental role on the
sterilization enhanced by
increasing temperature.
Banana
(2005)

57
3.7. CONCLUSION
In the last four decades, there has been a tremendous interest in the use
of supercritical fluid as solvent for extraction, separation and removing
purpose. The intensive study on extraction of food components by
using supercritical fluid began in early 1970s. Many patents resulted
from these studies such as for the extraction of hops, decaffeination of
coffee and tea, tobacco and spices. There is little doubt that
supercritical carbon dioxide extraction is very promising at it offers
greater selectivity, efficiency and environmental benefits than
conventional methods. The bioactive properties of the extract are also
preserved as degradation due to prolonged exposure to harsh
extraction condition is avoided. At present, supercritical extraction is
more feasible for high value phyto-chemicals and neutral-ceuticals. As
expected the 21st century is a tertiary peak with mounting interest in
the possible development of new material especially on the medicinal
plant industry. Also, supercritical fluid technology has become an
interdisciplinary field utilized by chemical engineers, chemists, food
scientists, materials scientists, agronomists and researcher in
biotechnology and environmental control.
REFERENCES
Anuar, O., 2002, Modification of palm kernel cake composition by

using supercritical carbon dioxide extraction method, MSc.
Thesis. Universiti Sains Malaysia.
58
Banana, A. A., 2005, Sterlization and extraction of palm oil fruit fiber
using supercritical carbon dioxide extraction. MS.c thesis.
Universiti Sains Malaysia, Penang, Malaysia.
Bisunadan, M.,1993, Extraction of oil from oil palm fruits using
supercritical carbon dioxide, MS.c thesis. Universiti Sains
Malaysia, Penang Malaysia.
Brunetti, L., Daghetta, A., Fedeli, E. and Zanderighi, L., 1989,
Deacidification of olive oils by supercritical carbon dioxide,
JAOCS, 66 (2): 209-217.
Brunner, G., Malchow, T., Sturken, K. and Gottschau, T., 1991,
Separation of tocopherol from deoderizer condensate by
countercurrent extraction with carbon dioxide, J. of
Supercritical Fluids, 4: 72-78.
Choo, Y. M., Ma, A. N., Yahaya, H., Yamauchi, Y., Bounoshita, M.
and Saito, M., 1996, Separation of crude palm components by
semi preparative supercritical fluid charomatography, JAOCS,
73(4): 523-525.
Daood, H. G., Illes, V., Gnayfeed, M. H., Meszaros, B., Horvath, G.
and Biacs, P. A., 2002, Extraction of pungent spice paprika by
supercritical carbon dioxide and subcritical propane. J. of
Supercritical Fluids. 23: 143-152.
De Franca, L. F., Reber, G., Meireles M. A., Machado, N. T. and
Brunner, G., 1999, Supercritical extraction of carotenoids and
lipids from buruti (Mauritia Flexuosa), a fruit from the
Amazon Region. J. of Supercritical Fluids, 14: 247-256.
De Valle, J. M., Jimenez, M. and De la Fuente, J. C., 2003, Extraction
kinetics of pre-pelletized jalapeno peppers with supercritical
CO2, J. of Supercritical Fluids, 25: 33-44.

59
Dean, J. R.,1993, Application of supercritical fluids in industrial

analysis. Blackie Academic & Professional Publisher,
Glasgow.
Fattori, M., Bulley, N. R. and Meisen, A., 1988, Carbon dioxide
extraction of canola seeds: oil solubility and effect of seeds
treatment, J. of Am. Oil Chem. Soc, 65 (6): 968-974.
Favati, F., King, J. W. and Mazzanti, M., 1991, Supercritical carbon
dioxide extraction of evening primrose oil, JAOCS. 68(6): 422427.
Gast, K., Jungfer, M., Saure, C. and Brunner, G., 2005, Purification of
tocochromanols from edible oil, J. of Supercritical Fluids,
34:17-25.
Gordillo, M. D., Blanco, M. A., Molero, A. and de la Ossa, E. M.,
1999, Solubility of the antibiotic penicilin G in supercritical
carbon dioxide, J. of Supercritical Fluids, 15:183-190.
Harcharan, S., Ng, S. C., Hoon, T. Y. and Ong. S. S., 2002,
Supercritical carbon dioxide extraction of black pepper oil.
Proceedings of the 2nd World Engineering Congress. Sarawak
Malaysia, August, 72-75.
Hassan, M. N., 2000, Extraction and Fractionation of palm kernel oil
using supercritical carbon dioxide as a solvent. PhD thesis.
Universiti Sains Malaysia, Penang Malaysia
Hassan, M. N., Ab. Rahman, N. N and Ab. Kadir, M. O., 1999, Major
chemical constituents of supercritical carbon dioxide extract of
pandanus odorus Leave, Journal of Natural Product Sciences,
5(2): 75-79.
Kallio, M. and Kerrola, K., 1992, Application of liquid carbon dioxide
to the extraction of essential oil of Coriander Lebensm, J. of
Agric. Food Chemistry, 41: 785-781.
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King, J. W., Favati, F., and Taylor, S. L., 1996, Production of

tocopherol concentrates by supercritical fluid extraction and
chromatography, J. of Separation Science and Technology, 13
(31): 1840-1847.
Kiriamiti, H. K., Rascol, E., Marty, A., and Condoret, J. S., 2001,
Extraction rates of oil from high oleic sunflower seeds with
supercritical carbon dioxide, J. of Chemical Engineering and
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Lang, Q., and Wai, C. M., 2001, Supercritical fluid fxtraction in herbal
and natural product studies a practical review, J. of Talanta,
53: 771-782.
Lee, H, Chung, B. H. and Park, Y. H., 1991, Concentration of
tocopherols from soybean sludge by supercritical carbon
dioxide, JAOCS, 8(68): 571- 574.
Lee. B. C., Kim. J. D., Hwang, K. Y. and Lee. Y. Y., 1994, Extraction
of oil from evening primrose seed with supercritical carbon
dioxide. In Supercritical Fluid Processing of Food and
Biomaterials (Rizvi S. S. H. eds). Blackie Academic &
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Lin. M. C., Tsai. M. J. and Wen. K. C., 1999, Supercritical fluid
extraction of flavonoids from scutellariae radix, J. of
Charomatography A, 830: 387-395.
Lucas de, A., Martinez de la. O. E., Rincon, J., Blanco, M. A. and
Gracia, I., 2002, Supercritical fluid extraction of tocopherol
concentartes from olive tree leaves, J. of Supercritical Fluids,
22: 221-228.
Mastura, M., Harcharan, S. and Mashitah, H., 2001, SC-CO2
fractionation of crude palm oil, J. of Supercritical Fluids, 20:
45-53.
McHugh, M. A. and Krukonis, V. J., 1986, Supercritical fluid
extraction: principles and practice. 2nd edition, Butterworth
Publishers, Boston

61
Mohd Azizi, C. Y., 2007, Extraction, identification and separation of

vitamin E and djenkolic acid from Pithecellobium jiringan
(jack) Prain seeds using supercritical carbon dioxide, PhD
Murga, R, Sanz, M. T., Beltrn S. and Cabezas, J. L., 2005, Solubility
of three hydroxycinnamic acids in supercritical carbon
dioxide, J. of Supercritical Fluids, 35, pp. 106-113.
Nik Norulaini, N. A., Zaidul, I. S. M., Anuar, O. and Mohd Omar, A.
K., 2004, Supercritical enchancement for separation of lauric
acid and oleic acid in palm kernel oil (PKO), J. of Separation
and Purification Technology, 35: 55-60.
Norhuda, I., 2005, Studies on mass transfer characteristics of palm
kernel oil extraction using supercritical carbon dioxide
extraction. Ph. D thesis. Universiti Sains Malaysia, Penang
Malaysia.
Perakis, C., Louli, V. and Magoulas, K., 2005, Supercritical fluid
extraction of black pepper oil, J. of Supercritical Fluids, 71:
386-393.
Perretti, G., Miniati, E., Montanari, L., and Fantozzi, P., 2003,
Improving the value of rice by products by supercritical fluid
extraction, J. of Supercritical Fluids, 26: 1-9.
Reverchon, E., 1997, Supercritical fluid extraction and fractionation of
essential oils and related products, J. of Supercritical Fluids,
10:1-37.
Reverchon, E., Ambruosi, A. and Senatore, F., 1994, Isolation of
peppermint oil using supercritical carbon dioxide extraction,
J. of Flavour and Fragrance, 9:19-25.
Saldana, M. D. A., Zetzl, C., Mohamed, R. S. and Brunner, G., 2002,
Decaffeination of guarana seeds in a microextarction column
using water-saturated CO2, J. of Supercritical Fluids,
(22):119-127.
62
Starmans, D. A. J. and Nijhuis, H., 1996, Extraction of secondary

metabolites from plant material: a review,Trends in Food
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Taylor, T. L., 1996, Supercritical fluids extraction: techniques in
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Tonthubthimthong, P., Chuaprasert, S., Douglas, P. and
Luewisutthichat, W., 2001, Supercritical CO2 extraction of
nimbin from neem seeds - an experimental study, J. of Food
Engineering, 47: 289-293.
Wahyu, B. S., Smith, R. and Inomata, H., 2003, Supercritical carbon
dioxide Extraction of cashew shell to obtain anacardic acid and
cardol. Proceedings of the International mini-Symposium on
Supercritical Fluid Extraction. Sendai Japan, January 16-17.
Weatherly, L. R., 1994, Near-critical fluid extraction for biological
and food products. Butterwoth Heinamann Publication, New
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Zaidul, I. S. M., 2003, Supercritical carbon dioxide as a process
fractionation technology of palm kernel oil for cocoa butter
quality fat. Ph. D thesis, Universiti Sains Malaysia, Penang
Malaysia.
Zosel, K., 1981, Process for the decaffeination of coffee. US Patent
4260639.
4
REGENERATION TECHNIQUE FOR THE
SPENT BLEACHING CLAY: OIL
EXTRACTION AND ACTIVATION
Abu Hanipah Nawi
Wahyu Bahari Setianto
Nik Norulaini Nik Abdul Rahman
Mohd Omar Abdul Kadir
4.1
INTRODUCTION
Malaysia has become the biggest palm oil producer and exporter with
total cultivated land area about 4.3 million hectares. It has the
capability to generate about 16.5 millions tonne of palm oil per year
(MPOB, 2007). In order to process the palm oil, it has 434 palm oil
mills operating throughout the Malaysia with 49 mills concentrating in
processing palm kernel and 66 mills for refinery process. In refinery
mill of crude palm oil, general processes of crude palm oil are
summarized in Figure 4.1.
Degumming
Alkali refining
Bleaching
Deodorization
Figure 1 General process in oil refinery mill.
64
The degumming process consists the removal of mucilages or

gums with phosphoric acid at 30-40C and the alkali refining is the
process of neutralisation by addition of caustic soda or by drawing
vapour distillation at vacuum and high tempature for removing the free
fatty acids. The bleaching or also called as decoloration is removing
process of coloring substances such as carotene, chloropiles and
suspension solution. The oil is placed with contact with the bleaching
clay where the pigments are absorbed. The deodorization process role
is to remove and neutralize the odorant and flavouring substances
(Blasco et. at., 2004).
The spent bleaching clay under study is the by product of the
bleaching process where it absorbs about 17-35% of the contacting
oil. Many attempt have been taken in order to remove the residual oil
so that it will not cause bad odor and water contamination through
dissipation from the landfill disposal.
4.2
MATERIAL AND METHODS
4.2.1 SPENT BLEACHING CLAY
Bleaching clay is composed mainly of smectite, an aluminosilicate

mineral.It is used in oils bleaching for edible purpose for removal of
variety of impurities by adsorption. The oil bleaching also removes
color to produce light-colored and stable oil. In palm oil refinery mills,
the usage of the bleaching clay is about 0.5-1% of the refining volume
of crude palm oil. Hence, in 2007 alone, the amount of spent bleaching
clay generated in order to produce 16.5 million MT crude palm oil are
about 165,000 MT.
This paper elaborates the trend of oil extraction by an adept
technology i.e. supercritical carbon dioxide extraction and observation
Regeneration Technique for the Spent Bleaching Clay
65
result on the regeneration of the deoiled spent bleaching clay. Several

researchers studied the possibilities of oil extraction from the spent
bleaching clay such as King et al., 1992, studied oil extraction of
spent bleaching clay used in soybean oil refinery mill using lab and
pilot plant supercritical fluid extraction. Ng et.al., 1996, used
supercritical carbon dioxide extraction to extract the oil before
applying heat and acid activation for the regeneration of de-oiled spent
bleaching clay. Foletto et al., 2002, used solvent extraction for oil
removal for clays used in vegetable oil processing industry and applied
the calcination to the de-oiled clay as regeneration method.
4.2.2
OIL EXTRACTION
For oil extraction, 5 kg of the spent bleaching clay were collected from
the palm oil refinery mill in Seberang Prai, Penang. Experiments using
the adept technique were performed with an apparatus that consisted
of a SC-CO2 extractor (ISCO,Inc.,Lincoln,NE,USA,model SFX 220)
a carbon cylinder, a chiller and a high pressure syringe pump. 2.5 ml
of sample was put into the extractor and carbon dioxide will be chilled
into liquid form before the high-pressure pump pumped it at desired
pressure. Temperature and pressure used in the experiment were
controlled by software integrated with the extractor. Extractions were
performed at temperatures 40, 60 and 80-degree Celsius with pressure
variation at 200, 300 and 400 bar. For comparative study, experiments
using soxhlet extraction were also conducted with extraction time
varied from 4-hour to24 hours. The schematic flow diagram for
supercritical fluid extraction is given in Figure 4.2.
66
4.2.3
REGENERATION
In order to reactivate the spent bleaching clays, it is necessary to do

regeneration process that the area and surface volume of the clay could
be maximized during the contact with the oil during the bleaching
process. In our study, we use heat and acid for the reactivation.
Several concentration of acid was used to seek the optimal condition
in getting the best combination for the regeneration process. Heating
period and acid treatment time were also varied for observation on the
effect of the regeneration performance.
3UHVVXUH
LQGLFDWRU
&RROHU
3UHVVXUH
UHJXODWRU
0HWH ULQJ
YDOYH
([WUDFWRU
2 YHQ
3XPS
&2
F\OLQGHU
3UHVVXUH
LQ GLFD WRU
: HWJDV
P HWHU
$QDO\WH
UHFHLYHU
Figure 4.2 Schematic diagram of supercritical fluid extraction using

carbon dioxide.

4.3
RESULTS AND DISCUSSIONS
4.3.1
OIL YIELD AND EXTRACTION TIME
67
Figure 4.3 shows the results of experiments performed using SC-CO2

and it comparative Soxhlet extraction. The SC-CO2 gave a better
performance of 37.5% (g oil/g-sample) even at low pressure. However
it gave the same oil yield percentage at quicker extraction time about
30 minutes at pressure of 400 bar. A Soxhlet extraction only managed
to give average of 27.9 % of oil yield.
40
30
20
(%)
10
(a)Extraction curves at various temperatures at 200 bar
68
40
30
20
(%)
10
(b) Extraction curves at various temperatures at 400 bar

G raphperc entag eaverag eofoilyieldag ains t
ex trac tiontime
A verag eperc entag e
(% )
28
27.5
27
26.5
average
26
25.5
25
4
16
24
E xtrac tiontime(hours )
(c) Extraction curves using Soxhlet extraction

Figure 4.3 Extraction curves of spent bleaching clay under various
temperature and pressure conditions.

4.3.2
69
ACID ACTIVATION
Applying heat performed regeneration process of the spent bleaching

clay and acid activation on the de-oiled spent bleaching clay. Figure
4.4 shows the effects on the regeneration performances due to the heat
and acid activation. It shows clearly that the heat and acid activation
revives the ability and performance of the spent bleaching clays.
Further studies were carried out to determine the optimal
condition of the heating temperatures and used acid concentration.
Figure 4.5 below shows the absorbance of the regenerated clay, which
were heated at 200 degree Celsius and activated with various acid
sulfuric concentrations. In the figure, the reading of absorbance for all
percentage of acid activation at first increased from 0 minutes to 30
minutes. Then its decreased at 60 minutes for all percentage of acid
treatment. After 150 minutes, 10% and 20% activated samples showed
the increasing reading in absorbance which indicates the defect in
getting the original clay characteristics.
70
G raphads orbanc eag ains ttimeofads orption

Absorbance (AU)
0.3
0.25
non
0.2
heat
0.15
ac id(20% )
0.1
0.05
0
0
30
60
90
120
150
180
T imeofAds orption(minutes )
Figure 4.4 Regeneration performances curve for various conditions of
deoiled spent bleaching clays.
Graph absorbance against time for 200C heating
value
absorbance(AU)
3.00
2.80
acid activation 10%
2.60
acid activation 20%

2.40
acid activation 30%
2.20
2.00
0
30
60
90
120
150
180
time(min)
Figure 4.5 Regeneration performances of the deoiled spent bleaching clay

under various heating temperature and acid concentration.
71
While for 30% acid treatment, the absorbance percentage decreased

until it reached 180 minutes. This result means that the treatment of
spent bleaching clay with 30% H2SO4 resulted a material, which was
most effective in removing collared pigments from crude palm oil.
4.3.2 SEM IMAGES
A few samples of the regenerated clay were sent for the

analysis under surface electron microscope (SEM). The images were
shown in Figure 4.6. In Figure 4.6 (a), the image shows the condition
of the SBC after the oil extraction and heat treatment. Although the
voids and pore inside the SBC were not equipped by oil or any
pigment, but it has least surfaces. Figure 4.6(b) shows that the acid
treatment had given extra and wider surface, which leads to a better
absorbance of color pigment when it has its contact with oil. This
confirms the absorbance test applied on the sample under the
regeneration.
72
(a)
Images of regenerated SBC with heat
(b)
Images of regenerated SBC with heat and 30% acid sulphuric
73
4.4. CONCLUSION
The study showed that SC-CO2 gave the best performance in oil yield
and extraction time compare to the conventional method. Average oil
yield is 35% of its original weight. The conditions of applying
temperature and pressure are 80 degree Celsius and 300 bars
respectively.
Observation on several heating and acid sulphuric
concentration combination expel that heating at 200 degrees Celsius
and 30% Acid Sulphuric is the best combination. At 3 hour retention
time, it finally gives the lowest value of absorbance which indicates
the capability of the material in removing the pigment colour when it
is in contact with oil.
REFERENCES
Foletto, E. L., Alves, C. C. A., Sganzerla, L. R. and Porto,L. M.,

2002, Regeneration and utilization of spent bleaching clay, Lat.
American Appl. Reserach, 32: 205-208
Jerry, W. K., Gary, R. L. and James H. J, 1992, Supercritical carbon
dioxide extraction of spent bleaching clays, J. Supercritical
Fluids, 5: 38-41.
Blasco, M., Capilla, V. and Subirats, S., 2004, State of the art book on
supercritical fluids. Published by AINIA, 333-341,
Malaysia Palm Oil Board, 2008, www.mpob.gov.my, accesed on 30
September, 2008
74
Ng, K. F., 1996, Supercritical carbon dioxide extraction of spent

bleaching clay from the palm oil industry, MSc Thesis,
Universiti Sains Malaysia.
5
ION EXCHANGE TECHNOLOGY FOR
WATER AND WASTEWATER
TREATMENT: PRINCIPLES AND
PROGRESS IN MATERIALS
DEVELOPMENT
Mohamed Mahmoud Nasef,
Hamdani Saidi
Zaini Ujang
ABSTRACT
Ion exchange technology is one of the most effective treatment

methods for water and wastewater that been employed in various
industries for many years. Today ion exchange processes are
successfully applied for desalination of both sea water and brackish
water, softening hard water, treating industrial effluents for removal of
heavy metals, dyes, nitrates, ammonia and fluoride and dealkalization.
In spite of ion exchange technology advanced stage of development,
various aspects remain a subject of intensive investigations in many
institutes to bring about new functional materials that can tolerate
various operating conditions to improve its efficiency and economy of
its applications. The objective of this chapter is to review the current
status of the development in ion exchange materials and their
applications in the field of water and waste water treatment. A brief
encounter of the basic fundamentals of ion exchange processes
76
including mechanism, types of ion exchange resins and systems,

configurations and mode of operation are also illustrated.
Keywords: Ion exchange processes, ion exchange resins, membranes,

system configurations, water and waste water treatment.
5.1 INTRODUCTION
The interest in water production has been dramatically increased in the

recent years due to the shortage of water supplies in many areas and
continuous diminishing of natural fresh surface water resources
following the climate changes caused by the increase of green gas
emission. Also, reliable production of high quality water for the
process and power industries are one of basic requirements in modern
industry, which have derived the progress in water purification
technologies in many developing and industrialized countries.
The increase in using metals and chemicals in various process
industries has resulted in generation of large quantities of effluents
containing high level of toxic heavy metals and their presence poses
environmental-disposal problems due to their non-degradable and
persistence nature. Leaking of these heavy metals to the soil poses a
risk of contamination to groundwater and surface water. This could
have adverse effects on human, animal and aquatic life. Environmental
engineers and scientists are faced with the challenging task to develop
appropriate low cost technologies for effluent treatment. Hence, the
degree of sophistication of separation methods has increased
tremendously.
Conventional methods for treatment of water and waste water
including removing metals from aqueous solutions involves various
technologies such as chemical precipitation, chemical oxidation or
reduction, ion exchange, filtration, electrochemical treatment, reverse
osmosis, membrane technologies and evaporation recovery
Ion Exchange Technology 77

(Dabrowski et al 2004). Each method has its merits and limitations in
application. Among all, ion exchange method and its associated
electrochemical applications have been used successfully in many
industries for more than fifty years for the removal of heavy metals
from waste effluents of various characteristics (Nasef, 2006). This was
prompted by the effectiveness of ion exchange processes for the
removal of heavy metal ions and precious metals especially with large
volumes containing trace quantities in addition to simplicity, elegance
and range of variable reaction conditions (Pohl, 2006).
The advantages of ion exchange technology in water treatment
processes can be summarized as follows: 1) capability of handling and
separating components from dilute wastes, 2) possibility of
concentrating pollutants, 3) capability of handling hazardous wastes,
4) possibility of recovery expensive materials from waste such
precious metals, 5) possibility of regenerating ion exchanger and 6)
possibility of recycling components present in the waste and/or
regenerating chemicals (Wentz 1989). However, ion exchange
processes have some limitations that include low concentration in the
effluent to be treated, lack of selectivity against specific target ions,
susceptibility to fouling by organic substances present in the
wastewater and generation of waste as a result of ion exchanger
regeneration and down time for regeneration upon using single column
configuration (Metcalf and Eddy, 1991).
Figure 5.1 Phenol formaldehyde resins.
78
Figure 5.2: Strong cation and anion exchange resins based on polystyrene
divinylbenzene copolymer.
5. 2 HISTORICAL PERSPECTIVE
Ion exchange phenomena have been known for many years. The first
examples of ion exchange were discovered by Thompson and Way
(1850) during investigations concerning the way in which soluble
manures were retained for long periods in the soil, instead of being
washed out by rain water. The importance of this discovery (in ion
exchange terms) was not fully understood until later in that decade
when this reaction was found to be reversible. This phenomenon was
caused by certain minerals in the soil as released in the latter half of
the nineteenth century. These minerals, called resins, are based on
tetrahedron structure of silicon and aluminum compounds called
zeolites. In 1905, synthetic zeolites were manufactured and utilized for
water treatment in a form of water softening ever since (Gans, 1905).
Synthetic cation exchange resins were developed during the 1930s
using certain types of coal treated with sulfuric acid (Liebknecht,
1935; Smith, 1935). This was an important evolution due to the fact
that the sulfonated coal would operate in a greater pH range, 1 to 10.
This made the sulfonated coal more versatile enabling it to be used in
many more industrial applications. However, these resins were found

to have serious deficiency caused by their lower exchange capacity
compared to the zeolites. A few years later the phenol formaldehyde
polymer resin from the type shown in Figure 5.1 was synthesized
(Adams and Holmes, 1935). This polymer was sulfonated to form a
strong acid cation. Using the same base polymer only functionalized
with an amine (NH2) produced the first weak base anion exchange
resins. The major development for the power industry came in the
U.S.A. in 1944 when strong acid and strong base resins from the types
shown in Figure 5.2 were produced based on divinylbenzene
crosslinked polystyrene which was treated with sulfuric acid to make a
strong acid cation or chloromethylated and then aminated to make
strong base resins (dAlelio, 1945, McBurney, 1952). These resins
much better characteristics than earlier resins. These new resins are
now used almost exclusively in water demineralisation plants for high
pressure boilers. Eventually, the macroporous and acrylic resins were
synthesized, with each resin having its ownniche in the water
treatment industry. Table 1 shows the most important milestones in the
development of ion exchange resins.
Table 5.1 The most important milestones in the development of ion
exchange resins.
Year
1850
1858
1905
1913
1935
1944
1946
Milestones
Agricultural chemists Harry Thompson & John Way discovered ion
exchange phenomena
German Chemist Eichom reported that ion exchange is a reversible
reaction.
Robert Gans introduced first process to soften water using zeolite (sodium
alumiosilicate)
American company (Permutit) introduced first commercial zeolites.
English chemists Adams and Holmes prepared first synthetic polymer
cation and anion exchangers (Phenol formaldehyde & polyamine
formaldehyde).
can scientist DAlelio developed cation exchange resins based on
polymerization of styrene and divinylbenzene.
Anion exchange resins based on polymerization of styrene and
divinylbenzene were developed.
80
5.3 OBJECTIVE AND SCOPE
The primary objective of this chapter is to review the current status of

the development of ion exchange materials in the field of water and
wastewater treatment. To understand the key drivers in ion exchange
technology, the basic fundamentals of its key components are
reviewed with some emphasis given to ion exchange resins, their
types, forms and functions. The chapter starts with a brief review of
basic principles of ion exchange processes covering their basic
operational chemistry, various ion exchange materials and their
specifications. This is followed by previewing the engineering
configurations, operating modes and systems for ion exchange
processes. An intensive discussion on the status of development of
various ion exchange materials in wide range of applications related to
treatment of water and industrial waste water is presented. The
strategy of the discussion follows the diversity of applications in water
and wastewater field regardless not only the variation in the forms and
the types of ion resins but also their configurations as well as operating
systems. Challenges that are facing the technology today and future
directions for research to develop new materials to improve the
efficiency of the technology and its economy also illustrated.

Table 5.2 Summary of applications of various ion exchange materials in
water and wastewater treatment (Zagorodni, 2007).
Categories
Applications
Water production for different uses
Production
of
pure
(demineralization of water).
Water softening.
Potable water production.
Removal of specific pollutants from

industrial streams
and
ultrapure
Dealkalization
Fluoride removal.
Removal of organic matter.
Removal colorants.
Oxygen removal.
Removal of Iron.
Removal of manganese.
Removal of cadmium ions from drinking
water.
Removal of nitrate ions.
Removal of ammonia.
Removal of radionuclides from drinking water.
Removal of other harmful ions from drinking
water.
Nuclear industry
Radioactive waste water decontamination.

Condensate polishing.
Decontamination and recuperation

of waste streams:
Pulp and paper industry
Removal of inorganic salts from liquors.
Recycling of industrial water.

Removal of heavy metal ions.
Recuperation of metals.
Ammonia removal.
Recovery of calcium aconitate.
Removal of radioactive substances.
82
54 FUNDAMENTALS OF ION EXCHANGE PROCESS
5.4.1. ION EXCHANGE
Ion exchange is a process used to purify solutions by removing the

dissolved ions by electrostatic sorption into ion exchange materials
(resins, membranes or fibers). The removed ions are replaced with
equivalent amounts of other ions of the same charge of the solutions.
The use of ion exchange reaction allows either all ions to be removed
from a solution or particular ions to be separated. Therefore, selective
removal of ionic contamination and complete deionization can be
distinguished. The choice between both depends mainly on the
composition of the solution and on the extent of decontamination
required (Dardel and Arden, 2005).
The applications for ion-exchange are numerous and cover wide
ranges of industries. Ion exchange is most commonly used for
purification purposes, but is also widely implemented in the separation
and extraction of valuable substances such as uranium and plutonium
from the nuclear industry waste (Badawy, 2003). Deionisation or
demineralization of water and water softening can be cited as the most
common application. However, the spectrum of applications varies
from large scale extraction of metals in hydrometallurgical and metal
finishing processes to recovery of precious metals (Harland, 1994;
Zagorodni, 2007). Applications of ion exchange materials in the field
of water and waste water treatment in various industries are
summarized in Table 5.2.

5.4.2. ION EXCHANGE MECHANISM AND KINETICS
Ion exchange process depends on a mechanism in which mobile ions

from an external solution are exchanged for ions that are
electrostatically bound to the functional groups contained within a
solid matrix. Metal ions initially contained in an aqueous solution are
exchanged with ions initially contained in solid materials (most of
them in an organic ion exchange resin). Such a process is called cation
exchange and can be illustrated by reaction
R-H+
+A+
R-A+
+H+
(5.1)
where, R is ion exchanger and A+ is a positively charged metal ion.

The bars indicate the phase of the ion exchanger. The equilibrium
constant (K) is given by next formula:
[ RA] [ H ]
K
x
(5.2)
[ RH ] [ A ]
The distribution of the two ions between the two solid and liquid
phases (resin and external solution) depends on reaction constant
value. A similar process involving anions is called anion exchange:
R+Y-
+B-
R+B-
+Y(5.3)
where, B- and Y- are anions or negatively charged metal ions. The

equilibrium constant (K) is given by next formula:
K
[ RB] [Y ]
x
[ RY ] [ A ]
(5.4)
Ion exchange reactions are reversible and follow stoichiometry as seen

in the following reaction:
84
2(R-SO3H) + NiSO4 = (R-SO3)2Ni + H2SO4
(5.5)
Contrary to many other chemical separations, reactions 1 and 3 can be

successfully used even if they are shifted to the "wrong" direction. By
taking advantage of the fact that, under certain conditions, ion
exchange media has a greater affinity for certain ionic species than for
others, a separation of these species can be made.
Chemical selectivity of reactions 1 and 3 is desirable but is not a
requirement. The selectivity (ion exchange) coefficient for reaction 1
expresses the relative distribution of the ions when a resin in the H+
form is placed in a solution containing A+ ions whereas, the selectivity
coefficient for reaction 3 expresses the relative distribution of the ions
when a resin in the Y- form is placed in a solution containing B- ions.
The selectivity coefficients for reaction (1) (
given by:
) and (2) (
) are
(5.6)
The selectivity coefficients in ion exchange systems are not constant
and vary with the operation conditions such as types of resins,
concentration of feed solution, temperature and the presence of other
ions in the solution. The selectivity coefficient provides a means of
determining what to expect when various ions are involved. However,
the determination of selectivity coefficients is practically complicated
task and is ordinarily not undertaken in the design of waste treatment
systems; most of these parameters can be extracted from
manufacturers data or research literature. More details on ion
exchange kinetics can be found in Korkisch (1989).

In ion chelation which depends on the affinity towards certain ion, the
metal ion of opposite charge forms a coordinate bond leading to metal
complex formation as follows:
Ion transfer takes places in the ion exchange resin (particles) through
the inter-phase boundary in a form of: 1) chemical reaction, 2)
diffusion inside the material, 3) diffusion in surrounding solution and
4) diffusion through the thin film formed around the particles. Ion
exchange process is controlled by the diffusion of counter-ions. The
two rate determining steps are considered: diffusion of ions inside the
material (particle diffusion) and diffusion of ions through the film
(film diffusion).
To achieve an efficient separation, column techniques are applied.
Depending on the type of the functional group, ion exchangers can be
divided into several types: strong acidic, strong basic, weak acidic and
weak basic as discussed next.
5.4.3. ION EXCHANGE MATERIALS
Ion exchange materials are available in different forms that vary in

their classifications as shown in Figure 5.3. On the material origin
basis, there is a wide variety of organic and inorganic ion exchange
materials. Only cation exchange inorganic materials (for example
zeolites and betonites) are known. These materials compose of
negatively charged porous structures with exchangeable cations
located in internal voids. Unlikely, organic ion exchange materials
86
can be both cation- and anion exchangers and this classifies them
based on the functional groups. Considering the separation function,
ion exchangers can be classified into various categories including ion
exchangers, chelating adsorbents, hydrogels, affinity polymers and
polymer electrolytes.
Figure 5..3 Schematic representation for various classifications of polymeric

ion exchange materials
On a material view point, organic (polymeric) ion exchange materials,

which consists of functional groups bound to different polymeric
frameworks available in various physical forms including gels, resins,
fibers, membranes and fabrics having widely differing chemical and
physical properties. The majority of these forms have synthetic
polymer structures i.e. such as polyethylene (PE), polystyrene (PS)
and polyvinyl fluoride (PVF) while some of them are obtained from
modified natural polymer sources including chitosan, starch, and
cellulose. Various types of ion exchange membranes (schematized in
Figure 5.4) are not only present in ion cation and anion exchange form
but also in a bipolar or combined form containing positively charge
groups on one side and negatively charged groups on the other side
forming a unique structure called amphoteric membranes. The
separation behaviour of each type of these membranes is determined
by the chemical nature of attached charged group. Unlike ion

exchange membranes, resins follow the main two classes i.e. cation
and anion exchange forms. A combination of cation and anion
exchanges form can be used to obtain a combined bipolar form of the
resins in a mixed bed column. More details on the various types of ion
exchange materials can be found in the most regarded book on ion
exchange materials was published by Helfferich (1962) and in the
recent published book on the same subject by Zagorondi (2007).
Since ion exchangers act in a similar way to conventional acids and
bases, the main classes of these materials i.e cation- and anion
exchangers can be further classified as follow:
cation exchanges are divided into strong and weak cation exchangers
depending on the type of functional group attached to the polymer.
Most typical strong acid exchangers contain sulfonic groups (-SO3-).
Such materials are active over the entire pH range. Most weak acid
exchangers have carboxylic groups (-COO-). The weak acid
exchangers are not active at pH values below 4-6 (this value
significantly differs for different materials). However, they often have
higher ion exchange capacities than the strong acid exchangers and
have other specific advantages as well (Zagorodni, 2007).
Figure 5.4 Classification of ion exchange materials in ion exchange

forms
The majority of strongly acid resins in use today have a styrenedivinylbenzene copolymer bead structure similar to that shown in
Figure 5.2. This crosslinked structure gives the ion exchange resin
bead certain physical properties including particle size and water
88
swelling. Another important resin bead structure for water treatment

resins is the acrylic resin structure. The operating properties for acrylic
resins are different from those of an equivalent styrene divinylbenzene
resin. One cannot say which resin structure is advantageous without
knowing the site-specific operating conditions. The preferred resin is
the one that has operating properties matching up best with the site's
operating parameters, thus maximizing operating efficiency and cost
effectiveness.
Strongly acid resins are commonly used in water softening and
demineralization applications. In softening applications, the resin is
used in the sodium form (regenerated with salt) and in
demineralization applications the resin is used in the hydrogen form
(regenerated with acid). These resins also can be used in a split-stream
dealkalization process. These resins can be purchased with different
percentages of crosslinking. The common crosslinking level in these
resin is 8 percent crosslinked. However, higher and lower crosslinked
resins are also available with less or more water content. On the other
hand, weak acid cation resins can be used in demineralization and
dealkalization systems. They are very efficient when matched up with
the proper influent water chemistry.
Anion exchangers are classified in a similar way into strong base
anion exchangers and weak base anion exchangers. Strong base
exchangers have quaternary ammonium groups (-NR3+). They are
active over the entire pH range. Weak base exchangers have primary (NH2), secondary (-NRH), and/or tertiary (-NR2) amine groups. The
weak base exchangers are not active at alkaline pH. However, they are
advantageous in many practical cases. Strong base anion resins are
used in ion exchange demineralization, dealkalization, desilicization
processes and also in organic trap applications.
Unlikely, weak base anion exchange resins can not be used for
demineralization as they allow the carbonate/bicarbonate and silica
ions to pass through. Therefore, they have to be used with strong base
resin to consider such application. However, weak base resins can
remove the anions of the strong mineral acids (sulfate, chloride and
nitrate).

A summary of the common functional groups and their negative
logarithm of the dissociation constant (pK) is presented in Table 1. It
can be clearly seen that each of these major resin classes has several
physical or chemical variations within the class. The variations impart
different operating properties to the resin.
With the wide number of resin types available in the marketplace, it is
highly possible that there is more than one technically effective
solution that meets all the system's design specifications. To have a
robust ion exchange system design a thorough knowledge of all
available resin types along with their various advantages and
disadvantages to ensure the efficiency and cost effectiveness of the ion
exchange system.
Table 5..3 Common functional groups of polymeric ion exchange materials
and their respective pK values
Figure 5.5 Schematic representation of various morphologies of ion

exchange polymeric materials and their respective operating systems
90
5.4.4. ION EXCHANGE PROCESS EQUIPMENT AND

OPERATION
The feasibility and success of separation and purification processes

depend on the nature of the separating polymer and the operating
conditions. In other words, the morphology of the separating polymer
dictates the selection of the process and its operating mode. As
mentioned earlier, the basic morphologies of ion exchange materials
include resins or beads, membranes, fibers and hydrogels. To bring
such polymer forms into applications, they have to be hosted in a
proper engineering system that provides a set of adjustable operating
parameters to control their performance. Accordingly, the separation
systems take various configurations depending primarily on the
physical form of separating polymers as indicated in the schematic
representation given in Figure 5.5 which depicts as a schematic
representation of various morphologies of ion exchange polymeric
materials and their respective operating systems. In addition, the
volume of the solution to be treated also plays a role in selection of
system configuration but to lesser extent.
Figure 5..6 Schematic representation of ion exchange operation cycle.
Figure 5.7: Schematic representation of fixed bed ion exchange column

system showing operation and regeneration modes.
When separating polymers are in a resins form, ion exchange

processing is conventionally accomplished by either a batch method or
a column method. Most commonly the ion exchange is performed in
cyclic operations. Each cycle is divided into three main stages: 1)
sorption, 2) elution and 3) regeneration as shown in the ion operation
cycle schematized in Figure 5.6. Details of ion operation cycle are as
follow:
1) Sorption: The solution containing the targeted ions is passed slowly
through to reaction vessel. The ions bind into the resins. Ions initially
contained in the exchanger are released.
2) Elution (stripping): The target ions are subsequently stripped from
the loaded resin with a small volume of an eluent. The eluent replaces
and hence also releases the target ions from the resin into the solution
phase. 3) Regeneration: Depending on the type of the ion exchanger
and the stripping agent, the ion exchanger sometimes has to be
regenerated. For example, if the sorption step uses a cation exchanger
loaded with H+ ions but the elution leaves Na+ ions in the exchanger
phase, the material has to be protonated. A strong acid could be
applied in order to convert (regenerate) the exchanger in the initial
state. It is important to mention that designing an optimized cyclic ion
92
exchange process requires efficient utilization of bed through selection

of solutions having high selectivity towards the target ions.
In most techniques, solutions are consequently pumped through
a vessel loaded with the ion exchange resin and represent one of four
basic operating configurations for (i) continuous stirred (batch) tank,
(ii) fixed bed column, (iii) fluidized bed column, and (iv) a moving
bed (closed loop) system.
In a batch method, the resin and solution are mixed in a batch
tank, the exchange reaction is allowed to come to equilibrium and then
the resin is separated from solution. The degree to which the exchange
takes place is limited by the preference the resin exhibits for the ions
in solution. Consequently, the use of the resins exchange capacity is
limited unless the selectivity for the ions in solution is far greater than
for the exchangeable ion attached to the resin. Because batch
regeneration of the resin is chemically inefficient, batch processing by
ion exchange has limited potential for industrial applications and often
used in laboratories for fundamental studies.
Figure 5. 8 Schematic diagram of a laboratory scale ion exchange

column.
The fixed bed column operation is the most usable ion exchange
configuration in industrial applications. It has a basic component of
which the beads column is considered to be analogous of several batch

reactors in series. The purpose of column operation is to work around
the limitation of selectivity. To favor high selectivity, more stages can
be used (multi-column system). Three types of column operation
modes are available: i) down flow, ii) up flow and iii) counter flow.
Most column beds operate with down flow operation as shown in
Figure 5.7 that depicts a schematic representation of fixed bed ion
exchange column system with operation and regeneration modes. This
is where feed and beads pass down through the resin bed. On the
contrary, up flow operation is used when the feed and resin are raised
through a bed. The final flow is counter flow and it consists of the feed
flowing down from the top and the regenerate flows up from the
bottom. Most industrial applications of ion exchange use fixed bed
column systems, the basic component of which is the resin column.
Figure 5.9 Typical example of ion exchange plant based on fixed bed
column system (Courtesy of Robert Mesick from REMCO Engineering
water systems and controls).
The column design must consider the following:

1. Contain and support the ion exchange resin
2. Uniformly distribute the service and regeneration flow through
the resin bed
94
Provide space to fluidize the resin during backwash

Include the piping, valves, and instruments needed to regulate
flow of feed, regenerant and backwash solutions.
The operating or breakthrough capacity of a column type ion
exchange system depends on its design and operating parameters, the
concentration of the ions being removed and the effects of interference
from other ions. In a column system this generally refers to the volume
of the solution that can be treated before a sharp increase in the
effluent concentration of the species being removed is observed. At
this point the ion exchange medium is considered to be spent and must
be replaced or regenerated. The operating or breakthrough capacity is
most significant parameter in the design of a column type ion
exchange system and is generally given as the number of bed volumes
(the ratio of the volume of liquid processed before the breakthrough
point to the volume of the settled bed of the exchanger). Some
important parameters that affect the breakthrough capacity include: i)
the nature of the functional group on the exchanger, ii) the degree of
crosslinking of the resins, iii) the concentration of the solution to be
treated, iv) the valence and the ionic size of ions to be exchanged, and
v) the operating temperature. Figure 8 shows an example of a
schematic diagram of a laboratory fixed bed column ion exchange
system whereas a typical example of ion exchange plant based on
fixed bed column system is shown in Figure 5.9. In practical
operations it is desirable to have the breakthrough curve as steep as
possible. A steeper breakthrough curve will increase the extent of
column utilization; that is, bring the ratio of the breakthrough capacity
to the total capacity closer to unity. If the required decontamination
factor is high the degree of column utilization can vary considerably
for different ion exchange media.
The fluidized bed column operation is used when the raw feed
contains suspended solids. The column is partially filled with the ion
exchange beds. The feed solution flows down up through the bed with
a speed sufficient to push up the beads up into the column causing
them to float and swirl around. The floating of the ion exchange beads
reduces the hydrodynamic resistance of the column. The presence of
3.
4.

free-pass ways between beads allow the solid suspended particles to be
transferred through the column giving fluidized bed column the
advantage of processing solid-containing solution without any
pretreatment. Therefore, fluidized beds are commonly used for
biochemical filtration such as fermentation broth where treated
solutions are highly containing suspended solids.
Table 5.3 Comparison between various ion exchange operating
techniques.
System configuration
Advantages
Disadvantages
Batch system
Simple to construct and

operate.
Good for small scale
applications.
A wide variety if ion
exchange media can be
used.
Easy to be customized for
specific treatment problems
Operate with resins of
various shapes
Manual operation may

be cumbersome to
operate with large
volumes of waste
The separation of liquid
and ion exchange media
is required
Can only be operated at
atmospheric pressures
and ambient
temperatures
Once through use only
Conventional
column system
Good throughput
Simple to operate
A wide variety of media are
available
Can be operated at elevated
temperatures and pressures
High decontamination
factors are possible
Large equipment can be

costly
The regeneration of
media may require extra
equipment
There are difficulties in
transporting inorganic
ion exchangers through
pipelines
Can not treat solid
containing solutions and
thus prefiltration is
necessary
Limited part of the bed
particle is involved in
96

ion exchange in the
same time.
Moving bed system
The process is continuous.

Very high decontamination
factors.
Less operation space.
Requires less number of
supplementary equipments.
Low pressure drop.
Can treat solid containing
solution without bed
plugging.
Membrane system
Can be used either as a

waste treatment or fluid
concentration technique.
Prefiltration is not
necessary.
Does not need granular
materials.
High decontamination
factors are possible.
Instability of the process.

Longitudinal mixing
problem.
Difficulty in providing
circular motion.
Extensive mechanical
deterioration of resin
beads.
Equipment can be costly

to construct and operate
on a large scale.
The moving beds are the most economical beds of ion exchange
systems. The principle of this operation relies on bringing the beads
and the solution to flow through the system. The beads are contacted
counter-currently with the exhausting stream and regenerated stream.
The advantage of this operation is featured by continuous product of
uniform quality at less space, capital and labor. However, the
complexity of the design poses a problem during operation.
With the wide number of resin types available in the marketplace, it is
probable that there is more than one technically effective solution that
will meet all the system's design specifications. This is where
experience and knowledge is required to help select the system design
that will do the job expected by the customer under the conditions
existing at the customer site. This experience will include a thorough
knowledge of all available resin types along with their various

advantages and disadvantages so they can be applied in ion exchange
systems that are both technically sound and cost effective. Sound
equipment design, effective use of available resins and readily
available services that meet all the expectations of the industrial
customer will lead to a win/win long-term customer relationship that
benefits both the supplier and the customer.
Similarly, putting ion exchange materials in a film or membrane
form into any practical application requires that the membrane to be
housed in a vessel called module. Membrane module provides support
and protection against operating pressures and daily wear and tear of a
production environment and to allow applying control strategy over
the system performance. More detail on the requirements for efficient
module designs to house various membrane types can be found
elsewhere (Baker 2004). The most common configurations of
membranes modules are: 1) Plate-and-frame, 2) spiral wound, 3)
tubular and 4) hollow fiber. Among all plate-and-Frame module is
most common module used processes utilizing ion exchange
membranes
Plate-and-Frame module consists of layers of membranes
separated by corrugated structural sheets. Corrugations run at right
angles in alternating layers with feed material flowing in and retentate
flowing out in one direction, while a carrier fluid flows in and
permeate out in the other direction. Advantages include ability to
accommodate low levels of suspended solids and viscous fluids and
the ability to change out membranes if needed (though it is difficult).
Disadvantages include relatively low packing density, high initial cost,
and difficult disassembly for cleaning. Electrodialysis and
electrochemical membranes use only this configuration; other
membrane types may also be so configured (Strathmann 2004,
Kariduraganavar et al. 2006). Table 5.3 shows a comparison between
the four configurations of ion exchange systems.
98
5.4.5. SELECTION OF ION EXCHANGERS AND SYSTEMS
There are three major factors affection of the selection of the ion
exchangers and the process to apply them. They are 1) waste
characteristics where the concentration of total suspended solids in the
waste should be less than 4 mg/L and the total dissolved salts content
is less than1 to 2 g/L, 2) ion exchange material and system where the
selection of an appropriate material s (resin, hydrogel, or fibers)
depends on the needs of the system. The ion exchange media must be
compatible with the chemical nature of the waste (such as the pH and
type of ionic species present), as well as the operating parameters,
notably temperature and pressure. However, if there are large
concentrations of chemically similar ions in the waste the process of
selection becomes more difficult and 3) cost consideration where the
total cost of operating a process is the sum of the capital costs, the
initial cost of the ion exchange media, the operating costs, and the
costs associated with the treatment and disposal of the spent ion
exchanger. The total costs can be reduced by a frequent regeneration
of the spent ion exchange material instead of using a once through
process.
A good ion exchange system designer not only will design the
system to meet all design specifications but also will utilize resins that
will allow the system to operate at peak efficiency and maximum cost
effectiveness.

Table 5.4: Various applications of ion exchange in water and waste water
treatment.
Application
Operating principle
Types or resins
Softening
Ca+2, Mn+2 and Fe+3

replaced by Na+ ions.
Cation exchange resins in Na+

form.
Deionization
All cations replaced by H+

ion and all anions replaced
by OH- ion.
Cation exchange resins in H+

form coupled with anion
exchange resins ion in OH- form
or mixture of them.
Metal removal
Selective removal Fe+3,

Ni+2, Zn+2, Cu+2, Pb+2 ions
replaced by H+.
Cation exchanage resins in H+

form
Nitrate removal
Nitrates replaced by
chloride
Anion exchange resins in Cl-form.
Dealkalization
Bicarbonate and
carbonates replaced by
chloride
Arsenic removal
Arsenic V replaced by
chloride
Dye removal
Ionic dyes in form cations

and anions
Cation exchange resins, anion

exchange resins or both
Solution
purification
Cr+3, Fe+3, Cu+2 and Ni+2

replaced by H+ to
decontaminate chromic
acid to extend it life and
improve product quality.
Cation exchange resins or

chelating resins
Precious metal
Recovery
Gold, silver replaced by

chloride
Cation exchange resins in H+

form or chelating resins
100
5.5. WATER AND WASTE WATER TREATMENT

APPLICATIONS
Ion exchange technology has been applied for many years in water and
wastewater operations and other activities involving the treatment of
radioactive waste of nuclear power plants. The early great success of
ion exchange in the area of water purification and water softening
prompted extending the use of the technology for various applications
including desalination of sea and brackish water, removal of heavy
metals from industrial streams, removal of dyes and colors, removal of
nitrate and ammonia, removal of fluoride and dealkalization. The
technique was commonly perceived as suitable almost only for water
purification. However, it can be successfully applied for almost any
separation of ionic, ionizable, or locally chargeable substances and
therefore was applied for recovery of precious metals such as silver,
gold and platinum. A summary of various applications of ion
exchange processes in water and waste water treatment, operating
principles and the types of utilized resins are presented in Table 5.4.
The technique is robust, allowing successful separations even in nonoptimized chemical systems. This is one of main advantages in
comparison with competing techniques, such as and adsorption and
solvent extraction.
The main limitation of current ion exchange technology is
economical. The separation process is inexpensive if operated with a
low concentration of ions. Increase of the concentration results in a
cost increase. Thus, competing techniques (membrane separations,
solvent extraction, etc) could be considered (but not necessarily
preferred) for treating concentrated solutions. To overcome such
limitations new ion exchange materials with appropriate
functionalities tolerating wider operational parameters to improve the
economy and the efficiency of ion exchange processes and extend
their applications to new fields are sought. The status of research on
development of ion exchange materials and their application in water
and wastewater treatment is reviewed in the following section. The

paste of presentation takes care of materials being researched for water
and waste water applications without considering their physical forms.
5.5.1 DESALINATION OF BRACKISH WATER
Production of water by desalination methods continues to receive an

increasing attention despite 4 decades of commercialization. The
essential requirement that determines the viability of any scheme
providing fresh water suitable for human use is the cost. Desalination
of brackish water by electrodialysis (ED) is considered to be the most
important large-scale application in terms of installation number (>
2000 plants worldwide with a total production capacity of >1,000,000
m3 of water per day are installed with > 1.5 million m2 membrane
area), which is competing with reverse osmosis (RO) and fractional
distillation (Tanaka, 199, Veza 20001). This process is also most
economical when used with water of relatively low salt concentrations
(< 5000 ppm). ED is also used for the production of potable water
(Nagarale et al 2006).
In principle, ED is a significant mass membrane separation
process where selective separation of certain ions forms water by
migration across ion exchange membranes under influence of electric
field applied on two electrodes bordering a number of cationic and
anionic membranes placed in alternating pattern. The degree of
separation is mainly determined by the properties of ion exchange
membranes used in the process whereas the economics of the process
is determined by the operating costs that involve the energy
consumption. The various process design parameters, such as flow
turbulence, limiting current density, effective cell pair area and cell
dimensions also have the significant effect on the energy consumption
and thus total operating cost. Recent developments in ion exchange
membranes properties have led to tremendous improvement in the
efficiency of ED (Xu, 2005).
102
Various grafted and/or crosslinked ion exchange membranes

prepared by grafting and crosslinking techniques are commercially
available. Styrene-divinylbenzene-based membranes are the bestknown in literature for the application of ED (Sata 1994).
The use of grafted and crosslinked anionic and cationic
membranes for sea water desalination by ED has not been genuinely
addressed. Only few articles reported in literature. Aminabhavi et al.
(2004) have developed homogeneous anion exchange membranes for
the conversion of sea and brackish water into potable water by
simultaneous polymerization of 4-vinylpyridine (4-VP) and
crosslinking with epichlorohydrin (EPI) and aniline non-woven
support cloth at different ratios. The resulting membranes were
quaternized with methyl iodide using hexane as a solvent. The
obtained membranes showed a promising performance.
Choi et al (2001) studied the desalination of water by ED with
two types of ion exchange membranes (IEMs) prepared by radiation
grafting of glycidyl methacrylate (GMA) onto PE and PP nonwoven
fabrics followed by treatment of the obtained grafts with triethylamine
(TEA) and phosphoric acid. Currently, research is focusing on
addressing remaining unsolved issues such as membranes organic
fouling, selective permeability for specific ions, instability of
quaternary ammonium exchange groups in alkaline solution in anion
exchange membranes (Lindstrand et al 2000 a and b, Lee et al 2002).
5.5.3. DESALINATION OF SEA WATER
Water desalination by RO is another field where membranes have

been served since their early days (Bohdziewicz et al. 1999). In this
application, membrane material plays a significant role in affecting the
system performance (Tanaka 1999; Matsuura, 2001). Neutral
membranes and IEMs have been utilized for such application.
However, the contribution of IEMs to industrial application is far
small compared to neutral membranes.

Graft copolymer cation exchange membranes prepared by
radiation grafting of acrylic monomers i.e acrylic acid (AAc)
methacrylic acid (MAc) and acrylamide (AAm) and vinyl monomers
(vinyl
acetate,
VAc)
onto
polyethylene
(PE)
and
poly(tetrafluorethylene) (PTFE) and poly(tetrafluorethylene-cohexfluoropropylene) FEP films were tested for water desalination with
a salt rejection of more than 90 % (Hegazy et al. 1981, 1986, 1989,
Higuchi and Lijimo 1985). The performance towards salt rejection was
further improved by using membranes obtained by grafting of
combined (binary) monomer systems of styrene/AAm onto various
polymer films (Vigo et al. 1981).
Anionic exchange membranes prepared by grafting and
subsequent quaternization (treatment with methyl bromide) of 2-VP
and 4-VP onto poly(vinyl chloride) (PVC) and poly[3,3bis(chloromethyl)oxetane], (penton) films demonstrated very good RO
properties including sufficient salt rejection (Bittencout et al. 1981
a,b). Similar quaternized poly(4-VP) grafted-PE and -PTFE
membranes also demonstrated a combined good flux and salt rejection
properties (Hegzay et al. 1986, Canepa et al. 1973, Kaur et al. 1997,
2001).
Cation exchange membranes having phosphoric acid groups
obtained by radiation induced grafting of GMA onto PE hollow fibers
followed by phosphorylation of the epoxy rings were also reported to
be capable of maintaining salt splitting capacity of 0.68 mol/kg it their
Na-form (Saito et al. 1989).
Commercial microfiltration PE hollow fiber membranes
modified by radiation induced grafting of diethylaminoethyl
methacrylate (DEAEMA) followed by amination with diethyl amine
(DEA) recorded a high pure water flux of 1.0 m.h-1 at filtration
pressure of 0.1 MPa and ion exchange capacity of 1 mol.kg-1
(Kobayashi et al. 1993).
104
5.5.4. SOFTENING OF HARD WATER

The presence of Ca2+ and Mg2+ in water results in water being
considered hard. Ca2+ and Mg2+ ions in water react with heat, metallic
plumbing, and chemical agents such as detergents to decrease the
effectiveness of nearly any cleaning task. Water softening (removal of
hardness) is one of the most classical applications of ion exchange
materials that have been established for many years. This treatment
involves the removal of hardness caused by presence of Ca2+ and Mg2+
ions from water solution by exchanging with Na+ ion available in ion
exchange resins. Various ion exchange resins are commercially
available for water softening application. However, there is a need to
replace conventional softening resins with other materials such as
functionalized microfiltration or nanofiltration membranes where the
operation does not produce by-products to the finished water and
therefore eliminates the by-product disposal cost.
Ion exchange membranes prepared by photografting of porous
ultrafiltration (UF) membranes have been tested for water softening.
Bequet et al (2000) reported a composite nanofiltration membrane for
water softening using a route consists of photografting of acrylic acid
(AAc) onto the surface of UF-porous polystyrene membranes with UV.
The surface modified membranes was stabilized by crosslinking with
N, N'-methylene bis-acrylamide. The obtained membranes showed
softening properties of 95% recovery when measured in dead-end
mode.
Similar membranes prepared by photografting of AAc and
sodium ally sulfonate (SAS) onto polyetherketone cardo (PEK-C) with
UV without photoinitiator showed unexpectedly large permeation flux
without loss of salt retention (Qiu et al 2007).
Treatment of natural nonpotable and irrigation un-fit water by
novel cellulose-based hydrogels synthesized from reactive networks
and graft copolymers of either AAm or AAc and a mixture of them in
presence of benzoyl peroxide as an initiator and glutaraldehyde (GL)
as a crosslinker were reported recently (Chauhan and La 2003). The

water treated with these hydrogels became potable and could be used
for agricultural purposes.
5.5.5 REMOVAL
OF
HEAVY
INDUSTRIAL WASTE WATER
METALS
FROM
Removal of toxic heavy metal ions such as Cu2+, Pb2+, Ni2+, Cd2+,
Zn2+, Mn2+, Co2+, Hg2+ and Fe3+ from industrial waste water is an
essential step from the standpoint of environmental pollution control.
This is to eliminate the direct toxic effect of such heavy metal ions on
human and animal health and aquatic life. Various ion exchange
materials, which are being researched to reduce the cost of existing
commercial counterparts and to tolerate a wide range of operating
conditions, have various forms (ion exchange resins and membranes,
affinity adsorbents and hydrogels) of grafted and crosslinked
structures based on synthetic or natural polymers containing functional
groups.
Grafting of hydrophilic monomers onto a variety of polymer
films offered various types of membranes having metal ion-binding
capacity (Fang et al. 1998; Hegazy et al. 1999, 2000, Gupta and
Anjum, 2001, Gupta et al 2004). To further enhance the metal
adsorption capacity of the membranes, grafting of binary monomer
mixtures was introduced. Cation/anionic graft copolymer membranes
prepared by radiation grafting of AAc and 4-VP onto low density PE
films showed a good potential for separation of Fe3+ and other heavy
metal ions such as Pb2+, Cd2+ and Cu2+ from contaminated water
resources (Hegazy et al. 1997). The stability of similar membranes
was improved by grafting of AAc and N-VP onto PTFE films. These
membranes were found to have potential for separation of Cu2+, Ni2+,
Pb2+, Cd2+, Zn2+, Mn2+, Co2+, Cr2+ and Fe3+ ions (Hegazy et al. 2000;
2001).
Double (strongly and weakly) acidic graft copolymer
membranes prepared by radiation grafting of styrene/AAc onto low
106
density PE films followed by sulfonation and alkaline treatment have

showed good affinity for adsorption of Pb2+ and Fe3+ ions (Hegazy et
al. 1999).
The binary grafting mixture of styrene/AAc was also used to
modify PVA films converting them to hydrogel membranes that
showed high selectivity towards Ni2+ and Co2+ ions as reported by Abd
El-Rehim et al (1999). The same authors investigated another
membrane by grafting styrene and maleic anhydride (MAn) monomers
into PE films followed by treatments that included conversion of the
membranes to carboxylic acid and amino groups by alkaline
hydrolysis and treatment with different alkylamines (Abd El-Rahim et
al. 2000). It was found that the grafted films treated with
thiosemicarbazide prefer Cu2+, the hydroxylamine (HA) hydrochloride
treatment prefers Cr3+ and NaOH treatment prefers Fe3+ in a mixture of
Cu2+, Cr3+ and Fe3+ at 70oC.
Chelating membranes prepared by radiation grafting of epoxygroups containing monomers such as GMA onto porous PE films
followed by conversion of expoxy groups to an iminodiacetate groups
have shown great potential for removal of Cu2+ from CuCl2 solution
(Saito et al. 2002).
Hollow fiber membranes prepared by radiation grafting of
GMA onto PE hollow fibres followed by amination reaction have
shown a great potential for the removal Pb2+ and Pd2+ ions (Choi and
Nho, 2000, Kim and Saito, 2000). Similar PE-g-poly(GMA) hollow
fibre membranes activated by iminodiacetation and sulfonation were
proposed for the removal of Co2+ and Cs+ (Choi and Nho, 1999 a,b),
Pd2+ ions (Li et al 1994) and germanium oxide (Ozawa et al. 2000)
from effluents produced by processing of spent catalysts and the
production of polyethyleneterephthalate (PET), respectively.
Strongly acidic cationic membranes prepared by radiation
grafting of styrene/DVB mixture onto FEP films followed by
sulfonation were early proposed for acid recovery (chromic acid) from
electroplating industry by separation of Cu2+, Ni2+ and Fe3+ ion from
the chromium plating solution (Kim and Saito, 2000). Weakly acidic
cation exchange membranes prepared by grafting of AAc onto PE,

PTFE, FEP and poly(tetrafluoroethylene-co-perfluorovinyl ether)
(PFA) films have showed remarkable capability to separate Cu2+, Ni2+,
Mo2+, Zn2+, Mn2+, Co2+, Cr2+ and Fe 3+ ions from low and
intermediate level waste water (Hegazy et al. 1999).
Removal of Hg2+ ion from solutions by radiation grafted PE-gpoly(AAm) membranes was reported by Gupta et al (2004). These
membranes have achieved 99 % separation when used to treat a
solution containing 200 ppm of Hg2+ ions.
Sugi et al (1981) prepared heavy metal removing resins by
immobilization of phenylalanine (Phe) into spherical macroreticular
styrene/DVB beads. The obtained resins were found to be capable of
separating Cu2+ from Co2+ and Ni2+ ions. These resins were also
capable of removing Cu2+ ions from seawater.
Photografted
membranes
composed
of
poly
(2hydroxyethylmeth- acrylate-co-methacrylamindophenylalanine) were
tested for Cu2+ ions adsorption (Denizli et al. a,b). The membranes
showed metal affinity in the order of Hg2+> Ni2+> Cu2+, with metal
adsorption increasing with increased pH, levelling off at pH 5. Grafted
phenylalanine (Phe)/PE hollow fiber membranes prepared by
radiation-induced grafting were also investigated for removal of Cu2+
ions (Kiyohara et al. 1996).
Kumar et al (2006) studied the removal of Co2+, Ni2+, Mn2+,
and Cd2+ ions from aqueous solution using grafted adsorbent obtained
by radiation grafting of acrylonitrile (AN) onto a non-woven thermally
bonded PP followed by amidoximation of nitrile groups to a chelating
amidoxime (AMO) groups. The adsorption capacities of the adsorbent
for the metal ions were found to follow the order
Cd2+>Co2+>Ni2+>Mn2+ ions. The kinetics of adsorption of these ions
indicated that the rate of adsorption of Cd2+ ions was faster than that of
other ions studied.
Trochimczuk and Streat (1999) prepared effective chelating
resins composed of copolymer of AN/EA/DVB for removal of Cd2+
ions from a solution in the presence of Ni2+ ions. The initial polymer is
modified by means of 7.5% w/w ethylenediamine thiophosphonate.
The chelating resins showed high selectivity for Cd2+ ions in the
108
presence of Ni2+ ions in aqueous solutions, independently of the

applied pH range (3.55.6).
Grafted and/or crosslinked natural polymers such as chitosan
and starch offer very promising materials for removal of toxic metal
ions from waste water. Particularly, chitosan has been subjected to
tremendous number of studies because of its excellent adsorption
capacity for heavy metal removal. For example, the saturation
adsorption capacity of Cd2+ ions on chitosan crosslinked with GA
decreased exponentially from 250 to 100 mg.g-1 as the extent of
crosslinking increased from 0 to 1.3 mol GA mol-1 of amine (Hsien
and Rorrer 1997). Similar study conducted by Rorrer et al (1993) with
saturation adsorption capacity of 518 mg.g-1 for Cd2+ ions on GAchitosan 1 mm diameter beads.
Beside GA, other crosslinking agents including EPI (Baba et al
1994), ethylene glycol diglycidyl ether (EGDGE), iminodiacetic acid
(Shim and Ryu 1998), nitriloacetic acid and organic diisocyanates
(Tikhonov et al 1996) have been used to crosslink chitosan adsorbents
to improve their stability and swelling. For example chitosan
crosslinked with EGDGE, diethylene glycol diglycidyl ether
(DEGDGE) and polyethylene glycol diglycidyl (PEGDGE) showed
interesting selectivity for Cu2+ over Ni2+ and Co2+ ions (Qu and Lui,
1996 a,b,c).
A polyaminated highly porous resins prepared from porous
beads of chitosan by crosslinking with EGDGE and then reacting the
crosslinked beads with EPI and polyethyleneimine (PEI), respectively.
The order of selectivity of adsorption of metal ions onto the resins at
pH 7 was Hg2+>UO2+>Cd2+>Zn2+>Cu2+ >Ni2+. Interestingly, Ca2+ Ga3+
As3+, Sr2+ were not adsorbed on the resins at all (Kawamura et al 1993,
1997).
GA or EPI-crosslinked chitosan membranes were tested for
removal of Hg2+ ions from aqueous solutions in a batch reaction under
various conditions. The maximum adsorbed amounts, at pH 6.0, were
30.3 and 75.5 mg.g-1, respectively, to EPI-crosslinked and GAcrosslinked chitosan membranes. The GA-crosslinked chitosan
showed lower % of recovery, but they were chemically stable at lower

values of pH, allowing their use in acidic solution and continuous
stages of adsorption and desorption (Vieira and Beppu 2006). Similar
crosslinked chitosan beads were used for the removal of Fe2+ and Fe3+
ions from aqueous solutions. The capacity of Fe3+ adsorption using
chitosan and crosslinked chitosan beads was greater than Fe2+ ions
adsorption (Wan Ngah et al 2005).
Chitosan grafted with poly(AN) has been further modified by the
reaction of cyano group of chitosan bead-g-poly(AN) copolymer with
HA, after crosslinking of chitosan with GA and AN grafting to yield
amidoximated crosslinked chitosan (Kang et al 1996), a derivative has
higher adsorption for Cu2+, Mn2+ and Pb2+ compared to crosslinked
chitosan. The adsorption capacity had a linear dependence on pH in
cases of Cu2+ and Pb2+ ions. However, a slight decrease in the
adsorption capacity was observed in case of Zn2+ and Cd2+ ions (Kang
et al 1999).
Chitosan-g-PVP resins synthesized by homogenous grafting of
4-VP onto chitosan were effectively used bind to Cu2+ ions and then
became soluble in dilute HCl (Yazdani and Retuert 1997). Amphoteric
flocculent, which can function as polycationic flocculent in acidic
media and polyanionic flocculent in basic media, with high adsorption
properties were synthesized by grafting alkanedioic, alkenedioic as
well as alkenoic acids onto chitosan (Kim et al. 1998). Good
adsorbents for Hg2+ and Cd2+ ions were also obtained by crosslinking
of chitosan with diisocyanates and nitriloacetic acid. More details on
prospective of crosslinked chitosan adsrbents can be found in the
review by Varma et al (2004).
Recently, other polysaccharide derivatives such as starch,
cyclodextrins (CD), cellulose and biomass have been considered for
developing biosorbent for heavy metal removal. Crosslinked and
carboxymethylated starch (CMS) has been proposed as sorbents for
the removal of divalent heavy metal ions (Cu2+, Pb2+, Cd2+ and Hg2+).
The removal efficiency of the metal ions depends on the
carboxymethyl groups and the starch content in the sorbent.
Chan et al (2001) studied the adsorption dynamics of Cu2+ ions
onto insoluble amphoteric starch containing quaternary ammonium
110
and phosphate groups. The Cu2+ ions sorption process was found to
occur in two stages: external mass transport occurs in the early stage
and intra-particle diffusion occurs in the long-term stage.
Zhang and Chen investigated crosslinked poly(dimethylaminoethyl methacrylate) starch grafted copolymers containing tertiary
amine groups, as adsorbents for Pb2+ and Cu2+ ions (Zhang and Chen,
2002). The crosslinked graft copolymer starch with 60% degree of
grafting achieved an equilibrium adsorption capacity of 2.09 and 2.12
mmol g1 (dry weight), for for Pb2+ and Cu2+ ions, respectively.
Guo et al (2006) investigated the adsorption of Cu2+ ions on
crosslinked starch phosphate carbamates adsorbent prepared from an
aqueous solution. The adsorbent achieved a maximum adsorption
capacity of 1.60 m mol.g-1 for Cu2+ ions with a regeneration capacity
of above 96% when treated with 1 N HCl solution for 1 h.
The use of biomass has been also reported for developing
biosorbent chelating materials for heavy metals removal. The biomass
of Penicillium chrysogenum was modified by grafting of AAc or
polyethylenimine (PEI) on the surface of ozonepretreated biomass
(Deng and Ting 2005 a,b). However, the preparation process was very
complex and is not cost effective.
Yu et al (2007 a) reported a simple process to prepare a
biosorbent with high sorption capacity by grafting of poly(amic acid),
which was obtained through reaction of pyromellitic dianhydride
(PMDA) and thiourea, onto the biomass (bakers yeast) surface at 50
C for 4 h. The adsorption capacity of the modified biomass increased
15- and 11-folds for Cd2+ and Pb2+ ions respectively compared with
the pristine biomass. The regenerated biomass can be used for at least
4 four times with little loss of uptake capacity. The same authors
extended their workby modification of the surface of dried biomass of
baker's yeast by crosslinking the cystine with GA. The adsorption
capacity of the modified biomass for Cd2+ and Pb2+ ions showed an
increasing trend compared with the pristine biomass due to the
presence of cystine on the biomass surface. The adsorption capacities
for Cd2+ and Pb2+ were 11.63 and 45.87 mg g1, respectively (Yu et al.,
2007 b).

The use of metal binding proteins (amino acids) such as glycine
(Gly) immobilized onto a solid support (crosslinked resins) as a metal
chelating agents have been investigated. The metal binding
capabilities of poly(AAm) crosslinked with N,N-methylene-bisacrylamide (NNMBA) immobilized with Gly was reported towards
heavy metals: Co2+, Cu2+, Fe2+, Fe2+, Ni2+ and Zn2+ ions. The metal
binding capacity increased with the increase in crosslinking up to 8%
beyond, which it declined (George et al.1999, George et al. 2000).
In another study, George and Mathew (2001) studied the metal
binding ability of DVB-crosslinked poly(AAm) supported Gly toward
Co2+, Cu2+, Ni2+ and Zn2+ ions. Interestingly, as the degree of
crosslinking increased from 2-20%, the metal complexation decreased
due to a decrease in the available carboxylate ligands for metal
binding with an increase in DVB content. The comparison between the
metal-ion complexation characteristics between Gly functionalities
supported on DVB-crosslinked poly(AAm) and NNMBA-crosslinked
poly(AAm) toward Co2+, Cu2+, Ni2+ and Zn2+ ions showed that the
latter to be more effective at metal complexation than the former while
inclusion of DVB showed an increased metal selectivity over
NNMBA.
Hydrogels of synthetic or natural polymer origins have been also
used for heavy metal removal. In particular, chelating radiation grafted
poly(4-VP/AAc) copolymer hydrogel was investigated for the removal
of Fe3+, Cu2+ and Mn2+ ions from aqueous solution in a batch
equilibration technique under various conditions. The removal of the
metal ions followed the order: Fe3+> Cu2+> Mn2+ ions (El-Hag et al
2003).
Merrifield et al. (2004) investigated the uptake of Hg2+ ions by
thiol-grafted chitosan gel beads. The gel beads were prepared by
dissolving chitosan and converting it into spherical beads using a
phase inversion technique followed by crosslinking to improve their
porosity and chemical stability. Cysteine was grafted onto the beads in
order to improve the adsorption affinity of Hg2+ ions to the beads. The
adsorption capacity was approximately 8.0 mmol-Hg2+ g-1-dry beads
at pH 7, and decreased with decreasing pH.
112
Graft copolymer hydrogels prepared by radiation grafting

composed of PVA/citric acid (CA)/water and PVA/Succinic acid
(SA)/water were investigated for heavy metal removal. The
incorporation of SA and CA groups onto PVA networks improved
remarkably the affinity of these hydrogels for Co2+ and Ni2+ ion uptake
(Bodugoz et al. 1999).
Graft copolymer PVA/ethylenediaminetetraacetic acid (EDTA)
hydrogels prepared by radiation grafting were tested for removal of
Pb2+, Cd2+ and Hg2+ ions. The incorporation of EDTA moieties onto
PVA network results in a hydrogel, which has a strong absorption
affinity for Pb2+ and Cd2+ ions. The maximum chelating capacities of
the hydrogel were found to be 8.5 mg/g for Pb2+ ions and 4.2 mg/g for
Cd2+ ions. No affinity for absorption of Hg2+ by the hydrogel was
observed (Sanju and Varshney 2005).
Decontamination of synthetic Pb2+, Zn2+, Cd2+ and Ni2+ ion
solutions was investigated, using crosslinked silica gels chemically
modified with poly(ethyleneimine) (PEI). It was revealed that the
sorption capacities depend on the pH, the initial concentration and to
some extent on the nature of the metal. The recovery of the metal
cations by the saturated sorbents was possible with diluted acid (Ghoul
et al 2003).
Hydrogels from cellulose water-soluble derivatives such as
chloromethylated (CM) cellulose and from crosslink CM-chitin and
CM-chitosan, crosslinked by high energy radiation at high
concentrated paste-like condition have show improved metal
adsorption capacity and can be suitable for heavy metal removal (Fei
et al 2000, Zhao et al 2003, Seko et al 2005).

5.5.6.
TREATMENT
OF
RADIOACTIVE WASTE
WATER
CONTAINING
Ion exchange is an effective treatment method for liquid radioactive

waste in nearly all phases of the nuclear fuel cycle, including the early
stages of uranium ore treatments, the chemical control of primary
circuit coolants during nuclear power plant operations and polishing
water effluents at spent fuel reprocessing plants (Hooper et al 1999).
Organic and synthetic ion exchangers in membranes and resins forms
have found their specific fields of application in different purification
and liquid radioactive waste treatment processes.
PE-g-poly(AAc)membranes prepared by radiation grafting of
AAc on PE, were tested for the recovery of uranium from simulated
waste solution. The membranes were found to be highly selective
towards Zr, which is the most important contaminating element in
uranium recovery (Hegazy et al. 1999 b).
Sulfonated hollow fibres membranes prepared by radiation
grafting of styrene onto hollow PE fibres followed by chemical
treatment with chlorosulfonic acid have shown good separation
properties for Co2+ ions (Choi and Nho, 2000). Yamagishi et al. (1991)
earlier removed Co2+ ions from its solution using similar chelating
radiation grafted porous hollow fibre membranes.
Grafted poly(AAm) membranes prepared by radiation grafting
of AAm onto PVC showed a great tendency to separate Co-60 from
liquid radioactive waste containing a mixture of Co-60 and Cs-137
(Maziad and Hegazy, 2002).
Amidoxime containing fibrous hydrogels prepared by radiation
induced grafting of AN onto PP fibers and non-woven fabrics
followed by subsequent amidoximation by HA have been found very
promising for the recovery of uranium and vanadium from sea water
(Suzuki et al. 2000). The adsorption for uranium and vanadium was
found to, respectively be 576 and 1800 g.g-1 fiber, from sea water
having concentrations of 3.3 and 1.5 g.g-1 sea water, respectively.
Similar potential adsorbent materials were prepared by graft
114
copolymerization of AN/N-VPn mixture followed by amidoximation

reaction with HA. An improved adsorption capacity of 0.75 g UO22+g-1
dry amidoximated adsorbent from its aqueous solution (e.g. 1400
ppm) was obtained (Pekel et al. 2001).
Amidoximated
AN/N-vinylimidazole
(VIm)
copolymer
hydrogels prepared by -radiation followed by amidoximation of the
AN moieties of copolymers with HA hydrochloride in basic medium
were used for separating UO22+ from aqueous solutions by a
complexation process. The adsorption capacity was found as high as
0.64 g UO22+ g1 dry amidoximated copolymer indicating that
amidoximated poly(AN/VIm) hydrogels are very suitable chelating
system for the adsorption of UO22+ ions from aqueous system (Pekel et
al 2003).
Separation of UO22+ was also attempted by Choi and Nho (2000)
using chelating agents prepared by grafting of AN, AAc, and AN/AAc
mixture. The AN unit of the grafted film was transformed into
amidoxime groups. This membrane was found to be good material for
UO22+ separation. In another study, GMA and AN were grafted into
PE and were transformed into IDA and oximes, respectively (Choi and
Nho, 1999). These membranes were effective for complexation with
UO22+ and Cu2+ ions. The separation of UO22+ ions by oximes has also
been demonstrated by similar membranes (Sekiguchi et al 1994).
Earlier, chitosan derivatives with ketoacids were found to be
effective in removal of Co-60 from nuclear plant waste water and for
separation of uranium from dilute solutions as well as from saline
water (Muzzarelli 1986).
5.5.7. RECOVERY OF WASTE ACIDS FROM WASTE

WATER
Recovery of spent acids from waste acids produced by several
industries such as pickling and surface treatment, pigments and
hydrometallurgy has become of major environmental concern.

Conventional technology for spent acid recovery includes either
neutralization, which leads to large amounts of sludges, or thermal
techniques such as distillation and evaporation or pyrolysis, which are
economically viable only for large volumes of effluents. Among the
possible alternatives ED with IEMs is an attractive technique for
treating waste acids (Yazicigil, 2007).
The recovery of sulfuric acid from zinc hydrometallurgy
industry has proved to be viable in laboratory and pilot scales ED cells
when tested with three crosslinked commercial IEMs: Neosepta CMS
(Tokuyama Soda Co.), Morgane CRA (Solvay), Selemion CHV
(Asahi Glass Co) in comparison with Nafion membranes (Boucher et
al. 1997).
Recovery of sulfuric acid produced in the diamond
manufacturing process by diffusion dialysis (DD) method using a
commercial crosslinked anion exchange membrane was conducted by
Joeng et al (2005). About 80% of the acid could be recovered from
waste sulfuric acid, which contained 4.5 M free-acid at the flow rate of
0.26 103 m3.h-1m-3. The concentration of recovered sulfuric acid
was 4.3 M and the total impurity was 2000 ppm. Preliminary
evaluation has revealed that dialysis system has high economical
perspective for such application.
Sulfonated and crosslinked polystyrene grafted ETFE
membranes prepared by radiation grafting showed an excellent
performance in acid recovery (Elmidaoui et al. 1992 a). Poly(4-VP)
grafted ETFE membranes also showed a comparable performance to
commercial IEMs (De Morgan) in acid recovery by dialysis
(Elmidaoui et al. 1992 b).
Also, membranes obtained by radiation grafting of vinyl
monomer such as MAc, AAm and N-VPn, 2-HEMMA onto various
films (PE, PP and ETFE) were found to be promising for acid
recovery by dialysis (Yuee and Tianyi, 1988, Fang et al. 1998).
116
5.7 RECOVERY OF PRECIOUS METALS FROM WASTE

WATER
Due to increasing demand, precious metals including silver (Ag), gold

(Au), palladium (Pd) and platinum (Pt) are recovered from a wide
variety of mining and indudtrial waste streams (Brooks 1991).
Biosorption with natural polymers such as modified chitosan offers
alternative promising technology to conventional ones (solvent
extraction and chemical precipitation) for the recovery of precious
metals (Guibal et al 2004). Details of the disadvantages and principels
of conventional method for recovery of precious metals were reviewed
elsewhere (Gksungur et al 2005).
Chitosan modified by GA crosslinking is an efficient sorbent for
Pt recovery from dilute acidic solutions. The sorption capacity reaches
levels as high as 300 mg Ptg-1 (~1.5 mmol Ptg-1) at pH range of 2.02.5. Uptake values of 100 mg Ptg-1 and higher were obtained at
concentrations as low as 0.2 mg Pt L-1 (Guibal et al 1999).
Guibal et al (2002) improved the sorption capacity of chitosan
based sorbent by grafting of thiourea and rubeanic acid
(dithiooxamide) for extraction of Pd from dilute solutions. The added
chelating sulfur functions were found to give the sorbent a dual anion
exchange/chelating structure enhancing the metal sorption capacity,
which makes these chitosan derivatives highly efficient in extraction
of Pd from dilute acidic solutions.
Crosslinked chitosan derivatives prepared by crosslinking with
GA or hexamethylene diisocyanate (HMDIC) followed by grafting of
sulfur compounds showed high efficiency for removal of Au from
dilute acidic solutions: Maximum uptake capacity reaches 600 mg Au
g1 (~3 mmol g1). The grafting of sulfur compounds and HMDIC
crosslinking was found to enhance the sorption kinetics of Au
(Arrascue et al 2003).

5.6 CONCLUDING REMARKS AND FUTURE DIRECTIONS
Ion exchange technology has played an important role in production of

water and treatment of waste water from various industries for the past
5 decades. The land mark change in using mixed bed ion exchange
resin columns has made the first large scale production of ion free
water became possible. This led to pure and ultrapure waters
production. Today, the application on ion exchanges technology
covers a variety of water and waste water treatment applications such
as water softening, water demineralization, removal of heavy metals,
removal of nitrate, removal of arsenic, removal of dyes, dealkalization,
and purification of water solutions and recovery of precious metals.
Ion exchange technology will continue to lead the way in water
and waste water treatment with the development of new materials and
processes capable of meeting the demands for water quality and
industrial waste water disposal standards efficiently and cost
effectively. A large number of chemically, radiochemically,
photochemically or plasma modified grafted and crosslinked ion
exchange materials of synthetic and natural origins have been
developed in various forms (resins, fibers, membrane and nonwoven
fabrics) and functionalities for specific water treatment applications.
Ion exchange materials based on natural polymers or derivatives are
promising and economically attractive. More efforts are needed to
bring many of newly developed ion exchange polymers from the
experimental level to the pilot scale production and also extend their
practical applications. This can be achieved by addressing several
aspects including boosting the selectivity without compromising flux
and stability, optimizing polymer modification techniques,
understanding the mechanism of transport, establishing structureproperty relationships for polymer forms, using the proper engineering
configuration, testing separation with actual samples under various
conditions and using cost effective materials.
118
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6
POTENTIAL OF BIO-PRODUCTS
ADSORPTION BY IMMOBILIZED METAL
ION AFFINITY
MESOPOROUS ADSORBENTS
Azizul Azri Mustaffa
6.1
IMPORTANCE OF BIO-PRODUCTS AND

BIOSEPARATION
A separation process which involves the extraction of bio-products

such as protein and antibiotics are called bio-separation processes.
Bio-products are chemical substances or a combination of chemical
substances that are made by living things and nature. They can be
derived or extracted from whole plants and animal or by synthesis in
bioreactors containing cells and enzymes. These bio-products are
valuable in terms of their chemical activity such as methanol for its
solvent activity, penicillin for its antibacterial activity, taxol for its
anti-cancer activity and many more to note. This wide variety of bioproducts with different nature produces a wide range of bio-separation
technologies and thus factors such as the nature of the products, purity,
yield and activity will determine which bio-separations technology are
the most suitable to apply. The biotechnology industry which started
in the late 1970s increases the importance of bio-products as many
products of biotechnology are proteins and antibiotics that are usually
hard to purify leading to high cost (Harrison et al., 2003). In addition,
of all the bio-separation technologies available today, some have their
limitations including poor product yield, inadequate separation
selectivity, high cost and many more. Therefore efforts to overcome
126
these limitations must be made by attempting to modify and improve

the existing bio-separation processes. Throughout this topic, the
potential of adsorption based bio-separation technology will be
discussed to extract protein and antibiotic by using mesoporous
adsorbents, MCM-41 and SBA-15 incorporated with intermediate
metal ions.
6.2
EXTRACTION OF PROTEIN AND ANTIBIOTICS
Bio-products such as protein and antibiotics have unique properties

including thermal stability, solubility, diffusivities, charge, isoelectric
pH, reaction rate constants and separation thermodynamics. Proteins
for example, are separated on the basis of differences in their
psychochemical properties, such as charge, adsorption characteristics,
solubility and heat stability. Their different sizes such as globular or
spherical, long or narrow also form a basis for separation (Scott,
1991).
6.3
ADSORPTION PROCESS TECHNOLOGY
Many processes take place at the boundary between two phases, while
others are initiated at the interface. The change in concentration of a
given substance as compared with the neighbouring phase is referred
to as adsorption. The systems can be categorized to the types of phases
in contact such as liquid-gas, solid-liquid and solid-gas (Dabrowski,
2001). Adsorption is a process in which a molecule is transferred from
solution to the surface of a solid where the molecule is bound by
physical and chemical forces. The solid is what we called as
Potential of Bio-Products Adsorption by Immobilized Metal

Ion Affinity Mesoporous Adsorbents
127
adsorbents and they are characterized by high surface area to provide
sufficient active sites to allow a high concentration of solute to adsorb.
Their porosity is also tailored to adequately provide access of surface
sites to diffusing molecules. The adsorbents may also be
functionalized in a number of ways: (1) attachment of ionogenic
functions renders an ion exchanger, which can differentiate between
proteins of different isoelectric points; (2) attachment of generic
affinity ligands can render it selectively sorptive for certain classes of
enzyme; (3) attachment of cell surface immunoglobin or (4)
attachment of specific monoclonal antibodies can render it uniquely
sorptive for one antigenic protein.
6.3.1 FUNDAMENTAL ASPECTS

ADSORPTION PROCESS
OF
BIO-PRODUCTS
There are several fundamental aspects of adsorption of bio-products

such as protein and antibiotic which are very important. These
fundamental aspects are very essential as they influenced the
adsorption behaviour of a certain types of bioproducts and should not
be underestimated. Generally, the adsorption of protein for instance is
influenced by the characteristics of the adsorbate, adsorbent and
solution.
128
6.3.2 CHARACTERISTICS OF THE ADSORBATE
Adsorbate is the bio-products to be adsorbed by the adsorbent. One

characteristic of the adsorbate that must be known is the chemical
properties of the adsorbate to determine the size and configuration of
the particular molecule to be adsorbed. Molecular size is important
because for any homologous series of organic molecules, as size
increases solubility generally decreases. A material which has low
solubility in water, will have a higher affinity for solid surfaces than
for water, and will therefore have a tendency to concentrate on those
surfaces or to adsorb. It is also important for the perspective that all
adsorbents depend upon internal surface area for the full use of the
adsorption capabilities. If the molecular size is too large, adsorption
will be hindered and adsorption capacity will decrease as very large
molecules block or cannot penetrate pores or pathways within the
adsorbent. Larger molecules will tend to diffuse more slowly from
solution and therefore require longer times for full equilibrium
adsorption capacity to be realized.
The adsorption capacities are also affected by the molecular
properties of the adsorbate and the removal of adsorbate from solution
by adsorption is effected by its ionic or neutral state, or whether it is a
branched isomer or isomer.
6.3.3 CHARACTERISTICS OF THE ADSORBENT
The chemical and physical properties of the adsorbent used to remove

a material from solution are quite important. The presence of ionized
or otherwise active functional groups on the adsorbent surface allow
chemical interactions of chemisorptions which usually produces

129
effects different from and less reversible than physical adsorption.
This effect may be advantageous or not, depending on a particular
application. The general ability of the chemical nature of the adsorbent
to change the chemical characteristics of the solution to be treated can
also have either beneficial or adverse effects on the adsorption
process.
The physical properties of the adsorbents are like-wise
important. The adsorbent can be in the form of granules or particles
which may have density near or very different from the solution to be
treated; or the adsorbent may be in very fine powdered form which
may be easily suspended in the solution to be treated. Some physical
properties such as the surface area, size and distribution of pores in the
particles directly affect adsorption performance by determining the
amount of adsorbent capacity available and the molecular sizes which
can be adsorbed.
6.3.4 CHARACTERISTICS OF THE SOLUTION
The three major solution characteristics which have particular impact

on the adsorption are: (1) the solution pH; (2) temperature; and (3) the
presence of competing adsorbate compounds. The pH of a solution is
of significance for its effect on the adsorbent, as well as on the
adsorbate. Both adsorbate and adsorbent may have chemical
characteristics which are affected by the concentration of hydrogen
ions (H+) in the solution. Some adsorbents have affinity for H+ or OHions and can directly affect the solution pH and therefore solubility
and adsorption capacity. Adsorption from solution can be highly pH
sensitive in aqueous system where an adsorbate exhibits an isoelectric
point or neutral point on the pH scale. It is at this point where
maximum adsorption can be achieved since solubility is minimized
130
and the non-polar adsorbent has greatest affinity for non-ionic

materials.
The temperature of a solution has two major effects on
adsorption. The rate of adsorption is usually increased at higher
temperatures. This is due to the increase rate of diffusion of adsorbate
molecules though the solution to the adsorbents. Further, since
solubility and adsorption are inversely related, as temperature affects
solubility it will therefore affect the extent of adsorption or capacity of
the adsorbent for the particular adsorbate.
A major influence of the solution character on adsorption is the
presence of competing adsorbate compounds. Few adsorbents
demonstrate controllable selectivity for specific compounds and
therefore all absorbable compounds present will compete for
adsorption sites. Further, since physical adsorption is a reversible
phenomenon, the presence of materials with a particularly high affinity
for the adsorbent will, under continued application, tend to displace
from the adsorbent materials of lesser affinity. The adsorption
technique utilizes van der Waals and hydrophobic forces, which bind
the sorbate to the surface (Ismail, 2005)
6.4
MESOPOROUS MATERIALS AND THEIR

POTENTIALS AS ADSORBENTS
6.4.1 TYPES OF MESOPOROUS MATERIALS
Ordered mesoporous silicates have attracted a great interest in the past

decade because of their use in catalysts, separations, sensors, drug
delivery and optical devices. In 1992, the M41S family of materials
ended the pore size constraints of zeolite where materials with pore
size more than 20 Anstroms and a sharp molecular sieve like pore size

131
distribution could be synthesized. In 1998, the family of highly
ordered mesoporous silicates have been extended by the synthesis of
Santa Barbara Amorphous (SBA) type materials (Zhao et al., 1998)
They have pore size ranging between 20 to 300 Anstroms and use non
ionic block copolymers as structure directing agents in highly acidic
media.
The initial members of the M41S family are MCM-41
(hexagonal phases), MCM-48 (cubic phase) and MCM-50 (a stabilized
lamellar phase). The most prominent and most extensively
investigated member of the M41S family is MCM-41. MCM-41 is a
hexagonal material containing a regular array of mesopores with
uniform diameter. By proper synthesis the pores can be varied from 2
to 10 nm (Weitkamp, 2000). However, M41S materials are limited to a
pore diameter of approximately 80 Anstroms and furhermore they
have significant external surface areas.
Meanwhile, one type of the Santa Barbara Amorphous (SBA)
is SBA-15 which was synthesized in 1998 using tri-block copolymer
poly (ethyleneoxide) poly (propylene oxide) poly (ethylene oxide),
which is commercially available as pluronics P123 (EO20PO70EO20)
and characterized by thicker silica walls. SBA-15 possess large BET
surface area (>700m2/g) with large pore diameter and large pore wall
thickness. The large wall thickness results in higher hydrothermal
stability than M41S materials. SBA-15 has proved to be very
promising for the size selective separation of large biomolecules,
because pore diameters are in the range required for these separations,
and the silica framework is well suited for the development of bonded,
selective sorption phases.
132
6.4.2 POTENTIAL MODIFICATION AND APPLICATION OF

MESOPOROUS MATERIALS
There has been efforts to incorporate catalytically active elements into

the mesoporous framework. The recent discovery of mesoporous
molecular sieves which have highly ordered pore systems with
tuneable pore sizes, large specific surface areas and pore volumes, and
high density of surface silanols provide excellent opportunities in
inclusion chemistry. A large number of functionalizing entities
including both organic and inorganic ligands have been introduced in
the channels to generate catalysts, adsorbents, and to improve
hydrothermal and mechanical stabilities.
All these modifications were achieved either by postmodification or by in-situ synthesis. Post-modification allows one to
design and synthesize custom-tailored materials. For example, postsilylation can improve the hydrothermal stability and mechanical
stability of MCM-41 materials due to enhanced surface
hydrophobicity, whereas thiol-functionalized MCM-41 adsorbents
show a high adsorption efficiency of heavy metal ions from water.
Although the detailed chemical reaction mechanism of postmodification has not been fully understood, it is believed that surface
silanols serve as reactive sites and can be replaced by other ligands or
modifiers. In such a way, a monolayer of a given modifier is attached
to the surfaces of MCM-41, resulting in the changes of not only
structural properties such as pore size, pore volume, and surface area,
but also surface properties such as hydrophobicity or hydrophilicity,
polarity, acidity and affinity. Many studies have been devoted to
structural characterization of organic-modified MCM-41 materials
using adsorption techniques.

133
6.4.3 SBA-15 AND MCM-41 AS ADSORBENTS
Mesoporous molecular sieves such as MCM-41 and SBA-15 have

many desirable properties for applications as adsorbents. Their high
surface area (~ 1000 m2/g) and tuneable uniform pore size of 1.8 40
nm make them ideal for size exclusion separations of proteins and
other biological molecules of importance in the biochemical and
pharmaceutical industries (Kisler et al., 2001). Although microporous
zeolite and zeotype materials with uniform and molecular size pores
have been widely used as industrial adsorbents, they can not adsorb
large biomolecules such as proteins, enzymes or vitamins, due to the
limitation of micropore size.
6.5
IMMOBILIZED METAL ION AFFINITY

CHROMATOGRAPHY (IMAC)
Immobilized-Metal Affinity Chromatography (IMAC) is a separation

technique where metal ion are incorporated on solid chromatographic
support by covalently bound compounds. It can be act as affinity
ligands for various proteins, making use of coordinative binding of
some amino acid residues exposed on the surface. IMAC have many
advantages over affinity chromatographic techniques, which have a
similar order of affinity constants and exploit affinities between
enzymes and their cofactors or inhibitors, receptors and their ligands
or between antigens and antibodies. The benefits of IMAC are ligand
stability, high protein loading, mild elution conditions, simple
regeneration and low cost which are decisive when developing largescale purification procedures for industrial applications (GabercPorekar and Menart, 2001).
134
6.5.1 METAL ION AFFINITIES AND THEIR ROLE IN

BIO-PRODUCT ADSORPTION
IMAC is a separation principle that utilizes the differential affinity of

proteins for immobilized metal ions to effect their separation. This
differential affinity derives from the coordination bonds formed
between metal ions and certain amino acid side chains exposed on the
surface of the protein molecules. Since the interaction between the
immobilized metal ions and the side chains of amino acids has a
readily reversible character, it can be utilized for adsorption and then
be disrupted using mild conditions.
Metal ions can be divided into three categories (hard,
intermediate and soft) based on their preferential reactivity towards
nucleophiles. To the group of hard metal ions belong Fe3+, Ca2+ and
Al3+, which show a preference for oxygen. Soft metal ions such as
Cu+, Hg2+, Ag+ prefer sulfur. Intermediate metal ions (Cu2+, Ni2+, Zn2+,
Co2+) coordinate nitrogen, oxygen and sulfur.
For the separation of bio-products, one important method is by
chromatography and the developments of mesoporous materials as
size-selective chromatographic supports which holds a potential for
more efficient separations. Chromatographic separations depend on
the differential partitioning of proteins or antibiotics between a
stationary phase (adsorbent) and mobile phase (buffer). The proper
selection of the stationary phase and mobile phase both will influence
the separation.

135
6.6
RECENT RESEARCH DEVELOPMENT
Recently (2006), a study have been conducted investigating the

adsorption characteristics of bovine serum albumin (BSA), a protein
and rifampicin, an antibiotics on immobilized metal ion affinity
mesoporous adsorbents. Intermediate metal ions Cu2+, Co2+ and Ni2+
have been immobilized into the MCM-41 and SBA-15 structure. The
effects of pH, adsorbents, metal ion concentration the adsorption
isotherms have been investigated.
It has been found that the amount of BSA and rifampicin
adsorbed on different adsorbents was significantly changed by
adjusting the solution pH and increased by the introduction of
intermediate metal ion (Cu2+, Co2+, Ni2+) into the pure silica materials.
For BSA adsorption, CuSBA-15 at pH 4 adsorbed the most BSA while
CoSBA-15 at pH 7 showed good adsorption capacity for rifampicin.
Modified SBA-15 and MCM-41 with different nickel ion
concentration also can affect and change the adsorption capacity of
BSA and rifampicin. The x-ray diffraction results and the Fourier
Transform Infrared (FTIR) analysis confirms the structure of the
modified MCM-41 and SBA-15 and the adsorption of BSA and
rifampicin into the adsorbents. The Langmuir isotherm model was
found to fit well with the BSA adsorption isotherm while it is revealed
that the adsorption of rifampicin fits well the Freundlich adsorption
isotherm model instead.
136
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Proteins on Mesoporous Molecular Sieve, Mater. Phys. Mech.,
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mesoporous silica structures, J. Am. Chem. Soc., 120: 60246036.
7
REVERSE MICELLE EXTRACTION
7.1
INTRODUCTION
Reverse micelle is an alternative method to conventional separation

and purification procedures due to their potential for large scale use
(Ono et al., 1996; Naoe et al., 2004; Shen and Yu, 2007; Moore and
Palepu, 2007), their potential application to continuous separation of
biological substances (Zhang et al., 1999; Hong and Kuboi, 1999;
Naoe et al., 2002; Noh and Imm, 2005; Juang et al., 2006; Majumdar
and Mahapatra, 2007), the process similarities to liquid-liquid
extraction (Naoe et al., 2004; Hasmann et al., 2007), and easy scale-up
(Chang et al., 1997; Rodrigues et al., 1999; Kilikian et al., 2000;
Mathew and Juang, 2007). Nishiki et al. (1998) showed that reverse
micelle extraction is able to recover proteins with little denaturation
because proteins can be hosted in a solubilised aqueous phase which is
shielded from the organic solvent by surfactant molecules;
solubilisation inside reverse micelle does not damage the native
protein structure (Yu et al., 2003; Hetch and Peled, 2006). Therefore,
in an ordinary liquid-liquid extraction, although it has been difficult to
extract large bioactive molecules such as proteins, the limitation can
be overcome by utilising a nanostructural molecular assembly like
reverse micelle (Aires-Barros and Cabral, 1991; Ono et al., 1996).
138
7.2
FUNDAMENTAL ASPECT OF REVERSE MICELLE
Reverse micelle are nanometre aggregates of surfactant molecules in a

polar solvents (Figure 7.1). They can be regarded as very simple
bioaggregates (Lee et al., 2001; Freda et al., 2002; Havre, 2002;
Debnath et al., 2007), dispersed in a continuous organic solvent
medium with the help of a surfactant (Castagnola and Dutta, 2000;
Kinugasa et al., 2003; Paul and Mitra, 2006).
Water-pool
Surfactant
Hydrophilic Biomolecule
Figure 7.1 Schematic representation of a reverse micelle.
Surface active reagents, better known as surfactants, are molecules

made up of hydrophobic and hydrophilic structures that are able to
modify the interfacial properties of the liquid (non-aqueous or
Reverse Micelle Extraction
139
aqueous) in which they are present (Yang, 2001). The hydrophilic and
hydrophobic regions are illustrated in Figure 7.2.
Hydrophobic tail
Hydrophilic head
Figure 7.2 Structure of an AOT.
Working in reverse micelle phases presents many advantages (Vyve

and Renken, 1999). The reverse micelle can dissolve molecules which
are normally not soluble in the bulk solvent. Also, due to the very
small size of a micelle, there is an enormous increase of the interfacial
area between the continuous and the dispersed pseudo-phases in
comparison to a mechanically stirred system without surfactants.
Mathew and Juang (2007) summarised the main advantages of using
reverse micelle as the alternative to conventional extraction method as
follows:
1) Solublisation behaviour of biomolecules in reverse micelle can be
regulated by varying the size and shape of the reverse micelle.
2) Selective partitioning of biomolecules can be achieved in the
extraction based on the hydrophobic nature of proteins due to the
fact that reverse micelle provide the hydrophobic and hydrophilic
environments to solutes simultaneously.
140
3) Recovery of biomolecules from the reverse micelle can be easily

facilitated by exploiting the de-assembling nature of reverse
micelle in the aqueous media. Moreover, the surfactants can be
separated from the system by filtration and recycled.
Reverse micelle extraction of biomolecule products is a relatively new
discovery. Kilikian et al. (2000) suggested that a growing number of
studies on reverse micelle techniques clearly demonstrate the scientific
interest in reversed micelles in the separation of biotechnology
products. A reverse micelle is a droplet containing an inner core of
water in an aqueous solution where size may vary in the order of 50 to
100 (Wang et al., 1995). Reverse micelle is based on a water phase
and a hydrocarbon phase, and is a thermodynamically stable mixture
of water, oil and surfactants where the water regions are separated
from oil by a monolayer of surfactants (Hong and Kuboi, 1999; Goto
et al., 1998; Pandit and Basu, 2002; Bong et al., 2004). Baier (1999)
found that reverse micelle is a group of individual surfactant
molecules arranged with the hydrophilic ends concentrated together
and surrounded by the hydrophobic ends. The aqueous phase is
stabilized in a polar environment with the presence of surfactant at the
interface. In this way small hydrophilic regions are created within the
organic phase. When a reverse micelle hydrocarbon phase is contacted
with an aqueous phase containing a bio-product, the bio-product may
be captured into the micelle.
To date, the reverse micelle system has been used for the
separation of proteins (Goklen and Hatton, 1987; Aires-Barros and
Cabral, 1991; Brandani et al., 1996; Pires et al., 1996; Su and Lee,
1999; Kinugasa et al., 2003; Bong et al., 2004; Jun et al., 2004; Noh
and Imm, 2005; Hecth and Peled, 2006; Shen and Yu, 2007), peptides
(Kishi and Furusaki, 1992), amino acids (Leodidis and Hatton, 1989;
Furusaki and Kishi, 1992; Adachi et al., 2000; Dvyap et al., 2006),
bovine serum albumin (BSA) (Zhang et al., 1999), enzymes (MorenoHagelsieb et al., 1999; Liu et al., 2004; Bong et al., 2004; Debnath et
al., 2007) and metals (Nakashio et al., 1992; Ismael and Tondre, 1992;
Zhang et al., 2006; Majumdar and Mahapatra, 2007) as well as in a
141
bioreactor to carry out biotransformation (Kamiya et al., 1995). Thus,

reverse micelle has been demonstrated to be capable of hosting large
quantities of biomolecules without causing denaturation by adjusting
operational parameters (Hatton, 1985; Paradkar and Dordick, 1994;
Goto et al., 1998; Cardoso et al., 1999; Shen and Yu, 2007).
7.3
FORMATION OF REVERSE MICELLE
Figure 7.3 shows the comparison between normal micelle and reverse
micelle. Water plays a decisive role in the formation of reverse
micelle. The surfactant molecules aggregate at interfaces forming a
middle phase in equilibrium with an excess organic phase and an
excess aqueous phase. The transition from micelle to reverse-micelle
system, as described by Esalah (1997), is generally driven by a
decrease of the surfactant hydrophilicity. Increasing the salinity of the
aqueous constituent by adding a medium-chained alcohol (cosurfactant) decreases the surfactant hydrophilicity.
The structures of biomacromolecules are very sensitive to the
organic solvent involved (Chang et al., 2000). Using reverse micelle
systems can therefore create a suitable environment for solubilising
the biomolecule solutes into the organic solvent region. Reverse
micelle can be formed in an organic phase by a contact method or a
titration method (Rabie et al., 1997). These two methods have been
found to be significantly different with respect to their solubilisation
capacities for water and different solutes such as salts, amino acids,
proteins, antibiotics, etc. Rabie (1997) found that the titration method
had been used widely to form reverse micelle as injection fluids for a
number of tertiary oil recovery processes and also as microreactors in
which guest molecules could be brought to react, often showing
reactivity that differed markedly from that observed in pure solvents.
The titration method on the other hand is a process where an aqueous
142
solution is titrated into an organic solvent containing a surfactant and a

co-surfactant. The maximum mass of aqueous solution that can be
solubilised is determined using the appearance of permanent turbidity.
In this method, there is no excess aqueous phase in equilibrium with
the organic phase containing the reverse micelle. Since there is no
excess aqueous phase present, the composition of the water pool is
identical to the composition of the titrant. The use of an electrolyte is
not required in the titration method but, if present in the titrant, its
concentration affects the maximum water solubilisation.
(a)
(b)
Organic
Phase
Aqueous
Phase
Figure 7.3 Aggregation of surfactants; (a) micelle, and (b) reverse micelle.
The contact method is a process where a bulk excess aqueous phase is

contacted with an organic solvent containing the surfactant and cosurfactant and identical to the basic step used in the extraction of
solutes from water into a reverse micelle organic phase. The excess
143
aqueous phase must contain an electrolyte to prevent the transfer of

the surfactant from the organic phase to the aqueous phase. After
mixing and settling, the organic phase equilibrates with an excess
aqueous phase. The amount of water solubilised is obtained by
measuring the water content of the organic phase, usually by a Karl
Fischer titration. The composition of the water pool is determined by
the exchange equilibrium of ions and solutes between the phases.
7.4
SURFACTANTS
Surfactants and soaps comprise a large category of amphiphilic,

surface active compounds used in industrial applications, household
cleansers and scientific research. Surfactant applications are present in
almost every chemical industry and can be found in every cleaning or
conditioning product and in the oil industry (Yang, 2001). Crude
production of these materials is easily accomplished by mixing animal
fats with wood ash, followed by saponification to create free fatty
acids. Toerne (2002) also mentioned that the production of surfactants
is thought to have been taking place for more than 2500 years, and that
the industrial synthesis of soaps in recent times has exceeded $10
billion per year in the United States alone.
Moreover, the
investigation and utilization of organized assemblies of surfactant
molecules is one of the most active areas of colloid chemistry and it is
well known that among the various kinds of assemblies, reverse
micelle have drawn considerable attention in recent years. The
surfactant plays an important role in forming a spherical shell
surrounding the micelle (Kilikian et al., 2000).
By way of example, Pandit and Basu (2002) studied the
comparison of dye removal from the aqueous phase with and without
using surfactant. They found that the dye removal efficiency without
surfactants was very low compared to the percentage removal when
144
using surfactant. In the absence of surfactant, the removal of methyl

orange from water to amyl alcohol follows a conventional solvent
extraction process and yields only 7% extraction of methyl orange.
The percentage removal of methyl orange increases drastically up to
80% in the presence of the HTAB surfactants due to encapsulation of
the dye in the reverse micelle formed by the alcohol.
7.5
SURFACTANT STRUCTURE
Most surfactants have a long hydrocarbon tail that can be linear or

branched and interact only very weakly with the water molecules in an
aqueous environment; hence the chain is called a hydrophobic tail.
The hydrophilic head is a relatively small ionic or polar group that
interacts strongly with the water via dipole-dipole or ion-dipole
interactions. As a surface-active reagent, the surfactant molecule is
capable of changing the interfacial free energy, which is the internal
energy of an interface. The molecules tend to aggregate at the
interface between an aqueous phase and another phase of the system
where they then reduce the surface or interfacial tension (Esalah,
1996).
The intermolecular forces that keep the aggregates together
arise from weak van der Waals forces, hydrophobic interactions,
screened electrostatic interactions and hydrogen bonding (Chang et al.,
2000). At relatively high concentrations in water, aqueous soluble
surfactants self associate to form micelles which are aggregates
characterized by a non-polar core formed by hydrocarbon groups and
hydrophilic groups facing outwards towards the water. When this
aqueous solution is contacted with an organic phase, the surfactant
molecules may migrate to the organic phase and aggregate, thus
forming reverse micelle.
145
7.5.1 CATEGORIES OF SURFACTANT
Surfactants can be divided into four categories: anionic, cationic,

nonionic and zwitterionic that contains both anionic and cationic
charges on the same molecule (Rabie and Vera, 1996; Chang et al.,
2000). Surfactants tend to migrate to surfaces and alter free energy
states or interfacial tensions (free energy per unit area), which can
result in the formation of emulsions and thus greatly increase the
interfacial area between immiscible liquids like oil and water.
Molecules that possess both the hydrophilic and hydrophobic parts are
termed amphiphiles or amphiphatics.
In solvents with different polarity, an orientation can be seen
whereby the surfactant molecules align to shield the hydrophobic
heads from the polar solvent. The non-polar tail of surfactant
molecules comes into contact with the organic phase and the polar
heads sequester together, forming a vesicle that encloses the water
pool inside the cavity (Chang et al., 2000). Havre (2002) postulated
that hydrophobic parts of the molecules are directed into the centre of
the micelles, while the water-soluble, hydrophilic part is directed
towards the water phase. The aggregation process is driven by an
increase in entropy. When a hydrocarbon chain is in contact with
water, the water molecules close to the chain order themselves thus
leading to a reduction in the entropy compared to the bulk of the
water. The entropy increases if the hydrocarbon chains are removed
from contact with the water, into the micelle.
Ionic reverse micelles are more frequently investigated
compared to cationic system.
For example, Sodium di(2ethylhexyl)sulfosuccinate (AOT), an anionic surfactant, has been used
widely because of its ability to form reverse micelle containing large
amounts of water (Rabie et al., 1997; Moore and Palepu, 2007).
Extraction using heptane as the oil can produce a maximum solubility
of water of about 70 molecules per AOT molecule at room
temperature (Bardez et al., 1996). Adachi et al. (2000) demonstrated
146
that reversed micelle composed of non-ionic surfactants interact

mildly with proteins, resulting in low denaturing of proteins and has
no capability of extracting water soluble proteins because of the lack
of strong interactions between the reverse micelle and proteins.
Most studies on reverse micelle extraction have employed an
anionic surfactant such as AOT (Goklen and Hatton, 1987; Dungan et
al., 1991; Naoe et al., 1999; Castagnola and Dutta, 2000; Franqueville
et al., 2003; Shin et al., 2003; Zhang et al., 2006; Majumdar and
Mahapatra, 2007; Mathew and Juang, 2007,) which forms stable
reverse micelle even with high water contents, in a wide variety of
solvents (Freda et al., 2002). Anionic molecules spontaneously form
spherical aggregates that have a narrow size distribution and a
negatively charged polar head group, thereby enabling the positively
charged solute to be extracted into the reverse micelle (Naoe et al.,
1999). Moreover, strong electrostatic interactions between the protein
and the polar head groups of the surfactant permits stable and speedy
extraction that is responsive to the operational parameters (Goklen and
Hatton, 1987).
As an anionic surfactant, AOT has the capacity to form large
micelles in apolar solvents without a co-surfactant (Andrews, 1993;
Kilikian et al., 2000; Paul and Mitra, 2006). Hossain et al. (1999)
suggested that the motivation to use an anionic surfactant AOT in their
studies was the large amount of published data on the physicochemical properties of AOT reverse micelle and the ease of reverse
micelle formation. Ionic surfactants such as AOT can spontaneously
form reverse micelle with a micro water pool in an apolar medium
thereby enabling the charged biomolecule to be solubilised into the
reverse micelle (Naoe et al., 1999). Yonker et al. (2003) used AOT in
their study because it is well known that the aqueous core regions of
the AOT micelle are capable of dissolving a broad range of highly
polar, ionic and/or high molecular weight species.

7.5.2
147
CRITICAL MICELLE CONCENTRATION (CMC)
Critical micelle concentration (CMC) is the minimum surfactant

concentration needed for micelle to form and represents the
concentration of free or unaggregated surfactant molecules in
equilibrium with the micelle (Frense et al., 1995; Kilikian et al., 2000;
Michaels et al., 2007). The CMC is a characteristic of the system and
dependent on temperature, pressure, solvent, and chemical structure of
the surfactant (Rabie and Vera, 1996). Therefore, if the concentration
of the surfactant is below the CMC value, then surfactant molecules
can only exist as free molecules in the solution without the formation
of micelles.
The CMC is often determined by measuring the surface tension
at different concentrations of surfactant (Havre, 2002). Upon addition
of a surfactant to an aqueous solution, the surface tension decreases
until the CMC is reached. Above this concentration, surfactant that is
added will form micelles and the concentration of monomeric
surfactant will remain practically constant, resulting in a constant
surface tension. The CMC can also be determined by measuring other
physicochemical properties that change at the CMC such as electric
conductivity, refractive index, osmotic pressure, turbidity, x-ray
diffraction, or viscosity. The CMC value is obtained by plotting the
values of one (or more) of these properties against the surfactant
concentration (Figure 7.4).
The CMC is denoted by a sharp change in the trends of these
physical parameters. Below this point, surfactants are thought to exist
predominately as monomers, while a population of free monomers at a
concentration near the CMC remains outside the aggregates (Toerne,
2002). The CMC is believed to be the point at which solution
interfaces are saturated with surfactant solubilisation into the bulk
solvent through the creation of micelle. Micelle formation solubilises
the surfactant into the bulk solution where its surface activity is
lowered, resulting in small surface tension changes above the CMC.
148
Physical property scale
CMC
Conductivity / mole
Surface
tension
Turbidity
Surfactant concentration
Figure 7.4 Effect of surfactant concentration on several solution properties,

and the consequence of micelle formation on these properties.
Addition of an ionic surfactant to water leads to a reduction in the

molar conductivity up to CMC, where the decrease becomes more
dramatic. This is due to the decreased electrophoretic mobility of
micelle. Moreover, the surface organization of surfactants exhibits
interesting characteristics at concentrations above and below the
CMC. Studies of various hydrophilic and hydrophobic surfaces at
widely varying surfactant concentrations have revealed the existence
of hemimicelle structures, monolayers or hexagonal packing of rods
absorbed into solution interfaces (Toerne, 2002).
149
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Pires, M. J., Aires-Barros, M. R. and Cabral, J. M. S., 1996, LiquidLiquid Extraction of Proteins with Reverse Micelles,
Biotechnol. Prog., 12: 290-301.
Rabie, H. R. and Vera, J. H., 1996, Extraction of Zwitterionic Amino
Acids with Reverse Micelles in the Presence of Different Ions,
Ind. Eng. Chem. Res., 35: 3665-3672.
Rabie, H. R., Helou, D., Weber, M. E. and Vera, J. H., 1997,
Comparison of the Titration and Contacts Methods for the
Water Solubilization Capacity of AOT Reverse Micelles in the
Presence of a Cosurfactant, J. Coll. Interface Sci., 189: 208215.
Rodrigues, E. M. G., Milagres, A. M. F. and Jr, A. P., 1999, Xylanase
Recovery: Effect of Extraction Conditions on the AOTReversed Micellar Systems Using Experimental Design, Proc.
Biochem., 34: 121-125.
Counter-Current Chromatography Using Reverse Micelle
Solvent Systems, J. Chromatography A, 1151: 164-168.
Shin, Y., Weber, M. E. and Vera, J. H., 2003, Comparison of Two
Methods to Recover Lysozyme from Reverse Micellar Phases,
Sep. Sci. Tech., 38(8): 1733-1748.
Su, W. D. and Lee, C. K., 1999, Reversed Micellar Extraction of
Vancomycin: Effect of pH, Salt Concentration and Affinity
Ligands, Sep. Sci. Tech., 34(8): 1703-1715.
155
Vyve, F. V. and Renken, A., 1999, Hydroformylation in Reverse

Micellar Systems, Cata. Today, 48: 237-243.
Wang, W., Weber, M. E. and Vera, J. H., 1995, Reverse Micellar
Extraction of Amino Acids Using Dioctydimethylammonium
Chloride, Ind. Eng. Chem. Res., 34: 599-606.
Yang, X., 2001, Synthesis and Use of Chiral Surfactants. East
Tennessee State University: Master Degree Thesis.
Yonker, C. R., Fulton, J. L., Phelps, M. R. and Bowman, L. E., 2003,
Membrane Separations Using Reverse Micelles in Nearcritical
and Supercritical Fluid Solvents, J. Supercritical Fluids, 25:
25-231.
Activity by Reverse Micelles, J. Coll. Interf. Sci., 267: 6064.
Zhang, T., Liu, H. and Chen, J., 1999, Affinity Extraction of BSA by
Mixed Reversed Micellar System with Unbound Triazine Dye,
Biochem. Eng. J., 4: 17-21.
Zhang, W., Qiao, X., Chen, J. and Wang, H., 2006, Preparation of
Silver Nanoparticles in Water-In-Oil AOT Reverse Micelles,
J. Coll Interf. Sci., 302: 370-373.
8
LIQUID-LIQUID SEPARATION OF
ANTIBIOTIC WITH REVERSE MICELLES
8.1
INTRODUCTION
Antibiotics are normally produced in aqueous environments, which

require further separation and purification steps. The steps are very
important and consume a huge part of the overall production cost.
Moreover, the production of antibiotics, which typically involves
fermentation process, is usually capital intensive because large and
complex fermentors and extensive equipment for multi-step
downstream processing are required to handle large volume
fermentation broths with a low product concentration (Lee et al.,
2004). Thus, improvement of the separation and purification methods
can make significant savings to the overall manufacturing costs. A
range of separation methods such as conventional solvent extraction,
ion-exchange, chromatography, crystallization, or a combination of
these methods have been used for the recovery of antibiotics. In the
separation of penicillin G from fermentation broth, a solvent extraction
method has been used by industry for many years. If the antibiotic is a
weak acid with a low pKa value, the pH used in the extraction should
be lower than the pKa value to obtain the antibiotic in its free acid
form (Gu, 2000). Nabais and Cardoso noted that the biggest concern in
current solvent separation technique is the frequent formation of stable
emulsions. These emulsions are difficult to be discarded with
conventional techniques such as gravitation or centrifugation. These
problems lead to other problems such as contamination of the final
158
product, low extraction yield, high solvent losses and clogging of the
equipment.
A reverse micelle extraction process is based on the
distribution of biomolecule between aqueous solution and reverse
micelle organic solution. The biomolecule in aqueous solution is
contacted with organic solution, which spontaneously forms reverse
micelles. This method is a kind of liquid-liquid solvent extraction
technique using reverse micelle as the extractant (Kinugasa et al.,
2003). The reverse micelle extraction technique has a great potential
to be an alternative to the conventional liquid-liquid extraction method
for the separation and purification of penicillin G. Since last few
decades, the technique has received researchers attention due to its
ability to solubilise many biomolecule without causing denaturation of
the product. It has been used successfully and widely in the separation
of proteins Goklen and Hatton, 1987; Aires-Barros and Cabral, 1991;
Brandani et al., 1996; Pires et al., 1996; Su and Lee, 1999; Kinugasa
et al., 2003; Bong et al., 2004; Jun et al., 2004; Noh and Imm, 2005;
Hecht and Peled, 2006; Shen and Yu, 2007), amino acids (Leodidis
and Hatton, 1989; Furusaki and Kishi, 1992; Adachi et al., 2000;
Dovyap et al., 2006), enzymes (Moreno-Hagelsieb et al., 1999, Bong
et al., 2004, Liu et al., 2004, Debnath et al., 2007) and metals
(Nakashio et al., 1992; Ismael and Tondre, 1992; Zhang et al., 2006;
Majumdar and Mahapatra, 2007). Mathew and Juang (2007) stressed
that in the case of protein separation, the reverse micelle technique is
more suitable than the conventional liquid-liquid extraction technique
or other separation method because the transfer of proteins into
solvents often results in the irreversible denaturation and loss of
biological activity.
Although penicillin G is considered as one of the most famous
antibiotics and has been widely investigated in detail for a long time,
surprisingly few studies have been attempted so far on its extraction
by the reverse micelle technique. Perhaps this is due to the difficulty in
the extraction and problem associated with the analysis work. Despite
the drawbacks of the liquid-liquid extraction technique as mentioned
by Chimuka et al. (2004), which include difficult to automate, easily
Liquid-Liquid Separation of Antibiotic with Reverse Micelles
159
form emulsion and environmentally unfriendly, however, according to

a review by Goto (2006), the liquid-liquid extraction technique is easy
to scale up without a loss in resolution capability, requires complex
equipment design, economic limitations, and the possibility to operate
continuously. A reverse micelle extraction technique, which follows
the liquid-liquid extraction principles, has great potential to be used in
the large scale antibiotic separation process. Moreover, biomolecule
solubilisation in the reverse micelles does not damage its native
structure and maintained its catalytic activity (Dekker et al., 1989; Yu
et al., 2003).
In this chapter, we investigate the parameters that affect
solubilisation of penicillin G molecules in the extraction process. The
effect of pH, salt concentration, surfactant concentration and initial
penicillin G concentration on the partitioning of the penicillin G
between the two phases are presented. In this study, we show that
penicillin G can solubilise from an aqueous phase to an organic phase
by controlling experimental parameters. We found that penicillin G is
strongly dependent on surfactant concentration in the reverse micelle
phase and pH of the aqueous phase.
8.2
MATERIALS AND METHODS
Reagent grade sodium di-2-ethylhexyl sulfosuccinate (AOT) was used

as the surfactant, the organic solvent used for the reversed micelles
was analytical grade isooctane, and the biomolecule was penicillin G
sodium salt. CaCl2 and KCl were used as salts in the aqueous phase.
All chemicals were supplied from Sigma Aldrich UK and were used as
received. All experiments were conducted at a temperature of 231oC.
5 ml of aqueous solution containing penicillin G and KCl was
contacted with an equal volume of isooctane containing AOT; the
phase transfer experiments are shown diagrammatically in Figure X.1.
In a sequence of experiments different AOT concentrations, pH, and
160
penicillin G concentrations were used. The pH of the aqueous phase

was adjusted using either NaOH or KOH. The solutions were then
mixed for 10 minutes using a magnetic stirrer at 400 rpm. Higher
stirrer speeds and longer stirring periods were not used since a stable
emulsion formed under those conditions. The mixture then was left
for the phases to separate. After 24 hours of settling (some samples
needed more than 24 hours to produce clear interface between the
aqueous and organic phases), samples of the organic phase were
removed carefully using a pipette or a syringe. For each experiment a
blank solution was prepared by contacting an aqueous solution without
penicillin G with an equal volume of initial micellar solution. After
settling, all samples of both phases were transparent to the naked eye.
All experiments were replicated from two to four times in order to
assess the reproducibility of the results.
The concentration of penicillin G in the separated organic and
aqueous phases was measured using the Kjeldahl method. A Buchi
Nitrogen Determination System (consisting of a distillation unit, a
scrubber unit, and a digestion unit) was used to measure the nitrogen
content of the samples; since nitrogen existed in only the penicillin G,
the nitrogen contents were calibrated against the penicillin G
concentration. Surface tension measurements were performed using a
Wilhelmy Ring connected to a tensiometer (White Electrical
Instrument Co. Ltd).
8.3
RESULTS AND DISCUSSION
The motivation for using AOT as the surfactant in this study

was due to its ease of forming reverse micelles, its stability in
comparison with other surfactants, and the fact that it has been used in
many published studies of other systems with success (Dekker et al.,
1989; Naoe et al., 1999; Jun et al., 2004; Shen and Yu, 2007; Mathew
and Juang, 2007). The critical micelle concentration (CMC), the
161
lowest concentration of surfactant needed for reversed micelle

formation, of the AOT in isooctane is 40 g/l. The CMC was measured
using a tensiometer by using a Wilhelmy Ring Method. Use of a
stirrer speed greater than 400 rpm caused formation of an emulsion
that made clear phases separation difficult. In some cases the
emulsion was short lived and disappeared after 24 hours settling time.
From our observations there were no significant differences between
24 hours and one week settling times indicating that the penicillin
solubilisation had reached the equilibrium state; more than 24 hours
settling time did not have an effect on the extraction efficiency.
8.3.1 ADDITIONAL VOLUME OF ORGANIC PHASE
When experiments were carried out using different AOT

concentrations, there was an increase in the volume of the organic
phase that was proportionate to a decrease of the aqueous phase
volume. The additional volume of the organic phase, shown in Figure
8.1, increased when concentrations of AOT >40 g/l were used. At
AOT concentrations less than the CMC, there was no visible increase
in the volume of the organic phase. When the AOT concentration was
increased to above about 40 g/l, the effect
Additional volume of organic phase (ml)
162
2.5
2.0
1.5
1.0
0.5
0.0
0
100
200
300
400
500
Concentration of AOT in organic phase, [S] g/l
Figure 8.1 Additional volume of the organic phase at different AOT

concentrations. (Initial volumes of the organic and aqueous phases were
each 5 ml; [KCl] = 10 g/l; temperature, 23r1C; stirring speed, 400 rpm (10
min); pH 7.6; initial penicillin G concentration in the aqueous phase, 3.6 g/l.)
of reverse micelle formation showed itself by an increase in the

volume of the organic phase as water was transferred into the reverse
micellar structures.
163
8.3.2 EFFECT OF AOT CONCENTRATION
A sought after effect of the AOT concentration on penicillin extraction

is to increase the amount of penicillin transferred to the reverse
micellar phase. It was known that the surfactant concentration
controls the number of reverse micelles and hence the capacity to
solubilise biomolecules into the reverse micelle pool (Pires et al.,
1996; Noh and Imm, 2005, Dovyap et al., 2006; Debnath et al., 2007).
Increasing the surfactant concentration increases the number of reverse
micelles (Yu et al., 2003), thus leading to improvement in extraction
capacity of the reverse micelles. Figure 8.2 shows that as the
concentration of AOT was increased from 0 to 445 g/l, the amount of
penicillin
extracted
into
Concentration of penicillin
in organic phase, [P]rm+o.f g/l
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0
100
200
300
400
500
Concentration of AOT in organic phase, [S] g/l
Figure 8.2 Concentration of penicillin G in the organic phase at different

AOT concentrations. ([KCl] = 10 g/l; temperature = 23r1oC; stirring speed
= 400 rpm (10 min); pH = 7.6; initial penicillin G concentration in the
aqueous phase = 3.6 g/l.)
164
the reverse micellar phase increased. There is no optimum AOT

concentration as the amount of penicillin extracted was almost in
proportion to the amount of AOT used. However, use of AOT
concentration >445 g/l produced an emulsion that was stable (when
settled under gravity), making it difficult to separate and analyse.
Haghtalab and Osfouri (2003) asserted that adding more surfactant to
the solution intensifies the interaction between the reverse micelles
and the protein molecules (in their case) and result in the number of
moles, and the maximum mole number of protein on the surface of the
reverse micelles, being changed.
8.3.3 EFFECT OF PH
To examine the effect of pH on the partitioning of penicillin G,

aqueous KCl solutions containing 3.6 g/l of penicillin and various pH
values were contacted with solutions of AOT of various
concentrations at volume ratios of 1:1. The results are shown in
Figure 8.3 as a relation between the concentration of penicillin G in
the organic phase and the pH value at the equilibrium state. For all
concentrations of AOT, the results obtained show a maximum
penicillin G concentration in the reverse micellar phase at low pH
values (as low as 1); this could be attributed to the high electrostatic
attraction between AOT anions and the positively charged penicillin G
aggregates at pH | 1. Many researchers found that for protein
extraction, pH plays important role in reverse micellar systems
(Nishiki et al., 1993; Pires et al., 1996; Ono et al., 1996; Hu and
Gulari, 1996; Tzeng et al., 1999; Kilikian et al., 2000; Yu et al., 2003).
Noble and Varley (1999), using protein as the biomolecule, pointed
out that the pH of the protein and surfactant solutions were could be
manipulated so that the protein would exhibit a net charge opposite to
165
that of the surfactant, thus allowing electrostatic interactions between

the protein and surfactant in the different phases.
Concentration of penicillin
in the organic phase, [P]rm+o.f g/l
1.8
[S ] = 88 g/l
[S ] = 222 g/l
[S ] = 445g/l
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0
10
12
Initial pH of aqueous phase
Figure 8.3 The effects of pH and AOT concentration on the amount of

penicillin extracted into the micellar phases. (Temperature = 23r1oC; stirring
speed = 400 rpm (10 min); initial penicillin G concentration in the aqueous
phase = 3.6 g/l.)
Kilikian et al. (2000) explained that the overall protein charge is

determined by the pH of the aqueous phase and the isoelectric point
(pI) of the biomolecule used (protein, in their case). If the pH of the
aqueous phase is higher than the pI, the charge is negative, but if the
pH is lower than the pI, the charge is positive. Using this postulate for
the anionic AOT, the penicillin G (pI = 2.74) is expected to have
strong attraction with the surfactant head group at pH < 2.74. Hence,
more penicillin G could be solubilized into the reverse micellar
solution at pH<pI. Ono et al. (1996), using anionic surfactant DOLPA
166
to extract hemoglobin (pI = 6.8) found that in case of the pH << pI, the
biomolecules tend to form a complex that binds to the surfactant. In
this study, we observed precipitation when high initial penicillin G
concentrations were used at low pH. Although extremely low pH
range were used, at [KCl] = 10 g/l and 400 rpm stirring speed, a
transparent two phases system was obtained with a clear interphase
when [P]aq.i < 10 g/l. From the results, it appears that the pH effect is
important for the penicillin G extraction, although the electrostatic
contribution is also relevant for the AOT reverse micelles which
achieved better binding at higher pH values.
8.3.4 EFFECTS OF SALT CONCENTRATION AND TYPE
The salt concentration of the aqueous phase influences the

solubilisation of penicillin into the reverse micellar phase. The salt
concentration was investigated by varying the KCl concentration in
the aqueous phase at fixed pH 7.6 (Figure 8.4). When the [KCl] > 10
g/l the penicillin concentration in the organic phase was found to
decrease, although increasing [KCl] from 0 to 10 g/l also show a
significant increase of penicillin in the organic phase. At [KCl] < 5 g/l
almost all of the surfactant migrated to the aqueous phase and no
reverse micelles were formed in the organic phase. This phenomenon
was observed visually, and made addition of salt to the aqueous
penicillin G solution very important to avoid the formation of a stable
emulsion. A minimum amount of salt is necessary to salt out the
surfactant from the excess aqueous phase into the organic phase, to
form reverse micelles (Wang et al., 1994; Rabie et al., 1997). Reverse
micelles will not form if there is too little salt in the aqueous phase;
however, if there is too much salt, the efficiency of surfactant
molecules to solubilise the penicillin G molecules will reduce. Our
experimental results showed that penicillin G could be extracted into
167
the reverse micellar phase quantitatively over a relatively small salt

concentration range. With increasing salt concentration over 10 g/l,
the extent of antibiotic transfer decreased in spite of the pH being in
the region where the electrostatic interaction between antibiotic and
surfactant is attractive. According to Dekker et al. (1989) the ionic
strength of the aqueous phase determines the degree of shielding of the
electrostatic potential imposed by a charged surfactant head group.
They also suggested that by increasing the ionic strength the
electrostatic interaction between the charged penicillin molecules and
the charged surfactant head groups is reduced.
Concentration of penicillin G
in organic phase, [P]rm+o.f g/l
1.0
[A O T ] = 2 67 g/l
[A O T ] = 2 22 g/l
0.8
0.6
0.4
0.2
0.0
0
20
40
60
80
C oncentration of K C l in aqueous phase, [K C l] g/l
Figure 8.4. Effect of KCl concentrations on the concentration of penicillin G

in the organic phase. (Temperature = 23r1oC; [AOT] = 267 g/l; stirring
speed = 400 rpm (10 min); pH = 7.6; initial penicillin G concentration in the
aqueous phase = 3.6 g/l.)
168
Salt type also plays important role in the extraction of biomolecule

from the aqueous to the revese micellar phases (Leodidis and Hatton,
1989; Goto et al., 1989; Cheng and Sabatini, 2001; Kinugasa et al.,
2003). We used two salts, KCl and CaCl2 dissolved in the aqueous
phase, and found that the concentration of penicillin G in the reverse
micellar phase is higher when using CaCl2 (Figure 8.5). This is
because a divalent salt like CaCl2 leads to smaller reverse micelle
droplets and the ability to solubilise more penicillin is increased.
However, it was observed that when
water content in organic phase, %
40
without penicilin G
with penicillin G
30
20
10
0
0
20
40
60
80
Concentration of KCl in aqueous phase, [KCl] g/l
Figure 8.5. The effect of KCl concentration on the water content.

(Temperature = 23r1 oC; [AOT] = 267 g/l; stirring speed = 400 rpm (10
min); pH = 7.6; initial penicillin G concentration in the aqueous phase = 3.6
g/l.)
using CaCl2 the solution became more sensitive to the stirring speed
and tended to emulsify more easily. This caused problems with
169
getting a good phase separation, and even when the phases can be
separated extra time was needed for the solutions to achieve
equilibrium.
8.4
CONCLUSION
Experiments on the solubilisation of penicillin G into reverse micelles

of AOT have been carried out and are reported in this chapter. The
results show that the concentration of penicillin G in the organic phase
depends primarily on the pH and surfactant concentration, with the salt
type and concentration in the aqueous phase also being important. The
solubilisation of the penicillin into the reverse micelles increases at
higher AOT concentrations.
But at any specified surfactant
concentration the potential extent of extraction is limited; there is a
saturation limit, determined by the surfactant concentration, of
penicillin in the organic phase above which the penicillin cannot be
transferred. Reverse micelle formation affects the volume of water
transferred into the organic phase (this water is held within the reverse
micelles). The additional volume increases by increasing AOT
concentration.
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9
EXTRACTION, IDENTIFICATION AND
SEPARATION OF DJENKOLIC ACID
FROM PITHECELLOBIUM JIRINGAN
SEEDS USING SUPERCRITICAL CARBON
Wahyu B. Setianto
9.1
INTRODUCTION
Pithecellobium jiringan jack (P.jiringan), commonly known as jering

in Malaysia and jengkol in Indonesia is a member of the family
leguminosae sub-family Mimosaceae. This plant is a south East Asian
origin and occurs in primary and secondary rain forests and in
evergreen forests (De Guzman and Siemonsma, 1999). On the
traditional medicinal usage, the plants part such as seed and pod were
used for treating different kinds of disease. The seed is used to treat
hypertension and anti diabetic effect, the old leaves burnt to ashes
were used against itching meanwhile the pod potentially be used for
flavor and fragrant product such as soaps, shampoos and detergent
(Vimala et al., 2003). The most important plant parts identified was
the seed, which contains several active constituents with high level of
therapeutic effects. The picture of fruits and seeds of P.jiringan are
shows in Figure 9.1. The chemical constituents contain in the seeds
mainly of group of fatty acids, vitamins (Vitamin A, B, C, D, and E),
protein, alkaloid, carbohydrate, fiber, calcium, phosphorus iron and
cystein (Abullah, 1999). It has been reported that P. jiringan seeds are
176
good source of natural antioxidants that could destroy excess free

radicals and prevent oxidative damage. P.jiringan seed also have high
antioxidant activity almost more than 85% in two antioxidant bioassay
experimental pathways superoxide free radical scavenging activity,
SS, and 1,2 diphenyl-2-picrylhydrazyl radical scavenging activity,
DPPH. As reported by Harborne (1980), the group of chemical
constituents of non-protein amino acid frequently occurs in high
concentration in seeds, particularly in those of the Leguminosae
family.
Figure 9.1 P.jiringan fruits and seeds.
This plant seeds is categorized as unpopular plant than most of

common plants like eurycoma longifolia jack (tongkat ali),
orthosiphon stamines benth (misai kucing) and andrographis
paniculata (hempedu bumi). The reasons P.jiringan seeds is
considered unpopular medicinal plant for human consumption because
the seeds contain of undesired compounds such as volatile oil
consisting of an ally sulfur compounds and toxic acid namely
Extraction And Separation Of Djenkolic Acid From Pithecellobium

Jiringan Seeds
177
djenkolic acid (Goh et al., 1993). The ally sulfur compounds, which is
a group of short chain hydrocarbon with presence of sulfur have very
strong noxious smell meanwhile excessive consumption of seeds may
lead `kejenkolan disease, caused by crystallization of djenkolic acid
in the kidneys and bladder with symptoms such as renal hyperemia,
oliguria, no urination and painful of flow urine.
The molecules structure of djenkolic acid was shows in Figure
9.2. Djenkolic acid is a sulfur containing amino acid, occurs in high
concentration in seeds particularly in those of the leguminosae family
and is a toxic compound (Harborne and Baxter, 1978). Djenkolic acid
is amphoteric, it can be an ion at the ordinary state and as neutral
molecule at pH around 7.0. Djenkolic acid is mildly soluble in acid
and alkali. The shape of djenkolic acid crystal is like fine sharp needle
at the both side. The crystals will deposit on the wall of urethral
calculus (urinary tract) causing the wall diameter to become smaller.
Hence, djenkolic acid crystals will exert force as the crystals
accumulate and the same time the urethral calculus gets blocked. This
situation is called `djenkolism symptoms.
178
Figure 9.2 Molecules structure of djenkolic acid presented in mode of space

filling display.
Areekul et al. (1976) was conducted the extraction djenkolic acid from
djenkol bean with 70 % ethanol and water. The amount of extracted
djenkolic acid was quantitatively determined by paper
chromatography. It was found that djenkol beans contain 0.3 to 1.3 mg
djenkolic acid. The toxicity of djenkol beans was studied in 5 rhesus
monkeys, 9 albino rats and 22 mice fed with 70% ethanol extract. The
observation showed that the total urinary output decreased and there
was an increase in specific gravity of the urine during the period of
feeding monkeys with djenkol beans. Urinary samples of the
experimental animals were turbid and contained some red cells, white
cell, epithelial cells, albumin and amorphous particles. Out of one
mice showed excreted sharp needle-shaped crystals in the urine on day
3 after feeding. Histological examination of kidneys of rats and mice
showed mild to severe acute tubular necrosis with some glomerular
cell necrosis.
On the toxicity to human, the effects caused tend to vary
among individuals, depends on age, sex and genetic background of the

Jiringan Seeds
179
consumers. As reported by Arrekul et al. (1978), an 8-year-old boy
was admitted into the hospital with symptoms of anuria after
consumption of 12 djenkol beans. Laparotomy showed a urethral
calculus, size 2.0 by 0.4 cm, which was found to contain djenkolic
acid 65 gm/100 gm stone with a small amount of protein, sodium,
potassium, ammonium salt, oxalate, carbonate, cysteine and fibrin.
Therefore very important to exploit SC-CO2 extraction method on the
extraction and simultaneously separation of medicinal and toxic
compounds from P.jiringan seeds in order to produce high quality
finished product.
The application of supercritical carbon dioxide extraction (SCCO2) is promising alternatives method for determination of chemical
constituents present especially for unexploded medicinal plants. In
addition, supercritical extraction has distinctive advantages acts two in
one, extraction and separation (Reverchon, 1997). The separation of
the extracted compound could be done by adjusting operational
parameters such as introducing the proportion of liquid modifier or by
altering the pressure and/or temperature of the supercritical condition.
As reported by Palma et. al (1999), on the investigation of active
phenol compounds in grape seeds, the extracted obtained was strong
antioxidant activity consisting mainly of fatty acids, aliphatic
aldehydes and sterol meanwhile by using CO2 with 20% ethanol as a
modifier, the low concentration of secondary and tertiary compound
such as gallic acid and epicatechin could be extract elsewhere.
The rationale of using carbon dioxide as a solvent in the
supercritical fluid extraction has been discussed in many publications.
The most important reason used of carbon dioxide is because it has a
best critical temperature and pressure of 31.3 o C and 7.38 MPa,
respectively. As mentioned by Dick and Herry (1996) no material with
critical parameters as mild as carbon dioxide. This means that
extraction can be conducted at temperatures that are low enough not to
harm the physiochemical properties of the extract. The clear
advantages over other organic solvents are chemically stable,
inflammable, radioactively stable, high purity, non-toxic and inert in
180
nature. Thus, there is no risk of side reactions such as oxidation. It has

been accepted by most European food and drugs acts as an extraction
medium for the isolation of food related compounds. Moreover, there
are no other residual solvents in any final extracted material since CO2
is the only gaseous at room temperature and pressure. Thus, with CO2,
the extracted oil can be easily separated by decompression and
subsequently, its recovery percentage is still high. Moreover, the
characteristics of the finished products is also generally better than the
conventional methods since it is free from inorganic salts, heavy
metals and micro bacterial life.
In this study, P.jiringan seeds was extracted at pressure ranges
from 20.68 MPa (3000 Psi) to 55.16 MPa (8000 Psi) and temperature
from 40o C to 90o C. The extraction process was conducted for 60
minutes. This ranging of temperature and pressure were chosen in
order to extract both, polar and non-polar compounds especially on the
extraction of djenkolic acid. The high performance liquid
chromatography, HPLC, was applied to quantify the amount of
djenkolic acid at different pressure and temperature ranges. The
quantitative analysis was performed under the isocratic method using
the external standard technique.
9.2
MATERIAL AND METHODS
9.2.1 MATERIAL
Fresh P. Jiringan seeds with commercial maturity (the colour of seed
testa is dark brown) were obtained from a local Kepala Batas market
Penang, Malaysia. The seeds were separated from the fruit, thoroughly
washed with tap water, rinsed with distilled water and then cut into
small pieces (2-3 cm diameter and 1 mm thickness). The seeds were

Jiringan Seeds
181
oven dried at 40 o C overnight. The dried seeds are then ground with a
dry mixer (Waring laboratory, US) and the particle size distribution
was determined by sieve analysis, Vibrator Steve Shaker (Retsch,
German). In this study, the particle size range used was 180 m 250
m. The purity of carbon dioxide used was 99 % (w/w) from Malaysia
Oxygen Penang, Malaysia. The standard of djenkolic acid with purity
of 80% was purchased from Sigma Adrich.
9.2.2 SUPERCRITICAL CARBON DIOXIDE EXTRACTION

Supercritical CO2 extraction was carried out using SFX TM 220
extraction system (ISCO, Lincoln, NE, US). The components of a SFX
TM
220 comprises of a CO2 cylinder, a chiller (Model Yih Der BI-730)
to liquefied CO2 gas, a high pressure syringe pump with maximum
operating pressure of 689.5 bar, an extractor with size 22.7 cm by 21.2
cm by 24.2 cm equipped with a 2.5 ml extraction cell, a heated
capillary restrictor with maximum operating temperature of 150 o C
with outside diameter 50 m to reduce solute deposition and a 30 ml
vial to collect the extract. The equipment set up of the extraction
process SFX TM 220 is shown in Figure 3.
182
Pump A
Chiller
CO2 tank
Collecting tube
Pump B
Sample cartridge
Extraction Chamber
Modifier reservoir
Controller
Figure 9.3 SFXTM 220 Extraction units.
9.2.3 HIGH PERFORMANCE LIQUID

CHROMATOGRAPHY (HPLC)
Chromatographic separation was carried out using a LiChrospher RP18 column (250 mm x 4.6 i.d. mm, 5m particle size) (Merck
Darmstadt, Germany). The Mobile phase used consists of methanol,
water and acetonitrile (20:70:10 v/v) by applying isocratic methods.
Methanol and acetonitrile were filtered through an FA 0.5 Pm filter
(Millipore) prior to use. The mobile phase was degassed and then
delivered at a flow rate of 0.3 mL/min at temperature 25 o C. The
injection volume of sample used was 15 PL. The peak of djenkolic

Jiringan Seeds
183
acid in the standard solution and extracted sample was detected at
wavelength 210 nm. The composition of djenkolic acid in the
extracted oils was calculated using the regression parameters obtained
from the calibration curve of standards. The peak of djenkolic acid
was determined by comparing its retention time with the standard of
djenkolic acid.
9.3
RESULTS AND DISCUSSIONS
9.3.1 EFFECT OF SC-CO2 ON THE OVERALL EXTRACTED

OIL YIELD
The effects of temperature and pressure on the overall oil yield are
shown in Figure 9.4 and Figure 9.5. The percentage of oil yield was
calculated by the mass of oil extracted per unit mass of sample used.
184
Figure 9.4 Percentage of oil yield obtained function of pressure at constant

temperature studied.
In general, the yield curves as shown in the Figure 9.4 and Figure 9.5
demonstrate similar trends for a range of different pressures at each
constant temperature over 60 minutes extraction time. The overall
extracted oil yield strongly increased with increase in pressure and
temperature. At each constant temperature, the highest overall
extracted oil yield was obtained at higher pressure of 55.16 MPa,
meanwhile the lower pressure of 20.68 MPa generated lowest overall
extraction yield.

Jiringan Seeds
185
Figure 9.5 Percentage of oil yield obtained function of temperature at

constant pressure studied.
This phenomenon was easily explained by the basic principles of

supercritical fluid extraction. As the pressure increases the density of
the supercritical CO2 approaches that of a liquid resulting in an
increase in the solvating power (the interaction of inter-molecular
solvent and solute). The solubility of oil in SC-CO2 increases due to
their high solvating power, which enhanced the extraction rate.
Nevertheless, at the high operating temperature, the vapour pressure of
extracted oils was increased; therefore the transportation of oil onto
the seed surfaces was easier.
The same trend was reported by Perretti et al. (2003) on the
improving the value of rice by product by SC-CO2, whereby the
extraction conducted at a high pressure of 10000 Psi (68.95 MPa) and
high temperature of 80o C gave the highest extraction yield of 4.93 g
of rice bran oil (24.65 %). Another study point out the highest
extracted oil yield was obtained at higher operating pressure and
temperature was given by Favati et al. (1991) on extraction of evening
186
primrose oil. Their results show that oil recovery above 90 % was
obtained when the extraction pressure was increased to 30.0 MPa, and
even higher yield was recorded when operating at pressures of 50.0
MPa and 70.0 MPa. Bisunadan (1993) also reported similar results on
the extraction of oil from palm oil fruits. The SC-CO2 extraction was
carried out at 300, 400 and 500 bar and 40o, 60o and 80o C. The
results show that the optimum condition for extraction of oil was
obtained at the higher pressure and temperature of 500 bar and 80o C.
At each constant pressure, the highest overall extraction yield
was obtained at highest temperature of 80o C, meanwhile the lowest
temperature of 40o C produced lowest overall extraction yield. These
results show that extraction yields increase with increasing
temperature over pressure ranges from 20.68 MPa to 55.16 MPa. This
is due to fact increase in oil volatility was more dominant than drops
of CO2 density in the temperature range from 40o to 80o C.
Particularly, at constant pressure, the diffusivity of the supercritical
fluid increases with increasing temperature (as the fluids tend towards
a more gas-like state) and as result the extraction rate will increase,
producing a faster extraction to reach asymptotic yield especially
when operating at higher pressure. Another reason is that at constant
pressure, an increase in temperature increases the mass transfer
coefficient, therefore decreases the internal and external mass transfer
resistance.
The trend that the extraction yield increased with increasing
temperature at constant pressure has been found in others studies.
Ozkel et al. (2005) was conducted the extraction yield of hazelnut oil
in SC-CO2 at extraction conditions of 15.0 to 60.0 MPa and 40 o to 60o
C. The extraction yield increased both with pressure and temperature.
It was higher than 50 % at all temperatures at pressure of 60.0 MPa.
Zaidul (2003) reported that the higher pressure range of 34.5 MPa to
48.3 MPa and high temperature of 80o C are preferred to extract palm
kernel oil in SC-CO2. Zhimin and Samuel (2000) showed that the
extraction yield from rice bran at constant pressure of 68.9 MPa were
significantly higher at 55, 60 and 75o C than at lower temperatures.

Jiringan Seeds
187
9.3.2 IDENTIFICATION OF DJENKOLIC ACID AT SCCO2 CONDITIONS
The compound of djenkolic acid is identified when their retention time
is under the tolerance of standard retention time. The presence of
djenkolic acid in extracted oil has been clearly observed at constant
temperature of 80o C and corresponding to a range of pressure from
20.68 MPa to 55.16 MPa. On the other hand, the absence of djenkolic
acid in extracted oil also has been clearly observed at constant
temperature of 40o C over pressure range from 20.68 MPa to 34.47
MPa.
mAU
3.503
Wavelength=210 nm (AZIZI\DJSTAD01)
700 Wavelength=210 nm (AZIZI\7P80T)
600
Djenkolic acid standard
500
400
Extract oil
3.766
300
0
0
2.5
7.5
10
17.5
20
22.5
23.785
22.725
22.027
20.401
17.353
15
18.672
15.696
12.5
13.571
14.083
11.738
12.868
9.633
7.203
7.835
8.408
8.898
10.173
4.843
5.917
6.318
2.456
2.809
1.841
0.762
100
4.303
3.962
200
min
Figure 9.6 Overlay of HPLC profiles of djenkolic acid standard and

extracted oil at SC-CO2 conditions of 20.68 MPa and 80o C.
188
Figure 9.6 shows the retention time of extracted djenkolic acid is

3.548 minute whereas the retention time of djenkolic acid standard is
3.503 minute. Their tolerance for the extracted djenkolic acid about
retention time of the standard is 0.045 minute. The retention time
tolerances accordance to WADA Technical Documents (2003) for
HPLC non-volatile compounds methods is 0.4 min, whereas US
EPA methods (2003) are 5.0 %. It can be seen that the tolerance for
the extracted djenkolic acid peak about retention time of the standard
peak is complying both, WADA and US FDA guidelines. Therefore,
the peak on the HPLC profile eluted at 3.548 minute at the SC-CO2
condition of 20.68 MPa and 80o C was confirmed as peak of djenkolic
acid. Figure 6.7 shows the overlay of HPLC profile of extracted oil
and peak of djenkolic acid standard at extraction condition of 20.68
MPa and 40o C. Regarding to WADA and FDA guidelines, at the
tolerance retention time of 0.4 minute and 5.0 % of the djenkolic
acid retention time standard of 3.503 min, there is no peak appears. In
other words, at this condition (20.68 MPa and 40o C) it is confirmed
djenkolic acid is absent in the extracted oil (Mohd Azizi, 2007).

Jiringan Seeds
189
mAU
3.503
Wavelength=210 nm (AZIZI\DJSTAD02)
Wavelength=210 nm (AZIZI\4P40T)
250
Djenkolic acid standard

200
4.887
150
100
7.5
10
12.5
15
21.594
16.426
13.458
12.161
9.290
8.692
7.852
3.152
3.693
4.078
4.582
2.5
6.460
1.441
0.437
2.440
50
17.5
20
22.5
min
Figure 9.7 Overlay of HPLC profiles of djenkolic acid standard and

extracted oil at SC-CO2 conditions of 20.68 MPa and 40o C.
9.3.3 EFFECT OF SC-CO2 ON EXTRACTED DJENKOLIC

ACID
The effect of extraction temperature on concentration of
djenkolic acid yield at constant pressure, and also the effect of
extraction pressure on concentration of at constant temperature were
illustrated in Figure 9.8 and Figure 9.9 respectively.
The
concentration djenkolic acid was defined as mg of djenkolic acid per g
of extracted oil during 60 minute extraction time.
C o n c e n tra tio n o f e x tra c te d

d je n k o lic a c id ,
m g d je n k o lic a c id / g o il
190
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
P=55.16 MPa
P=48.26 MPa
P=41.37 MPa
P=34.47 MPa
P=27.58 MPa
P=20.68 MPa
30
40
50
60
70
80
90
Temperature, (C)
Figure 9.8 Effect of extraction temperature at constant pressure on the

concentration of djenkolic acid during 60 minute extraction.
At constant pressure, the djenkolic acid concentration increases with

increase in temperature. The djenkolic acid concentration obtained in
the range of extraction conditions applied is following the same trend
as the overall extraction of oil yield. The djenkolic acid concentration
depends on a complex balance between the increase in the
supercritical carbon dioxide density as well as increase in vapor
pressure. The maximum amount of djenkolic acid concentration
obtained is 4.587 mg of extracted djenkolic acid per g of extracted oil
(0.459 wt %) at temperature 80o C and pressure 55.16 MPa, while the
low amount of djenkolic acid concentration is 0.76 mg of extracted
djenkolic acid per g of extracted oil (0.076 wt %) at temperature and
pressure of 50o C and 27.56 MPa, respectively. In this study, it can be
observed that the effect of temperature is more dominant than
pressure.

Jiringan Seeds
191
C o n cen tra tio n o f extra cted

d je n ko lic ac id ,
m g d jen ko lic acid / g o il
T=80 C
T=70 C
T=60 C
T=50 C
T=40 C
2
1
0
-1
10
20
30
40
50
60
Pressure, MPa
Figure 9.9 Effect of extraction pressure at constant temperature on the

concentration of djenkolic acid during 60 minute extraction.
At constant temperature, the results show that djenkolic acid

concentration increased with pressure (Figure 9.9). The same trend as
the previous was observed with the maximum amount of djenkolic
acid concentration obtained at the high extraction condition. This is
due to the fact that since the density of CO2 increases with pressure
causing a decrease of the intermolecular distances that increases the
solute-solvent interaction. As a result, the djenkolic acid concentration
increases when the strong intermolecular force of djenkolic acid-CO2
prevails (Mohd Azizi, 2007). Occasionally, an increased pressure not
only increases the solute solubility, but also facilitates its diffusion.
Therefore, the rate of solute diffusion through a matrix sample can be
raised by increasing the pressure. The result obtained in this study is in
good agreement with suggested by Luque et al. (1994) on the
properties of the solute. He suggested that solute with high molecular
192
weight containing polar groups call for high fluid densities in order to
make this solute more readily soluble in pure CO2.
It can be seen that the effect of temperature at 40o C does not
significantly apply in this study. This is because no djenkolic acid can
be extract over the pressure ranges and only in trace amount at the
pressure above 41.37 MPa. This is due to the fact that at 40o C the
desorption kinetics of the compounds from the matrix sample are very
slow. As temperature increases desorption is faster thus tending to
increase the solubility of djenkolic acid and hence resulted increase in
djenkolic acid concentration. Furthermore, as summarizes on the
method development for supercritical fluid extraction by Dean (1993)
for the extraction of non-volatile and mild-polar/polar compounds the
recommended extraction temperature must be done at least 15o C
higher than the critical temperature of the fluid.
9.4.
CONCLUSION
The overall extracted oil yield of P.jiringan seeds was increased with
increase in pressure at constant temperature. Likewise, the overall
extracted oil yield also increased with increase in temperatures at
constant pressure. The maximum overall extraction oil yield is 8.06 %,
at the highest supercritical conditions of pressure and temperature of
55.16 MPa and 80o C, respectively. The solubility of P.jiringan oil
increased with increase in temperature under constant pressure.
However, at constant temperature the solubility values were constant
during the entire extraction pressure range from 20.68 MPa to 34.47
MPa. Above 35.0 MPa, the solubility begins to increase until reaching
to maximum solubility value. Meanwhile, at constant temperature of
80o C, the solubility values increased with increase in temperature.
The maximum solubility of P.jiringan oil is 0.26 wt % (2.6 mg oil per
g CO2) under the supercritical condition of pressure and temperature

Jiringan Seeds
193
of 55.16 MPa and 80o C, respectively. On the other hand, the
minimum solubility of P.jiringan oil is 0.06 wt % (0.6 mg oil per g
CO2) was obtained at lowest pressure and temperature of 20.68 MPa
and 40o C, respectively.
The study shows that the concentration of extracted djenkolic
acid increased with increase in temperature under constant pressure in
60 minute extraction period. However, no djenkolic acid was extracted
from P.jiringan seeds under constant temperature of 40o C and
pressure range 20.68 MPa to 41.37 MPa and at extraction condition of
50o C and 20.68 MPa. It was also observed that at constant
temperature of 50o C with pressure range of 27.58 MPa to 48.26 MPa,
the amount of djenkolic acid extracted was only trace amount. The
maximum concentration of extracted djenkolic acid was 0.459 wt %
(4.59 mg/ g oil) at the highest pressure and temperature of 55.16 MPa
and 80o C, respectively.
The findings of this study show that if the extracted oil is
considered as finished product, the best SC-CO2 conditions is 20.68
MPa and 40o C. On the other hand, the SC-CO2 conditions of 55.16
MPa and 80o C is the best operational if residue sample is chosen as
the finished product.
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INDEX
Absorbance 68
Adsorbate 128
Antibiotic 16, 23, 132, 154
Biological 12, 23, 131, 155
Biomolecule 17, 24, 131
Bleaching 63, 68
Carbon dioxide 176
Chromatography 15, 16, 131,
177
Countercurrent 3, 54
Crude palm oil 33, 62
Degumming 63
Deodorization 63
Diffusivity 1, 35
Efficiency 12, 33, 47, 79, 99,
141
Energy 2, 11, 100, 142, 143
Equilibrium 3, 7, 20, 37, 81,
99, 126, 139, 158
Free fatty acid 53, 141
Heterogeneous 2, 3, 33
Homogeneous 2, 3, 101
Hydrophobic 20, 129
Hydrothermal 130
Isoelectric 124
Liquid-liquid extraction 6, 11,
20, 135, 155
Mass transfer 3, 21, 32, 38,
44, 182
Membrane 4, 8, 19, 21, 74,
85, 96, 100
Mesoporous 123, 132
Mobile phase 133, 179
Modifier 50, 130, 176
Molecular 1, 51, 129, 142,
187
Permeability 101
Pharmaceutical 12, 15, 18,
131
Protein 161, 173
P-T diagram 37
Reverse micelles 26, 155
198
Rifampicin 133
Selectivity 6, 18, 53, 56, 75,
90
Silica 63, 129
Solubility 1, 19, 41, 55, 128
Supercritical 33, 41, 46, 48,
64, 172
Surfactant 18, 135, 156
Thermal degradation 32
Thermodynamic 1, 7, 48, 138
Viscosity 5, 17, 38
Volatility 6, 182
Zeolite 77, 129
Index

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First Edition 2007

ZAINUDDIN ABDUL MANAN, MOHAMED MAHMOUD NASEF

Advances in separation process / [editors Zainuddin Abdul Manan,

Diterbitkan di Malaysia oleh / Published in Malaysia by

34 38, Jln. Kebudayaan 1,Taman Universiti

Lot. 47 & 48, Jalan SR 1/9, Seksyen 9,

Chapter 1 Introduction to Separation Processes

Chapter 2 Separation Methods in the Pharmaceutical 15

Chapter 4 Regeneration Technique for the Spent

Chapter 5 Ion Exchange Technology: Principles and 75

Chapter 7 Reverse Micelle Extraction

Chapter 8 Liquid-Liquid Separation of Antibiotic

Chapter 9 Extraction And Separation Of Djenkolic 175

Separation and purification technologies play a very vital role in a

separation processes. The information reported by the contributors

Zainuddin Abdul Manan

1.1 DEFINITION OF SEPARATION PROCESSES

Thermodynamic and transport

According to the second law of thermodynamics, which states

Advances in Separation Processes

inherently mixing processes which occur spontaneously. The reverse

1.2 HOMOGENEOUS SYSTEMS

The feedstocks to separation processes may be either homogeneous,

Introduction to Separation Processes

Homogeneous systems usually require more effort to separate.

1.3 EQUILIBRIUM PROCESSES

Homogeneous systems can be based on either equilibrium processes or

Advances in Separation Processes

heat to the liquid feed resulting in a vapour phase which is in

1.4 RATE-CONTROLLED PROCESSES

Rate-controlled (diffusional) processes involve differential rates of

Introduction to Separation Processes

1.5 HETEROGENEOUS SYSTEMS

Heterogeneous systems can be easily separated by mechanical

1.6 MECHANISM OF SEPARATION

Seader and Henley attributed separation to be caused by any five

Advances in Separation Processes

The most common industrial technique is the creation of a

Introduction to Separation Processes

use of cyclone separators for the removal of suspended dust particles

Table 1.2 A few examples of separation processes and their separation

Solvent (MSA) and

Removal of CO2 from

Advances in Separation Processes

Gas (MSA) and/or

Nitrogen from air

Introduction to Separation Processes

Electrical force field

Electrical force field

Electrical force field

(1) - (10): separation by phase addition or creation, (11) (17): separation by

Advances in Separation Processes

The selection of the proper process depends on several factors, for

Introduction to Separation Processes

Table 1.3 Factors that influence the selection of feasible separation

An optimum separation process involves a balance between energy

Table 1.4 Ease of scale-up of the most common separation operations.

Need for parallel

For regeneration cycle

Advances in Separation Processes

1.8 IMPORTANCE OF SEPARATION PROCESSES

Separation processes play an important role in petroleum, chemical,

Introduction to Separation Processes