Bioseparation Technology (BT-602)

Extraction
An extraction process makes use of the partitioning of a solute between two immiscible or partially miscible
phases. For example the antibiotic penicillin is more soluble in the organic solvent methyl isobutyl ketone (MIBK)
than in its aqueous fermentation medium at acidic pH values. This phenomenon is utilized for penicillin
purification. When the extraction takes place from one liquid medium to another, the process is referred to as
liquid-liquid extraction. When a liquid is used to extract solutes from a solid material, the process is referred to as
solid-liquid extraction or leaching. When a supercritical fluid is used as an extracting solvent, the process is
referred to as supercritical fluid extraction (SFE).

Typical applications of extraction in bioprocessing include:

Purification of antibiotics
Purification of alkaloids
Protein purification using aqueous two phase systems
Purification of peptides and small proteins
Purification of lipids
Purification of DNA

Solvent Systems:
Solvent extraction as used in the bio-industry can be classified into three types depending on the solvent systems used:
Aqueous/non-aqueous extraction
Aqueous two-phase extraction
Supercritical fluid extraction

Low and intermediate molecular weight compounds such as antibiotics, alkaloids, steroids and small peptides are
generally extracted using aqueous/non-aqueous solvent systems. Biological macromolecules such as proteins and
nucleic acids can be extracted by aqueous two-phase systems. Supercritical fluids are used as extracting solvents
where organic or aqueous solvents cannot be used satisfactorily.

Ideally, the two solvents involved in an extraction process

should be immiscible. However, in some extraction processes
partially miscible solvent systems have to be used. For partially
miscible solvent systems, particularly where the solute
concentration in the system is high, triangular or ternary phase
diagrams are used. In such diagrams the concentration of the
components are usually expressed in mole fraction or mass
fraction. The following figure shows the phase diagram for a
solute A, its initial solvent B and its extracting solvent C. Such
phase diagrams rely on the fact that all possible composition of
the three components can be represented by the area within
the triangle. The composition of the mixture represented by
point H on the diagram is such that content of A is proportional
to HL, content of B is proportional to HJ and content of C is

Arka Dhar
Department of Biotechnology, BCET
proportional to HK. The curve shown is called the binodal solubility curve. The area under the curve represents the
two-phase region. Any mixture represented by a point within this region will split up into two phases in
equilibrium with each other. For a mixture having an overall composition H, the composition of the two phases are
represented by points P and Q which are obtained from the points of intersection of the binodal solubility curve
with the tie line PQ which passes through H. The tie lines are straight lines which connect together the
compositions of the two phases, which are in equilibrium with one another. These tie lines are experimentally
determined. The point F on the binodal solubility curve is called the plait point. The area above the binodal
solubility curve represents the single phase region where all three components in the system are mutually
miscible.

Theory of Extraction:
The feed consists of the solute to be extracted in its original solvent e.g. penicillin in fermentation media. The
extracting solvent (e.g. MIBK) is the phase to which the solute (i.e. penicillin in this case) is to be transferred and
device within which this transfer of solute takes place is called an extractor. The raffinate is the spent feed while
the extract is the enriched extracting solvent.

The distribution of a solute between the raffinate and the extract can be expressed in terms of the partition
coefficient K:

Where, CR = Equilibrium solute concentration in raffinate (kg/m3)

CE = Equilibrium solute concentration in extracting solvent (kg/m3)

The value of K is often independent of the solute concentration, particularly at low solute concentrations. However,
at higher solute concentrations, deviation from the linearity between CR and CE may be observed in some systems.
The partition coefficient of a solute between two phases is usually determined by experimental methods. Once
equilibrium distribution is reached, the chemical potential of the solute in the two phases must be equal.
μR = μE
where, μR = chemical potential of solute in extract
μE = chemical potential of solute in raffinate

Therefore:

Where, x = Solute concentration in raffinate (mole fraction)

y = Solute concentration in extract (mole fraction)
μR0 = Standard reference potential in extract
μE0 = Standard reference potential in raffinate

Rearranging we get:

Arka Dhar
Department of Biotechnology, BCET
Where, Kyx = mole fraction partition coefficient

From the above equation it is evident that the greater the difference between the chemical potentials at standard
reference state, the better is the extraction. The equation also indicates that solute transfer is favored at lower
temperatures.

Liquid-liquid extraction involves transfer of solute from one liquid phase to another. The three basic steps common
to all liquid-liquid extraction processes are:
Mixing or Contacting: Transfer of solute between two partially or completely immiscible liquids requires
intimate contact of the two. This is usually achieved by dispersing one liquid (the dispersed phase) as tiny
droplets in the other liquid (i.e. the continuous phase). Solute transport rate depends on the interfacial
mass-transfer coefficient which depends on the hydrodynamic conditions in the system and on the
available contact area. Vigorous mixing can achieve both these requirements.
Phase separation or settling: Once the desired extent of solute transport has been achieved (i.e.
equilibrium has been reached), the next step is to allow the droplets of the dispersed phase to coalesce.
This eventually leads to the separation of the two liquids into distinct layers due to density difference. In a
liquid-liquid extraction process, it is important that the two phases (i.e. raffinate and the extract) should
have sufficient density difference to facilitate segregation of phases.
Collection of phases: After settling (or phase separation), the extract and raffinate phases are collected as
separate streams by appropriate means.

Aqueous two-phase Extraction

Aqueous two-phase extraction which is a special cased of liquid-liquid extraction involves transfer of solute from
one aqueous phase to another. The two immiscible aqueous phases are generated in-situ by addition of substances
such as polymers and salts to an aqueous solution. Two types of aqueous two-phase systems are commonly used:
Polymer-polymer two-phase system
Polymer-salt two-phase system
A polymer-polymer two phase system can for instance be
obtained by mixing dextran and PEG at a certain composition. By
adding specific amounts of these polymers to an aqueous feed
phase (which contains the solute), two aqueous phases, one rich in
PEG and the other rich in dextran can be obtained. Aqueous two-
phase systems can also be generated using a polymer (e.g. PEG or
dextran) and a salt such as sodium or potassium phosphate.
Aqueous two-phase separations take place at certain compositions
only. The following figure shows a PEG-dextran phase diagram
where a solubility curve separates the two-phase region (above
the curve) from the single phase region (below the curve). Such
"binary" phase diagrams which are based on the compositions of
the two polymers (or polymer and salt) only are used for determining the concentrations needed for an extraction
process. These phase diagrams also predict the polymer/salt content of the raffinate and the extract phases. The
composition of the individual phases generated can be obtained using tie-lines as shown in the figure.

Arka Dhar
Department of Biotechnology, BCET
The partition of a solute between the two aqueous phases depends on its physicochemical properties as well as
those of the two polymers (or polymer and salt). The partition coefficient of a biological macromolecule in an
aqueous two-phase system formed by two polymers A and B can be determined using equations of the form shown
below:
-------------------------------------------------(a)

Where, K = Partition coefficient

aA = Association constant for the macromolecule and polymer A
aB = Association constant for the macromolecule and polymer B
b = System constant
CA1 = Polymer A concentration in extract (kg/m3)
CA2 = Polymer A concentration in raffinate (kg/m3)
CB1 = Polymer B concentration in extract (kg/m3)
CB2 = Polymer B concentration in raffinate (kg/m3)

In PEG/dextran aqueous two-phase extraction of proteins, the partition behavior depends to a great extent on the
relative polymer composition. It also depends on the solution pH and the molecular weight of the protein.
Generally speaking, protein partitioning into the PEG rich phase is favored. When a polymer-salt combination is
used to generate the aqueous two-phase system, a protein partitions favorably into the polymer rich phase.

Batch Extraction

In a batch extraction process a batch of feed solution is mixed with a batch of extracting solvent in an appropriate
vessel. The solute distributes between the two phases depending on its partition coefficient. The rate at which the
transfer of solute takes place from the feed to the extracting solvent depends on the mixing rate. Once equilibrium
is attained, the mixing is stopped and the extract and raffinate phases are allowed to separate. Mixer-settler units
are usually used for large-scale batch extraction. The basic principle of batch extraction using a mixer settler unit is
shown below. The mixer unit must be able to generate high interfacial area, must provide high solute mass transfer
coefficient and cause low entrainment of air bubbles. The settler unit must have a low aspect ratio, i.e. be of flat
geometry, must allow easy coalescence and phase separation, and must allow for easy collection of raffinate and
extract as separate streams. The antibiotic penicillin partitions favorably in an organic solvent from an aqueous
fermentation media at acidic conditions. However, at a neutral pH, the partitioning from organic phase to aqueous
phase is favored. Thus the antibiotic could be purified by sequential reversed batch extraction, where the antibiotic
is moved from aqueous to organic phase and back again. This sequence is usually repeated a few times in order to
obtain highly pure antibiotic.

Arka Dhar
Department of Biotechnology, BCET
 Batch extraction using mixer settler unit Sequential reverse batch extraction

If a batch of feed containing R amount of initial solvent and an initial solute concentration of CR0 is mixed with E
amount of extracting solvent, the concentration distribution in the extract and the raffinate at equilibrium is given
by:
CE = KCR ------------------------------------------(b)

Where, CE = solute concentration in extract (kg/m3)

CR = solute concentration in raffinate (kg/m3)

By performing material balance, we get:

RCR0 = RCR + ECE ------------------------------------(c)

The extraction factor λ is defined as:

From equations (a), (b) and (c) we have

The fraction extracted is given by:

Where, p = fraction extracted.

Arka Dhar
Department of Biotechnology, BCET