ATPS

• To extract biologically active polymers such as

proteins, enzymes and nucleic acid it is necessary
to have a two phase system which will not
denature the biopolymers.
• In aqueous two phase extraction the basic
principle involves the differential partitioning of
desired protein in two immiscible phases
extracted from an aqueous feed containing a
complex mixture of proteins.
• The term aqueous was used as it contains
relatively high water content (80-85%)
Types of Aqueous two phase systems
Polymer – Polymer systems
PEG Dextran
Polyvinyl alcohol (PVA)
Polyvinyl pyrrolidone(PVP)
Ficoll
Polypropylene glycol Dextran
PVA
PVP
PVA Dextran
Methyl cellulose
PVP Dextran
Methyl cellulose
Polymer - Salt (low molecular weight ) systems
 
PEG Potassium phosphate
PVP Potassium phosphate
PPG Glucose
Dextran Propyl alcohol
Sodium dextran sulphate Sodium chloride
Polyelectrolyte –Non ionic polymer + Salt systems
 Sodium dextran sulphate PEG and Nacl
PVA and Nacl
Sodium Carboxy methyl cellulose PEG
PVA
 PHASE DIAGRAMS
TERNARY PHASE DIAGRAM

• PHASE DIAGRAMS
 
• TERNARY PHASE DIAGRAM
• The point A lies above the bimodal curve and hence exist as homogenous
phase
• Whereas the point B lies below the bimodal curve and hence it creates a
biphase
• For example one phase contains 90% water and 10% PEG and other phase
contain 80% water + 19% Dextran and 1% PEG
The properties of ATPS depends on factors such as
• Molecular weight of polymers
• Viscocity
• Density
• Interfacial tension and
• Temperature
• High molecular weight polymers give phase separation even
at low concentration
• Viscocity is an important parameter in bringing the mass
transfer between the phases
• Difference in density between two phases is preferable for
better contact and phase separation, however the ratio of the
density is mostly close to the unity in most cases.
• A low interfacial tension is preferable for effective dispersion
and creation of high interfacial area.
• Temperature influences the contacting process through
changes in viscocity and phase equilibrium
 THEORETICAL PRINCIPLES OF ATPS

• The partitioning coefficient K may be expressed as the ratio of the

concentration of the desired protein in the lighter phase to that in
the denser lower phase.
 
• The partition coefficient may be expressed as a function of five
important factors involving electrical, hydrophobic, hydrophilic,
conformational and ligand type interactions given by:
• ln K= ln Kelec + ln Khphob + ln Khphil + ln Kconf + ln Klig

The partition coefficient for a protein in ATPS is influenced by factors

such as
• The molecular weight of the phase forming polymer
• Type of salt and its concentration and
• pH
Examples
• Partitioning of pullulanase in PEG phase of PEG-
Dextran system- The K values decreased from 1.3 to
0.25 as the mol.wt of PEG increased from 1500 to 6000
• The partitioning of the glucose-6-dehydrogenase
increased in PEG-Cibacron blue- salt system due to the
presence of the affinity ligand cibacron in one of the
phase.
• The K values for L-Hydroxyisocaroate dehydrogenase
partitioning in PEG-Potassium sulphate system showed
that the K values increased as the pH increased from 5
to 7.2.
AQUEOUS TWOPHASE EXTRACTION
PROCESS
 EQUIPMENT FOR ATPS

The ATPS can be carried out in batch mode or

continuous mode. But the continuous mode
has several advantages compared to the batch
mode such as
• Product uniformity and purity
• Automated operation
• Smaller process equipment
• But the problems in continuous mode are
viscocity and the low interfacial tension.
• Hence taking both these factors into
consideration, the equipment used in ATPS
include
• inline or static mixers in combination with
centrifugal separators or disc-stack centrifuges,
nozzle separators or decantor extractors, used
for batch as well as continuous operations.
APPLICATIONS
 
• RECOVERY OF ENZYMES
– Recovery of fumarase from B.ammoniagenes
– Pencillin acylase from a recombinant E.coli
– Recovery of formate dehydrogenase
• Recovery of bulk proteins and valuable proteins from
waste stream
• Partioning of cell debris
• Affinity portioning