1. FACTORS EFFECTING PARTITION
IN
AQUEOUS TWO PHASE SYSTEM
Haris saddique
M.Phil scholar
Department of Biotechnology
University of Malakand
Kpk, Pakistan

2. AQUEOUS TWO PHASE SYSTEM
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ATPSs can be considered as an integrated
technique where extraction, concentration and
primary purification are in a single unit operation
The application of two-phase systems has been
focused on the recovery of biomaterials from
fermentation broths and biological extracts.
Normally, the biological products are present in
the broth at low levels or in dilute form and have
to be concentrated, isolated and purified from
other constituents of broth

3. AQUEOUS TWO PHASE EXTRACTION
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Use widely in separation of
proteins, enzymes, viruses, cells and cell
organelles etc .
not denature the biological entities as they might be
in organic solvents.
The proteins are partitioning between two aqueous
phases which contains nontoxic polymers or other
salts.

4. Factors effecting partition
Factors that affecting biomolecule partitioning in
Aqueous Two Phase System:
1.
2.
3.
1. Polymer(s) Concentration
2. Biomolecule Concentration
3. Surface properties of biomolecule
4. Phase composition
5. Salt effects
6. Affinity ligands attached to polymer
7. PH
8. Temperature

5. Biomolecule & Polymers concentration
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Increase in protein concentration and molecular
weight of protein decrease their separation in
the upper phase
In case of polymer concentration viscosity of the
phase play important role b/c polymer may
reduce space to protein sample
So for extreme partition high content of polymer
is to be chosen

6. Surface properties of biomolecules
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Surface net charge on protein and the presence
of different amino acids makes protein
hydrophobic or hydrophilic
Lysine and glutamic acid have relatively low
hydrophobicity, and they partition favorably in
the salt-rich phase in the PEG/salt ATP systems
Whereas tryptophan and phenylalanine, with
their aromatic side group, have relatively high
hydrophobicity and partition to the PEG rich
phase predominantly

7. PH
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PH also effects the partition
In PEG/Salt system as PH changes from acidic
to basic the protein becomes less positive or
more negative charge
In PEG/Salt system negative charge protein
prefer upper phase i.e. PEG layer b/c of
repulsion by salt anion

8. Temperature
At high temperature it is easy to form two
phase system with small concentration of
PEG and salt
 PEG/ dextran system will form two phase
system at lower temperature
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9. Affinity ligand attach to polymers
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PEG has many sites to which other group can
attach
When hydrophobic group is attach the
hydrophobic protein and amino acid will be
separated in the upper phase

10. Salt
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It was observed that salt ions partition differently
between the phases, causing an uneven
distribution in the system that generates a
difference in electrical potential between the
phases.
This difference in electrical potential would be
independent of salt concentration, but linearly
dependent on the partition behavior of the ions.
Polyvalent anions such as phosphate, sulphate
and citrate partitioned preferentially into dextranrich phases, while halides partitioned nearly
equally.

11. Concentration and Purification of
Viruses
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Ultracentrifugation in density gradients is very
efficient in the concentration and purification of
virions from culture fluids
But it does sometimes yield a low recovery of
infectious particles
The loosely attached components of the virus
surface do not withstand the shearing forces
involved in that procedure
In dextran sulphate and PEG system virIon
particle in bottom phase and can be adjusted to
layer using NaCl concentrations and virus
protein in top phase

13. CELL PARTITION
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ATPSs are gentle enough to isolate fragile cell types
without altering their surface structure
It is relatively inexpensive, both in terms of reagents
and apparatus,
Members of a functional subpopulation of cells often
possess similar functional properties but differ in size
and shape
Phase partitioning is one of the few techniques that
separates cells on the basis of surface properties
For best partition countercurrent distribution (CCD) is
used

15. ISOLATION OF THYLAKOID VESICLES

17. THANKS FOR YOUR KIND ATTENTION
&
HAVE A NICE DAY