FACTORS EFFECTING PARTITION

IN

AQUEOUS TWO PHASE SYSTEM

Haris saddique

M.Phil scholar

Department of Biotechnology

University of Malakand

Kpk, Pakistan

AQUEOUS TWO PHASE SYSTEM

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

AQUEOUS TWO PHASE EXTRACTION

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.

Factors effecting partition

Factors that affecting biomolecule partitioning in Aqueous Two Phase System:

1.

1. Polymer(s) Concentration 2. Biomolecule Concentration 3. Surface properties of biomolecule 4. Phase composition 5. Salt effects6. Affinity ligands attached to polymer

2. 7. PH

3. 8. Temperature

Biomolecule & Polymers concentration

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

Surface properties of biomolecules

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

PH

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

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

Affinity ligand attach to polymers

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

Salt

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 dextran-

rich phases, while halides partitioned nearly

equally.

Concentration and Purification of Viruses

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 virIonparticle in bottom phase and can be adjusted to layer using NaCl concentrations and virus protein in top phase

CELL PARTITION

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

ISOLATION OF THYLAKOID VESICLES

THANKS FOR YOUR KIND ATTENTION

&

HAVE A NICE DAY