Aqueous Two Phase System for the separation for Bio molecules

Examples:

The aqueous two-phase system is designed by a binodal curve which separates two-phase area from the single phase zone. The phase diagram gives the exact composition of the top and bottom phase system The composition represented by the points below the binodal curve are homogenous The two phases are formed only by the composition above the binodal curve Tie line The line joining points representing the top and bottom phase composition in the phase diagram is called tie line ( these points are not arbitrary points in phase diagram ) Any points in the same tie line will have same phase composition but of different in volume ratio of the phases A tie line TMB was fitted with in the phase diagram
Point T( node point) Top phase composition; Point B( node point) Bottom Phase composition; Point M Represent composition of phase system

Plate point ( critical point) The point P represent critical point, which is point at which the composition and volume of the two phases theoretically become equal. Composition System with a short tie line have phases whose composition differs less than those of a system on a longer tie line. System with higher tie lines, proteins as well as others partitioned substances will have an increasingly more one-sided partitioning towards on of the phases. This creates the possibility of adjusting the extraction via choice of tie line.

Phase diagram of different two-phase aqueous system :

Determination of Phase diagram( binodal points, critical point, tie line) of aqueous two-phase system:
Few grams of concentrated polymer P ( PEG) is kept into a test tube. A known concentration of polymer( D , Dextran) is then added drop wise. 1st, homogenous mixtures obtained, but after certain amount of polymer D addition, turbidity appears and tqo phase system will arise. The composition of the mixture in noted . 2nd , 1.0 ml water is added, the mixture becomes clear again 3rd, More solution of D is then added drop wise until turbidity and two phase system arises, The composition is again noted In this way a series of composition close to binodal points are obtained and if the concentration ( % w/w)of P is plotted against that of D for these composition.
Dextran

Peg (%w/w)

Peg (%w/w)

PEG %(w/w)

Dextran%(w/w)

How to determine the tie line ?

For this we have to determine the top phase and bottom phase composition for the two phase forming polymer. For example you should form a two phase aqueous system by mixing certain amount of PEG and Dextran, then analyze the composition of PEG & Dextran both Top and Bottom phase. Now put top phase composition and bottom phase composition in the binodal curve to get the points T and B ( binodal points) respectively. Joint TB to get the tie line.

Tie line and binodal curve determination :

Prepare a few systems with different concentrations of phase forming components. The systems are equilibrated at a given temperature and, when the phases have settled, they are collected and analyzed for the constituents. In PEG-Dextran systems the dextran concentration ( % w/w) is determined by polarimetry and refractive index method Polarimetry: A known weight, m( 4-10 g) of phase is diluted to a given volume, V( 25-50 ml), with water and the angle of the optical rotation, ( degrees), is measured using Na light ( 589 nm). With specific optical rotation 199g dm ml-1 degree-1 for dextran its concentration, CDx ( %w/w) in the phase is given by

[ ( degrees)]T = / ( l x d); T: temperature, : wave length ( nm). L: Path length ( dm),: specific rotation when l=1 dm, d=1g/ml The concentration of PEG can be determined from the dry weight(1100 C) of known amount of phase which is corrected for the amount of dextran. Refractive Index Method: A more reliable method is to determine the increment n, in refractive index ( relative to water) of the same dilution as used for the determination of dextran. The increment is 0.137 ml g-1 for PEG and 0.1515 ml g-1 for dextran at 200 C. The PEG concentration, CPEG ( % w/w) is obtained by using the equation

Calculation of volume ratio of TOP & BOTTOM phase from phase diagram:
PEG (% w/w) T A A B Dextran (% w/w)

A,A,B,T all the points have same total phase composition(same total amount of PEG and Dextran) The weight ratio ( %w/w) bottom phase/top phase is equal to the ratio of AT : AB ( at point A)
Volume of top phase(Vt ) AB Volume of bottom phase(Vb)

AT

mt - mass of top phase; mb - mass of phase; m0 - total mass Vt - volume of top phase;Vb - volume of bottom phase dt - density of top phase Ct - concentration of polymer P in ( % w/w) in top phase Cb - concentration of polymer P in ( % w/w) in bottom phase C0 - total concentration of polymer P in ( % w/w)

mt + mb = m0 (1) mt = Vt . dt .Ct (2) mb = Vb .db .Cb (3) m0 = (Vt . dt + Vb .db ) .C0..(4) Now, Substitute (2), (3), ( 4) in (1) Vt . dt .Ct + Vb .db .Cb = (Vt . dt + Vb .db ) .C0
Vt . dt

From the diagram

Cb C0 C0 - Ct

AB

Vb . db

Cb C0 C0 - Ct

AT AB

Hence

Vt . dt Vb . db Vt

= =
dt

AT AB

Vb

db

AT

The density of polymer phase are not very much different from water ( usually 1.00-1.01) and ratio of the volume of the two phases may be obtained therefore, approximately from the distance AB & AT on the tie line.

Relation among yield Y, the separtion factor G, depending on the volume ratio R, and K
In general phase diagrams are altered by addition of other components in sufficient concentration such as cell homogenate and different salts. Phase diagram is very useful for the selection of phase system ( choosing the Top and bottom phase volume for partitioning the protein of interest) The separation power of a system is characterized by the yield Y, the separtion factor G, depending on the volume ratio R, and K. The yield of top phase is defined as

YT =

NT NT + NB

K=

CT CB

N is the molar quantity of the desired protein in the volume of the top ( V T) and bottom phase ( V B), respectively. G is defined as the product of the partition coefficient K and the volume ratio R. Y, K, and R affect each other and usually YT increases if K is increased at a constant R or if R is increased at constant K.

Interdependence of yield (YT ), partition coefficient ( K) and volume ration ( R).

Separation of phase system : The separation power of phase system is characterized by the yield ( Y ), the sepaartion factor G, depending on the volume ration ( R ), and K. The yield in top phase is defined as N

YT =

NT + N B

Where N- molar quantity of desired protein in the volume of the top ( VT ) and the bottom phase ( VB ), respectively.

Partition of a proteins, bovine serum albumin, in two phase system with increasing tie line length, S ( i.e. increasing polymer concentration. The protein is partitioned at its isoelectric point in the system containing dextran T500 and PEG 8000( o) or PEG 3500(), Temperature 200 C.

Factors determining K:
The partition coefficient K does not depend on the concentration of the protein i so long as the concentration ci is much smaller than the concentration of the phase -forming polymers, and no complex formation takes place. There exists an exponential relation between K and surface area( molecular mass) of i component

The Effect of salt on partitioning of protein:

Effect of Temperature: The influence of temperature on the protein partitioning in two-phase system is complex. In general - On increasing temperature shorter tie line is formed when it is polymerpolymer type - For PEG-Salt system the tie line length is increased - The K0 and may also be strongly influenced Normally for the extraction process temperature is kept 00 C 250 C

Correlation of Charge, MW, K of protein in aquous two phase system

M.W. and K quite satisfactorily correlated with PEG/Polymer ratio, PEG/Salt ratio. The effect of M.W. of phase forming polymer is such that when the M.W. of one polymer is decreased, the protein tend to favour the phase rich in this polymer. Partitioning of high M.W. proteins are more affected by charges and the M.W. of the polymer than small M.W. proteins. The partition coefficient correlates directly with the difference in M.W. between two phase polymers, the greater the difference in M.W. larger the deviation of K from unity. The proteins favour the phase that contains more compatible polymer in terms of surface charge and hydrophobicity. K of native protein and denatured protein are different and K value of native ( globular) weakly depend on M.W., whereas K value of denature protein depends strongly on M.W.

Aqueous two phase extraction Process:

Aq. Feed containing cell debris & Proteins
Bottom phase

PEG-Salt mixture

1st stage extraction

Heavier salt phase ( removal of cell debris)

Top phase

Addition of Salt solution to top phase( pH, ionic

strength controlled)

2nd stage extraction

Bottom phase

Heavier salt phase ( removal of NA, bulk proteins, polysaccharides)

Top phase

Addition of salt solution to top phase

3rd stage Bottom phase Heavier salt phase extraction ( containing desired protein)

Top phase PEG ( residual polymer recycled)

Ultrafiltration (for protein recovery)

Affinity partitioning:

Polymers are carrier of affinity ligand. For most purposes it is enough to introduce the ligand ( 1 or 2 molecules per molecule of polymer) or a few percent of the actual polymer The ligand has a weak influence on the distribution of the polymer between the phases. The partitioning of the polymer-bound ligand can be regulated by changing the composition of the system across the tie lines. The presence of a ligand mainly in one phase tremendous effect on partitioning of ligand-binding proteins e.g. enzymes. This type of specific extraction is known as affinity partitioning.

logK = log [ K( with ligand)/ K( without ligand)

Influence of ligand concentration on protein partitioning

Lactate Dehydrogenase

Bulk protein

The concentration dependence in saturation curve: In this case, when the enzyme concentration was around 0.25g/kg(total) system and the concentration of bulk protein was 6kg/kg(total) system, not more than 2% of the PEG molecules have to carry one ligand molecule each in order to achieve maximal effect.

The maximum K value, Kmax :

The maximum K value, Kmax , for a protein in the presence of ( excess of) ligand-PEG is given by equation

n number of ( effective) binding sites on the protein molecule, KL-PEG - partition coefficient of ligand-PEG Kass,T , Kass,B - association constants in top and bottom phase respectively for the interaction between ligand and one binding site. The binding sites are assumed to be identical and independent. KL-PEG can reach values as high as 50 and if we assume equal association constants in the phases, that a single binding site on the protein molecule would give a 50-fold increase in the partition coefficient Two binding sites would give a 2500- fold increase etc. Therefore, a low number of binding sites ( e.g. one or two) is no limitation on an effective extraction. Experimental values for the maximal increase in the partition coefficient of an enzyme as a function tie line length. Variation of Kaff( fractional increase in K) for phosphofructokinase ( 4Kat l-1 ) from using Cibacron blue G3G-A-PEG with ssytems of increase tie-line lengths. Phase system: dextranT500, PEG8000 and 80mM Naphosphate buffer, pH 7.0. Temperature, 00 C.
S ( tie line length)

The partition coefficient of bulk protein is 0.01 100 The protein has two biding sites for the ligand.

Capacity of affinity partitioning in the PEG-ligand layer :

Kass of protein in the both phases ( Dextran and PEG) are same ( 106 M-1 ) and the extracion of protein ( in various concentration) ion the top phase containing various % of ligand-PEG is shown in the figure. The partition coefficient of bulk protein is 0.01 The partition coefficient with ligand in the system to be 100 and the two phases have equal volume and the protein has two binding sites for the ligand. Good recovery in the upper phase ( 95%) at 100% ligand -PEG can be obtained for protein concentration as high as500g L -1 Even if the association constant is reduced to 10 M , the enzyme present at a concentration of 100g/L is extracted by using half of the PEG as ligand.

Affinity Partitioning of protein:

Bulk protein K = 0.01, enzyme K for affinity ligand = 100 The upper phase is repeatedly washed with fresh lower phase, keeping the volume constant. The extraction is done with systems of phase volume ratio V ( top : Bottom) values 5, 1 and 0.2. By using small top phase ( one sixth of the system) to which enzyme is extracted, high purity is achieved after only one washing, but nearly 10% of the original enzyme is lost. With a large top phase ( five-sixths of the total system) 3- fold washing is necessary to reach the same purity but as much as 99% of enzyme recovery. Thus , volume ratio and number of washing steps are parameters which can be adjusted to achieve the most economic extraction process.

Enzyme purification by affinity partitioning :

In practice the bulk proteins have a broad range of partitioning constants. It is therefore useful to make a pre-extraction of one or two steps before introducing the affinity ligand.. By this pretreatment, proteins of high or moderate partition coefficient will be removed. The top phase used for pre-extraction is removed and replaced by the ligand-containing phase in order to extract the target enzyme. Further purification is obtained by washing this top phase a few times by equilibrating with new portion of fresh bottom phase in order to remove other weakly co-existacted enzymes and traces of bulk proteins. The final top phase in which the target enzyme and the ligand-PEG are associated has to be further treated to separate the ligandPEG, for recycling, and to obtain the free enzymes. This could be done by two ways:

Enzyme purification by affinity partitioning : Extraction of the enzyme from top phase by two ways

1. Addition of phosphate , sulphate or a citrate salt in the top phase, which result in a PEG-salt two phase system. The high salt concentration inhibits ( in most cases) the affinity of the ligand for the enzyme. Because of the high PEG content of the upper phase ( 30-45%) the enzyme is usually totally excluded and is found in salt-rich bottom phase in good yield ( 95-100%). The ligand-PEG remains in the concentrated top phase ( 95-97%) and can be used directly or a new set of extraction.

2. The enzyme can be collected in a dextran phase, added to the final top phase, by introducing free ligand which will compete with PEG-bound one for the enzyme. If the ligand bound to PEG is less specific the use of natural free ligand will also give rise to a further purification in this step.

Lactic dehydrogenase ( LDH) purification by using PEG-Procion yellow HE-3G as affinity ligand in PEG-Dextran system

System composition : 10% Dextran, 7.03% PEG, 0.07% Procion yellow HE-3G, 50mM sodium phosphate buffer, pH 7.9, and 25% muscle extract, temperature 00 C. After the affinity partitioning step the upper phase of this system has extracted twice with the same volume of pure lower phase. LDH activity is expressed as micromole of substrate converted per minute at 250 C.

Recovery and purification factor of LDH in upper phase, using systems with different concentrations of dextran 500 and PEG 800. Systems contained, besides the polymers, 25% ( w/w) protein extract and 12.5mM sodium phosphate buffer pH 7.9; 1% PEG carrying procion yellow HE-3G, temperature 00 C.

Large scale extraction process by using thermo reversible polymer :

PEG polymer is replaced by thermo separating random copolymer of ethylene oxide( EO) and propylene oxide( PO) and the dextran has been replaced with starch polymers. Solution of EOPO coplolymer separate into one water phase and one copolymer phase when heated. The copolymer phase will be depleted from protein which makes it possible to recycle the copolymer after thermo separation. The target protein is obtained in a water phase.

Problems in Aq. Two phase system:

1.Calculate the volume ratio of Top : Bottom phase of the following phase composition of dextran and PEG.
System Total System Dextran (%w/w) A B 0.20 6.67 PEG (%w/w) 6.51 4.76 Bottom phase Dextran (%w/w) 17.01 17.79 PEG (%w/w) 0.86 1.0 Top phase Dextran (%w/w) 0.01 0.03 PEG (%w/w) 6.56 7.03

2.Calculate the amount of Dextran and PEG in gram needed for the preparation of 1.0 litre of total phase having Top : Bottom phase volume ratio a) 2 : 8, b) 5 : 5, c) 8:2 The node point composition of the tie-line is following:
Dextran(%w/w) Top Bottom 0.08 15.59 PEG(%w/w) 6.02 0.80

1.Calculate the volume ratio of Top : Bottom phase of the following phase composition of dextran and PEG.
System Total System Dextran (%w/w) A 0.20 PEG (%w/w) 6.51 Bottom phase Dextran (%w/w) 17.01 PEG (%w/w) 0.86 Top phase Dextran (%w/w) 0.01 PEG (%w/w) 6.56

Mass of PEG in Top phase( mP,T ) + Mass of PEG in bottom phase ( mP,B ) = Total mass of PEG in a tie line ( mP,total ) m = volume x density x concentration .(1) mP,T = VT x T x CP,T ( %w/w); CP,T ( %w/w) = 6.56(2) mP,B = VB x B x CP,B ( %w/w); CP,B ( %w/w) = 0.86(3)
mP total = [( VT x T ) + ( VB x B )] CP total ( %w/w); CP,total ( %w/w) = 6.51..(4) ( VT x T x CP,T ) + (VB x B x CP,B ) = [( VT x T ) + ( VB x B )] CP total ( %w/w) ( VT x T ) ( CP,T - CP total ) = (VB x B ) ( CP,total - CP,B ) ( VT x T ) / (VB x B ) = ( CP,total - CP,B ) / ( CP,T - CP total ) = ( 6.51-0.86) / ( 6.56-6.51)

Problem related to affinity based partitioning:

3.The crude extract of 1.0l solution having alkaline phosphatase 5.0% of the total protein ( 30.0 g/l), is allowed to partitioned PEG ( contain 4% Porcian red HE-3G PEG ligand) and dextran phase of top : bottom phase volume ratio is a) 4:1; b)1:1; c) 1:4 respectively. The K value of alkaline phosphatase in PEG layer contain porcian red and dextran phase is 100 and rest of the protein is 0.01. How many times the upper PEG layer should be partioned with same volume of dextran phase to get 99% of pure alakaline phosphatase in PEG layer. How will you get back alakaline phosphatase from upper phase ( PEG layer) to lower phase dextran layer.