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ENGINEER IN BIOTECHNOLOGY – LABORATORY OF BIOPROCESSES

Practice # 3
Aqueous two-phase systems

Introduction
Aqueous two-phase systems (ATPS) are a primary recovery and purification technique. This
technique has the capacity to carry out the integration and intensification of processes and has proven
to be particularly effective for the recovery of biological compounds. There are several two-phase
systems, such as polymer-polymer aqueous phase systems, alternatively, polyethylene glycol (PEG)
forms a two - phase system when it is combined with certain salts, resulting in polymer - salt systems,
which, because of their low cost and short separation time, are often used. The most used and best
characterized ATPS are formed by PEG - potassium phosphate (PO4). Figure 1 shows a diagram
illustrating the use of aqueous phases for the recovery and primary purification of biological
compounds.

Figure 1. Application of two-phase aqueous systems for the recovery and primary purification
of biological compounds.

The characteristics of biological products, such as molecular weight, isoelectric point and
hydrophobicity influence their preference for each phase of the system. In addition, the parameters of
the aqueous two-phase system play a major role during the partitioning of the product of interest into a
particular phase. These parameters include:
Molecular weight and concentration of the polymer used to construct the system, nature and
concentration of the salts used, system pH, addition of molecules with biochemical affinity,
temperature, etc.
Binodal curve of systems
The formation of the phases of the system is described by the equilibrium diagram or binodal curve of
that particular system, which represents the boundary between the monophasic and biphasic regions.
The polymer - salt combinations below the curve will form a single homogeneous phase, while those
above the binodal curve will form a two - phase system. Figure 2 shows a binodal curve for 1450 g /
mol-phosphate PEG systems.

Figure 2. Representative example of a binodal curve for PEG 1450 g / mol-phosphate aqueous
two-phase systems.

Tie Line Length (TLL)

The composition of a ATPS can be expressed as a function of the concentration of the components in
each of the system phases across the tie line length (TLL). Taking as an example the systems in the
binodal curve shown in Figure 2, the cut line of the systems X1, X2 and X3 is represented by the line
AC. These systems differ in their relation of volumes. The TLL of a particular ATPS can be calculated
using the following equation:
TLL2 = (ΔPEG )2 + (ΔPO4 )2

where: TLL = Tie Line Length

ΔPEG = gradient of PEG concentrations between phases (% w/w)
ΔPO4 = gradient of phosphate concentrations between phases (% w/w)

Molecular weight of the polymer (MW)

The molecular weight (MW) of the polymer used to construct the ATPS plays an important role in the
partitioning behavior of the present compounds. This is due to two phenomena: a) increase in
hydrophobicity in the polymer phase, and b) increase in volume excluded. The compounds with high
molecular weights are influenced to a greater degree by the changes in molecular weight of the
polymer than those with smaller size.

Volumes between phases (VR)

The volume ratio of an ATPS is defined as the ratio of the volume of its upper phase to the volume of
its lower phase (VR). In Figure 2, the system X1 has a VR greater than X2 and X3 because the overall
composition of the system has a higher weight percentage of PEG, which generates a higher phase of
higher volume. Following the same principle, the system X3 has a VR smaller than X2 and X1
because the percentage of phosphates in that system is higher, which generates a lower phase of
greater volume.

The objective of this practice is to study the influence of the MW of the PEG, considering two MW of
PEG, 1000 and 8000, and two TLL, 25% and 45% w/w on the intracellular protein partitioning
behavior of Saccharomyces cerevisiae in PEG-PO4 ATPS.

Procedure
PART A: PREPARATION OF MATERIALS
Activity 1: Preparation of solutions and calculations of ATPS
• PEG 1000 g/mol 80% w/w
• PEG 8000 g/mol 50% w/w
• PO4 solution 40% w/w pH 8
Calculate the amount of standard solution required for each system according to Table 1.

Table 1. Composition of two-phase aqueous systems to study the effect of MW and

TLL. The given composition corresponds to systems with experimental V R close to 1, T of
25 °C and pH 8.
PO4 PEG (g) PO4 H2O (g) Sample System
System PEG TLL PEG (%w/w) 40% (g) (g) (g)
No. (g/mol) (% w/w) (% w/w)

1 1000 25 17.1 10.3 5

B1 1000 25 17.1 10.3 0 5

2 1000 42.9 22 12.3 5

B2 1000 42.9 22 12.3 0 5

3 8000 24.5 13.9 7.7 5

 B3 8000 24.5 13.9 7.7 0 5

4 8000 42.6 20 11.6 5

B4 8000 42.6 20 11.6 0 5

Option 1: defreeze the tube of the homogenate you had the highest concentration of protein (practice
2). Separate 10 ml for liquid-liquid extraction and store at -20 °C the rest.
Option 2: obtain 80 ml of homogenate from wet biomass obtained from S. cerevisiae by using
sonication: add 12 g to 80 ml of lysis buffer and carry out the cell rupture by sonication (8 seconds
pulses, pauses of 20 seconds) during 6 min. Remember to do it with an ice bath. Separate 10 ml for
the liquid-liquid fractionation and store the rest at - 20 °C.

Activity 2: Construction of two-phase aqueous systems

Build the ATPS from standard solutions of PEG 1000 and 8000 g/mol and potassium phosphate (40%
w/w, pH 8) based on the percentages calculated previously, by duplicate. It is usually recommended
to add 10% w/w of the sample to the ATPS. Prepare 3 tubes of each of the 4 systems, two tubes will
be for the samples and another for the blank. Construct 5 g total weight systems as described below:
weigh the amount of phosphate solution required for each system. Then add the amount of PEG
solution, carefully. Add the required amount of water. Do not add the sample yet, store at 4 ° C.
Report: a table with the weighed quantities of standard PEG and phosphate solution, water and
sample for each system, and experimental VR of the blank systems.

PART B: EFFECT OF TLL AND PEG MW ON PROTEIN FRACTIONATION

Activity 2: Determine the experimental VR, the total protein concentration in each phase of
the system and the partition coefficient
All the systems should be at RT before adding the sample. Add the sample to each system. Use the
vortex to agitate and homogenate each tube, so all the components come into contact. Centrifuge at
2,000 rpm for 5 min to accelerate the formation of the phases. Obtain the experimental V R. Recover
one sample from the upper and one bottom phase. Determine protein from each phase and the initial
protein solution clarified. Remember that, if the absorbance is above the upper limit of the standard
curve, it is necessary to dilute the sample. Use as blank the upper or lower phase of the blank
system, as the case may be. Determine the partition coefficient (Kp) of total protein considering the
concentration of protein in the upper and lower phases. The partition coefficient is defined as the
ratio of protein concentration in the upper phase and the protein concentration in the bottom phase.
Report: the experimental VR, the average concentration (including standard error) of protein in the
upper and lower phases, the partition coefficient, the phase in which cell debris is concentrated, and
the mass balance of the recovery process (initial protein = sum of protein in each of the phases).

Key question:
How much of protein did you recover in each phase? What is the best system to recover the
highest amount of protein?

Remember:
- To keep samples (100 ul) of the initial protein solution clarified and of each phase of all
the systems at -20°C for the SDS-PAGE.
- To store at - 20 °C the 70 ml of solution obtained after sonication for the next practice.