Determination of cloud-point temperatures for different copolymers

Determination of cloud-point temperatures for different copolymers

G.M.Campese^I; E.M.G.Rodrigues^II; E.B.TambourgiI^*; A.Pessoa Jr^II

^ISchool of Chemical Engineering, Campinas State University, Fax: (0055) (19) 3788-3946, P.O. Box 6066, 13081-970 Campinas - SP, Brazil, E-mail: [e-mail]

^IIBiochemical and Pharmaceutical Department, FCF/USP, P.O. Box 66083, 05315-970, São Paulo (SP), Brazil

ABSTRACT

This paper describes a novel system which has a great potential for use for extractions in biotechnological processes as it uses only polymers and can be operated at moderate temperatures and salt concentrations. The polymers used in this work are ethylene oxide-propylene oxide 10:90 (w/w) (EO10PO90) and ethylene oxide-propylene oxide 20:80 (w/w) (EO20PO80). The temperature required for thermoseparation decreases with increasing PO content of the copolymer and increasing buffer concentration.

Keywords: aqueous two-phase systems, cloud point, ethylene oxide–propylene oxide, thermoseparating polymers.

INTRODUCTION

Aqueous two-phase systems are increasingly being used for separation of biomolecules, cells and cell particles. The systems are composed of two incompatible polymers, e.g. dextran and polyethylene glycol (PEG), or of one polymer (PEG) in a high concentration of salt. These systems are suitable for biological samples because each phase contains 70-90% water, which means that biomolecules will not be denatured (Walter & Johansson, 1994). However, the PEG-salt systems have the disadvantage of low solubility for amphiphilic proteins, which have a high tendency to aggregate in water solutions. Recently the use of thermoseparating polymers in aqueous two-phase systems has been introduced. PEG is a thermoseparating polymer (Saeki et al., 1976), but its cloud point is too high (above 100 ° C) for use in thermoseparating processes for the separation of biomaterial. In the last few years random copolymers of ethylene oxide (EO) and propylene oxide (PO) have been used in aqueous two-phase systems because of their thermoseparating properties (Harris et al., 1991). The use of (EO)-(PO) copolymer instead of PEG in aqueous two-phase systems, will have two positive effects: 1) the copolymer can be recovered after thermal phase separation and reused in a new aqueous two-phase system and 2) the proteins can be recovered in a water phase. Ethylene oxide (EO)-propylene oxide (PO) random copolymers have lower cloud points and have been used in two-stage separation systems for protein purification (Alred et al., 1994; Harris et al., 1991; Persson et al., 1998).

Thermoseparating polymers are polymers that have decreased solubility at elevated temperatures. The cloud-point temperature (CTP) is the temperature at which phase separation starts. At this temperature the solution becomes turbid/cloudy due to the formation of polymer-rich emulsion droplets. Cloud-point is dependent on polymer concentration. The lowest cloud point is referred to as the lowest critical solution temperature (LCST) (Saeki et al., 1976). The (EO)-(PO) copolymers are thermoseparating, i.e., over a critical temperature (the cloud point) the polymer separates from the water solution and a new aqueous two-phase system is formed where the water phase is in equilibrium with the polymer phase. Many thermoseparating polymers contain ethylene oxide groups.

In this work, we present a new thermoseparating two-phase system formed with only one polymer in buffer solution. The polymers used were EO10PO90 and EO20PO80. The influence of pH, buffer concentration and EO/PO concentration on the cloud-point temperature was studied.

MATERIALS AND METHODS

Chemicals

Polymer stock solutions were prepared in potassium phosphate buffer. EO10PO90 (10% ethylene oxide, 90% propylene oxide, average molecular weight 2100 Da) and EO20PO80 (20% ethylene oxide, 80% propylene oxide, average molecular weight 2400 Da).

Two-Phase Systems

All polymer concentrations were calculated as % weight/weight (w/w). The two-phase systems were prepared by dissolving the pure polymers in the buffer solution. Systems with a final weight of 2 g were prepared. All experiments were performed in duplicate and the experimental data are average values.

Measurement of Cloud-Point Temperature

The cloud-point temperatures were taken in a thermostable bath by immersing the polymer solution in a capped glass tube. The temperature was gradually increased by 1° C increments and the CPT was taken at the first sign of turbidity.

RESULTS AND DISCUSSION

Cloud-point curves for the binary systems are shown in Fig. 1. Cloud-point temperature (CTP) varies as a function of additive concentration of PE62 in the potassium phosphate buffer (15mM) and pH. It was observed that the polymer has two cloud points, the lower and the upper, at the same concentration. At concentrations lower than 6%, the system had only one lower cloud-point. Moreover, for the values studied, pH did not influence cloud-point. Polymers have a high cloud-point temperature are not appropriate for utilization in processes that use thermosensitive biomolecules, such as enzymes, because of the denaturation phenomenon.

As a result, a new polymer, was chosen. The EO10PO90, has results showed that this polymer has only one cloud-point and that pH value does not influence its profile. Thus, it was decided to work at pH 7.0, varying only the concentration of the buffer. These results are shown in Fig. 2. Note that at concentrations of 30 and 45 mM, CPT values were similar, but they were different at 15 mM. The increase in concentration of the buffer resulted in a reduction in CPT. This salting-out behavior is well known and has been extensively described in the literature (Johansson et al., 1997; Galaev & Mattiasson, 1993).

CONCLUSIONS

The polymer EO20PO80 had two cloud-points, making its use difficult. Owing to the need to use high temperatures for separation of the phases, sensitive biomolecules can denature. However, the polymer EO10PO90 showed satisfactory initial results.

ACKNOWLEDGEMENTS

Eliana M.G. Rodrigues acknowledges the postdoctoral fellowship received from FAPESP. Gilsinei M. Campese thanks CNPq for the doctoral fellowship.

Received: September 10, 2002

Accepted: April 30, 2003

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*

To whom correspondece should be addressed

Publication Dates

• Publication in this collection
  01 Sept 2003
• Date of issue
  Sept 2003

History

• Accepted
  30 Apr 2003
• Received
  10 Sept 2002