and Potassium Phosphate - American Chemical Society

Article pubs.acs.org/jced

Interfacial Tension of Aqueous Two-Phase Systems Containing Poly(ethylene glycol) and Potassium Phosphate Cássio Carmo de Oliveira,† Jane Sélia dos Reis Coimbra,*,† Abraham Damian Giraldo Zuniga,‡ Joaõ Paulo Martins,§ and Antonio Marcos de Oliveira Siqueira∥ †

Department of Food Technology, Federal University of Viçosa, P. H. Rolfs Av., s/n, 36570-000, Viçosa, MG, Brazil Food Engineering Course, Federal University of Tocantins, NS 15 Av., ALCNO 14, 77123-360, Palmas, TO, Brazil § Federal Institute of South of Minas Gerais, Dr João Beraldo, 242, 37550-000, Pouso Alegre, MG, Brazil ∥ Department of Chemistry, Federal University of Viçosa, P. H. Rolfs Av., s/n, 36570-000, Viçosa, MG, Brazil ‡

ABSTRACT: The effect of poly(ethylene glycol) (PEG) molar mass, pH, and temperature on interfacial tension was investigated for aqueous two-phase systems composed of PEG with molar masses of (1500, 4000, 6000, and 8000) g·mol−1 and potassium phosphate. The temperatures tested were (293 and 313) K at the pH values of 6, 7, and 9. Interfacial tension was determined by using a spinning drop tensiometer and the Vonnegut equation. An increase in both the PEG molar mass and the temperature resulted in an increase of interfacial tension values. The interfacial tension varied from (0.05 to 3.11) mN·m−1 for systems composed by PEG 4000 (mass fraction; w = 0.12) + potassium phosphate (w = 0.11) + water (w = 0.77) at pH 6.0 and 293 K and PEG 4000 (w = 0.19) + potassium phosphate (w = 0.16) + water (w = 0.65) at pH 7.0 and 313 K, respectively.

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INTRODUCTION Aqueous two-phase systems (ATPS's), as an alternative separation technique for biological materials, are of substantial interest to the biotechnology industry. Biopharmaceutical companies faced with increasing product quality standards and stiffening economic competition are reconsidering ATPS's as an alternative to chromatography. These systems are formed using two polymers soluble in water or a water-soluble polymer and a component of low molar mass, like inorganic salts.1 Due to their low interfacial tension and high water content, the ATPS's provide mild separation conditions to preserve biological activities of labile compounds, such as proteins, cells, or other biological materials.1 ATPS's have been utilized in the determination of hydrophobic properties of cell membranes and extractive bioconversion of biocompounds, as well as the separation and purification of metals, proteins, enzymes, hormones, organelles, and cells.1−3 The acquisition of equilibrium data and the properties of ATPS's, such as interfacial tension, are necessary for the design of extraction processes and for the development of models that can predict biomolecule partitioning between phases. The interfacial tension between phases plays a decisive influence on the separation and partition mechanism of biomolecules and cells, as well as participates in dispersion, emulsification, flocculation, and solubilization processes. Interfacial tension influences the shape of fluid interfaces and controls their deformability. Data on interfacial tension and other physical properties allow for the prediction of system © 2012 American Chemical Society

behavior, velocity of phase formation, phase separation, and reagent composition for the system. The methods for the determination of interfacial tension can be divided as static (in the pendant drop technique) and dynamic (in the spinning drop technique) which was introduced in 1942 by Vonnegut.4 The spinning drop method4 was applied to determine interfacial tension of the ATPS poly(ethylene glycol) (PEG) + dextran (DEX) as reported by Ryden and Albertsson.5 The interfacial tension between two aqueous rich phases is usually very small, often between (0.001 and 1) mN·m−1. The use of standard methods for interfacial tension measurements in this range, such as the capillary or ring techniques is not easy; thus, the spinning drop technique appears to be a suitable technique for the determination of very low interfacial tension values. Forciniti et al.6 used the spinning drop technique to study the interfacial tension of PEG + DEX ATPS's. The authors reported a detailed investigation of interfacial tension as a function of temperature, polymer concentration, and PEG and DEX molar masses. Very low values for interfacial tension were found, between (1.5·10−3 and 0.35) mN·m−1, when compared to other organic extraction systems. For instance, systems composed of hexane + water, glycerin + hexane, and toluene + water presented interfacial tension values equal to (48.5, 34.9, and 35.7) mN·m−1, respectively.7 Bamberger et al.8 also studied Received: October 8, 2010 Accepted: May 25, 2012 Published: June 1, 2012 1648

dx.doi.org/10.1021/je1010104 | J. Chem. Eng. Data 2012, 57, 1648−1652

Journal of Chemical & Engineering Data

Article

obtained by reweighing the pycnometer filled with the phases and computing the mass difference with the empty pycnometer. The volume used in this calculation was equal to that of distilled water for calibration. The temperature control for each sample was maintained by a thermostatic water bath (TE-184, Tecnal, Brazil), accurate to 0.1 K. The procedure was repeated for the superior and inferior phases with unknown density. Phase density measurements were analyzed in triplicate. The expanded uncertainties13,14 of the density data, at 95 % confidence, ranged from (0.01 to 0.004) g·cm−3. Interfacial Tension. Interfacial tension data were obtained by using a spinning drop tensiometer (SITE 04, KRUSSGmbH, Hamburg, Germany). Experiments were conducted at (293 and 313) K and pH values of 6, 7, and 9. The pH was monitored using Digimed DM20 (Sao Paulo, Brazil) equipment, accurate to ± 0.01. All measurements were performed with at least three repetitions for each system and each rotational speed by using the following procedure: (a) The capillary tube temperature (3.5 mm i.d.) of the tensiometer was stabilized within ± 0.01 K by using a thermostatic oil bath (DC30/DL30, Haake, Frankfurt, Germany). (b) The heavy bottom phase rich in potassium phosphate was gently introduced into the capillary tube to avoid bubble formation and expel air. (c) The capillary tube rotation speed was adjusted with a speed controller, and 10 μL of the light top phase rich in polymer was injected in the capillary, using a microsyringe (Hamilton, Reno, NV, USA). The formed drop was centralized on the eyepiece. (d) The lens focus for drop visualization was found in each measurement. The scale zero point for the diameter reading was coincidentally adjusted with the drop bottom limit. (e) Rotation speed was adjusted for each operational velocity of (2000, 3000, 4000, 5000, and 7000) rpm, accurate to 1 rpm. After 10 min, the drop diameter was recorded as the scale diameter value (sdv) of the instrument, accurate to 0.03 sdv. Before starting the measurement, a drop of heavy phase was placed in the device several times until we visually observed the drop in the capillary. The PEG + potassium phosphate ATPS interfacial tension was determined by the Vonnegut equation,4 as recommended by the manufacturer of the equipment, as follows:

the interfacial tension behavior of PEG + DEX systems with different compositions. Whu and Zhu9 developed a model for interfacial tension prediction by using experimental data obtained by Forciniti et al.6 The model was able to predict low values of interfacial tension from (1.0·10−4 to 1.0) mN·m−1. Zuniga et al.10 reported the influence of PEG molar mass on interfacial tension values for ATPS composed of (4000, 6000, and 8000) g·mol−1 PEG and maltodextrin (MD), with a molar mass of 2800 g·mol−1. Interfacial tensions of ATPS containing (4000, 6000, and 20 000) g·mol−1 PEG and dipotassium hydrogen phosphate were studied by Mishima et al.11 using a shaking flask method and a drop volume method at (288.15, 298.15, and 308.15) K, obtaining interfacial tension values in the range of (1.0·10−3 to 1.26) mN·m−1. Kim and Rha12 reported interfacial tension values as low as 0.01 mN·m−1 in PEG + potassium phosphate ATPS's. The authors observed an increase in interfacial tension with the increase of the polymer and salt concentrations. In this work, the Vonnegut equation was used to determine interfacial tension of systems composed by PEG with molar masses of (1500, 4000, 6000, and 8000) g·mol−1, potassium phosphate, and water. The temperatures tested were of (293 and 313) K at pH values of 6, 7, and 9.

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EXPERIMENTAL SECTION Materials. The PEG (1500, 4000, 6000, and 8000) g·mol−1 (0.95 mass purity) used in the experiments was purchased from Sigma (Sigma-Aldrich, St. Louis, MO). Potassium phosphate mono and dibasic (0.98 mass purity) was supplied by Vetec (Brazil). Deionized water (Milli-Q, Millipore, USA) was utilized in all experiments (R ≥ 18.2 MΩ·cm−1). All of the other reagents were analytical grade with a minimum purity of 0.99. Aqueous Two-Phase Systems. Stock solutions of PEG mass fraction (w = 0.50) and potassium phosphate (w = 0.30) were prepared by the addition of deionized water (Milli-Q, Millipore, USA) to a predefined quantity of polymer and salt. Mixtures consisting of known masses of PEG and potassium phosphate stock solutions were prepared by weighing aliquots on an analytical balance accurate to 0.0001 g (M-310, Denver, USA). The mixing cell utilized for ATPS preparation was constructed of borosilicate glass and sealed with a Teflon cork with two openings, one for a thermometer and the other to feed the system. A water filled jacket with capacity for 50 mL surrounded the flask for temperature control. The system was agitated using a magnetic stirrer for 1 h and maintained at rest in a thermostatic bath water with an accuracy of ± 0.1 K (TE184, Tecnal, Brazil) for 12 h which was the time necessary to reach equilibrium. After this period the two phases became clear and transparent, and the interface was well-defined. Finally aliquots of each phase were withdrawn, and the density and interfacial tension were measured. The ATPS's were prepared at pH values of 6, 7, and 9. To adjust the pH, solutions of NaOH and HCl, supplied by Merck (Germany), were used to reach the desired pH value in the aqueous phase. The measurement of pH values was performed using a Digimed DM20 (Brazil) equipment, accurate to ± 0.01. All pH measurements were performed with at least three repetitions. Phase Density. Phase density was determined using a pycnometer. The technique uses a working fluid with wellknown density, such as water. Thus, the pycnometer was previously calibrated with distilled water at the operational temperatures of (293 and 313) K. The density (ρ) was

γ = e·(v ·d)3 ·n2 ·Δρ

(1) −1

where γ is the interfacial tension (mN·m ), e is a constant value (3.427·10−6 mN·min2·mm−1·g−3), v is a correction factor (0.167 mm·sdv−1), d is the drop diameter measured in the instrument unit (sdv), n is the rotational speed (rpm), and Δρ is the density difference between each phase (g·cm−3). Princen et al.15 when studying the relation between the spinning drop shape and interfacial tension concluded that the Vonnegut equation can be used for systems at high rotation speed. The expanded uncertainty12,13 of the interfacial tension performed within this work, with a level of confidence of approximately 95 %, was estimated to be between 3 % and 8 %, corresponding to the values ranging from (3.1 to 0.25) mN·m−1.

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RESULTS AND DISCUSSION Table 1 shows the PEG mass fraction (wPEG), potassium phosphate mass fraction (wPP), water mass fraction (wH2O), and PEG molar mass (M) for each analyzed system. The interfacial tension for ATPS's was calculated using five different rotation speeds. The average interfacial tension was determined as an arithmetic median between interfacial tension values at (2000, 1649



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Table 1. PEG + Potassium Phosphate ATPS Composition system 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

−1

M/g·mol 1500 1500 1500 1500 1500 4000 4000 4000 4000 6000 6000 6000 6000 8000 8000 8000 8000

100w PEG

100w PP

100w H2O

14 16 16 18 18 12 15 17 19 10 12 14 16 12 14 16 18

14 14 16 14 18 11 12 15 16 10 11 12 14 10 11 14 14

72 70 68 68 64 77 73 68 65 80 77 74 70 78 75 70 68

Table 2. Interfacial Tension of the PEG + Potassium Phosphate ATPS, at 293 K and pH 6 system 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

3000, 4000, 5000, and 7000) rpm. The influence of the rotation speed on interfacial tension is shown in Figure 1, in which the systems presented were taken as an example since the behavior for all systems was the same.

(Δρ ± uncertainty)

(γAV ± uncertainty)

σ

g·cm−3

mN·m−1

mN·m−1

0.086 0.114 0.128 0.118 0.164 0.044 0.077 0.122 0.146

± ± ± ± ± ± ± ± ±

0.002 0.002 0.003 0.002 0.003 0.001 0.002 0.002 0.003

0.049 ± 0.001 0.071 ± 0.001 0.103 ± 0.002 0.045 ± 0.001 0.083 ± 0.002 0.098 ± 0.002

0.151 ± 0.007 0.281 ± 0.011 0.493 ± 0.018 0.401 ± 0.015 0.959 ± 0.032 0.050 ± 0.002 0.249 ± 0.009 0.737 ± 0.024 1.037 ± 0.033 no phase formation 0.100 ± 0.004 0.260 ± 0.010 0.421 ± 0.015 no phase formation 0.100 ± 0.004 0.601 ± 0.019 0.567 ± 0.019

0.023 0.016 0.012 0.012 0.008 0.015 0.011 0.009 0.011 0.010 0.011 0.007 0.011 0.002 0.006


Figure 1. Interfacial tension versus rotational speed (n) for the PEG + potassium phosphate ATPS: ▼, system 1, pH 7; ○, system 3, pH 7; ●, system 4, pH 9 at 293 K.



σ

g·cm−3

mN·m−1

mN·m−1

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.030 0.010 0.011 0.007 0.007 0.012 0.029 0.021 0.023 0.022 0.022 0.028 0.016 0.011 0.018 0.006 0.013

0.093 0.107 0.118 0.120 0.149 0.057 0.077 0.012 0.150 0.039 0.060 0.076 0.107 0.049 0.068 0.107 0.121

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.002 0.002 0.002 0.002 0.003 0.001 0.002 0.000 0.003 0.001 0.001 0.002 0.002 0.001 0.001 0.002 0.002

0.289 0.359 0.542 0.551 1.007 0.120 0.350 0.881 1.255 0.040 0.189 0.369 0.768 0.150 0.331 0.822 1.013

0.011 0.013 0.019 0.019 0.032 0.005 0.012 0.020 0.039 0.002 0.007 0.013 0.024 0.006 0.011 0.026 0.031

Our experimental data of interfacial tension varying from (0.05 to 3.11) mN·m−1 are in good agreement with the range of values found by Mishima et al.11 for the PEG + dipotassium hydrogen phosphate ATPS between (0.098 and 1.26) mN·m−1. This demonstrates the reliability of the Vonnegut method for measurements of interfacial tension. Figure 2 shows that the interfacial tension increases with the rise of PEG molar mass for systems with the same composition for PEG (w = 0.16) and PP (w = 0.14). Kim and Rha12 also used the spinning drop technique to study the interfacial tension of PEG + potassium phosphate systems, verifying a rise in interfacial tension for the increase in both PEG and salt concentrations, as well as the increase of PEG molar mass. Mishima et al.11 analyzed the effect of polymer molar masses of (4000, 6000, and 20 000) g·mol−1 and temperatures of (288.15, 298.15, and 308.15) K on the interfacial tension for the PEG + dibasic potassium phosphate ATPS, using the drop volume

The rotational speed range for interfacial tension measurement of each phase was greater than 2000 rpm to guarantee that gyrostatic equilibrium was reached.6 The increase of the rotation speed presents a minimal (7 %) elevation in interfacial tension obtained by using eq 1 for the system tested; small variations in interfacial tension values are probably caused by the deformation of the drops inside the tensiometer.16 Kim and Rha,12 studying the PEG + potassium phosphate system, observed that the interfacial tension became constant for rotational speeds greater than 2500 rpm. Tables 2 to 5 show the phase density difference (Δρ) ± the uncertainty for each PEG + potassium phosphate (PP) ATPS analyzed, the average interfacial tension (γ AV ) ± the uncertainty, and their corresponding standard deviation (σ). The mean standard deviation found for the density experimental data was 0.001 g·cm−3 (data not shown). 1650



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σ

g·cm−3

mN·m−1

mN·m−1

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.011 0.011 0.009 0.015 0.007 0.010 0.006 0.005 0.003 0.005 0.009 0.007 0.005 0.003 0.006 0.004 0.008

0.108 0.117 0.140 0.130 0.172 0.066 0.091 0.134 0.157 0.045 0.064 0.083 0.114 0.056 0.073 0.118 0.127

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.002 0.002 0.003 0.003 0.003 0.001 0.002 0.003 0.003 0.001 0.001 0.002 0.002 0.001 0.001 0.002 0.003

0.543 0.677 1.065 0.879 1.696 0.289 0.653 1.333 1.734 0.149 0.359 0.619 1.114 0.301 0.529 1.195 1.355

0.019 0.022 0.033 0.028 0.051 0.010 0.021 0.040 0.051 0.006 0.012 0.020 0.033 0.010 0.017 0.036 0.040

Figure 2. Influence of PEG molar mass (M) on the interfacial tension (γ) of systems 2, 13, and 16: ●, pH 6, 293 K; ○, pH 7, 293 K; ▼, pH 9, 293 K; △, pH 7, at 313 K.




σ

g·cm−3

mN·m−1

mN·m−1

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.004 0.003 0.006 0.002 0.010 0.004 0.004 0.006 0.014 0.002 0.040 0.008 0.012 0.001 0.007 0.006 0.003

0.164 0.192 0.202 0.207 0.215 0.112 0.140 0.188 0.210 0.083 0.106 0.164 0.196 0.092 0.117 0.145 0.146

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.003 0.004 0.004 0.004 0.004 0.002 0.003 0.004 0.004 0.002 0.002 0.003 0.004 0.002 0.002 0.003 0.003

1.675 2.212 2.808 2.656 2.919 1.216 1.696 2.593 3.108 0.848 1.201 2.037 2.924 1.051 1.406 2.965 1.599

0.050 0.064 0.079 0.076 0.082 0.036 0.049 0.073 0.087 0.025 0.035 0.058 0.081 0.031 0.041 0.079 0.047

Figure 3. Effect of pH on the interfacial tension (γ), at 293 K: ●, system 6; ○, system 7; ▼, system 8; △, system 9.

influence of ionic strength and pH on interfacial tension, verifying that interfacial tension increased at high pH values. A linear fit was also applied to evaluate the influence of pH on the interfacial tension and provides a good fit to the experimental data since their R2 (0.99) values are closer to 1 than for all other systems tested. An increase in temperature induces increases in interfacial tension (Tables 2 and 5). Mishima et al.11 also reported a smooth interfacial tension increase with the temperature rise for the PEG + dibasic potassium phosphate ATPS.

technique. The authors observed that the interfacial tension increases with an increase in tie-line length and PEG molar mass and the values of interfacial tension varied from (1.0·10−3 to 1.26) mN·m−1. Similar behavior was reported by Zuniga et al.10 for PEG + MD systems. These authors reported values ranging from (0.55 to 0.33) mN·m−1. We use a linear fit for the correlation between interfacial tension data and polymer molar mass (M). Since the corresponding determination coefficients were higher than 0.93 it is possible to conclude that the linear fit used provides a good fit for the experimental data. Figure 3 shows the effect of pH on interfacial tension for systems with PEG molar mass of 4000. Tables 2, 3, and 4 demonstrate that for all analyzed systems the higher the pH value, the higher the interfacial tension values. The increase of interfacial tension as a function of pH causes a growth in the phase separation region and variation in the slope of the tie-lines.6 Schluck et al.17 also determined the

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CONCLUSIONS

The Vonnegut equation was appropriate to fit the interfacial tension data of ATPS's composed of PEG and potassium phosphate. It was also observed that interfacial tension values increased with the elevation of PEG molar mass, temperature, and pH value. 1651



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+ dextran biphasic systems: Part II. Biotechnol. Bioeng. 1995, 47, 252− 260.

AUTHOR INFORMATION

Corresponding Author

*Fax: 55-31-38992208. Phone: 55-31-38991618. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors wish to acknowledge the National Council of Technological and Scientific Development (CNPq) and the Research Foundation of the State of Minas Gerais (FAPEMIG) for their financial support.

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REFERENCES

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