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Separation and Purification Technology 78 (2011) 69–75

Contents lists available at ScienceDirect

Separation and Purification Technology

journa l homepage: www.e lsev ier .com/ locate /seppur

Study of biomolecules partition coefficients on a novel continuous separatorusing polymer-salt aqueous two-phase systems

Patricia Vázquez-Villegas, Oscar Aguilar, Marco Rito-Palomares ∗

Departamento de Biotecnología e Ingeniería de Alimentos, Centro de Biotecnología-FEMSA, Tecnológico de Monterrey, Campus Monterrey,Ave. Eugenio Garza Sada 2501 Sur, Monterrey, NL 64849, Mexico

a r t i c l e i n f o

Article history:Received 28 September 2010Received in revised form 17 January 2011Accepted 21 January 2011

Keywords:Continuous aqueous two-phase systemSeparatorProtein isolate

a b s t r a c t

A novel experimental prototype device was evaluated for the continuous operation of an aqueous two-phase extraction protocol. As proof-of-concept, preliminary tests were performed using an organic blueand Gentian violet dyes, to demonstrate the capacity of the system for solute partitioning and continuousphase separation. Different experimental configurations were established by changing tubular separatorlength (3.0 and 5.0 m) and static mixer configurations (single Beads-filled tube, zig-zag, 3 Serial Beads-filled and tube Spiral/single Beads-filled tube). The use of different static mixer configurations ensuredtotal organic dyes transfer from bottom to top phase with shorter equilibration time (1.0–4.0 min) thanbatch system. The turbulent regime created at the entrance of the device by the static mixer, contributedto an efficient mass transfer that resulted in improvement of product partition coefficient (Kp) previ-ously obtained from batch systems, when bovine serum albumin (BSA) was used as a model protein. Acomplex protein extract, namely whey protein extract (WPI), characterized by the presence of two mainproteins (�-lactalbumin and �-lactoglobulin) with different phase affinities was successfully processed,resulting in differential protein partitioning with total recoveries up to 90% (top + bottom phase) andminimal protein precipitation at the interface. The obtained results suggested that the concentration ofprotein products in a defined phase could be feasible, by controlling the PEG-rich or salt-rich phase flowrates. These findings demonstrated the potential application of the proposed separator device for thecontinuous protein recovery using aqueous two-phase systems.

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

Since late 1950s aqueous two-phase systems (ATPS) havedemonstrated its potential to separate biomolecules and cellularcomponents [1]. Extensive reports have highlighted the usefulnessof batch systems with one or more extraction steps for the recoveryof proteins from different biological origin [2]. There are numerousreports about the behavior of bioparticles at lab and bench scale,and the advantages of scaling-up and practical applications of ATPShave been clearly stated by several authors [1,3–5]. However, thosestudies have raised the need of the design of continuous operationsthat can be integrated to other process steps.

During the last decades, in order to increase the attentiontowards the industrial application of ATPS studies addressing therecycling and low cost of chemicals have been documented [6].In general, ATPS applications are limited by the understanding ofphase equilibrium, system selectivity and partition of proteins [4].Benavides and Rito-Palomares, proposed a strategy that favors the

∗ Corresponding author. Tel.: +52 81 8328 4132; fax: +52 81 8358 1400.E-mail address: mrito@itesm.mx (M. Rito-Palomares).

predictive design of batch ATPS process for biomolecules recoveryand purification [7]. However, in order to reduce the reluctance ofindustries to adopt this technology, the continuous operation ofATPS extraction stages is required.

In an attempt to address the continuous operation of ATPS,the use of vertical settlers, columns, centrifuges and coalescersfor recovering of biomolecules has been suggested [4,8–10]. To alesser degree, horizontal continuous settlers have also been docu-mented [11]. The most recurrent experimental prototype consistsof perforated rotating disc contactors (PRDC) because of their majoroperational flexibility than conventional ones; gravity being themain driving force for column feeding [12,13]. Possibilities and lim-itations of hydrodynamic gravity settler modeling, such as columnflooding, have been pointed out in previous reviews [14]. The spraycolumn and the Graesser extractor are other examples of equip-ment that have shown to be suited for ATPS [15,16]. Procedureswith variation of TLL and settler entrance configuration have beencarried out in decantation tanks, including back extraction stepsand loads of more than 1.0 kg [17].

Nonetheless, the scaling of these systems based on thermody-namic conditions is difficult to generalize and understand, given thegreat variability on system compositions and components. Parame-

1383-5866/$ – see front matter © 2011 Elsevier B.V. All rights reserved.doi:10.1016/j.seppur.2011.01.023

70 P. Vázquez-Villegas et al. / Separation and Purification Technology 78 (2011) 69–75

Fig. 1. Simplify scheme of continuous separator. (a) Pre-equilibrated phases and sample solution; (b) peristaltic pumps; (c) phase mixer and turbulence generator; (d)continuous tubular separator; (e) phase collector with interface harvesting port.

ters as initial concentration of neutral salts, feed flow, fresh solventand volume ratios have been defined as crucial for the employedequipment design [11]. The settling rate of a particular system isalso crucial parameter. The use of commercial centrifuges to dimin-ish the time of phase separation observed in sedimentation orgravity separation settlers has been disadvantaged due to its highcost, therefore the employment of extraction column is preferred.At laboratory scale, the kinetics of phase separation in aqueous two-phase systems has been investigated as a function of the systemphysical properties [18,19].

In the present work we have evaluated the use of a novelcontinuous system to perform aqueous two-phase partitioningof biomolecules with the aim of replicate the partition coeffi-cients obtained from batch experiments. The proposed prototypeexploits the tubular reactor approach with a large and adjustablelength/diameter ratio to reduce settling time with minimal manip-ulation of phases, and without the need for external equipmentsuch as centrifuges for phase separation. An experimental appara-tus was specifically designed for this operation, and characterizedusing organic blue dye, Gentian violet and BSA as a model protein.A complex protein extract, namely whey protein extract (WPI) wastested. The system designed does not present any column floodingand can be operated with a continuous feed of industrial turbulentfermentation streams.

2. Experimental

2.1. Chemicals

Blue dye was obtained from MANE, S.A. de C.V. (Méx-ico, D.F.), Gentian violet, monobasic and dibasic phosphate,poly(ethyleneglycol) (PEG), Bradford reagent, and bovine serumalbumin (BSA) were purchased from Sigma-Aldrich Chemicals (StLouis, MO). Whey protein isolate (WPI) was gently donated byTECSA® (Monterrey, México).

2.2. ATPS formulation

Aqueous two-phase system composition was formulated to give17% (w/w) PEG 1000 and 15.2% (w/w) potassium phosphate for

dyes and BSA partitioning. The system composition and tie-linelengths (TLLs) were calculated from phase diagrams reported byZaslavsky [21]. A second system composition, 14% (w/w) PEG 1000and 18% (w/w) potassium phosphate, was obtained from Chen [20]for partitioning experiments involving bovine whey protein isolate(WPI) as a complex protein mixture. ATPS for batch and continuousmode were prepared at pH 7, according to the methodology welldescribed elsewhere [22,23]. Briefly, the correspondent amountof distillated water and solids, namely PEG and phosphates, wereweighted separately and mixed together until total dissolution.After 15 min equilibration, the formed phases were separated usingperistaltic pumps to different containers. Interphase contaminantswere discarded when appeared.

2.3. Continuous separator

A simplified scheme of the separator is depicted in Fig. 1. Thesystem consisted of a couple of storage tanks for the continuoussupply of the pre-equilibrated top and bottom phases, a series ofpumps providing a continuous flow of 50 mL/min (60 mL/min forblue dye) to a tubular reactor of either 3.0 or 5.0 m long with 0.6 or0.9 cm cross-sectional diameter, respectively. A turbulence gener-ator, consisting of 10 cm long bead-filled tubing or 15–30 cm longzig-zag/spiral-shape tubing with internal bumps, was placed beforethe separator to improve mass transfer. Top and bottom phaseswere collected individually and used for further analysis. Reten-tion time distribution (RTD) for top and bottom phase was definedas the concentration profile of a dye or protein particle over time insteady state conditions. An additional port was available to harvestthe interface when necessary.

2.4. Operation procedure

Dye samples (1–3 �g/mL) were pre-dissolved in the bottomphase and introduced to the system continuously by switch-ing between clear and colored solutions. Protein samples wereinjected as discrete pulses, bottom phase inlet switched fromblank to protein-containing phase (1.0 mg/mL) at the sameflow (50 mL/min). Different samples and system configurationsemployed are listed in Table 1. Prior to the injection of the

Table 1Operation conditions used for continuous separator and samples employed.

Configuration Static mixer Tubular separatorlength (cm)

Inlet flow (mL/min) Sample type

A None 500 60 Blue dyeB Beads-filled tube 500 60 Blue dyeC Zig-zag 500 60 Blue dyeD Beads-filled tube 300 50 Gentian violetE Zig-zag 300 50 Gentian violetF 3 Serial Beads-filled tube 500 50 BSAa

G Spiral and Beads-filled tube 500 50 WPIb

a Bovine serum albumin.b Bovine milk whey protein isolate.

P. Vázquez-Villegas et al. / Separation and Purification Technology 78 (2011) 69–75 71

Fig. 2. Retention time distribution (RTD) of blue dye in continuous aqueous two-phase system using different static mixer configurations. (—) Top phase; (––) bottom phase.(A) Configuration A (see Table 1); (B) Configuration B; (C) Configuration C.

phases, deionized water was circulated through the system inorder to avoid leaks and air bubbles. Pre-equilibrated phases wereemployed to avoid uncertainty from the final system compositioncoming from injecting stock solutions into the system. Phases wereintroduced simultaneously at the same flow rate to have a totalvolume ratio (VR) = 1.0, verified visually through connectors andtubular separator. The system was kept at constant room temper-ature of 25 ◦C. Samples from top and bottom phases were takensimultaneously at the exit of the separator every 10 s for blue dyeand 20 s for the rest and placed in 96-well microplate and used toquantify solute partitioning. All experimental data are the averageof duplicate experiments.

2.5. Kp estimations

Dye partitioning was monitored measuring phase absorbanceat a 640 nm for Blue dye and 580 nm for Gentian violet (Bio-TekInstruments, VT, U.S.A.). Total protein determination for BSA andWPI samples was made using the microplate version of Bradfordreaction [24], using BSA as standard. All dyes and protein deter-minations included calibration curves using proper solvents andblank ATPS for correction of any interference from phase-formingcomponents.

2.6. Batch experiments

For Gentian violet, BSA and WPI solute distribution in batchATPS, solute previously dissolved in pre-equilibrated bottom phasewas mixed with an equal volume of pre-equilibrated top phase togive a 50 g system. After 15 min of gentle agitation on a vertical

rotary mixer (50 rpm, Labnet, Intl., NJ, U.S.A.), solute distributionwas monitored every 1, 2, 3, 4, 5, 10, 15, 20, 30 and 40 min by tak-ing carefully 0.3 mL samples from the center point of every phase,out of the inversion zone, with constriction pipette. Samples wereused to measure absorbance at 580 nm in the case of Gentian vio-let or for protein determination by Bradford method. Systems andmeasurements were run by triplicate.

2.7. SDS–PAGE

Samples from top and bottom phase were taken from systemrun with WPI and analyzed by SDS–PAGE electrophoresis accord-ing to the Laemmli protocol [25]. Acrylamide gels (15%) were run at150 V during 80 min in a Hoefer miniVE vertical electrophoresis sys-tem (GE Healthcare). The gels were removed from the cassette andvisualized by staining with 0.1% Coomassie blue G-250 dissolved in40% (v/v) methanol, 10% (v/v) acetic acid, 50% distilled water. After1.0 h staining, gels were distained with the same methanol–aceticacid–water solution (without Coomassie dye) for 3 h. Gels werescanned using a flatbed Image Scanner (GE Healthcare) at 600 dpiin transmissive mode.

3. Results and discussion

A modular system for the processing of aqueous two-phasesystems was designed with the final purpose of allowing the contin-uous harvesting of individual phases after a two-phase partitioningoperation. The scheme in Fig. 1 shows the general configurationemployed. A set of initial experiments was designed as a proof-of-concept to demonstrate the applicability of such a system for

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Fig. 3. Batch separation time distribution versus RTD of Violet Cristal in continuous ATPS using two different configurations. (—) Top phase; (––) bottom phase. A. 50 g batchsystem; B. Configuration D (see Table 1); C. Configuration E.

the partitioning of a top phase preferring product. Different con-figurations were employed as they are further described (Table 1).Fig. 2 shows the partition behavior of blue dye with the 5 m longconfigurations. The amount of colorant in the plots is referred tothe concentration of blue dye present in either phase at a certaintime, and relative to the initial concentration of colorant introducedduring the experiment run. Concentrations greater than 100% aredue to experimental errors, resulting from some displacements insample timing or cross contamination between the phases duringsampling. The three plots show a corresponding void time of 4 minresulting in a total operation volume of 240 mL. In the absence ofthe turbulence generator (Fig. 2A) the distribution coefficient ofblue dye at the system equilibrium remains more or less close tounity. The 5 m tubular separator employed was not enough to allowa complete transfer of the product to the top phase. The use of aturbulence generator (glass bead-filled tubing and zig-zag-shapetubing; Fig. 2B and C, respectively) demonstrated that intimatephase dispersion is necessary to improve mass transfer, and suchsolute interchange occurs immediately during dispersion. This wascorroborated by the absence of blue dye in bottom phase, simulta-neously with the increase in top phase absorbance. More than 50%of the product is transferred to the top phase after the first minuteof color detection out of the tubular separator.

With the on-line sampling method employed simultaneoussamples were taken every 10 s from top and bottom phase out-

lets. A clear plug-flow behavior could be observed as the hills andvalleys are coincidental, indicating a laminar flow corresponding toviscous liquids in small diameter tubes [26]. As it can be observedin plots B and C, the droplet dispersion created by the on-line staticmixer, greatly improved mass transfer between the phases. The dif-ferent mixer configurations employed ensured the creation of aninterfacial surface area that allowed bulk homogenization ensuringthat all flow components distributed uniformly in the cross-sectionand exposed to similar levels of turbulence.

Previous experiments performed with Gentian violet (data notshown) showed a higher affinity for top phase than blue dye. Thissuggested a lower retention time necessary for top phase transfer.Mass transfer experiments performed in batch system are depictedin Fig. 3A, where the amounts of the violet dye rapidly decreasedto zero after the first minute of monitoring. The percentage of col-orant for every plot refers to the amount of dye determined in thephases at the same time, compared with the maximum amount ofcolorant in the system observed during the total sampling time.Total mass transfer was achieved after 3 min when 100% of thecolorant was homogeneously distributed in top phase. Due to itsapparently higher preference for polymeric phase, Gentian violetwas tested with a shorter tubular separator (3 m long) at 50 mL/min.Fig. 3B and C shows the same set of experiments than those withthe blue dye, but in this case, the use of a higher affinity top-phasedye decreased the retention time to 1.0 min with an immediate

P. Vázquez-Villegas et al. / Separation and Purification Technology 78 (2011) 69–75 73

Fig. 4. Batch separation time distribution versus RTD of BSA in continuous ATPS using two different configurations. (—) Top phase; (––) bottom phase. (A) 50 g batch system;(B) Configuration F (see Table 1).

transfer of color to the top phase using either of the static mixerspreviously mentioned. A highly top phase preference compoundallowed a considerable tubular settler length reduction and a signif-icantly shorter residence time resulting in lower solvent and energyrequirements.

It is important to point that with a batch ATPS, every unit oper-ation is realized in a discontinuous independent manner, withconsequent loss of sample and mainly time for analysis. With thepresent proposed device, the manipulation of samples betweensteps is diminished and the partition time is decreased withouta significant effect on partition coefficients.

According to these results with dye partitioning, it was decidedto test the proposed device with a series of three glass bead-filledstatic mixers using BSA as a model protein. In order to increase thetime of intimate contact between the phases before tubular sep-arator (5 m long) a triple static mixer was used. This set up camefrom the batch experiment (shown in Fig. 4A), where a remarkablylarge partition time for BSA was observed. For the batch system,the protein exhibited a partition coefficient of 1.5 after 15 min ofshaking. With the use of continuous system (Fig. 4B), the equilib-rium time and phase separation was lower than batch system. Thisreflects a clear advantage on processing times and an improvedmass transfer between the phases reflected by a higher Kp in thecontinuous system: Kp = 3.6 (at minute 16–18) versus Kp = 1.5 (atminute 35) for the batch system. At the same time, the serial con-nection of the static mixer corroborates the need to increase thecontact area between phases and so the mass transference [9]. Withthe use of the continuous system it was demonstrated that proteinpartitioning could be significantly improved, allowing the continu-ous harvesting of product from both phases. A minor disadvantageof the use of three in-line static mixers is the flow restriction andpower adjustments needed to surpass the glass packing of the mix-ers; however such restriction is necessary to maximize transfer ofthe desired product.

After partitioning of dyes and BSA as a model protein, the ideaof implementation to recover different protein fractions from acomplex extract, was obtained from previous reports of the useof ATPS for differential partitioning of components from milkwhey [20,27,28]. These authors have reported differential parti-tioning performed in PEG/salt and PEG/dextran systems to obtainenriched phases of �-lactalbumin (�-La) and �-Lactoglobulin (�-Lg). PEG/phosphate systems usually reported for whey proteinsshow partition coefficients minor to unity for �-Lg and around unityfor �-La, also more susceptible to phase shifts with the polymer

molecular weight [20,28]. Fig. 5 shows the result of injecting wheyprotein isolate (WPI) dissolved in bottom-phase into a 5-m longsystem. In order to decrease flow restrictions caused by a seriesof three glass bead-packed mixers, a spiral-shape mixer was fitbefore a single glass beads-filled mixer without compromise masstransfer.

Considering the initial amount of protein added to bottom phase(1.0 mg/mL), an average recovery of 72% was measured from thecollected bottom phase, and 30% from the top phase when the sys-tem reached a steady state, allowing the continuous harvesting ofthe phases. Given the different affinity previously reported for themain whey components, it was reasonable to suggest that differ-ent protein profile could be found in each phase. Fig. 6 shows theSDS–PAGE images from ATPS samples taken at different times dur-ing the run, two main bands corresponding to �-Lg (18 kDa) and�-La (14 kDa) can be clearly recognized. Given the apparent rela-tive abundance of �-Lg in the sample, it is now clear that the highestrecoveries observed in Fig. 5 came from bottom phase samples com-posed mainly of �-Lg, as confirmed in Fig. 6. Important �-Lg partialdepletion can be successfully achieved from the bovine whey withour proposed system.

The use of constant and controllable flows enabled simple andreproducible generation of stable interfaces and a plug-like two-

Fig. 5. Residence time distribution of whey protein isolate (WPI) in continuous ATPS.(—) Top phase; (––) bottom phase. Configuration G (see Table 1).

74 P. Vázquez-Villegas et al. / Separation and Purification Technology 78 (2011) 69–75

Fig. 6. SDS–PAGE image of whey proteins partitioned in batch and continuous ATPS. Lane (1) M.W. marker; (2) top phase from batch system; (3) bottom phase from batchsystem; Lanes (4–7) top phase from continuous system at min 4.5, 6.0, 7.5 and 10.5 respectively. Lanes (8–11) bottom phase from continuous system at min 4.5, 6.0, 7.5 and10.5 respectively.

phase flow. Since the majority of whey proteins (namely �-Lg)showed a higher affinity for bottom phase, a maximum averageKp = 0.5 was obtained. The change in WPI Kp during operation timeof the continuous system is depicted in Fig. 7 for the G configura-tion previously described. The observed variability is mainly due tomanual sampling of the phases, or pulses generated by the pump-ing system, however once the system evolved, a quasi-steady statewas reached with average Kp values lower than those obtainedwith batch system. An additional clear advantage of the continu-ous device was a significantly lower accumulation at the systeminterface as it can be calculated from Fig. 5. Total soluble pro-tein recoveries around 90% (top + bottom phases) and higher wereobtained, compared with 33% ±0.05 of total protein recovered inthe batch system. A higher proportion of bottom-phase proteins (�-Lg) partitioned in the continuous system could explain the lowerKp values obtained, due mainly to the dynamic nature of the inter-face of two moving liquids, minimizing protein precipitation as it

Fig. 7. Partition behavior of WPI in continuous ATPS and batch system (—); Kpobtained in 50 g batch system (––); moving average from continuous system (�).Configuration G (see Table 1).

occurred with batch systems. In general, the proposed system canbe tailor-made for different industrial purposes, from the continu-ous extraction of dyes from textile wastewater effluents, treatmentof whey from lactic plants and further processing and commercial-ization of crude fermentation broths.

4. Conclusions

The present research described a functional prototype for thecontinuous two-phase partitioning of solutes, demonstrating itssuitability for the continuous partitioning of biomolecules. As aproof-of-concept, the use of different in-line static mixers previousto a tubular separator, proved to be an efficient system for the masstransfer of solutes between the two phases and allowed differen-tial partitioning of proteins from complex feedstock such as milkwhey protein isolate. Also, the length of the tubular separator pro-vided additional time to increase the interfacial contact betweenthe phases and protein distribution with minimal accumulationat the system interface. Recoveries up to 90% were possible withthe continuous system, lowering the amount of proteins lost at thesystem interface typically observed in batch ATPS. However thegeneral performance of the proposed prototype device may dependon the ATPS composition and the load capacity of each system. Thefindings reported here open the potential generic application ofstrategy for the ATPS separation in continuous processes.

Conflict of interest

The authors declare no conflict of interests.

Acknowledgements

The authors wish to acknowledge the financial support ofTecnológico de Monterrey, Biotechnology research chair (GrantCAT161) and the CONACYT Doctoral Fellowship No. 295236 forPatricia Vázquez-Villegas.

P. Vázquez-Villegas et al. / Separation and Purification Technology 78 (2011) 69–75 75

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