Synthesis of pH-responsive polymers forming recyclable aqueous two-phase systems and application to the extraction of demeclocycline

Accepted Manuscript Title: Synthesis of pH-responsive polymers forming recyclable aqueous two-phase systems and application to the extraction of demeclocycline Authors: Zhiliang Gai, Junfen Wan, Xuejun Cao PII: DOI: Reference:

S1369-703X(18)30309-7 https://doi.org/10.1016/j.bej.2018.08.016 BEJ 7027

To appear in:

Biochemical Engineering Journal

Received date: Revised date: Accepted date:

30-5-2018 25-8-2018 27-8-2018

Please cite this article as: Gai Z, Wan J, Cao X, Synthesis of pH-responsive polymers forming recyclable aqueous two-phase systems and application to the extraction of demeclocycline, Biochemical Engineering Journal (2018), https://doi.org/10.1016/j.bej.2018.08.016 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Synthesis of pH-responsive polymers forming recyclable aqueous two-phase

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systems and application to the extraction of demeclocycline

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Zhiliang Gai1, Junfen Wan1, Xuejun Cao*

State Key Laboratory of Bioreactor Engineering, Department of Bioengineering, East

Both authors contributed equally to this work.

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China University of Science and Technology, Shanghai, China.

Corresponding author: Prof. Xuejun Cao; State Key Laboratory of Bioreactor

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Engineering, Department of Bioengineering, East China University of Science and Technology, 130 Meilong Rd, Shanghai 200237, China; [email protected]; +86 21

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64252695

Highlights

A new pH recycling ATPS has been developed for demeclocycline extraction.

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The recoveries of polymers could reach over 95.0%.

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The addition of MgSO4 to ATPS can improve the extraction of demeclocycline.

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Reaction temperature and initiator affected the polymer molecular weight.

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Phase formation mechanism have been studied using Low-field NMR.

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Abstract

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The application of aqueous two-phase systems (ATPS) to bioseparation and

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biocatalyst engineering has attracted interest. However, despite the distinct advantages of the technique, the scaling up of ATPS is limited by the cost of the system components.

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In this study, two novel recyclable pH-responsive polymers were synthesized and tested

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for the partition of demeclocycline. The recoveries of two recyclable pH-responsive polymers could reach over 95.0% by only adjusting the solution pH to the isoelectric

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points of the polymers. The control variate method was used to investigate the effects of polymerization conditions on polymer synthesis. Polymer PADB4.6 was firstly

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synthesized in 500L reactor with two-stage heating method (25-40 ℃ and 40-62 ℃), as the heating rate is an important factor in the scale-up process. The polymer characteristics (molecular weight, viscosity, surface tension, and zeta potential) were studied to understand their influences on the synthetic process. Furthermore, the phase formation mechanism was studied with low-field nuclear magnetic resonance (LF-

NMR). The optimal partition coefficient and recovery of demeclocycline using the recyclable ATPS were 0.24 and 82.9% at pH 6.30 in presence of 20 mmol/L MgSO4.

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Abbreviations ATPS, aqueous two-phase systems; RSM, response surface methodology; LF-NMR,

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low-field nuclear magnetic resonance; PEG, polyethylene glycol; CDs, cyclodextrins; AA, Acrylic acid; BMA, butyl methacrylate; DMAEMA, 2-dimethylaminoethyl

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methacrylate; APS, ammonium persulfate; pI, isoelectric point; FT-IR, Fourier

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transform infrared.

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Keywords: Aqueous Two-Phase Systems; Bioseparations; Recycling; Liquid-Liquid

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Extraction; Industrial Biotechnology

1.

Introduction

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Since the 1970s, the two-phase extraction technology has been applied in process

of biological separation[1]. Aqueous two-phase systems (ATPS) usually comprise incompatible polymer/polymer, polymer/salt, ionic liquid/salt, alcohol/salt, or cationic/anionic surfactants in aqueous solution above certain concentrations[2-6].

ATPS has great potential for product recovery in biotechnology owing to its advantages such as higher water content, mild reaction conditions, lower surface tension, rapid mass transfer, environmental friendly features[7, 8].

used.

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extractive

bioconversion

with

Bacillus

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In previous works on ATPS[3, 9-12], polyethylene glycol (PEG) has been widely cereus cyclodextrin

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glycosyltransferase in a PEG 20000/dextran T500 ATPS was investigated for the

synthesis and recovery of cyclodextrins (CDs). CDs were efficiently extracted into the PEG-rich top phase, whereas the enzymatic conversion occurred mainly in the bottom

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phase[10]. The partition of ficin was conducted in an ATPS under conditions of 20%

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PEG 1000 and 19% (NH4)2SO4, and the partition coefficient was 3.6[11]. However, the

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scaling up of ATPS is limited by the cost of phase-forming polymers and the difficulty in isolating the target products from the polymer phase[13]. Therefore, the use of low-

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cost phase-forming polymers and their recycling have been thoroughly considered.

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Demeclocycline, a secondary metabolite of Streptomyces aureofaciens[14], has

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an antibacterial effect against both gram-negative and gram-positive bacteria. Generally, antibiotic concentration is low in fermentation broth, the extraction of demeclocycline is usually carried out with a large number of ethyl acetate and butyl

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alcohol. The recovery of organic solvents used consume massive energy, the use of organic solvents can also cause environmental pollution. Therefore, simple, environmental friendly and energy-efficient access to high-purity antibiotic is a problem to be solved in process of antibiotic separation. Lately, much work has gone

into studying recycling ATPS that has one of the phase components a “smart” polymer, which is readily recovered from solution as it undergoes phase separation by a moderate change in an environmental condition, like temperature or pH[2, 15].

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In this paper, two pH-responsive polymers, PADB4.0 and PADB4.6 (where the number in subscripts indicate pI of polymer), were synthesized using Acrylic acid (AA), 2-

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dimethylaminoethyl methacrylate (DMAEMA) and butyl methacrylate (BMA) to form

ATPS. Monomer AA and DMAEMA are used as the main monomers to endow that the two polymers are pH-response and to regulate the pI. Monomer DMAEMA and BMA

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are able to regulate the hydrophobicity of polymers to form ATPS. The control variate

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method was used to investigate the effects of polymerization conditions on polymer

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synthesis. Then, polymer PADB4.6 was synthesized in a 500 L reactor. Both pHresponsive polymers displayed high recoveries (> 95%). Information on the phase

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properties (viscosity, surface tension, zeta potential and molecular weight) help

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elucidate the phase separation behavior and can be used for guidance in polymer synthesis. Low-field nuclear magnetic resonance (LF-NMR) was applied to analyze the

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phase formation mechanism through measurements of the water-binding ability of the polymers. Furthermore, this ATPS was used for the extraction of demeclocycline to

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investigate the performance of the polymers.

2. Materials and methods

2.1. Materials and reagents

Acrylic acid (AA), butyl methacrylate (BMA), sodium bisulfate (NaHSO3) and sodium hydroxide were purchased from Ling Feng Chemical Co., Ltd. (Shanghai, China); 2-dimethylaminoethyl methacrylate (DMAEMA) was obtained from Wanduofu Fine Chemical Co., Ltd. (Shandong, China); ammonium persulfate (APS)

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and MgSO4 were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China); and ethanol and hydrochloric acid were purchased from Shanghai Titan

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Scientific Co., Ltd. (Shanghai, China). All the reagents were of analytical grade (AR).

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2.2. Methods

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2.2.1. Synthesis of copolymers PADB4.0, PADB4.6 I, PADB4.6 II and PADB4.6 II-500L

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The ATPS employed in this study were formed from PADB4.0 and PADB4.6, the structures of which are shown in Fig. 1. Copolymer PADB4.0 was synthesized as follows:

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0.0729 mol of AA, 0.0267 mol of DMAEMA, and 0.0013 mol of BMA were added to

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a 250 ml conical flask containing 120 ml of water. Then, 0.14 g of ammonium persulfate and 0.14 g of sodium bisulfate were added to the flask as initiators. The

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copolymerization was carried out in a water bath shaker at 55 °C and 200 rpm for 12 h under N2 protection. When the reaction was finished, ethanol was used to remove the

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initiators and the unreacted monomers via polymer precipitation, and then the polymer was dried in a vacuum oven at 55 °C to a constant mass.

The synthesis of copolymer PADB4.6 was similar to that of PADB4.0: 0.0801 mol of AA, 0.0475 mol of DMAEMA, and 0.0031 mol of BMA were added into a 250 ml

conical flask containing 120 ml of water. The copolymerization of P ADB4.6 I was carried out at 55 °C and 250 rpm, and 0.21 g of ammonium persulfate and 0.21 g of sodium bisulfate were added as initiators. The copolymerization of PADB4.6 II was carried out at 62 °C and 200 rpm, and 0.24 g of ammonium persulfate and 0.24 g of sodium bisulfate

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were added as initiators.

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Copolymer PADB4.6 II-500L was synthesized as follows: 120.15 mol of AA, 71.25 mol of DMAEMA, and 4.64 mol of BMA were added into a 500 L enamel reactor containing 180 L of water. Then, 360 g of ammonium persulfate and 360 g of sodium

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bisulfate were added as initiators. The copolymerization was carried out at 100 rpm and

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62 °C with a two-stage heating method (25-40 °C and 40-62 °C) for 12 h under N2

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2.2.2. Polymer characterization

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protection.

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The chemical structure of the polymers was evaluated by Fourier transform infrared (FT-IR) spectroscopy (Nicolet MagnalR550 infrared equipment, Thermo,

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USA). The viscosity and average molecular weight of the polymers were measured by the Ubbelohde viscometer method[17, 18]. Polymer solutions of different known

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concentrations were prepared, and the time for a certain volume of polymer solution to flow through a capillary at 25 °C was recorded. The internal diameter of the capillary was 0.5-0.6 mm.

2.2.3. Preparation of PADB4.0/PADB4.6 ATPS

First, 0.6 g of PADB4.0 (6 wt%) was dissolved in 10 ml of NaOH (200 mmol/L), and 0.8 g of PADB4.6 (8 wt%) was dissolved in 10 ml of deionized water. After all the components were completely dissolved, equal volumes of these two solutions were mixed. Then, the mixture was transferred to a graduated centrifuge tube until the phase

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interface was well defined at room temperature (25 °C). The illustration of phase

2.2.4. The determination of the conditions for recycling

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formation of ATPS was showed as Fig. 2.

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As pH-responsive polymers, PADB4.0 and PADB4.6 can be precipitated from solution

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by adjusting the pH of the solution to their isoelectric points[19]. Polymers were

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dissolved in a 50 ml centrifuge tube at certain concentrations, and the pH was adjusted

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by dropwise addition of HCl (100 mmol/L) or NaOH (100 mmol/L) to the

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abovementioned polymer solution. Then, the polymers were separated from the solution by centrifugation at 8000 rpm for 30 min and dried to constant weight in a

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vacuum oven at 55 °C. The pI was determined using a Zetasizer Nano ZEN3600

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(Malvern). When the zeta potential was zero, the pH of the polymer solution was the pI[20]. First, 10 ppm polymer solutions were prepared, the pH was adjusted to different values, and then the zeta potentials were measured. Each solution at a different pH value

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was subjected to three trials.

2.2.5. LF-NMR relaxation time measurement

The measurements of transverse relaxation time (T2) were conducted on a Niumag Benchtop Pulsed NMR analyzer (Niumag PQ001; Niumag Electric Corporation, Shanghai, China) with a working resonance frequency of 21 MHz. Carr-PurcellMeiboom-Gill (CPMG) sequences, a multipulse sequence applied to protons, were

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employed to measure the spin-spin relaxation time T2[21, 22]. The data were analyzed by a multiexponential model with MultiExp Inv Analysis software (Niumag Electric

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Corporation, Shanghai, China) using the inverse Laplace transform algorithm. To investigate the interaction between copolymers and water, 6 wt% PADB4.0, 8 wt% PADB4.6

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I, 8 wt% PADB4.6 II and deionized water were prepared for measurement. ΔT was defined

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(1)

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ΔT = 𝑇21 (𝑃𝐴𝐷𝐵4.6 ) − 𝑇21 (𝑃𝐴𝐷𝐵4.0 )

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as expressed in Eq. (1):

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2.2.6. Surface tension measurements

Surface tension measurements were conducted on an interfacial tensiometer

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(JK99B, Shanghai Zhongchen Digital Technic Apparatus Co., Ltd) by the Wilhelmy

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plate method at room temperature. First, 6 wt% PADB4.0, 8 wt% PADB4.6 I, 8 wt% PADB4.6 II and deionized water were prepared for measurement, and then 1 ml samples were removed and diluted to afford different ratio solutions. All experiments were repeated

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in triplicate.

2.2.7. Partition of demeclocycline

In this experiment, demeclocycline was partitioned in the PADB4.0 and PADB4.6 system to study the partition performance. The initial concentration of demeclocycline employed in the ATPS was 5 mg/ml. Several inorganic salts (MgSO4, LiCl, KBr, MgCl2, KCl, LiBr) were selected with different concentrations ranging from 5 to 30 mmol/L to

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investigate the partition behavior. Additionally, the effect of pH on the partition of demeclocycline was examined. When phase separation occurred, top and bottom phases

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were removed via pipetting, respectively. Then, the pH values of the top and bottom

phase were adjusted by dropwise addition of HCl (100 mmol/L) to the isoelectric points

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of the polymers. The polymers could be precipitated from the top or bottom phase to

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keep only demeclocycline in the resulting solution. After phase separation, a 200 μl

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sample was taken from both the top and bottom phase of the ATPS and then diluted

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with deionized water to 1 ml. The concentration of demeclocycline in both phases was

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measured by a UV spectrophotometer (UV mini-1240, Shimadzu Corporation, Japan) at 350 nm.

(2)

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K = 𝐶𝑡 ⁄𝐶𝑏

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The partition coefficient (K) is defined as follows (Eq. (2)):

where Ct and Cb are the concentration of demeclocycline in top phase and bottom phase,

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respectively.

The phase volume ratio (R) was defined as Eq. (3):

R = 𝑉𝑡 ⁄𝑉𝑏

Where Vt and Vb represent the volumes of top and bottom phase, respectively.

(3)

The recovery (Y) of demeclocycline in top and bottom phase were calculated from Eq. (4). All experiments were performed with 3 replicates. 𝑌 = 𝐶𝑏 𝑉𝑏 ⁄(𝐶𝑏 𝑉𝑏 + 𝐶𝑡 𝑉𝑡 ) × 100%

(4)

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3. Results and discussion

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3.1. Selection of the optimum reaction conditions

Reaction conditions such as temperature, initiator concentration and rotational speed can influence the synthesis of a polymer[23]. Generally, as the reaction

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temperature and initiator concentration increase, the polymer molecular weight

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decreases[24]. The results of a single-factor experiment shown in Fig. 3 reveal that the

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fastest phase formation time was achieved at a temperature of 65 °C, an initiator ratio of 2% and a rotational speed of 150 rpm, respectively. As shown in Fig. 3 (a),

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polymerization cannot occur below 35 °C, and the phase formation time is related to

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the polymer molecular weight. Fig. 3 (b) shows that polymers could not form an ATPS when the molecular weight was too large or too small. These results showed that the

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phase-forming ability first decreased and then increased as the polymer molecular weight decreased. Similar trends have been reported in ionic liquid/salt ATPS[4, 25], in

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which an increase in the alkyl side-chain length enhances the phase-forming ability, whereas very long alkyl side chains are unfavorable for separation.

Because free-radical polymerization is very complex, the interactions between independent process variables such as the thermal decomposition rates of initiators

should be considered[26]. Therefore, the optimization of these factors is necessary to obtain the optimum conditions. The optimum conditions for polymer synthesis are a temperature of 62 °C, a rotational speed of 200 rpm and an initiator concentration of 1.75%, and these conditions led to the polymers forming an ATPS in 1.5 h, the rapid

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phase formation can reduce the inactivation of bioproduct during extraction process.

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3.2. The determination of the conditions for recycling

Both PADB4.0 and PADB4.6 contain -COOH groups from the monomer AA and -

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N(CH3)2 from the monomer DMAEMA. In aqueous solution, the functional groups will

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ionize:

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-N(CH3)2 + H+ ⇋ -NH+(CH3)2

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-COOH ⇋ -COO- + H+

(5) (6)

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When NaOH / HCl was added dropwise to the copolymer solution, the ionization

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balance was changed. When the surface charge of PADB4.0 and PADB4.6 is completely neutralized, the repulsive forces of like charges disappear, and the polymers precipitate

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from the solution. The phase-forming components were recycled in the process by pI precipitation[19], and the maximum recoveries occurred when the pH of solution was

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near the pI. The recoveries of PADB4.0 and PADB4.6 at various pH values are shown as Fig. 4. The maximum recoveries of PADB4.0 and PADB4.6 are 98.42% at pH 4.00 and 96.26% at pH 4.50, respectively. If the surface charge of the polymers became positive or negative again, the solubility of the polymers also increased. The recycled polymers could be recovered efficiently by only adjusting pH and reused to form new ATPS.

The isoelectric point (pI) is defined as the pH at which the surface charge of a polymer is zero[20]. The surface charge of a polymer is negative at pH higher than its pI and positive at pH lower than its pI. The zeta potentials of PADB4.0, PADB4.6 I and PADB4.6 II, as shown in Fig. 5, were measured at different pH. The isoelectric points of

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PADB4.0, PADB4.6 I and PADB4.6 II were 4.05, 4.60 and 4.62, respectively. In the polymer synthesized by Liu and Cao[15] with a pI of 6.29, the monomers included 0.0729 mol

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of AA, 0.0594 mol of DMAEMA and 0.0031 mol of BMA. Different ratios of AA and

DMAEMA produce polymers with different isoelectric points, indicating that PADB4.6 I

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and PADB4.6 II have similar monomer ratios. The pH at which the polymers had the

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maximum recoveries were close to their pI values (Fig. 4).

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3.3. Characterization and analysis of PADB4.0 and PADB4.6

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The FT-IR spectra of PADB4.0, PADB4.6 I and PADB4.6 II are presented in S5. The results of the analysis are as follows: 4000-3300 cm-1 (the peak shift and peak areas of

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O-H in hydroxyl groups); 3300-2500 cm-1 (O-H in carboxylic acid groups); 3000-2850

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cm-1 (C-H in methyl and methylene groups); 1760-1710 cm-1 (C=O in carboxyl and ester group); 1465-1380 cm-1 (C-H in –(CH2)n- groups); 1300-1200 cm-1 (C-O-C in ester groups); 1250-1020 cm-1 (C-N in tertiary amine groups). The results illustrate the

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presence of AA, DMAEMA and BMA in the polymers.

The intrinsic viscosity ([η]) and average molecular weight (Mη) were determined by viscometer using the Fuoss equation and Mark-Houwink equation, respectively, as follows[27]:

(𝜂⁄𝑐)−1 = ([𝜂])−1 + 𝐵([𝜂])−1 𝑐1/2

(7)

[η] = K ∗ Mη𝛼

(8)

where c is the concentration of the solution (g/ml), B is a constant parameter, and

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the parameters α and K are 0.86 and 1.94×10-2 ml/g[27], respectively. According to the intrinsic viscosity calculated in S6, the average molecular weight was determined as

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shown in Table 1. The average molecular weight of PADB4.0 was 27 kDa, whereas the

average molecular weight of PADB4.6 I and PADB4.6 II were 457 kDa and 222 kDa, respectively. The average molecular weight of PADB4.6 II-500L was 204 kDa, which is

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similar to that of PADB4.6 II. The results of this study indicate that the polymerization

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conditions affected the degree of polymerization and average molecular weight. S3

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shows that the molecular weight of PADB4.6 decreased as the reaction temperature and initiator concentration increased. In the process of free-radical polymerization,

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temperature can influence the decomposition rate of the initiator and the polymerization

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rate and degree of polymerization of the polymers[23]. Increasing the initiator concentration causes a significant increase in the polymerization rate[28]. The phase

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formation time of the polymers is significantly affected by their molecular weight: the lower the molecular weight is, the faster the molecular motion. When the molecular

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weight of PADB4.6 was less than a certain value, the ability of PADB4.6 to form a phase with PADB4.0 decreased because the influence of the volume exclusion of the two polymers was decreased[29]. On the contrary, when the molecular weight of P ADB4.6 was more than a certain value, the molecular motion will be slow, even can’t form ATPS.

3.4. Scaling-up of PADB4.6 in different size reactors

The characterization results of PADB4.6 synthesized in a flask and 5 L and 50 L reactors are illustrated in Table 2. As the reactor size increased, the molecular weight

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of PADB4.6 increased, and the phase formation time was also partially affected. Some experimental T-t (temperature vs. time) curves are shown in Fig. 6, where the maximum

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temperature was 55 °C and the flask reached the maximum temperature within 4.5 min.

Clearly, the heating rate of the flask is higher than those of the 5 L and 50 L reactors. The effects of reaction scale on the polymerization are actually the same as the effects

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of temperature on the polymerization[26]. Polymerization cannot occur below 35 °C

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(Fig. 3a), so PADB4.6 was synthesized in a 500 L reactor with a two-stage heating method

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(25-40 °C and 40-62 °C), and the heating rate was controlled at 2 °C/min above 40 °C.

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3.5. Phase formation mechanism analysis

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3.5.1. Surface tension

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The ATPS has two incompatible phases, and surface tension measurements provide important insight into the phase formation mechanism of the two polymers[30]. In Fig. 7, the X-axis represents the dilution ratio, while the Y-axis represents the surface

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tension values. The surface tension values of the two polymer solutions increased with increasing dilution ratio. However, when the polymer solutions were diluted 500-fold, the surface tension of PADB4.6 increased to 72.3 mN/m, which was almost equal to that of deionized water[31]. By contrast, the surface tension of PADB4.0 increased to 58.7

mN/m. The dynamic surface tension results indicated the interactions between the polymers and water and revealed the water mobility behavior in the two-phase system[30]. This behavior results in incompatibility, and because of steric exclusion, the polymers start to separate into two different phases. Because the molecular weight

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of PADB4.6 I higher than that of PADB4.6 II (Table 1), the molecular movement became

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slower, and the phase formation time was longer.

3.5.2. LF-NMR relaxation measurements

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In this paper, we investigated the water mobility in the two phases by the transverse

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relaxation time (T2) measurements using LF-NMR. The LF-NMR T2 relaxation curves

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for the ATPS samples showed a multiexponential distribution with two states of water,

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namely, tightly bound water (T21, 10-100 ms) and weakly bound water (T22, 1000-5000

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ms)[32]. The T2 relaxation time reflects the interaction between the environment and protons in the samples, which is related to the binding force and degree of freedom of

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water. Water species with a longer relaxation time bind more loosely to macromolecules

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than those with a shorter relaxation time[33]. As presented in Fig. 8, the T2 relaxation curve of water has only one peak, and that of the polymer solution has two peaks. The peak of T22 is higher peak than that of T21 because of there is much more free water

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than bound water in the polymer solutions. The tightly bound water in the polymer sample reflects the interaction between the polymer and water molecules; thus, T21 was selected to investigate this interaction.

The T21 of PADB4.0 is shorter than that of PADB4.6, indicating that the water-binding ability of PADB4.0 is stronger than that of PADB4.6. The water molecules tended to move to the top phase. The difference leads to exclusion phenomena reflecting the feasibility of phase formation. The result was consistent with that of the surface tension

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experiments shown in Fig. 7. Moreover, the T21 of PADB4.6 I is shorter than that of PADB4.6 II, ΔTI is less than ΔTII, and the molecular weight of PADB4.6 I is higher than that PADB4.6

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II (Table 1); therefore, the phase formation time of PADB4.6 I was longer than that of PADB4.6 II.

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3.6. Partition of demeclocycline in the ATPS

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Salt additives can influence the partitioning of target molecules in an ATPS. In the

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experiment, several inorganic salts were investigated to optimize the partition

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coefficient, and the salt concentration was varied from 5 to 30 mmol/L at pH 6.30. The results indicated that demeclocycline displayed different partition behaviors in

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PADB4.0/PADB4.6 ATPSs depending on the type and concentration of ionic species present

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in the system (Fig. 9). In general, the addition of salts to ATPS promotes hydrophobic interactions due to generation of an electrical potential difference between the two phases[34]. The increase in hydrophobicity is related to the decrease in the amount of

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bound water resulting from the need to maintain electrical neutrality[12]. The waterbinding ability of PADB4.0 is stronger than that of PADB4.6 (Fig. 8), KBr, MgCl2 and LiBr can promote the hydrophobic interactions in the top phase at low concentrations[34]. The higher the hydrophobicity of the top phase is, the stronger the ability to form two

phases, and the stronger the interactions between the polymers and demeclocycline. Therefore, demeclocycline preferentially partitioned to the top phase. However, the water-binding ability of PADB4.0 decreased in the presence of high salt concentrations, and the partition coefficients of demeclocycline decreased as the KBr, MgCl2 and LiBr

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concentrations increased. Among these inorganic salts, MgSO4 was the most helpful in improving the partition coefficient. The fact that the optimal partition coefficients were

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achieved in the presence of MgSO4 were attributed to the generation of an electrical potential difference. In the ATPS, the surface charges of the polymers were negative,

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and the zeta potential of PADB4.0 was higher than that of PADB4.6 (Fig. 5). Demeclocycline,

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which was positively charged in the ATPS, was preferentially partitioned to the bottom

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phase. The partition coefficient of the PADB4.6 II system is better than that of the PADB4.6

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I system (Fig. 9). Polymer/polymer and polymer/salt ATPS are well known to promote

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the size-dependent partitioning of solutes[35]. As these systems are constructed using polymers, the free volume available in the polymer-rich phases is limited. A system

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with a high-molecular-weight polymer will result in a change in partition coefficient

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due to the influence of the volume exclusion of the two polymers and demeclocycline[29]. The optimal partition coefficient was 0.24 in the PADB4.6 II system

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with 20 mmol/L MgSO4.

Additionally, pH affects the electrostatic interaction between the two pH-

responsive polymers and target biomolecules and thus influences the partitioning behavior. Several samples at different pH were used to explore the influence of pH on the partition coefficient and recovery (Fig. 10). The pH range was limited from 5.00 to

6.50 because the systems could not form two phases when the pH was below 5.00 or above 6.50. The partition coefficient decreased with the pH in the range of 5.00-6.50, and the higher the pH of the system was, the higher the electrical potential difference between the two phases. Thus, demeclocycline preferentially partitioned to the bottom

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phase. The minimal partition coefficient and highest recovery with 20 mmol/L MgSO4 at pH 6.30 are 0.24 and 82.9%, respectively. In other words, after two step extraction

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process, 97% of demeclocycline can be extracted from fermentation broth. This result

showed that the demeclocycline is enriched in bottom phase in the PADB4.0/PADB4.6

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system and thus is easier to separate.

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4. Conclusions

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The recyclable pH-responsive polymers PADB4.0 and PADB4.6 were synthesized and

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characterized in this study. Both polymers have high recoveries over 95% upon only adjusting the pH of the solution to their respective pI values. The optimal partition

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coefficient and highest recovery are 0.24 and 82.9% in the presence of 20 mmol/L

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MgSO4 at pH 6.30, which shows that demeclocycline could be effectively separated. Furthermore, LF-NMR was used to investigate the phase formation mechanism, revealing that the phase separation is attributable to the difference in water-binding

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ability. Polymer molecular weight, which governs the partitioning behavior in an ATPS, is easily controlled by the synthetic process. The current findings provided insight into the influence of different factors on the polymer synthetic process. PADB4.6 was first synthesized in a 500 L reactor, and the heating rate was found to be a key consideration

in the scale-up process. The novel ATPSs are low cost and easily recyclable by a moderate change in an environmental condition and show significant potential for industrial separation.

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Acknowledgments

This work was financially supported by the National Natural Science Foundation

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of China (No. 21376078).

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Figure captions

Fig. 1. Synthesis of PADB4.0 and PADB4.6. x, y, and z denote the monomer ratios of AA,

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DMAEMA, BMA, respectively.

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Fig. 2. Illustration of phase formation of PADB4.0/PADB4.6 ATPS.

Fig. 3. The effect of operating conditions (a. temperature; b. initiator concentration; c.

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rotational speed) on phase formation time and yield.

Fig. 4. Recovery of the polymers (a. PADB4.0; b. PADB4.6) at different pH values. The

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recovery was determined using 8% PADB4.0/PADB4.6 ATPS.

Fig. 5. The zeta potentials of PADB4.0, PADB4.6 I and PADB4.6 II at different pH. PADB4.6 I was synthesized at 55 °C, 250 rpm and 0.21 g initiators; PADB4.6 II was synthesized at

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62 °C, 200 rpm and 0.24 g initiators.

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Fig. 6. Experimental T-t curves in different size reactors.

Fig. 7. The surface tension of polymers at different dilution ratios. PADB4.6 I was synthesized at 55 °C, 250 rpm and 0.21 g initiators; PADB4.6 II was synthesized at 62 °C,

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200 rpm and 0.24 g initiators.

Fig. 8. T2 relaxation times of PADB4.0, PADB4.6 I, PADB4.6 II and water. PADB4.6 I was synthesized at 55 °C, 250 rpm and 0.21 g initiators; PADB4.6 II was synthesized at 62 °C,

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200 rpm and 0.24 g initiators.

Fig. 9. Effect of different salts at different concentrations on the partition coefficient of demeclocycline. (I) indicates partition in the PADB4.0/PADB4.6 I ATPS, and (II) indicates

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partition in the PADB4.0/PADB4.6 II ATPS.

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Fig. 10. Effect of pH on the partition coefficient and recovery of demeclocycline.

Table 1 The average molecular weight of PADB4.0, PADB4.6 I, PADB4.6 II and PADB4.6 II500L.

Intrinsic

viscosity

[η] Mη (kDa)

PADB4.0

123.46

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PADB4.6 I

1428.57

457

PADB4.6 II

769.23

PADB4.6 II-500L

714.28

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(ml/g)

222

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Table 2 Characterization of PADB4.6 synthesized in different size reactors.

Systems

Intrinsic viscosity Mη (kDa)

Phase formation time (h)

769.23

222

1.5

5L

909.09

270

3

50 L

1250.00

391

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Flask

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[η] (ml/g)