One-step preparative isolation of aristolochic acids by strong ion-exchange centrifugal partition chromatography

Separation and Purification Technology xxx (2015) xxx–xxx

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One-step preparative isolation of aristolochic acids by strong ion-exchange centrifugal partition chromatography Abdelgadir A. Abdelgadir a,b, Leslie Boudesocque-Delaye a,⇑, Isabelle Thery-Koné a, Alain Gueiffier c, Elhadi M. Ahmed b, Cécile Enguehard-Gueiffier c a b c

UMR INRA 1282 Infectiologie et Santé Publique, Université de Tours, UFR des Sciences Pharmaceutiques, 31 avenue Monge, 37200 Tours, France Department of Pharmacognosy, Faculty of Pharmacy, University of Gezira, Sudan Inserm UMR 1069 Nutrition, Croissance et Cancer, Université de Tours, UFR des Sciences Pharmaceutiques, 31 avenue Monge, 37200 Tours, France

a r t i c l e

i n f o

Article history: Received 15 July 2015 Received in revised form 13 October 2015 Accepted 14 October 2015 Available online xxxx Keywords: Aristolochic acids Aristolochia bracteolata Ion-exchange Counter-current chromatography Centrifugal partition chromatography

a b s t r a c t Aristolochic acids are biologically active and highly toxic secondary metabolites that recently became an important pharmacological tool for in vivo models of acute kidney disorders. Only aristolochic acid I and a mixture of aristolochic acids I and II are currently commercially available. A method based on the strongion exchange displacement mode in centrifugal partition chromatography was developed to extract and purify arictolochic acids, naturally present in Aristolochia bracteolata Lam. (Aristolochiaceae). The extraction and purification of aristolochic acids were performed at a flow rate of 2 mL min 1 in the descending mode by using the biphasic solvent system methyl-ter-butylether, acetone, methanol and water (3:1:1:3, v/v/v/v). Trioctylmethylammonium chloride (Aliquat 336Ò) was used as the anion exchanger in the organic stationary phase and sodium iodide as the displacer in the aqueous mobile phase. From 5 g of crude extract of A. bracteolata, 45.5 mg, 77.2 mg and 35.1 mg of aristolochic acids I, II and IIIa were obtained respectively with purities greater than 95%. Ó 2015 Elsevier B.V. All rights reserved.

1. Introduction Aristolochic acids (AAs) are nephrotoxic secondary metabolites (Fig. 1) present in many Aristolochia species. ‘‘Chinese Herb Nephropathy” and endemic (Balkan) nephropathy are both major renal toxicities linked to the consumption of Aristolochia sp. in herbal remedies [1]. AA are responsible of progressive interstitial kidney fibrosis leading to the need for dialysis or transplant in most of the cases [2]. Moreover, AAs are also associated with high prevalence of urothelial carcinomas (up to 45%) [3]. Recently research has focused on the comprehension of mechanisms of AAassociated nephrotoxicity [4,5]. Reduced forms of AAs I and II form covalent adducts with DNA and RNA, which explains their important role in carcinomas. Nevertheless, the specific mechanisms by which AA-associated nephropathy develops remains unclear. AAs are now used to develop new nephrotoxicity models in zebrafish [6], in mice [7] or to cause acute kidney injury [8] in order to elu-

Abbreviations: AAs, aristolochic acids; MP, mobile phase; Lc, liter of column; SIX-CPC, Strong Ion eXchange Centrifugal Partition Chromatography; SP, stationary phase. ⇑ Corresponding author. E-mail address: [email protected] (L. Boudesocque-Delaye).

cidate AA-specific mechanisms. Only AA I and a mixture of AAs I and II are currently commercially available even though four major AAs are commonly described in the literature (AA I, II, IIIa and IVa). Isolation of those polar metabolites that exhibit closely-related structures by classical chromatography methods is usually time consuming and highly challenging, involving multistep process. Sequential solid/liquid extraction followed by two chromatographic steps of flash chromatography on silica gel [9], or on LH20 resin [10] and then semi preparative HPLC [10] or preparative TLC [11] were usually reported to obtain pure AAs. To isolate milligrams of different AAs, support-free liquid–liquid chromatography techniques such as counter-current chromatography [12] and centrifugal partition chromatography (CPC) [13] could be an innovative option. These techniques are well adapted to the purification of polar secondary metabolites, when carried out either in the elution or in the pH-zone refining [14] and the ion-exchange displacement modes [15]. The Strong Ion eXchange mode (SIX-CPC), is particularly suitable for the purification of ionized or ionizable metabolites such as hydroxycinnamic acids [16], rosmarinic acid [17], glucosinolates [18,19], anthocyanins [20], saponosides [21] or peptides [22,23]. In SIX-CPC, the exchanger is permanently ionic and keeps its positive (cationic exchanger) or negative (anionic exchanger) charge at any pH value [24]. Anionic

http://dx.doi.org/10.1016/j.seppur.2015.10.033 1383-5866/Ó 2015 Elsevier B.V. All rights reserved.

Please cite this article in press as: A.A. Abdelgadir et al., One-step preparative isolation of aristolochic acids by strong ion-exchange centrifugal partition chromatography, Separ. Purif. Technol. (2015), http://dx.doi.org/10.1016/j.seppur.2015.10.033

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A.A. Abdelgadir et al. / Separation and Purification Technology xxx (2015) xxx–xxx

(Rhodano, Italy). Methyltrioctylammonium chloride (Aliquat 336Ò), sodium iodide (NaI), sodium hydroxide (NaOH), mixture of aristolochic acids I and II standards were purchased from Sigma Aldrich (Saint Quentin, France). Water was purified by a Milli-Qsystem (Millipore Corporation, Bedford, MA, USA). A. bracteolata samples were collected during August 2013 from the Southern Gezira area (Sudan) and taxonomically identified in the Department of Pharmacognosy, University of Gezira. A voucher specimen is deposited in the Faculty of Pharmacy, University of Gezira. Fig. 1. Chemical structures of aristolochic acids.

2.2. Preparation of A. bracteolata crude extract exchangers are principally quaternary ammonium salts, such as benzalkonium chlorides or methyltrioctylammonium chloride (Aliquat 336Ò). When using this mode, ionic analytes are directly captured into the stationary phase by forming stable ion-pairs with the exchanger. The displacer-free mobile phase is not able to elute the extracted analytes. However, when adding the displacer, analytes return to the mobile phase (Fig. 2). The separation mechanisms in SIX-CPC have been well described and modeled by Maciuk et al. [16] using separation of phenolic acid regioisomers as methodological support. All analytes moved along the column with the same velocity depending on displacer concentration. This typical column organization is called an isotachic train (Fig. 2). SIXCPC provides high resolution purification based on competition between analytes to form ion-pairs with the exchanger. The aim of this work is to demonstrate that SIX-CPC is an innovative tool for the preparative isolation of AA I, II and IIIa from the Sudanese Aristolochia bracteolata L.

Crushed whole plants of A. bracteolata (289 g) were extracted by 1.25 L of MeOH in a Soxhlet apparatus during 20 h. MeOH was evaporated under reduced pressure and the extract was then freeze dried (71.2 g of freeze dried crude extract).

2.3. CPC apparatus Centrifugal partition chromatography (CPC) separations were performed on a FCPCÒ Preparative 200 Kromaton Technologies apparatus (Rousselet Robatel, Annonay, France) using a rotor made of 20 circular partition disks (840 partition twin-cells; total column capacity: 205 mL, dead volume: 32.3 mL). Rotation speed could be adjusted from 200 to 3000 rpm, thus producing a centrifugal force field in the partition cells of about 120g at 1000 rpm and 480g at 2000 rpm. The solvents were pumped by a preparative Ecom Beta 50 Gradient pump binary low-pressure gradient pump (Praha, Czech Republic). The samples were introduced into the column through a PEEK dual mode preparative scale sample injector 3725i (Rheodyne, Rohnert Park, CA, USA) equipped with a 20 mL sample loop. Effluent content was monitored by an Ecom Flash 06 DAD 600 detector equipped with a preparative flow cell (45 lL internal volume, optical path 0.3 mm). Fractions were collected by an Advantec Super Fraction collector (Otowa, Japan).

2. Experimental 2.1. Chemicals and reagents Acetone, acetonitrile, chloroform (CHCl3), methanol (MeOH), methyl-ter-butylether (MtBE), trifluoroacetic acid (TFA) were purchased in chromatographic grade solvents from Carlo Erba

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Please cite this article in press as: A.A. Abdelgadir et al., One-step preparative isolation of aristolochic acids by strong ion-exchange centrifugal partition chromatography, Separ. Purif. Technol. (2015), http://dx.doi.org/10.1016/j.seppur.2015.10.033

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A.A. Abdelgadir et al. / Separation and Purification Technology xxx (2015) xxx–xxx

2.4. CPC general procedure

100 90

Experimental conditions are summarized in Table 1. Before each experiment, the column was washed with MeOH/water (50:50, v/v) in the ascending mode at 20 mL min 1 with a 600 rpm rotation speed. Two column volumes (410 mL) of the organic stationary phase (SP) containing the exchanger were then pumped in the descending mode at the same flow rate and rotation speed. The sample was injected through the sample loop at 2 mL min 1 at 1200 rpm. Displacer-free mobile phase (MP) (40 mL on average) was pumped at 2 mL min 1 in order to allow column equilibration. Finally, the aqueous mobile phase containing the displacer was pumped at 2 mL min 1, and the fractions were collected every minute. The experiment was monitored at k = 254 nm. Stationary phase retention was about 73% on average. The pressure was approximately 20 bars. The experiments were conducted at room temperature (22 ± 2 °C). 2.5. Preparation of the biphasic solvent system for CPC separations The biphasic system (2 L) was prepared by mixing MtBE, acetone, MeOH and water in proportions (3:1:1:3, v/v/v/v) in a separatory funnel. The solvents were vigorously shaken and then allowed to settle until the phases became limpid. The pH of the aqueous phase was adjusted to 7 by adding the appropriate amount of NaOH 0.1 M. After phase separation, Aliquat 336Ò was added to the organic stationary phase. The mobile phase was prepared by adding the appropriate amount of displacer NaI. 2.6. Pseudoternary diagram construction A solubility isotherm (or a binodal curve at 22 ± 2 °C) of the exchanger Aliquat 336Ò in the MtBE, acetone, MeOH and water pH 7 (3:1:1:3, v/v) system was generated. Pre-defined ratios (w/ w) of stationary phase to mobile phase were successively added to 500 mg of Aliquat 336Ò until the appearance of the conjugated phase. The coordinates of these points are plotted to design the ternary diagram, presented here in its orthogonal form (Fig. 3).

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70 60 50 40 30

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40

50

60

70

80

90

100

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Fig. 3. Pseudo-ternary diagram of the system ionic exchanger—aqueous phase– organic phase (in%, w/w) for the initial biphasic system MtBE/acetone/MeOH/water pH 7 (3:1:1:3, v/v/v/v).

oven and a UHPLC+ DAD-3000 diode array detector (ThermoFisher SA, Voisins le Bretonneux, France). The system was fitted with an Accucore aQ (15 cm  3 mm i.d., 2.6 lm particle size) column, itself protected by an Accucore aQ defender guard 13  3.0 mm cartridge (ThermoFisher SA, Voisins le Bretonneux, France). The mobile phases were solvent A 0.1% TFA in water, solvent B acetonitrile. The gradient was set as follow: initial acetonitrile content was 0%, it was raised to 100% in 15.25 min and maintained for 2 min; the flow rate was 1 mL min 1, the oven temperature was set at 40 °C and the chromatogram was monitored at 254 nm. The collected CPC fractions were analyzed by TLC on Merck 60 F254 silica gel plates. Mobile phase was lower phase of solvent system CHCl3– MeOH–Water (13:7:2, v/v) and observed at 254 nm. 1H, 13C and 2D NMR experiments were performed at 300 MHz (1H) and 75 MHz (13C) on a Brucker-Avance 300 MHz spectrometer (Bruker Biospin, Wissenbourg, France). 3. Results and discussion

2.7. Preparation of sample solutions The crude A. bracteolata extract was dissolved in 18 mL of NaIfree aqueous phase and pH adjusted to 7 by adding the appropriate amount of NaOH 0.1 M. This aqueous solution was then resaturated by 2 mL of Aliquat 336Ò-free organic phase.

A. bracteolata is a widespread Aristolochiaceae specie intensively used in traditional medicine in India and in Sudan [1]. Sudanese A. bracteolata grows in southern area of Gezira (Sudan) and contains high amounts of AAs I and II versus other species [11]. This represents an interesting raw material for large scale isolation of AAs.

2.8. Fractions analyses

3.1. Operating conditions selection

HPLC-DAD analysis were performed on a Dionex UHPLC U3000RS system equipped with a LPG-3400RS quaternary pump, a RSLC WPS-300T RS automated injector, a TCC-300SD column

In neutral solutions (pH = 7), AAs are present as anions (pKa  3) and can then interact with an anion-exchanger, such as Aliquat 336Ò to form ion-pairs.

Table 1 Experimental conditions for CPC runs. CPC run

1

2

3

4

5

6

7

Crude extract sample Estimated content of AAs Biphasic solvent system Pumping mode Aliquat 336Ò concentration in organic phase Washing step duration NaI in aqueous phase Estimated Aliquat 336Ò/AA ratio Aliquat 336Ò/NaI ratio Stationary phase retention Back pressure Collection time

1g

1g

1g

1g

1g

5g

1.25 mM 65 min 0.625 mM 2 2

1.25 mM 65 min 0.31 mM 2 4

1.25 mM 65 min 0.125 mM 2 10

1g 3% (w/w) MtBE/acetone/MeOH/water (3:1:1:3, v/v/v/v) Descending 1.25 mM 65 min 0.06 mM 2 20 70–73% 20–24 bars 1 min (2 mL)

2.4 mM 60 min 0.12 mM 4 20

3.6 mM 50 min 0.18 mM 6 20

18 mM 50 min 0.9 mM 6 20

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Fig. 4. CPC chromatograms of run 1, 4 and 6 (A) and corresponding TLC fractograms (B) (For experimental conditions see Table 1 and Section 2.8).

Classical SIX-CPC solvent systems were screened including CHCl3/n-BuOH/water pH 7 [17], AcOEt/n-BuOH/water pH 7 [18] or MtBE/MeOH/water pH 7 at several ratios without any satisfying results. We observed unfavorable AAs distribution, i.e. mostly in favor of the exchanger free organic stationary phase, or poor solubility of the extract. Finally, the solvent system MtBE/acetone/ MeOH/water (3:1:1:3, v/v/v/v) was selected, which allowed a good solubilization of the crude extract combined with a favorable distribution of AAs. Two cationic exchangers were screened-Aliquat 336Ò and benzalkonium chloride. They were already used to purify glucosinolates [19] and rosmarinic acid [17], respectively. Benzalkonium chloride was not able to extract AAs in the organic phase of the MtBE/acetone/MeOH/water pH 7 (3:1:1:3, v/v/v/v) solvent system whereas Aliquat 336Ò extracted AAs in organic stationary phase at

low concentrations, and was then selected as exchanger in this work. As the stability of a biphasic solvent system is based on equilibrium between the two phases, adding an ion exchanger could disturb this equilibrium and reduce system stability [18]. To investigate the influence of Aliquat 336Ò on the MtBE/acetone/MeOH/ water pH 7 (3:1:1:3, v/v/v/v) solvent system, pseudo-ternary diagrams of organic phase/aqueous phase/Aliquat 336Ò were built. Fig. 3 illustrates that Aliquat 336Ò has a minor disturbing effect on the biphasic system MtBE/acetone/MeOH/water (3:1:1:3, v/v). This allows the addition of large amounts of the exchanger (up to 50% (w/w)), which is favorable to pilot scale studies. The solvent system MtBE/acetone/MeOH/water pH 7 (3:1:1:3, v/v/v/v) and cationic exchanger Aliquat 336Ò were selected. In SIX-CPC, the displacer is a lipophilic ion-here a lipophilic anion. Sodium iodide was so selected as the displacer.

Please cite this article in press as: A.A. Abdelgadir et al., One-step preparative isolation of aristolochic acids by strong ion-exchange centrifugal partition chromatography, Separ. Purif. Technol. (2015), http://dx.doi.org/10.1016/j.seppur.2015.10.033

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5

Fig. 5. SIX-CPC chromatogram of the purification of AAs (Experimental conditions see Run 7, Table 1), and HPLC-DAD analysis of pure AAs.

3.2. SIX-CPC optimization SIX-CPC optimization includes the determination of both ratiosthe exchanger (Aliquat 336Ò)/analytes (AAs) ratio and the exchanger (Aliquat 336Ò)/displacer (NaI) ratio. First, the Aliquat 336Ò/AAs ratio was set at 2, since an excess of exchanger was usually needed to achieve a total extraction during equilibration step. The Aliquat 336Ò/NaI ratio was then optimized (see Runs 1–4, Table 1). The first Aliquat 336Ò/NaI ratio tested (Run 1, Table 1) led to isolation of a small amount (3.7 mg) of AA IIIa with high purity (96%); other AAs were eluted as a mixture. Although this first result was highly promising, as AA IIIa was described and isolated for the first time from A. bracteolata, the experimental conditions have to be optimized. The TLC-fractogram (Fig. 4) of the eluent showed that AAs were eluted as a series of shocklayers, highlighting an excess of displacer. The concentration of NaI was then decreased from 0.625 mM to 0.125 mM (Runs 2 and 3, Table 1). Purification of AA IIIa was improved and mixing zones between the four AAs were gradually reduced. Decreasing the NaI concentration to 0.06 mM (Run 4, Table 1), AA IIIa (4.9 mg) and AA I (8.5 mg) were obtained with purity over 95%, but in small yields. Under these conditions, the mixing zone between all AAs was again reduced, but high mixing zones between AA IIIa and AA IVa, and between AA I and II still remained (Fig. 4). Other CPC experiments were performed using lower NaI concentrations (data not shown) without any improvement in AAs separation. The displacer does not seem to be responsible for the poor efficiency of the purification process.

One hypothesis is that the exchanger concentration is not sufficient to achieve an optimal organization of the isotachic train during the washing step. The lack of exchanger could not allow proper analytes extraction along the whole column. The AAs then would emerge from the column following both displacement and elution mechanisms explaining the important mixing zones. By increasing the Aliquat 336Ò concentration, the number of theoretical plates would increase as well as the resolution. Increasing the Aliquat 336Ò/AAs ratio to 4 (Run 5, Table 1), the purification of AA IIIa (4.2 mg) and AA I (11.1 mg) was slightly improved and a latency period between AAs and other lipophilic metabolites elution was visible, but large mixing zones were still noticedespecially between AAs II, IIIa and IVa (4.5 mg). The Aliquat 336Ò concentration was thus increased again (Run 6, Table 1) to reach an Aliquat 336Ò/AAs ratio of 6. Under these conditions, we were delighted to find excellent separation between AAs IIIa and IVa, and AAs I and II, which was achieved for the first time. Moreover, AAs IIIa (7.2 mg), II (7 mg) and I (11.5 mg) were mainly isolated with purity superior to 95%. AA IVa was obtained as a mixture with AA IIIa, probably due to its small amount insufficient to form a large isotachic zone. These results illustrated the high resolution of SIX-CPC as AAs I, II and IIIa differ from each other by only one methoxyl group. At this stage, the goal was mainly achieved since three of the four AAs present in A. bracteolata were purified. 3.3. SIX-CPC scale up A scale up was then performed with a 5 g sample injection (Run 7, Table 1). As usually observed in displacement CPC, separation

Please cite this article in press as: A.A. Abdelgadir et al., One-step preparative isolation of aristolochic acids by strong ion-exchange centrifugal partition chromatography, Separ. Purif. Technol. (2015), http://dx.doi.org/10.1016/j.seppur.2015.10.033

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was improved leading to isolation of AA IIIa (35.1 mg, purity 98.7%), AA II (77.2 mg, purity 96.7%) and AA I (45.5 mg, purity 95.0%) in one step. AA IVa was still obtained in mixture with AA IIIa (see Fig. 5). Moreover, mixing zones were largely reduced compared to pure compound elution zones. The productivity of the process was calculated considering each AAs: 21 mg h 1 Lc 1 (AA IIIa), 45 mg h 1 Lc 1 (AA II) and 27 mg h 1 Lc 1 (AA I). It is important to note that the process was not pushed over the edge here, due to lack of crude extract. It is then reasonable to think that injection could be increased, almost ten times, reaching usual scale up Aliquat 336Ò concentrations (between 100 and 400 mM) in a 200 mL CPC column [18,19], without damaging the stability of the system. Also, the use of a liquid ion exchanger with tensioactive properties limits the flow rate of the mobile phase to low values, usually 2 or 4 mL min 1, which contribute to reduce the productivity.

[8]

[9]

[10]

[11]

[12] [13]

4. Conclusion AA IIIa and AA IVa were described for the first time in A. bracteolata. SIX-CPC was shown to be an efficient tool for one step isolation of AAs, with high resolution. AA I, AA II and AA IIIa were isolated from crude extracts in one step at a high level of purity in large amounts. This process allows access of new AA standards as AAs II and IIIa for further extensive pharmacological studies. Productivity is in accordance with traditional SIX-CPC values. It might be improved using Centrifugal Partition Extraction (CPE). CPE is a prototype similar to CPC with a smaller number of larger cells, allowing the use of high flow rates. Ion pairing extraction (IX-CPE) was already described for glucosinolates isolation [25] with a flow rate of 30 mL min 1 that significantly improved productivity.

[14]

[15]

[16]

[17]

[18]

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Acknowledgments Abdelgadir A. Abdelgadir thanks the French embassy in Sudan and the University of Gezira for Financial support. References [1] M. Heinrich, J. Chan, S. Wanke, C. Neinhuis, M.S.J. Simmonds, Local uses of Aristolochia species and content of nephrotoxic aristolochic acid 1 and 2-A global assessment based on bibliographic sources, J. Ethnopharmacol. 125 (2009) 108–144, http://dx.doi.org/10.1016/j.jep.2009.05.028. [2] F.D. Debelle, J.-L. Vanherweghem, J.L. Nortier, Aristolochic acid nephropathy: a worldwide problem, Kidney Int. 74 (2008) 158–169, http://dx.doi.org/ 10.1038/ki.2008.129. [3] R.L. Luciano, M.A. Perazella, Aristolochic acid nephropathy: epidemiology, Clin. Present., Treatment, Drug Saf. 38 (2015) 55–64, http://dx.doi.org/10.1007/ s40264-014-0244-x. [4] B. Jelakovic´, X. Castells, K. Tomic´, M. Ardin, S. Karanovic´, J. Zavadil, Renal cell carcinomas of chronic kidney disease patients harbor the mutational signature of carcinogenic aristolochic acid, Int. J. Cancer 136 (2015) 2967–2972, http:// dx.doi.org/10.1002/ijc.29338. [5] E.M.K. Leung, W. Chan, Comparison of DNA and RNA adduct formation: significantly higher levels of RNA than DNA modifications in the internal organs of aristolochic acid-dosed rats, Chem. Res. Toxicol. 28 (2015) 248–255, http://dx.doi.org/10.1021/tx500423m. [6] Y.-J. Ding, Y.-H. Chen, Developmental nephrotoxicity of aristolochic acid in a zebrafish model, Toxicol. Appl. Pharmacol. 261 (2012) 59–65, http://dx.doi. org/10.1016/j.taap.2012.03.011. [7] T.C. Huang, S.M. Chen, Y.C. Li, J.A. Lee, Increased renal semicarbazide-sensitive amine oxidase activity and methylglyoxal levels in aristolochic acid-induced

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Please cite this article in press as: A.A. Abdelgadir et al., One-step preparative isolation of aristolochic acids by strong ion-exchange centrifugal partition chromatography, Separ. Purif. Technol. (2015), http://dx.doi.org/10.1016/j.seppur.2015.10.033