Salt Aqueous

Mar 27, 2017 - (3, 4) Compared with a common type of ATPSs, IL-based ATPSs own the advantages of negligible viscosity, no emulsion formation, negligib...
1 downloads 15 Views 152KB Size
Download PDF
Research Article pubs.acs.org/journal/ascecg

Hexafluoroisopropanol-Based Deep Eutectic Solvent/Salt Aqueous Two-Phase Systems for Extraction of Anthraquinones from Rhei Radix et Rhizoma Samples Wen-Wen Deng, Yu Zong, and Yu-Xiu Xiao* Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Ministry of Education) and School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, China S Supporting Information *

ABSTRACT: Deep eutectic solvents (DESs), a new class of green and sustainable solvents, have shown a promising application prospect in the aqueous two-phase system (ATPS) extraction. In the present work, hexafluoroisopropanol (HFIP) was introduced as a hydrogen-bond donor to synthesize DESs with choline chloride (ChCl), decyltrimethylammonium bromide, dodecyltrimethylammonium bromide, and tetradecyltrimethylammonium bromide (hydrogen-bond acceptor), respectively. These HFIP-based DESs (HFIP-DESs) can form ATPS with various inorganic salts, and their phase separation ability is significantly stronger than that of small molecule aliphatic alcohols, HFIP, and traditional DESs. The ATPSs based on HFIP-DESs were first used to extract anthraquinones (AQs) from Rhei Radix et Rhizoma samples. Under the optimal conditions, more than 92% of AQs (including aloe-emodin, rhein, emodin, chrysophanol, and physcion) was enriched in the DES-rich phase of the ATPS composed of ChCl-HFIP DES and Na2SO4. Compared with the traditional chloroform extraction method (60 mL chloroform consumption), the ChCl-HFIP DES/ Na2SO4 ATPS extraction method attained high extraction efficiency but only consumed 0.5 mL of organic solvent (HFIP). KEYWORDS: Deep eutectic solvent, Aqueous two-phase system, Hexafluoroisopropanol, Phase separation, Anthraquinones, Liquid−liquid extraction

■

INTRODUCTION An aqueous two-phase system (ATPS), as a mild, biocompatible, and environmentally friendly liquid−liquid extraction technology, has been extensively used in the extraction, separation, and purification of bioactive components.1 Up until now, most ATPSs applied in extraction are based on polymer/salt, polymer/polymer, and small molecule aliphatic alcohol/salt systems.1,2 However, most of the phase-forming polymers have high viscosity and can be emulsified easily, which increases the extraction time and interferes with the detection of analytes.3 Small molecule alcohol/salt systems require a large volume of volatile organic solvent, which is detrimental to the activity of biosamples and the health of the environment. In the past decade, ATPSs based on ionic liquid (IL) have been proposed and used for the extraction and purification of various compounds.3,4 Compared with a common type of ATPSs, ILbased ATPSs own the advantages of negligible viscosity, no emulsion formation, negligible volatility, quick phase separation, high extraction efficiency, etc. due to the excellent characteristics of IL. Nonetheless, the following two disadvantages of ILs hinder the development of IL-based ATPSs. One is that the preparation of ILs is expensive, tedious, and purification-difficult. Another is related to their toxicity, © 2017 American Chemical Society

biodegradability, and bioaccumulation, which have aroused increasing public attention to the harmful influence of ILs on the environment.5 A deep eutectic solvent (DES), emerging as a new generation of green and sustainable solvents, has attracted attention in many fields.6 Generally, DESs are prepared by mixing two starting components, i.e. a hydrogen-bond donor (HBD) and a hydrogen-bond acceptor (HBA), with the aid of strong hydrogen-bonding interaction. DESs have a melting point lower than that of each component. Compared to ILs, the synthesis of DESs is relatively simple, and the products require no purification with a 100% atom utilization rate, which greatly cut the costs. Besides, DESs retain the excellent physicochemical properties of traditional ILs. Thus far, DESs have been used as extraction media for extracting active ingredients in traditional Chinese medicine7−9 and phase-forming components of ATPS for protein and DNA extraction.10−13 In 2014, Wang et al. first found that a DES composed of choline chloride (ChCl) and urea could form ATPS with K2HPO4, and the Received: January 26, 2017 Revised: March 19, 2017 Published: March 27, 2017 4267

DOI: 10.1021/acssuschemeng.7b00282 ACS Sustainable Chem. Eng. 2017, 5, 4267−4275

Research Article

ACS Sustainable Chemistry & Engineering Table 1. Compositions, Structures, and Physicochemical Properties of the Studied DESs

a

Data obtained using SciFinder Scholar from Chemical Abstracts Service. bData obtained by analysis of DSC.

DES-based ATPS could efficiently extract bovine serum albumin from aqueous solutions.10 Soon after, they further extracted protein and DNA by the ATPS based on ChClglycerol DES, betaine-urea DES, and tetrabutylammonium bromide-ethylene glycol DES, respectively.11−13 These researches indicate that DES-based ATPS is a promising extraction method. However, the phase separation capability of the DESs reported so far is a bit poor.12 Therefore, it is of great significance to synthesize the novel DESs with strong phase separation ability for broadening the practical applications of DES-based ATPS. Hexafluoroisopropanol (HFIP) is a perfluorinated alcohol with strong hydrogen bond donor ability (1.96, larger than that of water 1.02) and high dissolving power. It has been proved that HFIP can efficiently induce the aggregation and liquid− liquid phase separation in aqueous solutions of various amphiphiles14 and displays promising prospects in the analytical field.15,16 Recently, Wang et al. prepared a novel DES based on HFIP and ChCl and employed it as a green reaction medium for the rapid and efficient synthesis of phenols and halogenation of 4,4-difluoro-4-bora-3a,4a-diaza-s-indacenes.17,18 This research highlights the application of HFIP in DES preparation. Meanwhile, considering the superior ability of HFIP to induce liquid−liquid phase separation in aqueous solutions of amphiphiles, HFIP-based DES (HFIP-DES) is expected to have excellent phase-separation capability as a phase component of ATPS. So far, ATPS based on HFIP-DES and its application have not yet been reported. Rhei Radix et Rhizoma (RRR) belongs to the Polygonaceae family of plants. It is a famous traditional Chinese herbal medicine with many pharmacological activities such as antifungal, antiviral, antioxidant, anticancer, and antimutagenicity. Anthraquinones (AQs) including aloe-emodin, rhein, emodin, chrysophanol, and physcion are the main bioactive constituents of RRR.19 The traditional method for extraction of AQs from RRR samples is as follows: RRR powder was extracted by heat reflux with methanol, the obtained methanol solution was dried, and then the residue was treated by acid hydrolysis and liquid−liquid extraction with chloroform.20 This method is time-consuming and requires a great deal of toxic chloroform. Li et al. investigated the application of green DESs in the extraction of AQs from RRR; however, the extraction yield was unsatisfactory due to the weak polarity of AQs.9

In this work, HFIP was introduced to prepare novel DESs, and HFIP-DESs were used as phase-forming components to develop ATPSs with an aqueous salt solution for the first time. The phase behaviors were investigated to evaluate the phase separation ability of HFIP-DESs. ATPSs based on HFIP-DES were applied for extracting AQs from RRR samples. The results indicate that the ChCl-HFIP DES based ATPS extraction method is highly efficient and very reliable for the extraction of AQs in RRR samples. What is more, this new extraction method is much greener than the traditional chloroform extraction for AQs.

■

EXPERIMENTAL SECTION

Materials and Reagents. RRR was purchased from a local traditional Chinese medicine drugstore (Wuhan, China). Aloe-emodin, chrysophanol, emodin, physcion, and rhein were purchased from Chengdu Must Biotechnology Co., Ltd. (Chengdu, China), and their physical and chemical properties are shown in Table S1. HFIP and 1,8dihydroxyanthraquinone were purchased from Aladdin Chemistry Co. (Shanghai, China). Na2SO4, (NH4)2SO4, Na2HPO4, K2HPO4, magnesium acetate, ChCl, decyltrimethylammonium bromide (DeTAB), dodecyltrimethylammonium bromide (DTAB), tetradecyltrimethylammonium bromide (TTAB), urea, glycerol, and ethylene glycol were obtained from Sinopharm Chemical Reagent Co. (Shanghai, China). Deionized water was obtained from a Milli-Q system (Millipore, Molsheim, France). All other reagents used are of analytical grade. Preparation and Characterization of DESs. HFIP was used as HBD to synthesize DESs with ChCl, DeTAB, DTAB, and TTAB (HBA), as shown in Table 1. The DESs were prepared by following a simple procedure. HFIP was mixed with a HBA at a proper molar ratio. The mixture was heated at 80 °C with constant magnetic agitation until the formation of a clear, homogeneous, and stable liquid, which is called DES. The melting point of DESs was measured by differential scanning calorimetry (DSC) (STA449C 200P, Germany), with temperature increasing from −120 to 50 °C at a rate of 10 °C/min under nitrogen. The viscosity of DESs was determined using a Rheometer (DISCOVERY HR-2, USA) at constant temperature. The 1H−1H-nuclear overhauser spectroscopy (NOESY) and FT-IR spectra were examined by an NMR spectrometer (Bruker DPX-400, Germany) and an FT-IR spectrometer (PerkinElmer, USA), respectively. Preparation of Phase Diagrams. The phase diagrams were prepared by the method of cloud point titration at room temperature.21 A salt aqueous solution with known concentration was added in a 50 mL conical flask. Then, a DES or alcohol was added into the conical flask drop by drop. When the mixture became turbid, 4268

DOI: 10.1021/acssuschemeng.7b00282 ACS Sustainable Chem. Eng. 2017, 5, 4267−4275

Research Article

ACS Sustainable Chemistry & Engineering

Figure 1. NOESY spectra of ChCl-HFIP DES (a) and DTAB-HFIP DES (b). deionized water was added until the turbidity disappeared. The above procedure was repeated to acquire adequate data about the concentrations of a DES (or alcohol) and salt at different turbid points, which was used to construct the phase diagrams. Aqueous Two-Phase System Extraction. The extraction procedure of ATPS based on HFIP-DES is illustrated in Figure S1. An appropriate amount of a DES was added in a 15 mL centrifuge tube. Then, a 5 mL salt solution with a certain concentration was added into the tube. Upon the addition of a certain volume of RRR samples, the mixture was shaken vigorously for 20 s using a vortex mixer and then centrifuged at 3500 rpm for 10 min. At this time, the mixture was separated to a liquid−liquid two phase system. The bottom was the HFIP-DES-rich phase, while the top was the HFIPDES-poor phase (i.e., the aqueous phase or the salt-rich phase). The volumes of the top phase and the bottom phase were recorded. The bottom phase was withdrawn for spectrometric determination or HPLC analysis. The volume ratio of the HFIP-DES-rich phase versus the aqueous phase is defined as the phase ratio (R). The phase ratio, enrichment factor (EF), and extraction efficiency (EE) of AQs were calculated by the following equations

R=

VD VA

ATPS, the RRR sample, or the reference RRR sample was carried out on a Shimadzu HPLC system equipped with two LC-20AT pumps, a SPD-20A UV detector, and a column temperature control oven. Separation was performed on an Amethyst C18 column (250 mm × 4.6 mm, 5 μm). The mobile phase was composed of 0.1% H3PO4 (eluent A) and methanol (eluent B) with a gradient elution. The gradient program was as follows: 0−10 min, 70%−75% B; 10−11 min 75%−85% B; 11−30 min 85%−95% B. The flow rate was set at 1.0 mL/min, the detection wavelength was set at 254 nm, and the column temperature was set at 25 °C. The injection volume was 10 μL. The validation results of spectrometry and the HPLC analysis method for quantification of AQs are shown in Table S2 and Table 3S, respectively. Preparation of RRR Samples. A RRR sample was prepared according to ref 20 and Chinese Pharmacopoeia published in 2015 with appropriate modifications. A 0.5 g sample of powdered RRR herb (65 mesh) was extracted with 25 mL of methanol by refluxing for 60 min. The obtained solution was cooled to room temperature and then filtered. The filtrate was transferred to a flask and evaporated to dryness. The residue was mixed with 10 mL of 8% (v/v) HCl solution. The mixed solution was sonicated for 2 min and then hydrolyzed for 60 min in an oil bath at 80 °C. The hydrolyzed solution was dried under the reduced pressure. The residue was dissolved with methanol and transferred into a 10 mL volumetric flask, and methanol was added to the mark. The RRR sample was used for HFIP-DES based ATPS extraction, and the obtained DES-rich phase was used for detection of AQs by HPLC or spectrometry. To obtain a reference RRR sample, 0.5 g of powdered RRR was used to obtain the mixed solution (with HCl) according to the steps mentioned above. After sonication for 2 min, 10 mL of chloroform was added into the mixed solution. The resulting solution was refluxed for 60 min in an oil bath for hydrolyzation. After cooling to room temperature, the hydrolyzed solution was transferred to a separating funnel and extracted three times with 10 mL of chloroform. The combined extraction solution was evaporated to dryness under reduced pressure. The residue was dissolved with methanol and transferred to a 10 mL volumetric flask. Then, methanol was added to the mark. The reference RRR sample was directly used for detection of AQs by HPLC.

(1)

EF =

CD CT

(2)

EE =

C D × VD C T × VT

(3)

where VD, VA, and VT are the volume of the DES-rich phase, the aqueous phase, and total solution, respectively, and CD and CT represent the concentration of AQs in the DES-rich phase and the original concentration of AQs, respectively. Spectrometry and HPLC Analysis of AQs Samples. The concentration of total AQs in the DES-rich phase of ATPS or the RRR sample was determined by spectrometry.22 A certain volume of the DES-rich phase or the RRR sample was added in a 5 mL volumetric flask and diluted to the mark using a 0.5% magnesium acetate solution. AQs were changed to pink at weak alkaline condition and could be measured at 512 nm by a UV−vis spectrophotometer (Shimadzu, UV2100, Japan). A calibration curve was established with 1,8dihydroxyanthraquinone standard solutions to calculate the total AQs concentration. HPLC analysis of AQs in the DES-rich phase of

■

RESULTS AND DISCUSSION Characterization of HFIP-DESs. The melting points of the four HFIP-DESs are all below −24 °C, which is obviously less

4269

DOI: 10.1021/acssuschemeng.7b00282 ACS Sustainable Chem. Eng. 2017, 5, 4267−4275

Research Article

ACS Sustainable Chemistry & Engineering

Figure 2. Phase diagrams of ATPSs at room temperature. (a) ATPSs composed of ChCl-HFIP and various salts; (b) ATPSs composed of K2HPO4 and different alcohols or ChCl-HFIP; (c) ATPSs composed of K2HPO4 and various DESs.

Figure 3. Effect of the amount of ChCl-HFIP DES (a) and salt concentration (b) on the phase ratio of the ATPS. (a) K2HPO4 concentration was set at 0.3 g/mL. (b) The amount of ChCl-HFIP DES was set at 0.2 g.

(H4′) of DTAB. This might be due to the hydrogen-bonding interaction between the −OH of HFIP and Br− of DTAB as well as the hydrophobic interaction between the trifluoromethyl group of HFIP and methylene of DTAB (Figure 1b). According to the results of FT-IR and NOESY analyses, we can confirm the formation of a hydrogen-bond between HFIP and QACs in the synthesis of HFIP- DESs. Phase Diagram of ATPS Based on HFIP-DESs. Liquid− liquid equilibrium data of ATPS are necessary for the design of an extraction process. In the phase diagram of ATPS, above the curve is the two-phase area, while below the curve is the homogeneous phase area. The curve is closer to the origin, and the phase separation ability of the compositions of ATPS is stronger.28−30 In this work, five inorganic salts were selected as phase-forming salts to construct phase diagrams with ChClHFIP DES. As shown in Figure 2a, ChCl-HFIP DES can form ATPS with all five salts, and the phase separation ability of these salts is on the order of Na2SO4 > K2HPO4 > Na2HPO4 > (NH4) 2SO4 > KH2PO4. Li et al. used three salts (K2HPO4, Na2HPO4, and KH2PO4) to form ATPSs with the six DESs they prepared (betaine-methylurea, betaine-urea, betaine-ethylene glycol, betaine-glucose, betaine-sorbitol, betaine-glycerol), but only K2HPO4 could form ATPSs with these DESs.12 This indicates that ChCl-HFIP DES has superior phase separation ability compared to the six DESs. To further compare the phase separation ability of HFIPDESs with that of alcohols and common DESs (ChCl-ethylene glycol, ChCl-glycerol, and ChCl-urea as representatives), the phase diagrams of aqueous mixed systems consisting of HFIPDESs/various alcohols/common DESs and K2HPO4 were

than that of their compositions (Table 1). The decrease in the melting point might be due to the disruption of the crystalline structure of quaternary ammonium compounds (QACs) caused by the hydrogen-bond interaction between HFIP and QACs.23 To confirm hydrogen bonds in HFIP-DESs, FT-IR spectra and NOESY of two representative HFIP-DESs (ChCl-HFIP and DTAB-HFIP) were examined. The results are shown in Figure S2 and Figure 1, respectively. In the FT-IR spectra of the DESs, the O−H stretching vibration peaks shift to a lower wavenumber (3383.76 cm−1, 3416.27 cm−1) compared to the O−H stretching vibration of pure HFIP (3446.90 cm−1), which might be caused by the transfer of the electron cloud of an oxygen atom to hydrogen bonding and thus the decrease in force constant.24,25 This phenomenon shows the formation of the hydrogen bond between HFIP and QACs. In addition, no new peak is observed, indicating that there is no chemical reaction occurrence in the preparation of DESs. The NOESY has been successfully applied to research the hydrogen-bonding interaction in DESs and ILs. Mele et al. observed the intermolecular and intramolecular hydrogen-bonding interaction of 1-n-butyl-3-methylimidazolium tetrafluoroborate molecules through the NOESY.26 Dai et al. confirmed the hydrogenbonding interaction between 1,2-propanediol and ChCl (two components of DES) by the NOESY.27 In our study, the NOESY spectrum of ChCl-HFIP DES proves that the proton of −OH of HFIP interacts intensely with the proton of −OH of ChCl. This implies that the −OH of HFIP can form a hydrogen-bond with ChCl (Figure 1a). The NOESY spectrum of DTAB-HFIP DES reveals that the proton of −OH of HFIP interacts with the protons of methyl (H2′) and methylene 4270

DOI: 10.1021/acssuschemeng.7b00282 ACS Sustainable Chem. Eng. 2017, 5, 4267−4275

Research Article

ACS Sustainable Chemistry & Engineering

Figure 4. Effect of type of HFIP-DES (a), type of salt (b), salt concentration (c), HFIP-DES amount (d), the quantity of RRR samples (e), temperature and the pH of the aqueous solution (f) on the extraction efficiency of total AQs. In part (f): 1, 2, 3, and 4 on the x-axis stand for temperature at 25, 35, 45, and 55 °C; 1, 2, 3, 4, and 5 on the x-axis stand for pH at 3, 5, 7, 9, 11, and 13.

mL, the salt-outing force reaches saturation, thus the phase ratio remains nearly unchanged. Optimization of Extraction Conditions. The ATPSs based on HFIP-DESs and inorganic salts were used to extract total AQs from the RRR samples. Many factors, including the type and amount of HFIP-DESs and inorganic salts, the quantity of the RRR sample, temperature, and solution pH, were optimized to acquire high extraction efficiency. The total AQs were determined by spectrometry. Type of HFIP-DES. Four kinds of HFIP-DESs were chosen as phase-forming components of ATPS to investigate the influence of different HFIP-DESs on the extraction efficiency of total AQs. According to the phase diagrams (Figure 2c), 0.5 g of HFIP-DES and 5 mL of 0.2 g/mL K2HPO4 solution were selected to ensure the formation of ATPSs. A RRR sample of 100 μL was added into the ATPS. As shown in Figure 4a, the ATPS based on HFIP-ChCl DES presents the highest extraction efficiency. The reason may be ascribed to the structure of ChCl with the −OH group, which can provide additional hydrogen-bonding interaction with AQs. Furthermore, the extraction efficiency decreases with increasing the alkyl chain length of HBAs in the other three DESs. This is possibly relevant to the increase of the viscosity of a DES (Table 1), which would hinder the AQs diffusing into the DESrich phase. ChCl-HFIP was selected as the phase-forming DES to construct ATPSs in the following experiments. Type of Inorganic Salt. Five kinds of common salts ((NH4)2SO4, Na2SO4, Na2HPO4, K2HPO4, KH2PO4) were employed to examine the effect of different salts on the extraction efficiency of total AQs. The ATPS was composed of ChCl-HFIP DES (0.5 g) and the salt solution (0.2 g/mL, 5 mL). A RRR sample of 100 μL was added. The results indicate that the phase separation ability of KH2PO4 is too weak to form ATPS under the above conditions. As shown in Figure 4b, the

recorded. As shown in Figure 2b and c, ChCl-HFIP DES has a stronger phase separation ability than aliphatic alcohols, HFIP, and the three common DESs based on ChCl. It has been proved that the phase separation ability of a phase-forming solvent would increase with increasing the interaction force between the molecules of the solvent and with decreasing the interaction force between the solvent and water molecules.31 Therefore, the excellent phase separation ability of ChCl-HFIP DES may be ascribed to the strong hydrogen-bond interaction between HFIP and ChCl, which can, in turn, inhibit the interaction between ChCl-HFIP DES and the water molecules. Furthermore, because HFIP owns stronger hydrophobicity than common HBDs (such as urea, ethylene glycol, glycerol), HFIPDESs can be separated more easily from the water solution by the salt-outing force, which also contributes to the stronger phase separation ability of ChCl-HFIP DES. Meanwhile, Figure 2c shows that the phase separation ability of HFIP-DESs with long alkyl chain QACs (DeTAB-HFIP, DTAB-HFIP, TTABHFIP) is more excellent than that of ChCl-HFIP DES with a short alkyl chain. This is because the strong hydrophobicity of long alkyl chain QACs can greatly reduce the interaction force between the DESs and water molecules.32 Phase Ratio. The effects of the amount of ChCl-HFIP DES and the concentration of the salt solution on the phase ratio of ChCl−HFIP DES based ATPS were investigated at room temperature. From Figure 3a, the phase ratio increases linearly (r = 0.9957) as the amount of ChCl-HFIP DES increases from 0.05 to 0.3 g. As seen in Figure 3b, with the increase of salt concentration, the phase ratio increases at first and then remains almost unchanged over 0.3 g/mL. This is because the salt-outing force can be strengthened with an increasing salt concentration, making a greater amount of ChCl-HFIP DES be salted out from the water solution. As a consequence, the phase ratio is increased. As the salt concentration is larger than 0.3 g/ 4271

DOI: 10.1021/acssuschemeng.7b00282 ACS Sustainable Chem. Eng. 2017, 5, 4267−4275

Research Article

ACS Sustainable Chemistry & Engineering

Figure 5. Chromatograms of (a) the DES-rich phase obtained after ATPS extraction for the RRR sample solution, (b) the aqueous phase obtained after ATPS extraction for the RRR sample solution, (c) the RRR sample solution, (d) the standard solution of AQs (each AQ at 10 μg/mL), and (e) the DES-rich phase of ATPS in the absence of the RRR sample (blank control). Peak identification: 1, aloe emodin; 2, rhein; 3, emodin; 4, chrysophanol; 5, physcion.

displays a fast increase from 0.2 to 0.35 g, a slow increase from 0.35 to 0.65 g, and a little decline over 0.65 g. The explanation is that the number and size of DES aggregates in the DES-rich phase might increase rapidly at first and then tend to be saturated as the added DES amount varies from 0.2 to 0.65 g.11 With the DES addition amount being larger than 0.65 g, an excessive DES would dissolve in the aqueous phase, leading to the slight decline of the AQs extracted into the DES-rich phase. The optimum mass of DES added in ATPS was selected at 0.65 g. Quantity of the RRR Sample. ChCl-HFIP DES (0.65 g)/ Na2SO4 (0.25 g/mL, 5.0 mL) ATPS was used for the optimization of the quantity of the RRR sample added in ATPS. As the RRR sample increases from 50 to 300 μL, the extraction efficiency presents a gradual rise at first, then a constant value, and a decrease at last (Figure 4e). In the aqueous phase of DES-based ATPS, there are still bits of DES, which can dissolve some AQs and lead to low extraction efficiency at a small quantity of the sample. As the sample quantity increases, many more AQs would be extracted into the DES-rich phase due to the very limited dissolving capacity of the DES in the aqueous phase. However, because the extraction capacity of the DES-rich phase is also limited, the excessive addition of the sample would make excessive AQs precipitate, hence the extraction efficiency decreases. The optimized volume was chosen as 250 μL. Temperature and Solution pH. In order to investigate the influence of extraction temperature and pH value of the aqueous solution on the extraction efficiency, ChCl-HFIP DES (0.65 g)/Na2SO4 (0.25 g/mL, 5.0 mL) ATPS was used to extract total AQs from the RRR sample (250 μL). As shown in Figure 4f, the extraction efficiency is not apparently affected by the variation of both temperature (25−55 °C) and pH (3−11). Although the pH of the aqueous solution is influential for the charged status of AQs and thus affects the interactions (hydrophobic, electrostatic, and hydrogen-bonding interactions) of AQs with the DES-rich phase, Na2SO4 with a very strong salt-outing force (Figure 2a) can strip both ionized and

Na2SO4-based ATPS has the highest extraction efficiency. This is because of the strongest phase separation ability of Na2SO4 (Figure 2a). That means more DES can aggregate in the DESrich phase of the Na2SO4-based ATPS. As a result, the more AQs were extracted into the DES-rich phase by the interaction forces between AQs and DES as well as the strong salt-outing force of Na2SO4. However, although Na2HPO4 and K2HPO4 show a stronger phase separation ability than (NH4)2SO4, their ATPSs give the relatively lower extraction efficiency than the (NH4)2SO4-based ATPS. The explanation is that the AQs with pKa values less than 6.6 (Table S1) can ionize in both Na2HPO4 and K2HPO4 aqueous alkaline solutions, leading to the decrease of hydrophobic interaction of AQs with the DESrich phase and thus the lower extraction efficiency. Na2SO4 was selected as a phase-separation salt for further investigation. Concentration of Salt. The effect of Na2SO4 concentration was investigated using ChCl-HFIP DES (0.5 g)/Na2SO4 (5.0 mL) ATPS with 100 μL of the RRR sample added. The results are shown in Figure 4c. The extraction efficiency increases rapidly at first, then tends to plateau, and finally decreases a bit with increasing Na2SO4 concentration in the range of 0.1−0.3 g/mL. The possible reason is that both the salting-out effect and the phase separation ability are much enhanced with the increase of salt concentration from 0.1 to 0.15 g/mL, leading to the obvious increase of the amount of DES in the DES-rich phase. Consequently, many more AQs were extracted into the DES-rich phase. However, the amount of DES in the DES-rich phase might tend to saturation when the salt concentration is over 0.15 g/mL, thus the extraction efficiency increases very slowly. As the salt concentration is greater than 0.25 g/mL, the increased viscosity of the salt aqueous solution would reduce the diffusion of AQs into the DES-rich phase, resulting in the slight decrease of the extraction efficiency. The optimal Na2SO4 concentration was set as 0.25 g/mL. Addition Amount of HFIP-DES. Figure 4d shows the influence of the amount of ChCl-HFIP EDS added in ATPS on the extraction efficiency of total AQs. As the DES amount varies in the range of 0.2−0.8 g, the extraction efficiency 4272

DOI: 10.1021/acssuschemeng.7b00282 ACS Sustainable Chem. Eng. 2017, 5, 4267−4275

Research Article

ACS Sustainable Chemistry & Engineering Table 2. Comparison of ATPS Based on ChCl-HFIP DES with a Traditional Extraction Method amount of extracted AQs ± SD (mg/g RRR powder, n = 3) extraction method ChCl-HFIP DES/Na2SO4 ATPS extraction chloroform extraction

aloe-emodin

rhein

emodin

chrysophanol

physcion

total AQs

organic solvent consumption/ 1.0 g RRR powder

1.07 ± 0.46

1.30 ± 0.05

3.23 ± 0.06

4.08 ± 0.06

1.43 ± 0.02

11.11 ± 0.06

DES:1.30 g (HFIP: 0.5 mL)

1.02 ± 0.22

1.60 ± 0.02

2.93 ± 0.02

4.48 ± 0.10

1.36 ± 0.03

11.40 ± 0.17

CHCl3: 60 mL

Figure 6. Laser confocal micrograph of the DES-rich phase of ATPS based on ChCl-HFIP DES/Na2SO4 with Rhodamine B (a) and BODIPY (b) as fluorescent probes. Exciting wavelength is 561 nm for Rhodamine B and 575 nm for BODIPY. Emission wavelength is 587 nm for Rhodamine B and 665 nm for BODIPY.

ChCl-HFIP DES-based ATPS extraction method are very close to those by the conventional method. In addition, the proposed method did not use a large volume of toxic chloroform and just needed 0.5 mL of HFIP to extract AQs from 1 g of RRR herb. Therefore, the ChCl-HFIP DES-based ATPS extraction method is reliable, effective, and greener and can replace the conventional method to extract AQs from RRR samples. Extraction Mechanism. The salting-out effect and the solubilization of extraction solvents for target compounds are often regarded as main extraction mechanisms of ATPS.33 In the present study, the salting-out experiment and the laser confocal microscopy examination (Leica-LCS-SP8-ATED, Germany) were employed to explore the extraction mechanism of ChCl-HFIP DES-based ATPS. As seen in Figure S3, 1,8dihydroxyanthraquinone (DHAQ) was precipitated from its solution by the salt-outing force of Na2SO4. Upon further adding ChCl-HFIP DES in the tube, an ATPS formed and DHAQ was dissolved in the DES-rich phase. The results indicate that the salt-outing effect plays an important role in the extraction of AQs, and ChCl-HFIP DES is an ideal solvent for AQs. As we know, DHAQ is pink (anionic form) in dilute alkaline solution but is yellow (neutral form) in neutral and acidic environments.34 Given that DHAQ is yellow in the DESrich phase, it is concluded that most DHAQ is dissolved in the DES aggregates (neutral environment) of the DES-rich phase. Figure 6 shows the laser confocal microscopy results. As seen, there are spherical DES aggregates (100 nm−5 μm in diameter) in the DES-rich phase. Both water-soluble Rhodamine B and water-insoluble BODIPY (Figure S4) can be partitioned into the DES aggregates in the DES-rich phase after ATPS extraction. This phenomenon reveals that ChCl-HFIP DES has a strong solubilizing capability in that it can provide various interactions with target analytes, such as hydrogenbonding interaction, hydrophobic interaction, electrostatic interaction, and so on. In short, the salting-out effect and the

nonionized AQs from the aqueous solution to the DES rich phase. As a result, the effect of pH on the extraction efficiency is negligible. For the sake of convenience, room temperature was adopted, and the pH of the aqueous solution was not adjusted in all the extraction experiments. In summary, the optimized ATPS extraction conditions for total AQs are as follows: the addition amount of ChCl-HFIP DES is 0.65 g, the concentration of the Na2SO4 aqueous solution (5 mL) is 0.25 g/mL, and the RRR sample quantity is 250 μL; the extraction experiments were carried out at room temperature. At these conditions, the extraction efficiency of total AQs reaches 93.5%. Analysis of the Components of Extracted Total AQs. The HPLC-UV method was employed to analyze the constituents of total AQs in the DES-rich phases obtained after extracting the RRR sample solution under optimal conditions. As shown in Figure 5, the total AQs extracted by ChCl-HFIP DES/Na2SO4 ATPS include aloe-emodin, rhein, emodin, chrysophanol, and physcion. As seen in Table S4, the extraction efficiency of each AQ is in the range of 92.1%− 97.4%, averaging 94.5% which is almost equal to the extraction efficiency of total AQs determined by spectrometry. Meanwhile, the concentration of each AQ was enriched by ca. 15− 16-fold after extraction. Actually, if the enrichment factor, rather than the extraction efficiency, was used as a criterion for optimizing extraction conditions, the higher enrichment effect would be obtained since the phase ratio can be controlled at a very low value (Figure 3). Comparison of HFIP-DES/Salt ATPS Extraction with Conventional Chloroform Extraction. Figure 5 shows that the total AQs extracted by the ATPS contain the five constituents, being the same as the components of the total AQs obtained by the conventional chloroform extraction. The two extraction methods were used for determination of the AQs in real RRR herb, and the results are listed in Table 2. For both the total AQs and each AQ, the results derived by the 4273

DOI: 10.1021/acssuschemeng.7b00282 ACS Sustainable Chem. Eng. 2017, 5, 4267−4275

ACS Sustainable Chemistry & Engineering

■

strong solubilizing capability of ChCl-HFIP DES are the main extraction mechanisms of ChCl-HFIP DES/salt ATPS for AQs.

CONCLUSIONS Four HFIP-DESs were prepared and used to construct ATPSs with inorganic salts. Compared to traditional DESs (not based on HFIP), small molecule aliphatic alcohols, and HFIP, HFIPDESs show a much stronger phase separation ability. Thus, the amount of both HFIP-DESs and salts required to form ATPS can be reduced significantly. Moreover, the ATPSs composed of HFIP-DESs and salts were employed to extract the AQs from RRR samples. The high extraction recovery was obtained for each AQ (>92%) by use of the ATPS composed of ChClHFIP DES and Na2SO4. The salt-outing force and the solubilizing capability of ChCl-HFIP DES contribute to the high extraction efficiency. In comparison with the traditional chloroform extraction method, the ChCl-HFIP DES-based ATPS extraction method attained almost the same results for extracting the AQs from RRR samples but just consumed a very small amount of organic solvent. The proposed method is reliable, effective, and greener and can replace the conventional method to extract AQs from RRR samples. This study holds promise for further utilization of the ATPSs based on HFIPDESs in extraction of bioactive natural products. ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.7b00282. Chemical structure and physical property of AQs, analytical performance of spectrometry and HPLC analysis method for detection of total AQs, extraction efficiency and enrichment factor of each AQ, the procedure of HFIP-DES based ATPS for extraction AQs from RRR samples, FT-IR spectra of DESs, the effect of salting-out and DES solubilization, and structures of BODIPY and Rhodamine B (PDF)

■

REFERENCES

(1) Glyk, A.; Scheper, T.; Beutel, S. PEG-salt aqueous two-phase systems: an attractive and versatile liquid-liquid extraction technology for the downstream processing of proteins and enzymes. Appl. Microbiol. Biotechnol. 2015, 99 (16), 6599−6616. (2) Feng, Y. C.; Li, W. L.; He, F. M.; Kong, T. T.; Huang, X. W.; Gao, Z. H.; Lu, N. H.; Li, H. L. Aqueous two-phase system as an effective tool for purification of phenolic compounds from Fig Fruits (Ficus caricaL.). Sep. Sci. Technol. 2015, 50 (12), 1785−1793. (3) Ventura, S. P. M.; de Barros, R. L. F.; de Pinho Barbosa, J. M.; Soares, C. M. F.; Lima, Á . S.; Coutinho, J. A. P. Production and purification of an extracellular lipolytic enzyme using ionic liquid-based aqueous two-phase systems. Green Chem. 2012, 14, 734−740. (4) Lin, X.; Wang, Y.; Liu, X.; Huang, S.; Zeng, Q. ILs-based microwave-assisted extraction coupled with aqueous two-phase for the extraction of useful compounds from Chinese medicine. Analyst 2012, 137, 4076−4085. (5) Couling, D. J.; Bernot, R. J.; Docherty, K. M.; Dixon, J. K.; Maginn, E. J. Assessing the factors responsible for ionic liquid toxicity to aquatic organisms via quantitative structure−property relationship modeling. Green Chem. 2006, 8 (1), 82−90. (6) Jeong, K. M.; Lee, M. S.; Nam, M. W.; Zhao, J.; Jin, Y.; Lee, D. K.; Kwon, S. W.; Jeong, J. H.; Lee, J. Tailoring and recycling of deep eutectic solvents as sustainable andefficient extraction media. J. Chromatogr. A 2015, 1424, 10−17. (7) Zhao, B. Y.; Xu, P.; Yang, F. X.; Wu, H.; Zong, M. H.; Lou, W. Y. Biocompatible deep eutectic solvents based on choline chloride: characterization and application to the extraction of rutin from sophora japonica. ACS Sustainable Chem. Eng. 2015, 3 (11), 2746− 2755. (8) Cvjetko Bubalo, M.; Curko, N.; Tomasevic, M.; Kovacevic Ganic, K.; Radojcic Redovnikovic, I. Green extraction of grape skin phenolics by using deep eutectic solvents. Food Chem. 2016, 200, 159−166. (9) Duan, L.; Dou, L. L.; Guo, L.; Li, P.; Liu, E. H. Comprehensive evaluation of deep eutectic solvents in extraction of bioactive natural products. ACS Sustainable Chem. Eng. 2016, 4 (4), 2405−2411. (10) Zeng, Q.; Wang, Y.; Huang, Y.; Ding, X.; Chen, J.; Xu, K. Deep eutectic solvents as novel extraction media for protein partitioning. Analyst 2014, 139 (10), 2565−2673. (11) Xu, K.; Wang, Y.; Huang, Y.; Li, N.; Wen, Q. A green deep eutectic solvent-based aqueous two-phase system for protein extracting. Anal. Chim. Acta 2015, 864, 9−20. (12) Li, N.; Wang, Y.; Xu, K.; Huang, Y.; Wen, Q.; Ding, X. Development of green betaine-based deep eutectic solvent aqueous two-phase system for the extraction of protein. Talanta 2016, 152, 23−32. (13) Li, N.; Wang, Y.; Xu, K.; Wen, Q.; Ding, X.; Zhang, H.; Yang, Q. High-performance of deep eutectic solvent based aqueous bi-phasic systems for the extraction of DNA. RSC Adv. 2016, 6 (87), 84406− 84414. (14) Khaledi, M. G.; Jenkins, S. I.; Liang, S. Perfluorinated alcohols and acids induce coacervation in aqueous solutions of amphiphiles. Langmuir 2013, 29 (8), 2458−2464. (15) Chen, D.; Zhang, P.; Li, Y.; Mei, Z.; Xiao, Y. Hexafluoroisopropanol-induced coacervation in aqueous mixed systems of cationic and anionic surfactants for the extraction of sulfonamides in water samples. Anal. Bioanal. Chem. 2014, 406 (24), 6051−6060. (16) Tian, Y.; Li, Y.; Mei, J.; Deng, B.; Xiao, Y. Hexafluoroisopropanol-modified cetyltrimethylammonium bromide/sodium dodecyl sulfate vesicles as a pseudostationary phase in electrokinetic chromatography. J. Chromatogr. A 2015, 1404, 131−140. (17) Wang, L.; Dai, D. Y.; Chen, Q.; He, M. Y. Rapid and green synthesis of phenols catalyzed by a deep eutectic mixture based on fluorinated alcohol in water. J. Fluorine Chem. 2014, 158, 44−47. (18) Wang, L.; Zhu, K. Q.; Chen, Q.; He, M. Y. Facile and environmentally friendly halogenation of BODIPYs in deep eutectic solvent. Dyes Pigm. 2015, 112, 274−279. (19) Arvindekar, A. U.; Pereira, G. R.; Laddha, K. S. Assessment of conventional and novel extraction techniques on extraction efficiency

■

■

Research Article

AUTHOR INFORMATION

Corresponding Author

*Phone: +86 027 68759892. Fax: +86 027 68759850. E-mail: [email protected], [email protected]. Corresponding author address: School of Pharmaceutical Sciences, Wuhan University, 185# Donghu Road, Wuchang District, Wuhan 430071, China. ORCID

Yu-Xiu Xiao: 0000-0002-9125-1184 Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS A fluorescent dye called BODIPY was kindly provided by Prof. Xuechuan Hong in School of Pharmaceutical Sciences at Wuhan University. We thank the National Natural Science Foundation of China (Grant nos. 81373045 and 81673394) and the Provincial Natural Science Foundation of Hubei of China (Grant no. 2015CFA139). 4274

DOI: 10.1021/acssuschemeng.7b00282 ACS Sustainable Chem. Eng. 2017, 5, 4267−4275

Research Article

ACS Sustainable Chemistry & Engineering of five anthraquinones from Rheum emodi. J. Food Sci. Technol. 2015, 52 (10), 6574−6582. (20) Zhang, H. F.; Shi, Y. P. Temperature-assisted ionic liquid dispersive liquid-liquid microextraction combined with high performance liquid chromatography for the determination of anthraquinones in Radix et Rhizoma Rhei samples. Talanta 2010, 82 (3), 1010−1016. (21) Wu, X.; Liang, L.; Zou, Y.; Zhao, T.; Zhao, J.; Li, F.; Yang, L. Aqueous two-phase extraction, identification and antioxidant activity of anthocyanins from mulberry (Morus atropurpurea Roxb.). Food Chem. 2011, 129 (2), 443−453. (22) Tan, Z.; Li, F.; Xu, X. Isolation and purification of aloe anthraquinones based on an ionic liquid/salt aqueous two-phase system. Sep. Purif. Technol. 2012, 98, 150−157. (23) Francisco, M.; van den Bruinhorst, A.; Kroon, M. C. Lowtransition-temperature mixtures (LTTMs): a new generation of designer solvents. Angew. Chem., Int. Ed. 2013, 52 (11), 3074−3085. (24) Khezeli, T.; Daneshfar, A.; Sahraei, R. A green ultrasonicassisted liquid-liquid microextraction based on deep eutectic solvent for the HPLC-UV determination of ferulic, caffeic and cinnamic acid from olive, almond, sesame and cinnamon oil. Talanta 2016, 150, 577−585. (25) Guo, W.; Hou, Y.; Wu, W.; Ren, S.; Tian, S.; Marsh, K. N. Separation of phenol from model oils with quaternary ammonium saltsvia forming deep eutectic solvents. Green Chem. 2013, 15 (1), 226−229. (26) Mele, A.; Tran, C. D.; De Paoli Lacerda, S. H. The structure of a room-temperature ionic liquid with and without trace amounts of water: the role of C[bond]H···O and C[bond]H···F interactions in 1n-butyl-3-methylimidazolium tetrafluoroborate. Angew. Chem., Int. Ed. 2003, 42 (36), 4364−4366. (27) Dai, Y.; Van Spronsen, J.; Witkamp, G. J.; Verpoorte, R.; Choi, Y. H. Natural deep eutectic solvents as new potential media for green technology. Anal. Chim. Acta 2013, 766, 61−68. (28) Martins, J. P.; Carvalho, C. de P.; Silva, L. H. M. da; Coimbra, J. S. dos R.; Silva, M. do C. H. da; Rodrigues, G. D.; Minim, L. A. Liquid−liquid equilibria of an aqueous two-phase system containing poly(ethylene) glycol 1500 and sulfate salts at different temperatures. J. Chem. Eng. Data 2008, 53, 238−241. (29) Tan, Z.; Wang, C.; Yi, Y.; Wang, H.; Li, M.; Zhou, W.; Tan, S.; Li, F. Extraction and purification of chlorogenic acid from ramie (Boehmeria nivea L. Gaud) leaf using an ethanol/salt aqueous twophase system. Sep. Purif. Technol. 2014, 132, 396−400. (30) Xie, X.; Wang, Y.; Han, J.; Yan, Y. Extraction mechanism of sulfamethoxazole in water samples using aqueous two-phase systems of poly(propylene glycol) and salt. Anal. Chim. Acta 2011, 687 (1), 61−66. (31) Wang, Y.; Yan, Y.; Hu, S.; Han, J.; Xu, X. Phase diagrams of ammonium sulfate + ethanol/1-propanol/2-propanol + water aqueous two-phase systems at 298.15 K and Correlation. J. Chem. Eng. Data 2010, 55, 876−881. (32) Freire, M. G.; Pereira, J. F.; Francisco, M.; Rodriguez, H.; Rebelo, L. P.; Rogers, R. D.; Coutinho, J. A. Insight into the interactions that control the phase behaviour of new aqueous biphasic systems composed of polyethylene glycol polymers and ionic liquids. Chem. - Eur. J. 2012, 18 (6), 1831−1839. (33) Wang, Y.; Han, J.; Xu, X.; Hu, S.; Yan, Y. Partition behavior and partition mechanism of antibiotics in ethanol/2-propanol−ammonium sulfate aqueous two-phase systems. Sep. Purif. Technol. 2010, 75 (3), 352−357. (34) Wang, Y.; Wang, L.; Shi, L. L.; Shang, Z. B.; Zhang, Z.; Jin, W. J. Colorimetric and fluorescence sensing of Cu2+ in water using 1,8dihydroxyanthraquinone-beta-cyclodextrin complex with the assistance of ammonia. Talanta 2012, 94, 172−177.

4275

DOI: 10.1021/acssuschemeng.7b00282 ACS Sustainable Chem. Eng. 2017, 5, 4267−4275