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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 Yuxiu Xiao ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b00282 • Publication Date (Web): 27 Mar 2017 Downloaded from http://pubs.acs.org on April 3, 2017

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ACS Sustainable Chemistry & Engineering

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Hexafluoroisopropanol-Based Deep Eutectic Solvent / Salt

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Aqueous Two-Phase Systems for Extraction of

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Anthraquinones from Rhei Radix et Rhizoma Samples

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Wen-Wen Deng, Yu Zong, and Yu-Xiu Xiao*

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Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Ministry of Education), and School of

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Pharmaceutical Sciences, Wuhan University, Wuhan 430071, China

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The full mailing address: Professor Yu-Xiu Xiao

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School of Pharmaceutical Sciences, Wuhan University

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185# Donghu Road, Wuchang District

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Wuhan 430071, China

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ACS Paragon Plus Environment

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ABSTRACT: Deep eutectic solvents (DESs), a new class of green and sustainable solvents, have shown a

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promising application prospect in the aqueous two-phase system (ATPS) extraction. In the present work,

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hexafluoroisopropanol (HFIP) was introduced as a hydrogen-bond donor to synthesize DESs with choline

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chloride

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tetradecyltrimethylammonium bromide (hydrogen-bond acceptor), respectively. These HFIP-based DESs

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(HFIP-DESs) can form ATPS with various inorganic salts and their phase separation ability is significantly

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stronger than that of small molecule aliphatic alcohols, HFIP and traditional DESs. The ATPSs based on HFIP-

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DESs were first used to extract anthraquinones (AQs) from Rhei Radix et Rhizoma samples. Under the optimal

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conditions, more than 92% of AQs (including aloe-emodin, rhein, emodin, chrysophanol and physcion) was

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enriched in the DES-rich phase of the ATPS composed of ChCl-HFIP DES and Na2SO4. Compared with

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traditional chloroform extraction method (60 mL chloroform consumption), the ChCl-HFIP DES / Na2SO4 ATPS

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extraction method attained high extraction efficiency, but only consumed 0.5 mL organic solvent (HFIP).

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KEYWORDS：Deep eutectic solvent, Aqueous two-phase system, Hexafluoroisopropanol, Phase separation,

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Anthraquinones, Liquid−liquid extraction

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INTRODUCTION

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Aqueous two-phase system (ATPS), as a mild, biocompatible and environmentally-friendly liquid-liquid

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extraction technology, has been extensively used in the extraction, separation and purification of bioactive

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components.1 Up until now, most ATPSs applied in extraction are based on polymer/salt, polymer/polymer and

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small molecule aliphatic alcohol/salt systems.1,2 However, most of phase-forming polymers have high viscosity

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and can be emulsified easily, which increases the extraction time and interferes with the detection of analytes.3

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Small molecule alcohol/salt systems require large volume of volatile organic solvent, which is detrimental to the

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activity of biosamples and the health of environment. In the past decade, ATPSs based on ionic liquid (IL) have

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been proposed and used for the extraction and purification of various compounds.3,4 Compared with common

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type of ATPSs, IL-based ATPSs own the advantages of negligible viscosity, no emulsion formation, negligible

(ChCl),

decyltrimethylammonium

bromide,

dodecyltrimethylammonium

bromide,

and


2

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volatility, quick phase separation, high extraction efficiency etc. due to the excellent characteristics of IL.

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Nonetheless, the following two disadvantages of ILs hinder the development of IL-based ATPSs. One is that the

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preparation of ILs is expensive, tedious and purification-difficult. Another is related to their toxicity,

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biodegradability and bioaccumulation, which have aroused increasing public attention to the harmful influence

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of ILs on the environment.5

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Deep eutectic solvent (DES), emerging as a new generation of green and sustainable solvent, has attracted

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attention in many fields.6 Generally, DESs are prepared by mixing two starting components, i.e. a hydrogen-

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bond donor (HBD) and a hydrogen-bond acceptor (HBA), with the aid of strong hydrogen-bonding interaction.

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DESs have a melting point lower than that of each component. Compared to ILs, the synthesis of DESs is

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relatively simple, and the products require no purification with 100% atom utilization rate, which greatly cut the

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costs. Besides, DESs retain the excellent physicochemical properties of traditional ILs. Thus far, DESs have

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been used as extraction media for extracting active ingredients in traditional Chinese medicine7-9 and phase-

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forming components of ATPS for protein and DNA extraction.10-13 In 2014, Wang et al. first found that DES

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composed of choline chloride (ChCl) and urea could form ATPS with K2HPO4, and the DES-based ATPS could

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efficiently extract bovine serum albumin from aqueous solutions.10 Soon after, they further extracted protein and

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DNA by the ATPS based on ChCl-glycerol DES, betaine-urea DES and tetrabutylammonium bromide-ethylene

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glycol DES, respectively.11-13 These researches indicate that DES-based ATPS is a promising extraction method.

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However, the phase separation capability of the DESs reported so far is a bit poor.12 Therefore, it is of great

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significance to synthesize the novel DESs with strong phase separation ability for broadening the practical

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applications of DES-based ATPS.

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Hexafluoroisopropanol (HFIP) is a perfluorinated alcohol with strong hydrogen bond donor ability (1.96,

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larger than that of water 1.02) and high dissolving power. It has been proved that HFIP can efficiently induce the

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aggregation and liquid-liquid phase separation in aqueous solutions of various amphiphiles,14 and displays

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promising prospects in analytical field.15,16 Recently, Wang et al. prepared a novel DES based on HFIP and

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ChCl, and employed it as a green reaction medium for the rapid and efficient synthesis of phenols and

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halogenation of 4,4-difluoro-4-bora-3a,4a-diaza-s-indacenes.17,18 These researches highlight the application of

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HFIP in DES preparation. Meanwhile, considering the superior ability of HFIP to induce liquid-liquid phase


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separation in aqueous solutions of amphiphiles, HFIP-based DES (HFIP-DES) is expected to have excellent

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phase-separation capability as a phase component of ATPS. So far, ATPS based on HFIP-DES and its application

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have not yet been reported.

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Rhei Radix et Rhizoma (RRR) belongs to polygonaceae plants. It is a famous traditional Chinese herbal

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medicine with many pharmacological activities such as antifungal, antiviral, antioxidant, anticancer and

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antimutagenicity. Anthraquinones (AQs) including aloe-emodin, rhein, emodin, chrysophanol and physcion are

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the main bioactive constituents of RRR.19 The traditional method for extraction of AQs from RRR samples is as

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follows: RRR powder was extracted by heat reflux with methanol; the obtained methanol solution was dried,

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then the residue was treated by acid hydrolysis and liquid–liquid extraction with chloroform.20 This method is

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time consuming and requires a great deal of toxic chloroform. Li et al. investigated the application of green DESs

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in the extraction of AQs from RRR; however, the extraction yield was unsatisfactory due to the weak polarity of

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AQs.9

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In this work, HFIP was introduced to prepare novel DESs, and HFIP-DESs were used as phase-forming

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components to develop ATPSs with aqueous salt solution for the first time. The phase behaviors were

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investigated to evaluate the phase separation ability of HFIP-DESs. ATPSs based on HFIP-DES were applied

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for extracting AQs from RRR samples. The results indicate that the ChCl-HFIP DES based ATPS extraction

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method is highly efficient and very reliable for the extraction of AQs in RRR samples. What is more, this new

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extraction method is much greener than the traditional chloroform extraction for AQs.

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EXPERIMENTAL SECTION

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Materials and Reagents. RRR was purchased from a local traditional Chinese medicine drugstore (Wuhan,

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China). Aloe-emodin, chrysophanol, emodin, physcion and rhein were purchased from Chengdu Must

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Biotechnology Co., Ltd. (Chengdu, China) and their physical and chemical properties are shown in Table S1.

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HFIP, 1,8-dihydroxyanthraquinone were purchased from Aladdin Chemistry Co. (Shanghai, China). Na2SO4,

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(NH4)2SO4, Na2HPO4, K2HPO4, magnesium acetate, ChCl, decyltrimethylammonium bromide (DeTAB),

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dodecyltrimethylammonium bromide (DTAB), tetradecyltrimethylammonium bromide (TTAB), urea, glycerol


4

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and ethylene glycol were obtained from Sinopharm Chemical Reagent Co. (Shanghai, China). Deionized water

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was obtained from a Milli-Q system (Millipore, Molsheim, France). All other reagents used are of analytical

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grade.

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Preparation and Characterization of DESs. HFIP was used as HBD to synthesize DESs with ChCl,

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DeTAB, DTAB and TTAB (HBA), as shown in Table 1. The DESs were prepared by following simple procedure.

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HFIP was mixed with a HBA at a proper molar ratio. The mixture was heated at 80 °C with constant magnetic

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agitation until the formation of a clear, homogeneous and stable liquid, which is called DES. The melt point of

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DESs was measured by differential scanning calorimetry (DSC) (STA449C 200P, Germany), with temperature

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increasing from −120 oC to 50 oC at a rate of 10 oC/min under nitrogen. The viscosity of DESs was determined

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using a Rheometer (DISCOVERY HR-2, USA) at constant temperature. The 1H–1H-nuclear overhauser

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spectroscopy (NOESY) and FT-IR spectra were examined by NMR spectrometer (Bruker DPX-400, Germany)

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and FT-IR spectrometer (PerkinElmer, USA), respectively.

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Preparation of Phase Diagrams. The phase diagrams were prepared by the method of cloud point titration

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at room temperature.21 Salt aqueous solution with known concentration was added in a 50 mL conical flask. Then,

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a DES or alcohol was added into the conical flask drop by drop. When the mixture became turbid, deionized

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water was added until the turbidity disappeared. The above procedure was repeated to acquire adequate data

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about concentrations of DES (or alcohol) and salt at different turbid points, which was used to construct the

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phase diagrams.

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Aqueous Two-Phase System Extraction. The extraction procedure of ATPS based on HFIP-DES is

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illustrated in Figure S1. An appropriate amount of DES was added in a 15 mL centrifuge tube. Then, 5 mL salt

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solution with certain concentration was added into the tube. Upon the addition of a certain volume of RRR

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samples, the mixture was shaken vigorously for 20 s using a vortex mixer, and then centrifuged at 3500 rpm for

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10 min. At this time, the mixture was separated to liquid-liquid two phase system. The bottom was HFIP-DES-

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rich phase, while the top was HFIP-DES-poor phase (i.e. aqueous phase or salt-rich phase). The volumes of the

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top phase and the bottom phase were recorded. The bottom phase was withdrawn for spectrometric determination


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or HPLC analysis. The volume ratio of HFIP-DES-rich phase versus aqueous phase is defined as phase ratio (R).

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Phase ratio, enrichment factor (EF) and extraction efficiency (EE) of AQs were calculated by the following

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equations: 𝑉D

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𝑅=

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𝐸𝐹 =

𝐶D

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𝐸𝐸 =

𝐶D × 𝑉D

𝑉A

𝐶T

𝐶T × 𝑉T

(1)

(2)

(3)

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where VD, VA and VT are the volume of DES-rich phase, aqueous phase and total solution, respectively; CD and

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CT represent the concentration of AQs in DES-rich phase and the original concentration of AQs, respectively.

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Spectrometry and HPLC Analysis of AQs samples. The concentration of total AQs in the DES-rich phase

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of ATPS or the RRR sample was determined by spectrometry.22 A certain volume of the DES-rich phase or the

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RRR sample was added in 5 ml volumetric flask, and diluted to the mark using 0.5% magnesium acetate solution.

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AQs were changed to pink at weak alkaline condition and could be measured at 512 nm by a UV–Vis

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spectrophotometer (Shimadzu, UV-2100, Japan). A calibration curve was established with 1,8-

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dihydroxyanthraquinone standard solutions to calculate the total AQs concentration. HPLC analysis of AQs in

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the DES-rich phase of ATPS, the RRR sample or the reference RRR sample was carried out on a Shimadzu

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HPLC system equipped with two LC-20AT pumps, a SPD-20A UV detector and a column temperature control

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oven. Separation was performed on an Amethyst C18 column (250 mm × 4.6 mm, 5 µm). The mobile phase was

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composed of 0.1% H3PO4 (eluent A) and methanol (eluent B) with a gradient elution. The gradient program was

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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

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1.0 mL/min, the detection wavelength 254 nm and the column temperature 25°C. The injection volume was 10

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µL. The validation results of spectrometry and HPLC analysis method for quantification of AQs are shown Table

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S2 and Table 3S, respectively.


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Preparation of RRR Samples. A RRR sample was prepared according to the reference20 and Chinese

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pharmacopoeia published in 2015 with appropriate modifications. A 0.5 g sample of powdered RRR herb (65

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mesh) was extracted with 25 mL methanol by refluxing for 60 min. The obtained solution was cooled to room

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temperature and then filtered. The filtrate was transferred to a flask and evaporated to dryness. The residue was

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mixed with 10 mL of 8% (v/v) HCl solution. The mixed solution was sonicated for 2 min, and then hydrolyzed

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for 60 min on an oil bath at 80 °C. The hydrolyzed solution was dried under the reduced pressure. The residue

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was dissolved with methanol and transferred into a 10 mL volumetric flask, and methanol was added to the mark.

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The RRR sample was used for HFIP-DES based ATPS extraction, and the obtained DES-rich phase for detection

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of AQs by HPLC or spectrometry.

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To obtain a reference RRR sample, 0.5 g powdered RRR was used to obtain the mixed solution (with HCl)

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according to the steps mentioned above. After sonication for 2 min, 10 mL chloroform was added into the mixed

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solution. The resulting solution was refluxed for 60 min on an oil bath for hydrolyzation. After cooling to room

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temperature, the hydrolyzed solution was transferred to a separating funnel and extracted three times with 10

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mL of chloroform. The combined extraction solution was evaporated to dryness under reduced pressure. The

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residue was dissolved with methanol and transferred to a 10 mL volumetric flask. Then, methanol was added to

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the mark. The reference RRR sample was directly used for detection of AQs by HPLC.

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RESULTS AND DISCUSSION

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Characterization of HFIP-DESs. The melting point of the four HFIP-DESs are all below -24 oC, which

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is obviously less than that of their compositions (Table 1). The decrease in the melting point might be due to the

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disruption of the crystalline structure of quaternary ammonium compounds (QACs) caused by the hydrogen-

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bond interaction between HFIP and QACs.23

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To confirm hydrogen bonds in HFIP-DESs, FT-IR spectra and NOESY of two representative HFIP-DESs

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(ChCl-HFIP and DTAB-HFIP) were examined. The results are shown in Figure S2 and Figure 1, respectively.

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In the FT-IR spectra of the DESs, the O-H stretching vibration peaks shift to lower wave number (3383.76 cm-

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1,

3416.27 cm-1) compared to the O-H stretching vibration of pure HFIP (3446.90 cm-1), which might be caused


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by the transfer of the electron cloud of oxygen atom to hydrogen bonding and thus the decrease in force constant.

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24,25

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peak is observed, indicating that there is no chemical reaction occurrence in the preparation of DESs. The

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NOESY has been successfully applied to search hydrogen-bonding interaction in DESs and ILs. Mele et al.

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observed intermolecular and intramolecular hydrogen-bonding interaction of 1-nbutyl-3-methylimidazolium

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tetrafluoroborate molecules through the NOESY.26 Dai et al. confirmed the hydrogen-bonding interaction

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between 1, 2-propanediol and ChCl (two components of DES) by the NOESY. 27 In our study, the NOESY

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spectrum of ChCl-HFIP DES proves that the proton of -OH of HFIP interacts intensely with the proton of -OH

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of ChCl. This implies that the -OH of HFIP can form a hydrogen-bond with ChCl (Figure 1a). The NOESY

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spectrum of DTAB-HFIP DES reveals that the proton of -OH of HFIP interacts with the protons of methyl (H2')

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and methylene (H4') of DTAB. This might be due to the hydrogen-bonding interaction between the -OH of HFIP

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and Br- of DTAB as well as the hydrophobic interaction between trifluoromethyl group of HFIP and methylene

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of DTAB (Figure 1b). According to the results of FT-IR and NOESY analyses, we can confirm the formation of

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hydrogen-bond between HFIP and QACs in the synthesis of HFIP- DESs.

This phenomenon shows the formation of hydrogen bond between HFIP and QACs. In addition, no new

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Phase Diagram of ATPS Based on HFIP-DESs. Liquid–liquid equilibrium data of ATPS are necessary

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for the design of an extraction process. In the phase diagram of ATPS, above the curve is the two-phase area,

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while below the curve is the homogeneous phase area. The curve is closer to the origin, the phase separation

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ability of the compositions of ATPS is stronger.28-30 In this work, five inorganic salts were selected as phase-

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forming salt to construct phase diagrams with ChCl-HFIP DES. As shown in Figure 2a, ChCl-HFIP DES can

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form ATPS with all the five salts, and the phase separation ability of these salts are in the order of Na2SO4 >

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K2HPO4 > Na2HPO4 > (NH4) 2SO4> KH2PO4. Li et al. used three salts (K2HPO4, Na2HPO4 and KH2PO4) to form

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ATPSs with the six DESs they prepared (Betaine-Methylurea, Betaine-Urea, Betaine-Ethylene glycol, Betaine-

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glucose, Betaine-sorbitol, Betaine-Glycerol), but only K2HPO4 could form ATPSs with these DESs.12 This

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indicates that ChCl-HFIP DES has superior phase separation ability compared to the six DESs.

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To further compare the phase separation ability of HFIP-DESs with that of alcohols and common DESs

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(ChCl-Ethylene glycol, ChCl-Glycerol and ChCl-Urea as representatives), the phase diagrams of aqueous mixed


8

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systems consisting of HFIP-DESs /various alcohols /common DESs and K2HPO4 were recorded. As shown in

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Figure 2b and c, ChCl-HFIP DES has stronger phase separation ability than aliphatic alcohols, HFIP and the

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three common DESs based on ChCl. It has been proved that the phase separation ability of a phase-forming

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solvent would increase with increasing the acting force between the molecules of the solvent and with decreasing

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the interaction force between the solvent and water molecules.31 Therefore, the excellent phase separation ability

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of ChCl-HFIP DES may be ascribed to the strong hydrogen-bond interaction between HFIP and ChCl, which

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can in turn inhibit the interaction between ChCl-HFIP DES and the water molecules. Furthermore, because HFIP

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owns stronger hydrophobicity than common HBDs (such as urea, ethylene glycol, glycerol), HFIP-DESs can be

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separated more easily from water solution by salt-outing force, which also contributes to the stronger phase

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separation ability of ChCl-HFIP DES. Meanwhile, Figure 2c shows that the phase separation ability of HFIP-

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DESs with long alkyl chain QACs (DeTAB-HFIP, DTAB-HFIP, TTAB-HFIP) are more excellent than that of

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ChCl-HFIP DES with short alkyl chain. This is because the strong hydrophobicity of long alkyl chain QACs can

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greatly reduce the interaction force between the DESs and water molecules.32

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Phase Ratio. The effects of the amount of ChCl-HFIP DES and the concentration of salt solution on the

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phase ratio of ChCl–HFIP DES based ATPS were investigated at room temperature. From Figure 3a, the phase

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ratio increases linearly (r=0.9957) as the amount of ChCl-HFIP DES increases from 0.05 g to 0.3 g. As seen in

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Figure 3b, with the increase of salt concentration, the phase ratio increases at first, and then remains almost

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unchanged over 0.3 g/mL. This is because the salt-outing force can be strengthened with increasing salt

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concentration, making the more amount of ChCl-HFIP DES be salted out from water solution. As a consequence,

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the phase ratio is increased. As the salt concentration is larger than 0.3 g/mL, the salt-outing force reaches

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saturation, thus the phase ratio keeps nearly unchanged.

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Optimization of Extraction Conditions. The ATPSs based on HFIP-DESs and inorganic salts were used

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to extract total AQs from the RRR samples. Many factors, including the type and amount of HFIP-DESs and

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inorganic salts, the quantity of the RRR sample, temperature and solution pH, were optimized to acquire high

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extraction efficiency. The total AQs was determined by spectrometry.


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Type of HFIP-DES. Four kinds of HFIP-DESs were chosen as phase-forming components of ATPS to

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investigate the influence of different HFIP-DESs on the extraction efficiency of total AQs. According to the

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phase diagrams (Figure 2c), 0.5 g HFIP-DES and 5 mL of 0.2 g/mL K2HPO4 solution were selected to ensure

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the formation of ATPSs. A RRR sample of 100 µL was added into the ATPS. As shown in Figure 4a, the ATPS

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based on HFIP-ChCl DES presents the highest extraction efficiency. The reason may be ascribed to the structure

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of ChCl with –OH group, which can provide additional hydrogen-bonding interaction with AQs. Furthermore,

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the extraction efficiency decreases with increasing the alkyl chain length of HBAs in the other three DESs. This

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is possibly relevant to the increase of the viscosity of DES (Table 1), which would hinder the AQs diffusing into

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the DES-rich phase. ChCl-HFIP was selected as the phase-forming DES to construct ATPSs in the following

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experiments.

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Type of Inorganic Salt. Five kinds of common salts ((NH4)2SO4, Na2SO4, Na2HPO4, K2HPO4, KH2PO4)

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were employed to examine the effect of different salts on the extraction efficiency of total AQs. The ATPS was

227

composed of ChCl-HFIP DES (0.5 g) and salt solution (0.2 g/mL, 5 mL). A RRR sample of 100 μL was added.

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The results indicate that the phase separation ability of KH2PO4 is too weak to form ATPS under the above

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conditions. As shown in Figure 4b, the Na2SO4-based ATPS has the highest extraction efficiency. This is because

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of the strongest phase separation ability of Na2SO4 (Figure 2a). That means the more DES can aggregate in the

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DES-rich phase of the Na2SO4-based ATPS. As a result, the more AQs were extracted into the DES-rich phase

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by the interaction forces between AQs and DES as well as the strong salt-outing force of Na2SO4. However,

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although Na2HPO4 and K2HPO4 show stronger phase separation ability than (NH4)2SO4, their ATPSs give the

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relatively lower extraction efficiency than the (NH4)2SO4-based ATPS. The explanation is that the AQs with pKa

235

values less than 6.6 (Table S1) can ionize in both Na2HPO4 and K2HPO4 aqueous alkaline solution, leading to

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the decrease of hydrophobic interaction of AQs with the DES-rich phase and thus the lower extraction efficiency.

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Na2SO4 was selected as phase-separation salt for the further investigation.

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Concentration of Salt. The effect of Na2SO4 concentration was investigated using ChCl-HFIP DES (0.5

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g)/Na2SO4 (5.0 mL) ATPS with 100 μL RRR sample added. The results are shown in Figure 4c. The extraction

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efficiency increases rapidly at first, then tends to a plateau and finally decreases a bit with increasing Na2SO4

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concentration in range of 0.1-0.3 g/mL. The possible reason is that both the salting-out effect and the phase


10

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separation ability is much enhanced with the increase of salt concentration from 0.1 to 0.15 g/mL, leading to the

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obvious increase of the amount of DES in the DES-rich phase. Consequently, the much more AQs were extracted

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into the DES-rich phase. However, the amount of DES in the DES-rich phase might tend to saturation when the

245

salt concentration is over 0.15 g/mL, thus the extraction efficiency increases very slowly. As the salt

246

concentration is greater than 0.25 g/mL, the increased viscosity of salt aqueous solution would reduce the

247

diffusion of AQs into the DES-rich phase, resulting in the slight decrease of the extraction efficiency. The optimal

248

Na2SO4 concentration was set as 0.25 g/ mL.

249

Addition Amount of HFIP-DES. Figure 4d shows the influence of the amount of ChCl-HFIP EDS added

250

in ATPS on the extraction efficiency of total AQs. As the DES amount varies in range of 0.2-0.8 g, the extraction

251

efficiency displays fast increase from 0.2 to 0.35 g, slow increase from 0.35 to 0.65 g and a little decline over

252

0.65 g. The explanation is that the number and size of DES aggregates in the DES-rich phase might increase

253

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

254

addition amount being larger than 0.65 g, excessive DES would dissolve in the aqueous phase, leading to the

255

slight decline of the AQs extracted into the DES-rich phase. The optimum mass of DES added in ATPS was

256

selected at 0.65 g.

257

Quantity of the RRR Sample. ChCl-HFIP DES (0.65 g)/Na2SO4 (0.25 g/mL, 5.0 mL) ATPS was used

258

for the optimization of the quantity of the RRR sample added in ATPS. As the RRR sample increases from 50 to

259

300 µL, the extraction efficiency presents a gradual rise at first, then a constant value, and a decrease at last

260

(Figure 4e). In the aqueous phase of DES-based ATPS, there is still bits of DES, which can dissolve some AQs

261

and lead to low extraction efficiency at a small quantity of the sample. As the sample quantity increases, the

262

much more AQs would be extracted into the DES-rich phase due to the very limited dissolving capacity of the

263

DES in the aqueous phase. However, because the extraction capacity of the DES-rich phase is also limited, the

264

excessive addition of the sample would make excessive AQs precipitate, hence the extraction efficiency

265

decreases. The optimized volume was chosen as 250 μL.

266

Temperature and Solution pH. In order to investigate the influence of extraction temperature and pH

267

value of the aqueous solution on the extraction efficiency, ChCl-HFIP DES (0.65 g)/Na2SO4 (0.25 g/mL, 5.0 mL)

268

ATPS was used to extract total AQs from the RRR sample (250 μL). As shown in Figure 4f, the extraction


11


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269

efficiency is not apparently affected by the variation of both temperature (25-55 oC) and pH (3-11). Although the

270

pH of the aqueous solution is influential for the charged status of AQs and thus affects the interactions

271

(hydrophobic, electrostatic and hydrogen-bonding interactions) of AQs with the DES-rich phase, Na2SO4 with

272

very strong salt-outing force (Figure 2a) can strip both ionized and non-ionized AQs from the aqueous solution

273

to the DES rich phase. As a result, the effect of pH on the extraction efficiency is negligible. For the sake of

274

convenience, room temperature was adopted and the pH of the aqueous solution were not adjusted in all the

275

extraction experiments.

276

In summary, the optimized ATPS extraction conditions for total AQs are as follows: the addition amount of

277

ChCl-HFIP DES is 0.65 g, the concentration of Na2SO4 aqueous solution (5 mL) 0.25 g/mL, and the RRR sample

278

quantity is 250 μL; the extraction experiments were carried out at room temperature. At these conditions, the

279

extraction efficiency of total AQs reaches 93.5%.

280

Analysis of the Components of Extracted Total AQs. HPLC-UV method was employed to analyze the

281

constitutes of total AQs in the DES-rich phases obtained after extracting RRR sample solution under optimal

282

conditions. As shown in Figure 5, the total AQs extracted by ChCl-HFIP DES/ Na2SO4 ATPS include aloe-

283

emodin, rhein, emodin, chrysophanol and physcion. As seen in Table S4, the extraction efficiency of each AQ is

284

in range of 92.1% ~ 97.4%, averaging 94.5% which is almost equal to the extraction efficiency of total AQs

285

determined by spectrometry. Meanwhile, the concentration of each AQ was enriched by ca.15-16 fold after

286

extraction. Actually, if the enrichment factor, rather than the extraction efficiency, was used as a criterion for

287

optimizing extraction conditions, the higher enrichment effect would be obtained since the phase ratio can be

288

controlled at very low value (Figure 3).

289

Comparison of HFIP-DES/Salt ATPS extraction with Conventional Chloroform Extraction. Figure 5

290

shows that the total AQs extracted by the ATPS contain the five constituents, being the same as the components

291

of the total AQs obtained by the conventional chloroform extraction. The two extraction methods were used for

292

determination of the AQs in real RRR herb, and the results are listed in Table 2. For both the total AQs and each

293

AQ, the results derived by the ChCl-HFIP DES-based ATPS extraction method are very close to those by the

294

conventional method. In addition, the proposed method did not use large volume of toxic chloroform, and just


12

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295

needed 0.5 mL HFIP to extract AQs from 1 g RRR herb. Therefore, the ChCl-HFIP DES-based ATPS extraction

296

method is reliable, effective, greener, and can replace the conventional method to extract AQs from RRR samples.

297

Extraction Mechanism. The salting-out effect and the solubilization of extraction solvents for target

298

compounds are often regarded as main extraction mechanisms of ATPS.33 In present study, salting-out

299

experiment and laser confocal microscopy examination (Leica-LCS-SP8-ATED, Germany) were employed to

300

explore the extraction mechanism of ChCl-HFIP DES-based ATPS. As seen in Figure S3, 1,8-

301

dihydroxyanthraquinone (DHAQ) was precipitated from its solution by salt-outing force of Na2SO4. Upon

302

further adding ChCl-HFIP DES in the tube, an ATPS formed and DHAQ was dissolved in the DES-rich phase.

303

The results indicate that salt-outing effect plays an important role in the extraction of AQs, and ChCl-HFIP DES

304

is an ideal solvent for AQs. As we know, DHAQ is pink (anionic form) in dilute alkaline solution, but is yellow

305

(neutral form) in neutral and acidic environment.34 Given DHAQ is yellow in the DES-rich phase, it is concluded

306

that most DHAQ is dissolved in the DES aggregates (neutral environment) of the DES-rich phase.

307

Figure 6 shows the laser confocal microscopy results. As seen, there are spherical DES aggregates (100 nm

308

~ 5 µm in diameter) in the DES-rich phase. Both water-soluble Rhodamine B and water-insoluble BODIPY

309

(Figure S4) can be partitioned into the DES aggregates in the DES-rich phase after ATPS extraction. This

310

phenomenon reveals that ChCl-HFIP DES has strong solubilizing capability in that it can provide various

311

interactions with target analytes, such as hydrogen-bonding interaction, hydrophobic interaction, electrostatic

312

interaction and so on. In short, the salting-out effect and the strong solubilizing capability of ChCl-HFIP DES

313

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

314

CONCLUSIONS

315

Four HFIP-DESs were prepared and used to construct ATPSs with inorganic salts. Compared to traditional DESs

316

(not based on HFIP), small molecule aliphatic alcohols and HFIP, HFIP-DESs show much stronger phase

317

separation ability. Thus, the amount of both HFIP-DESs and salts required to form ATPS can be reduced

318

significantly. Moreover, the ATPSs composed of HFIP-DESs and salts were employed to extract the AQs from

319

RRR samples. The high extraction recovery was obtained for each AQ (> 92%) by use of the ATPS composed


13


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Page 14 of 24

320

of ChCl-HFIP DES and Na2SO4. The salt-outing force and the solubilizing capability of ChCl-HFIP DES

321

contribute to the high extraction efficiency. In comparison with traditional chloroform extraction method, the

322

ChCl-HFIP DES-based ATPS extraction method attained almost the same results for extracting the AQs from

323

RRR samples, but just consumed a very small amount of organic solvent. The proposed method is reliable,

324

effective and greener, and can replace the conventional method to extract AQs from RRR samples. This study

325

holds promise for further utilization of the ATPSs based on HFIP-DESs in extraction of bioactive natural

326

products.

327

ASSOCIATED CONTENT

328

Supporting Information. Chemical structure and physical property of AQs, analytical performance of

329

spectrometry and HPLC analysis method for detection of total AQs, the extraction efficiency and enrichment

330

factor of each AQ, the procedure of HFIP-DES based ATPS for extraction AQs from RRR samples, FT-IR spectra

331

of DESs, the effect of salting-out and DES solubilization, and structure of BODIPY and Rhodamine B (PDF).

332

AUTHOR INFORMATION

333

Corresponding Author

334

*Yu-Xiu Xiao. E-mail: [email protected], [email protected] Tel.: +86 027 68759892; fax: +86 027 68759850.

335 336

Notes

337

The authors declare no competing financial interest.

338

ACKNOWLEDGMENTS

339

A fluorescent dye called BODIPY was kindly provided by Prof. Xuechuan Hong in School of Pharmaceutical

340

Sciences at Wuhan University. We thank the National Natural Science Foundation of China (Grant Nos.


14

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341

81373045 and 81673394) and the Provincial Natural Science Foundation of Hubei of China (Grant no.

342

2015CFA139).

343

REFERENCES

344

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443 444 445 446 447 448 449 450 451


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Table 1. The compositions，structures and physicochemical properties of the studied DESs

DES

HBD

HBA

Viscosity （Pa.s)

2:3

0.04

1.39

302

–40.0

1:2

0.03

1.28

233–235

–24.9

1:2

0.04

1.22

1:2

0.05

1.14

ChCl-HFIP

DeTAB-HFIP

Melting point: – 4 oC

DTAB-HFIP

TTAB-HFIP

453

a Data

454

b

Melting point (oC) Density a DESb (g/mL) HBA

Molar ratio (HBA:HBD)

228–230

247.5

–29.0

–33.4

obtained using SciFinder Scholar from Chemical Abstract Service.

Data obtained by analysis of DSC.

455 456 457

Table 2. Comparison of ATPS based on ChCl-HFIP DES with traditional extraction method The amount of extracted AQs ± SD (mg /g RRR powder, n=3)

Organic solvent

Extraction method

consumption /1.0 g Aloeemodin

Rhein

Emodin

Chrysophanol

Physcion

Total AQs

1.07

1.30

3.23

4.08

1.43

11.11

±0.46

±0.05

±0.06

±0.06

±0.02

±0.06

Chloroform

1.02

1.60

2.93

4.48

1.36

11.40

extraction

±0.22

±0.02

±0.02

.±0.10

±0.03

±0.17

ChCl-HFIP DES/Na2SO4 ATPS extraction

RRR powder

DES:1.30 g (HFIP : 0.5 mL)

CHCl3 : 60 mL

458 459


20

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460 Figure 1. NOESY spectra of ChCl-HFIP DES (a) and DTAB-HFIP DES (b).

461 462 463

464 465

Figure 2. Phase diagrams of ATPSs at room temperature. (a) ATPSs composed of ChCl-HFIP and various salts;

466

(b) ATPSs composed of K2HPO4 and different alcohols or ChCl-HFIP; (c) ATPSs composed of K2HPO4 and

467

various DESs.

468 469 470


21


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Page 22 of 24

471 472

Figure 3. Effect of the amount of ChCl-HFIP DES (a) and salt concentration (b) on the phase ratio of the

473

ATPS. (a) K2HPO4 concentration was set at 0.3 g/mL. (b) The amount of ChCl-HFIP DES was set at 0.2 g.

474 475 476

477

Figure 4. Effect of type of HFIP-DES (a), type of salt (b), salt concentration (c), HFIP-DES amount (d), the

478

quantity of RRR samples (e), temperature and the pH of the aqueous solution (f) on the extraction efficiency of

479

total AQs. In figure (f): 1, 2, 3 and 4 in x-axis stand for temperature at 25 oC, 35 oC, 45 oC and 55 oC; 1, 2, 3, 4 and

480

5 in x-axis stand for pH at 3, 5, 7, 9, 11 and 13.


22

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481 482

Figure 5. Chromatograms of (a) the DES-rich phase obtained after ATPS extraction for RRR sample solution, (b) the

483

aqueous phase obtained after ATPS extraction for RRR sample solution, (c) RRR sample solution, (d) the standard

484

solution of AQs (each AQ at 10 µg/mL) and (e) the DES-rich phase of ATPS in absence of RRR sample (blank control).

485

Peak identification: 1, aloe emodin; 2, rhein; 3, emodin; 4, chrysophanol; 5, physcion.

486

487 488

Figure 6. Laser confocal micrograph of the DES-rich phase of ATPS based on ChCl-HFIP DES/ Na2SO4 with

489

Rhodamine B (a) and BODIPY (b) as fluorescent probe. Exciting wavelength is 561 nm for Rhodamine B and

490

575 nm for BODIPY. Emission wavelength is 587 nm for Rhodamine B and 665 nm for BODIPY.

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TOC/Abstract graphic image

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SYNOPSIS

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DESs have been regarded as a class of green and sustainable solvents to replace common organic solvents or

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even ionic liquids. HFIP-DESs have excellent phase separation ability, thus the amount of both HFIP-DESs and

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salts required to form ATPS can be reduced significantly. ChCl-HFIP DES based ATPS obtained high extraction

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efficiency (>92%), but just needed 0.5 mL HFIP to extract AQs from 1 g RRR herb, while the conventional

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method needed 60 mL toxic chloroform for extracting AQs from 1 g RRR herb.

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Hexafluoroisopropanol-Based Deep Eutectic ... - ACS Publications

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