Salt Aqueous

Subscriber access provided by University of Newcastle, Australia

Article

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Sustainable Chemistry & Engineering is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Sustainable Chemistry & Engineering

1

Hexafluoroisopropanol-Based Deep Eutectic Solvent / Salt

2

Aqueous Two-Phase Systems for Extraction of

3

Anthraquinones from Rhei Radix et Rhizoma Samples

4

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

5

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

6

Pharmaceutical Sciences, Wuhan University, Wuhan 430071, China

7

8 9

The full mailing address: Professor Yu-Xiu Xiao

10

School of Pharmaceutical Sciences, Wuhan University

11

185# Donghu Road, Wuchang District

12

Wuhan 430071, China

13

14

15

ACS Paragon Plus Environment

1


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 24

16

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

17

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

18

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

19

chloride

20

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

21

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

22

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

23

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

24

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

25

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

26

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

27

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

28

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

29

Anthraquinones, Liquid−liquid extraction

30

INTRODUCTION

31

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

32

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

33

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

34

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

35

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

36

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

37

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

38

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

39

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

(ChCl),

decyltrimethylammonium

bromide,

dodecyltrimethylammonium

bromide,

and


2

Page 3 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


40

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

41

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

42

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

43

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

44

of ILs on the environment.5

45

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

46

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

47

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

48

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

49

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

50

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

51

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

52

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

53

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

54

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

55

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

56

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

57

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

58

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

59

applications of DES-based ATPS.

60

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

61

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

62

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

63

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

64

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

65

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

66

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


3


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 24

67

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

68

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

69

have not yet been reported.

70

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

71

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

72

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

73

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

74

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

75

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

76

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

77

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

78

AQs.9

79

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

80

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

81

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

82

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

83

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

84

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

85

EXPERIMENTAL SECTION

86

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

87

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

88

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

89

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

90

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

91

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


4

Page 5 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


92

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

93

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

94

grade.

95

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

96

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

97

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

98

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

99

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

100

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

101

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

102

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

103

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

104

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

105

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

106

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

107

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

108

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

109

phase diagrams.

110

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

111

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

112

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

113

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

114

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

115

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

116

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


5


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 24

117

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

118

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

119

equations: 𝑉D

120

𝑅=

121

𝐸𝐹 =

𝐶D

122

𝐸𝐸 =

𝐶D × 𝑉D

𝑉A

𝐶T

𝐶T × 𝑉T

(1)

(2)

(3)

123

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

124

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

125

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

126

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

127

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

128

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

129

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

130

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

131

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

132

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

133

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

134

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

135

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

136

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

137

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

138

S2 and Table 3S, respectively.


6

Page 7 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


139

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

140

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

141

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

142

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

143

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

144

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

145

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

146

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

147

of AQs by HPLC or spectrometry.

148

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

149

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

150

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

151

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

152

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

153

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

154

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

155

RESULTS AND DISCUSSION

156

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

157

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

158

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

159

bond interaction between HFIP and QACs.23

160

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

161

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

162

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

163

1,

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


7


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 24

164

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

165

24,25

166

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

167

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

168

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

169

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

170

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

171

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

172

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

173

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

174

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

175

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

176

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

177

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

178

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

179

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

180

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

181

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

182

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

183

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

184

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

185

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

186

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

187

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

188

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

189

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


8

Page 9 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


190

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

191

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

192

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

193

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

194

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

195

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

196

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

197

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

198

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

199

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

200

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

201

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

202

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

203

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

204

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

205

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

206

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

207

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

208

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

209

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

210

saturation, thus the phase ratio keeps nearly unchanged.

211

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

212

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

213

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

214

extraction efficiency. The total AQs was determined by spectrometry.


9


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 24

215

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

216

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

217

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

218

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

219

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

220

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

221

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

222

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

223

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

224

experiments.

225

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

226

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.

228

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

229

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

230

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

231

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

232

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

233

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

234

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

236

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

237

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

238

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

239

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

240

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

241

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


10

Page 11 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


242

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

243

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

244

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


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 24

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

Page 13 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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

Page 15 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


341

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

342

2015CFA139).

343

REFERENCES

344

(1) Glyk, A.; Scheper, T.; Beutel, S. PEG-salt aqueous two-phase systems: an attractive and versatile liquid-

345

liquid extraction technology for the downstream processing of proteins and enzymes. Appl. Microbiol.

346

Biotechnol. 2015, 99 (16), 6599–6616. DOI: 10.1007/s00253-015-6779-7.

347

(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

348

two-phase system as an effective tool for purification of phenolic compounds from Fig Fruits (Ficus caricaL.).

349

Sep. Sci. Technol. 2015, 50 (12), 1785–1793. DOI: 10.1080/01496395.2015.1014054.

350

(3) Ventura, S. P. M.; de Barros, R. L. F.; de Pinho Barbosa, J. M.; Soares, C. M. F.; Lima, Á. S.; Coutinho, J.

351

A. P. Production and purification of an extracellular lipolytic enzyme using ionic liquid-based aqueous two-

352

phase systems. Green Chem. 2012, 14, 734–740. DOI: 10.1039/C2GC16428K.

353

(4) Lin, X.; Wang, Y.; Liu, X.; Huang, S.; Zeng, Q. ILs-based microwave-assisted extraction coupled with

354

aqueous two-phase for the extraction of useful compounds from Chinese medicine. Analyst 2012, 137, 4076–

355

4085. DOI: 10.1039/C2AN35476D.

356

(5) Couling, D. J.; Bernot, R. J.; Docherty, K. M.; Dixon, J. K.; Maginn, E. J. Assessing the factors responsible

357

for ionic liquid toxicity to aquatic organisms via quantitative structure–property relationship modeling. Green

358

Chem. 2006, 8 (1), 82–90. DOI: 10.1039/B511333D.

359

(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.

360

Tailoring and recycling of deep eutectic solvents as sustainable andefficient extraction media. J. Chromatogr. A

361

2015, 1424, 10–17. DOI:10.1016/j.chroma.2015.10.083.

362

(7) Zhao, B.Y.; Xu, P.; Yang, F. X.; Wu, H.; Zong, M.H.; Lou, W.Y. Biocompatible deep eutectic solvents

363

based on choline chloride: characterization and application to the extraction of rutin from sophora japonica. ACS


15


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

364

Page 16 of 24

Sustainable Chem. Eng. 2015, 3 (11), 2746–2755. DOI: 10.1021/acssuschemeng.5b00619.

365

(8) Cvjetko Bubalo, M.; Curko, N.; Tomasevic, M.; Kovacevic Ganic, K.; Radojcic Redovnikovic, I. Green

366

extraction of grape skin phenolics by using deep eutectic solvents. Food Chem. 2016, 200, 159–166. DOI:

367

10.1016/j.foodchem.2016.01.040.

368

(9) Duan, L.; Dou, L.L.; Guo, L.; Li, P.; Liu, E. H. Comprehensive evaluation of deep eutectic solvents in

369

extraction of bioactive natural products. ACS Sustainable Chem. Eng. 2016, 4 (4), 2405–2411. DOI:

370

10.1021/acssuschemeng.6b00091.

371 372 373 374

(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. DOI: 10.1039/c3an02235h. (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. DOI: 10.1016/j.aca.2015.01.026.

375

(12) Li, N.; Wang, Y.; Xu, K.; Huang, Y.; Wen, Q.; Ding, X. Development of green betaine-based deep eutectic

376

solvent aqueous two-phase system for the extraction of protein. Talanta 2016, 152, 23–32. DOI:

377

10.1016/j.talanta.2016.01.042.

378

(13) Li, N.; Wang, Y.; Xu, K.; Wen, Q.; Ding, X.; Zhang, H.; Yang, Q. High-performance of deep eutectic

379

solvent based aqueous bi-phasic systems for the extraction of DNA. RSC Adv. 2016, 6 (87), 84406–84414. DOI:

380

10.1039/c6ra17689e.

381 382

(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. DOI: 10.1021/la303035h.

383

(15) Chen, D.; Zhang, P.; Li, Y.; Mei, Z.; Xiao, Y. Hexafluoroisopropanol-induced coacervation in aqueous

384

mixed systems of cationic and anionic surfactants for the extraction of sulfonamides in water samples. Anal.

385

Bioanal. Chem. 2014, 406 (24), 6051–6060. DOI: 10.1007/s00216-014-8031-1.


16

Page 17 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


386

(16) Tian, Y.; Li, Y.; Mei, J.; Deng, B.; Xiao, Y. Hexafluoroisopropanol-modified cetyltrimethylammonium

387

bromide/sodium dodecyl sulfate vesicles as a pseudostationary phase in electrokinetic chromatography. J.

388

Chromatogr. A 2015, 1404, 131–140. DOI: 10.1016/j.chroma.2015.05.036.

389

(17) Wang, L.; Dai, D.Y.; Chen, Q.; He, M.Y. Rapid and green synthesis of phenols catalyzed by a deep

390

eutectic mixture based on fluorinated alcohol in water. J. Fluorine Chem. 2014, 158, 44–47. DOI:

391

10.1016/j.jfluchem.2013.12.006.

392 393

(18) Wang, L.; Zhu, K.Q.; Chen, Q.; He, M.Y. Facile and environmentally friendly halogenation of BODIPYs in deep eutectic solvent. Dyes and Pigments 2015, 112, 274–279. DOI: 10.1016/j.dyepig.2014.07.024.

394

(19) Arvindekar, A. U.; Pereira, G. R.; Laddha, K. S. Assessment of conventional and novel extraction

395

techniques on extraction efficiency of five anthraquinones from Rheum emodi. J. Food Sci. Technol. 2015, 52

396

(10), 6574–6582. DOI: 10.1007/s13197-015-1814-3.

397

(20) Zhang, H. F.; Shi, Y. P. Temperature-assisted ionic liquid dispersive liquid-liquid microextraction

398

combined with high performance liquid chromatography for the determination of anthraquinones in Radix et

399

Rhizoma Rhei samples. Talanta 2010, 82 (3), 1010–1016. DOI: 10.1016/j.talanta.2010.06.008.

400

(21) Wu, X.; Liang, L.; Zou, Y.; Zhao, T.; Zhao, J.; Li, F.; Yang, L. Aqueous two-phase extraction,

401

identification and antioxidant activity of anthocyanins from mulberry (Morus atropurpurea Roxb.). Food Chem.

402

2011, 129 (2), 443–453. DOI: 10.1016/j.foodchem.2011.04.097.

403 404

(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. DOI: 10.1016/j.seppur.2012.06.021.

405

(23) Francisco, M.; van den Bruinhorst, A.; Kroon, M. C. Low-transition-temperature mixtures (LTTMs): a

406

new generation of designer solvents. Angew. Chem. Int. Ed. 2013, 52 (11), 3074–3085. DOI:

407

10.1002/anie.201207548.

408

(24) Khezeli, T.; Daneshfar, A.; Sahraei, R. A green ultrasonic-assisted liquid-liquid microextraction based on


17


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 24

409

deep eutectic solvent for the HPLC-UV determination of ferulic, caffeic and cinnamic acid from olive, almond,

410

sesame and cinnamon oil. Talanta 2016, 150, 577–585. DOI: 10.1016/j.talanta.2015.12.077.

411

(25) Guo, W.; Hou, Y.; Wu, W.; Ren, S.; Tian, S.; Marsh, K. N. Separation of phenol from model oils with

412

quaternary ammonium saltsvia forming deep eutectic solvents. Green Chem. 2013, 15 (1), 226–229. DOI:

413

10.1039/c2gc36602a.

414

(26) Mele, A.; Tran, C. D.; De Paoli Lacerda, S. H. The structure of a room-temperature ionic liquid with and

415

without trace amounts of water: the role of C[bond]H...O and C[bond]H...F interactions in 1-n-butyl-3-

416

methylimidazolium tetrafluoroborate. Angew. Chem. Int. Ed. 2003, 42 (36), 4364–4366. DOI:

417

10.1002/anie.200351783.

418

(27) Dai, Y.; Van Spronsen, J.; Witkamp, G. J.; Verpoorte, R.; Choi, Y. H. Natural deep eutectic solvents as

419

new potential media for green technology. Anal. Chim. Acta 2013, 766, 61–68. DOI: 10.1016/j.aca.2012.12.019.

420

(28) Martins, J. P.; Carvalho, C. de P.; Silva, L. H. M. da; Coimbra, J. S. dos R.; Silva, M. do C. H. da;

421

Rodrigues, G. D.; Minim, L. A. Liquid–liquid equilibria of an aqueous two-phase system containing

422

poly(ethylene) glycol 1500 and sulfate salts at different temperatures. J. Chem. Eng. Data 2008, 53, 238–241.

423

DOI: 10.1021/je700538z.

424

(29) Tan, Z.; Wang, C.; Yi, Y.; Wang, H.; Li, M.; Zhou, W.; Tan, S.; Li, F. Extraction and purification of

425

chlorogenic acid from ramie (Boehmeria nivea L. Gaud) leaf using an ethanol/salt aqueous two-phase system.

426

Sep. Purif. Technol. 2014, 132, 396–400. DOI: 10.1016/j.seppur.2014.05.048.

427

(30) Xie, X.; Wang, Y.; Han, J.; Yan, Y. Extraction mechanism of sulfamethoxazole in water samples using

428

aqueous two-phase systems of poly(propylene glycol) and salt. Anal. Chim. Acta 2011, 687 (1), 61–66. DOI:

429

10.1016/j.aca.2010.12.012.

430

(31) Wang, Y.; Yan, Y.; Hu, S.; Han, J.; Xu, X. Phase diagrams of ammonium sulfate + ethanol/1-propanol/2-

431

propanol + water aqueous two-phase systems at 298.15 K and Correlation. J. Chem. Eng. Data 2010, 55, 876–


18

Page 19 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

432


881. DOI: 10.1021/je900504e.

433

(32) Freire, M. G.; Pereira, J. F.; Francisco, M.; Rodriguez, H.; Rebelo, L. P.; Rogers, R. D.; Coutinho, J. A.

434

Insight into the interactions that control the phase behaviour of new aqueous biphasic systems composed of

435

polyethylene glycol polymers and ionic liquids. Chem. Eur. J. 2012, 18 (6), 1831–1839. DOI:

436

10.1002/chem.201101780.

437

(33) Wang, Y.; Han, J.; Xu, X.; Hu, S.; Yan, Y. Partition behavior and partition mechanism of antibiotics in

438

ethanol/2-propanol–ammonium sulfate aqueous two-phase systems. Sep. Purif. Technol. 2010, 75 (3), 352–357.

439

DOI: 10.1016/j.seppur.2010.09.004.

440

(34) Wang, Y.; Wang, L.; Shi, L. L.; Shang, Z. B.; Zhang, Z.; Jin, W. J. Colorimetric and fluorescence sensing

441

of Cu2+ in water using 1,8-dihydroxyanthraquinone-beta-cyclodextrin complex with the assistance of ammonia.

442

Talanta 2012, 94, 172–177. DOI: 10.1016/j.talanta.2012.03.013.

443 444 445 446 447 448 449 450 451


19


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

452

Page 20 of 24

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

Page 21 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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

Page 23 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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.

491 492


23


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 24

493

494

TOC/Abstract graphic image

495 496 497

498

SYNOPSIS

499

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

500

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

501

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

502

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

503

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

504


24

Salt Aqueous

Recommend Documents