Accepted Manuscript

Extraction techniques with deep eutectic solvents

Sara C. Cunha, José Fernandes

PII: S0165-9936(18)30118-3
DOI: 10.1016/j.trac.2018.05.001
Reference: TRAC 15145

To appear in: Trends in Analytical Chemistry

Received Date: 12 March 2018

Revised Date: 1 May 2018
Accepted Date: 1 May 2018

Please cite this article as: S.C. Cunha, J. Fernandes, Extraction techniques with deep eutectic solvents,
Trends in Analytical Chemistry (2018), doi: 10.1016/j.trac.2018.05.001.

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1 Extraction techniques with deep eutectic solvents

2 Sara C. Cunha* and José Fernandes*

4 *Corresponding author:

5 José O. Fernandes and Sara C. Cunha: Tel: +351-220428639; Fax: +351-226093390;

6 E-mail: josefer@ff.up.pt, sara.cunha@ff.up.pt.

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8 LAQV-REQUIMTE, Laboratório de Bromatologia e Hidrologia, Faculdade de Farmácia,

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9 Universidade do Porto, Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal.

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11 ABSTRACT

12 In last years, a plethora of extraction techniques has emerged as environmental-

13 friendly alternatives to conventional extraction procedures. In this particular field, a
14 novel class of solvents known as deep eutectic solvents (DES) has arisen as a new
15 and very promising tool. Compared with conventional organic solvents, DES as well as
16 the so-called natural deep eutectic solvents (NADES) have attracted considerable
17 attention due to the fact that they not only are eco-friendly, non-toxic, and

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18 biodegradable organic compounds but also have a low cost, being easy to produce in
19 the own laboratory. The present review provides a critical and organized overview of

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20 novel extraction techniques using DES as extracting solvents that were applied in food,
21 biological and environmental sample analysis. An evaluation of how these

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22 DES/NADES could improve extraction yields of a variety of analytes and advantages
23 and limitations of each proposal will be discussed and compared with earlier studies.
24 Keywords: Sample Preparation, Deep Eutectic Solvents, Natural Deep Eutectic

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25 Solvents, extraction, Chemical Analysis
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27 Abbreviations: AALLME, air-assisted liquid-liquid microextraction, AA-DLME, air-

28 assisted dispersive liquid-liquid microextraction, AAS, flame atomic absorption

29 spectrometry AAS, CPEs, carbon paste electrodes, DA, daidzein, DAGL, daidzin,

30 DEHP, di(2-ethylhexyl)phthalate, DLLME, dispersive liquid-liquid microextraction, DNA,

31 Deoxyribonucleic acid, ELISA, Enzyme-Linked Immunosorbent Assay, FAAS, flame

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32 atomic absorption spectrometry, FT-IR, fourier-transform infrared spectroscopy, GA-

33 DLLME, gas air-dispersive liqui-lquid microextraction, GC-ECD, gas chromatography-

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34 electron capture detector, GC-FID, gas chromatography- flame ionization detector, GT,

35 genistein, GTGL, genistin, HBA, hydrogen-bond acceptor, HBD, hydrogen-bond donor,

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36 HPLC-DAD, high performance liquid chromatography- diode array detection, HPLC-

37 UV, high performance liquid chromatography-ultraviolet, HS-SME, headpsace solid-

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single microextraction, HF-LPME, hollow fiber-liquid phase microextraction IAA, Indole-
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39 3-acetic acid, IBA, 3-indolebutyric acid, 4-IPOAA, 4-iodophenoxyacetic acid, LGH,

40 lactic acid–glucose, LIT, lithospermic acid, LPME, liquid phase microextraction, MAE,
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41 microwave-assisted extraction, MAME microwave microextraction, MDES, magnetic

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42 deep eutectic solvents, MIPs, Molecularly imprinted polymers, MMWCNT, magnetic

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43 multi-walled carbon nanotube nanocomposite, NIPs, non-imprinted polymers, NMR,

44 nuclear magnetic resonance, OCPs, organochlorine pesticides Ox, oxalic acid,

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45 PAHs, polycyclic aromatic hydrocarbons, PDA, photodiode array, PCH, 1,2-

46 propanediol–choline chloride, PT-SPE, pipette-tip solid-phase extraction, ROS,

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47 rosmarinic acid, RSD, relative standard deviation, SAA, salvionalic acid A, SAB,
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48 salvionalic acid, SDME, single drop microextraction, SPME, solid-phase

49 microextraction, S-SIL-MSPD silica-supported ionic liquid-based matrix solid phase

50 dispersion, SPE, solid-phase extractions, UAE, ultrasound-assisted extraction,

51 UHPLC-UV, ultra-high performance liquid chromatography with ultraviolet detection,

52 UAME, ultrasound assisted microextraction, UALPME ultrasound assisted liquid phase

53 microextraction USAEME, ultrasound-assisted emulsification microextraction, UPLC-

54 TOF-MS, Ultra Performance Liquid Chromatography-Time-of-flight Mass Spectrometry,

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55 UV-VIS, ultraviolet-visible spectrophotometry, TIIA, tanshinone IIA, THF,

56 tetrahydrofuran, VWD, variable wavelength detector.

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57 1. INTRODUCTION

58 Sample preparation is a common step in trace analytical methods, involving the

59 extraction of an analyte or a class of analytes from its original matrix into a solvent in
60 order to allow its further analysis by a sensitive and selective way. Conventional
61 sample preparation techniques such as multi-step liquid-liquid extraction, Soxhlet
62 extraction and solid-phase extraction, among others, are usually time-consuming, labor
63 intensive and involve the use of a large volume of organic solvents, which is expensive

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64 and generate a considerable amount of waste harmful for human health and the
65 environment. To overcome these drawbacks, numerous efforts have been made over

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66 the last decades, to streamline sample extraction procedures and also to reduce or
67 eliminate the use or generation of hazardous substances in agreement with the

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68 principles of the green chemistry [1]. New techniques miniaturizing solid-phase
69 extraction or liquid-liquid extraction have appeared such as SPME and LPME,
70 respectively. SPME is a solventless extraction technique very popular in recent years
71
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that use different fiber materials in various configurations for the extraction of a wide
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72 range of volatile analytes [2]. LPME comprises a range of slightly different techniques
73 characterized by using low amounts of sample matrices and small volumes of organic
74 solvents. Often it can employ different types of kinetic energy such as ultrasound
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75 (UAME) or microwave (MAE) in order to improve extraction efficiency, enrichment

76 factor and simultaneously reduce extraction time. Progresses made in the last decade
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77 (2006-2017) in the field of sample extraction are confirmed by the increasing number of
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78 published scientific articles on SPME, LPME, UAME and MAE. In the ISI Web of
79 knowledge there are references for that period of about 11,133 published scientific
80 papers on SPME, 1,351 on LPME, 1,790 on UAME and 2,803 on MAE for
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81 determination of different organic and inorganic compounds, of which more than 60%
82 were published in the last five years. The keywords used for this research were: “solid-
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83 phase microextraction”, “liquid phase microextraction”, “ultrasound assisted extraction”

84 and “microwave-assisted extraction”.
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85 Until some years ago, solvents used in LPME techniques were always associated with
86 a certain degree of toxicity, but the recent development of a remarkable generation of
87 new extracting solvents, i.e. DES is changing this scenario. These solvents are
88 commonly composed of two non-toxic components, one of them with capacity to be a
89 HBA (quaternary ammonium, tetralkylammonium or phosphonium salts) while the other
90 (acids, alcohols, amines and carbohydrates) possess HBD properties [3]. Generally,
91 DES have much lower melting point than that of any of its individual components,
92 which is due to the formation of intramolecular hydrogen bonds [4], and possess some

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93 noteworthy chemical properties, such as low vapor pressure, relatively wide liquid-
94 range, non-flammability and unreactivity towards water [5]. Moreover, DES are easy to
95 be prepared not requiring purification steps, are made from low cost compounds,
96 present low or negligible toxicity and are biodegradable and easily recyclable. These
97 features make DES preferable over the conventional solvents used in extraction
98 procedures.

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99 Similar properties to DES have the so-called NADES, solvents which are prepared
100 using natural components produced by cell metabolism. Based on the hypothesis that
101 the existence of NADES in living organisms can explain many biological processes,

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102 several NADES composed of simple molecules always present in living cells (urea,
103 amino acids, sugars and choline) have been synthesised and studied in what respect

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104 their solvent properties [6].

105 Indeed, DES and NADES showed to have excellent potential as extracting solvents in

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106 several sample preparation procedures such as LPME, UAME and MAE. Literature
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107 research in ISI Web of knowledge indicates a total of 1,303 references about DES and
108 56 regarding NADES, of which 321 and 31, respectively, are related with extraction
109 procedures. Since 2011 the number of publications has increased considerably, arising
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110 462 in 2017, 443 of which related to DES and 19 to NADES (Figure 1). According to
111 the Web Science Categories, most of these references are within the scope of
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112 multidisciplinary chemistry, followed by chemistry physical or chemistry analytical,

113 respectively. The reason for the increase number of publications in the analytical field
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114 can be attributed to the unique features of these new liquids such as high thermal
115 stability, low thermal conductivity, low volatility and adjustable miscibility as well as the
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116 capacity to be combined with advanced separation techniques like HPLC and GC. The
117 present review, cover applications reported until the end of 2017 and is mainly focused
118 on microextraction techniques using DES/NADES as extracting solvents for food,
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119 biological and environmental sample analysis. The potential of each modern extraction
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120 technique will be discussed and compared with earlier studies and an evaluation of
121 how these techniques could improve extraction efficiency for a variety of analytes will
122 be made.
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124 2. DEEP EUTECTIC SOLVENTS (DES): A BRIEF OVERVIEW

125 In the year of 2001, Abbot and co-workers reported that a mixture of a choline chloride
126 and a metal salt (zinc chloride) could form a liquid, at temperatures below 100°C [7].
127 Two years later, the same group developed a mixture of ChCl with an hydrogen-bond

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128 donor (urea) [8] designating it as DES. In the subsequent years other DES have been
129 reported namely that formed by mixing ChCl with different carboxylic acids (oxalic,
130 malonic, and succinic acids) [9]. Another major family of DES that have been widely
131 explored are those that combine a carbohydrate (or a reduced derivative as is the case
132 with sorbitol and mannitol), an urea derivative (N,N’-dimethylurea), and a chloride salt
133 (ammonium chloride)[10]. Generally, the melting point of the DES is lower than the
134 melting points of each of its starting components. One of the attractive features of

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135 these novel solvents is the possibility of having, simply by changing one or both
136 components, a huge number of eutectic mixtures with different chemical properties.

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137 Recently, a new class of DES have been synthetized, based on combinations of
138 decanoic acid with various quaternary ammonium salts [11] or DL-menthol with several

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139 natural acids [12]. This new class of DES showed a high hydrophobic behavior in
140 contrast to the previous hydrophilic DES [11]. Many extraction procedures can become
141 more effective by using these hydrophobic DES due their capability of extracting

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142 analytes hydrophobic from aqueous solutions. Additionally, these new solvents have
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143 the ability to extract both dissociated and undissociated forms of acidic compounds,
144 broadening the application of hydrophobic DES for different pH environments [11].
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145 DES are easily produced by just mixing two or more compounds and heating them to
146 around 80 °C [8, 13] or freeze-drying [14], without the subsequent need of any complex
147 purification step, thus reaction conditions are accessible for any laboratory. However,
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148 owing to the extreme hygroscopicity of the HBA (e.g. ChCl, tetrabutylammonium, DL-
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149 menthol), attracting moisture to preparations, a careful manipulation through using

150 vacuum conditions is advised. Although ChCl is the most employed quaternary
151 ammonium salt because of its low cost and biodegradability (it is indeed produced in
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152 large scale and added to the chicken feed as nutrient), many other halides
153 (methyltriphenylphosphonium bromide, benzyltriphenylphosphonium chloride,
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154 acetylcholine chloride, tetramethylammonium chloride among others) are suitable for
155 making DES [5].
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156 One of the main features of DES is their possibility to be used as extracting solvent for
157 a wide range of chemical different solutes [3]. Their uses as solvents in extraction
158 procedures depends on their physical properties, such as viscosity, density, miscibility
159 and polarity. It is convenient to select solvents with low viscosity to facilitate mixing but
160 with a large density difference from the matrix in order to easy the separation of phases
161 [15]. The main physical properties of DES including freezing point, density, viscosity,
162 conductivity, and polarity are briefly discussed below in this section and some DES are
163 highlighted in Table 1. More detailed information on the properties of DES can be

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164 found in recent reviews by Zang et al. [3], Tang and Row [16], Smith et al. [5], and
165 references cited therein.

166 The DES freezing point is dependent on DES components (type of HBD and HBA) and
167 its molar ratio. For instance, the freezing point of a choline salt-derived DES combined
168 with urea decreases in the order F- > NO3- > Cl- > BF4- indicating a correlation with the
169 hydrogen bond strength [3, 17]. The organic salt/HBD molar ratio has also significant

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170 effect on the freezing point of a DES. For instance, when ChCl was mixed with urea in
171 a molar ratio of 1:1 and 1:2, the resulting DES showed a freezing point >50 °C and 12
172 °C, respectively [3].

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173 DES exhibit in general higher density values than water with levels ranging from 1.041

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174 g cm-3 to 1.63 g cm-3 [3]. This feature helps the rapid settling of DES phase in phase
175 separation devices used in DLLME techniques.

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176 Most of the DES exhibit a relatively high viscosity at room temperature (> 100 cP) [3].
177 This can be of substantial benefit when carrying out SDME, as the high viscosity
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178 facilitates the suspension of larger drops at the tip of a needle. However, in some
179 extraction procedures such as DLLME this characteristic can affect negatively the
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180 diffusion of analytes. To surpass this drawback some authors, increase the
181 temperature during extraction or, alternatively, increase the ChCl concentration, which
182 are reported in literature to reduce the viscosity of some eutectic mixtures.
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183 In general, DES show poor conductivity (lower than 2 mS cm-1 at room temperature) [3]
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184 due to their high viscosity. However, conductivities of DES increase significantly as the
185 temperature increases due to a decrease of the respective viscosity [3, 17, 18].
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186 Furthermore, the successive addition of ChCl to glycerol lowers the viscosity and
187 increases the conductivity (from 0.74 mS cm-1 for a molar ratio of 1-4 ChCl˗glycerol to
188 1.30 mS cm-1 for a molar ratio of 1-2 ChCl˗glycerol) [18] due to more available charge
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190 Polarity is one of the most important distinguishing characteristics of DES, in view of
191 their extraction ability and their miscibility with other solvents. Nevertheless, few studies
192 related to DES polarity are published. Abbott and co-workers reported that the polarity
193 scale of ChCl-glycerol was similar to RNH3+X-, R2NH2+X-, and imidazolium ionic liquids
194 [17, 18]. In DES composed of an ammonium salt and carboxylic acids the acidity is
195 mainly provided by the organic acid present in the mixture, and an increase of the alkyl
196 side chain of both compounds leads to a lower ability of the solvent to donate protons
197 [19]. Carbohydrate derivative DES present higher polariry than those observed for

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198 short chain alcohols (e.g. ethanol, 2-propanol) and some polar aprotic solvents (e.g.
199 dimethylsulfoxide and dimethylformamide) [10].

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201 3. NATURAL DEEP EUTECTIC SOLVENTS: A BRIEF OVERVIEW

202 Recently, Choi and collaborators have explored natural products as a source of DES

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203 solvents, including primary metabolites common in living cells (sugars, sugar alcohols,
204 organic acids, amino acids, amines) as well as water [6]. NADES can be obtained by
205 heating a mixture of two or three components in certain molar ratios in the presence of

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206 water, which decrease their viscosity and allows the occurrence of extensive inter
207 molecular interactions e.g. H-bonds or ionic bonds [20](Table 2). The usually

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208 components of NADES as HBA are amines (ChCl, ammonium chloride) or amino acids
209 (alanine, proline, glycine, betain) while as HBD the more common are organic acids

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210 (oxalic acid, lactic acid, malic acid, etc.) or carbohydrates (glucose, fructose, maltose,
211 etc.).
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212 As a whole NADES possess excellent properties as solvents [20], e.g., negligible
213 volatility, a very low melting point (they are liquid even below -20 oC), a broad polarity
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214 range and high solubilization power of a wide range of compounds, especially poorly
215 water-soluble compounds [6, 20]. The high solubility of scarce water-soluble
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216 metabolites and macromolecules (e.g. DNA, proteins, cellulose, and amino acids) has
217 been demonstrated [20-22] as well as their suitability as media for enzymatic reactions
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218 [6, 23] and biotransformations [24]. It was verified that NADES composed by 1 mol of
219 ChCl and 2 mol of 1,2-ethanediol, glycerol, malonic acid, or urea, the two first present a
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220 more dipolar nature than the last ones, which can be attributed to the presence of
221 alcohol functionalities on 1,2-ethanediol and glycerol [25]. Despite this data, detailed
222 information about chemical and physical properties is still scarce, and only few
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223 applications involving NADES in the analytical field, e.g. extraction of organic
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224 compounds, are reported. Therefore, a deep research on the preparation of NADES for
225 specific applications is still required.

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227 4. LIQUID-PHASE MICROEXTRACTION

228 LPME extraction procedures correspond to efficient alternatives to traditional liquid-

229 liquid extraction, offering numerous advantages such as a high degree of concentration

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230 (or enrichment factor), and low consumption of organic solvents while requesting
231 minute amounts of samples.

232 In LPME techniques, usually few microliters of the extracting solvent (usually
233 designated as acceptor phase) are placed directly into the aqueous sample containing
234 analytes (donor phase) or in its headspace and later the extracting solvent is collected.

235 In the last few years numerous LPME procedures have been developed such as

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236 SDME, HF-LPME and DLLME. In SDME a drop of extractive solvent is suspended in
237 the tip of a syringe which can be immersed in the aqueous phase of the sample or

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238 exposed to the respective headspace (HS-SME). In HF-LPME the extracting phase is
239 placed inside the lumen of a porous polypropylene hollow fiber, to improve the stability

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240 and reliability of extraction [26]. In DLLME a cloudy solution of small droplets of
241 extracting solvent (microliters of a water-immiscible high density organic solvent) is
242 formed and dispersed throughout the aqueous phase using a dispersive solvent,

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243 miscible in both extracting and aqueous phases. Currently some DLLME procedures
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244 do not require the use of dispersive solvent being in those cases the cloud solution
245 obtained either by the injection of air bubbles (GA-DLLME), formation of air bubbles
246 using a vortex, or by the mechanic action of sucking and injecting the aqueous solution
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247 with a syringe (AALLME and AA-DLME). Overall, in all these procedures, extraction
248 efficiency is dependent of many factors such as partition coefficient, type and volume of
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249 extracting and dispersive solvents, sample volume, analyte properties, agitation, ionic
250 strength, extraction time, temperature, etc. A detailed discussion of the importance of
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251 each parameter can be found in the literature [27].

252 LPME techniques using DES or NADES as extracting solvent had been used for
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253 extraction of several polar nonvolatile and volatile compounds from food and water
254 matrices (Table 3).
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255 Tang et al. [28] optimized a two phase HS-SME technique where a DES (ChCl and
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256 ethylene glycol, 1:4 molar ratio) drop suspended in a tip of a microsyringe needle was
257 exposed for 30 min to the headspace of leaf samples to extract bioactive terpenoids.
258 The use of a 2 µl DES drop was enough to allow the adsorption of the target
259 compounds volatilized from the samples heated at 100 °C and ultrasonicated at 70 W.
260 Once stopped the procedure, the suspended drop was retracted back into the
261 microsyringe and injected into a GC-FID.

262 In the same year 2014, Gu et al. [29] employed a similar HS-SME procedure for the
263 extraction of phenolic compounds from crude oils; they also used ChCl and ethylene

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264 glycol (1:3, molar ratio) as DES and ultrasonication to reduce the extraction time
265 needed and to improve the yields [29].

266 A three phase HS-SME procedure using DES (methyltriphenylphosphonium iodide and
267 ethylene glycol, 1:4 molar ratio) plus 20% v/w methanol as an acceptor phase and n-
268 dodecane as extraction solvent was applied to extract steroidal hormones
269 (dydrogesterone and cyproterone acetate) from biological samples (urine and plasma)

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270 [30]. The analytes were further analyzed by LC-UV, and the obtained performance in
271 what respect range of linearity and LOD have compared positively to those reported in
272 literature using more sensitive detectors.

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273 Recently, Yousefi et al.[31] developed a two phase HS-SME procedure using an

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274 hydrophobic magnetic bucky gel formed by combining DES (ChCl:chlorophenol, 1:2
275 molar ratio) and magnetic multiwalled carbon nanotubes for the extraction of volatile
276 hydrocarbons from water and urine samples. The extraction was successfully

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277 completed in 10 min using a drop of 20 µL that was placed in the headspace of the vial
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278 containing the aqueous sample. The extraction temperature was kept at 30 °C and the
279 vial was stirred at 1200 rpm. The use of a large drop compared with other HS-SME
280 based DES procedure showed to provide a higher sensitivity. When compared with
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281 conventional solvents DES showed more adequate to form stable drops for HS-SME
282 due to its higher thermal stability, higher viscosity, lower volatility and adjustable
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283 miscibility.
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284 Concerning to DLLME conventional process using DES as extracting solvent it was
285 used just by Ferrone et al. [32] for determination of oxyprenylated, phenylpropanoids in
286 vegetable oils. The extraction was performed with 45 µL of DES (phenylacetic acid:
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287 betaine, 2:1 molar ratio) and isopropanol (200 µL) as dispersive solvent; following
288 agitation and centrifugation, 5 µL of the bottom phase were injected in a UHPLC-PDA
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289 system. This method allowed the achievement of good limits of detection, linearity and
290 reproducibility similar or better to those reported in literature for the same analytes.
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291 A novel rapid and efficient GA-DLLME procedure characterized by the absence of
292 dispersive solvent was developed by Farajzadeh et al. [33] for the extraction of 9
293 pesticide residues from fruit and vegetable juices. In the optimized extraction
294 procedure, air was bubbled into a test tube to disperse the extracting DES (ChCl: 4-
295 chlorophenol, 1:2 molar ratio) into the aqueous solution, in order to obtain a cloudy
296 solution. After centrifugation, an aliquot of the sedimented phase is injected into GC-
297 FID for the separation and determination of the enriched pesticide residues [33].

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298 Another new type of DLLME is the AALLME. Similar to GA-DLLME, in this technique
299 the cloud solution is obtained by sucking and injecting several times the mixture of
300 extracting solvent and aqueous sample. Lamei et al. [34] used this technique
301 employing a mixture of ChCl and 5,6,7,8-Tetrahydro-5,5,8,8-tetramethylnaphthalen-2-ol
302 (1:2 molar ratio) as DES extractor and THF as a demulsifier agent, for the extraction of
303 methadone from water and biological samples. Therefore, the dispersion of the
304 aggregated DES droplets into aqueous phase was achieved by the effect of sucking

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305 and injecting (10 times) the mixture of sample, extracting solvent and demulsifier agent.
306 After a brief centrifugation the uplayer was directly analyzed by GC-FID. A pronounced

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307 advantage in comparison to the other LPME described in the literature for methadone
308 extraction is the low toxicity and low cost of the extracting solvent used.

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309 AA-DLLME was developed and optimized by Ge et al. [35] for pre-concentration and
310 separation of 6 benzophenones from different types of waters. Using a hydrophobic

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311 DES (mixture of DL-menthol and decanoic acid, 2:1 molar ratio) as extractor solvent,
312 which was several times injected into the aqueous solutions, the authors were able to
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313 extract efficiently the benzophenones without the use of dispersive solvents.

314 In a similar way, Zheng et al. [36] used a hydrophobic DES with 1-decyl-3-
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315 methylimidazolium chloride and 1-dodecanol (1:2, molar ratio) for the extraction of
316 benzoylureas from waters, followed by HPLC-UV analysis. Various conditions were
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317 optimized, namely the type and volume of extractor, salt addition, vortex time,
318 temperature of extraction and pH of the aqueous sample. The authors concluded that
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319 8.0 mL of sample solution with 450 mg sodium chloride and pH at 4-6 provided the best
320 recoveries when extracted with 40 µL of DES during 3 min in vortex at 40 °C.
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321 Since the discovery of NADES by the Choi group [6] several works have been
322 published by the same research group exploring the potential of NADES as solvents to
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323 extract bioactive compounds such as antocyanins, and phenols from several solid
324 samples (Table 3) [20, 37, 38]. In general, a low amount of sample (<100 mg) is
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325 extracted with a small volume of the chosen NADES (< 3 ml) at controlled temperature
326 (40 ºC) using vortex stirring. A pronounced advantage of the use of NADES in LPME in
327 comparison to the conventional extracting solvents is the higher stability of the extract
328 obtained and, of course, the use of a solvent sustainable and environmental-friendly.

329 In 2013, Dai et al. [20] tested different NADES and a multivariate data analysis to
330 demonstrate that the extractability of a wide range of phenolic compounds (hydrophilic
331 and hydrophobic metabolites of safflower) were greater with NADES than with
332 conventional solvents. This high extractability was attributed to H-bonding interactions

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333 between molecules of NADES and phenolic compounds. In 2014 the same group
334 continued the previous work, exploring the ability of different NADES to stabilize
335 unstable phenolic compounds extracted from safflower such as carthamin [38]. They
336 found that carthamin is more stable in glucose-ChCl and sucrose-ChCl than in acidic
337 NADES such as proline-malic acid or lactic-acid-glucose and the stabilization aptitude
338 of NADES increases with increasing viscosity (low water content). Lately, Dai et al [39]
339 tested diverse NADES for the extraction of anthocyanins from purple and orange petals

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340 of Catharanthus roseus. Among the NADES evaluated, LGH and PCH showed to
341 present a similar extraction power for anthocyanins as conventional organic solvents,

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342 with the additional advantage exhibited by LGH to provide at least three times higher
343 stability capacity for cyanidins than acidified ethanol, facilitating their extraction and

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344 further analysis by UPLC-TOF-MS.

345 These works clearly showed that both DES and NADES have a high potential as

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346 extracting solvents for a wide range of analytes of low to medium polarity and
347 demonstrated a bright future for the application of these novel classes of extracting
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348 solvents in LPME field.

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350 5. ULTRASOUND ASSISTED MICROEXTRACTION

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351 In UAME techniques the extraction procedure takes place under ultrasonic energy
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352 (typical systems use a frequency energy of 40 kHz), which favors the contact between
353 sample and extracting solvent. Ultrasounds radiation can be applied by means of
354 water-baths or ultrasonic probes, being the last ones able to deliver higher energy input
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355 per volume due to a more focused and uniform power input. Conversely, ultrasonic
356 probes provide an entire control over the most important sonication parameters,
357 leading to more reproducible results [40].
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358 In general, extraction efficiency of UAME techniques depends on different factors such
359 as extracting solvent (type, pH, volume), ultrasonic conditions (temperature, amplitude
360 of sonication, sonication time), and sample features (matrix, amount, particle size) [40,
361 41].

362 The main advantage of using UAME compared with conventional DLLME is that no
363 dispersive solvent is needed to achieve a high surface area of contact between the
364 extracting solvent and sample. Additionally, UAME can promote a greater penetration
365 of the extracting solvent in solid samples matrices.

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366 DES based UAME have been applied mainly for extraction of organic compounds from
367 liquid samples, although their application to solid samples or to the extraction of
368 inorganic analytes has also been proposed (Table 3). Some works have explored the
369 use of magnetic DES or NADES as extracting solvents with the purpose to simplify the
370 process and improve yields. In general, UAME involves lower volumes of DES (less
371 than 500 µL) and shorter times of extraction (less than 15 min), than the previous
372 mentioned LPME techniques. Like LPME, UAME also did not allow automation of the

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373 extraction process.

374 Cvjetko Bubalo et al. [42] used a ChCl-based DES containing oxalic acid as a

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375 hydrogen bond donor with 25% of water, for the extraction of grape skin phenolic
376 compounds. To increase extraction yield, samples were sonicated in a water bath

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377 during 50 min at 65 °C. According to the authors, t he extraction efficiency obtained by
378 UAME was higher than those obtained with microwave-assisted extraction or

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379 conventional extraction methods. AN
380 Recently, Zhuang et al. [43] combined DES-UAME with HPLC-UV for the simultaneous
381 determination of flavonoid glycosides and respective aglycones in Platycladi Cacumen.
382 In the optimized conditions, 1 ml of DES (ChCl- laevulinic acid with 75% of water) was
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383 mixed with 25 mg of sample during 30 min at 50°C un der an ultrasound bath (200 W,
384 40 kHz); the extract was further diluted 8 times with acetonitrile before HPLC-UV
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385 analysis. Additionally, the authors evaluated the possibility to recover the target
386 analytes from the DES extracts, in order to enable their further application in
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387 pharmaceutical or food industry. For that purpose, several macroporous resins were
388 evaluated, being the best results for both flavonoid glycosides and aglycones obtained
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389 with LX-38.

390 Khezeli et al. [44] have reported a simple and highly reproducible UAME method for the
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391 determination of ferulic, caffeic and cinnamic acids in olive, almond, sesame, and
392 cinnamon oil samples, using ChCl and ethylene glycol (1:2, molar ratio) as DES
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393 extracting solvent. The solvent was added to the sample dissolved in n-hexane, then
394 the mixture was placed in an ultrasound bath for 5 min, to form an emulsion of
395 microdroplets, and consequently increase the contact surface between DES and
396 sample. Once completed the extraction, the DES phase containing the analytes was
397 separated by centrifugation, and submitted to HPLC-UV analysis. The results indicated
398 that the novel approach have shown the advantages of good sensitivity (limits of
399 detection between 0.39 and 0.63 µg/L), reproducibility (RSD <5.1%), convenience
400 (extraction time less than 15 min), and high accuracy (recoveries from 95-105%). Tan

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401 et al. [45] employed also UAME-DES followed by HPLC/UV for determination of plant
402 growth regulators from several vegetable oil samples, using as DES a
403 tetramethylammonium chloride–ethylene glycol mixture (1:3 molar ratio). When
404 compared with other techniques such as SDME, SPME and S-SIL-based MSPD the
405 UAME-DES proposed in the works above referred are more sensible, fast and simple
406 of execution [39, 40].

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407 The applicability of hydrophobic DES for extraction of analytes dissolved in raw waters
408 such as UV-filters have been evaluated by Wang et al [46]. After optimization of diverse
409 parameters (for examples sample volume, salt addition, sample pH, ultrasonic time) the

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410 authors proposed the use of a mixture of trioctylmethylammonium chloride with
411 decanoic acid (1:3 molar ratio) as DES, using sonication for 5 min in an ultrasonic bath

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412 (150 W; 40 kHz) for pre-concentration and separation of three benzophenones. This
413 method has the great advantage of enabling the extraction and purification in one step

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414 without the use of toxic solvents. AN
415 The use of a magnetic deep eutectic solvent (MDES) formed by the mixture of ChCl
416 with phenol and anhydrous FeCl3 (1:2:1 molar ratio) was recently proposed by Khezeli
417 et al. [47] for the extraction of thiophene from a heptane solution. The MDES was
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418 dispersed into the solution by ultrasounds during 5 min, in order to enhance the mass
419 transfer of thiophene from n-heptane to MDES phase. After that, microdroplets of
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420 MDES were collected by a magnet and the remained concentration of thiophene in n-
421 heptane was analyzed by GC-FID. The method provided very good results in the
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422 elimination of thiophene from the initial solution (close to 100%), with the great
423 advantage of eliminating the centrifugation step usually required in any LPME
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424 extraction. According to the authors, the developed MDES are able to be reused after
425 four runs without losses of extraction efficiency.
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426 A novel UAME application based on DES, named by the authors as emulsification
427 liquid-liquid microextraction (ELLME-DES) was introduced by the group of Khezeli [48]
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428 for the simultaneous extraction of BTE (benzene, toluene, and ethylbenzene) and
429 seven polycyclic aromatic hydrocarbons from water samples. Here, the ultrasound
430 helps both the mass transfer between phases and the formation of tiny emulsified
431 droplets, increasing the contact area. Other novelty was the use of a water miscible
432 aprotic solvent, THF for instance, able to decrease the tendency of water molecules to
433 interact with DES and thus promoting the self-aggregation of DES microdroplets.
434 Briefly, 100 µL of DES (mixture of ChCl:phenol in 1:2, 1:3, and 1:4 molar ratios) were
435 added to 1.5 mL of sample and a homogeneous solution was formed immediately. The

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436 further injection of 100 µL of THF followed by sonication for 20 min in an ultrasonic bath
437 provided a turbid state in which DES microdroplets are entirely disperse along all the
438 water phase, so enhancing the transference rate of the compounds from the water to
439 the DES phase. After a centrifugation step (10 min at 3000 rpm) the DES upper phase
440 was withdrawn through a micro-syringe and analyzed by HPLC/UV. Very good results
441 were obtained in what concerns linearity, precision, and accuracy. These approach
442 could be very useful for the multi-analysis of hydrocarbons in environmental water

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443 samples, with the advantages of simplicity, low cost, and high sensitivity providing by
444 the high enrichment factor of the extraction procedure, while avoiding the use of

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445 chlorinated solvents.

446 Similar approach was used by Aydin et al. [49] for the extraction of malachite green

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447 from farmed and ornamental aquarium fish water samples followed by a simple UV–
448 VIS spectrometry analysis. The developed method presented good extraction

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449 recoveries (≥95%) and high precision (RSD < 4% for real samples) for evaluation of
450 this anti-parasite which its illegally used in the aquaculture industry to prevent fish
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451 diseases caused by external parasites and fungus. Moreover, the method showed a
452 good selectivity for malachite green along with other advantages such as simplicity, low
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453 cost, rapid separation and ease of operation. The same authors proposed a ELLME-
454 DES combined vortex for separation and pre-concentration of curcumin in herbal tea
455 samples followed by UV-Vis determination using 400 µL of DES (ChCl: phenol, molar
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456 ratio 1:4) as extracting solvent, 400 µL of THF as emulsifier agent and 2 min of
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457 ultrasonication to achieve the best extraction[50].

458 The same principles were at the base of the application of DES as extracting solvent
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459 on UAME procedures for extraction of inorganic compounds, namely cobalt (II) and
460 chromium (III/VI) ions from aqueous matrices, performed by the group of Soylak [51]
461 [52]. In both works, the metals were previously complexed by adding as ligands 1-
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462 nitroso-2-naphthol and sodium diethyldithiocarbamate, respectively, allowing the

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463 formation of very stable and hydrophobic complex and consequently promoting the
464 extraction efficiency. The DES solvents were mixtures of ChCl with phenol (4:1, and
465 3:1 molar ratios, respectively) and THF was added to allow the self-aggregation and full
466 separation of the DES molecules from the water phase. Following sonication for 2 min
467 and centrifugation, the authors were able to recollect a DES aggregate containing the
468 complexed metals, suitable for quantification by AAS. Several parameters affecting the
469 extraction performance of the analytes were optimized, namely sample amount, pH,
470 type and volume of DES, of complexing agent, and of emulsifier solvent, and
471 ultrasonication time. The results obtained under the optimized conditions are

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472 comparable or even better to those reported in literature for the same analytes, apart
473 from presenting a high enrichment factor and consequently low detection limits.

474 A comparable approach to those previously described was used by Panhwar et al. in
475 two works aiming the pre-concentration of inorganic compounds from water and food
476 samples, namely hydrophobic chelates of Se(IV) with 3,3′-diaminobenzidine [53] and Al
477 (III) with 8-hydroxyquinoline [54] before ETAAS analysis. In both works mixtures of

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478 ChCl:phenol were used as extractors with a molar ratio of 1:3 and 1:4, respectively,
479 and THF was the emulsifier agent. The authors compared the proposed methods with
480 other ETAAS procedures previously used, in which the pre-concentration was done

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481 with LLE, SPE or conventional DLLME, and found no significant differences between
482 the performance data achieved, with the advantages already indicated that

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483 characterize the use of DES.

484 The use of NADES in UAME is been increasing in last years as mentioned for other

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485 microextraction techniques. Lores et al. [55] used a fructose-citric acid NADES mixture
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486 in combination with UAE for fast and green gluten determination by ELISA. The
487 NADES-UAME developed allows the replacement of the ethanol-water solution
488 commonly used for gluten extraction with success, once good recoveries (62–135%)
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489 and high precision RSD < 15%) were obtained. In another work, Bajkacz and Adamek
490 [56] used NADES combined with UAME for the isolation of isoflavones (daidzin,
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491 genistin, genistein, daidzein) from soy products followed by UPLC-UV analysis. The
492 best results were obtained using a mixture of ChCl:citric acid (1:1 molar ratio) with a
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493 water content of 30%, and a ratio of NADES volume to sample amount of 3 The
494 extraction time was 60 min at 60ºC and the ultrasonic power was 616 W. Bosiljkov et
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495 al. [57] used chemometric tools, namely a response surface methodology to optimize
496 NADES-UAME/HPLC-DAD method for the quantification of anthocyanins in wine lees.
497 The maximum amount of extracted compounds were achieved using ChCl:malic acid
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498 (1:1 molar ratio) with 35.4% (w/w) of water in a ultrasound bath at 341.5 W during 30.6
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499 min. Recently, Huang et al [58] used a NADES-UAME/HPLC-UV method for the
500 extraction of rutin from from tartary buckwheat hull. The authors proved that the
501 solubility of rutin increased by 660–1577 times in ChCL-glycerol-based NADES when
502 compared to water.

503 The increasing number of works using DES and NADES in UAME, in recent years
504 make us believe that this novel class of extracting solvents can be used in removal and
505 extraction of a wide range of analyte in environmental and food matrices. Despite all
506 the advantages, the majority of the DES extracting solvents used in UAME described

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507 procedures requires the use of HPLC for organic compounds, or ETAAS and AAS for
508 inorganic compounds, as quantitative final technique, due to the incompatibility with GC
509 systems due to DES low volatility.

510

511 6. MICROWAVE-ASSISTED EXTRACTION

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512 MAE employs non-ionized electromagnetic irradiation (in a frequency range of 0.3-300
513 GHz) to heat both solvent and samples by movements of ions and rotation of molecular
514 and atomic dipoles, in order to increase extraction kinetics [59].

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515 MAE may be performed in closed vessels under pressure (pressurized MAE) or in

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516 open vessels at atmospheric pressure (focused MAE). The first ones are most used in
517 food analysis since usually provide enhanced extraction yields. The extraction
518 efficiency is dependent on the solvent (nature and solvent/sample ratio), temperature

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519 and pressure, extraction time, radiation power, sample composition (moisture mainly),
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520 and particle size (preferably 0.1 – 2 mm)[41, 59].

521 MAE using DES as extracting solvent was employed by Chen et al. [60] to extract 5
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522 active ingredients namely rosmarinic acid, lithospermic acid, salvionalic acid B,
523 salvionalic acid A, and tanshinone II A from Radix Salviae miltiorrhizae followed by
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524 HPLC-VWD quantification. Among twenty-five DES tested, the authors concluded that
525 ChCl-1,2-propanediol (1:1, molar ratio) showed the best yields. Other extraction
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526 factors, including temperature, time, power of microwave, and solid/liquid ratio, were
527 systematically investigated by response surface methodology. The hydrophilic and
528 hydrophobic compounds were extracted simultaneously under the optimized
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529 conditions: 20 vol% of water in ChCl-1,2-propanediol as solvent, microwave power of

530 800 W, temperature at 70°C, time at 11.11 min, and solid/liquid ratio of 0.007 g/ml. The
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531 proposed DES combined with the MAE provided a prominent advantage for fast and
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532 efficient extraction of active compounds compared with conventional procedures.

533 Moreover, in comparison to the techniques previously used, MAE allows the
534 automation of the extraction (40 samples per batch). The main limitations of MAE
535 based techniques are the higher costs of the equipment and the usual presence on the
536 extracts of many interfering compounds due to the exhaustive extraction process,
537 which requires a cleanup step prior to the analysis.
538
539 7. OTHER APPLICATIONS OF DES/NADES IN EXTRACTION TECHNIQUES

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540 DES have been also demonstrated to be an interesting alternative as reaction solvents
541 in the production of new sorbents [61] or polymeric phases [62] used in solid-phase
542 extractions (SPE), which enhance the range of possible DES applications in
543 environmentally friendly extraction procedures. Another application is using DES as
544 dissolution solvent [63] as a new alternative to the conventional Soxhlet extraction.
545
546 7.1. DES as solvents on liquid-liquid extraction

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547 Guo et al. [64] used a DES based on tetrahylammonium chloride and phenol (0.8:1,
548 molar ratio) to extract phenol (99.9%) from model oil (toluene). The extraction was

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549 achieved by mixing directly the DES with the matrix by 30 min at room temperature,
550 avoiding the use of alkali and acids and the production of phenol containing waste

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551 water, common in the traditional methods. The same group previously has verified that
552 the addition of ChCl allowed a formation of DES due to the presence of phenols
553 (phenol, cresols) in model oils (hexane, toluene, and p-xylene) [65]. The extraction of

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554 phenolic compounds from virgin olive oil using DES as extracting solvent was
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555 evaluated by García et al. [66]. The best results for the two most abundant secoiridoid
556 derivatives (oleacein and oleocanthal) were obtained with ChCl: xylitol (2:1, molar ratio)
557 and chlorine chloride:1,2 propanediol (1:1, molar ratio) as DES solvents. The better
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558 conditions were achieved by mixing the extractive solvent directly with the samples
559 inside a bath at 40°C with agitation for 1 h. Excel lent extraction yields were obtained
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560 with the tested DES compared with the commonly used mixture of methanol/water.
561 Despite all the advantages due to the use of DES such as low cost and low toxicity, the
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562 extraction time was not reduced.

563 The group of Ghanemi [67] for determination of PAHs by HPLC-UV used DES (ChCl–
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564 Ox, 1:2 molar ratio) at 55°C for 30 min to dissolve fish samples. After dissolution, PAHs
565 were quantitatively extracted from ChCl-Ox solution with 5 ml of cyclohexane and
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566 stirring for 20 min [67]. These procedures provide some operational advantages when
567 compared with the conventional Soxhlet extraction such as simplicity, low-cost and
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568 eco-sustainability of the eutectic solvent, relatively high-speed sample preparation and
569 low consumption of both sample and organic solvents. Similar advantages were also
570 described by Bi et al. [68] who applied a mixture of ChCl and 1,4-butanediol (1:5 molar
571 ratio) with 35% of water at 70°C for 40 min for the extraction of antioxidant flavonoids
572 (myricetin and mentoflavone) from leave plants, using a sample/volume ratio of 1 g/10
573 ml.

574
575 7.2. DES as sorbent on solid-phase extraction

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576 Solid-phase extraction (SPE) is one of the most popular extraction techniques, making
577 use of a wide range of supporting packings commercially available with different
578 sorbents. Depending on the nature of the sorbent, a number of mechanisms of solute
579 retention can be assumed to rule the process. Partitioning of analytes between a liquid
580 solution (sample matrix or extract) and a viscous liquid that is immobilized on a solid
581 support (sorbent phase) is the most usual retention mechanism for SPE process.
582 Liquid/solid adsorption as well as ion exchange or size exclusion are also possible

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583 mechanisms in various separations [69]. SPE extraction is a safe, efficient and
584 reproducible separation technique that can be automatized.

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585
586 Wang et al. [61] employed DES (ChCl and urea, 1:2 molar ratio) as reaction solvent to

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587 prepare a new nitro-substituted tris(indolyl)methane modified silica phase. This new
588 sorbent was used in the extraction of organic acids (benzoic, p-anisic, salicylic and
589 cinnamic acids) from grape juice and mineralized drinking water followed by HPLC-

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590 DAD analysis. Owing the multiple intermolecular forces of the new phase, such as π–
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591 π, hydrogen bonding and hydrophobic interactions, good extraction efficiency
592 (recoveries >68%) was achieved for the selected organic acids. Another important
593 advantage of this SPE sorbent is the possibility to be reused, which decrease the cost
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594 of the analysis.

595
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596 Liu et al. [70] used a DES (ChCl: ethylene glycol) to modify graphene. Compared with
597 conventional graphene, DES-graphene had a large winkle and formed a structure with
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598 a high specific surface area because linked groups were introduced into the parent
599 graphene structure what give it a higher adsorption ability. Two milligrams of DES-
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600 graphene were employed to pipette-tip SPE of sulfamerazine from river waters followed
601 by HPLC analysis. The optimization of PT-SPE included the evaluation of volume of
602 washing and elution solvent. Under the optimum conditions this method showed good
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603 recoveries (91-97%) and higher precision intraday (RSD range from 1.6 to 3.5%) and
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604 interday (RSD range from 0.7-3.8%).

605
606 7.3. DES as dissolution solvent

607 DES have been used by the group of Ghanemi [63] for the dissolution of marine
608 biological samples, which facilitated the quantitative extraction of some metals under
609 study with small volumes of dilute nitric acid for determination by FAAS. Briefly, in this
610 method, 100 mg of the sample were dissolved in ChCl–Ox (1:2, molar ratio) at 100 °C
611 for 45 min. Then, after addition of 5.0 mL HNO3 (1.0 M) the mixture was centrifuged

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612 and the supernatant analyzed. This procedure provided some clear advantage when
613 compared with other techniques (microwave or ultrasonic acid digestion) such as low-
614 cost and simplicity of the sample preparation while avoiding the formation of highly
615 carcinogenic nitrous vapors.

616

617 7.4. DES as carbon paste electrode

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618 Carbon paste prepared through a mixture of carbon (graphite) powder and a binder
619 (pasting liquid), has been used in the preparation of various electrodes, sensors, and

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620 detectors [65]. Currently, CPEs represent a popular type of electrode, because carbon
621 pastes are easily obtainable at minimal costs and can be modified simply to obtain

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622 quantitatively new sensors with the desired properties. The basic requirements for a
623 pasting liquid are its practical insolubility in the solution under measurement, a low

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624 vapour pressure to ensure both mechanical stability and long lifetime, and further, in
625 the case of voltammetric and amperometric applications, its electrochemical inactivity
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626 in the potential window of interest [71].

627 Zhu et al. [62] used a NADES composed of ChCl and urea (2:1 molar ratio) mixed with
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628 graphite power to prepare a CPE, which was used to directly extract DEHP from
629 polymer films. The microextraction process follows an exponential association function
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630 with the apparent first order rate constant of 6.35×10-4 s-1.
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631

632 7.5. DES as modified molecular imprinted polymers (MIP)

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633 MIPs are crosslinked polymers containing specific recognition sites (involved in various
634 types of interactions- covalent, non-covalent and semi-covalent) with a predetermined
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635 selectivity for a target analyte. This technique provides several advantages, such as
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636 high selectivity and great stability to heating and pH shifts when compared with SPE
637 and LLE. MIPs have been commonly employed in the preconcentration of analytes,
638 acting as the selective adsorbent of SPE [72]. However, MIP’s preparation can pose
639 some problems, like inconsistent molecular recognition, polymer swelling in
640 unfavorable solvents, slow binding kinetics, and potential sample contamination by
641 template bleeding [72].

642 Li et al. [73] compared the efficiency of a DES (synthesized with ChCl and glycerol, 1:2
643 molar ratio) modified molecular imprinted polymer a DES modified non-imprinted
644 polymers (without template), a MIP (without DES) and a NIP (without DES and without
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645 template) for the purification of chlorogenic acid from honeysuckles. The extract of
646 honeysuckles was previous obtained using ultrasound (20 min), ethanol at 60% and a
647 ratio of liquid to material (15 ml/g). Among the polymers proposed the DES-MIPs used
648 as SPE showed the highest adsorption capacity owing to its specific imprinted
649 recognition and the DES improved affinity, selectivity and adsorption in purification.

650 A similar approach was applied by the same group more recently using DES formed by

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651 ChCl and glycerol (1:2 molar ratio) to synthetize a novel MIP/SPE. The application was
652 successfully applied in the purification of chloromycetin and thiamphenicol from milk
653 [74].

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654 8. COMBINATION OF ANALYTICAL TECHNOLOGIES WITH

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655 MICROEXTRACTION TECHNIQUES

656 A broad range of analytical extractive and microextractive techniques have been

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657 developed using DES/NADES as extracting solvents for many quantitative analytical
658 purposes such as ETAAS determination of inorganic compounds, and HPLC and GC
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659 determination of organic compounds. In the majority of the cases, when DES/NADES
660 are employed as extracting solvent in various DLLME, UAME, MAE and other
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661 microextractions procedures, HPLC is preferred to GC as final quantitative technique

662 since the low volatility of the DES/NADES hinder the GC analysis. Nevertheless, Tang
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663 et al. [28], Farajzadeh et al. [33], Lamei et al.[34] and Khezeli et al. [47] have
664 successfully employed GC-FID for quantification of several organic compounds using
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665 the direct injection of DES extract. The possible contamination of the GC system
666 namely column and injector due to insufficient volatilization of DES extracts was not
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667 mentioned in these works. Therefore, the advantages of the use of DES with
668 microextraction techniques, avoiding the toxicity associated with the use of organic
669 solvents, are added to the analytical possibilities of GC. Furthermore, DES as extractor
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670 possess an extensive range of polarity/volatility which can be useful in many pre-
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671 concentration and separation processes.

672

673 9. OUTLOOK OF DES/NADES SOLVENTS WITHIN GREEN ANALYTICAL

674 CHEMISTRY

675 According to the twelve principles that govern the concept of Green Chemistry
676 proposed by Anastas and Warner[1], analytical methods should reduce or eliminate
677 hazardous substances used in or generated by a method. The use of DES/ NADES as

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678 extracting solvents in sample preparation techniques fits perfectly with this approach
679 due to their low toxicity and high biodegradability. Additionally, strategies that combine
680 the use of low volumes of these solvents such as LPME, MAE and UAME with green
681 analytical techniques like thermogravimetric, electrochemical or immunoassays
682 analysis [75, 76] are regarded as very interesting green analytical approaches. Despite
683 most of the described sample preparation methodologies presented in the previous
684 sections can be fully considered as environmental-friendly, especially due to low green

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685 solvent consumption, the further use of a chromatographic analytical determination
686 commonly associated increases energy costs of the analysis which is somehow a

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687 drawback for the analytical method greenness (Table 3). According the existing green
688 analytical evaluation tools (National Environmental Methods Index and Eco-scale) [75,

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689 76] the most energy-consuming techniques are NMR, GC-MS, LC-MS, and X-ray
690 diffractometry as opposed to immunoassay, spectroscopy, and electrochemical
691 techniques that are the more energy saved. Additional penalty points on the scales of

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692 assessment of analytical methods greenness comes from the requirements of
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693 calibration and validation of the methods, since the use of calibration solutions, internal
694 and external standards, standard reference materials or isotope dilution enhance
695 reagents consumption and waste generation [76]. In summary, ideal green analytical
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696 methods will be those that eliminate or minimize the use of hazardous solvents, reduce
697 energy use and generate minute quantities of wastes. Therefore, clear efforts are still
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698 needed to be made to encourage the development of sustainable analytical methods

699 based on the use of DES and NADES solvents. Information regarding the performance
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700 of (micro)extraction techniques based on application of DES/NADES are highlighted in

701 Table 4.
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702 10. CONCLUSIONS AND FUTURE PERSPECTIVES

703 In sample preparation, the choice of the right extracting solvent is essential to achieve
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704 a near-complete extraction of the analytes of interest, simultaneously with minimizing

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705 the amount of interferents. For this purpose, a new class of alternative and
706 environmental-friendly DES and NADES solvents have been employed in several novel
707 microextraction techniques such as LPME, UAME and MAE as well as in more
708 conventional extraction procedures like SPE. Indeed, by replacing conventional
709 solvents with DES the main merits of microextraction techniques such as simplicity of
710 operation, low cost, and environmental safety were enhanced. Additionally, the
711 selective and sensitive achieved by the new analytical methods designed for food and
712 environmental analysis was often better than those obtained by conventional extraction
713 techniques.

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714 There is no doubt that the application of DES and NADES solvents in food and
715 environmental analysis will grow in a near future. Forthcoming studies and
716 developments utilizing DES may focus on the following areas; (I) synthesis of new DES
717 and NADES solvents with different polarities and their exploitation in the development
718 of novel extraction techniques for multi-analyte food and environmental analyses; (II)
719 enhancement of the performance and selectivity of the several microextraction
720 techniques to help the execution of the extraction and separation procedure in the

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721 analytical laboratories; and (III) employment of new DES and NADES as extracting
722 solvents, sorbents or selective binding agents for the trace analysis of contaminants in

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723 both food and environmental samples.

724

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725 ACKNOWLEDGMENTS

726 Sara C. Cunha and José Fernandes thanks REQUIMTE, FCT (Fundação para a
727 Ciência e a Tecnologia) and FEDER through the project UID/QUI/50006/2013 –
728 POCI/01/0145/FEDER/007265 with financial support from FCT/MEC through national
729 funds and co-financed by FEDER, under the Partnership Agreement PT2020. Sara C.
730 Cunha acknowledges FCT for the IF/01616/2015 contract.

731

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732 References

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758 liquids?, Green Chemistry, 14 (2012) 2969.
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759 [11] D.J.G.P. van Osch, L.F. Zubeir, A. van den Bruinhorst, M.A.A. Rocha, M.C. Kroon,
760 Hydrophobic deep eutectic solvents as water-immiscible extractants, Green Chem., 17 (2015)
AC

761 4518-4521.
762 [12] B.D. Ribeiro, C. Florindo, L.C. Iff, M.A.Z. Coelho, I.M. Marrucho, Menthol-based Eutectic
763 Mixtures: Hydrophobic Low Viscosity Solvents, ACS Sustainable Chemistry & Engineering, 3
764 (2015) 2469-2477.
765 [13] G. Imperato, E. Eibler, J. Niedermaier, B. Konig, Low-melting sugar-urea-salt mixtures as
766 solvents for Diels-Alder reactions, Chem Commun (Camb), DOI 10.1039/b414515a(2005) 1170-
767 1172.
768 [14] M.C. Gutierrez, M.L. Ferrer, C.R. Mateo, F. del Monte, Freeze-drying of aqueous solutions
769 of deep eutectic solvents: a suitable approach to deep eutectic suspensions of self-assembled
770 structures, Langmuir, 25 (2009) 5509-5515.

25
 ACCEPTED MANUSCRIPT
771 [15] H.D.W. Jonathan G. Huddleston, Richard P. Swatloski, Ann E. Visser and Robin D. Rogers
772 Room temperature ionic liquids as novel media for ‘clean’ liquid–liquid extraction, Chem.
773 Commun., DOI 10.1039/A803999B( 1998) 1765-1766
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775 Monatshefte für Chemie - Chemical Monthly, 144 (2013) 1427-1454.
776 [17] B. Tang, H. Zhang, K.H. Row, Application of deep eutectic solvents in the extraction and
777 separation of target compounds from various samples, J Sep Sci, 38 (2015) 1053-1064.
778 [18] A.P. Abbott, R.C. Harris, K.S. Ryder, C. D'Agostino, L.F. Gladden, M.D. Mantle, Glycerol
779 eutectics as sustainable solvent systems, Green Chem., 13 (2011) 82-90.

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780 [19] A.R.R. Teles, E.V. Capela, R.S. Carmo, J.A.P. Coutinho, A.J.D. Silvestre, M.G. Freire,
781 Solvatochromic parameters of deep eutectic solvents formed by ammonium-based salts and
782 carboxylic acids, Fluid Phase Equilibria, 448 (2017) 15-21.
783 [20] Y. Dai, J. van Spronsen, G.J. Witkamp, R. Verpoorte, Y.H. Choi, Natural deep eutectic

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784 solvents as new potential media for green technology, Anal Chim Acta, 766 (2013) 61-68.
785 [21] I. Mamajanov, A.E. Engelhart, H.D. Bean, N.V. Hud, DNA and RNA in anhydrous media:
786 duplex, triplex, and G-quadruplex secondary structures in a deep eutectic solvent, Angew

SC
787 Chem Int Ed Engl, 49 (2010) 6310-6314.
788 [22] F. Cardellini, M. Tiecco, R. Germani, G. Cardinali, L. Corte, L. Roscini, N. Spreti, Novel
789 zwitterionic deep eutectic solvents from trimethylglycine and carboxylic acids: characterization
790 of their properties and their toxicity, RSC Adv., 4 (2014) 55990-56002.

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791 [23] H. Zhao, G.A. Baker, S. Holmes, New eutectic ionic liquids for lipase activation and
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792 enzymatic preparation of biodiesel, Org Biomol Chem, 9 (2011) 1908-1916.
793 [24] M.a.C. Gutiérrez, F. Rubio, F. del Monte, Resorcinol-Formaldehyde Polycondensation in
794 Deep Eutectic Solvents for the Preparation of Carbons and Carbon−Carbon Nanotube
795 Composites, Chemistry of Materials, 22 (2010) 2711-2719.
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796 [25] A. Pandey, R. Rai, M. Pal, S. Pandey, How polar are choline chloride-based deep eutectic
797 solvents?, Phys Chem Chem Phys, 16 (2014) 1559-1568.
798 [26] M. Ghambarian, Y. Yamini, A. Esrafili, Developments in hollow fiber based liquid-phase
D

799 microextraction: principles and applications, Microchimica Acta, 177 (2012) 271-294.
800 [27] J.O.F.a.M.B.P.P.O. Sara C. Cunha, Current Trends in Liquid-Liquid Microextraction for
TE

801 Analysis of Pesticide Residues in Food and Water, in: M. Stoytcheva (Ed.) "Pesticides -
802 Strategies for Pesticides Analysis", https://www.intechopen.com, 2011, pp. 27.
803 [28] B. Tang, W. Bi, H. Zhang, K.H. Row, Deep Eutectic Solvent-Based HS-SME Coupled with GC
804 for the Analysis of Bioactive Terpenoids in Chamaecyparis obtusa Leaves, Chromatographia, 77
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805 (2013) 373-377.

806 [29] T. Gu, M. Zhang, T. Tan, J. Chen, Z. Li, Q. Zhang, H. Qiu, Deep eutectic solvents as novel
807 extraction media for phenolic compounds from model oil, Chem Commun (Camb), 50 (2014)
808 11749-11752.
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809 [30] M.M. Khataei, Y. Yamini, A. Nazaripour, M. Karimi, Novel generation of deep eutectic
810 solvent as an acceptor phase in three-phase hollow fiber liquid phase microextraction for
AC

811 extraction and preconcentration of steroidal hormones from biological fluids, Talanta, 178
812 (2018) 473-480.
813 [31] S.M. Yousefi, F. Shemirani, S.A. Ghorbanian, Enhanced headspace single drop
814 microextraction method using deep eutectic solvent based magnetic bucky gels: Application to
815 the determination of volatile aromatic hydrocarbons in water and urine samples, J Sep Sci, DOI
816 10.1002/jssc.201700807(2017).
817 [32] V. Ferrone, S. Genovese, M. Carlucci, M. Tiecco, R. Germani, F. Preziuso, F. Epifano, G.
818 Carlucci, V.A. Taddeo, A green deep eutectic solvent dispersive liquid-liquid micro-extraction
819 (DES-DLLME) for the UHPLC-PDA determination of oxyprenylated phenylpropanoids in olive,
820 soy, peanuts, corn, and sunflower oil, Food Chem, 245 (2018) 578-585.
821 [33] M.A. Farajzadeh, M. Sattari Dabbagh, A. Yadeghari, Deep eutectic solvent based gas-
822 assisted dispersive liquid-phase microextraction combined with gas chromatography and flame

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823 ionization detection for the determination of some pesticide residues in fruit and vegetable
824 samples, J Sep Sci, DOI 10.1002/jssc.201700052(2017).
825 [34] N. Lamei, M. Ezoddin, K. Abdi, Air assisted emulsification liquid-liquid microextraction
826 based on deep eutectic solvent for preconcentration of methadone in water and biological
827 samples, Talanta, 165 (2017) 176-181.
828 [35] D. Ge, Y. Zhang, Y. Dai, S. Yang, Air-assisted dispersive liquid-liquid microextraction based
829 on a new hydrophobic deep eutectic solvent for the preconcentration of benzophenone-type
830 UV filters from aqueous samples, J Sep Sci, DOI 10.1002/jssc.201701282(2017).
831 [36] H. Zeng, K. Qiao, X. Li, M. Yang, S. Zhang, R. Lu, J. Li, H. Gao, W. Zhou, Dispersive liquid-

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832 liquid microextraction based on the solidification of deep eutectic solvent for the
833 determination of benzoylureas in environmental water samples, J Sep Sci, 40 (2017) 4563-
834 4570.
835 [37] Y. Dai, J. van Spronsen, G.J. Witkamp, R. Verpoorte, Y.H. Choi, Ionic liquids and deep

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836 eutectic solvents in natural products research: mixtures of solids as extraction solvents, J Nat
837 Prod, 76 (2013) 2162-2173.
838 [38] Y. Dai, R. Verpoorte, Y.H. Choi, Natural deep eutectic solvents providing enhanced stability

SC
839 of natural colorants from safflower (Carthamus tinctorius), Food Chem, 159 (2014) 116-121.
840 [39] Y. Dai, E. Rozema, R. Verpoorte, Y.H. Choi, Application of natural deep eutectic solvents to
841 the extraction of anthocyanins from Catharanthus roseus with high extractability and stability
842 replacing conventional organic solvents, J Chromatogr A, 1434 (2016) 50-56.

U
843 [40] H.M. Santos, Lodeiro, C. and Capelo-Martínez, J.-L. , The Power of Ultrasound, in
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844 Ultrasound in Chemistry: Analytical Application, Wiley-VCH Verlag GmbH & Co. KGaA,
845 Weinheim, Germany2008.
846 [41] R. Cruz, S.C. Cunha, A. Marques, S. Casal, Polybrominated diphenyl ethers and metabolites
847 – An analytical review on seafood occurrence, TrAC Trends in Analytical Chemistry, 87 (2017)
M

848 129-144.
849 [42] M. Cvjetko Bubalo, N. Curko, M. Tomasevic, K. Kovacevic Ganic, I. Radojcic Redovnikovic,
850 Green extraction of grape skin phenolics by using deep eutectic solvents, Food Chem, 200
D

851 (2016) 159-166.

852 [43] B. Zhuang, L.-L. Dou, P. Li, E.H. Liu, Deep eutectic solvents as green media for extraction of
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853 flavonoid glycosides and aglycones from Platycladi Cacumen, Journal of Pharmaceutical and
854 Biomedical Analysis, 134 (2017) 214-219.
855 [44] T. Khezeli, A. Daneshfar, R. Sahraei, A green ultrasonic-assisted liquid-liquid
856 microextraction based on deep eutectic solvent for the HPLC-UV determination of ferulic,
EP

857 caffeic and cinnamic acid from olive, almond, sesame and cinnamon oil, Talanta, 150 (2016)
858 577-585.
859 [45] T. Tan, M. Zhang, Y. Wan, H. Qiu, Utilization of deep eutectic solvents as novel mobile
860 phase additives for improving the separation of bioactive quaternary alkaloids, Talanta, 149
C

861 (2016) 85-90.

862 [46] H. Wang, L. Hu, X. Liu, S. Yin, R. Lu, S. Zhang, W. Zhou, H. Gao, Deep eutectic solvent-based
AC

863 ultrasound-assisted dispersive liquid-liquid microextraction coupled with high-performance

864 liquid chromatography for the determination of ultraviolet filters in water samples, J
865 Chromatogr A, 1516 (2017) 1-8.
866 [47] T. Khezeli, A. Daneshfar, Synthesis and application of magnetic deep eutectic solvents:
867 Novel solvents for ultrasound assisted liquid-liquid microextraction of thiophene, Ultrason
868 Sonochem, DOI 10.1016/j.ultsonch.2016.08.023(2016).
869 [48] T. Khezeli, A. Daneshfar, R. Sahraei, Emulsification liquid-liquid microextraction based on
870 deep eutectic solvent: An extraction method for the determination of benzene, toluene,
871 ethylbenzene and seven polycyclic aromatic hydrocarbons from water samples, J Chromatogr
872 A, 1425 (2015) 25-33.

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873 [49] F. Aydin, E. Yilmaz, M. Soylak, A simple and novel deep eutectic solvent based ultrasound-
874 assisted emulsification liquid phase microextraction method for malachite green in farmed and
875 ornamental aquarium fish water samples, Microchemical Journal, 132 (2017) 280-285.
876 [50] F. Aydin, E. Yilmaz, M. Soylak, Vortex assisted deep eutectic solvent (DES)-emulsification
877 liquid-liquid microextraction of trace curcumin in food and herbal tea samples, Food Chem,
878 243 (2018) 442-447.
879 [51] M.B. Arain, E. Yilmaz, M. Soylak, Deep eutectic solvent based ultrasonic assisted liquid
880 phase microextraction for the FAAS determination of cobalt, Journal of Molecular Liquids, 224
881 (2016) 538-543.

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882 [52] E. Yilmaz, M. Soylak, Ultrasound assisted-deep eutectic solvent based on emulsification
883 liquid phase microextraction combined with microsample injection flame atomic absorption
884 spectrometry for valence speciation of chromium(III/VI) in environmental samples, Talanta,
885 160 (2016) 680-685.

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886 [53] A.H. Panhwar, M. Tuzen, T.G. Kazi, Ultrasonic assisted dispersive liquid-liquid
887 microextraction method based on deep eutectic solvent for speciation, preconcentration and
888 determination of selenium species (IV) and (VI) in water and food samples, Talanta, 175 (2017)

SC
889 352-358.
890 [54] A.H. Panhwar, M. Tuzen, T.G. Kazi, Deep eutectic solvent based advance microextraction
891 method for determination of aluminum in water and food samples: Multivariate study,
892 Talanta, 178 (2018) 588-593.

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893 [55] H. Lores, V. Romero, I. Costas, C. Bendicho, I. Lavilla, Natural deep eutectic solvents in
AN
894 combination with ultrasonic energy as a green approach for solubilisation of proteins:
895 application to gluten determination by immunoassay, Talanta, 162 (2017) 453-459.
896 [56] S. Bajkacz, J. Adamek, Evaluation of new natural deep eutectic solvents for the extraction
897 of isoflavones from soy products, Talanta, 168 (2017) 329-335.
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898 [57] T. Bosiljkov, F. Dujmić, M. Cvjetko Bubalo, J. Hribar, R. Vidrih, M. Brnčić, E. Zlatic, I.
899 Radojčić Redovniković, S. Jokić, Natural deep eutectic solvents and ultrasound-assisted
900 extraction: Green approaches for extraction of wine lees anthocyanins, Food and Bioproducts
D

901 Processing, 102 (2017) 195-203.

902 [58] Y. Huang, F. Feng, J. Jiang, Y. Qiao, T. Wu, J. Voglmeir, Z.-G. Chen, Green and efficient
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903 extraction of rutin from tartary buckwheat hull by using natural deep eutectic solvents, Food
904 Chemistry, 221 (2017) 1400-1405.
905 [59] S. Moldoveanu, V. David, Chapter 7 - Solid-Phase Extraction, Modern Sample Preparation
906 for Chromatography, Elsevier, Amsterdam, 2015, pp. 191-286.
EP

907 [60] J. Chen, M. Liu, Q. Wang, H. Du, L. Zhang, Deep Eutectic Solvent-Based Microwave-
908 Assisted Method for Extraction of Hydrophilic and Hydrophobic Components from Radix
909 Salviae miltiorrhizae, Molecules, 21 (2016).
910 [61] N. Wang, J. Wang, Y. Liao, S. Shao, Preparation of a nitro-substituted tris(indolyl)methane
C

911 modified silica in deep eutectic solvents for solid-phase extraction of organic acids, Talanta,
912 151 (2016) 1-7.
AC

913 [62] J.G. X. Zhu, J. Lang, H. Zhang, Y. Zhu, Liquid phase microextarion of Di2 ethylexyl phthalate,
914 International Journal of Innovative Research in Science, Engineering and Technology

915 2(2013) 6622-6628.

916 [63] E. Habibi, K. Ghanemi, M. Fallah-Mehrjardi, A. Dadolahi-Sohrab, A novel digestion method
917 based on a choline chloride–oxalic acid deep eutectic solvent for determining Cu, Fe, and Zn in
918 fish samples, Analytica Chimica Acta, 762 (2013) 61-67.
919 [64] W. Guo, Y. Hou, W. Wu, S. Ren, S. Tian, K.N. Marsh, Separation of phenol from model oils
920 with quaternary ammonium saltsvia forming deep eutectic solvents, Green Chem., 15 (2013)
921 226-229.

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922 [65] I. Švancara, K. Vytřas, K. Kalcher, A. Walcarius, J. Wang, Carbon Paste Electrodes in Facts,
923 Numbers, and Notes: A Review on the Occasion of the 50-Years Jubilee of Carbon Paste in
924 Electrochemistry and Electroanalysis, Electroanalysis, 21 (2009) 7-28.
925 [66] A. García, E. Rodríguez-Juan, G. Rodríguez-Gutiérrez, J.J. Rios, J. Fernández-Bolaños,
926 Extraction of phenolic compounds from virgin olive oil by deep eutectic solvents (DESs), Food
927 Chemistry, 197, Part A (2016) 554-561.
928 [67] Z. Helalat-Nezhad, K. Ghanemi, M. Fallah-Mehrjardi, Dissolution of biological samples in
929 deep eutectic solvents: an approach for extraction of polycyclic aromatic hydrocarbons
930 followed by liquid chromatography-fluorescence detection, J Chromatogr A, 1394 (2015) 46-

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931 53.
932 [68] W. Bi, M. Tian, K.H. Row, Evaluation of alcohol-based deep eutectic solvent in extraction
933 and determination of flavonoids with response surface methodology optimization, J
934 Chromatogr A, 1285 (2013) 22-30.

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935 [69] S.M.a.V. David, Modern Sample Preparation for Chromatography, in: S.D. Moldoveanu, V.
936 (Ed.), 2015, pp. 33-51.
937 [70] L. Liu, W. Tang, B. Tang, D. Han, K.H. Row, T. Zhu, Pipette-tip solid-phase extraction based

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938 on deep eutectic solvent modified graphene for the determination of sulfamerazine in river
939 water, J Sep Sci, 40 (2017) 1887-1895.
940 [71] K. Vytřas, I. Svancara, R. Metelka, Carbon paste electrodes in electroanalytical chemistry,
941 Journal of the Serbian Chemical Society, 74 (2009) 1021-1033.

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942 [72] V.L. Pereira, J.O. Fernandes, S.C. Cunha, Mycotoxins in cereals and related foodstuffs: A
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943 review on occurrence and recent methods of analysis, Trends in Food Science & Technology,
944 36 (2014) 96-136.
945 [73] G. Li, W. Wang, Q. Wang, T. Zhu, Deep Eutectic Solvents Modified Molecular Imprinted
946 Polymers for Optimized Purification of Chlorogenic Acid from Honeysuckle, J Chromatogr Sci,
M

947 54 (2016) 271-279.

948 [74] G. Li, T. Zhu, K.H. Row, Deep eutectic solvents for the purification of chloromycetin and
949 thiamphenicol from milk, J Sep Sci, 40 (2017) 625-634.
D

950 [75] L.U.G. L. H. Keith, J.L.Young, Green Analytical Methodologies, Chem Rev, 107 (2007) 2695-
951 2708.
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952 [76] A. Gałuszka, Z.M. Migaszewski, P. Konieczka, J. Namieśnik, Analytical Eco-Scale for
953 assessing the greenness of analytical procedures, TrAC Trends in Analytical Chemistry, 37
954 (2012) 61-72.
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955 Figure Capitations

956 Figure 1- Time-trend (2006-2017) representation of the DES and NADES application
957 and their distribution in the different Web Science Categories.
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958
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959

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Table 1- Density, viscosity, condutivity and spectroscopic polarity index (ETN)of some DES at
specific temperatures [3-5,16]

Density Viscosity Condutivity

DES (molar ratio) 3 2 ET N
g/cm mm /s (mS cm-1)
Urea:ChCl (2:1) 1.25 (25°C) 750 (25°C) 0.20 (40°C) 0.84
Ethylene Glycol: ChCl (2:1) 1.12 (25°C) 37 (25°C) 7.61 (20°C) 0.8
Glycerol: ChCl (2:1) 1.18 (25°C) 359 (25°C) 1.05 (20°C ) 0.86

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Malonic: ChCl (1:1) 1.25 (25°C) 721 (25°C) 0.55 (25°C) -
1,4- butanediol: ChCl (3:1) 1.06 140 (20°C) 1.64 (20°C ) -
Urea: ethylammonium chloride (1.5:1) 1.041 128 (40 °C ) - -

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Acetamide: ethylammonium chloride (1.5:1) 1.14 64 (40 °C) 0.69 (40 °C) -
2,2,2-trifluoroacetamide: ChCl (2:1) 1.342 77 (40 °C) 0.286 (40 °C) -
Water 0.992 1 250 1

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(-) not found

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Table 2- Density, viscosity, decomposition temperature (Td) and glass transition temperature
(Tg) of some NADES at specific temperatures [4,20]

Density (40°C) Viscosity (40°C) T d/°C T g/°C

NADES (molar ratio) 3 2
g/cm mm /s
Fructose: ChCl: water (2:5:5) 1.2078 280.8 160 -84.58
Glucose: ChCl: water (2:5:5) 1.2069 397.4 170 -83.86
Lactic acid: glucose:water (5:1:3) 1.2497 37 135 -77.06

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Levulinic acid: ChCl (2:1) 1.14 226.8 176.6 -11.9
Malic acid: ChCl: water (1:1:2) 1.2303 445.9 201 -71.32
Sorbitol: ChCl: water (2:5:6) 1.1854 138.4 >200 -89.62

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Sucrose: ChCl: water (1:4:4) 1.2269 581 >200 -82.96
Xylitol: ChCl: water (1:2:3) 1.17841 86.1 >200 -93.33

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Water 0.992 1
Td -decomposition temperature; Tg -glass transition temperature; ENR= hcNA /λmax=28591/λmax

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Table 3- Summary of the application of DES and NADES in extraction techniques from 2006-2017

Sample Extraction
DES - NADES

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Techniques Other features Analyts extracted Instrumental analysis LOQ LOD Ref.
Composition Molar ratio Volume Type amount Time
LPME ChCl -1,4-butanediol 1:5 2.0 ml Leaves (Chamaecyparis 200 mg 40 min Heating 70°C- 30% vol Flavonoids (myricetin, amentoflavone) HPLC-UV Myricetin-0.07 µg/ml; 68
obtusa) water amentoflavone-0.09 µg/ml
LPME ChCl -xylitol 2:1 14 g Olive oil 14 g 1h Heating 40 °C Phenolic compounds (hydroxytyrosol, HPLC-DAD 66
tyrosol, oleacein, oleocanthal, oleuropein

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agylcon,ligstroside aglycon)
DLLME ChCl-urea 1:2 50 µl Water samples (farm 50 ml 1 min 5 mg MMWCNTs, 150 ml Pesticides (alpha-HCH, beta-HCH, GC-ECD alpha-HCB 0.83 ng/l; beta-HCH alpha-HCH 0.25 ng/l; beta-HCH 31
water, rural water, lake acetonitrile, ultrasonic bath gamma-HCH, delta-HCH, heptachlor, 0.20 ng/l; gamma-HCH 0.50 ng/l; 0.06 ng/l; gamma-HCH 0.15 ng/l;
water and river water) aldrin, heptachlor-endo-epoxide, alpha- delta-HCB-0.17 ng/l; heptachlor- delta-HCH- 0.05 ng/l; heptachlor-
endosulfan, dieldrin, 4,4′ –DDE, endrin, 0.89 ng/l; aldrin 0.73 ng/l; 0.27 ng/l; aldrin- 0.22 ng/l;

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betaendosulfan, 4,4′ –DDD, endrin- heptachlor endoepoxide 0.46
aldehyde, endosulfan-sulfate, 4,4′ –DDT, ng/l; alpha-endosulfan 0.13 ng/l;
endrin-ketone and methoxychlor) dieldrin- 0.10 ng/l; 4,4′ -DDE-
0.17 ng/l; endrin- 0.17 ng/l; beta-
endosulfan 0.17 ng/l; 4,4′ -DDD-
heptachlor endoepoxide 0.14 ng/l;
alpha-endosulfan- 0.04 ng/l;
dieldrin- 0.03 ng/l; 4,4′ -DDE 0.05
ng/l; endrin- 0.05 ng/l; beta-
endosulfan 0.05 ng/l; 4,4′ -DDD

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0.17 ng/l; endrin-aldehyde 0.36
ng/l; endosulfan-sulfate 0.43 ng/l;
4,4′ -DDT- 0.73 ng/l; endrin-
ketone- 0.63 ng/l; methoxychlor-
0.05 ng/l; endrin-aldehyde 0.11
ng/l; endosulfan-sulfate 0.13 ng/l;
4,4′ -DDT- 0.22 ng/l; endrin-
ketone- 0.19 ng/l; methoxychlor-

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0.36 ng/l 0.11 ng/l

AG-DLLME ChCl-4-Chlorophenol 1:2 190 µl Fruit and vegetable juice 5 ml 6 min pH 2; water bath at 40°C for Pesticides (Penconazole, hexaconazole, GC-FID Diazinon 4.2 µg/l; penconazole Diazinon 1.4 µg/l; penconazole 33
3 min, ice and NaCl bath diniconazole, tebuconazole, 0.75 µg/l; haloxyfop-R-methyl 1.4 0.25 µg/l; haloxyfop-R-methyl
diazinon, fenazaquin, clodinafop- µg/l; hexaconazole 0.84 µg/l; 0.45 µg/l; hexaconazole 0.28 µg/l;

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propargyl, and haloxyfop- diniconazole 1.3 µg/l; clodinafop- diniconazole 0.43 µg/l; clodinafop-
R-methyl) propargyl 3.9 µg/l; tebuconazole propargyl 1.3 µg/l; tebuconazole
1.9 µg/l; bromopropylate 0.71 0.64 µg/l; bromopropylate 0.24
µg/l; fenazaquin 2.7 µg/l µg/l; fenazaquin 0.90 µg/l

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AAELLME ChCl-5,6,7,8-Tetrahydro-5,5,8,8- 1:2 100 µl Water, urine and plasma Urine added with 3 min pH 10; 100 µl THF; 10 times Methadone GC-FID 0.70 µg/l 2.30 µg/l 34
tetramethylnaphthalen-2-ol NaOH solution (2 pulling and pushing

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mol/l) until pH 10 and
dilluted to 10 ml with
water. The
supernatant of 1 ml
plasma with 0.5 ml
zinc sulfate solution
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(0.7 mol/l) and 0.1 ml
sodium hydroxide
solution (1 mol/l) was
dilluted to 10 ml with
water
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HPLME ChCl-ethylene glycol 1:3 10 µl Gasoline, diesel fuel, 1 ml 3 min Ultrasonic bath Phenolic (phenol, ρ -cresol, β-naphthol) HPLC-UV - phenol-0.025 µg/l; ρ -cresol-0.05 29
kerosene µg/l; β-naphthol
HPLME ChCl-ethylene glycol 1:4 2 µl Leaves of 300 mg 30 min Heating 100 °C ultrasonic Bioactive terpenoids (Linalool, α- GC-FID Linalool- 6.687 ng/ml; α-terpineol- Linalool- 2.006 ng/ml; α-terpineol- 28
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Chamaecyparis obtusa irradion 70W terpineol, terpinyl-acetate) 10.50 ng/ml; terpinylacetate- 3.150 ng/ml; terpinylacetate-
7.099 ng/ml 2.129 ng/ml
LPME Lactic acid–glucose-water 5:1:3 1.5 ml Orange petals of 50 mg 30 min Stirring at 40 °C Anthocyanins UPLC-TOF- M S 39
Catharanthus roseus
LPME Sucrose-ChCl:water 1:4:4 1.5 ml Safflower (Carthamus 100 mg 1h Stirring at 40 °C Phenolic compounds (hydro xysafflor HPLC-DAD 37
tinctorius L. ) Yellow A, cartormin, and carthamin)
LPME Sucrose-ChCl:water 1:4:4 3 ml Safflower (Carthamus 50 mg 30 min Stirring at 40 °C Colorants (carthamin) H PLC-DAD 38
tinctorius L. )
 ACCEPTED MANUSCRIPT

Sample Extraction
Techniques DES - NADES Other features Analyts extracted Instrumental analysis LOQ LOD Ref.
Composition Molar ratio Volume Type amount Time
UAME ChCl-ethylene glycol 1:2 50 µl Olive, almond, sesame 2 ml (1 ml oil -1 ml 5 min Ultrasonic bath Phenolic acids (ferulic, caffeic, and HPLC-UV Ferulic acid -1.30-1.86 µg/l; Ferulic acid -0.39-0.56 µg/l; 44
and Cinnamon oils hexane) cinnamic acids) caffeic acid - 1.43-2.10 µg/l; caffeic acid - 0.43-0.63 µg/l ;

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cinnamic acid 1.60-2.10 µg/l cinnamic acid 0.48-0.63 µg/l

UALME ChCl-phenol 1:4 500 µl Tea Residue resulted of 2 min Ultrasonic bath; pH >6; 1 ml Cobalt (II) FAAS 3.60 µg/l 1.1 µg/l 51
wet digestion (0.5 g) nitroso-2-naphthol; 0.5 ml
added with 10 ml THF
water

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UALME ChCl-phenol 1:3 450 µl Waters (tap, lake, 10 ml 2 min Ultrasonic bath; 0.375 ml Chromium (III/VI) FAAS 18.2 µg/l 5.5 µg/l 52
waste) 0.5M H2SO4; 0.4 ml
0.125% sodium
diethyldithiocarbamate; 0.45

SC
ml THF
UALME ChCl:phenol:FeCl3 1:2:1 25 µl n-heptane 1.5 ml 5 min Ultrasonic bath (35 kHz) Thiophene GC-FID 47
25°C
UALME ChCl-phenol 1:2 100 µl Waters (tap and 1.5 ml 20 min Ultrasonic bath; 100 µl THF Benzene, toluene, ethylbenezene, PAHs HPLC-UV Benzene- 21.0 µg/l; toluene- 22.0 Benzene- 6.2 µg/l; toluene- 6.8 48
industrial wastewater) (biphenyl, fluorene, phenanthrene, µg/l; ethylbenzene-2.90 µg/l; µg/l; ethylbenzene-0.8 µg/l;
anthracene, pyrene, chrysene and biphenyl- 2.30 µg/l; fluorene- 2.20 biphenyl- 0.7 µg/l; fluorene- 0.7

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benzo[a]pyrene ) µg/l; phenanthrene- 0.30 µg/l; µg/l; phenanthrene- 0.09 µg/l;
anthracene- 0.06 µg/l; pyrene- anthracene- 0.02 µg/l; pyrene-
0.26 µg/l; chrysene- 0.70 µg/l; 0.07 µg/l; chrysene- 0.21 µg/l;

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benzo[a ]pyrene- 0.28 µg/l benzo[a ]pyrene- 0.08 µg/l
UALME ChCl-phenol 1:4 500 µl Waters (farmed and 2 ml 3 min Ultrasonic bath; pH =3; 500 Malachite green UV-VIS 11.8 µg/l 3.6 µg/l 49
ornamental aquarium µl THF
fish)
UALME ChCl-Ox 1:1 1 ml Grape skin 100 mg freeze dried 50 min Ultrasonic bath: 65 °C, 35 Phenolics ((+)-Catechin, delphinindin-3- HPLC-UV (+)-Catechin- 1.24 mg/l, (+)-Catechin- 0.37 mg/l, 42

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skin kHz; 25% water O - glucoside, Cyanidin-3-O- glucoside, delphinindin-3-O- glucoside- 0.60 delphinindin-3-O- glucoside- 0.18
petunidin-3-O -glucoside, peonidin-3-O - mg/l, Cyanidin-3-O-glucoside- mg/l, cyanidin-3-O-glucoside-0.21
glucoside, malvidin-3-O -glucoside, 0.71 mg/l, petunidin-3-O- mg/l, petunidin-3-O-glucoside-
quercetin-3-O -glucoside) glucoside- 0.80 mg/l, peonidin-3- 0.24 mg/l, peonidin-3-O-glucoside-
O-glucoside-0.65 mg/l, malvidin- 0.19 mg/l, malvidin-3-O-glucoside

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3-O-glucoside - 0.90 mg/l, - 0.30 mg/l, quercetin-3-O-
quercetin-3-O-glucoside -0.46 glucoside -0.05 mg/l
mg/l

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UALME tetramethylammonium 1:3 30 µl Saflower, olive,camellia, 1 ml (10 % oil - 90% 7 min Ultrasonic bath -50°C Plant growth regulators ( IAA, IBA, 4- HPLC-UV IAA -0.05 µg/ml; IBA -0.06 µg/ml; 45
chloride–ethylene glycol colza and soybean oils hexane) IPOAA) 4-IPOAA 0.75 µg/ml

UALME Choline Chloride-Laevulinic 1:2 1 ml Platycladi Cacumen 25 mg 30 min Ultrasonic bath: 50°C, 200 Flavonoid glycosides and aglycones HPLC-UV 43
Acid W; 75% water (myricitrin, quercitrin, amentoflavone,
EP
hinokiflavone)
UAME Choline chloride-malic acid 1:1 1 ml Wine lees 33.3 mg 30.6 min Ultrasaound bath: 341.5 W, Anthocyanins HPLC-DAD Delphinidin-3-O glucoside- 0.87 Delphinidin-3-O glucoside- 0.28 57
37 kHz, 35°C ; 35.4% water mg/l; cyanidin-3-O glucoside- mg/l; cyanidin-3-O glucoside-
0.52 mg/l; petunidin-3-O 0.17 mg/l; petunidin-3-O
glucoside- 0.44 mg/l; peonidin-3- glucoside- 0.15 mg/l; peonidin-3-
C

O glucoside- 0.62 mg/l; malvidin- O glucoside- 0.2 mg/l; malvidin-3-

3-O glucoside- 0.81 mg/l O glucoside- 0.25 mg/l

UAME Choline chloride-glycerol 1:1 1 ml tartary buckwheat hulls 40 mg 1h Ultrasaound bath: 20 kHz, Flavonoid (rutin) HPLC-UV 58
AC

200 W, 40 °C; 20% water

UAME Fructose-citric acid 1:1 1 ml Raw and processed 25 mg 15 min Ultrasound bath (100 w 42 Gluten Enzyme Linked 55
food (spaghetti, biscuts, kHz) 40% amplitude; 20% ImmunoSorbent Assay
and ham) water
UAME ChCl:citric acid 1.1 600 µl Soy products 200 mg 60 min Ultrasonic bath: 440 W, Isoflavones (GT, DA, GTGL, daidzin UHPC-UV DAGL- 0.40 µg/g; GTGL- 0.45 DAGL- 0.12 µg/g; GTGL- 0.14 56
(soybeans, flour, pasta, 60°C; 30% water DAGL) µg/g; DA- 0.25 µg/g; GT 0.20 µg/g; DA- 0.08 µg/g; GT 0.06
breakfast cereals, µg/g µg/g
cutlets, tripe, soy drink,
soy nuts, soy cubes and
three different dietary
supplements)
MAE ChCl-1,2-propanediol 1:1 10 ml Radix Salviae 50 mg 11.11 min Microwave: 800W, 70°C Bioactive compoun ds (ROS, LIT, SAB, HPLC-DAD ROS- 0.80 µg/ml; LIT- 1.37 ROS- 0.24 µg/ml; LIT- 0.49 µg/ml; 60
miltiorrhizae SAA, TIIA) µg/ml; SAB- 1.96 µg/ml; SAA- SAB- 0.62 µg/ml; SAA- 0.31
0.87 µg/ml; TIIA- 1.45 µg/ml µg/ml; TIIA- 0.48 µg/ml
 ACCEPTED MANUSCRIPT

Sample Extraction
Techniques DES - NADES Other features Analyts extracted Instrumental analysis LOQ LOD Ref.
Composition Molar ratio Volume Type amount Time
UAME ChCl-ethylene glycol 1:2 50 µl Olive, almond, sesame 2 ml (1 ml oil -1 ml 5 min Ultrasonic bath Phenolic acids (ferulic, caffeic, and HPLC-UV Ferulic acid -1.30-1.86 µg/l; Ferulic acid -0.39-0.56 µg/l; 44
and Cinnamon oils hexane) cinnamic acids) caffeic acid - 1.43-2.10 µg/l; caffeic acid - 0.43-0.63 µg/l ;

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cinnamic acid 1.60-2.10 µg/l cinnamic acid 0.48-0.63 µg/l

UALME ChCl-phenol 1:4 500 µl Tea Residue resulted of 2 min Ultrasonic bath; pH >6; 1 ml Cobalt (II) FAAS 3.60 µg/l 1.1 µg/l 51
wet digestion (0.5 g) nitroso-2-naphthol; 0.5 ml
added with 10 ml THF
water

RI
UALME ChCl-phenol 1:3 450 µl Waters (tap, lake, 10 ml 2 min Ultrasonic bath; 0.375 ml Chromium (III/VI) FAAS 18.2 µg/l 5.5 µg/l 52
waste) 0.5M H2 SO4 ; 0.4 ml
0.125% sodium
diethyldithiocarbamate; 0.45

SC
ml THF
UALME ChCl:phenol:FeCl3 1:2:1 25 µl n-heptane 1.5 ml 5 min Ultrasonic bath (35 kHz) Thiophene GC-FID 47
25°C
UALME ChCl-phenol 1:2 100 µl Waters (tap and 1.5 ml 20 min Ultrasonic bath; 100 µl THF Benzene, toluene, ethylbenezene, PAHs HPLC-UV Benzene- 21.0 µg/l; toluene- 22.0 Benzene- 6.2 µg/l; toluene- 6.8 48
industrial wastewater) (biphenyl, fluorene, phenanthrene, µg/l; ethylbenzene-2.90 µg/l; µg/l; ethylbenzene-0.8 µg/l;
anthracene, pyrene, chrysene and biphenyl- 2.30 µg/l; fluorene- 2.20 biphenyl- 0.7 µg/l; fluorene- 0.7

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benzo[a]pyrene) µg/l; phenanthrene- 0.30 µg/l; µg/l; phenanthrene- 0.09 µg/l;
anthracene- 0.06 µg/l; pyrene- anthracene- 0.02 µg/l; pyrene-
0.26 µg/l; chrysene- 0.70 µg/l; 0.07 µg/l; chrysene- 0.21 µg/l;

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benzo[a ]pyrene- 0.28 µg/l benzo[a ]pyrene- 0.08 µg/l
UALME ChCl-phenol 1:4 500 µl Waters (farmed and 2 ml 3 min Ultrasonic bath; pH =3; 500 Malachite green UV-VIS 11.8 µg/l 3.6 µg/l 49
ornamental aquarium µl THF
fish)
UALME ChCl-Ox 1:1 1 ml Grape skin 100 mg freeze dried 50 min Ultrasonic bath: 65 °C, 35 Phenolics ((+)-Catechin, delphinindin-3- HPLC-UV (+)-Catechin- 1.24 mg/l, (+)-Catechin- 0.37 mg/l, 42
skin kHz; 25% water O - glucoside, Cyanidin-3-O- glucoside, delphinindin-3-O- glucoside- 0.60 delphinindin-3-O- glucoside- 0.18

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petunidin-3-O -glucoside, peonidin-3-O - mg/l, Cyanidin-3-O-glucoside- mg/l, cyanidin-3-O-glucoside-0.21
glucoside, malvidin-3-O -glucoside, 0.71 mg/l, petunidin-3-O- mg/l, petunidin-3-O-glucoside-
quercetin-3-O -glucoside) glucoside- 0.80 mg/l, peonidin-3- 0.24 mg/l, peonidin-3-O-glucoside-
O-glucoside-0.65 mg/l, malvidin- 0.19 mg/l, malvidin-3-O-glucoside

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3-O-glucoside - 0.90 mg/l, - 0.30 mg/l, quercetin-3-O-
quercetin-3-O-glucoside -0.46 glucoside -0.05 mg/l
mg/l

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UALME tetramethylammonium 1:3 30 µl Saflower, olive,camellia, 1 ml (10 % oil - 90% 7 min Ultrasonic bath -50°C Plant growth regulators (IAA, IBA, 4- HPLC-UV IAA -0.05 µg/ml; IBA -0.06 µg/ml; 45
chloride–ethylene glycol colza and soybean oils hexane) IPOAA) 4-IPOAA 0.75 µg/ml

UALME Choline Chloride-Laevulinic 1:2 1 ml Platycladi Cacumen 25 mg 30 min Ultrasonic bath: 50°C, 200 Flavonoid glycosides and aglycones HPLC-UV 43
Acid W; 75% water (myricitrin, quercitrin, amentoflavone,
EP
hinokiflavone)
UAME Choline chloride-malic acid 1:1 1 ml Wine lees 33.3 mg 30.6 min Ultrasaound bath: 341.5 W, Anthocyanins HPLC-DAD Delphinidin-3-O glucoside- 0.87 Delphinidin-3-O glucoside- 0.28 57
37 kHz, 35°C ; 35.4% water mg/l; cyanidin-3-O glucoside- mg/l; cyanidin-3-O glucoside-
0.52 mg/l; petunidin-3-O 0.17 mg/l; petunidin-3-O
glucoside- 0.44 mg/l; peonidin-3- glucoside- 0.15 mg/l; peonidin-3-
O glucoside- 0.62 mg/l; malvidin- O glucoside- 0.2 mg/l; malvidin-3-
C

3-O glucoside- 0.81 mg/l O glucoside- 0.25 mg/l

UAME Choline chloride-glycerol 1:1 1 ml tartary buckwheat hulls 40 mg 1h Ultrasaound bath: 20 kHz, Flavonoid (rutin) HPLC-UV 58
AC

200 W, 40 °C; 20% water

UAME Fructose-citric acid 1:1 1 ml Raw and processed 25 mg 15 min Ultrasound bath (100 w 42 Gluten Enzyme Linked 55
food (spaghetti, biscuts, kHz) 40% amplitude; 20% ImmunoSorbent Assay
and ham) water
UAME ChCl:citric acid 1.1 600 µl Soy products 200 mg 60 min Ultrasonic bath: 440 W, Isoflavones (GT, DA, GTGL, daidzin UHPC-UV DAGL- 0.40 µg/g; GTGL- 0.45 DAGL- 0.12 µg/g; GTGL- 0.14 56
(soybeans, flour, pasta, 60°C; 30% water DAGL) µg/g; DA- 0.25 µg/g; GT 0.20 µg/g; DA- 0.08 µg/g; GT 0.06
breakfast cereals, µg/g µg/g
cutlets, tripe, soy drink,
soy nuts, soy cubes and
three different dietary
supplements)
MA ChCl-1,2-propanediol 1:1 10 ml Radix Salviae 50 mg 11.11 min Microwave: 800W, 70°C Bioactive compou nds (ROS, LIT, SAB, HPLC-DAD ROS- 0.80 µg/ml; LIT- 1.37 ROS- 0.24 µg/ml; LIT- 0.49 µg/ml; 60
miltiorrhizae SAA, TIIA) µg/ml; SAB- 1.96 µg/ml; SAA- SAB- 0.62 µg/ml; SAA- 0.31
0.87 µg/ml; TIIA- 1.45 µg/ml µg/ml; TIIA- 0.48 µg/ml
 ACCEPTED MANUSCRIPT

Table 4- Overall performance of extraction techniques based on the use DES/NADES solvents

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Technique Time Simplicity Low Solvent Volume Cost Common instrument analysis Greenness
LPME - + + + HPLC +
DLLME ++ ++ + + HPLC and GC +

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AG-DLLME and AAELLME + ++ ++ ++ HPLC and GC +
HPLME + + ++ + HPLC and GC +

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UAME + ++ ++ + HPLC, spectrophotometry, Immunoassay ++
MAE + ++ ++ - HPLC +
(-), less favourable; (+), favourable; (++), more favourable

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LPME, liquid-phase microextraction; DLLME, dispersive liquid-lquid microextraction; AG-DLLME, gas air-dispersive liqui-lquid microextraction; AAELLME,air-assisted liquid-liquid

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microextraction; HPLME, hollow fiber-liquid phase microextraction; UAME, ultrasound-assisted microextraction; MAE, microwave-assisted Extraction; HPLC, high performance
liquid chromatography; GC, gas chromatography

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 ACCEPTED MANUSCRIPT

Fig 1
DES NADES
500 20

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450 18
Number of publications

Number of publications
400 16
350 14

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300 12
250 10

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200 8
150 6
100 4

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50 2
0 0

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2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017

year year

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Web of Science Categories Web of Science Categories

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ENERGY FUELS Chemistry AppliedCHMEISTRY APPLIED

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ELECTROCHEMISTRY BIOCHEMISTRY MOLECULAR BIOLOGY
CHEMISTRY ANALYTICAL PLANT SCIENCES
THERMODYNAMICS FOOD SCIENCE TECHNOLOGY
EP
PHYSICS ATOMIC MOLECULAR CHEMICAL CHEMISTRY MEDICINAL
MATERIALS SCIENCE MULTIDISCIPLINARY GREEN SUSTAINABLE SCIENCE…
C

GREEN SUSTAINABLE SCIENCE TECHNOLOGY PHARMACOLOGY PHARMACY

ENGINEERING CHEMICAL
AC

ENGINEERING CHEMICAL
CHEMISTRY PHYSICAL CHEMISTRY ANALYTICAL
CHEMISTRY MULTIDISCIPLINARY CHEMISTRY MULTIDISCIPLINARY
0 5 10 15 20 25 30 35 0 5 10 15 20 25 30
% of publication from a total of 1303 % of publication from a total of 56
 ACCEPTED MANUSCRIPT
Highlights

DES and NADES are environmentally-friendly alternative to organic solvents

Novel applications of DES and NADES in microextraction techniques for food, biological and
environmental analysis are described

Recent microextraction techniques based in DES/NADES are compared with earlier approaches

Future perspectives of DES/NADES in microextraction techniques are stated

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