Microextraction Techniques with Deep Eutectic Solvents

Version	Summary	Created by	Modification	Content Size	Created at	Operation
1		Victoria Samanidou	--	4420	2023-07-17 18:19:29

This entry is adapted from the peer-reviewed paper 10.3390/molecules25246026

The development and application of sustainable solvents has been a hot topic in different scientific and technological areas. Deep eutectic solvents (DESs), were introduced in 2001 as an alternative to ionic liquids (ILs). These showed a stronger ecofriendly profile, with easier and cheaper production, while having similar properties. DESs contain large, asymmetrical ions that have low lattice energy and, thus, low melting points. They are often acquired by the complexation of a quaternary ammonium salt with a metal salt or hydrogen bond donor (HBD).

deep eutectic solvents microextraction solid phase extraction solid phase microextraction stir bar sorptive extraction liquid phase microextraction liquid–liquid microextraction single drop microextraction dispersive liquid–liquid microextract

1. Introduction

Sample preparation is considered to be the bottleneck of the whole analytical process, because it covers a plethora of operations that are essential to modify the sample, to make it amenable for chromatographic analysis, or to improve the analytical parameters, such as precision and accuracy. ^[1]^[2] Furthermore, it eliminates typical problems like possible interferences and low sensitivity. Samples can be considered as being made from two distinct parts, the analytes of interest and the matrix. The analytes of interest are the compounds to be determined, while the matrix is the rest of the sample, which not only does not necessitate analysis, but also may contain interfering substances. Sample preparation may focus on the analytes’ extraction, on the matrix transformation, or on both, in order to achieve dissolution, cleanup, preconcentration, or chemical modifications of the sample, so as to obtain better analytical results ^[3].

In recent years, more and more attention has been devoted to replacing conventional extraction techniques with the so-called “green” extraction techniques. This started with the introduction of green analytical chemistry (GAC) and the 12 principles, formulated by P. Anastas in 1998, the main concern of which was to create environmentally friendly analytical techniques, especially extraction techniques ^[4]. Greener approaches to extraction techniques include more inexpensive, quicker, and environmentally safe practices in environmental, clinical, and food analysis ^[5]. The adverse environmental impact of analytical procedures has been reduced in three different ways: reduction of the volume of solvents required in sample preparation; reduction in the amount and the toxicity of solvents and reagents employed in the measurement step, especially with automation and miniaturization; and the development of different direct analytical techniques not requiring solvents or reagents ^[6]. Thus, newly developed extraction techniques, which have a greener approach, are on the rise. Moreover, microextraction techniques, for example solid phase microextraction (SPME) and liquid phase microextraction (LPME), which will be discussed below, are considered to be green extraction techniques, owing to the use of very small quantities of solvents of the extraction phase in relation to the volume of the sample or to the solvent volume used in classical approaches ^[7].

The development and application of sustainable solvents has been a hot topic in different scientific and technological areas ^[8]^[9]^[10]^[11]^[12]^[13]^[14]^[15]. In this regard, remarkable advances towards the replacement of volatile organic solvents have been achieved by a group of organic salts with melting points below 100 °C, generally referred to as ionic liquids (ILs) ^[16]. Nevertheless, the problems with their biodegradability, toxicity, stability, and the expensive synthesis make them less than perfect solvents ^[17]. Therefore, deep eutectic solvents (DESs), were introduced in 2001 as an alternative to ILs. These showed a stronger ecofriendly profile, with easier and cheaper production, while having similar properties ^[18]. DESs contain large, asymmetrical ions that have low lattice energy and, thus, low melting points. They are often acquired by the complexation of a quaternary ammonium salt with a metal salt or hydrogen bond donor (HBD). The charge delocalization occurring through hydrogen bonding between, for instance a halide ion and the hydrogen-donor moiety, is responsible for the decrease in the melting point of the mixture, in relation to the melting points of the individual components ^[19]^[20]. Since 2001, many scientists around the globe pursed the utilization of DESs and published a variety of studies ^[21]^[22]^[23]^[24]^[25]^[26]^[27]^[28]^[29]^[30]^[31].

2. Synthesis and Properties of Deep Eutectic Solvents

2.1. Synthesis of Deep Eutectic Solvents

One of the approaches used to synthesize DESs is by mixing and heating two or more salts to form a homogenous solution. Typically, ambient temperature molten salts have been formed by mixing quaternary ammonium salts, a hydrogen bond acceptor (HBA) with metal salts ^[32]^[33]. Hence, four main different types of DES occur: I. quaternary salt with a metal chloride, II. quaternary salt with a hydrated metal chloride, III. quaternary salt (HBA) with an HBD compound and IV. metal chloride with an HBD. The respective general formulas of the four types are shown at Table 1. The first two types are used to synthesize hydrophilic DESs, whilst the other two for hydrophobic DESs ^[20]^[34]. Due to the stability in the aquatic solutions which type III and IV provide, they are often utilized in many papers, with choline chloride (ChCl) being the most used HBA. In Figure 1, some common HBA and HBD are shown.

Figure 1. Structures of some halide salts (HBAs) and hydrogen bond donors (HBDs) used in the formation of deep eutectic solvents.

Table 1. The four types of deep eutectic solvents.

Type	Formula	Terms
I	Cat⁺X⁻ + zMCl_x	M = Zn, Sn, Al, Ga, Fe, In
II	Cat⁺X⁻ + zMC_lx · yH₂O	M = Co, Cu, Ni, Fe, Cr
III	Cat⁺X⁻ + zRZ	Z = OH, COOH, CONH₂
IV	MCl_x + RZ = MCl_x−1⁺ · RZ + MCl_x+1 ⁻	M = Zn, Al and Z = OH, CONH₂

Cat⁺ = any phosphonium, ammonium or sulfonium cation, X⁻ = a Lewis base, generally a halide anion, MCl_x = metal chloride, RZ = organic compound.

2.2. Properties of Deep Eutectic Solvents

Various interactions (such as anion exchange, weak non-covalent interactions, π-π and/or hydrogen bonding) take place, amongst an HBD and an HBA in various combinations and molar ratios that contribute to some of the physicochemical properties discussed below ^[35]. Those properties are viscosity, density, conductivity, acidity, surface tension, volatility, and melting or freezing point. There is also an intensive study explaining the effect of hydrogen bonding proton transfer mechanism on the physical and chemical properties ^[36]. Other properties taken into account before selecting the optimal DES are biodegradability, toxicity, and thermal stability ^[37]. Moreover, both ILs and DESs can exhibit different physicochemical properties as binary mixtures, depending on the selected co-solvent, such as water or organic solvents. The interactions that may occur from the solute–solvent combination are defined as solvatochromic properties, which can further aid in the comprehension of chemical reactions through preferential solvation (PS) ^[38]. A useful tool to investigate the PS parameters and interactions, like hydrogen bond between molecules and ions, is the molecular dynamics (MD) simulation, which can be used as completion and confirmation of experimental results. For instance, Aryafard et. al. conducted an extensive investigation for three different DESs and their binary mixtures with polyethylene glycol (PEG 400) as co-solvent, through MD simulations analysis, while showing that PEG 400 can make a strong hydrogen bond with DESs ^[39].

3. Microextraction Techniques Using DES

3.1. Solid Phase Microextraction

3.1.1. Solid Phase Extraction

Solid phase extraction (SPE) is an efficient technique commonly used to handle aqueous samples and extract semi-volatile or non-volatile analytes, due to it being easy, inexpensive, and possible to be automated. SPE was firstly introduced in the 1950s ^[40], and it uses appropriate sorbents so as to isolate the target compounds from the sample. Currently, there is a wide variety of available sorbents to cover almost all the possible interactions with the compounds. Nevertheless, analytes and interferents could coelute from the SPE sorbent because of the limited selectivity of conventional solid sorbents, such as silica-based, carbon-based, and clay-based resins ^[41]^[42]. Furthermore, due to the consumption of significant quantities of sample, organic solvents, and sorbents, the SPE technique is still improving ^[41].

There are many modifications to the conventional SPE technique, including magnetic SPE (MSPE), pipette-tip SPE (PT-SPE) and dispersive SPE (dSPE or DSPE) ^[43]. In MSPE, magnetic nanoparticles are utilized, such as magnetite (Fe₃O₄) and maghemite (γ-Fe₂O₃), and afterwards, they are embedded in a suitable coating of inorganic substances, mainly in graphene ^[44]. PT-SPE is a miniaturized form of SPE that resembles the procedure of conventional SPE in terms of conditioning, sample loading, washing, and elution ^[45]. DSPE is based on the dispersion of a solid sorbent in liquid samples for the extraction, isolation, and cleaning of various target compounds from complex matrices ^[46].

Jeong et al. presented the idea of tailoring and recycling of deep eutectic solvents as a sustainable and efficient extraction media to maximize the extraction efficiencies of several compounds found in ginseng. While experimenting with different solvents and ratios, they found that the combination of three effective DES components of sucrose, L-proline, and glycerol at a molar ratio of 1:4:9 provided the best results. The recovery of this study was determined to be 102.6 ± 4.1% (n = 5), and the recycled solvents’ extraction efficiencies were 91.9 (±2.9%), 85.4 (±2.3%), and 82.6 (±4.7%) for every recurring recycle, indicating that the DES can be recycled and reused at least three times, so as to achieve a high level of extraction yields ^[47].

N. Fu et al. experimented with environmentally friendly and non-polluting solvents, in order to extract polyphenols (such as protocatechuic acid, catechins, caffeic acid and epicatechin) from palm samples by using different ChCl DES. They tried eight different compounds with the ChCl to optimize the conditions of enrichment and purification of SPE, so as to acquire high yields. The optimal DES for the enrichment step was ChCl-FA, with a molar ratio of 1:1, and for the purification step, they utilized a ChCl-Ph DES-modified absorbent for the SPE cartridges and ChCl-Urea-H₂O (1:1:5), ChCl-Gly (1:1), ChCl-FA-H₂O (1:1:5), and H₂O as eluents. The recoveries of the four analytes were 100% on the first cycle of usage of the DES, and they maintained a percentage between 60–117% throughout the five cycles studied in this work ^[48].

G. Li et al. tested DES modified molecular imprinted polymers (DES-MIPs) and DES modified non-imprinted polymers (DES-NIPs) for the purification of chlorogenic acid (CA) from honeysuckle. The preparation of these absorbents is explained in their paper. The DES used in this work had a ChCl base and glycerol, with a molar ratio of 1:2. The recoveries of MIPs, NIPs, DES-MIPs, and DES-NIPs were determined by the comparison of the peak areas of CA before and after the usage of SPE and were found to be 69.34%, 60.08%, 72.56%, and 64.79%, respectively ^[49].

Wang et al. utilized graphene (G) and graphene oxide (GO) whilst modifying them with DES and supporting them on silica as absorbents for SPE, for the preconcetration and extraction of three chlorophenols (4-chlorophenol, 2,4-dichlorophenol, and 2,4,6-trichlorophenol) from environmental water samples. The DESs used in this work were ChCl with FA, acetic acid, propionic acid, urea, Gly, ethylene glycol, and 1,4-butanodiol at the molar ratios of 1:2 ^[50].

In another study, G. Li et al. worked with Fe₃O₄/MIPs which were modified by ChCl based DES for the swift purification of alkaloid isomers (theophylline and theobromine) from green tea samples. MSPE was used as the purification method of the two compounds. The washing solution used was a mixture of methanol and water (80:20 v/v), and the eluent consisted of methanol/acetic acid (80:20 v/v) at pH 3 and with a volume of 4 mL. The recoveries of theophylline and theobromine in green tea were 87.51% and 92.27%, respectively. The optimal DES for this study was ChCl-Urea (1:2) ^[51].

X. Li et al. applied ternary DES in MIPs for the purification of levofloxacin, a known quinolone. The mixture of DES contained Caffeic Acid, ChCl, and FA at different molar ratios and were prepared by the heating method. The DES that exhibited the highest recovery rate (91.4%) was the one with a molar ratio of 1:3:1.5 ^[52].

Chen et al. utilized ternary hydrosulphonyl-based DES (THS-DES) modified by magnetic GO for the removal of mercury (Hg²⁺) from water, by using MSPE. The THS-DES was consisted of ChCl-itaconic acid-3-mercaptopropionic acid (2:1:1), and the removal efficiency achieved was 99.91% under optimized conditions. The THS-DES@M-GO absorbent retained a high removal efficiency of 90.23% even after seven MSPE cycles ^[53].

Ma et al. synthesized two types of MIPs based on magnetic chitosan with different DES (Fe₃O₄-CTS@DES-MIPs) and applied them as adsorbents in MSPE for the selective separation of catechins ((+)-catechin, (−)-epicatechin, and (−)-epigallocatechin gallate) in black tea samples. The two DESs were formed by ChCl/methacrylic acid (1:2 molar ratio) and betaine/methacrylic acid/H₂O (1:2:1). The DES which gave the highest recovery rates was the first one, showing outstanding recognition, selectivity, specificity and magnetism. The recovery rates of the three catechins were 95.4%, 92.4%, and 90.3%, respectively ^[54].

Another study by Ma et al. that utilized MSPE was about extracting catechins from green tea, by using DES-MIP as a functional monomer and magnetic molybdenum disulfide as a base (Fe₃O₄@MoS₂@DES-MIP). The catechins examined were the three mentioned above plus (−)-epigallocatechin and (−)-epicatechin gallate. The DES consisted of vinyl pyrrolidone and malonic acid (molar ratio 1:1). Both intra-day and inter-day yielded recoveries of the five analytes between 80–98% ^[55].

Liu et al. used DES modified graphene for the determination of sulfamerazine in river water samples, with the PT-SPE as the extraction technique. In particular, they tested the use of ChCl as a base and ethylene glycol, Gly, or urea as an HBD (at a 1:2 molar ratio with every solvent combination). Afterwards, they synthesized the DES-graphene and placed it in a 100 μL pipette tip using degrease cotton at both ends as frits. A 1 mL PT was inserted into the 100 μL PT, and the adsorbent was activated with 1 mL methanol and 1 mL of water. The extraction recovery by DES-G was 95.38%, whilst the G presented a 26.98% recovery rate ^[56].

Sun et al. utilized DES modified by a heteroatom doped GO for the application of PT-SPE and the extraction of two flavonoids, myricetin and rutin. Two different types of GO were prepared, one doped with nitrogen (N-GO), and the other with boron (B-GO). Subsequently, they prepared the N-GO-DES and B-GO-DES, with the DES comprising of ChCl-ethylene glycol. Finally, N-GO-DES was selected as the optimum adsorbent for this method, due to its permeability and high recoveries (that of myricetin was in the range of 70.61–99.77%, and rutin in between 76.58–98.14%) ^[57].

Yousefi et al. utilized dSPE for the ultra-trace analysis of organochlorine pesticides by using hydrophilic DES magnetic bucky gels (MBG) and magnetic multi-walled carbon nanotube nanocomposites (MMWCNT), prior to GC-μECD. The DES acted as a carrier and, at same time, as a disperser of the MMWCNTs and MBG. Thus, they opted for ChCl:Urea, 1:2 molar ratio, which had proper stearical interactions with MMWCNT, and it was miscible with water, so as to improve the dispersion of the sorbent. The LOD of this method was found to be 0.04–0.27 ng/L, and the enhancement factors were between 270–340, which are better than other techniques cited in the study ^[58].

Lamei et al. also used dSPE with magnetic GO nanoparticles coated with DES and ultrasound for the preconcentration of methadone in water and biological samples followed by GC-MS and GC-FID. The developed magnetic GO-DES (Fe₃O₄@GO-DES) was dispersed by ultrasound in the mixture of deionized water, NaOH and the sample solution that contained methadone to extract and preconcentrate the analyte. The DES studied in this work were ChCl with TNO (5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-ol), glycerol, and phenol at different molar ratios. The most efficient of the three was TNO, at molar ratio of 1:2 (ChCl:TNO), due to the high recoveries it provided. The LOD by using this method with GC-FID was 0.8 μg/L, whilst with GC-MS it was 0.03 μg/L. The recoveries of methadone in different samples were as follow: in distilled water, 98.4–101.5%; in urine, 95.3–99.3%; and in plasma, 95.1–96.4% ^[59].

Zarei et al. applied DES based magnetic colloidal gel for dSPE of ultra-trace amounts of some nitroaromatic compounds in water samples, followed by GC-μECD. The magnetic colloidal gel consisted of MMWCNTs and a deep eutectic solvent (ChCl:urea, ChCl:phenol, ChCl:acetic acid, or ChCl:gly at a molar ratio of 1:2). The optimal DES that provided the highest extraction efficiency was ChCl:urea. Then, the mixture was sonicated for half an hour to obtain the black paste or gel. The compounds examined in this work were 2-NT (2-nitrotoluene), 3-NT (3-nitrotoluene), 2,6 DNT (2,6-dinitrotoluene), 2,4 DNT (2,4-dinitrotoluene), and TNT (2,4,6-trinitrotoluene). The LODs of each compound analyzed with this method were 12.4 ng/L, 8.9 ng/L, 0.9 ng/L, 1.6 ng/L, and 0.8 ng/L, respectively, and the relative recovery rates were 90–105%, 90–110%, 95–110%, 90–110%, and 95–107%, respectively, for each compound in the different water samples ^[60].

Majidi et al. developed an alcohol-based DES as a carrier of SiO₂@Fe₃O₄, a sorbent used in magnetic d-μSPE. The aim of this study was to apply the above for the preconcentration and determination of morin in grape and apple juices, in diluted and acidic extracts of dried onion and in green tea infusion samples. Different molar ratios of tetramethylammonium chloride and EG were tested, and the optimal ratio was 1:3. The addition of 20% v/v methanol in the DES substantially enhanced the extraction efficiency of morin. At optimal conditions, the LOD of the analyte was observed as 0.91 μg/L, while the recoveries of morin were 82.80–90.56% in apple juice samples, 91.06–95.56% in commercial red grape juice samples, 93.00–98.93% in onion samples, and 94.26–95.30% in green tea samples ^[61].

Li G. et al. used a mixture of three DES in MIPs for the separation of two xanthines (theobromine and theophylline), (+)-catechin hydrate and caffeic acid from green tea samples via d-μMSPE. The ternary DES synthesized were ChCl-OA-EG, with molar ratios of 1:1:1, 1:1:2, and 1:1:3, ChCl-OA-Gly (1:1:1) and ChCl-Caffeic Acid-EG (1:1:1). ChCl-OA-Gly (1:1:1) gave the highest percentages of recovery for the four analytes, being 89.72–92.25% for theophylline, 87.15–91.86% for theobromine, 87.16–90.17% for (+)-catechin hydrate, and 86.17–92.32% for caffeic acid ^[62].

Lastly, in an additional study, Li G. et al. synthesized hydrophilic MI chitosan (HMICS) based on DESs that were used as a template and as a functional monomer for the enrichment of gallic acid from red ginseng tea samples by SPE. From the three different molar ratios studied in this work, the best was ChCl-Gallic acid (1:2), as it was the only one that had a stable form. The adsorption capacity of gallic acid on HMICS with DES was 10.13 mg/g, 2.36 times higher than that of NICS (non-imprinted chitosan) without DES ^[63].

3.1.2. Solid Phase Microextraction

Solid phase microextraction (SPME) is a sample preparation technique developed by Pawliszyn in 1990, where a fiber, with a small diameter, coated with a sorbent, is placed in aqueous solutions, so as to extract the analytes ^[64]. There are mainly two approaches: headspace SPME (HS-SPME), when the coated fiber was exposed to the gas phase, absorbing volatile compounds in the headspace of gaseous, gas or liquid samples, and direct immersion SPME (DI-SPME), in which the fiber is immersed in a small volume of the liquid sample, extracting non-volatile and semi-volatile analytes. When the equilibrium between the fiber and the analytes is completed, the SPME fiber is inserted into a GC for thermal desorption or into a desorption solvent to a LC ^[65]. Other approaches to this technique are thin-film SPME (TF-SPME), in-tube SPME, hollow fiber SPME (HF-SPME), and in-needle SPME ^[43].

Wang et al. utilized the in-tube method of SPME, as well as a poly-DES monolithic column coupled online to HPLC for the determination of anti-inflammatory and non-steroidal drugs (ketoprofen, flurbiprofen and diclofenac). In fact, they prepared a polymer monolithic column based on the DES monomer of ChCl and itaconic acid, (molar ratio 3:2) and connected it on the six-port valve of the HPLC, in order to build up an online SPME-HPLC device. The linearity of the proposed method was proved by a determination coefficient of 0.9997 in the range of 0.05–50 ng/mL for the first two compounds, and 0.25–500 ng/mL for sodium diclofenac. The LODs are 0.01 ng/mL, 0.01 ng/mL, and 0.05 ng/mL, respectively, and the recoveries of these drugs in lake water samples are 91.6–113.7% for ketoprofen, 87.3–109.5% for flurbiprofen, and 91.4–109.2% for sodium diclofenac, while, in human plasma samples, the recovery rates were 84.5–105.5%, 84.7–104.5%, and 87–99.9%, respectively, for each NSAID ^[66].

Li Tiemei et al. produced three hydrophobic DES, consisting of ethyl 4-hydroxybenzoate and methyl trioctyl ammonium chloride, with the molar ratios of 2:1, 1:1, and 1:2, which were used as an effective additive of the sol-gel sorbent coating of a PDMS fiber. This additive could create plenty of neat pores in the surface of the PDMS fiber, substantially improving its extraction efficiency. The new PDMS-DES fiber was evaluated by HS-SPME and GC-FID for the determination of VOCs, such as toluene, ethylbenzene, and o-xylene. The optimal molar ratio of the three that were tested was 2:1, as it exhibited the highest extraction efficiency. The linearity ranges of the new fiber were between 10–1000 μg/L, and the LOD was in the range of 0.005–0.025 μg/L ^[67].

Recently, Mirzajani et al. fabricated hollow fiber and monolithic fiber based on MOFs-DES/MIPs for the extraction of phthalate esters in yoghurt, bottled water, and soybean oil under termed hollow fibers liquid membrane-protected SPME (HFLMP-SPME), followed by GC-FID. Under optimal conditions, LODs of dimethylphthalate, diethylphthalate, di-isobutylphthalate, and n-dibutylphthalate were 0.008 μg/L, 0.012 μg/L, 0.030 μg/L, and 0.030 μg/L, respectively, while the mean recoveries of the analytes varied from 95.5–100.0%. The DES consisted of ChCl-Gly (2:1), which was then used for the preparation of the MOF-DES/MIPs SPME fibers ^[68].

3.1.3. Stir Bar Sorptive Extraction

Stir bar sorptive extraction (SBSE) was originally developed and published by Baltussen et al. in 1999. They presented a novel approach on sample enrichment by using the sorbent PDMS coated onto stir bars with a length of 10 mm and 40 mm (coated with 55 μL and 219 μL of PDMS liquid phase, respectively). The former is more suitable for stirring sample volumes from 10–50 mL, and the latter is suited for volumes up to 250 mL ^[69]. When the adsorption process completes, the back-extraction of the analytes takes place, by liquid desorption, into an appropriate organic solvent. Benedé and her colleagues introduced a combination of SBSE and d-μSPE in 2014, which was called stir bar sorptive-dispersive microextraction (SBSDME). They utilized a neodymium-core stirring bar physically coated with a hydrophobic magnetic nanosorbent (MNP). Depending on the stirring speed, the magnetic nanosorbent either acts as a coating to the stir bar, rendering extraction feasible like SBSE, or as a dispersed medium for the extraction of the target analytes, like d-μSPE. To extract the analytes from the sample, the same methodology with SBSE was carried out ^[70].

In a study, Zarei et al. prepared a DES magnetic nanofluid, which was coupled with SBSDME as a hyphenated sample technique for the efficient extraction of ultra-trace nitroaromatic explosives in wastewater. Out of the four DESs, the one that was the most compatible for this work was ChCl:Resorcinol (1:2 molar ratio). The extraction solvent used was acetone, and the extract was then injected into the GC instrument for analysis of the compounds. The LOD of the analytes were 4.92 for 2-NT, 3.19 for 3-NT, 0.25 for 2,6-DNT, 0.45 for 2,4-DNT, and 0.22 ng/L for TNT. The related linear ranges were between 20–1500 ng/L, 15–1500 ng/L, 1–1500 ng/L, 2–1500 ng/L, and 1–1500 ng/L, respectively, for each target compound. It is also mentioned that the developed method is both user and environmentally friendly, according to the principles of the green analytical chemistry ^[71].

Nemati et al. developed a SBSE method coupled to solidification of floating droplets in DLLME based on DESs for the effective derivatization and simultaneous extraction of acidic pesticides from tomato samples. Initially, the target compounds are adsorbed on a coated stir bar from tomato juice filled in a narrow tube. Then, the stir bar is removed upon extraction and put in a water-miscible DES (ChCl:EG 1:2) to elute the analytes. Finally, a derivatization agent and a water-immiscible DES (ChCl:Butyric acid 1:2) are added to the eluent. The LODs of the five acidic pesticides were 7 ng/L for dalapon, 9 ng/L for MCPA, 14 ng/L for 2,4-dichlorophenoxyacetic acid 7 for fenoxaprop, and 11 ng/L for haloxyfob. Lastly, their mean relative recoveries were 92–95%, 94%, 93–95%, 92–98%, and 94–99% for each compound, respectively ^[72].

3.2. Liquid Phase Microextraction

Liquid phase microextraction (LPME) is a well-known term referring to a group of miniaturized techniques, based on the fundamentals of liquid–liquid extraction, while overcoming many of its drawbacks. LPME has been developed at the late 1990s, following the principal introduction to microextraction after the establishment of SPME ^[73]^[74]. Those techniques focus on the extraction and enrichment of analytes during the sample preparation with the use of fewer resources, primarily by reducing the quantities of solvents to less than 100 μL, thereby harmonizing with the principles of green analytical chemistry (GAC). LPME can be classified into three different types: (1) hollow fiber liquid phase microextraction (HF-LPME), (2) single drop microextraction (SDME), and (3) dispersive liquid–liquid microextraction (DLLME). In addition to the tendency of reducing the use of solvents and the produced waste, in recent years, there have been new applications for specific techniques that include green solvents, such as ionic liquids and deep eutectic solvents, in order to substitute toxic organic reagents and minimize the cost ^[75]^[76].

In the recent years, there are many reports for LPME combined with DESs for the extraction of several compounds, with different applications based on the three different categories mentioned previously. One of the first applications of DES with LPME was introduced by Karimi et al. for the extraction, preconcentration and determination of lead and cadmium in edible oils, coupled with electrothermal atomic absorption spectrometry (ETAAS). The selected DES was a mix of ChCl-Urea (4:1 molar ratio) mixed with a 2% solution of nitric acid. The limits of detection resulted to be 0.008 ng g⁻¹ for Pb and 0.0002 ng g⁻¹ for Cd, results that were comparable or even better than those of similar methods. Additionally, the method was applied to edible oils, such as olive oil, sunflower oil, sesame oil, and soybean oil, with good recoveries (95–104%) from spiked samples ^[77].

Furthermore, the utilization of DES on ultrasound assisted LPME is substantial, with most applications referring to the determination of metals in water and food samples. The ultrasonication effect is used so that the aggregated DES droplets will gradually break into tiny droplets, while the use of an emulsifier for further enhancement of the process (usually with addition of THF) is possible too and is also referred to as emulsification liquid phase microextraction (ELPME). The stage that follows is centrifugation to achieve separation between the aqueous and DES rich phase. The DES phase is isolated, filled with extra volume of a proper solvent, if necessary, and analyzed with the selected method. The main combination chosen as an extraction solvent is ChCl-phenol at molar ratios of 1:3 and 1:4, applied to different samples for the determination of several analytes such as: cobalt Co (II) in a pharmaceutical supplement and tea samples by a microsample injection system, coupled with flame atomic absorption spectrometer (MS-FAAS) ^[78], Cr(III), and Cr(VI) in water samples with MS-FAAS ^[79], malachite green (MG) in farmed and ornamental aquarium fish water samples by UV-VIS spectrometry ^[80], cadmium (Cd) in water and ice tea samples by electrothermal atomic absorption spectrometry (ETAAS) ^[81], Patent Blue V analysis in syrup and water samples by UV-VIS ^[82], Hg²⁺ and CH₃Hg⁺ in water samples and freshwater fish samples with ETAAS ^[83], and pesticide residues in different traditional Chinese medicines (TCMs) with HPLC-DAD ^[84].

References

Moldoveanu, S.; David, V. The Role of Sample Preparation. Modern Sample Preparation for Chromatography; Elsevier: Amsterdam, The Netherlands, 2015; pp. 33–49.
Wen, Y. Recent advances in solid-phase extraction techniques with nanomaterials. In Handbook of Nanomaterials in Analytical Chemistry; Elsevier: Amsterdam, The Netherlands, 2020; pp. 57–73.
Moldoveanu, C.S.; David, V. Preparatory Information. J. Chromatogr. Libr. 2020, 65, 3–111.
Armenta, S.; Garrigues, S.; Esteve-Turrillas, F.A.; De La Guardia, M. Green extraction techniques in green analytical chemistry. TrAC Trends Anal. Chem. 2019, 116, 248–253.
Armenta, S.; Garrigues, S.; De La Guardia, M. The role of green extraction techniques in Green Analytical Chemistry. TrAC Trends Anal. Chem. 2015, 71, 2–8.
Armenta, S.; Garrigues, S.; De La Guardia, M. Green Analytical Chemistry. TrAC Trends Anal. Chem. 2008, 27, 497–511.
Lord, H.; Pawliszyn, J. Microextraction of drugs. J. Chromatogr. A 2000, 902, 17–63.
Sanderson, K. Chemistry: It’s not easy being green. Nature 2011, 469, 18–20.
Ávalos, M.; Babiano, R.; Cintas, P.; Jiménez, J.L.; Palacios, J.C. Greener Media in Chemical Synthesis and Processing. Angew. Chem. Int. Ed. 2006, 45, 3904–3908.
Clark, J.H. Chemistry goes green. Nat. Chem. 2009, 1, 12.
Ranke, J.; Stolte, S.; Störmann, R.; Aming, A.J.; Jastorff, B. Design of Sustainable Chemical Products the Example of Ionic Liquids. Chem. Rev. 2007, 107, 2183–2206.
Kunz, W.; Maurer, E.; Klein, R.; Touraud, D.; Rengstl, D.; Harrar, A.; Dengler, S.; Zech, O. Low Toxic Ionic Liquids, Liquid Catanionics, and Ionic Liquid Microemulsions. J. Dispers. Sci. Technol. 2011, 32, 1694–1699.
Rogers, R.D.; Seddon, K.R. CHEMISTRY: Ionic Liquids—Solvents of the Future? Science 2003, 302, 792–793.
Zhao, H.; Xia, S.; Ma, P. Use of ionic liquids as ‘green’ solvents for extractions. J. Chem. Technol. Biotechnol. 2005, 80, 1089–1096.
Li, Z.; Pei, Y.; Wang, H.; Fan, J.; Wang, J. Ionic liquid-based aqueous two-phase systems and their applications in green separation processes. TrAC Trends Anal. Chem. 2010, 29, 1336–1346.
Pena-Pereira, F.; Namieśnik, J. Ionic Liquids and Deep Eutectic Mixtures: Sustainable Solvents for Extraction Processes. ChemSusChem 2014, 7, 1784–1800.
Romero, A.; Santos, A.; Tojo, J.; Rodríguez, A. Toxicity and biodegradability of imidazolium ionic liquids. J. Hazard. Mater. 2008, 151, 268–273.
Kissoudi, M.; Samanidou, V. Recent Advances in Applications of Ionic Liquids in Miniaturized Microextraction Techniques. Molecules 2018, 23, 1437.
Abbott, A.P.; Capper, G.; Davies, D.L.; Munro, H.L.; Rasheed, R.K.; Tambyrajah, V. Preparation of novel, moisture-stable, Lewis-acidic ionic liquids containing quaternary ammonium salts with functional side chains. Chem. Commun. 2001, 1, 2010–2011.
Smith, E.L.; Abbott, A.P.; Ryder, K.S. Deep Eutectic Solvents (DESs) and Their Applications. Chem. Rev. 2014, 114, 11060–11082.
Shishov, A.; Pochivalov, A.; Nugbienyo, L.; Andruch, V.; Bulatov, A. Deep eutectic solvents are not only effective extractants. TrAC Trends Anal. Chem. 2020, 129, 115956.
Kabeer, M.; Hakami, Y.; Asif, M.; Alrefaei, T.; Sajid, M. Modern solutions in magnetic analytical extractions of metals: A review. TrAC Trends Anal. Chem. 2020, 130, 115987.
Xu, K.; Wang, Y.; Huang, Y.; Li, N.; Wen, Q. A green deep eutectic solvent-based aqueous two-phase system for protein extracting. Anal. Chim. Acta 2014, 864, 9–20.
Yao, X.-H.; Zhang, D.-Y.; Duan, M.-H.; Cui, Q.; Xu, W.-J.; Luo, M.; Li, C.-Y.; Zu, Y.-G.; Fu, Y.-J. Preparation and determination of phenolic compounds from Pyrola incarnata Fisch. with a green polyols based-deep eutectic solvent. Sep. Purif. Technol. 2015, 149, 116–123.
Wang, M.; Wang, J.; Zhang, Y.; Xia, Q.; Bi, W.; Yang, X.; Chen, D.D.Y. Fast environment-friendly ball mill-assisted deep eutectic solvent-based extraction of natural products. J. Chromatogr. A 2016, 1443, 262–266.
Werner, J.; Grześkowiak, T.; Zgoła-Grześkowiak, A.; Stanisz, E. Recent trends in microextraction techniques used in determination of arsenic species. TrAC Trends Anal. Chem. 2018, 105, 121–136.
Moghadam, A.G.; Rajabi, M.; Asghari, A. Efficient and relatively safe emulsification microextraction using a deep eutectic solvent for influential enrichment of trace main anti-depressant drugs from complicated samples. J. Chromatogr. B 2018, 1072, 50–59.
Kanberoglu, G.S.; Yilmaz, E.; Soylak, M. Application of deep eutectic solvent in ultrasound-assisted emulsification microextraction of quercetin from some fruits and vegetables. J. Mol. Liq. 2019, 279, 571–577.
Li, L.; Liu, Y.; Wang, Z.; Yang, L.; Liu, H. Development and applications of deep eutectic solvent derived functional materials in chromatographic separation. J. Sep. Sci. 2020.
Doğan, B.; Elik, A.; Altunay, N. Determination of paracetamol in synthetic urea and pharmaceutical samples by shaker-assisted deep eutectic solvent microextraction and spectrophotometry. Microchem. J. 2020, 154, 104645.
Othman, Z.A.A.L.; Habila, M.A.; Yilmaz, E.; Alabdullkarem, E.A.; Soylak, M. A novel deep eutectic solvent microextraction procedure for enrichment, separation and atomic absorption spectrometric determination of palladium at ultra-trace levels in environmental samples. Measurement 2020, 153, 107394.
Abbott, A.; Boothby, D.; Capper, G.; Davies, D.L.; Rasheed, R.K. Deep Eutectic Solvents Formed between Choline Chloride and Carboxylic Acids: Versatile Alternatives to Ionic Liquids. J. Am. Chem. Soc. 2004, 126, 9142–9147.
Sereshti, H.; Jamshidi, F.; Nouri, N.; Nodeh, H.R. Hyphenated dispersive solid- and liquid-phase microextraction technique based on a hydrophobic deep eutectic solvent: Application for trace analysis of pesticides in fruit juices. J. Sci. Food Agric. 2020, 100, 2534–2543.
Abbott, A.; Capper, G.; Davies, D.L.; Rasheed, R.K.; Tambyrajah, V. Novel solvent properties of choline chloride/urea mixtures. Chem. Commun. 2003, 70–71.
Chen, J.; Liu, M.; Wang, Q.; Du, H.; Zhang, L. Deep Eutectic Solvent-Based Microwave-Assisted Method for Extraction of Hydrophilic and Hydrophobic Components from Radix Salviae miltiorrhizae. Molecules 2016, 21, 1383.
Doi, H.; Song, X.; Minofar, B.; Kanzaki, R.; Takamuku, T.; Umebayashi, Y. A New Proton Conductive Liquid with No Ions: Pseudo-Protic Ionic Liquids. Chem. A Eur. J. 2013, 19, 11522–11526.
Aryafard, M.; Karimi, A.; Harifi-Mood, A.R.; Minofar, B. Molecular Dynamics Simulations, Solvatochromic Parameters, and Preferential Solvation in Aqueous Solutions of Ethaline, Ethylene Glycol, and Choline Chloride. J. Chem. Eng. Data 2020, 65, 4556–4566.
Aryafard, M.; Abbasi, M.; Řeha, D.; Harifi-Mood, A.R.; Minofar, B. Experimental and theoretical investigation of solvatochromic properties and ion solvation structure in DESs of reline, glyceline, ethaline and their mixtures with PEG 400. J. Mol. Liq. 2019, 284, 59–67.
Florindo, C.; Oliveira, F.S.; Rebelo, L.P.N.; Fernandes, A.M.; Marrucho, I.M. Insights into the Synthesis and Properties of Deep Eutectic Solvents Based on Cholinium Chloride and Carboxylic Acids. ACS Sustain. Chem. Eng. 2014, 2, 2416–2425.
Liska, I. Fifty years of solid-phase extraction in water analysis—Historical development and overview. J. Chromatogr. A 2000, 885, 3–16.
Makoś, P.; Słupek, E.; Gębicki, J. Hydrophobic deep eutectic solvents in microextraction techniques—A review. Microchem. J. 2020, 152, 104384.
Keçili, R.; Büyüktiryaki, S.; Dolak, I.; Hussain, C.M. The use of magnetic nanoparticles in sample preparation devices and tools. In Handbook of Nanomaterials in Analytical Chemistry; Elsevier: Amsterdam, The Netherlands, 2020; pp. 75–95.
Hou, X.; Tang, S.; Wang, J. Recent advances and applications of graphene-based extraction materials in food safety. TrAC Trends Anal. Chem. 2019, 119, 115603.
Wierucka, M.; Biziuk, M. Application of magnetic nanoparticles for magnetic solid-phase extraction in preparing biological, environmental and food samples. TrAC Trends Anal. Chem. 2014, 59, 50–58.
Mirzaee, M.T.; Seidi, S.; Alizadeh, R. Pipette-tip SPE based on Graphene/ZnCr LDH for Pb(II) analysis in hair samples followed by GFAAS. Anal. Biochem. 2020, 612, 113949.
Khezeli, T.; Daneshfar, A. Dispersive micro-solid-phase extraction of dopamine, epinephrine and norepinephrine from biological samples based on green deep eutectic solvents and Fe3O4@MIL-100 (Fe) core–shell nanoparticles grafted with pyrocatechol. RSC Adv. 2015, 5, 65264–65273.
Jeong, K.M.; Lee, M.S.; Nam, M.W.; Zhao, J.; Jin, Y.; Lee, D.-K.; Kwon, S.W.; Jeong, J.H.; Lee, J. Tailoring and recycling of deep eutectic solvents as sustainable and efficient extraction media. J. Chromatogr. A 2015, 1424, 10–17.
Fu, N.; Lv, R.; Guo, Z.; Guo, Y.; You, X.; Tang, B.; Han, D.; Yan, H.; Row, K.H. Environmentally friendly and non-polluting solvent pretreatment of palm samples for polyphenol analysis using choline chloride deep eutectic solvents. J. Chromatogr. A 2017, 1492, 1–11.
Li, G.; Wang, W.; Wang, Q.; Zhu, T. Deep Eutectic Solvents Modified Molecular Imprinted Polymers for Optimized Purification of Chlorogenic Acid from Honeysuckle. J. Chromatogr. Sci. 2016, 54, 271–279.
Wang, X.; Li, G.; Row, K.H. Graphene and Graphene Oxide Modified by Deep Eutectic Solvents and Ionic Liquids Supported on Silica as Adsorbents for Solid-Phase Extraction. Bull. Korean Chem. Soc. 2017, 38, 251–257.
Li, G.; Wang, X.; Row, K.H. Magnetic Solid-phase Extraction with Fe3O4/Molecularly Imprinted Polymers Modified by Deep Eutectic Solvents and Ionic Liquids for the Rapid Purification of Alkaloid Isomers (Theobromine and Theophylline) from Green Tea. Molecules 2017, 22, 1061.
Li, X.; Row, K.H. Application of novel ternary deep eutectic solvents as a functional monomer in molecularly imprinted polymers for purification of levofloxacin. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 2017, 1068–1069, 56–63.
Chen, J.; Wanga, Y.; Wei, X.; Xu, P.; Xu, W.; Ni, R.; Meng, J. Magnetic solid-phase extraction for the removal of mercury from water with ternary hydrosulphonyl-based deep eutectic solvent modified magnetic graphene oxide. Talanta 2018, 188, 454–462.
Ma, W.; Dai, Y.; Row, K.H. Molecular imprinted polymers based on magnetic chitosan with different deep eutectic solvent monomers for the selective separation of catechins in black tea. Electrophoresis 2018, 39, 2039–2046.
Ma, W.; Row, K.H. Solid-Phase Extraction of Catechins from Green Tea with Deep Eutectic Solvent Immobilized Magnetic Molybdenum Disulfide Molecularly Imprinted Polymer. Molecules 2020, 25, 280.
Liu, L.; Tang, W.; Tang, B.; Han, D.; Row, K.H.; Zhu, T. Pipette-tip solid-phase extraction based on deep eutectic solvent modified graphene for the determination of sulfamerazine in river water. J. Sep. Sci. 2017, 40, 1887–1895.
Sun, L.; Deng, Q.; Zhu, T. Optimization of heteroatom doped graphene oxide by deep eutectic solvents and the application for pipette-tip solid-phase extraction of flavonoids. J. Sep. Sci. 2019, 42, 2371–2378.
Yousefi, S.M.; Shemirani, F.; Ghorbanian, S.A. Deep eutectic solvent magnetic bucky gels in developing dispersive solid phase extraction: Application for ultra trace analysis of organochlorine pesticides by GC-micro ECD using a large-volume injection technique. Talanta 2017, 168, 73–81.
Lamei, N.; Ezoddin, M.; Ardestani, M.S.; Abdi, K. Dispersion of magnetic graphene oxide nanoparticles coated with a deep eutectic solvent using ultrasound assistance for preconcentration of methadone in biological and water samples followed by GC–FID and GC–MS. Anal. Bioanal. Chem. 2017, 409, 6113–6121.
Zarei, A.; Nedaei, M.; Ghorbanian, S.A. Application of deep eutectic solvent based magnetic colloidal gel for dispersive solid phase extraction of ultra-trace amounts of some nitroaromatic compounds in water samples. J. Mol. Liq. 2017, 246, 58–65.
Majidi, S.M.; Hadjmohammadi, M.R. Alcohol-based deep eutectic solvent as a carrier of SiO2 @Fe3O4 for the development of magnetic dispersive micro-solid-phase extraction method: Application for the preconcentration and determination of morin in apple and grape juices, diluted and acidic extract of dried onion and green tea infusion samples. J. Sep. Sci. 2019, 42, 2842–2850.
Li, G.; Row, K.H. Ternary deep eutectic solvent magnetic molecularly imprinted polymers for the dispersive magnetic solid-phase microextraction of green tea. J. Sep. Sci. 2018, 41, 3424–3431.
Li, G.; Rwo, K.H. Hydrophilic molecularly imprinted chitosan based on deep eutectic solvents for the enrichment of gallic acid in red ginseng tea. Polymers (Basel) 2019, 11, 1434.
Arthur, C.L.; Pawliszyn, J. Solid phase microextraction with thermal desorption using fused silica optical fibers. Anal. Chem. 1990, 62, 2145–2148.
Hasan, A.K.; Xin, D.; Manjree, A.; Oh, Y.J.; Lin, R.Y. Application of Direct Immersion Solid-Phase Microextraction (DI-SPME) for Understanding Biological Changes of Mediterranean Fruit Fly (Ceratitis capitata) During Mating Procedures. Molecules 2018, 23, 2951.
Wang, R.; Li, W.; Chen, Z. Solid phase microextraction with poly(deep eutectic solvent) monolithic column online coupled to HPLC for determination of non-steroidal anti-inflammatory drugs. Anal. Chim. Acta 2018, 1018, 111–118.
Li, T.; Song, Y.; Xu, J.; Fan, J. A hydrophobic deep eutectic solvent mediated sol-gel coating of solid phase microextraction fiber for determination of toluene, ethylbenzene and o-xylene in water coupled with GC-FID. Talanta 2019, 195, 298–305.
Mirzajani, R.; Kardani, F.; Ramezani, Z. Fabrication of UMCM-1 based monolithic and hollow fiber—Metal-organic framework deep eutectic solvents/molecularly imprinted polymers and their use in solid phase microextraction of phthalate esters in yogurt, water and edible oil by GC-FID. Food Chem. 2020, 314, 126179.
Baltussen, E.; Sandra, P.; David, F.; Cramers, C. Stir bar sorptive extraction (SBSE), A novel extraction technique for aqueous samples: Theory and principles. J. Microcolumn Sep. 1999, 11, 737–747.
Benedé, J.L.; Chisvert, A.; Giokas, D.L.; Salvador, A. Development of stir bar sorptive-dispersive microextraction mediated by magnetic nanoparticles and its analytical application to the determination of hydrophobic organic compounds in aqueous media. J. Chromatogr. A 2014, 1362, 25–33.
Zarei, A.; Nedaei, M.; Ghorbanian, S.A. Deep eutectic solvent based magnetic nanofluid in the development of stir bar sorptive dispersive microextraction: An efficient hyphenated sample preparation for ultra-trace nitroaromatic explosives extraction in wastewater. J. Sep. Sci. 2017, 40, 4757–4764.
Nemati, M.; Farajzadeh, M.A.; Mohebbi, A.; Khodadadeian, F.; Mogaddam, M.R.A. Development of a stir bar sorptive extraction method coupled to solidification of floating droplets dispersive liquid–liquid microextraction based on deep eutectic solvents for the extraction of acidic pesticides from tomato samples. J. Sep. Sci. 2020, 43, 1119–1127.
He, Y.; Lee, H.K. Liquid-Phase Microextraction in a Single Drop of Organic Solvent by Using a Conventional Microsyringe. Anal. Chem. 1997, 69, 4634–4640.
He, Y. Recent advances in application of liquid-based micro-extraction: A review. Chem. Pap. 2014, 68, 995–1007.
Sarafraz-Yazdi, A.; Amiri, A. Liquid-phase microextraction. TrAC Trends Anal. Chem. 2010, 29, 1–14.
Anthemidis, A.N.; Mitani, C. Advances in Liquid Phase Micro-Extraction Techniques for Metal, Metalloid and Organometallic Species Determination. Curr. Anal. Chem. 2013, 9, 250–278.
Karimi, M.; Dadfarnia, S.; Shabani, A.M.H.; Tamaddon, F.; Azadi, D. Deep eutectic liquid organic salt as a new solvent for liquid-phase microextraction and its application in ligandless extraction and preconcentraion of lead and cadmium in edible oils. Talanta 2015, 144, 648–654.
Arain, M.B.; Yilmaz, E.; Soylak, M. Deep eutectic solvent based ultrasonic assisted liquid phase microextraction for the FAAS determination of cobalt. J. Mol. Liq. 2016, 224, 538–543.
Yilmaz, E.; Soylak, M. Ultrasound assisted-deep eutectic solvent based on emulsification liquid phase microextraction combined with microsample injection flame atomic absorption spectrometry for valence speciation of chromium(III/VI) in environmental samples. Talanta 2016, 160, 680–685.
Aydin, F.; Yilmaz, E.; Soylak, M. A simple and novel deep eutectic solvent based ultrasound-assisted emulsification liquid phase microextraction method for malachite green in farmed and ornamental aquarium fish water samples. Microchem. J. 2017, 132, 280–285.
Zounr, R.A.; Tuzen, M.; Deligonul, N.; Khuhawar, M.Y. A highly selective and sensitive ultrasonic assisted dispersive liquid phase microextraction based on deep eutectic solvent for determination of cadmium in food and water samples prior to electrothermal atomic absorption spectrometry. Food Chem. 2018, 253, 277–283.
Kanberoglu, G.S.; Yilmaz, E.; Soylak, M. Developing a new and simple ultrasound-assisted emulsification liquid phase microextraction method built upon deep eutectic solvents for Patent Blue V in syrup and water samples. Microchem. J. 2019, 145, 813–818.
Thongsaw, A.; Udnan, Y.; Ross, G.M.; Chaiyasith, W.C. Speciation of mercury in water and biological samples by eco-friendly ultrasound-assisted deep eutectic solvent based on liquid phase microextraction with electrothermal atomic absorption spectrometry. Talanta 2019, 197, 310–318.
Li, X.; Wang, M.; Zhao, J.; Guo, H.; Gao, X.; Xiong, Z.; Zhao, L. Ultrasound-assisted emulsification liquid phase microextraction method based on deep eutectic solvent as extraction solvent for determination of five pesticides in traditional Chinese medicine. J. Pharm. Biomed. Anal. 2019, 166, 213–221.

© Text is available under the terms and conditions of the Creative Commons Attribution (CC BY) license; additional terms may apply. By using this site, you agree to the Terms and Conditions and Privacy Policy.

Upload a video for this entry

Information

Subjects: Chemistry, Analytical

Contributors MDPI registered users' name will be linked to their SciProfiles pages. To register with us, please refer to https://encyclopedia.pub/register :

Orfeas-Evangelos Plastiras

Eirini Andreasidou

Victoria Samanidou

View Times: 252

Update Date: 17 Jul 2023

Table of Contents

Video Upload Options

Confirm