The presented paper is the first to show thin layer chromatography (TLC) analysis based on eutectic mobile phases (Deep Eutectic Solvents – DES). During the experiment 25 eutectic mixtures were investigated for their chromatographic properties.
Journal of Chromatography A 1621 (2020) 461044 Contents lists available at ScienceDirect Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma Short communication Thin-layer chromatography with eutectic mobile phases—preliminary results Danuta Raj Department of Pharmacognosy and Herbal Medicines, Wroclaw Medical University, Borowska 211a, 50-556, Wrocław, Poland a r t i c l e i n f o Article history: Received 10 October 2019 Revised 10 March 2020 Accepted 11 March 2020 Available online 14 March 2020 Keywords: Chelidonium Chromatography Eutectic solvents Isoquinoline alkaloids NADES TLC a b s t r a c t The presented paper is the first to show thin layer chromatography (TLC) analysis based on eutectic mobile phases (Deep Eutectic Solvents – DES) During the experiment 25 eutectic mixtures were investigated for their chromatographic properties Most of them belong to the natural deep eutectic solvents (NADES) group Also, new eutectic liquids based on phenolics and terpenes, not previously employed in analytical practice, were tested The eutectic liquids were investigated as pure or diluted with solvents used in chromatographic routine: methanol, water, acetone, chloroform or diethyl ether The analyses were carried out using classic and high performance silica gel plates The working solution was a mixture of five alkaloids found in genus Chelidonium, namely sanguinarine, coptisine, chelerythrine, chelidonine, and berberine, with UV light detection of 366 nm This report proves that eutectic TLC is possible and that the eutectic interactions play a crucial role in the separation process In most of the tested modifications at least partial separation was achieved The most successful mobile phase, which enabled separation of all the tested alkaloids, was the equimolar mixture of menthol and phenol with a 35% addition of methanol The system was also effective in separating alkaloids in the real Chelidonium maius extract sample © 2020 The Author(s) Published by Elsevier B.V This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Introduction From the physicochemical point of view DES is a class of liquids composed of hydrogen bonds donors and acceptors, which after mixing show strong melting point depression compared to the pure components The mixture is characterized by minor vapor pressure, overall high solvation capacity and – in many cases – low toxicity and eco-friendliness [3] In particular, the solubilization properties exhibited by DES drew the attention of phytochemists, as these solvents are able to efficiently extract a wide range of compounds, including alkaloids, ginkgolides, ginsenosides, flavonoids, xanthones, catechins and essential oils [4–6] This led to the question of the possibility of employing eutectic solvents in chromatographic techniques Successful assays were performed in countercurrent separation [7,8] One publication indicates that NADES not disrupt LC systems [9], however it does not consider the attempt of eutectic separation Several reports can be found regarding a DES used as a mobile phase modifier in HPLC analyses [10–13] In these cases eutectic mixtures were added to a mobile phase in a maximum 4% concentration, far below the 50% concentration limit pointed to in the literature data as the moment of disE-mail address: danuta.raj@umed.wroc.pl ruption of intramolecular bondings which create an eutectic matrix [6] To the Author’s best knowledge, at this time there is no report about the chromatographic system involving stationary phase and pure eutectic solvents In this work, I present the results of a preliminary investigation of DES being employed as mobile phases in thin layer chromatography (TLC) The work intends to present that eutectic liquids allow chromatographic separation of mixtures of natural compounds, with particular regard to alkaloids For this purpose several eutectic solvents have been selected, which are recognized as eutectic mixtures and classified as NADES They were employed to separate a mixture of selected natural compounds, either as pure or after dilution with methanol, water, acetone, chloroform or diethyl ether Materials and methods 2.1 Preparation of mobile phases The eutectic mobile phases were prepared according to one of three procedures [2,6] (Table 1) Procedure (P1) consisted of simply mixing the components with subsequent spontaneous liquefying, resulting in a homogenous and stable liquid Procedure https://doi.org/10.1016/j.chroma.2020.461044 0021-9673/© 2020 The Author(s) Published by Elsevier B.V This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) D Raj / Journal of Chromatography A 1621 (2020) 461044 Table The list of pure DES prepared within the experiment Components Molar ratio No Obtaining procedure Camphor + phenyl salicylate camphor + chloral hydrate phenol + chloral hydrate phenol + menthol phenol + thymol choline chloride + lactic acid choline chloride + malonic acid choline chloride + raffinose choline chloride + rhamnose choline chloride + xylitol choline chloride + malic acid + proline citric acid + fructose citric acid + xylitol citric acid + raffinose citric acid + L-α -alanine citric acid + proline citric acid + sorbitol citric acid + glucose malic acid + L-α -alanine malic acid + xylitol malic acid + sorbitol malic acid + glucose malic acid + fructose malic acid + sucrose proline + glucose 1:1 1:1 1:1 1:1 1:1 1:1 1:1 11:2 2:1 5:2 1:1:1 1:1 1:1 3:1 1:1 1:1 1:1 1:1 1:1 1:1 1:1 1:1 1:1 1:1 1:1 D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 D11 D12 D13 D14 D15 D16 D17 D18 D19 D20 D21 D22 D23 D24 D25 P1 P1 P1 P1 P1 P1 P1 P2 P2 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 (P2) consisted of mixing the components and subsequent heating to 50 °C until a homogenous and stable liquid was formed Procedure (P3) consisted of mixing the components with the addition of water, using the smallest amount of water necessary for dissolution of the components, and subsequent evaporation of water using a rotary evaporator (R-210, Büchi, Germany) at 60 °C to the stable mass The eutectic liquids were used as pure or diluted with the addition of 10, 20, 30 or 40% water, methanol, chloroform, diethyl ether, or acetone (W, M, C, E or A, respectively), which was marked in their names as a suffix containing an abbreviation of the solvent and degree of dilution (e.g., D1 -W10 means that D1 DES contains 10% water) The dilutions were made using wt/wt ratio (Table 2) Additional information on the methodology for obtaining the working solution, a real Chelidonium extract sample and chromatographic parameters is included in Supplementary material Results and discussion DES are characterized by a relatively high viscosity, which is probably the reason why researchers have not yet attempted stationary phase-based chromatographic separations using DES However, the literature data regarding DES point at two favorable pieces of information: specific eutectic mixtures differ significantly in viscosity, in a range of 20–10 0 times more than water [15] and they can be diluted up to 50% without breaking the intramolecular interactions, which would allow further a further decrease in viscosity and presumably could facilitate chromatography on a stationary phase Moreover, dilution with water was mentioned as a way of fine-tuning the polarity of DES [6], which would ease the optimization of a mobile phase’s elution strength As the working solution, a mixture of alkaloids from genus Chelidonium were selected (sanguinarine, coptisine, chelerythrine, chelidonine, berberine – Fig S1), given that the plant alkaloids possess numerous pharmacological effects, including spasmolytic, antiinflammatory, antimicrobial, antiviral, cholagogue, and antiproliferative, and it is widely used in traditional phytotherapy [14] Moreover, the selected alkaloids are visible in 366 nm UV light [14], which enables detection without derivatization The working mixture is also significantly complex, as the mentioned alkaloids were only recently separated on silica gel plates with a single mobile phase [16] The mobile phases tested within the experiment have lowvolatility [6] After development they did not evaporate from the chromatographic plates, and during the preliminary stage of experiment the resulting impregnation of silica gel with mobile phase, in many cases, blocked wetting it with spraying reagents In some cases, reactions between mobile phase components and a spraying reagent did not allow to form visualized bands (data not presented) Thus, UV 366 mn detection was applied 3.1 Preparation of mobile phases In order to select DES for the purpose of the experiment, literature data was reviewed for the information on eutectic mixtures possible to be employed in the chromatographic process During the search the assumption was made that eutectic mixtures to be included, must be liquid and stable in ambient temperature, should contain nature-derived compounds or simple chemicals and shall be simple in preparation Part of the examples of creating eutectic mixtures were defined as pharmaceutical incompatibilities (D1 –D5 [17]) and these were not previously employed in analytical practice, e.g extraction Other eutectic mixtures were found in the literature describing NADES (D6 –D25 [2,6,9,15]) For the NADES-based eutectics, the main including criterion was relatively low viscosity according to the data presented in the source papers Furthermore, DES with water being the essential component (e.g., choline chloride:fructose:water 5:2:5; [9]) were excluded, as the initial water amount would interfere with the projected dilution steps The classic eutectic mixture containing choline chloride:urea 1:2 [18] was also excluded based on the preliminary experiments, as it proved to solidify during the chromatographic process DES used within the experiment were created using the molar ratio suggested in the literature data In the absence of such information a default ratio 1:1 was applied based on the available information [1,6] The preparation procedures for the specific eutectic liquids were taken from the literature data [2,6,17] Where possible, spontaneous liquefaction was employed (P1) or liquefaction supported by heating (P2) In the remaining cases water addition and subsequent evaporation was necessary (P3) [2,6] Table DESs dilutions and their chromatographic properties Modification Methanol D1 D2 D3 D4 D5 D6 D7 D9 D10 D11 D12 DES D13 D14 D15 D16 D17 D18 D19 D20 D21 D22 D23 D24 D25 D1 -M10 – D2 -M10 D3 -M10 D4 -M10 D5 -M10 D6 -M10 D7 -M10 D8 -M10 D9 -M10 D10 -M10 D11 -M10 D12 -M10 n.s D13 -M10 n.s D14 -M10 n.s D15 -M10 n.s D16 -M10 n.s D17 -M10 n.s D18 -M10 n.s D19 -M10 n.s D20 -M10 n.s D21 -M10 n.s D22 -M10 n.s D23 -M10 n.s D24 -M10 n.s D25 -M10 n.s D1 -M20 – D2 -M20 D3 -M20 D4 - M20 D5 -M20 D6 -M20 D7 -M20 D8 -M20 D9 -M20 D10 -M20 D11 -M20 D12 -M20 n.s D13 -M20 n.s D14 -M20 n.s D15 -M20 n.s D16 -M20 n.s D17 -M20 n.s D18 -M20 n.s D19 -M20 n.s D20 -M20 n.s D21 -M20 n.s D22 -M20 n.s D23 -M20 n.s D24 -M20 n.s D25 -M20 n.s D1 -M30 – D2 -M30 D3 -M30 D4 -M30 D5 -M30 D6 -M30 D7 -M30 D8 -M30 D9 -M30 D10 -M30 D11 -M30 D12 -M30 n.s D13 -M30 n.s D14 -M30 n.s D15 -M30 n.s D16 -M30 n.s D17 -M30 n.s D18 -M30 n.s D19 -M30 n.s D20 -M30 n.s D21 -M30 n.s D22 -M30 n.s D23 -M30 n.s D24 -M30 n.s D25 -M30 n.s Water D1 -M40 – D2 -M40 D3 -M40 D4 -M40 D5 -M40 D6 -M40 D7 -M40 D8 -M40 D9 -M40 D10 -M40 D11 -M40 D12 -M40 n.s D13 -M40 n.s D14 -M40 n.s D15 -M40 n.s D16 -M40 n.s D17 -M40 n.s D18 -M40 n.s D19 -M40 n.s D20 -M40 n.s D21 -M40 n.s D22 -M40 n.s D23 -M40 n.s D24 -M40 n.s D25 -M40 n.s D1 -W10 – D2 -W10 – D3 -W10 – D4 -W10 – D5 -W10 – D6 -W10 D7 -W10 D8 -W10 D9 -W10 D10 -W10 D11 -W10 D12 -W10 n.s D13 -W10 n.s D14 -W10 n.s D15 -W10 n.s D16 -W10 n.s D17 -W10 n.s D18 -W10 n.s D19 -W10 n.s D20 -W10 n.s D21 -W10 n.s D22 -W10 n.s D23 -W10 n.s D24 -W10 n.s D25 -W10 n.s D1 -W20 – D2 -W20 – D3 -W20 – D4 -W20 – D5 -W20 – D6 -W20 D7 -W20 D8 -W20 D9 -W20 D10 -W20 D11 -W20 D12 -W20 D13 -W20 D14 -W20 D15 -W20 n.s D16 -W20 D17 -W20 n.s D18 -W20 n.s D19 -W20 n.s D20 -W20 n.s D21 -W20 D22 -W20 n.s D23 -W20 n.s D24 -W20 D25 -W20 D1 -W30 – D2 -W30 – D3 -W30 – D4 -W30 – D5 -W30 – D6 -W30 D7 -W30 D8 -W30 D9 -W30 D10 -W30 D11 -W30 D12 -W30 D13 -W30 D14 -W30 D15 -W30 D16 -W30 D17 -W30 D18 -W30 D19 -W30 D20 -W30 D21 -W30 D22 -W30 D23 -W30 D24 -W30 D25 -W30 Acetone D1 -W40 – D2 -W40 – D3 -W40 – D4 -W40 – D5 -W40 – D6 -W40 D7 -W40 D8 -W40 D9 -W40 D10 -W40 D11 -W40 D12 -W40 D13 -W40 D14 -W40 D15 -W40 D16 -W40 D17 -W40 D18 -W40 D19 -W40 D20 -W40 D21 -W40 D22 -W40 D23 -W40 D24 -W40 D25 -W40 D1 -A10 D2 -A10 D3 -A10 D4 -A10 D5 -A10 D1 -A20 D2 -A20 D3 -A20 D4 -A20 D5 -A20 D1 -A30 D2 -A30 D3 -A30 D4 -A30 D5 -A30 Chloroform D1 -A40 D2 -A40 D3 -A40 D4 -A40 D5 -A40 D1 –C10 D2 –C10 D3 –C10 D4 –C10 D5 –C10 D1 –C20 D2 –C20 D3 –C20 D4 –C20 D5 –C20 D1 –C30 D2 –C30 D3 –C30 D4 –C30 D5 –C30 Diethyl ether D1 –C40 D2 –C40 D3 –C40 D4 –C40 D5 –C40 D1 -E10 D2 -E10 D3 -E10 D4 -E10 D5 -E10 D1 -E20 D2 -E20 D3 -E20 D4 -E20 D5 -E20 D1 -E30 D2 -E30 D3 -E30 D4 -E30 D5 -E30 D1 -E40 D2 -E40 D3 -E40 D4 -E40 D5 -E40 D Raj / Journal of Chromatography A 1621 (2020) 461044 D8 Pure Pure Pure Pure Pure Pure Pure Pure Pure Pure Pure Pure n.s Pure n.s Pure n.s Pure n.s Pure n.s Pure n.s Pure n.s Pure n.s Pure n.s Pure n.s Pure n.s Pure n.s Pure n.s Pure n.s Numbers refer to the number of bands possible to distinguish in the chromatogram: – no separation; – all the compounds were detectable; n.s – the mobile phase not suitable for chromatographic purposes (timeout); - – the mobile phase components were not miscible For the sake of readability of the Table, the non-miscible D6 –D25 dilutions with acetone, chloroform, and diethyl ether were excluded D Raj / Journal of Chromatography A 1621 (2020) 461044 The experiments were projected to investigate the chromatographic properties of both pure DES and their dilutions It was decided that 10%, 20%, 30% and 40% diluting solvent would be added to the initial DES The 50% dilution is indicated as a boundary for the eutectic properties [2,6], and thus it was excluded Apart from water, which was the main dilution agent in the literature data [6], methanol, chloroform, acetone and diethyl ether, the solvents widely used in TLC routine, were included D1 –D5 did not mix with water in any proportion Methanol was expected to be a proper solvent as the solubility of all the chemicals included in D1 –D5 in the alcohol is at least good Surprisingly, D1 did not mix with methanol up to a concentration of 60%, while further addition of methanol allowed the components to dissolve Apart from water (and regarding the case mentioned above) the D1 –D5 were miscible with the tested solvents In turn, the choline chloride-based DES (D6 –D11 ) were fully miscible only with water and methanol and to some extent with acetone (from 10 to 20%, depending on the particular DES) Since the acetone dilutions were not possible to obtain within the whole investigated range, they were excluded from the experiment The D12 –D25 were miscible only with water and methanol, in every investigated ratio 3.2 Chromatography with pure DES TLC analyses performed with pure eutectic solvents were able to carry out the separation of the investigated mixture depending on the properties of the individual tested DES (Table 2) For the pure DES the whole chromatographic process is relatively long and lasts from to more than 12 h In the case of development lasting longer than 12 h, it was assumed that the specific mobile phase (either pure or diluted DES) is unsuitable for the TLC purposes D1 - D5 had a development time between 180 and 240 They enabled forming bands, and the initial separation could be seen, with up to two recognizable bands (Table 2, Fig 1A - B) D1 , D4 and D5 as pure enabled low retention which indicates low polarity, while D2 and D3 DES were moving the investigated compounds close to the solvent front D6 –D11 had development time of 120–210 They were characterized by too high polarity for the tested standards, which resulted in moving the investigated compounds to the solvent front (Fig 1I) The pure D12 –D25 were unsuitable for chromatographic purposes due to excessive time of development 3.3 Chromatography with diluted DES The addition of diluting solvents to DES influenced to varying degrees the resolution and time of development In order to ensure comparability of the results, the chromatograms of the pure solvents were also presented (Fig 1N-R) For the D1 –D5 dilutions had an impact on the chromatogram development time, but interestingly, not all the diluting solvents decreased it The 40% methanol and acetone dilutions were the quickest, allowing 70–90 development, while the diethyl ether proportionally slowed it down, reaching 410 for the 40% dilution The addition of polar compounds, like acetone or methanol, significantly improved the resolution, whereas non-polar ones (chloroform, diethyl ether) did not Nevertheless, chloroform managed to shorten the development time, which may be an advantageous observation for future eutectic-TLC use For acetone and methanol, the ability to fine-tune the elution strength was proved and resulted in good resolution of the alkaloids The most efficient were DES containing a terpenoid and compound with phenolic ring (D1 , D4 , D5 ) The differences could be better observed after dilution due to increased Rf in such cases The most efficient were D1 -A40, D4 -M30, D4 -M40, D4 -A40, D5 -M40 and D5 -A40 (Fig 1C–H) Comparison of 40% dilution with acetone of D1 , D4 and D5 indicates their rank according to polarity as follows: D1 < D5 < D4 D4 and D5 differ only in the saturation of the ring of the terpenoid compound (in menthol the ring is saturated while in thymol it is aromatic), and the unsaturation is associated with lower polarity Moreover, the lowest polarity is noted for D1 which contains phenyl salicylate that includes two aromatic rings The observation is contrary to the standard eluotropic series, where a saturated ring indicates a much lower elution strength than an aromatic one (e.g., cyclohexane vs benzene) This is noteworthy, considering methanol dilutions, neither the pure DES nor pure solvent were able to move efficiently the investigated compounds (Fig 1A, N) It was only the mixture of both that managed to move the alkaloids towards the solvent front (Fig 1E) Thus, it may be concluded that the diluted DES was more polar than either the pure eutectic or solvent individually The nature of the phenomenon has yet to be investigated The D6 –D11 were diluted with water or methanol, which in every case accelerated the chromatographic process Regarding polarity of the solvents, the standards could not be withdrawn from the solvent front For D12 –D24 water dilution decreased the development time in a concentration-dependent manner (Table 2) – 40% dilutions were developed between 240 and 360 The same DES mixed with methanol, however, were much slower (800 and more) and were disqualified as mobile phases The degree of separation was differential The best chromatographic properties, regarding both time and separation, were observed for D18 -W40 and D23 -W40 modifications (Fig 1K – L) Water had a negligible impact on resolution Given that D4 -M30 and D4 -M40 gave promising results, it was decided to lower the threshold of dilution, testing also D4 -M27.5, D4 -M32.5, D4 -M35 and D4 -M37.5 The best results were achieved with D4 -M35, which interestingly had a different pattern of the standards compared to D4 -M30 and D4 -M40 Using the HPTLC plate for the selected mobile phase further improved the results D4 -M35 was subsequently tested with the real sample obtained from the Chelidonium maius herb and proved to be efficient (Fig 1T; Table S1-S2) and is considered to be applicable The influence of methanol and diethyl ether on a development time was ambiguous In silica gel-based TLC, due to the low viscosity, they generally enabled short-lasting analyzes Thus, the mentioned solvents were supposed to decrease the development time Actually, their effect was incongruous Diethyl ether slowed down the process in every investigated case, but methanol either improved (D2 –D11 ) or worsened (D12 –D25 ) the parameter The observed phenomenon could be linked with the viscosity changes of the mobile phase, but the parameter was not tested during the investigation and the explanation needs additional experiments Water addition had little impact on the elution strength of the tested DES This is contrary to what was expected regarding the information presented in the literature data [6], where water was being used for modifications of DES polarity The phenomenon may result from additional interactions between water and stationary phase’s functional groups that not occur during extraction from plant material It was a matter of interest whether other solvents apart from water would dilute DES without disrupting the eutectic matrix At the initial stage of the experiment, it was taken under consideration that after dilution the separation may depend only on chromatographic properties of the solvents, leaving DES as an inert part of the mobile phase That would give the same separation pattern for all same-solvent dilutions (e.g., 40% acetone dilutions would be similar regardless of DES used) However, chromatograms obtained with different eutectics and the same solvent were not alike but had similar features to the specific pure DES (Fig 1E, G) This means that the eutectic matrix was preserved after dilution with the tested solvents in the investigated range and that the matrix had a substantial impact on the chromatographic process Additional support for that conclusion is an observation D Raj / Journal of Chromatography A 1621 (2020) 461044 Fig Chromatograms obtained with the selected mobile phases, using TLC Si60 plates, unless otherwise noted All the pictures were taken at 366 nm Mobile phases, left to right: A–D4 ; B–D5 ; C–D1 -A40; D–D4 -M30; E–D4 -M40; F–D4 -A40; G–D5 -M40; H–D5 -A40; I–D7 ; J–D7 -W40; K–D18 -W40; L–D23 -W40; M–D4 -M35 on HPTLC plate The working solution developed in pure solvents: N – methanol; O – water; P – acetone; Q – chloroform; R – diethyl ether; S – the working solution developed in D4 -M60; T – Chelidonium maius root extract developed with D4 -M35 mobile phase on HPTLC plate Alkaloids marked in the chromatograms: Be – berberine, Ce – chelerythrine, Ci – chelidonine, Co – coptisine, Sa – sanguinarine made at the preliminary stage of the experiment: a chromatogram made using 60% dilution (Fig 1S) was significantly different from the one obtained within the eutectic range while similar to the pure methanol one (Fig 1N) The issue observed during the experiment is problematic downregulation of specific DES elution strength While DES polarity can be easily up-regulated with the addition of polar solvents, the nonpolar ones not change the elution Lowering DES content along with replacing it with less polar compounds in case of eutectic mobile phases is not effective Thus, the solution to the presented problem may be using different DES with lower polarity as a starting point matographic mobile phases based on non-classical interactions to be widely employed Author contribution I am the only Author of the manuscript, therefore I am responsible for the: Conceptualization, Methodology, Validation, Investigation, Resources, Writing - Original Draft, Writing - Review & Editing, Visualization, Supervision, Project administration Declaration of Competing Interests None Conclusions CRediT authorship contribution statement Eutectic TLC is possible and can result in good separation, even for the complicated matrices that proved to be problematic for a classic TLC It is most probable that, in a short time, new DES types with low viscosity will emerge, allowing for a new class of chro- Danuta Raj: Conceptualization, Investigation, Methodology, Validation, Visualization, Writing - original draft, Writing - review & editing 6 D Raj / Journal of Chromatography A 1621 (2020) 461044 Acknowledgments ´ I would like to thank Dr Sylwia Zielinska for advising and supplying with standards, and Natalia Maryniak for her technical support Supplementary materials Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.chroma.2020.461044 References [1] Y Liu, J.B Friesen, J.B McAlpine, D.C Lankin, S.-N Chen, G.F Pauli, Natural deep eutectic solvents: properties, applications, and perspectives, J Nat Prod 81 (2018) 679–690, doi:10.1021/acs.jnatprod.7b00945 [2] Y.H Choi, J van Spronsen, Y Dai, M Verberne, F Hollmann, I.W.C.E Arends, G.J Witkamp, R Verpoorte, Are natural deep eutectic solvents the missing link in understanding cellular metabolism and physiology? 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