Interactions of basic compounds with ionic liquids used as oils in microemulsion liquid chromatography

Aqueous microemulsions (MEs), where an oil coexists with water in the presence of the anionic surfactant sodium dodecyl sulphate (SDS), have been proposed as a solution to decrease the amount of organic solvent in the mobile phase needed in reversed-phase liquid chromatography (RPLC).

Journal of Chromatography A 1674 (2022) 463142 Contents lists available at ScienceDirect Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma Interactions of basic compounds with ionic liquids used as oils in microemulsion liquid chromatography N Pankajkumar-Patel, E Peris-García, M.J Ruiz-Angel, M.C García-Alvarez-Coque∗ Departament de Química Analítica, Universitat de València, c/Dr Moliner 50, Burjassot, Spain a r t i c l e i n f o Article history: Received 14 February 2022 Revised 10 May 2022 Accepted 10 May 2022 Available online 13 May 2022 Keywords: Oil-in-water microemulsion liquid chromatography Sodium dodecyl sulphate Ionic liquids β -adrenoceptor antagonists a b s t r a c t Aqueous microemulsions (MEs), where an oil coexists with water in the presence of the anionic surfactant sodium dodecyl sulphate (SDS), have been proposed as a solution to decrease the amount of organic solvent in the mobile phase needed in reversed-phase liquid chromatography (RPLC) However, the oil phase of a typical ME is volatile, toxic and flammable, and although it is added in a small amount, it would be desirable to avoid it from an environmental perspective This is the reason for the proposal of Peng et al (J Chromatogr A 1499 (2017) 132–139) to replace the oil in microemulsion liquid chromatography (MELC) by the apolar ionic liquid (IL) 1-hexyl-3-methylimidazolium hexafluorophosphate ([C6 C1 IM][PF6 ]), to analyse neutral phenolic acids at acidic pH Based on this report, an MELC procedure is here proposed for β -adrenoceptor antagonists, which are basic compounds where the dominant species is cationic To verify the formation of MEs containing SDS and IL, and elucidate the interactions between the cationic basic compounds with the SDS anion, and the cation and anion in the IL, an extensive study was carried out with several methylimidazolium ILs containing the cations [C2 C1 IM]+ , [C4 C1 IM]+ , or [C6 C1 IM]+ , combined with the anions Cl– , BF4 – , or PF6 – , using 1-butanol as co-surfactant The behaviour was compared with that observed in classical MELC with octane, micellar liquid chromatography with SDS and 1-propanol, and RPLC with mobile phases containing an IL and acetonitrile © 2022 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 Microemulsions (MEs) are thermodynamically stable and transparent colloidal solutions that contain an organic phase (oil) and an aqueous phase (water), the latter stabilised by a surfactant above its critical micelle concentration (CMC) and an organic solvent performing as co-surfactant [1,2] Oil-in-water (O/W) MEs are made up of oil droplets dispersed in the aqueous medium containing the surfactant, so they have high water content, low viscosity and high solubilising power that makes them suitable as mobile phases in reversed-phase liquid chromatography (RPLC) This chromatographic mode, known as microemulsion liquid chromatography (MELC), provides unique selectivity for both hydrophilic and hydrophobic substances and has gained relevance in recent years [3,4] Common reagents in O/W MELC are the surfactants sodium dodecyl sulphate (SDS, anionic) and polyoxyethylene(23) lauryl ether (Brij-35, non-ionic), the oils heptane, octane, cyclohexane, diiso- ∗ Corresponding author E-mail address: celia.garcia@uv.es (M.C García-Alvarez-Coque) propylether and ethyl acetate, and the alcohols 1-propanol, 1butanol and 1-pentanol In MELC systems, the mobile phases require lower concentration of organic solvent compared to conventional RPLC (below 1% and 10% v/v for the oil phase and cosurfactant, respectively) Since any change in the nature and concentration of the reagents (surfactant, oil and co-surfactant) can significantly affect the chromatographic behaviour of the solutes [4,5], a detailed systematic investigation is usually required to obtain successful separations In general, the replacement of harmful and volatile solvents, traditionally used in many processes, has generated great interest in recent years Ideally, the best solvent would be no solvent, considering health hazards, waste generation and treatment, as well as economic reasons [6] Since the absence of solvent is not always possible, several more environmentally friendly alternatives have been proposed to decrease the impact and overall risk of chemical exposure to conventional organic solvents Amongst these alternatives are ionic liquids (ILs) [7], which are salts with low melting points (usually below 100 °C), formed by a bulky organic cation associated with a smaller inorganic/organic anion to get electrical neutrality [8–10] https://doi.org/10.1016/j.chroma.2022.463142 0021-9673/© 2022 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/) N Pankajkumar-Patel, E Peris-García, M.J Ruiz-Angel et al Journal of Chromatography A 1674 (2022) 463142 The interest in ILs can be attributed to the wide range of interactions with solutes (strong and weak ionic interactions, hydrogen bonding, and van der Waals, dispersive, n-π and π -π interactions) All these interactions give rise to interesting solvation properties, compared to conventional organic solvents [11] Other attractive features of ILs are their low volatility and flammability, high thermal stability, and low toxicity, which have led to the replacement of conventional polluting solvents by ILs, which have earned the label of benign or green solvents Some recent reports have shown, however, that some ILs (as those based on fluorinated anions), are not as safe and non-toxic as claimed [12,13], but their toxicity (as is the case for other physico-chemical properties) can be modulated by the appropriate selection of the IL cation and anion In the analytical field, ILs have been widely applied in sample preparation [14,15] and chromatographic analysis [16,17] They have also been used immobilised in stationary phases in gas chromatography [18,19] and liquid chromatography [20–22], and as mobile phase additives in the hydro-organic mobile phases used in RPLC [22,23] In RPLC analysis, ILs lose their physical characteristics as solvents, being just salts that dissociate in aqueous medium [24] A relevant advantage of the addition of ILs to the mobile phase is that ion exchange interactions of cationic solutes with residual anionic silanols, which are present in conventional silica stationary phases, are minimised This improves peak performance, which has been explained by the adsorption of the IL cation and anion onto the stationary phase, creating an asymmetric bilayer, positively or negatively charged that masks the silanols The effect is stronger for ILs with a cation of larger size [25] Recently, alkyl-methylimidazolium ILs, associated to tetrafluoroborate (BF4 – ) and hexafluorophosphate (PF6 – ), were proposed to prepare ionic liquid-in-water (IL/w) MEs (also called aqueous ILbased MEs), for the MELC analysis in acidic medium (pH = 2.5) of hydrophilic phenolic compounds (danshensu, caffeic acid, protocatechualdehyde, rosmarinic acid and salvianolic acid B) in Danshen samples (a traditional Chinese medicinal herb) [26] The procedure produced excellent selectivity and adequate resolution In this work, the effect of the addition to IL/w MEs of alkylmethylimidazolium ILs with alkyl chains of diverse lengths and associated with the anions Cl– , BF4 – and PF6 – (the most common in RPLC [23]), on the retention and peak profile behaviours of cationic basic compounds (β -adrenoceptor antagonists), is investigated at acidic medium The results are compared with those found with MELC mobile phases containing SDS, 1-butanol and octane as oil, and with RPLC mobile phases containing SDS and 1-propanol, or ILs and acetonitrile 3-methylimidazolium hexafluorophosphate ([C4 C1 IM][PF6 ]), 1hexyl-3-methylimidazolium hexafluorophosphate ([C6 C1 IM][PF6 ]), 1–butyl–3-methylimidazolium tetrafluoroborate ([C4 C1 IM][BF4 ]), 1-hexyl-3-methylimidazolium tetrafluoroborate ([C6 C1 IM][BF4 ]), and 1-hexyl-3-methylimidazolium chloride ([C6 C1 IM][Cl]), all from Sigma Molar concentrations were used for the surfactant and ILs Volumetric fractions (expressed as percentage) were used for acetonitrile, 1-butanol and octane The mobile phases considered in our study contained: (i) SDS, 1-butanol and IL, (ii) SDS, 1-butanol and octane (data taking from Ref [27]), (iii) SDS and 1-propanol (data taking from Ref [28]), or (iv) IL and acetonitrile (data taking from Ref [28]) The pH of the MELC mobile phases with either IL or octane was fixed at 1.35 with 0.05% trifluoroacetic acid from Thermo Fisher Scientific (Loughborough, UK) The pH of the other mobile phases was buffered at 3.0 with 0.01 M citric acid monohydrate and sodium hydroxide from Panreac (Barcelona) The pH metre was calibrated with aqueous buffers, while the pH of the mobile phases was always fixed in the presence of the organic solvent β -Adrenoceptor antagonists have a strong basic character (pKa ≥ 9), which means that at the acidic pH of the mobile phases the cationic species are dominant The solutions of the β -adrenoceptor antagonists and mobile phases were filtered through 0.45 μm Nylon membranes from Micron Separations (Westboro, MA, USA) Nanopure water obtained with an Adrona system (Riga, Latvia) was used throughout 2.2 Apparatus and columns An Agilent (Waldbronn, Germany) chromatograph was used, equipped with a quaternary pump (Series 1200), an automatic injector (Series 1260 Infinity II), a thermostatted column compartment (Series 1290 Infinity II), and a diode array detector (Series 1100) The β -adrenoceptor antagonists were monitored at 225 nm, except for timolol, which was detected at 300 nm Uracil was detected at 254 nm The retention data were obtained at 25 °C, using isocratic conditions with a flow rate of ml/min Duplicate injections of 20 μl were made The chromatographic system was controlled with an OpenLAB CDS LC Chemstation (Agilent B.04.03) The mathematical treatment was carried out with Excel (Microsoft Office 2010, Redmond, WA, USA) Chromatographic peaks were processed with the MICHROM software to obtain the peak parameters (retention times and peak half-widths) [29] An XTerra-MS C18 column from Waters (Milford, MA, USA), which replaces one out of every three silanols with a methyl group, was used with the MELC mobile phases of SDS, 1-butanol and IL or octane, and the mixtures of IL and acetonitrile, and SDS and 1-propanol The characteristics of the column were as follows: 150 mm × 4.6 mm i.d., μm particle size, 120 A˚ mean pore diameter, 175 m²/g surface area, and 12 wt% total carbon A Kromasil C18 column from Análisis Vínicos (Ciudad Real, Spain) with 150 mm × 4.6 mm i.d., μm particle size, 110 A˚ average pore diameter, 320 m2 /g surface area, and 19% carbon load, was also used when working with micellar mobile phases and mobile phases containing ILs and acetonitrile [28] In all cases, the analytical columns were preceded by similar 30 mm guard columns for mobile phase protection Mobile phases were recycled between runs and also during analysis to reduce reagent consumption and wastes The low evaporation risk of organic solvents in the mobile phases with additives makes recycling possible, as long as a sufficiently low number of injections is made The mobile phase was renewed each week when the composition was not changed A small flow rate of 0.1 ml min–1 was used between analyses The chromatographic system was periodically rinsed with pure water and methanol or 2-propanol (around 30 ml) to remove surfactant and IL from the Experimental 2.1 Reagents Seven β -adrenoceptor antagonists (atenolol, acebutolol, carteolol, metoprolol, timolol, oxprenolol, and propranolol), all from Sigma (St Louis, MA, USA) were used as probe compounds The drugs were dissolved in mL of methanol from VWR International (France), with the aid of an Elmasonic S15 H ultrasonic bath from Elma (Singen, Germany), and diluted with water The concentration of the stock solutions of the probe compounds, which were stable during at least two months at °C, was approximately 100 μg/mL These solutions were diluted with water to a final concentration of 20 μg/mL, prior to injection into the chromatograph Uracil from Acros Organics (Geel, Belgium) was used as hold up time marker The reagents used to prepare the mobile phases were sodium dodecyl sulphate from Merck (99% purity, Darmstadt, Germany), acetonitrile and 1-butanol from Scharlab (Barcelona, Spain), octane from Alfa Aesar (Kandel, Germany), and the ILs 1-ethyl-3methylimidazolium hexafluorophosphate ([C2 C1 IM][PF6 ]), 1–butyl– N Pankajkumar-Patel, E Peris-García, M.J Ruiz-Angel et al Journal of Chromatography A 1674 (2022) 463142 stationary phase Over the weekend, the column was maintained with 2-propanol Results and discussion 3.1 Formation of transparent mixtures of [C6 C1 IM][PF6 ], SDS and 1-butanol In a recent study, Peng et al assayed alkyl-methylimidazolium ILs formed with [C4 C1 IM]+ , [C6 C1 IM]+ and [C8 C1 IM]+ , associated to BF4 – , PF6 – and bis[(trifluoromethyl) sulfonyl] imide (NTf2 – ) to be used in MELC [26] These authors observed that due to the low solubility of [C6 C1 IM][PF6 ] in water it can replace the oil to form a ME composed of SDS, IL and 1-butanol This IL was found suitable to analyse a group of neutral phenolic compounds, based on the short analysis time and good separation selectivity In this work, this ME has been taken as starting point for the analysis of the cationic β -adrenoceptor antagonists To verify the conditions for the formation of a transparent medium to be used as mobile phase, or the presence of two well differentiated phases, we prepared several mixtures with different amounts of SDS, [C6 C1 IM][PF6 ] and 1-butanol The effect of the addition of [C6 C1 IM][PF6 ] was checked in the 0.01–0.10 M range, in solutions containing a fixed amount of SDS (0.10 M) and varying 1butanol (2–12% v/v), or fixed 1-butanol (8.15% v/v) and varying SDS (0.02–0.25 M) Once the reagents were mixed, the mixtures were allowed to stand for at least 12 h, and then centrifuged When transparent mixtures were obtained, they were left to rest for several weeks to check its long-term stability The formation of clear and stable solutions was visually verified at room temperature, at least during two weeks In a previous study [5], the formation of an emulsion containing SDS, with increasing octane or decreasing 1-butanol, gave rise to an upper phase that increased in thickness and turned whitish, an effect that was more intense at the smallest assayed concentrations of the surfactant When the oil was replaced with [C6 C1 IM][PF6 ], phase separation was not so clearly observed, being only evidenced by the observation of a whitish drop of IL falling through the solution However, in most tested mixtures, a clear solution was obtained The composition of the transparent mixtures, and of mixtures giving rise to the possible formation of an emulsion, is represented in Fig 1a and 1b Using 0.10 M SDS (Fig 1a), stable mixtures were always formed, with a maximal concentration of [C6 C1 IM][PF6 ] close to 0.08 M at both lower (1.81% ) and upper (12.7%) extreme concentrations of 1-butanol This means that the surfactant was able to solubilise the IL without the need of a large amount of co-surfactant When the concentration of 1-butanol was fixed at 8.15% v/v (Fig 1b), increasing amounts of [C6 C1 IM][PF6 ] required a higher concentration of SDS to obtain stable solutions Maximal concentrations of 0.10 M and 0.25 M were tested for [C6 C1 IM][PF6 ] and SDS, respectively It should be noted that the concentration range for [C6 C1 IM][PF6 ] in the mobile phase used in RPLC is usually narrow and with an upper value below 0.04 M to avoid high viscosity The ability of SDS to solubilise this IL can be explained by the formation of a stable ME, where the IL would act as oil (IL/w ME) However, the formation of a neutral ion pair or any other structure between the anionic SDS micelles and the alkyl-methylimidazolium cation must also be considered This could also explain the secondary role of 1-butanol in the solubilisation of the IL The results in Fig 1a and 1b should be compared with those shown in previous work with an SDS/octane/1-butanol system, where the role of the co-surfactant (1-butanol) was relevant for the solubilisation of octane [5] At 0.10 M and 0.18 M SDS concentra- Fig Concentration range for: (a) 1-butanol and [C6 C1 IM][PF6 ] in the presence of 0.10 M SDS, and (b) SDS and [C6 C1 IM][PF6 ] in the presence of 8.15% 1-butanol The circles correspond to the compositions that gave rise to the formation of clear solutions, whereas the crosses correspond to the compositions that produced phase separation tions, a high concentration of 1-butanol solubilised higher amounts of octane 3.2 Retention behaviour of basic compounds with mobile phases containing SDS, [C6 C1 IM][PF6 ] and 1-butanol In a chromatographic system with mobile phases containing SDS and IL, the stationary phase should probably be coated by layers of surfactant monomers, IL cation and, to a lesser extent, IL anion Alkyl-methylimidazolium cations with sufficiently long alkyl chains (such as [C6 C1 IM]+ ), associated to chaotropic anions (such as PF6 – ), have been reported to be significantly adsorbed on the stationary phase [30] The adsorbed reagents, which are ionic, change the nature of the stationary phase from an apolar (hydrophobic) to a polar charged (hydrophilic) surface The charge sites in the stationary phase produced by this adsorption serve as ion exchangers for cationic solutes The multiple possible effects (interactions of the anionic surfactant and IL cation and anion with the stationary phase, and of the cationic solutes with the surfac3 N Pankajkumar-Patel, E Peris-García, M.J Ruiz-Angel et al Journal of Chromatography A 1674 (2022) 463142 Table Half-width plots parameters for several chromatographic systems: slopes of the left (mA ) and right (mB ) half-widths, sum of slopes and slope ratio Without additivea SDS/1-butanol/IL [C2 C1 IM][PF6 ] [C4 C1 IM][PF6 ] [C6 C1 IM][PF6 ] [C6 C1 IM][BF4 ] [C6 C1 IM][Cl] SDS/1-butanol/octane IL/acetonitrile without SDSb [C2 C1 IM][PF6 ] [C4 C1 IM][PF6 ] [C2 C1 IM][BF4 ] [C4 C1 IM][BF4 ] [C6 C1 IM][BF4 ] [C2 C1 IM][Cl] [C4 C1 IM][Cl] [C6 C1 IM][Cl] a b mA mB mA + mB mB /mA 0.021 0.047 0.068 2.3 0.028 0.027 0.031 0.026 0.023 0.043 0.025 0.028 0.033 0.039 0.027 0.044 0.053 0.055 0.064 0.065 0.049 0.087 0.9 1.0 1.0 1.5 1.2 1.0 0.026 0.026 0.018 0.020 0.022 0.017 0.019 0.020 0.038 0.040 0.022 0.022 0.017 0.023 0.019 0.016 0.064 0.066 0.040 0.042 0.039 0.041 0.039 0.036 1.5 1.5 1.2 1.1 0.8 1.3 1.0 0.8 Acetonitrile-water, from Ref [32] From Refs [25,32] ME formed by SDS, 1.14% octane and 8.15% 1-butanol (Fig 2b), reported in Ref [27] The trend produced by increasing the SDS concentration is similar, but with lower retention when octane is used instead of [C6 C1 IM][PF6 ] Observe that with either IL or octane, the decrease in the retention factors at the highest SDS concentrations is no more relevant for all probe compounds 3.3 Effect of the IL cation and anion on retention In order to gain more insight on the effect of hybrid systems of SDS and IL on the retention of the β -adrenoceptor antagonists, several mobile phases containing 0.05 M SDS, 8.15% 1-butanol, and alkylimidazolium ILs with different cations and anions (and therefore, different solubility in water [30]) were assayed On the one hand, the effect of different anions using the same IL cation (1-hexyl-methylimidazolium) ([C6 C1 IM][Cl], [C6 C1 IM][BF4 ], and [C6 C1 IM][PF6 ]) was studied, and on the other, the effect of different alkyl lengths in the IL cation using the same IL anion (hexafluorophosphate) ([C6 C1 IM][PF6 ], [C4 C1 IM][PF6 ] and [C2 C1 IM][PF6 ]) The selected concentrations were 0.01 and 0.03 M for all ILs In previous work, the amount of the anions adsorbed on a Kromasil C18 column with mobile phases containing 30% acetonitrile and 0.05 M NaCl, NaBF4 or NaPF6 , was measured [30]: Cl– showed low affinity to the C18 stationary phase (∼2.5 μmol), whereas the affinity of BF4 – and PF6 – was moderate (∼15 μmol) and strong (∼32 μmol), respectively Fig 3a depicts the effect of the addition of different ILs, in the presence of 0.05 M SDS and 8.15% 1-butanol, on the behaviour of metoprolol, which shows intermediate retention amongst the studied β -adrenoceptor antagonists (similar trends were observed for the other compounds) The retention decreased with increasing concentration of the ILs, being the effect stronger as the alkyl chain in the IL increased: [C2 C1 IM]+ < [C4 C1 IM]+ < [C6 C1 IM]+ This decreasing trend was also observed in mobile phases containing the ILs without SDS, when combined with the anions BF4 – and Cl– , which are weakly adsorbed (Fig 3b) This can be explained by considering that the stronger adsorption of the more hydrophobic IL cation with a longer alkyl chain repels the cationic solutes significantly [28] Note that the IL cation dissolved in the mobile phase will also repel the cationic solute, but this would be shifted towards the stationary phase, increasing the retention (i.e., the opposite effect) Furthermore, a stronger adsorbed IL anion would attract the cationic solutes (also increasing the retention) Fig Change in retention at increasing concentration of SDS, in the presence of 8.15% 1-butanol and: (a) 0.01 M [C6 C1 IM][PF6 ], and (b) 1.14% octane Solute identity: ( ) acebutolol, (◦) atenolol, (♦) carteolol, ( ) metoprolol, (●) oxprenolol, ( ) propranolol, and ( ) timolol (acknowledgement is given to The Royal Society of Chemistry for the reproduction of Fig 2a from Ref 27) tant and IL ions in the mobile phase and adsorbed on the stationary phase) complicate the interpretation of the retention mechanism The retention factors for the β -adrenoceptor antagonists obtained with mobile phases containing 0.01 M [C6 C1 IM][PF6 ], 8.15% 1-butanol, and SDS in the range 0.05–0.25 M, are depicted in Fig 2a As observed, the addition of an increasing concentration of surfactant produced the expected decrease in retention, since there is a maximal amount of surfactant adsorbed on the C18 column that attracts the cationic solutes, while the concentration of SDS micelles in the mobile phase (which also interact with the solutes) increases [31] Therefore, the cationic solutes undergo a progressive distribution into an increased volume of microemulsion droplets (micelles containing IL in its core or surface), which increases the elution strength The observed behaviour must be compared with the changes in retention observed for the β -adrenoceptor antagonists with the N Pankajkumar-Patel, E Peris-García, M.J Ruiz-Angel et al Journal of Chromatography A 1674 (2022) 463142 Fig Retention behaviour of metoprolol in different RPLC systems containing: (a) 0.05 M SDS and 8.15% 1-butanol, with increasing concentration of diverse ILs, (b) several ILs in the presence of fixed 15% acetonitrile, and (c) SDS in the presence of fixed 15% 1-propanol In (b), the retention times are identical for [C6 C1 IM][PF6 ], [C6 C1 IM][BF4 ], and [C6 C1 IM][Cl] Assayed ILs: ( ) C2 C1 IM][PF6 ], ( ) [C4 C1 IM][PF6 ], ( ) [C4 C1 IM][BF4 ], (●) [C6 C1 IM][PF6 ], ( ) [C6 C1 IM][BF4 ], and (◦) [C6 C1 IM][Cl] on the stationary phase As commented above, once the stationary phase is saturated with SDS, the amount of surfactant in the mobile phase (forming micelles) is increased This makes the elution strength stronger due to the attraction of the cationic solutes to the anionic micelles A similar behaviour is observed with the mobile phases that contain an increased concentration of SDS in the presence of fixed amounts of IL and 1-butanol (Fig 2a), or octane and 1-butanol (Fig 2b), although the retention is globally smaller due to the presence of the organic solvents or IL A comparison of the trends in retention at increasing concentration of IL, in the presence of SDS (Fig 3a) (MELC with IL), and without SDS (Fig 3b) (RPLC with IL), can also help to interpret the possible interactions We should indicate that the behaviour could only be studied in the presence of [C2 C1 IM][PF6 ], [C4 C1 IM][PF6 ], [C4 C1 IM][BF4 ], [C6 C1 IM][BF4 ], and [C6 C1 IM][Cl], since the solubility of [C6 C1 IM][PF6 ] in the absence of SDS was too low The decreased retention of basic solutes at a higher concentration of IL, in the range of to 0.03 M, suggested that the interaction of the cationic basic compounds with the imidazolium cations (electrostatic repulsion with the adsorbed IL cation) should prevail over the association with the adsorbed IL anions on the stationary phase (which would cause the attraction of the basic compounds), whose concentration also changes when the IL is added to the mobile phase Therefore, the strongly adsorbed SDS should hinder the adsorption of the IL anion (even for PF6 – ) In Fig 3a, note that in the presence of SDS, the retention times for [C6 C1 IM][PF6 ], [C6 C1 IM][BF4 ] and [C6 C1 IM][Cl] are identical In the presence of SDS, the behaviour for [C4 C1 IM][PF6 ] and [C4 C1 IM][BF4 ] will probably be also similar Fig 3c shows the retention of metoprolol with a mobile phase with SDS in the 0.01–0.15 M range, containing also 15% 1-propanol The high retention at low concentration of the surfactant reveals the attraction of the cationic solutes towards the adsorbed SDS N Pankajkumar-Patel, E Peris-García, M.J Ruiz-Angel et al Journal of Chromatography A 1674 (2022) 463142 Fig Half-width plots (left, A (◦) and right, B (●)), built with the data obtained for the set of β -adrenoceptor antagonists with mobile phases containing 0.05 M SDS, 8.15% 1-butanol, and the following ILs at 0.01 and 0.03 M concentrations: (a) [C6 C1 IM][PF6 ], (b) [C4 C1 IM][PF6 ], (c) [C2 C1 IM][PF6 ], (d) [C4 C1 IM][BF4 ], and (e) [C4 C1 IM][Cl] N Pankajkumar-Patel, E Peris-García, M.J Ruiz-Angel et al Journal of Chromatography A 1674 (2022) 463142 Without surfactant (Fig 3b), the retention was significantly affected by the presence of specific IL cations and anions, which should be explained by their particular adsorption capability on the C18 stationary phase The adsorption of some cations and anions is stronger and also the saturation of the stationary phase towards these ions As discussed above, the adsorption on the stationary phase of the cation in the IL increases with increasing length of its alkyl chain, whereas the adsorption of PF6 – is significantly stronger compared to BF4 – and Cl– This explains the similar trend in retention with mobile phases containing [C6 C1 IM][BF4 ] and [C6 C1 IM][Cl], where the retention decreases with increasing IL, which is the behaviour observed in the presence of SDS (Fig 3a) Meanwhile, the combined effect of BF4 – and an IL with shorter alkyl length ([C4 C1 IM][BF4 ]), in the absence of SDS, results in a nearly constant retention at increasing amount of the IL This behaviour is produced by the smaller adsorption of [C4 C1 IM]+ , compared to [C6 C1 IM]+ (both with a decreasing effect on retention), the latter being more competitive with respect to the adsorption of BF4 – (which would increase the retention) For [C4 C1 IM][PF6 ] added to mobile phases without SDS, the combined effect of cation and anion resulted in increased retention at low IL concentration and decreased retention at higher concentration (Fig 3b) The interpretation of this behaviour is not easy, due to the significant amount of both cation ([C4 C1 IM]+ ) and anion (PF6 – ), adsorbed on the stationary phase and dissolved in the mobile phase, giving rise to repulsion and attraction of the cationic solutes, respectively In this regard, the trend observed for [C2 C1 IM][PF6 ] is interesting, since the lower adsorption of an IL cation with shorter alkyl length ([C2 C1 IM]+ ) is combined with an anion that shows strong adsorption (PF6 – ) In this case, the retention increased at least until reaching the maximal concentration tested, which indicates that the adsorption of the anion (which attracts the cationic solutes to the stationary phase) is dominant Note that, in contrast, for [C4 C1 IM][PF6 ] and [C2 C1 IM][PF6 ], the retention always decreases in the presence of SDS, with the addition of IL 3.4 Effect of the IL cation and anion on the peak profiles Peak profiles in liquid chromatography are characterised by their height, position, width and skewness, the two latter depending on the values of the left and right peak half-widths The observation of the trend of peak half-widths is useful to evaluate the interaction kinetics of the solutes with the stationary phase Also, equations that allow predicting the profiles of the peaks in the chromatograms can be obtained, which are useful for optimisation purposes Fortunately, simple correlations can be established between peak half-widths and retention times, which in isocratic elution can be approximated to straight-lines When all solutes experience the same kinetics, such plots can be obtained with the half-widths/retention time data obtained with a mobile phase of fixed or variable composition [32,33] When the solutes experience different resistance to mass transfer to/from the column, the plots will exhibit significant scattering Half-width plots for the set of β -adrenoceptor antagonists are plotted in Fig for mobile phases containing SDS/1-butanol and five ILs with diverse cations and anions The plots were drawn with the information obtained for the set of solutes eluted with mobile phases of variable composition Table collects the characteristics of the plots: the slopes of the left (mA ) and right (mB ) half-widths, the sum of slopes (which describes the relationship of the width with the retention times) and the ratio of slopes (which is related to the asymmetry) The values should be compared with the results obtained with mobile phases of acetonitrilewater, SDS/1-propanol, and IL/acetonitrile [28] The presence of the additives (SDS and/or IL), in all cases, gave rise to a significant im- Fig Half-width plots (left, A (◦) and right, B (●)), built with the data obtained for the set of β -adrenoceptor antagonists with mobile phases containing: (a) 0.114 M SDS, 0.28% octane, and 8.15% 1-butanol, and (b) 0.156 M SDS, 0.28% octane, and 8.15% 1-butanol provement in the peak profiles with respect to the classical hydroorganic RPLC with acetonitrile-water This can be explained by the masking effect of the free anionic silanols in the silica-based stationary phases by the ionic additives (SDS and IL) In the presence of IL, the peaks were significantly more symmetrical compared to acetonitrile-water mixtures, especially for [C2 C1 IM][PF6 ], [C4 C1 IM][PF6 ] and [C6 C1 IM][PF6 ], in the presence of SDS, and for [C4 C1 IM][BF4 ] and [C4 C1 IM][Cl] without SDS (B/A = 0.9–1.1, see Table 1) The parameters of the half-width plots in Fig should also be compared with those obtained for an MELC mobile phase with octane, where the mean value of B/A = 1.0 For comparison purposes, Fig represents the plots for particular mobile phases in MELC with octane: 0.28% octane/8.15% 1-butanol in the presence of 0.114 M or 0.156 M SDS, for which the B/A values were 1.1 and 0.9, respectively N Pankajkumar-Patel, E Peris-García, M.J Ruiz-Angel et al Journal of Chromatography A 1674 (2022) 463142 Fig Experimental chromatograms obtained for mixtures of β -adrenoceptor antagonists with: (a) 0.10 M SDS, 0.01 M [C6 C1 IM][PF6 ], and 8.15% 1-butanol, (b) 0.114 M SDS, 1.14% octane, and 8.15% 1-butanol, and (c) 0.10 M SDS and 0.02 M [C6 C1 IM][Cl] Solute identity: (1) Atenolol, (2) carteolol, (3) acebutolol, (4) metoprolol, (5) oxprenolol, and (6) propranolol Column: XTerra-MS C18 (150 mm × 4.6 mm i.d., μm) (acknowledgement is given to The Royal Society of Chemistry for the reproduction of Fig 4f from Ref [27]) 3.5 Retention of basic compounds with SDS / IL mobile phases without organic solvent of 0.114 M SDS/1.14% octane/8.15% 1-butanol, optimised in previous work (Fig 6b) It was thus evident that the co-surfactant (1butanol) did not help to achieve the needed chromatographic resolution for the basic compounds when [C6 C1 IM][PF6 ] was added instead of octane Meanwhile, the studies in Section 3.3 indicated that the separation was dominated by attraction of the cationic solutes to the adsorbed SDS monomers and repulsion from the adsorbed IL cation in the stationary phase Therefore, the possibility of removing the alcohol from the mobile phase was considered We thought that the combined effect of both reagents (i.e., attraction to the anionic SDS monomer and repulsion to the IL cation) would be able to modulate the separation of the analytes, and yield an adequate separation without the need of the co-surfactant We must remember that the purpose of adding 1-butanol is to help with the stabilisation of MEs, but when an IL is used instead of an apolar organic solvent as octane, the presence of 1butanol does not seem to be so relevant in the formation of clear mixtures useful for RPLC (see Section 3.1) On the other hand, the retention of β -adrenoceptor antagonists using mobile phases containing 0.10 M SDS/0.01 M [C6 C1 IM][PF6 ]/8.15% 1-butanol was too short (below 10 min), and a significant overlap of the peaks of the set of compounds was observed (Fig 6a) The separation was indeed poorer compared to that achieved with the mixture N Pankajkumar-Patel, E Peris-García, M.J Ruiz-Angel et al Journal of Chromatography A 1674 (2022) 463142 According to this, a mixture containing only SDS and [C6 C1 IM][Cl] in aqueous solution was prepared, which gave rise to a clear solution able to be used as mobile phase with an RPLC column It should be noted that the retention times for the β adrenoceptor antagonists are rather long with aqueous mobile phases containing only micellar SDS or [C6 C1 IM][Cl] (without organic solvent) Thus, the retention times for atenolol, carteolol, acebutolol, metoprolol, oxprenolol and propranol eluted with 0.10 M SDS (without IL) from the XTerra column were 9.9, 14.3, 16.8, 34.8, 57.1 and 83.5 min, respectively, while with 0.02 M [C6 C1 IM][Cl] (without SDS), atenolol and carteolol eluted at 3.5 and 11.6 min, respectively, and metoprolol, oxprenolol and propranolol needed above 60 Fig 6c shows the chromatogram obtained for the mixture of six β -adrenoceptor antagonists, using an isocratic mobile phase containing 0.10 M SDS and 0.02 M [C6 C1 IM][Cl], without organic solvent The separation suggests that the aqueous mixtures of SDS and [C6 C1 IM][Cl] can be successful in the separation of mixtures of the β -adrenoceptor antagonists, with a favourable effect on the resolution and an analysis time below 30 However, the most remarkable aspect is that the separation was achieved in aqueous medium, using an IL with chloride as anion, without the need an organic solvent in the mobile phase The formation of transparent and stable MEs prepared with surfactant (SDS), co-surfactant (1-butanol), and apolar solvent (IL or octane), to be used in RPLC, was found less dependant on the concentration of co-surfactant, when octane was replaced with an IL Furthermore, SDS allowed more concentrated solutions of the ILs, which suggested the formation of stable structures In view of this behaviour and the fact that the addition of 1-butanol to the ME formed with SDS and [C6 C1 IM][PF6 ] yielded too short retention times, and poor resolution in the separation of the group of β adrenoceptor antagonists, the elimination of the co-surfactant from the mobile phase was proposed, the detailed study of which will be the subject of future work Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper Acknowledgements Work supported by Grant PID2019–106708GB-I00 funded by MCIN (Ministery of Science and Innovation of Spain)/AEI/10.13039/50110 011033 Ester Peris-García thanks the University of Valencia for the post-doctoral grant UV INVPREDOC16F1–384313 Conclusions In the literature, ILs seem ideal for replacing organic solvents used as oil phase in MEs, due to their attractive physico-chemical properties and lower toxicity However, reported MEs formed by IL, water and surfactant (and in some cases, an alcohol as cosurfactant) are usually prepared with non-ionic surfactants, such as Brij-35 and Triton X-100, instead of the anionic SDS [34] The work by Peng et al., published in 2017 [26], pioneered the use of MEs in RPLC, where the oil was replaced with an insoluble IL ([C6 C1 IM][PF6 ]), and the anionic surfactant SDS (quite unusual for the preparation of IL/w MEs) was combined with 1-butanol as cosurfactant The authors developed an analytical procedure for neutral phenolic acids In this work, the feasibility of using the IL/w ME recommended by Peng et al as mobile phase, for the RPLC analysis of a group of basic compounds (β -adrenoceptor antagonists), which are positively charged, was investigated The research was focused on the effect on retention times and peak profiles produced by imidazolium ILs with alkyl chains of increasing length (with n = 2, and 6), associated to Cl– , BF4 – , or PF6 – The research group had previously developed a detailed work on the interactions of cationic solutes with RPLC C18 columns using mobile phases containing aqueous solutions of imidazolium ILs, in the presence of acetonitrile Here a comparison is made of the effect of the cation and anion in diverse ILs, in the presence of SDS and 1-butanol, with respect to our previous work with mobile phases containing IL and acetonitrile instead of 1-butanol in the absence of SDS The study gives some insight on the retention mechanisms The anionic surfactant SDS was found to compete with the IL anions for column adsorption, the behaviour being similar to that found without SDS, when an IL cation showing strong adsorption is associated with a weakly adsorbed anion In these situations, the retention decreased by addition of an increasing concentration of IL Meanwhile, in the absence of SDS, the addition of an IL with a weakly adsorbed cation or a strongly adsorbed anion makes retention to remain constant or increase with a maximum at a particular concentration of IL On the other hand, with all tested ILs, the peak profiles of the basic compounds were improved, but the effect was stronger in the presence of SDS The peaks were completely symmetrical (B/A = 1.0–1.1) for [C4 C1 IM][BF4 ] and [C4 C1 IM][Cl], indicating an efficient masking of the silanol effect References [1] 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García-Alvarez-Coque, M.J Ruiz-Angel, Gaining insight in the behaviour of imidazolium-based ionic liquids as additives in reversed-phase liquid chromatography for the analysis of basic compounds, J Chromatogr... determination of solvent properties of room temperature ionic liquids, J Chromatogr A 1037 (2004) 49–82 ´ [10] U Domanska, General review of ionic liquids and their properties, in: Ionic Liquids in. .. cation in the IL increases with increasing length of its alkyl chain, whereas the adsorption of PF6 – is significantly stronger compared to BF4 – and Cl– This explains the similar trend in retention