Use of Amino Acid-Based Ionic Liquids in Capillary Electrophoresis

Ionic liquids (ILs) are unique solvents with melting points at or below 100 oC. They have drawn scientific interest due to their unique properties that involve good thermal stability, miscibility in different solvents, tunable viscosity, conductivity, negligible vapor pressure, non-flamma‐ bility, and low toxicity. They have often been called designer solvents in order to indicate their large structural variability due to the cation or the anion of the salt. This, in turn, broadens their application area (chemical reactions, catalysis, electrochemistry, separation, etc.) [1-5].


Introduction
Ionic liquids (ILs) are unique solvents with melting points at or below 100 ºC. They have drawn scientific interest due to their unique properties that involve good thermal stability, miscibility in different solvents, tunable viscosity, conductivity, negligible vapor pressure, non-flammability, and low toxicity. They have often been called designer solvents in order to indicate their large structural variability due to the cation or the anion of the salt. This, in turn, broadens their application area (chemical reactions, catalysis, electrochemistry, separation, etc.) [1][2][3][4][5].
In the last few years, a big number of chiral ILs (CILs), have been designed, synthesized and used for applications in electrophoretic and chromatographic chiral discrimination [4,6]. They play a key role in enantioselective analysis because they combine the advantages of ILs with the properties of a chiral moiety, which can be anionic and/or cationic. Their utility in separation science as chiral selectors, additives, chiral ligands, and chiral stationary phases is becoming increasingly important [7].
Although a large number of reviews have been provided on the synthesis of CILs [8][9][10], a surprisingly limited number of articles have been published on their utility in analytical separations, and particularly in electrophoretic separations [7,11]. Capillary electrophoresis (CE) has been extensively used in chiral separations by using various chiral selectors, such as cyclodextrins (CDs), cyclofructans, oligo-and polysaccharides, polymeric surfactants, and others [12]. Some of the problems that limit their use involve low solubility, instability at high temperatures and/or low pH values, time-consuming organic synthesis procedures and high cost. The use of CILs is considered a potential alternative because they can dissolve various polar and nonpolar analytes, they may provide chiral selectivity, and their synthesis procedure is simple. In CE, the CILs are mainly used as BGE additives, and secondarily as chiral ligands and chiral selectors.
A new class of CILs, called amino acid ester-based ILs (AAILs), was synthesized and characterized in 2005 by Tao et al. [13]. AAILs consist of cations, which are derived from amino acids or amino acid esters, such as glycine (Gly), alanine (Ala), alanine methyl ester (AlaC 1 ), and alanine ethyl ester (AlaC 2 ), and commonly used anions, such as nitrate (NO 3 ), tetrafluoroborate (BF 4 ), lactate (Lac) and bis(trifluromethane)sulfonamide (NTf 2 ). Bwambok et al. [14] used a simple metathesis reaction to synthesize L-and D-alanine tert butyl ester-based ILs with several anions, and they studied their enantiomeric recognition properties by using fluorescence and nuclear magnetic resonance spectroscopy.
In this chapter, the ability of AAILs to be used as chiral and achiral media in CE is investigated. In particular, some representative studies that involve the utility of AAILs as background electrolyte (BGE) additives and as sole chiral selectors in electrophoretic separations are reported and discussed. These studies involve synthesis procedure, establishment of optimum separation conditions and method validation. The first part of this chapter involves the application of AAILs as BGE additives and the evaluation of their performance in both chiral and achiral analysis, while the second part demonstrates their chiral recognition ability for the enantioseparation of 1,1'-binaphthyl-2,2-diylhydrogenphosphate (BNP).

AAILs as background electrolyte additives
Most of the applications of AAILs ( Figure 1) in electrophoretic separations have been as additives in BGEs [15][16][17][18][19][20][21][22][23]. Hadjistasi et al. [15] were the first to report the use of an AAIL as an additive in the BGE to improve electrophoretic separations. In particular, the addition of the CIL D-alanine tert-butyl ester lactate (D-AlaC 4 Lac) into the BGE improved the resolution of the pipecolic acid enantiomers. This CIL provided an increase in resolution from 1.41, obtained when β-cyclodextrin (β-CD) was used as the sole chiral selector, to 1.87, obtained when both chiral selectors were added in the BGE. In 2013, two novel AAILs, tetramethylammonium-Larginine (TMA-L-Arg) and tetramethylammonium-L-aspartic acid (TMA-L-Asp), were applied, for the first time, in CE, with glycogen as the chiral selector, in order to evaluate their potential synergistic effect [16]. Glycogen is an electrically neutral and branched polysaccharide that has, over the years, proven its chiral recognition abilities for the enantioseparation of various basic and acidic drug compounds. In their study, it was observed that, when the TMA-L-Arg/glycogen and TMA-L-Asp/glycogen systems were applied, both resolution and selectivity were significantly improved in comparison to the single glycogen separation system. This, in turn, suggested the existence of the synergistic effect. In a more recent study, the vancomycin-based synergistic system with L-alanine and L-valine tert butyl ester bis(trifluoromethane)sulfonamide (L-AlaC 4 NTf 2 and L-ValC 4 NTf 2 ) as additives was evaluated in CE for the enantioseparation of five profens [18]. When the binary systems were applied, all enantioseparations were considerably improved, and the resolution values were greatly higher than in the case where vancomycin was used as the sole chiral selector. In this part of the chapter, two representative studies performed recently in chiral and achiral CE are briefly described [17,22].

Chiral analysis
The use of AAILs as BGE additives, for improved resolutions, selectivity factors, and efficiencies in chiral analysis, is demonstrated further here by providing a more in-depth analysis of a research work that was performed by Zhang et al. [17]. In their study, two AAILs (L-AlaC 4 NTf 2 and L-ValC 4 NTf 2 ) were applied as additives and β-CD derivatives (methyl-β-CD, hydropropyl-β-CD, glucose-β-CD) as chiral selectors in CE for the enantioseparation of six anionic racemic drug compounds.
The synthesis of both AAILs was accomplished by use of a one-step anion exchange reaction of the corresponding amino acid ester chloride and the bis(trifluoromethane)sulfonimide lithium salt [14]. Briefly, an appropriate amount of L-alanine and L-valine tert butyl ester hydrochloride and an equimolar amount of bis(trifluoromethane)sulfonimide lithium salt were separately dissolved in distilled water. Then, the two solutions were mixed and stirred for 2 h at room temperature. The mixture resulted in two layers, of which the lower layer was separated and dried under vacuum overnight. The resulted products were colorless liquids.
The main objective of their study was to evaluate the synergistic effect of the AAILs with the β-CD derivatives. It was proven to be significant for half of the analytes examined, and particularly for naproxen, pranoprofen and warfarin. Figure 2 demonstrates the electropherograms obtained when β-CD derivatives were used as the sole chiral selectors [(a)], and when AAILs were used as additives [(b) and (c)]. The novel synergistic system was optimized by using methyl-β-CD/AAILs as model systems. An important factor affecting the enantioseparation is the concentration of the chiral selector and the CIL, since both concentrations will determine the equilibria between the chiral selector, the CIL and the enantiomers. Initially, at a fixed concentration of 20 mM methyl-β-CD, as the concentrations of L-AlaC 4 NTf 2 and L-ValC 4 NTf 2 increased from 5 to 15 mM both resolution and effective selectivity factor improved. However, at the concentration of 20 mM, no peak was observed, even at 60 min. The electrophoretic mobility decreased dramatically, possibly due to the adsorption of the CIL cations onto the capillary walls.
The effect of the chiral selector´s concentration on enantioseparation was also studied by varying the concentration of methyl-β-CD from 10 to 50 mM. In the single methyl-β-CD separation system, resolution values increased upon increasing the concentration, due to an increase in the complexation between the chiral selector and the enantiomers. On the other hand, in the methyl-β-CD/AAILs systems, resolution was initially increased, and then decreased mainly due to the gradual complex saturation. Therefore, the optimum concentration of methyl-β-CD is lower with the presence of the synergistic effect in comparison to the single methyl-β-CD system.
The optimum BGE composition was also determined by examining two different systems (sodium sodium acetate-acetic acid and citrate-citric acid). The first BGE provided deformed peaks, while the use of the second one resulted in better peak shapes and better resolutions. The BGE pH was also investigated for the chiral recognition process, because the degree of protonation in the analytes and the AAILs depends on this parameter. It was observed that at pH values below 4.4 or above 5.6, resolutions of all the understudy chiral compounds were reduced. Therefore, a pH value of 5.0 was considered the optimum. According to the authors, at the particular optimum pH, in the present system of analyte/AAIL/methyl-β-CD, the following bindings occur: (a) hydrogen bonding among the hydroxyl function in methyl-β-CD, carboxyl group in the drug compounds and amino function in the AAILs and (b) ionic interactions between the carboxyl group in the analytes and the amino function in the AAILs.
Another important observation, in this study, was the improvement of both resolution and effective selectivity factor with the addition of an organic modifier, possibly due to a decrease in electroosmotic mobility, which, in turn, increases the interactions between the AAIL, methyl-β-CD and analyte. Other parameters, such as composition and pH of buffer system and applied voltage were also examined, and the optimum conditions included 15 mM AAIL, 20 mM methyl-β-CD, 30 mM sodium citrate/citric acid (pH 5), and 20 kV.

Achiral analysis
In a different study, the synergistic effect of sodium dodecyl sulfate (SDS) and L-alanine tert butyl ester lactate (L-AlaC 4 Lac) was examined by Mavroudi et al. [22] for the separation of 2arylpropionic acid non-steroidal anti-inflammatory drugs ( Figure 3). The BGE was supported with either SDS, or an AAIL or SDS/AAIL, and their performance was evaluated by comparing migration times, efficiencies and %RSD values. Many analytical CE methods have, so far, been used for the separation of a wide variety of NSAIDs, by applying different modes of CE. These include micellar electrokinetic chromatography (MEKC) with SDS as an additive, capillary electrochromatography (CEC) with poly(stearyl methacrylate-divinyl benzene) monolithic columns and non-aqueous CE [24][25][26]. In the study reported by Maria et al., the AAIL L-AlaC 4 Lac was applied in CE as a sole additive for the simultaneous separation of NSAIDs for the first time. A very important consideration in this study was whether the use of an AAIL as a sole additive resulted in more effective separations than in the case of using a common surfactant. Therefore, a comparison was performed by adding L-AlaC 4 Lac or SDS into the BGE (100 mM Tris/10 mM tetraborate decahydrate).
For the synthesis of L-AlaC 4 Lac, appropriate amounts of the corresponding amino acid ester hydrochloride and silver lactate were separately mixed with methanol. The amino acid ester solution and the suspended silver lactate were then mixed and stirred. Subsequently, the precipitate was filtered and removed, and the remained solution was evaporated in vacuo and purified by being crystallized in methanol/ether [14].
In the single SDS system, separation is achieved by differential partitioning of analytes between the hydrophilic core of the surfactant and the bulk aqueous phase via electrostatic and hydrophobic interactions, and hydrogen bonding. SDS was initially examined as a sole additive, by varying its concentration from 10 to 30 mM. It was observed from Figure 4 that, even though the migration times of all analytes increased along with the increase of SDS concentration, resolution values decreased dramatically. At 30 mM, a coelution of indoprofen and ketoprofen was observed, and carprofen did not elute, even at 30 min. This is because the predominant population, at pH 8, is in the anionic form, which is expected to be repelled by the negatively charged headgroup of the anionic surfactant.
For comparison purposes and for further optimization of the separation, an AAIL was used as an additive. The effect of its concentration on the separation of NSAIDs was first examined. The optimum concentration was determined according to resolution, efficiency and analysis time. As demonstrated in Figure 5, when a 20-mM concentration was used, the Rs values for the peak pairs carprofen-ketoprofen and flurbiprofen-ibuprofen were 1.3 and 1.2, respectively. Concentrations above 30 mM provided baseline separations with Rs values higher than 1.5. In addition, the total analysis time, in the cases of 20, 30 and 40 mM L-AlaC 4 Lac, was not altered (~ 9.5 min), while, from 50 to 70 mM, it was increased to ~ 12 min. Efficiency was determined by calculating the number of theoretical plates (N) for all peaks. It was observed that at a concentration of 40 mM, N for all peaks was very high, in comparison to the ones obtained when the other concentrations were applied ( Figure 6). Another important observation involved the elution order of NSAIDs, which was different from the elution order observed when the SDS was added in the BGE, probably due to the different types of interactions

Field Effect Electroosmosis -A Novel Phenomenon in Electrokinetics and its Applications in Capillary Electrophoresis
between the additive and the analytes. In the case of SDS, the interactions are based on hydrophobicity, while in the case of the additive L-AlaC 4 Lac, the separation is based on electrostatic interactions. pH is another important parameter that is necessary to be optimized because, firstly, alterations in pH can affect the analyte charge and, secondly, the primary amine group of the cation of the AAIL can be positively charged or neutral in several pHs. An increase in pH from 8.0 to 8.5, the resolution decreased from 2.9 to 1.5 for peaks carprofen-ketoprofen and from 1.7 to 1.3 for peaks flurbiprofen-ibuprofen. A further increase in pH (9.0 and 9.5) resulted in two coelutions and shorter migration times due to fewer electrostatic interactions between the AAIL and the negatively charged analyte, since the amount of the positively charged amino group is decreased. In addition, at high pH values, the AAIL may undergo ester hydrolysis, which results in the lack of the tert butyl group in the cation.
The reproducibilities were also evaluated and compared by calculating the relative standard deviation (RSD) values of the electroosmotic flow (EOF) and the migration times of all the analyte peaks. In particular, in both SDS and L-AlaC 4 Lac cases, the run-to-run RSD values were obtained from 10 consecutive electrophoresis runs. In the case of SDS, the RSD of the EOF was 2.1%, and the RSD values of the analytes ranged from 2.8% to 11.7%. In the case of L-AlaC 4 Lac, the RSD of the EOF was 0.4% and the RSDs of the NSAIDs ranged from 1.2% to 1.3% (Table  1). In the same table, a comparison between the two additives in regard to efficiency is also demonstrated. The efficiency of all analyte peaks was above 102,000 for L-AlaC 4 Lac, in comparison to SDS, which provided efficiency values between 47,000 and 76,000 theoretical plates.   A last consideration for this study involved the effect of the addition of both SDS and L-AlaC 4 Lac into the BGE on the separation of NSAIDs. A concentration of 10 mM SDS and different concentrations of L-AlaC 4 Lac were added into the BGE (1-40 mM). An increase in the concentration of L-AlaC 4 Lac resulted in a more effective separation of NSAIDs, in regard to efficiency and resolution, probably due to the synergistic effect of the SDS/L-AlaC 4 Lac system. However, the elution order and analysis time (~ 23 min) were similar to the ones obtained when the SDS was used as the sole additive. Therefore, it was clear from this study that the additive L-AlaC 4 Lac is considered an effective alternative to SDS for a reproducible, baseline, high efficient and fast separation of NSAIDs.

AAILs as chiral selectors
Although numerous studies reported the use of AAILs in electrophoretic enantiomeric separation, only one study was performed by using AAILs both as co-electrolytes and chiral selectors [27]. However, other CILs, that are not amino-acid based, have been added into the BGE and used as sole chiral selectors for the enantioseparation of a number of analytes [28][29][30][31].
Yuan et al. [28] used the IL (R)-N,N,N-trimethyl-2-aminobutanol-bis(trifluoromethanesulfon)imidate as the sole chiral selector in CE, GC and HPLC for the separation of fifteen enantiomeric compounds. It was used as an additive in a BGE of 20 mM Na 2 HPO 4 -NaH 2 PO 4 or 20 mM Na 2 B 4 O 7 . The resolution values varied from 0.60 for di-O,O' p-toluyl-tartaric acid to 6.80 for 3-benzyloxy-1,2-propane diol.
In another study by Tran and Mejac [29], the CIL S- The optimum separation conditions were established by altering different important parameters, such as the alkyl ester group, the anion, the configuration and the concentration of the AAIL. In their first study, the influence of steric hindrance on the enantiomeric separation of BNP was examined by applying separately as sole chiral selectors the AAILs L-AlaC 1 Lac, L-AlaC 2 Lac and L-AlaC 4 Lac at concentrations of 60 mM and 100 mM. It was observed that as the length and the bulkiness of the ester group increased, the resolution of BNP increased. In particular, the first AAIL did not demonstrate any enantioselectivity, while the second one was able to provide partial enantioseparation (R S : 1.09). However, when L-AlaC 4 Lac was used at both concentrations, a baseline separation was achieved with R S values of 1.94 and 2.43 ( Table 2). It is, therefore, concluded that the enantioseparation of BNP is favored in the presence of tert-butyl group, and steric hindrance is involved in the enantioseparation mechanism.
Two very important considerations in this study involved the effect of the anion and the configuration of the cation on resolution. Two different anions were used (Lac and NTf 2 ), which provided baseline separation (Table 2). In particular, the resolution obtained by use of L-AlaC 4 NTf 2 was slightly lower (R S =1.72) than the one obtained with L-AlaC 4 Lac (R S =1.94), possibly due to the low solubility of the first in water, which provides fewer free cations and less interaction with the analyte molecules. In addition, the results obtained with NTf 2 were not reproducible and provided an unstable baseline. As far as the cation configuration is concerned, the D-and L-AlaC 4 Lac were used at a concentration of 60 mM in order to compare their enantioseparation ability. It was observed that the R S values obtained were very similar (1.95 and 1.94), while the elution order was different. As expected, the chiral center of the cation is considered the main active center of the chiral selector. Therefore, the elution order of the enantiomers of the analyte is based on the configuration of the CIL. In another study for the optimization of the BNP enantioseparation, the effect of the concentration of L-AlaC 4 Lac on resolution was investigated. As demonstrated in Figure 7, resolution increased significantly from ~ 0.4 to 2.43 with increasing the concentration from 20 to 100 mM. It was also observed that an increase in the AAIL concentration resulted in a decrease in the electroosmotic flow mobility, probably due to the coating of the AAIL cations onto the capillary wall Figure 8. The electrophoretic mobility, μ EOF , was calculated according to: where L is the total capillary length, l is the effective capillary length (from the injection end to the detector), t nm is the migration time of the neutral marker and V is the applied voltage.
The last parameter examined was the BGE pH, which was important, since the cation of the CILs used seems to be pH depended. Resolution decreased from 1.94 to 1.29, upon increasing the pH from 8 to 8.5, while at higher pHs (9 and 10), no enantioseparation was observed. As mentioned in Section 2.2, an increase in the pH decreases the amount of the positively charged amino group, which consequently reduces the electrostatic interactions between the AAIL and the negatively charged analyte. In addition, at high pH values, and particularly pH 9 and 10, the AAIL may undergo ester hydrolysis. This results in the lack of the tert butyl group in the cation, which is an important factor for the particular enantioseparation. Finally, according to the authors, it is concluded from the above-mentioned studies that the enantioseparation  mechanism for this particular application is based on: (a) steric hindrance (tert butyl group), (b) electrostatic interactions (between the cation of the CIL and the negatively charged analyte) and (c) hydrogen bonding (hydrogen-bonding capability of the phosphate group in BNP).

Concluding remarks
In this chapter, the suitability of the AAILs in chiral and achiral CE analysis was evaluated. These new AAILs, which can be easily synthesized from commercially available reagents, proved to be efficient chiral additives for the enantioseparation of different analytes. Even though only a limited number of studies have, so far, applied AAILs as BGE additives and chiral selectors, it is easy to conclude from the data demonstrated in this chapter that the future of AAILs in separation science has a great deal of potential, and it is expected to expand significantly. Further research though is required in order to understand the chiral recognition mechanisms between the AAILs, the common chiral selectors and the enantiomers. This will, in turn, help us design even more effective AAILs for applications in chiral electrophoretic and chromatographic recognition.