**4. Chromatographic Techniques**

#### *4.1. High Performance Liquid Chromatography*

#### 4.1.1. IL Additives to the Mobile Phase

Liquid chromatography is the most commonly used technique for determining pharmaceuticals. Most of them are basic and their separation takes place in a reversed-phase using a silica-based column [140–142]. Unfortunately, this involves several serious problems during the analysis. The literature data indicate the main reason to be the presence of free silanol groups, which are negatively charged and can interact with positively charged basic analytes in an ion exchange reaction. Based on experimental research, it can be observed that this is often associated with problems with the resolution and shape of chromatographic peaks or a high retention factor. To prevent or minimize these deleterious effects, a mobile phase is used with additives for blocking free silanols [140]. The most popular additives are various types of amines, such as triethylamine (TEA), dimethyl-octylamine (DMOA) or buffers. The first researchers who noticed that ILs may also have suppressing properties against silanol groups were Kaliszan et al. [143]. In 2005 they published a paper in which they used an additive IL to the mobile phase in drug detection by thin layer chromatography (TLC) and reversed-phase liquid chromatography (RPLC) techniques. Since then, new publications have appeared systematically on similar topics (Table 3). However, considering the topic of ILs in drug determination, it should be highlighted that these works mainly focus on explaining the function of ILs in the suppression process and the drugs are less important as analytes. In addition, only a few works use biological [140,141,144–146] or environmental [142] samples as the matrices; in one, tablets were analyzed [147], but most often they were aqueous solutions [6,143,147–154].



[(C2H5)4N]: tetraethylammonium; [C8H17(SO4)]: octylsulfate; [C12MIM]: 1-dodecyl-3-methylimidazolium. (The other abbreviations explained under Table 2).

Focusing on the addition of an IL to the mobile phase in LC, it should be noted that the interpretation of the results requires consideration of the influence of both the IL anion and cation. Although the use of the term IL suggests that one large molecule is responsible for the effect, it should be remembered that in the mobile phase the IL dissociates into both the cation and anion, so their combined effect determines the final results. It should also be highlighted that despite the involvement of other physical and chemical factors in the separation process, the largest changes in the chromatogram can be seen when using different kinds of ILs [142]. Their basic mechanism during pharmaceutical analysis is the reaction of IL cations with free silanol groups, the repulsion of IL cations with cations of basic analytes, as well as the reaction of IL anions with cations of analytes [149]. Depending on their type, the mechanism may be a little different than described. The choice of cation, as in the case of extraction (see the Section 3.2) focuses on the selection of an appropriate imidazolium cation with a different alkyl chain length. In one work, the analysis of the imidazolium cation with two methyl substituents was also carried out [143]. The effect of the cation was studied by Herrera et al. [142]. They performed analyses for ILs with the same anion [BF4] and different cations (Table 3). The results showed that an IL with a longer alkyl chain causes a decrease in the retention factor and an increase in efficiency. The effect of changing the retention time is similar in all analyses of basic analytes. The explanation for this effect may be an increase in hydrophobicity along with an increase in the length of the alkyl chain [144]. In turn, in a publication concerning the analysis of β-lactam antibiotics, an increase in the length of the alkyl chain caused an increase in retention. Han et al. [152] highlighted that a different effect may be the result of weak acidic properties and large analyte structures (the decrease in retention in other publications concerned basic analytes). The ester moiety of the antibiotic competed more strongly with the IL used for adsorption, and therefore despite the use of the long alkyl chain of the cation, retention increased. Ubeda-Torres et al. [149] also suggested that the size of the cation is more important than its nature. To study the effects of the IL anion, in other experiments, the same cation but a different anion was used during optimizing the IL selection. The number of anions tested is much greater than cations. The most commonly used are [PF6], [Cl] and [BF4] anions, but the less popular [CH3(SO4)], octylsulfate ([C8H17(SO4)]) and [Nf2T] have also been tested (Table 3). The analysis provided several important facts. First, the [PF6] anion showed very strong adsorption on the column and had a stronger effect on the parameters than the present cation. This is probably the result of its strongly chaotropic character [148]. The [BF4] anion is also a chaotropic ion, but with less adsorption than [PF6]. For this reason [BF4] more often qualified for further parts of the experiment. The next popular anion [Cl] belongs to strongly hydrated ions, and does not react with the analyte and the stationary phase; in its presence, the cation is mainly responsible for the mechanism [151]. Although the literature data provide information on the effects of the use of individual anions and cations, their choice is not obvious, not only because of the often antagonistic effect of ions. The use of an ionic liquid, which significantly reduces the retention time, is often associated with a poorer peak shape or resolution. In addition, too short a retention time for biological samples is not recommended because of the interference of analytes and background signals. In turn, improving the shape of the peak is possible at the expense of a higher retention factor. Figure 7 shows the change in retention after the use of two different ILs. Despite the shortening of the retention time by [C6MIM][BF4], an IL with [Cl] was chosen for the study due to better resolution. Therefore, the choice of IL is a kind of compromise, and the choice depends on many factors, including the type and number of analytes, and the type of matrices [147]. The results also show the influence of other factors on the final results. One of the most important modifications is the change in IL concentration. It was observed that a higher concentration leads to an improvement in the shape of the peak and reduces the retention time. However, this effect is more complex. First of all, the crucial factor here is whether the anion or cation has a stronger impact. For example, if the [PF6] anion is used, which has strong adsorption on the stationary phase, the retention time increases, but if the low affinity [Cl] anion and the long alkyl chain cation are used, the retention time decreases. However, it was noted that both with increasing and decreasing retention the effect occurs already at a very low IL concentration, and occurs until the column is completely filled with the IL. When column saturation occurs, a further increase in the IL concentration in the mobile phase has less of an e ffect on the results. The retention time is constant or the e ffect is the opposite to the current one. The mechanism of action of the aforementioned [PF6] in such a situation is explained by the reaction in the stationary phase until the column is saturated, and the reaction of this ion in the mobile phase after its saturation, and consequently, to a decrease in retention time [148,150]. The e ffect of pH on ILs was also analyzed, and it was found that at a lower pH the retention time is high because a larger number of [H+] ions react with the IL anions and the elution power decreases. This can be both a disadvantage and an advantage, because on the one hand, the separation improves, but on the other hand, the analysis time is too long [152]. In another study, the purpose of which was to assess the e ffect of bu ffers on separation parameters in the presence and absence of ILs, it was observed that the IL is mainly responsible for the retention time, while the bu ffers more strongly a ffect the final e ffect without the addition of the IL. However, it should be mentioned that the IL [C6MIM] with a strongly adsorbing cation was used in the experiment [7]. Another publication also suggests that retention is a ffected by the ratio of unprotonated to protonated silanols [6]. As mentioned above, ILs are an alternative to other mobile phase additives. For this reason, the results of studies with the addition of these compounds and with the addition of ILs are compared. ILs are better than other additives in all tests, but it must be highlighted that TEA the most popular compound, also gives good separation results [151]. In addition, the competitive advantage of ILs over other additives is the lack of e ffect on pH and, as shown in the literature, the involvement of both cations and anions in suppressing the silanol interaction and improving the results. In addition, the use of ILs is also possible in hydrophilic interaction liquid chromatography (HILIC). The increase in the stationary phase surface polarity obtained after the addition of an IL is responsible for improving the retention and e fficiency parameters [146]. The verification of the positive e ffect of ILs was also presented in studies focusing more on the kind of columns used.

**Figure 7.** Simulated chromatograms for mixtures of the six TCAs using the C18 (**<sup>a</sup>**–**<sup>c</sup>**) and C8 (**d**–**f**) columns. Mobile phase composition: (**<sup>a</sup>**,**d**) 30% acetonitrile, (**b**,**<sup>e</sup>**) 30% acetonitrile/10 mM HMIM·Cl, and (**c** and **f**) 30% acetonitrile/10 mM HMIM·BF4. Peak identity: (1) doxepin, (2) imipramine, (3) nortryptiline, (4) maprotiline, (5) amitryptiline, and (6) clomipramine. Figure adopted from reference [147] with permission of the copyright holder.

The analyses were performed on monolithic columns [144], popular C8 and C18 columns (Figure 7) [147] and six commercially available stationary phases [148]. In each analysis, the addition of ILs improved the results. It was also noted that the results depend on the production process of columns, which decide about the number of free silanol groups. Thus, the best results during the application of ILs in LC are obtained for columns for which the result was worse when using a traditional mobile phase without the addition of an IL [148]. As already mentioned, the application of ILs in LC focuses on the reaction mechanism, and drug determination is not essential here. Apart from a small number of analyses for real samples, the quality of the developed methods is not confirmed by determining the validation parameters. Therefore, the aspects of linearity, repeatability or reproducibility are ignored. To our knowledge, only one work has performed validation [147]. Moreover, only a few anions and cations have been tested in the analyses. There is no information on the e ffects of less common ILs. In addition, not all works compare the results obtained for ILs with other popular additives. The application of ILs also has several limitations. Although they extend the life of the column by protecting the surface of the stationary phase, conditioning is necessary for several hours to remove adsorbed IL ions and return the column to the starting position [143]. Due to the involvement of both anions and cations in the separation mechanism, other unknown interactions with their participation may occur. In addition, the choice of detector is an important issue during the application of ILs to the mobile phase. The following detectors can be used: FL, UV or *diode array detector* (DAD), but it should be remembered that ILs have a natural ability for ultraviolet absorption, which may a ffect the final results or prevent the selection of the optimal wavelength for analytes [148,155]. Moreover, the use of mass spectrometry is very problematic, here. However, despite the inconveniences described above, the popularity of ILs is constantly increasing and they are being used in subsequent experiments

#### 4.1.2. Ionic Liquid Stationary Phases

The application of ILs as an addition to mobile phases is not the only way to use them in liquid chromatography. In 2004, the first stationary phase appeared with ILs immobilized on the silica surface [156]. However, despite progress in this area, the use of IL stationary phases is much less popular than IL additives to mobile phases. Several-stage binding reactions are the first step of column preparation, producing as a final product modified IL-silica adsorbents, which finally coat the stationary phase (detailed reaction descriptions can be found in the original papers) [157–159]. Based on previous experience, several similarities can be observed to the previous section. First, the use of an IL stationary phase is the result of the incorrect peak shape, separation, e fficiency and retention time obtained on traditional columns. As already mentioned, the same reasons concerned the use of ILs in the mobile phase. Secondly, research shows that both the cation and anion can be involved in the separation process. The imidazolium cation (single or multiple) is most commonly used to modify the stationary phase surface [160]. Furthermore, analytes were also separated on a column prepared using polymeric or chiral ILs [161,162]. There are many ways in the literature for obtaining IL-modified stationary phases based on various chemical reactions and substrates. However, their application is still not common. As mentioned, the number of publications is much smaller than the number of publications describing the suppression of free silanol groups by ILs present in the mobile phase.

This review focuses primarily on the determination of pharmaceuticals in biological and environmental samples, so it should be strongly highlighted here that the number of publications related to the determination of such analytes on IL columns by LC is negligible. Two such articles were published by Rahim et al. [163,164]. They prepared a stationary phase based on β-cyclodextrin and 3-benzylimidazolium tosylate as ILs for the enantioseparation of β-blockers and NSAIDs. The results confirmed an enhanced enantioseparation and better enantioresolution on the novelty stationary phase. Another publication in accordance with the criteria adopted in the review was published in 2019 by Xian et al. [165]. The stationary phase was prepared with photo-initiated thiol-ene click chemistry using the imidazolium cation and anion [Nf2T]. Then, on the prepared column, the sulfonamides were separated by mixed-mode HPLC (MHPLC). The results confirmed good performance and separation selectivity, and additional research on commercial columns proved that the IL is responsible for a shorter separation time (Figure 8). Although the determination of drugs using IL column modifiers is very rare, their application in the determination of vitamins [166], flavonoids [167], amino acids [168] and many other compounds shows that perhaps in subsequent years these methods will be extended also for such analytes.

**Figure 8.** Separation of a mixture of nucleosides and nucleic bases, sulfonamides and inorganic anions on Sil-NIM-CFS and Acclaim™ Mixed-Mode WAX-1 columns. (1). uracil; (2). uridine; (3). cytosine; (4). adenine; (5). cytidine; (6). sulfanilamide; (7). sulfamethoxypyridazine; (8). sulfadiazine; (9). sulfathiazole; (10) sulfamethoxazole.; (11). sulfacetamide; (12). potassium bromate; (13). potassium bromide; (14). potassium iodate; (15). sodium iodide; (**a**) Mobile phase for Sil-NIM-CFS: ACN/10 mM ammonium formate (92:8, *v*/*v*); Mobile phase for Acclaim™ Mixed-Mode WAX-1 columns: ACN/10 mM ammonium formate (80:20, *v*/*v*); Ph = 5.6, flow rate: 0.6 mL/min, detection wavelength: 254 nm. (**b**) Mobile phase for Sil-NIM-CFS: ACN/H2O (50:50, *v*/*v*), flow rate: 0.8 mL/min; Mobile phase for Acclaim™ Mixed-Mode WAX-1 columns: ACN/H2O (60:40, *v*/*v*), flow rate: 1.0 mL/min; detection wavelength: 254 nm. (**c**) Mobile phase for Sil-NIM-CFS: ACN/5 mM Na2SO4 (5:95, *v*/*v*), pH = 4.28; Mobile phase for Acclaim™ Mixed-Mode WAX-1 columns: ACN/50 mM Na3PO4 (50:50, *v*/*v*), Ph = 6.0; flow rate: 0.6 mL/min; detection wavelength: 210 nm; Injection volume: 40 μL, column temperature: 25 ◦C. Figure adopted with permission from [165].

#### *4.2. Other Chromatographic Techniques*
