4.2.2. Thin-Layer Chromatography

ILs are used in Thin-layer Chromatography (TLC) as stationary phase modifiers, especially in the separation of basic drug compounds. The separation of drug compounds is carried out normally with the use of silica-based stationary phases; however, it is often impossible because of the effect of free silanols on their chromatographic retention [196]. In order to remove this undesirable phenomenon, methods are used such as protonation, the addition of traditional amino quenchers, and changes in the mobile phase composition in order to increase ionic strength. The solution to this problem may be the application of ILs as silanol suppressing agents. The first study on improving separation in TLC by using ILs as mobile phase modifiers was presented by Kaliszan et al. [197]. The e fficacy of using ILs as stationary phase modifiers in TLC has also been confirmed in research by Marszałł et al. [198]. The aim of this study was the application of imidazolium-based ILs to reduce the deleterious e ffects of free silanols on the LC separation of naphazoline nitrate. The authors used [C2MIM][BF4] and [C6MIM][BF4] as modifiers of the mobile phase. The results showed that ILs with short alkyl-chain lengths are e fficient suppressors of free silanols, which are considered to be responsible for the troublesome and irreproducible chromatographic determinations of basic compounds. In the next study, Kaliszan et al. [199] also reported that ILs of the imidazolium tetrafluoroborate class when added to mobile phases blocked silanols and provided excellent TLC separations of strongly basic drugs which were otherwise not eluted, even with neat acetonitrile as the mobile phase. The ILs used by Marszałł et al. [198] as mobile phase modifiers were also tested in the studies reported by Mieszkowski et al. [196,200]. In the first study, 1-alkyl-3-methylimidazolium-based ILs (tetrafluoroborate [C2MIM][BF4], L-(+)-lactate [C2MIM][LAC] and ethyl sulfate [C2MIM][ETOSO3]) were used as the mobile phase [196]. The subject of the research was the development of a new HPTLC method for the determination of perazine in oral tablets, and a comparative study between these three di fferent ILs with the same cation but di fferent counterions as additives to the mobile phase. In e ffect, among the selected ILs, the optimum distribution parameters, such as shape and quality of spots,

high precision, and accuracy in qualitative and quantitative determination, characterize the system, with [C2MIM][BF4] as the mobile phase modifier. Summarizing this study, it can be concluded that [C2MIM][BF4] is a valuable and e fficient suppressor of free silanols, which are responsible for unwanted interactions of chromatographic stationary phases in the determination of the above compounds. In the second study, the authors compared two TLC methods for the determination of haloperidol in oral drops—the pharmacopeia method (European Pharmacopeia 7.0) and an alternative with IL modifiers of the mobile phase. The addition of [C2MIM][BF4] to the mobile phase gave similar separation and quantitative results with no peak tailing compared to the mobile phase suggested by the European Pharmacopeia 7.0 [200]. Besides the silanol-suppressing potency of [C2MIM][BF4], a lack of interaction and interference with UV densitometric detection was observed. Research on the use of ILs in TLC was also conducted by Lu et al. [201], who used ILs as mobile and stationary phases of TLC to analyze berberine hydrochloride, tetrahydropalmatine and related Chinese patent medicine. In this study, the shape and value of target spots together with the developing duration were compared regarding four mobile phases which were a combination of the ILs ([C4MIM][OH]), [C4MIM][BF4], [C4MIM][Br], [C4MIM][PF6]) and methanol. Moreover, these IL mobile phases were compared with two traditional developing reagents, *n*-hexane-chloroform-methanol and *n*-butanol-acetic acid-water. As a result, it was found that [C4MIM][OH]-methanol has a simpler composition and is more suitable for the simultaneous analysis of two target constituents in a plate. Besides any extra pH additives, the shape of spots was ideal and no tailing occured. [C4MIM][OH] was also used as the stationary phase, which was synthesized based on silica gel. The quantitative method for this kind of IL stationary phase showed a good correlation coe fficient ( *R<sup>2</sup>* = 0.9971–0.9976), good repeatability (%RSDs of berberine hydrochloride and tetrahydropalmatine were 0.88% and 0.79%, respectively) and method accuracy in terms of 95.91–104.85% (berberine hydrochloride) and 96.02–102.18% (tetrahydropalmatine). Research into the application of ILs as mobile phases in TLC was published by Tuzimski and Petruczynik [202]. The aim of the study was the separation of ten components of a mixture of isoquinoline alkaloids: allocryptopine, berberine, boldine, chelidonine, papaverine, emetine, columbamine, magnoflorine, palmatine and coptisine, using a 2D-TLC (two-dimensional TLC) method. The first dimension used an aqueous mobile phase (RP) (80% methanol–water–0.05 <sup>M</sup>/L−diethylamine), and in the second dimension a normal phase (NP) (75% methanol, 24.75% ethyl methyl ketone–0.25% IL [C4MIM][BF4]). The addition of ILs to conventional mobile phases caused a decrease in zone broadening and improved the chromatographic resolution. As shown in the results of the experiments, very symmetrical spots and peaks and high system e fficiency were obtained. In conclusion, the authors proposed that mobile phase systems containing ionic liquids can be applied to the separation of isoquinoline alkaloids in other natural samples. The use of ILs as stationary phase modifiers can be an e ffective and more "green" alternative to classical mobile phases such as amines.

## 4.2.3. Supercritical Fluid Chromatography

Among the numerous applications of ILs, they can also be used in solvent systems composed of ILs and supercritical fluids with an emphasis on supercritical carbon dioxide (scCO2). The specificity of IL–supercritical fluid biphasic systems follows from the availability of several mechanisms for tuning the solvent properties of such systems—apart from the wide selection of IL cations and IL anions to tailor the IL properties, the operating temperature and pressure are also available as variables to adjust the density and the solvent power of the supercritical fluid phase [203]. In an ILs-scCO2 system the product recovery process is based on the principle that scCO2 is soluble in ILs, but ILs are not soluble in scCO2. Since most organic compounds are soluble in scCO2, with the high solubility of scCO2 in ILs, these products are transferred from the IL to the supercritical phase [204]. Ji et al. [99] applied the IL [C8MIM][PF6] and methanol as the extraction and dispersion solvents in a method for the determination of four NSAIDs—nabumetone, ibuprofen, naproxen and diclofenac—in tap water and drinks. The method was based on ultrasound-assisted ionic liquid dispersive liquid–liquid microextraction (US-ILDLLME) followed by ultra-high performance supercritical fluid chromatography (UHPSFC) coupled to a photo-diode array detector (PDA). The developed method showed rapid separation (2.1 min), good recoveries (81.37–107.47%) and enrichment factors (126–132). The LODs for the analytes were from 0.62 (naproxen) to 7.69 (ibuprofen) ng/mL. This developed procedure was applied to real water samples, tap water, soda, lemon juice and green tea drink. In soda drink, ibuprofen was detected with detection levels of 16.43 ng/mL.

Because SFC can be performed with both polar and nonpolar stationary phases, columns that are marketed for HPLC can be used in SFC [205]. The application of immobilized ionic liquids (IILs) as a class of stationary phases for packed column SFC was studied by Smuts et al. [206]. The authors studied the cation and anion effect. The research was conducted on different IILs: tripropylphosphonium, tributylphosphonium, methylimidazolium, benzylimidazolium, triphenyl-phosphonium and 4,4-bipyridyl while keeping the counteranion constant, and an immobilized tributylphosphonium with five different anions: acetate, trifluoroacetate (TFA), [Cl], perchlorate and [Nf2T]. The best stationary phase in terms of low retention and good separation efficiency was the IIL tributylphosphonium with the TFA counter anion. Furthermore, the acetate anion exhibited the worst retention time and repeatability, and took the longest to reach baseline stability. [Nf2T]- displayed poor efficiency in separations for tributylphosphonium-based stationary phases. Chou et al. [207] used covalently bonded 1-octyl-3-propylimidazolium chloride on a silica gel column for the simultaneous separation of acidic, basic and neutral compounds (fenoprofen, ibuprofen, acetaminophen, metoprolol, naphthalene and testosterone) using carbon dioxide subcritical/supercritical fluid chromatography. The data indicated that the IL-modified column, in terms of resolution, was clearly superior to commercial C18 columns. Also, the simultaneous separation of acidic, basic and neutral compounds via SFC was successful with a co-solvent content of 20% MeOH, a pressure of 110 bar, and a column temperature of 35 ◦C (Figure 9).

**Figure 9.** Separation of acidic, basic, and neutral compounds via SFC using the IL-modified column and a commercial C18 column. Figure adopted with copyright permission from [207].

In conclusion, it should be stated that ILs seem to be good replacements for volatile organic solvents, and the development of new applications utilizing ILs will increase. However, the high cost of ILs and lack of complete data on e.g., toxicity should be noted.
