Chapter 1
Analysis and Separation Techniques

1.1 High Performance Liquid Chromatography

1.1.1 Ionic Liquids as Mobile Phase Additives

The popularity of ionic liquids has grown in several analytical separation techniques. Thus, the reports concerning the applications of ionic liquids are still increasing. The use of ionic liquids, mainly imidazolium-based, associated with chloride and tetrafluoroborate as mobile phase additives in high performance liquid chromatography (HPLC) has been reviewed (1).

Mostly, ionic liquids just function as salts, but keep several kinds of intermolecular interactions, which are useful for chromatographic separations. Both cation and anion can be adsorbed on the stationary phase, creating a bilayer. This gives rise to hydrophobic, electrostatic and other specific interactions with the stationary phase and solutes, which modify the retention behavior and peak shape (1).

1.1.1.1 Imidazolium Compounds

The beneficial effects of several ionic liquids as mobile phase additives in HPLC using an electrochemical detection for the determination of heterocyclic aromatic amines have been evaluated (2). The tested ionic liquids were 1-butyl-3-methylimidazolium tetrafluoroborate, 1-hexyl-3-methylimidazolium tetrafluoroborate, and 1-methyl-3-octylimidazolium tetrafluoroborate. These compounds are shown in Figure 1.1.

Figure 1.1 Ionic liquids.

Several chromatographic parameters have been evaluated in the presence or absence of ionic liquids, or using ammonium acetate as the most common mobile phase additive, with three different C18 stationary phases. The effect of the acetonitrile content was also studied. Acetonitrile is shown in Figure 1.2.

Figure 1.2 Acetonitrile.

Best resolution, lower peak-widths, and lower retention factors were obtained when using ionic liquids rather than ammonium acetate as mobile phase additives. The best chromatographic conditions were found when using 1-butyl-3-methylimidazolium tetrafluoroborate as the mobile phase additive (2).

1-Butyl-3-methylimidazolium chloride, cf. Figure 1.3, 1-octyl-3-methylimidazolium chloride, and 1-decyl-3-methylimidazolium chloride were used as mobile phase additives in the HPLC to simultaneously separate phenoxy acid herbicides and phenols at neutral pH (3). It was found that when using 1-butyl-3-methylimidazolium chloride, a good baseline separation and good chromatograms for all the acid compounds were obtained on a normal reversed phase C18 column.

Figure 1.3 1-Butyl-3-methylimidazolium chloride.

The retention time of the target acid compounds was shortened with the increase of the alkyl chain length and the concentrations of ionic liquids, probably due to the delocalization of the positive charge on the imidazolium cation, the repulsion between chlorine ions of ionic liquids and the acid compounds, as well as the stereohindrance effect (3).

Extraction of Sudan Dyes.   Sudan dyes are typically used as coloring additives in the manufacturing of wax, textile, and floor and shoe polishes (4, 5). Sudan I has been classified as a category 3 carcinogen by the International Agency for Research on Cancer (IARC). Also, Para Red could be a genotoxic carcinogen (6). The structures of the coloring additives are shown in Figure 1.4. The chemical names of the dyes are summarized in Table 1.1.

Figure 1.4 Sudans and Para Red.

Table 1.1 Chemical names of the dyes.

A method for the analysis of such dyes has been developed. The method is based on coupling of ionic liquid-based extraction with HPLC. In this way, Sudan dyes and Para Red in chili powder, chili oil, and food additive samples can be found.

Two ionic liquids, i.e., 1-butyl-3-methylimidazolium hexafluorophosphate, cf. Figure 1.5, and 1-octyl-3-methylimidazolium hexafluorophosphate have been compared as extraction solvents. It was found that 1-octyl-3-methylimidazolium hexafluorophosphate showed higher recoveries for each analyte.

Figure 1.5 1-Butyl-3-methylimidazolium hexafluorophosphate.

Also, the conditions for the extraction of Sudan dyes and Para Red were optimized. Under optimal conditions, a good reproducibility of extraction performance was obtained, with relative standard deviation values of 2.0-3.5% (7).

The ionic liquids were prepared according to a previously reported method (8, 9). The Sudan dyes and Para Red standard solutions were obtained from Zhejiang Entry-Exit Inspection and Quarantine Bureau (Hangzhou, China).

The detection limits and the recoveries are summarized in Table 1.2.

Table 1.2 Detection limits and recoveries for Sudan dyes and Para Red (7).

Nucleotides Separation.   A method for the separation of nucleotides has been developed (10). These nucleotides include 5´-monophosphate adenosine, 5´-monophosphate cytidine, 5´-monophosphate uridine, 5´-monophosphate guanosine, and 5´-monophosphate inosine. Some of these compounds are shown in Figure 1.6.

Figure 1.6 Nucleotides.

The essential feature of the method is that 1-alkyl-3-methylimidazolium salts are used as mobile phase additives, resulting in a baseline separation of nucleotides without the need for gradient elution and organic solvent addition, as usually used in reversed phase HPLC (10).

Amine Separation.   By varying the lengths and branching of alkyl chains of the anionic core and the cationic precursor, it is possible to design solvents for specific applications. Because of these characteristic properties, ionic liquids are widely used as new solvent media in heterogeneous catalysis, synthesis, electrochemistry, sensors, battery applications, analysis and separation techniques (11).

Some amines, including benzidine, benzylamine, N-ethylaniline and N,N´-dimethylaniline could be separated using ionic liquids as additives for the mobile phase in HPLC (12).

The compounds 1-ethyl-3-methylimidazolium tetrafluoroborate ([EMIm][BF4]), 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIm][BF4]), 1-hexyl-3-methylimidazolium tetrafluoroborate ([HMIm][BF4]) and 1-butyl-3-methylimidazolium bromide ([BMIm]-[Br]) were used as ionic liquids. Some of these compounds are shown in Figure 1.7. Some properties are summarized in Table 1.3.

Figure 1.7 Ionic liquids.

Table 1.3 Properties of certain ionic liquids (8, 12, 13).

The effects of the length of alkyl chain or counterions on different ionic liquids and their concentrations on the separation of these analytes have been assessed (12).

The differences between the ionic liquids and tetrabutyl ammonium bromide on the separation of o-phthalic acid, m-phthalic acid, and p-phthalic acid have been compared. The results indicated that ionic liquids act as ion-pair reagents, although their hydrophobicity and hydrogen bonding also play important roles (12).

Catecholamines.   The use of 1-alkyl-3-methylimidazolium salts and N-butyl-pyridinium salts as mobile phase additives for the separation of catecholamines in reversed phase HPLC has been reported (14). As catecholamines, norepinephrine, epinephrine and dopamine were investigated. These compounds are shown in Figure 1.8.

Figure 1.8 Catecholamines.

A good separation could be achieved with these additives. The effects of pH of the mobile phase, the...