HILIC-ESI-ITMS

Hydrophilic interaction liquid chromatography (HILIC) is a technique used to separate small polar compounds (e.g. (Hemström and Irgum, 2006; Buszewski and Noga, 2012).

HILIC was originally developed in 1990 (Alpert, 1990), although it wasn't until 2009 that this type of chromatography was used to characterise ambient aerosol (Stone et al., 2009).

Since 2009, however, this technique has remained relatively redundant within aerosol science, with very few studies reporting the use of HILIC for the characterisation of ambient aerosol (Kitanovski et al., 2011; Kitanovski et al., 2012a; Kitanovski et al., 2012b).

HILIC is considered to be a variant of normal phase liquid chromatography (NP-LC) and the opposite of reverse phase liquid chromatography (RP-LC) (e.g. (Guo and Gaiki, 2011;

Jandera, 2011; Moldoveanu and David, 2013)). Both NP-LC and HILIC consist of a polar stationary phase. However, unlike NP-LC, HILIC (analogous to RP-LC) uses aqueous organic solvents for the mobile phase composition, allowing this technique to be successfully coupled with ESI-MS (Cubbon et al., 2007). In contrast to RP-LC, HILIC elutes analytes in order of increasing hydrophilicity through increasing polar aqueous content (Alpert, 1990; Cubbon et al., 2007). The HILIC separation mechanism is still under debate (Wang and He, 2011; Xu et al., 2014). However, it is generally accepted that separation is achieved through liquid-liquid partitioning of an analyte from the hydrophobic mobile phase into a "water enriched" layer, close to the surface of the polar stationary phase (Alpert, 1990; Naidong, 2003; Buszewski and Noga, 2012). Once in this layer, the analyte can have intermolecular attractions (i.e. dipole-dipole, hydrogen bonding) with the polar stationary phase, resulting in retention. Increasing the mobile phase water content, results in the expansion of the water enriched layer; decreasing the strength of the intermolecular attractions between the analyte and the stationary phase, resulting in elution.

The three main types of HILIC stationary phases used include; neutral (e.g. cross-linked diol, cyano-propyl and amide), charged (e.g. amino) and zwitterionic (e.g. sulfobetaine and phosphorylcholine) (for further information see Jandera (2011) and Wang and He (2011)).

The neutral, cross-linked diol stationary phase is considered to be one of the best for HILIC (Olsen and Pack, 2013). The majority of HILIC stationary phases are bonded to a silica surface which have un-bonded residual silanols present (Buszewski and Noga, 2012). Un-bonded silanols can influence compound separation through the absorption of analytes, and in some cases, can result in irreversible absorption (Jandera, 2011). During the manufacturing process of the cross-linked diol stationary phase, a silylating reagent is used to block the un-bonded silanols (Jandera, 2011; Olsen and Pack, 2013). Furthermore, its

Chapter 2 - Development of HPLC-ITMS for Complex Samples

31 polymeric structure (Figure 2.1) effectively shields the un-bonded silanols from the analyte (Guo and Gaiki, 2011); minimising unwanted secondary interactions observed with other HILIC stationary phases (Guo and Gaiki, 2005; Hao et al., 2008; Jandera, 2011; Buszewski and Noga, 2012).

Figure 2.1 - Chemical structure of the HILIC cross-linked diol stationary phase with ether functionality. Displaying the hydrophobic (acetonitrile, ACN) and water enriched layer, and the shielding of a silanol by the polymeric cross-linked diol structure.

In this study, a LUNA HILIC cross-linked diol stationary phase was used to investigate its suitability for the separation of the small polar compounds shown in Table 2.1. Initially, a HILIC compatibility test was performed as recommended by the manufacture instructions.

The HILIC compatibility test consists of a gradient elution program, where the retention time of a compound dictates whether HILIC is suitable for its separation. The gradient elution compatibility test is shown in Figure 2.2. Any compounds eluted between 0 to 2.5 minutes (elution region 1) have little to no interaction with the stationary phase, and are thus not well suited for separation using HILIC. Conversely, compounds eluted between 2.5 to 10 minutes (elution region 2) are ideally suited for the application of HILIC. Finally, any compounds eluted between 10 to 12.5 minutes (elution region 3) have strong interactions with the stationary phase, and through modification of the water content, may also be suitable for application of HILIC.

CH₃

Chapter 2 - Development of HPLC-ITMS for Complex Samples

32 Figure 2.2 - Luna HILIC compound compatibility test, as recommend by manufacture instructions. Mobile phase; (A) = 90% acetonitrile, 10% of 50 mM ammonium formate or ammonium acetate; (B) = 80% water, 10% acetonitrile, 10%, 50 mM of ammonium formate or ammonium acetate. Compound elution region; (1) = little to no retention; (2) = ideal elution region; (3) = compounds strongly retained.

The HILIC compatibility test can be performed using either ammonium formate or ammonium acetate as the buffer. Initially 50 mM of ammonium formate was used. RP-LC was used prior to the investigation of HILIC. Of the seven external standards previously observed using RP-LC, only 4 were observed with the use of HILIC. These were; maleic acid (compound 1), citraconic acid (compound 2), citraconic anhydride (compound 4) and 5-methylfuran-2(3H)-one (compound 8). Only the deprotonated molecular species was observed for citraconic acid, and no adduct formation was observed for any of the identified species. All of these compounds eluted between 0 to 2.5 minutes, as shown in Figure 2.3.

Compounds 4 and 8 were observed to elute at the same time as the extra-column volume, at 1.4 minutes into the gradient elution. The extra-column volume (also referred to as dead volume) is the time it takes for the sample to reach the detector from its injection point if there are no interactions; including the connecting lines and the column. Thus, the elution of compounds 4 and 8 in the extra-column volume suggests there is no interaction of these compounds with the stationary phase. The other two compounds, maleic acid and citraconic acid, displayed a slight interaction with the stationary phase, although both compounds co-eluted at 2 minutes into the gradient. The use of 50 mM of ammonium acetate provided slightly better separation of maleic and citraconic acid. However, as observed with the use

0 2 4 6 8 10 12 14

Chapter 2 - Development of HPLC-ITMS for Complex Samples (compound 2, purple, m/z 129 (M-H)^-). B = Citraconic anhydride (compound 4, m/z 111 (M-H)^-). C = 5-methylfuran-2(3H)-one(compound 8, m/z 99 (M+H)⁺).

The lack of chromatographic separation is most likely the reason why only four compounds were observed using HILIC. This is supported by the observation of a higher intensity peak eluted in extra-column volume when the external standards were run individually, in comparison to the prior blank samples (Figure 2.5). This suggests that due to the lack of chromatographic separation, the concentration of the external standards were too low to be identified (i.e. masked by higher intensity species which are also eluting in the extra-column volume).

Vial 2 HILIC_93_01_4084.d: EIC 99 +All MS, Smoothed (2.29,1,GA)

A

Vial 1 HILIC_92_01_4082.d: EIC 111 -All MS, Smoothed (2.28,1,GA)

0 2 4 6 8 10 Time [min]

Vial 1 HILIC_92_01_4082.d: EIC 115 -All MS, Smoothed (2.28,1,GA) Vial 1 HILIC_92_01_4082.d: EIC 129 -All MS, Smoothed (2.28,1,GA)

Chapter 2 - Development of HPLC-ITMS for Complex Samples

The poor retention and resolution obtained with this column for the investigated standards has previously been observed in another study, with compounds of a similar chemical speciation, (see Nováková et al. (2009) for further information). In their study, the retention of ascorbic acid and dehydroascorbic acid was investigated using four HILIC columns of differing stationary phases; one of which was the LUNA HILIC. It was found that the LUNA HILIC displayed the lowest retention capability of all the stationary phases investigated (Nováková et al., 2009). However, it was noted that the retention of ascorbic acid and dehydroascorbic acid increased with very high concentrations of ammonium acetate, in excess of 100 mM (Nováková et al., 2009). In this study, numerous parameters such as the buffer concentration, pH, mobile phase composition and gradient elution were investigated. However, the best retention and resolution achieved was that shown in Figure

0 2 4 6 8 10 Time [min]

Vial 1 HILIC_92_01_4094.d: EIC 115 -All MS, Smoothed (2.23,1,GA) Vial 1 HILIC_92_01_4094.d: EIC 129 -All MS, Smoothed (2.23,1,GA)

0 2 4 6 8 10 Time [min]

Vial 1 HILIC_92_01_4094.d: EIC 111 -All MS, Smoothed (2.23,1,GA)

0 2 4 6 8 10 Time [min]

Vial 2 HILIC_93_01_4096.d: EIC 99.0 +All MS, Smoothed (2.23,1,GA)

A

B

C

Chapter 2 - Development of HPLC-ITMS for Complex Samples

35 2.4. Consequently, HILIC was deemed unsuitable for the separation of the small polar compounds shown in Table 2.1, resulting in the return to RP-LC.

Figure 2.5 - An example of the higher intensity peak observed in the extra column volume when the external standards were run individually, in-comparison to the prior blank sample.

Black = Total ion chromatogram (TIC) of 3-methyl-2(5H)furanone in acetonitrile at a concentration of 20 ppm (compound 5). Blue = TIC of acetonitrile (blank sample).