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May/June 2013


HILIC if the retention mechanism is consistent, in which case the relationship between ln k’ and 1/T is linear [6].


Although it has been demonstrated that the organic modifier/aqueous ratio is the predominant factor in providing the necessary separation selectivity in HILIC [6], the choice of stationary phase is also very important. Chirita et al. suggested a column selection scheme and applied it to neurotransmitters analysis [7]. They advocated choosing HILIC columns according to the nature of the interactions between analyte and stationary phase. Fine- tuning the separation by optimising the organic solvent content, the buffer concentration and the mobile phase pH should follow in the decision tree. This approach to HILIC method development highlights the importance of column selection. Given the fact that the stationary phases used in HILIC are quite diverse (Hemström and Irgum described more than forty separation materials used for HILIC applications [1]), choosing the optimal column can be very challenging.


Systematic studies of HILIC materials chemistries and the roles of their functional groups have been limited. A combination of these factors has lead to confusion and difficulties during HILIC column selection for method development. Pontén recently commented on the fact that users are under the impression that ‘HILIC columns’ are interchangeable, despite the difference in the chemical structure of the various HILIC stationary phases [8]. This misconception has probably been encouraged by the increasing interest and demand for HILIC methodologies which in turn resulted in an increase in the ‘HILIC’ branded products. HILIC column comparison studies have been undertaken in the last few years; however, these studies concerned specific classes of compounds and probed specific interaction modes, but without discussing partial structure selectivity [4, 9-12].


More recently, both Ikegami’s [13] and Irgum’s [14] groups, independently suggested two comprehensive and seminal characterisation studies to classify HILIC columns and investigate HILIC retention mechanisms, focusing on specific interactions. Irgum and co-workers designed a method based on selectivity factors for pairs of similar chemical compounds, one with properties promoting the particular interaction being assessed and the second one lacking such properties. The HILIC interactions characterised by Irgum and his


group were: hydrophilic, hydrophobic, electrostatic, hydrogen bonding, dipole-dipole,


-interaction and shape selectivity [14].


Ikegami’s group followed a similar approach but using different test compounds in their HILIC characterisation work [13]. The method they suggested could probe specific secondary interactions, namely: degree of hydrophilicity, selectivity for hydrophilic- hydrophobic groups, selectivity for positional and configurational isomers, evaluation of electrostatic interactions and evaluation of the acidic-basic nature of the stationary phases. The data from this study showed structure-selectivity relationship for the various HILIC phases and represent a good approach to HILIC column selection for when targeting separations whose analytes possess some of the same structural characteristics.


This testing scheme was applied in our laboratory, for examining columns with the following chemistries: bare silica, zwitterionic-, amino-, amide-, mixed-mode diol-, mixed-mode RP/anion- exchange/cation exchange (** in Table 1, Nanopolymer Silica Hybrid, NSH)- phases and a silica phase covalently modified with an hydrophilic group and an anion- exchanger (^ in Table 1).


The work presented in this paper can be divided into two main categories: the stationary phase characterisation and the investigation into the chromatographic parameters that have a major role in the HILIC selectivity for acidic and basic compounds.


Materials and Methods Chemicals and reagents


HPLC grade acetonitrile, water and toluene, analytical grade ammonium acetate, ammonium formate and Optima grade acetic acid were obtained from Fisher Scientific (Loughborough, UK). Uridine, 5- methyluridine, 2’-deoxyuridine, adenosine, vidarabine, 2'-deoxyguanosine, 3’- deoxyguanosine, uracil, sodium p- toluenesulfonate, N,N,N-


trimethylphenylammonium chloride, theobromine, theophylline, cytosine, cytidine, salicylamide, salicylic acid, aspirin and 3,4-dyhydroxyphenylacetic acid were purchased from Sigma-Aldrich (Poole, UK).


Chromatographic tests The chromatographic conditions were kept unaltered throughout the column


characterisation study; the mobile phase consisted of 90:10 (v/v)


acetonitrile:ammonium acetate (20mM on the column, pH 4.7). The flow rate was fixed at 0.5mL/min. UV detection was carried out at 254nm. The injection volume was 5µL. All runs were done with active thermostatting of the columns at 30°C. The columns assessed in this study are reported in Table 1. They cover a range of surface chemistry and physical properties (with regards to particle size and pore size). All the columns were from Thermo Scientific (Runcorn, UK).


Retention factors were determined as the average of six injections and toluene was used as an unretained marker (t0).


The chromatographic conditions used to investigate the effect of other experimental factors on retention had various mobile phase compositions, which were prepared by mixing the desired volumes of acetonitrile and stock buffer solutions. The pH of the salt solutions was adjusted before mixing with acetonitrile, but for the pH study only. The salt concentrations reported in the individual results sections refer to the final concentrations of the salt on the column. The flow rate was fixed at 1.0mL/min. UV detection was carried out at 228nm for the acid mixture and 248nm for the basic mixture. The injection volume was 5µL. The column temperature was maintained at 30°C. The columns assessed in this study are also reported in Table 1.


Instrument Chromatographic experiments were carried out on an Accela UHPLC system (Thermo Scientific, San Jose, USA). ChromQuest 5.0 (Thermo Scientific, San Jose, USA) was used to control the UHPLC system, and to process the chromatographic data.


Test Mixtures Characterisation tests


All the stock solutions for the individual test probes were prepared in mobile phase at 1mg/mL. The test mixtures comprised selected pairs of compounds that were expected to vary in their interactions with the


stationary phases, plus the t0 marker. A total of seven test mixtures were prepared and they were:


• test mixture 1: t0, uridine (U), 5- methyluridine (5MU)


• test mixture 2: t0, uridine, 2’-deoxyuridine (2dU) • test mixture 3: t0, adenosine (A), vidarabine (V)


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