Abstract
The kinetic performance of a bare silica and C18 phase prepared from the same sub-2μm and 3.5μm base materials were compared in the HILIC and RP mode using both charged and neutral solutes. The HILIC column was characterised using the neutral solute 5-hydroxymethyluridine, the weak base cytosine, and the strong base nortriptyline, the latter having sufficient retention also in the RP mode to allow comparison of performance. Naphthalene was also used as a simple neutral substance to evaluate the RP column alone. The retention factors of all substances were adjusted to give similar values (k′∼5.5) at their respective optimum linear velocities. Reduced van Deemter b-coefficients (determined by curve fitting and by the peak parking method, using a novel procedure involving switching to a dummy column) were significantly lower in HILIC for all substances compared with those found under RP conditions. Against expectation, c-coefficients were always lower in RP when compared with HILIC using sub-2μm particles. While measurement of these coefficients is complicated by retention shifts caused by the influence of high pressure and by frictional heating effects, broadly similar results were obtained on larger particle (3.5μm) phases. The mechanism of the separations was further investigated by examining the effect of buffer concentration on retention. It was concluded that HILIC can sometimes show somewhat inferior performance to RP for fast analysis at high mobile phase velocity, but clearly shows advantages when high column efficiencies, using longer columns at low flow velocity, are employed. The latter result is attributable to the lower viscosity of the mobile phase in HILIC and the reduced pressure requirement as well as the lower b-coefficients.
Highlights
Hydrophilic interaction chromatography (HILIC) has been gaining rapid acceptance for the analysis of hydrophilic/polar/charged solutes since the early work of Alpert et al [1] in 1990
In both HILIC and RP, the mobile phase strength was adjusted to give similar k (∼5.5) near their respective optimum linear velocities in order to minimise the corrections for extra-column bandspreading
Adsorption is a component of the retention mechanism in HILIC that is likely to be encouraged on bare silica columns which have less extensive water layers [4], especially with the rather low mobile phase water concentrations used in the present experiments [3]
Summary
Hydrophilic interaction chromatography (HILIC) has been gaining rapid acceptance for the analysis of hydrophilic/polar/charged solutes since the early work of Alpert et al [1] in 1990. In addition to the enhanced retention of polar compounds, there are several distinct advantages of HILIC over RP These include the volatility of ACN-rich mobile phases (ease of coupling and better sensitivity with mass spectrometry [11]), low operating pressures to achieve a given linear velocity [12], and different retention selectivity compared with RP [13], offering for example the potential for two-dimensional separations. Simple van Deemter plots show that sometimes, superior mass transfer is obtained in HILIC for basic compounds, due to the lower viscosity of the mobile phases used and resultant enhanced solute diffusivity, and that longer columns were applicable generating high numbers of theoretical plates in a reasonable analysis time [12]. The effect of buffer concentration on retention was investigated in order to inform the interpretation of the retention mechanism in these experiments
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