Hydrophilic interaction chromatography in nonaqueous elution mode for separation of hydrophilic analytes on silica‐based packings with noncharged polar bondings*

Abstract

Chromatographic effects of dedicated stationary and mobile phase variations in hydrophilic interaction chromatography (HILIC) were investigated using a set of nucleobases, nucleosides and deoxynucleosides as polar test solutes. Retention and selectivity profiles were comparatively mapped on four in-house developed silica materials modified with short alkyl chains (C4, C5) which carry hydroxyl functionalities (including diol motifs) as well as embedded sulphide or sulphoxide groups. These data were complemented by results obtained with two commercially available diol-type phases and a bare silica column. Besides elucidation of packing-related aspects this work concentrated specifically on extending aqueous HILIC (AQ-HILIC) to nonaqueous polar-organic elution conditions herein termed NA-HILIC. The exchange of the polar modifier water by various alcohols in ACN-rich mobile phases containing 5 mM ammonium acetate decreased the eluotropic strength of the resulting eluents. The gain in retention largely followed the order ethanol (EtOH)>methanol (MeOH)>1,2-ethanediol (Et(OH)2) and was accompanied by distinct effects on chromatographic selectivity. For example, on the most polar home-made packing the purine nucleoside selectivity guanosine/adenosine increased from 2.25 in the AQ-HILIC (kguanosine=8.3) to 7.33 (kguanosine=59) in the NA-HILIC mode when EtOH was employed as NA modifier while this value was 5.84 and 2.93 with MeOH and Et(OH)2, respectively (eluent: 5 mM ammonium acetate in ACN/modifier 90:10 v/v). Besides the type of protic modifier its percentage as well the retention and selectivity effects upon varying the ammonium acetate concentration and column temperature, respectively, were also investigated. Notable inter-column differences were found for all of these elution parameters. A mixed-mode retention model composed of partitioning and adsorption is proposed for both AQ- and NA-HILIC retention processes. The potential of (i) the implementation of novel polar bondings (such as ones containing sulphoxide functionalities) and (ii) the comprehensive exploitation of elution variables (type of protic modifiers, salt, etc.) for providing new selectivity increments to the separation of polar analytes in HILIC is emphasised.

Selectivity of arginine chromatography in promoting different interactions using synthetic oligonucleotides as model

“…Temperature is a parameter that might significantly influence oligonucleotide interactions with arginine matrix and consequently affect the retention, or even the chromatographic selectivity [28]. The elution profiles obtained when loading an oligonucleotide sample (e. g. monoA or polyA30) onto the arginine-agarose column have shown that oligonucleotide retention increased when the temperature was increased (Table 1).…”

Development and evaluation of new imidazolium-based zwitterionic stationary phases for hydrophilic interaction chromatography

“…It was reported that the negative values of H • indicated an exothermic, partitioning retention process and an endothermic process based on adsorptive interactions superimposed on HILIC partitioning mechanism would result in positive H • values [33,50,51]. However, the above discussion on the effect of pH and water content in mobile phase indicated that the adsorptive interaction exists authentically between the stationary phase and the solutes, especially for ionized solutes.…”

LC–MS Analysis of Sertraline and Its Active Metabolite in Human Serum Using a Silica Column with a Non-Aqueous Polar Mobile Phase

“…The column used was unmodified silica, 100 9 3.2 mm (3 lL) while the mobile phase consisted of acetonitrile/methanol modified with ammonium hydroxide and glacial acetic acid (80/20, v/v) at a flow rate of 0.5 mL min -1 . Technically speaking, these conditions are defined as non-aqueous hydrophilic interaction liquid chromatography or NA-HILIC [18] which, in general, is used to chromatograph highly polar compounds on normal phase columns under conditions conducive to LC-MS, i.e., without the use of ion pair reagents or other harsh chemicals. In this instance, neither compound of interest is particularly polar, nor is the problem that of a lack of hydrophobicity under reverse-phase conditions.…”