No abstract

We have characterized a sulfobetaine stationary phase based on 1.7 μm ethylene‐bridged hybrid organic–inorganic particles, which is intended for use in hydrophilic interaction chromatography. The efficiency of a column packed with this material was determined as a function of flow rate, demonstrating a minimum reduced plate height of 2.4. The batch‐to‐batch reproducibility was assessed using the separation of a mixture of acids, bases, and neutrals. We compared the retention and selectivity of the hybrid sulfobetaine stationary phase to that of several benchmark materials. The hybrid sulfobetaine material gave strong retention for polar neutrals and high selectivity for methyl groups, hydroxy groups, and configurational isomers. Large differences in cation and anion retention were observed among the columns. We characterized the acid and base stability of the hybrid sulfobetaine stationary phase, using accelerated tests at pH 1.3 and 11.0, both at 70°C. The results support a recommended pH range of 2–10. We also investigated the performance of columns packed with this material for metal‐sensitive analytes, comparing conventional stainless steel column hardware to hardware that incorporates hybrid surface technology to mitigate interactions with metal surfaces. Compared to the conventional columns, the hybrid surface technology columns showed a greatly improved peak shape.

We compared the separation selectivities of 19 different hydrophilic interaction chromatography columns. The stationary phases included underivatized silica and hybrid particles, cyano-bonded silica, materials with neutral ligands such as amide, diol, pentahydroxy, and urea, zwitterionic sorbents, and mixed-mode materials with amine functionalities. A set of 77 small molecules was used to evaluate the columns. We visualized the retention behavior of the different columns using retention time correlation plots. The analytes were classified as cations, anions, or neutral based on their estimated charge under the separation conditions. This involved adjusting the dissociation constants of the analytes for the acetonitrile content of the mobile phase and experimentally determining the pH of the mobile phase containing 70% acetonitrile. The retention correlation plots show that the selectivity differences strongly depended on ionic interactions. Comparisons of the neutral stationary phases (e.g., diol vs. amide) showed more similar selectivity than did comparisons of neutral columns versus columns with cation or anion exchange activity (bare silica or amine columns, respectively). The zwitterionic columns did not behave as perfectly neutral. The correlation plots indicated that they exhibited either cation or anion exchange activity, although to a lesser degree than the silica and amine-containing stationary phases.

Stability as a function of mobile phase pH is an important consideration when selecting a chromatographic column. While the pH stability of reversed-phase columns is widely studied, there are relatively few reports of the stability of hydrophilic interaction chromatography (HILIC) columns. We evaluated the stability of silica and ethylene-bridged hybrid HILIC columns when used with mobile phases containing basic buffers. The predominant mode of column degradation observed in our studies was a decrease in efficiency due to voiding, resulting from the hydrolysis of the silica particles. Associated with this were increases in tailing factors. Retention factor changes were also noted but were smaller than the efficiency losses. The dependence of the rate of efficiency decrease on the key variables of temperature, mobile phase pH and water content were studied for an unbonded silica column. The effect of the acetonitrile concentration on the pH of the mixed aqueous/acetonitrile mobile phases was also investigated. Using conditions found to cause a 50% decrease in efficiency after approximately five hours of exposure to the basic solution, we evaluated eight different commercially available HILIC columns containing silica or ethylene-bridged hybrid particles. The results show large differences between the stability of the silica and ethylene-bridged hybrid particle stationary phases, with the latter exhibiting greater stability.

Interactions of certain analytes with metal surfaces in high performance liquid chromatography (HPLC) instruments and columns cause a range of deleterious effects, including peak broadening and tailing, low peak areas, and the formation of new peaks due to chemical reactions. To mitigate these effects, we have developed a novel surface modification technology in which a hybrid organic/inorganic surface based on an ethylene-bridged siloxane chemistry is applied to the metal components in HPLC instruments and columns. We demonstrate the impact of this technology on peak symmetry, peak area, and injection-to-injection and column-to-column reproducibility for several metal-sensitive analytes. We also show an example of the mitigation of an on-column oxidation reaction. A variant of this technology has recently been developed for size-exclusion chromatography of proteins. An example is shown demonstrating the use of this variant applied to size-exclusion columns for the separation of a monoclonal antibody monomer and higher molecular weight species. Together, these results highlight the importance of preventing interactions of analytes with metal surfaces in HPLC in order to achieve accurate and precise results.