|A Practical, Selectivity Based Hydrophilic Interaction Liquid Chromatography (HILIC) Method Development Protocol
|Fundamentals in Separation Science & Sample Prep
|Dr Alan McKeown
|Advanced Chromatography Technologies Ltd
Abstract Information :
The ever increasing demands on chromatographers to rapidly produce fit-for-purpose separations require more efficient and methodical work processes. Whilst there are multiple common reversed-phase workflows available for method development, increasing numbers of scientists are using Hydrophilic Interaction Chromatography (HILIC), which is less understood practically and mechanistically. There is therefore a need to produce a rationally designed practical method development HILIC protocol, based on selectivity, to adequately explore the design space for a given analytical separation and maximise the chances of successful method development.
According to the resolution equation, selectivity is the most powerful parameter to optimise for successful method development. Any good method development procedure should therefore fully explore column and mobile phase selectivity to help identify suitable analytical conditions for the method. The HILIC protocol presented in this poster utilises acidic, basic and neutral stationary phase platforms, specially designed to maximise selectivity differences and suitable for a wide range of HILIC compatible analytes (i.e. logP ≈0 or less). Utilising these three stationary phases along with a variety of mobile phase pH's this protocol allows the analyst to methodically explore the analytical design space and identify a suitable column and mobile phase combination for the separation
The method development protocol was evaluated using a caffeine mixture spiked with related substances at 0.5% w/w and for the separation of methylmalonic acid and succinic acid. The compounds were injected onto three orthogonal stationary phases with two different pH values, specifically selected due to the physico-chemical properties of the analytes. The combination of mobile and stationary phases produced multiple chromatograms from which a suitable stationary phase/mobile phase combination can be selected. Further optimisation of parameters such as temperature and buffer concentration can then be assessed to obtain a final separation.