Hydrophilic interaction liquid chromatography, or HILIC, is a powerful mode of liquid chromatography (LC) that uses polar stationary phases and high-organic initial mobile phase conditions for the separation of polar analytes. Buffers are very important for HILIC because the inozation state of both the analytes and stationary phases will impact the separation. Here we walk through a routine to optimize your buffer concentrations.
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Now that you are starting HILIC LC method development, you have to decide on the mobile phases.
A good HILIC LC performance depends on the ionization state of both stationary phases and analytes, which are controlled by the pH of mobile phases. For a fast starting point, we recommend preparing at least three aqueous mobile phases with different pHs. The first one is 10 mM ammonium formate adjusted to pH 3 with formic acid solution, the second one is 10 mM ammonium acetate adjusted to pH 5 with acetic acid solution, and the third one is 10 mM ammonium acetate adjusted to pH 6.8 with acetic acid solution. By running isocratic or gradient elution starting with 90 or 95% acetonitrile, you should be able to tell which pH solution is better by just looking at the analyte peak shapes. With the pH of the mobile phase determined, you can then fine-tune the method by adjusting the buffer concentration and elution program. Sometimes you will find that higher buffer concentration can give rise to better peak shapes, but be aware that the buffer salt can precipitate by mixing with high content of organic solvent. And finally, for gradient elution, a good practice is to equally buffer the aqueous and organic mobile phases to keep a constant ionic strength during the run. If you can take good care of the pH selection and buffer concentration of the mobile phases, you are off to a good start. If you enjoy these videos, please like, share, and subscribe. You can post your ideas for a future tech tip in the comments below.
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