There are numerous factors we have to consider when selecting an LC column. Among them is the stationary phase, but how do we pick between all of the available choices? If we understand our analytes, our analysis requirements, and can identify the best mode of separation, we are sure to get excellent chromatography.
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With so many stationary phase choices, it’s sometimes difficult to determine which is best for your analysis. When should you choose a reversed-phase stationary phase such as C18, Biphenyl, or FluoroPhenyl? When is it appropriate to use HILIC chromatography?
An important factor to consider when choosing stationary phases or techniques relates to the inherent properties of your target analytes. Common retention mechanisms in reversed-phase chromatography are dispersive interactions, polarizable pi-pi interactions, and hydrogen bonding. It is important to consider the characteristics of the stationary phase and the inherent properties of your analytes in order to drive purposeful method development.
If your compounds are hydrophobic and you do not need to separate any critical pairs, there’s a good chance that any reversed-phase stationary phase could meet your needs. However, if you need to separate isomeric analytes, you can take advantage of the unique shape-selective characteristics of C18, Biphenyl, or FluoroPhenyl. If your compounds are aromatic and moderately polar, such as morphine and its metabolites, Biphenyl is perfectly suited for your needs. If you need to retain low-molecular-weight compounds that can carry a positive charge, such as metformin, FluoroPhenyl can be used under high-organic starting conditions to take advantage of its cation-exchange capabilities.
If your compounds are too polar for reversed-phase chromatography, HILIC stationary phases such as HILIC-Si could be your answer. HILIC-Si uses a synergistic combination of both cation-exchange characteristics and polar partitioning to retain compounds such as paraquat, creatinine, and melamine.
Before choosing the stationary phase of your next analytical column, consider analyte properties and what mode of separation to use to effectively pair your column with your analysis. If you enjoy these videos, please like, share, and subscribe. You can post your ideas for a future tech tip in the comments below. Thanks for watching, and we’ll see you next time.