HPLC problems with very polar molecules

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Very polar molecules will cause problems if the correct HPLC column is not being used.

There’s a specific type of HPLC column for polar molecules. They are known as the polar embedded phase.

Currently, every major column company has developed their version of this technology. You can easily spot which columns have polar embedded phases because their names have to do with water – which is of course the most famous polar molecule. Here’s a few examples: aqua, aquasil, hydro, hydroselect, and AQ.  These columns are crafted with specific applications in mind.

Design and Composition  

The design of these columns is unique. Primarily, we can consider them as a type of C18 column. However, a distinct feature lies at the base of the carbon structure: a polar linkage, which might be a carbamate or an amide. This polar linkage is the key feature of polar embedded columns as it enables the column to function effectively in 100% pure water. Why? The polar embedded phase allows the hydrophobic carbon chains to stand up straight instead of collapsing. This feature is particularly beneficial for certain analytical applications.

Functionality in Water  

The ability to operate in 100% water sets these columns apart. They are not just capable of running in a fully aqueous environment; they excel in it. The columns maintain their efficiency throughout the day, a testament to their robust design. This makes them the preferred choice for analyzing highly polar molecules. These molecules, which often elute in the void volume during reverse-phase chromatography, are more effectively separated using these polar embedded phases.

Application for Weak Acids  

An intriguing aspect of these columns is their versatility, especially when dealing with weak acids. For instance, substances like acetic acid, typically challenging to separate via reverse phase, are easily handled by these columns. This broadens the scope of compounds that can be analyzed using a single chromatographic method.

Versatility of Use  

The versatility of the polar embedded phase columns is further highlighted by their compatibility with both polar and non-polar compounds. They are not limited to just aqueous solutions but can also handle up to 100% organic solvents. This dual capability allows for the analysis of a wide range of compounds in a single run, making these columns highly efficient and versatile.

Comparison with Other Columns  

While these polar embedded phase columns are highly efficient, it’s important to note that they are typically considered a secondary choice. The primary preference often lies with a high-quality base-deactivated C18 columns, particularly those with a clean surface and devoid of any residual silanols. These C18 columns are generally the first choice due to their broad applicability and reliable results.

Polar Endcap Phases  

An alternative to the polar embedded phases are the polar endcap phases. Though slightly different in technology, as they use a polar endcap instead of a polar linkage, they achieve a similar outcome. These columns are also capable of running in 100% water, further emphasizing the versatility and adaptability of modern chromatographic columns in handling a diverse range of analytical requirements.

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