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Why Column Equilibration, and Factors Affecting RPLC Column Equilibration



Greetings and welcome,


I'm Bhaskar Napte, representing Pharma Growth Hub, and today, we'll delve into the various factors that play a pivotal role in column equilibration, particularly in the context of reverse-phase liquid chromatography.


Column equilibration is an essential step that must be completed before embarking on an analysis or when working with a new column. In this article, we'll elucidate why column equilibration is indispensable and explore the key factors influencing this process.


First and foremost, it's crucial to replace the column's storage solvent. When dealing with a new column, it may still contain remnants of the shipping solvent, which needs to be replaced. Even for older columns, before storage, you should replace the mobile phase with the storage solvent. Subsequently, this storage solvent must be substituted with the mobile phase. This emphasizes the necessity of column equilibration.


The second critical factor is establishing equilibrium between the mobile phase and the stationary phase. The mobile phase interacts with the stationary phase, and achieving equilibrium is pivotal. To attain this equilibration, it is imperative to subject the column to equilibration with the mobile phase.


The third point is especially intriguing and significant – it involves preparing the stationary phase to facilitate analyte partitioning. In the case of reverse-phase liquid chromatography, the column inherently possesses hydrophobic or nonpolar properties. If the mobile phase doesn't sufficiently interact with this hydrophobic stationary phase, the analyte cannot reach the active sites within the bonded stationary phase, such as C8 or C18. Properly preparing this bonded phase through effective column equilibration with the mobile phase is indispensable for generating reliable results. This influences factors like consistent retention time, response resolution, and baseline quality. Thus, the need for proper column equilibration is paramount to achieving dependable outcomes.


What happens if column equilibration is not executed correctly? Improper column equilibration can lead to a variety of issues, including baseline disturbances, variations in retention time, significant shape distortions, poor resolution, fluctuating response, and increased pressure, which may result in ghost peaks or spikes during chromatographic runs.

Now, let's delve into the factors that influence the column equilibration process. You might have heard that a minimum of 10 to 20 column volumes is necessary for proper column equilibration.


The first factor to consider is the nature of the stationary phase. Stationary phases with bulky groups, like phenyl, require longer equilibration due to their resistance to the passage of the mobile phase through the stationary phase, resulting in extended equilibration times. In contrast, faster column equilibration is preferred for stationary phases with lower surface areas.


Pore size is another crucial factor. Columns with wider pore sizes have lower surface areas and thus shorter column equilibration times. For instance, compare a column with a pore size of 70 angstroms to one with 400 angstroms – the latter will require less equilibration time due to its wider pore size.


The composition of the mobile phase also plays a significant role. Mobile phases with high buffer and ion pair reagent concentrations necessitate more time for column equilibration, as it takes longer for equilibrium to be established between the stationary and mobile phases. In cases where ion pair reagents are present, additional equilibration time is essential.

The amount of aqueous phase in the mobile phase is a critical factor in reverse-phase liquid chromatography.


More aqueous phase content can delay the wetting of the stationary phase, requiring a longer equilibration time to ensure proper wetting of the entire stationary phase.


Column temperature is another factor to consider. Elevated column temperatures expedite column equilibration by increasing the mobility of the mobile phase, reducing its viscosity.

For gradient elution, maintaining buffer concentration throughout the entire run by including buffer or salt in both mobile phase reservoirs ensures faster column equilibration between runs and a more stable baseline.


A useful tip: when initiating column equilibration with a buffer-containing mobile phase, begin with a proportionate mixture of water and organic solvent for a period before transitioning to the exact mobile phase. This can significantly reduce column equilibration time.


Thank you for your attention, and eager to hear your thoughts on this article.


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