Does Smaller Column Particle Size Always Lead to Better Resolution in Liquid Chromatography?

Column selection plays a pivotal role in achieving optimal separation in liquid chromatography (LC) method development. Key parameters of a column, including stationary phase, column length, diameter, particle size, and pore size, directly influence resolution.

It is adhered that for columns of the same length, smaller pore size generally results in better separation. But is it always true under identical conditions? Let’s explore this question with two real-world examples.

In this analysis using an Xtimate® C18 column (2.1 × 100 mm, 1.8 µm), the retentions of these five compounds are weak, and DA and MN are not separated at all on the chromatogram (Fig 1). While these compounds could be resolved by mass spectrometry, clinical testing demands chromatographic separation for accurate quantification.

Switching to an Xtimate® C18 column of the same dimensions but with a larger particle size (2.1 × 100 mm, 3 µm) significantly improved retention and achieved complete separation (Fig 2). This outcome demonstrates that increasing particle size can enhance resolution when polar analytes exhibit weak retention on smaller-particle columns.

In this case, impurities A and B were analyzed using an Ultisil® AQ-C18 column (4.6 × 250 mm, 3 µm). Despite extensive optimization, the separation factor between these two impurities lingered around 1.2, insufficient for regulatory compliance (Fig 3).

Switching to an Ultisil® AQ-C18 column with a larger particle size (4.6 × 250 mm, 5 µm) and slightly adjusting the flow rate (0.8 -> 1.0) led to a significant improvement in separation, with a resolution of 1.6 (Fig 4). This change satisfied analytical requirements.

The Van Deemter equation offers insight into these results. The first term in the equation, eddy diffusion, is influenced by particle size. Larger particles create a more tortuous path for analyte molecules, increasing the retention time and improving separation under certain conditions.

For Application 1, the polar catecholamines exhibited weaker elution strength on the 3 µm UHPLC column, enabling better resolution compared to the 1.8 µm one. The reduced elution strength allowed for longer retention and enhanced separation.

For Application 2, differences in optimal flow rates between the columns were critical. The 5 µm column’s optimal flow rate of 1 mL/min was achievable with conventional HPLC systems, while the 3 µm column required a higher flow rate (1.67 mL/min), exceeding the system’s pressure limits. Consequently, the 3 µm column operated far below its optimal flow rate, compromising resolution. The 5 µm column, operating at its optimal flow rate, outperformed the 3 µm column in resolution.

These examples illustrate that particle size alone does not dictate resolution. When separation challenges arise, try experimenting with both larger and smaller particle sizes, and unexpected benefits may be yielded.