Improve resolution with eXtended Performance (XP) columns

Mia Summers, Kenneth Berthelette and Kenneth J. Fountain

aims

XP 2.5 μm columns have demonstrated better separation performance than traditional HPLC particle sizes in response to separation challenges.

background

It has become a consensus to shift the method to a smaller particle size to shorten the analysis time. In fact, this can also improve the resolution. However, as the particle size becomes smaller, the column pressure will increase. Use sub 2μm UPLC ^® column system may be required, but the user by HPLC HPLC method was transferred to eXtended Performance (XP) on 2.5 μm column, the same degree of separation achieved significant improvement aspects. This is especially advantageous for the separation of complex mixtures, which can be utilized to identify impurities or target compounds with high resolution provided by small particle size columns without the need to increase column length and run time.

Use the extended erformance (XP) column to increase the resolution of related compounds.

Taking a mixture of abacavir-related substances as an example, it was confirmed that the XP 2.5 μm column can improve the resolution. Abacavir is a nucleoside reverse transcriptase inhibitor that is used in the treatment of HIV. The mixture of abacavir-related substances contains five compounds, the main component of which is abacavir, here the glutarate (Figure 1). The separation of abacavir from its trans isomer is very difficult. This example demonstrates a comprehensive improvement in the separation of abacavir from its related substances by comparing a 3.5 μm column with a highly efficient XP 2.5 μm column.

solution

In order to completely separate the relevant substances while avoiding the development of a large number of HPLC methods, high-efficiency columns with higher separation capacity should be selected. XP columns are packed with 2.5 μm particles and filled under high pressure in ultra-high performance chromatography hardware. The back pressure range is also suitable for use on HPLC systems.

To demonstrate the improved separation performance with XP columns, we tested a mixture of abacavir-related substances using a 100 mm 3.5 μm, XSelect ^® CSHTM C ₁₈ column. Run the same method on an XP 2.5 μm column of the same chemical nature and size. The separation effect comparison is shown in Figure 2.

In this case, simply replacing a 3.5 μm column with an XP 2.5 μm column can significantly improve separation performance. In the end, the overall peak capacity increased by 31% and the peak height increased by as much as 42%. An increase of 28% resolution indicates that the separation between abacavir and trans abacavir, which is difficult to separate, can be improved by using a small particle size column. This example demonstrates that XP columns improve sensitivity and resolution, making identification and quantification of target compounds such as impurities more accurate.

to sum up

Transfer HPLC to an XP 2.5 μm column for improved resolution and sensitivity. Through the analysis of abacavir-related substances, after converting a 3.5 μm column to an XP 2.5 μm column, the peak capacity increased by 31% overall, the peak height increased by 42%, and the resolution increased by 29%. The use of XP columns can help HPLC users maximize the separation performance of HPLC systems and reduce the need for further method development, resulting in increased cost efficiency and asset utilization.