How to judge when the column should be replaced with a new one.

Chromatographers often encounter such problems. When should the column be changed? Can the number of columns in the column reflect the state of the column? Determining the standard that a column should be “retired”, empirically, When the number of plates in a column is reduced to the percentage of the new column, you can think that the column can no longer be used. How do you judge that a column can no longer be used in your lab? What?

It is believed that every chromatographer cares about resolution. Resolution is an indicator of how well a column separates a particular sample. The core task of chromatography is to separate each peak. In practice, we need to obtain a narrow peak with a reasonable peak width in order to obtain a lower detection limit (this is no longer a problem, and now the column can meet this requirement) .

A better approach is to use a mixture of pressure, resolution, and peak shape to examine the state of a column. Pressure measurement is the easiest, and usually we determine the chromatographic method to meet the following requirements: with a new column, the column pressure is no higher than 2500 psi, and in any case the pressure does not exceed 3000 psi. Although modern LC systems can be used at higher pressures, system losses and leakage problems due to high pressure can be severe. When the pressure exceeds 3000 psi, consider replacing the 0.5 mm aperture online filter between the injector and the column. If the pressure is still high after replacing the filter, the filter or column of the column is already contaminated, which prompts us to change the column. Statistics show that more than 60% of column damage is due to excessive pressure.

Resolution is a very important indicator for our investigation of the column. As long as the two components can be separated from the baseline, there is no problem with the quantification of the analyte. For a symmetric Gaussian peak, a resolution of 1.5 results in a baseline separation; if Rs is between 1.7 and 2.0, it is better. If the peak tail is severe, a higher resolution is required. The benefit of using resolution as a benchmark for the column is that we can visually see the separation of adjacent peaks.

Another criterion for examining the quality of a column is the peak tailing. The peak shape of the manufacturer's test report is very beautiful, but this does not mean that the column analyzes the actual sample. The actual sample always contains components that cause more or less peak tailing. Most notable are compounds containing nitrogen atoms that bind very tightly to the free silanol groups on the silica backbone. As the column is used for longer, the bonded phase is lost and the tailing is more severe. However, the change in the tailing situation is not immediately apparent as the resolution changes. Based on this, it is best to set an upper limit for the tailing factor, such as the US Pharmacopoeia stipulates that the tailing factor does not exceed 1.8.

The best time to determine the column “discard” criteria is when we develop analytical methods or method validation. Usually, during the development of the analytical method you will test several columns and analyze enough actual samples. You know what happens when the separation deteriorates. I tend to use a wide range at the beginning. Our experience is this: if you tighten the system suitability requirements at the beginning, it is very difficult to relax the requirements after the method is certified. System suitability checks are performed prior to analysis of the actual sample to ensure that the chromatographic method meets the criteria acceptable to the analyst.