Peak Shape Changes for a Previously Used Column

As discussed in this series, changes in peak shape are a common problem in HPLC analyses. Ideally, peaks should be symmetrical, with a Gaussian shape [D. R. Stoll, LC-GC N. Am. 39 (2021), pp. 353–362]. The symmetry of a peak may be quantified by calculating the USP tailing factor (T), as illustrated in Figure 1. A tailing factor of 1 indicates perfect symmetry, while values less than 1 are referred to as fronting and values greater than 1 as tailing. Many methods require that the tailing factors for all peaks must be within a specified range. Tailing factors that deviate significantly from 1 may decrease the resolution of peaks that elute close together, making integration more difficult [D. R. Stoll, LC-GC N. Am. 39 (2021), pp. 353–362].

When setting up to run an established method on a previously used column, a suitability test should be carried out to check whether the column is in good condition. This test should assess the ability of the column and system to produce acceptable pressure, retention times, peak areas, peak widths, and peak symmetry. A significant change in any of these responses from the results previously obtained on the column could indicate a problem with it. An example is shown in Figure 2, which employed Waters reversed-phase QC reference material (p/n: 186006363). This reference material, which contains seven compounds, was analyzed using a gradient method. Initially (Figure 2A), the USP tailing factors ranged from 0.90 to 1.27, in an acceptable range. After the column was removed from the system then later reconnected, the same test method was carried out, producing the chromatogram shown in Figure 2B. While the retention times, peak areas and column pressure were similar to the initial results, the USP tailing factors for all seven peaks were higher, ranging from 1.32 to 1.65.

As discussed in part 1, there are several possible causes of changes in peak symmetry, including issues with the HPLC system, the mobile phase, the sample, and the column [J. W. Dolan and L. R. Snyder, Troubleshooting LC Systems, Springer Science+Business Media, New York, 1989, pp. 385–420]. As previously discussed, a good starting point for troubleshooting is to carefully analyze the chromatograms to observe whether the change in peak shape is seen for all the peaks, or only some of them. When all the peaks in a chromatogram show similar peak shape changes, as in Figure 2B, possible causes include the presence of a void in the column, accumulation of sample matrix constituents in the column or a poor connection of the column to the HPLC system. Because the problem hadn’t been seen when using the column previously, it is unlikely that the column had a void or an accumulation of sample matrix. That leaves a poor connection to the HPLC system as the most probable cause. The column connections were reseated, and the suitability test method was carried out again. As shown in Figure 2C, the USP tailing factors were restored to the initial values. This confirms that the problem was caused by an improper column connection, causing a gap between the column and the tubing connecting it to the system (see Figure 3). Even a small gap is enough to cause peak shape distortion. For demonstrations of how to connect columns with different types of end fittings to several HPLC systems, see the series of videos at this URL: How to Connect HPLC Columns to Waters LC Systems