Troubleshooting Peak Shape Problems in HPLC

Troubleshooting Peak Shape Problems in HPLC

Peak Shape Changes While Testing a Group of Samples

Peak Shape Changes While Testing a Group of Samples

One common problem in HPLC analyses is a change in peak shape. 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 state a requirement that the tailing factors for all peaks must be less than a specified limit. Higher tailing factors may decrease the resolution of peaks that elute close together and make integration more difficult [D. R. Stoll, LC-GC N. Am. 39 (2021), pp. 353–362].

Figure 1. Calculation of the USP tailing factor from the full peak width measured at 5% of the peak height (W0.05) and the width of the front segment of the peak at 5% of the peak height (f).

When using an established method to analyze a batch of samples, sometimes the peak shape may change over a series of injections. This may cause the tailing factors to fail the requirement. There are several possible causes of this problem, 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]. When troubleshooting, a good starting point 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. In the latter case, look at the properties of the analytes whose peaks have changed, and determine how they differ from those of the analytes whose peaks didn’t change. Are the problematic analytes basic or acidic, while the others are not? If so, the cause could be a change in the surface charge of the stationary phase or a change in the mobile phase pH. If the analytes showing the peak shape change are basic while the others are not, one likely root cause in reversed-phase methods is the loss of endcapping groups from the stationary phase, which increases the concentration of silanol groups. Ionized silanol groups on the base particle are a common cause of peak tailing for basic analytes [D. V. McCalley, Chem. Comm. 59 (2023), pp. 7887–7899].

In the case where all the peaks in a chromatogram show similar peak shape changes, one possible cause could be slippage of the tubing connecting the column to the HPLC system. This can happen when using PEEK finger-tight fittings. Another possible cause is the presence of a void in the column, which can occur when a column is exposed to rapid pressure changes or is used under pH and/or temperature conditions that result in hydrolysis of the stationary phase support particles. This is common when silica-based columns are used with basic (pH>7) mobile phases, particularly at elevated temperatures (>30 °C) [J. J. Kirkland, M. A. van Straten, H. A. Claessens, J. Chromatogr. A 691 (1995), pp. 3–19., H. A. Claessens, M. A. van Straten, J. J. Kirkland, J. Chromatogr. A 728 (1996), pp. 259–270]. A third potential cause is the accumulation of sample matrix constituents in the HPLC system and/or the column. Many samples contain components that can precipitate or strongly adsorb on the surfaces in the system and column, such as proteins, lipids, polysaccharides, and surfactants. When these components build up on the surfaces of the system and/or the column, they may disrupt the flow distribution, resulting in peak shape changes for all peaks.

Consider the chromatograms shown in Figure 2. The initial chromatogram shows five peaks with good tailing factors, ranging from 1.01 to 1.09. After 200 sample injections, the chromatogram now shows that all five peaks have increased tailing factors, ranging from 1.61 to 1.97. The column back pressure showed only a small (3.5%) increase over the 200 injections.

Figure 2. Chromatograms comparing the first and 200th separations of five analytes. The USP tailing factors are shown above the peaks. After replacing the guard column, the chromatogram labelled as injection 215 was obtained.

The analytes are all neutral under the separation conditions, so a change in the silanol group concentration on the surface of the stationary phase can’t be the cause of the tailing peaks. The mobile phase contained only water and acetonitrile and the column temperature was 40 °C, so column voiding is not likely. What about the buildup of sample matrix components in the column? The samples contained proteins, as well as fats and sugars. The column used for this example had a removeable integral guard column. When the guard column was replaced with a new one, the tailing factors for all five components were restored to values near 1, as shown in the bottom chromatogram. This confirms that the cause of the peak tailing was the accumulation of sample matrix components in the guard column. When working with samples that contain matrix components that may precipitate or strongly adsorb on the column, using a guard column can be a relatively inexpensive way to maximize the lifetime of a column. As shown in this example, it’s also a useful tool for identifying the root cause of peak shape problems.

Related

Industry-leading HPLC columns supported by innovative chemistries and technologies. Maximize your laboratory performance by optimizing method development or purification.

Achieve full method development flexibility with XBridge HPLC/UHPLC Columns designed with BEH technology for stability and lifetime, even in high pH/low pH, high temperatures, and other harsh operating conditions.

Maximize resolution and peak capacity for LC separations with CORTECS solid-core columns in 1.6 and 2.7 µm across 7 column chemistries

Get superior selectivity and peak shape for basic compounds with XSelect HPLC and UHPLC columns. Discover our CSH and high-strength silica columns.

Get predictable analytical performance and ensure batch-to-batch reproducibility with high purity silica-based Symmetry HPLC Columns.

Get exceptionally high mass loading for both analytical and preparative columns with SunFire HPLC Columns. Discover SunFire Silica, C8, and C18 columns.

Increase polar analyte retention over a wider pH range and mitigate unwanted analyte/surface interactions with Atlantis Premier Columns.
Back To Top Back To Top