Troubleshooting Basics, Part IV: Peak Shape Problems

Jul 01, 2012
Volume 30, Issue 7, pg 564–569

What do you do when the peak shape changes?

A change in peak shape is one of the most common observations of problems with a liquid chromatography (LC) method. Because of this, most system suitability tests include a measure of peak shape, so a quantitative value of peak shape can be tracked over time. Poor peak shape can compromise the results of an analysis by degrading resolution between closely eluted peaks and reducing precision and accuracy of measuring peak area, especially for small peaks. A change in peak shape is one of the first signs that the column is failing, but there are other causes of peak tailing, as well. This month we look at several aspects of peak tailing as we continue our "Troubleshooting Basics" series of column installments (1–3).

Measuring Peak Tailing

Figure 1: Measurement of tailing factor and asymmetry factor.
The ideal chromatographic peak will have a Gaussian shape, but it is rare that a perfectly symmetric peak is observed in real chromatograms. Most peaks tail slightly, and as the column ages, tailing typically increases. However, there are several other potential causes of peak tailing (or fronting) as well, so it is a good idea to track the peak shape over time to anticipate when practical problems will occur. As a result, nearly all system suitability tests include a measurement of peak shape.

Figure 2: Examples of tailing peaks.
The two most popular methods of measuring peak shape are illustrated in Figure 1. Other methods to measure peak shape are used much less often. The pharmaceutical industry uses the tailing factor, TF, which is determined by measuring the entire peak width at 5% of the height and dividing it by twice the front half-width. Nonpharmaceutical laboratories often use the asymmetry factor, A s, which is calculated by measuring the back half-width of the peak at 10% of the peak height and dividing it by the front half-width. You can see that if the peak is perfectly symmetric, the front and back half-widths will be the same, no matter where they are measured relative to the peak height, so for such peaks, TF ≡ A s . As tailing increases, however, the two numbers diverge, with A s growing faster than TF, but for peaks with a value less than 2 there is not a very noticeable difference. There is no inherent value in using one technique versus the other for measuring peak shape; rather, it is important to choose one technique and use it to look for changes in peak shape over time.

Most LC peaks tail or front a bit, so column manufacturers typically set their column-release specifications at 0.9 < TF < 1.2 as normal performance. As can be seen in Figure 2, when tailing increases, several practical problems can arise. The peaks are harder to integrate because the transition from the baseline to the peak or peak to baseline is much more gradual, and on noisy or sloping baselines the peak limits are difficult to determine. Generally, the peak area stays constant, so increased peak tailing translates into shorter peaks, and peak height is the limiting factor in determining detection limits, so method limits can suffer with tailing peaks. Tailing peaks also take a larger time window to be eluted, so to achieve baseline resolution between peaks, the run time must be longer. And tailing peaks are aesthetically less pleasing. You can see that all these factors favor symmetric peaks. From a practical standpoint, peak tailing is difficult to eliminate, however, for many applications peaks with TF ≤ 1.5 are acceptable. When TF ≥ 2, usually corrective action should be taken to identify and eliminate the source of tailing.

When peak tailing occurs, it usually shows up for one or just a few peaks in the chromatogram, but sometimes all the peaks in the run tail. The appearance of peak fronting is much less common. Most often, these three behaviors are caused by three different sources. We will look at each of the three problems next.

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