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
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 1: Measurement of tailing factor and asymmetry factor.
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
. 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.
Figure 2: Examples of tailing peaks.
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
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.