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 (1–3) of column instalments.
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, As, 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 ≡ As. As tailing increases, however, the two numbers diverge, with As growing faster than TF, but for peaks with a value < 2 there is not a very noticeable difference. There is no inherent value
in using one technique vs. 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 of 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 favour 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 behaviours are caused by three
different sources. We will look at each of the three problems next.
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LCGC COLUMNISTS 2014
Sample Prep Perspectives | Ronald E. Majors: LCGC Columnist Ron Majors, established authority on new column technologies, keeps readers up-to-date with new sample preparation trends in all branches of chromatography and reviews developments in existing technology lines.
History of Chromatography | Industry Veterans: With each installment of this column, a different industry veteran covers an aspect of the evolution and continued development of the science of chromatography, from its birth to its eventual growth into the high-powered industry we see today.
MS — The Practical Art| Kate Yu:
Kate Yu is the editor of 'MS-The Practical Art' bringing her expertise in the field of mass spectrometry and hyphenated techniques to the pages of LCGC. In this column she examines the mass spectrometric side of coupled liquid and gas-phase systems. Troubleshooting-style articles provide readers with invaluable advice for getting the most from their mass spectrometers.
LC Troubleshooting | John Dolan: LC Troubleshooting sets about making HPLC methods easier to master. By covering the basics of liquid chromatography separations and instrumentation, John Dolan, Vice President of LC Resources and world renowned expert on HPLC, is able to highlight common problems and provide remedies for them.