Peaks with strange shapes represent one of the most common problems that can arise in the LC/HPLC laboratory. A distortion that peaks show quite often is peak tailing. It should be first explained why tailing is bad :
- Peaks with tailing can be hard to integrate
- The precision and the reliability of assay methods involving distorted – peaks with tailing – is often poor when compared to good chromatography
- Resolution is low when peaks show tailing
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| Fig. 1: Peaks showing tailing |
How do we solve the problem of tailing peaks?
We should at first consider the various general causes of peak tailing such as:
- Bad column
- Sample overload
- Strong retention sites (normal-phase or ion-exchange chromatography)
- Not enough buffering
We should investigate which of the above causes create peak tailing and attempt to rule out causes that do not seem relevant. Often it is possible to quickly rule out a potential cause of peak tailing and go to the next possibility.
Bad column
A bad column is a column with defects in the flow characteristics. There are many causes for this such as:
- A blocked inlet frit caused by particulates
- The packing material may have settled causing a void at the column inlet
- Contamination of the stationary phase by strongly-attached compounds from previous samples
- Active sites that strongly retain acids, bases or ionized molecules
- A low plate-number
- Change in retention characteristics
The first sign of a bad column is band tailing or distortion that is similar for every peak in the chromatogram.
The general approach for fixing a column that shows peak tailing or distorted peaks is as follows:
Reverse and Flush: The column is removed from the system and reconnected with the ends reversed. Then the column is flushed with the mobile phase for 30 min (1-3 ml/min) and the sample that gave tailing peaks is injected and run. In almost 50 % of the cases the column will not show peak tails anymore.
Replace Frit: If the above mentioned column reversal and flushing does not fix the problem the original inlet-end of the column should be opened and the frit should be removed. The column packing should be examined for any settling (1mm or more) or holes in the column surface. If the column packing is smooth and level with the top of the column, a new frit should be inserted and the column should be reinstalled in the system (normal direction) and washed with mobile phase for 30 min (1-2 ml/min). The above method usually solves the problem in almost 30% of the cases.
Fill Void and Reverse: When the void is filled, the column bed will be more stable if the column is operated with the direction of flow reversed from its original direction.
If the above did not solve the peak tailing problem the column may be discarded.
Sample Overload
When one or more bands within the chromatogram tail, and these bands are larger than normal, the column may be overloaded. In cases like this, the total mass of solute is large enough and the linear capacity of the column is exceeded. The result is a decrease in retention time and change in band shape.
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| Fig 1: Sample overload and band (peak) tailing. (A) Normal sample (B) Tailing sample (C) Sample (B) diluted four times and rerun after decreasing the detector attenuation four-fold. |
The above problem can be corrected by diluting the sample fourfold and re-injecting it – after decreasing the detector attenuation four-fold. The result is shown in Fig. 1C where the original tailing peaks of run (B) are more symmetrical and their retention times have increased to the values of the normal sample of Fig. 1A.
References
L.R. Snyder. J.J. Kirkland, “Introduction to Modern Liquid Chromatography”, 2nd edition, Wiley, 1979
A. Weston and P. Brown, “HPLC and CE Principles and Practice”, Academic Press, 1997
J.W. Dolan, L.R. Snyder, “Troubleshooting LC Systems”, Humana Press, 1989
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