An ideal chromatography peak is a nice sharp symmetrical shape, a Gaussian peak, on a flat baseline. A peak can deviate from this ideal in several different ways. It can become asymmetrical, flatten and become broader, or the baseline can rise.
One of the common shifts away from a Gaussian peak is when the back half of the peak falls away. If the peak were split into two, vertically, the later half would be wider than the first half of the peak. This effect is most clearly seen close to the baseline and is known as peak tailing.
Peak tailing has been the most common peak shape problem in RP-HPLC. Most peak tailing is due to interaction with acidic or ionized silanol groups on the surface of the silica particles within the column. The low-purity silica (acidic silica) has a high content of acidic silanol (-Si-OH) groups and the presence of metal impurities (especially iron and aluminum) further increases the ionization of these groups to –Si-O-, which provides cation exchange sites.
The pKa of these materials is in the pH 4 - 5 region, meaning that at pH>6 most of the silanol groups are ionized. Efforts to improve the purity and lower the acidity of silica led to higher purity silica particles ("Type-B") and since their initial introduction, the purity of these silica packings has improved. High-purity silica has a pKa of >8, so there is minimal silanol ionization in the pH-stable range of 2<pH<8 for most columns.
Basic compounds are the most susceptible to silanol tailing and because a high proportion of sample molecules contain basic nitrogen functional groups, few compounds are completely immune to silanol interactions.
Insufficient buffer or mobile phase additive also can result in peak tailing. A common characteristic of buffers and other mobile phase additives is that their effect (e.g. reduction of peak tailing, stabilization of retention times) begins at low concentrations and continues as the concentration is increased, but gradually levels off into a plateau.
Select an additive concentration on the plateau for stable operation; excessive concentrations can cause solubility problems. Additives in the 10-25 mM region usually are sufficient for most applications, but it is a good idea to determine this on a case-by-case basis.
It is very difficult to remove all of the tailing from a peak, even for new columns. The quality of separation and the analytical data can be affected by tailing. Consequently, if tailing is quantified, it is possible to place an acceptable limit on the amount a peak can tail.
There are two main methods for defining peak tailing:
Tailing factor (Tf) – widely used in the pharmaceutical industry.
Let a and b be the peak half-widths at 5% of the peak height,
a is the front half-width,
b is the back.
Tf = (a + b) / 2a
Asymmetry factor (As) – used in most other industries.
As in Tf, a and b are the peak half-widths, but at 10% of the peak height.
As = b / a
Either method of measuring tailing can be used – unless it is defined in a method or standard – but note the methods are not interchangeable.
Acceptable Tailing
Since most columns exhibit some peak tailing, what is considered an acceptable As value?
A new column is considered acceptable if the As value is 0.9 - 1.2 (0.9 indicates slight fronting).
In practical terms, an As value below 1.5 is usually OK to work with, and up to As = 2.0 may be acceptable depending on the separation and resolution of the peaks.
If the As value is greater than 2.0, then there is a problem that needs to be identified and fixed.
Peak Problems
Two of the analytical issues as a result of peak tailing are:
- Peaks with a large As value might have a significantly reduced peak height. This affects the analysis when wide ranges of different concentrations of chemicals are being detected.
- Data analysis systems might not accurately assign the end of a peak, so the peak area is mis-calculated.
Stop Tailing at the Start
There are many possible causes of peak tailing and many different fixes.
ALSO READ: Chromatographic Calculations
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