A chromatographic peak with an asymmetric shape that has a gentle leading edge and a steep trailing edge is referred to as a leading peak. If the symmetry factor of a peak is between 0.95 and 1.05, it is considered normal; if it is less than 0.95, it is a leading peak, and if it is greater than 1.05, it is a tailing peak. Typically, chromatographic peaks with a symmetry factor of 0.9 to 1.2 are acceptable.
In the process of routine liquid chromatography analysis, encountering leading peaks in chromatograms is a common issue. Today, let’s learn about the reasons behind leading peaks and how to deal with them.
01 Impact of Leading Peaks
(1) Effect on Peak Height Calculation
When the peak height of leading peaks differs greatly due to their leading edge, it can affect the calculation of sample results, especially when calculating the detection limit.
(2) Effect on Peak Area Calculation
When calculating the peak area, the start and end points of the peak are generally determined by the slope of the chromatographic peak. The flatter the baseline of the leading edge of the peak, the more difficult it is to determine the starting point, leading to inaccurate peak area quantification.
(3) Impact on Confirmation of Trace Components
When the separation of the leading peak is not ideal, the sample peak can easily be hidden at the leading edge of the subsequent peak, making it difficult to calculate.
02 Analysis and Solutions for Leading Peaks
(1) Column Overloading
Each chromatography column has a maximum sample load capacity. When the sample load exceeds the capacity, overloading occurs, including mass overload and volume overload. We can reduce the sample load or concentration to see if the peak shape improves. Increasing the column diameter and using higher-capacity stationary phases can also help to solve the problem.
(2) Inappropriate Solvent Selection
When the elution power of the sample solvent is significantly stronger than that of the mobile phase, leading peaks can occur. Specifically, the sample moves uniformly forward in the chromatographic column, and we usually obtain a normally distributed peak. However, when the sample solution reaches the chromatographic column shortly after injection, before it has been sufficiently diluted by the mobile phase, the strong elution power of the sample solvent can cause the sample to be washed out rapidly, leading to a leading peak. For example, when using acetonitrile as the sample solvent in reversed-phase chromatography, leading peaks may occur if the elution power of the mobile phase is weak. We can choose a solvent that closely matches the mobile phase or a solvent with a lower elution power as the sample solvent.
(3) Column Damage
Chromatographic columns can dissolve the silica gel filling material and cause bed collapse after long-term use, leading to a decrease in column efficiency and leading peaks. If the chromatographic column damage is detected, it is recommended to replace the chromatographic column directly.
(4) Peak Interference
Two compounds co-elute, resulting in a small peak appearing before the main peak, and the chromatographic peaks are not separated. We can increase the sample purification program and adjust the mobile phase to improve the separation.