In high-performance liquid chromatography (HPLC), chromatographic peaks represent individual components eluting from the column over time. In some cases, the peak shape may exhibit splitting, where a single Gaussian peak appears as two or more peaks, usually conjoined while sharing the same base. An additional peak is often described as a ‘shoulder’ or a ‘twin.’ Understanding the causes of peak splitting and how to overcome it can be helpful in achieving accurate and reliable results in HPLC analysis. It is worth noting that peak splitting differs from other peak shape problems such as peak tailing and peak fronting, which each require their own set of troubleshooting measures.
There are a few key causes of HPLC peak splitting:
- Method parameters
- A blocked frit
- Voids or contamination in the stationary phase
Let’s look at each of these in more detail, along with possible solutions.
1. Method Parameters
If the peak splitting is only observed in a single peak, there is likely an issue with the separation itself. A simple way to confirm this is to try a smaller sample injection volume. If you observe two separate peaks, it’s likely that two different components are eluting very close together. Adjusting parameters such as temperature, mobile phase composition, column selection, or flow rate can usually resolve this issue.
For example, Chromatography Forum (CF) user Gerhad Kratz noted that the difference between the mobile phase temperature and the HPLC column temperature could lead to a double peak shape. He recommends using a water bath to adjust the solvent temperature.
Another forum user, Kreall, suggests that the organic concentration of the sample solvent or solvent used to wash the sample loop could cause a split peak issue. This user recommends the troubleshooting step of lowering the concentration of organic solvent as much as possible.
2. A Blocked Frit
The frit is a key component of the HPLC system that keeps the packing material inside the column while ensuring the sample flow remains consistent and uniform. A blockage in the column frit can disrupt the flow path of the analyte, altering retention time and causing peak splitting. If this is the cause, the splitting of all peaks will likely be observed. Alternatively, there may be contamination in the frit, causing an uneven flow. To combat this problem, the frit (or column) may need to be replaced.
3. Voids or Contamination in the Stationary Phase
Void spaces or channels in the column packing material can also contribute to peak splitting. A column void—often resulting from uneven packing density—can disrupt the flow path of the eluent, causing multiple retention times for the same component. Similarly, as pointed out by CF user Danko, contamination could occur in the stationary phase, leading to varying elution times for a single analyte. As flushing is unlikely to resolve these issues, replacement of the stationary phase or column may be necessary.
Conclusion
Peak splitting in HPLC can pose significant challenges to analytical accuracy and reproducibility. By understanding the various causes of peak splitting and implementing proactive measures and troubleshooting steps, chromatographers can minimize peak splitting and achieve reliable results.