Troubleshooting Problems with Peak Shape (Peak Tailing, Split Peaks, Peak Fronting)

High-Performance Liquid Chromatography (HPLC) is a widely used analytical technique that separates and quantifies compounds in a mixture. One critical aspect of HPLC analysis is the shape of the chromatographic peaks, as it can directly impact the accuracy and reliability of your results. In this article, we will delve into three common peak shape issues encountered in HPLC: 

1. Peak Tailing
2. Split Peaks
3. Peak Fronting

1. Peak Tailing

Peak tailing, also known as asymmetric peaks, occurs when the trailing side of a chromatographic peak is more extended and distorted than the leading side. It can be caused by several factors:

1. Column Overloading: Loading a sample with a concentration that exceeds the column's capacity can result in peak tailing. As the analyte concentration increases, it can interact with the stationary phase for a more extended period, leading to tailing.
2. Sample Matrix Interference: Impurities or matrix components in the sample can interact with the stationary phase and cause peak tailing. This is common when analyzing complex samples like biological fluids.
3. pH Effects: In some cases, pH variations can affect peak shape. For example, acidic or basic analytes may tail if the pH of the mobile phase is not optimized for their ionization state.
4. Column Bed Irregularities: Uneven packing of the HPLC column or voids in the column bed can lead to peak tailing.

Troubleshooting Problems with Peak Tailing

Possible Cause	Prevention/Solution
Interaction with active silanols	Use ultra-high purity silica-based stationary phase Add basic mobile phase additive (eg. TEA) Not needed with ultra-high purity phases
Chelation with metal ions in the stationary phase	Use ultra-high purity silica-based stationary phase Add basic mobile phase additive (eg. TEA) Not needed with ultra-high purity phases
Wrong mobile phase pH	Decrease mobile phase pH to suppress silanol ionization Increase buffer concentration.
Blocked frit	Reverse flush the column Use in-line filter
Column void	Reverse flush the column Replace the column
Unswept dead volume	Minimize the number of connections Use shorter connection tubing Check all fittings are tight

ALSO READ: What is Peak Tailing?

To mitigate peak tailing, one can consider the following strategies:

• Optimize the sample concentration to avoid overloading the column.
• Use sample preparation techniques, such as filtration or solid-phase extraction, to remove matrix components.
• Adjust the mobile phase pH to improve analyte ionization and reduce tailing.
• Ensure proper column maintenance and installation to prevent bed irregularities.

2. Split Peaks

Split peaks, also called shoulder peaks, occur when a single chromatographic peak splits into two or more distinct peaks. This phenomenon can be attributed to several factors:

1. Sample Overloading: Overloading the column with a high concentration of analytes or impurities can lead to split peaks. Excessive solute concentration can saturate the column's binding sites, causing multiple interactions and separations within the column.
2. Inadequate Column Resolution: If the column lacks sufficient resolution capacity to separate closely eluting compounds, it can result in split peaks.
3. Column Temperature: Temperature fluctuations can influence peak shape. An inappropriate temperature can cause split peaks if analytes partially interact with the stationary phase at certain temperatures.

Troubleshooting Problems with Split Peak

Possible Cause	Prevention/Solution
Contamination on guard or analytical column inlet	Remove guard cartridge and carry out analysis – replace guard if necessary The reverse flush analytical column For strongly retained contaminants, try the regeneration procedure Replace column
Blocked frit	Reverse flush the column Use in-line filter
Sample solvent incompatible with the mobile phase	Inject sample in the mobile phase
Simultaneous elution of the second component	Use sample clean-up prior to injection Change selectivity by changing mobile phase or column phase
Column overloaded	Use higher capacity stationary phase Increase column diameter Decrease sample amount

To address split peaks:

• Reduce the sample concentration to avoid overloading the column.
• Optimize the column and method parameters to enhance resolution.
• Maintain a stable and appropriate column temperature.

ALSO READ: SOP for HPLC Column Maintenance and Washing Procedure

3. Peak Fronting

Peak fronting, or symmetric fronting, is the opposite of peak tailing. It occurs when the leading edge of a chromatographic peak is broader and more distorted than the trailing edge. Peak fronting can be caused by various factors:

1. Column Overpacking: Excessive packing of the HPLC column can result in peak fronting by causing uneven analyte distribution.
2. Column Age and Degradation: A column that is old or deteriorating may exhibit peak fronting as the stationary phase loses its efficiency and becomes less homogeneous.
3. Sample Solvent Incompatibility: If the solvent used to dissolve the sample is not compatible with the mobile phase, it can lead to peak fronting.

Troubleshooting Problems with Peak Fronting

Possible Cause	Prevention/Solution
Formation of channels in column	Replace the column Operate within recommended pH limits of the column
Column overloaded	Inject a smaller volume or more dilute sample solution Use higher capacity stationary phase
Sample solvent incompatible with the mobile phase	Inject sample in the mobile phase
Low temperature	Increase column temperature

To combat peak fronting:

• Ensure proper column packing to avoid overpacking.
• Regularly replace and maintain columns to prevent degradation.
• Choose a compatible solvent for sample preparation and injection.

Conclusion

Understanding and addressing peak shape issues in HPLC is crucial for obtaining accurate and reliable analytical results. Peak tailing, split peaks, and peak fronting can arise from a variety of factors, including sample preparation, column conditions, and analyte properties. By optimizing method parameters, maintaining columns, and carefully preparing samples, analysts can overcome these challenges and achieve sharper and more symmetric chromatographic peaks, ultimately improving the quality of their HPLC analyses.

ALSO READ: Abnormal Peak Shapes