Peak splitting in chromatography refers to the appearance of shoulders or twin peaks within a single Gaussian peak. It can occur due to various factors, impacting the quality of separation. Let’s explore some common reasons for peak splitting:
Improper Connections: Issues in the flow path between the injector and the detector can lead to peak splitting. Examples include:
Tubing slippage when using PEEK fingertight fittings.
Incorrect ferrule depth on stainless steel fittings or other connectors.
Small voids inside the connection.
If all peaks are affected, the dead volume is often after the column1.
Particulates: Particles lodged in the column inlet can cause peak splitting. These particulates may originate from:
Contaminants in the sample.
Contaminants in the mobile phase.
Microbial contamination1.
Sample Solvent Effect: The sample solvent composition can impact peak shapes. For instance:
If the sample diluent contains a higher percentage of nonpolar solvent (reverse-phase) compared to the starting mobile phase.
If the sample diluent contains a higher percentage of polar solvent (normal phase; HILIC) compared to the starting mobile phase.
Dirty Emitter (for Mass Spectrometry): In mass spectrometry, a dirty emitter can cause peak shape issues. Additionally, dead volumes in the probe (non-tool-free versions) may contribute to splitting.
Sugars in HILIC Chromatography: When analyzing sugars, they can form anomers (open chain vs. closed chain), leading to peak splitting.
Column Buildup: Accumulation of sample matrix on the column can result in peak splitting.
Improper Connections: Issues in the flow path between the injector and the detector can lead to peak splitting. Examples include:
Tubing slippage when using PEEK fingertight fittings.
Incorrect ferrule depth on stainless steel fittings or other connectors.
Small voids inside the connection.
If all peaks are affected, the dead volume is often after the column1.
Particulates: Particles lodged in the column inlet can cause peak splitting. These particulates may originate from:
Contaminants in the sample.
Contaminants in the mobile phase.
Microbial contamination1.
Sample Solvent Effect: The sample solvent composition can impact peak shapes. For instance:
If the sample diluent contains a higher percentage of nonpolar solvent (reverse-phase) compared to the starting mobile phase.
If the sample diluent contains a higher percentage of polar solvent (normal phase; HILIC) compared to the starting mobile phase.
Dirty Emitter (for Mass Spectrometry): In mass spectrometry, a dirty emitter can cause peak shape issues. Additionally, dead volumes in the probe (non-tool-free versions) may contribute to splitting.
Sugars in HILIC Chromatography: When analyzing sugars, they can form anomers (open chain vs. closed chain), leading to peak splitting.
Column Buildup: Accumulation of sample matrix on the column can result in peak splitting.
Remember that peak splitting indicates deficiencies in method development, and addressing these factors is crucial for achieving accurate and reliable chromatographic results.
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