Why Does an Excessive Peak Appear Within 5 Min in Protein LC with 0.1% TFA and TFA/Acetonitrile?
In liquid chromatography (LC) analysis for proteomics, the occurrence of a peak significantly higher than the target peak within 5 minutes may be due to several factors:
System Peaks or Baseline Drift
At the initial stage of analysis, system peaks may arise due to the equilibration of the solvent components in the mobile phase with the column. This is particularly common when using mobile phases containing organic solvents (such as acetonitrile) and 0.1% trifluoroacetic acid (TFA).
Impurities in the Sample
Improper sample preparation may introduce impurities such as salts or small molecules, which can elute early and form large peaks.
Solvent Mismatch
If the sample solvent composition does not match the mobile phase, solvent effects may occur in the initial stage, leading to pronounced peaks.
Injection Volume of Solvent and Sample
Excessive sample injection or improper solvent volume may also contribute to the initial peak formation.
Insufficient Column Equilibration
If the column is not adequately equilibrated before analysis, abnormal peaks may appear.
If filtering the sample through an organic-phase membrane significantly reduces the solvent peak, this suggests a high level of impurities in the sample. In such cases, sample purification is recommended to remove high concentrations of salts or other small-molecule contaminants.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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