Reasons for HPLC Column Peak Fork and No Indication

In High-Performance Liquid Chromatography (HPLC), when you observe a “peak fork” phenomenon on the chromatogram and there’s no clear indication for its cause, it can be challenging to pinpoint the exact reason. However, there are several potential factors that could contribute to this issue:

  • HPLC column no indication:
    • The pump seal gasket is worn;
    • A large number of air bubbles enter the pump body. Treatment For this case, replace the gasket; for the second case, use a 50 ml glass syringe to help extract air at the pump outlet while the pump is acting.
  • HPLC column peak fork
    • The HPLC column is contaminated;
    • The stigma packing collapses. Treatment For this case, first flush the column with pure water, then replace it with methanol, then rinse the column with methanol + isopropanol (4+6) (the length of the rinse time is determined by the contamination of the sample), and then Rinse with methanol, rinse with pure water, and finally rinse with methanol for more than 30 minutes.

If the peak is still poor after rinsing, consider the second case. For the second case, unscrew the column head and check that the HPLC column packing is indented or collapsed.

Remove the indurated part (contaminated packing), fill in the new packing, drop a drop of methanol, fill the packing, refill, press with a smooth stainless steel rod with the same inner diameter of the column, fill it again, drop the methanol, and then press it again. Once, until it is filled and filled. Rinse the HPLC column head with methanol, wipe the packing on the outer wall of the column, tighten the column head, and rinse with pure methanol for more than 30 minutes.

  • Mobile Phase Issues:
    • Inadequate Solvent Mixing: Poor mixing of solvents can result in uneven flow rates through different parts of the column, leading to peak splitting.
    • Degassing Issues: If the mobile phase isn’t properly degassed, dissolved gases can cause bubbles and irregular flow patterns, affecting peak shapes.
    • Buffer Concentration: Inconsistent buffer concentration or pH can alter analyte retention times and peak shapes.
  • Sample-Related Factors:
    • Sample Concentration: Overloading the column with too much sample can lead to broadened or split peaks.
    • Sample Contamination: Contaminants or impurities in the sample can cause unexpected peak splitting or distortion.
    • Sample Matrix: The sample matrix might interact with the column or mobile phase in unforeseen ways, affecting peak shapes.
  • Instrument Issues:
    • Detector Malfunction: Problems with the detector, such as noise or baseline drift, can affect peak shapes.
    • Injection Variability: Inconsistent sample injection can result in irregular peak shapes.
    • Flow Rate Variability: Inaccurate or inconsistent flow rates can lead to peak distortion.
  • Method Development and Optimization:
    • Gradient Program: Incorrect gradient program parameters can cause unexpected changes in elution patterns and peak shapes.
    • Column Temperature: Inadequate temperature control can lead to retention time shifts and peak distortion.
    • Mobile Phase Flow Rate: Incorrect flow rate settings can affect peak shapes and separation.
  • Other Factors:
    • Tubing and Connection Issues: Leaks, dead volumes, or clogs in tubing or connections can lead to irregular flow patterns.
    • Sample Viscosity: High-viscosity samples can affect flow rates and peak shapes.
    • Instrument Calibration: Incorrect calibration of the system can lead to inaccurate retention times and peak shapes.

To troubleshoot this issue, consider the following steps:

  • Check the condition of your column and ensure it’s properly packed and clean.
  • Verify the proper preparation and degassing of the mobile phase.
  • Inspect your sample for impurities or contamination.
  • Check instrument settings and ensure proper maintenance.
  • Review your method parameters, including gradient program and flow rate.

If the issue persists, it might be helpful to consult with experienced chromatographers or HPLC instrument specialists who can provide further guidance based on their expertise.