Troubleshooting peak area variation between injections

Document created by paolalab Employee on Sep 23, 2020Last modified by paolalab Employee on Oct 19, 2020
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Agilent Support Insights


This Information Applies To: All Agilent Autosamplers



Non-reproducible peak areas between injections.



When different peak areas between one injection and another are observed using the same sample and the same experimental conditions, there are few steps to follow which will allow troubleshooting the issue.



The issue may come from different sources, it is recommended to verify all the following ones for a successful troubleshooting.

Safety WarningsWhen opening capillary or tube fittings, solvents may leak out.

Observe appropriate safety procedures (for example, wear goggles, safety gloves and protective clothing) as described in the material handling and safety data sheet supplied by the solvent vendor, especially when toxic, or hazardous solvents are used.

  • Vials
    • Use only Agilent vials, the shape, and the material is optimized for maximizing instrument precision.
    • Do not overfill the vials. Fill the vial up to 1/2 to 2/3 of the total volume.
  • Experimental conditions
    • Make sure that the same solvents were used between the runs.
    • Keep the room temperature constant and under 25 degrees Celsius. 
  • Acquisition method settings
    • Optimize the draw speed decreasing it down to 50µL/min. The default settings are at 200µL/min, but this speed may be too high for viscous samples.
  • Hardware
    • The issue may reside at the Autosampler. A leakage is one of the frequent causes, make sure that all the autosampler connections are tight. 


If the problem persists, contact Agilent Technologies for further guidance.


Learn more on how to effectively troubleshoot your LC Autosampler:

HPLC-0GEN-2201e - Chromatographic Troubleshooting for HPLC e-Learning course on Agilent University 



Keywords: hplc autosampler, reproducibility, peak area, troubleshoot, draw speed, vials