HPLC Troubleshooting

If any problem persists during HPLC analysis, don’t scared about it, think of little things there are the basic possible source of chromatographic troubles are Mobile Phase, Column, Guard Column, Pump, Injector, Detector, In-Line Filter, Tubing and fittings. At the beginning of HPLC analysis to follow basic practices like Filter the mobile phase, Sonication, preparation of buffer properly, Purge the system, wash the column and equilibrate it with sufficient time, then start the analysis.

If you pay attention to these things, then you will have low problems during the analysis.Here are mentioned. some troubles and possible reasons and solutions.

TROUBLE	POSSIBLE CAUSES	SOLUTIONS
No solvent flow	Empty Reservoirs Leakage, Air bubbles	Refill the solvent bottles, Find the leak and repair it, Purge the system
Noisy baseline or spiking on the baseline	Aging of the column Air bubbles Detectors problem	Replace the column Degas the mobile phase, Purge the system Check the detector
Mobile phase leaking	Loose inlet or outlet fittings Loose valve	Tighten the fittings Tightening the check valve
Pressure fluctuation	Dirty check valve Air entrap in the pump The issue with piston seal	Clean the valve Purge the system Check/Replace the seal
Inconsistent Retention time	Flow rate change Problem with column Leakage Air bubble	Check the flow rate Change the column Find and repair the leakage Remove air bubbles
Detector not response	Issue with wavelength Issue with lamp Dirty-flow cell Leakage Mobile phase contaminate	Select the correct wavelength Check the lamp Clean the flow cell Find and repair the leakage Replace the mobile phase
Results are not Reproducible	The issue with column efficiency An issue with the mobile phase An issue with injector valve	Wash and equilibrate the column properly. Prepare fresh mobile phase Check the rotor seal
Broad Peaks	Contaminated guard column Mobile phase composition Buffer pH An issue with column efficiency	Replace/Remove guard column Select the proper composition of Mobile phase Adjust the proper pH of a Buffer Check the column performance
Baseline Drift	Unstable mobile phase Dirty column Issue with lamp	Change with the stable mobile phase Wash/replace the column Check the lamp
High Backpressure	High Flow rate Guard column blocked Improper column Viscous buffer Buffer Precipitate Blocked Injector In line filter clog	Use the proper flow rate Replace/Remove guard column Wash/Replace column Use the correct Mobile phase System wash with warm water Wash blockage Wash with warm water
Low back pressure	Low flow rate Leakage High column temperature Improper column	Use the proper flow rate Find and repair the leakage Set correct the Temperature Wash/Replace column
Peak Tailing	pH of Mobile phase Mobile phase composition Column inefficiency	Use Adjust the proper pH of the mobile phase Select the proper composition of Mobile phase Wash/replace the column
Split peaks	Incompatible Mobile phase Column inefficiency	Change the mobile phase or MP composition Wash/replace the column
Impurities in sample or mobile phase	Impurities in samples or mobile phase Impurities in column	Prepare fresh mobile phase Wash and equilibrate the column

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