Troubleshooting Guide for Liquid Chromatography

Fault Phenomena and Solutions

• No Peaks: Possible causes include detector setting issues, compounds being retained longer than the run time, sample problems, blocked needles, and instrument problems. Solutions such as checking the relevant parts of the detector, adjusting the run time or solvent strength, preparing fresh samples, clearing or replacing the needle, and checking the instrument's mobile phase and air bubbles.

• Split Peaks: Reasons include contamination of the guard column or column inlet, incompatibility of the sample diluent with the mobile phase, and analyte properties. Solutions are to replace relevant parts, change the sample diluent, and adjust the analysis conditions.

• Tailing Peaks: May be caused by secondary interactions, dead volume, column degradation, column void, interfering peaks, incorrect mobile phase pH, sample chelation, insufficient buffering, and sample overload. Solutions include adjusting the pH, reducing dead volume, replacing the column, filling the void, optimizing the method, using ion pair reagents, increasing the buffer concentration, and reducing the sample concentration.

• Fronting Peaks: Causes include column degradation, incompatibility of the mobile phase/sample diluent, and sample overload. Solutions are to replace the column, adjust the mobile phase composition, and reduce the sample concentration.

• Broad Peaks: May be due to sample loading, column problems, column temperature settings, mobile phase, and instrument settings. It can be solved by reducing the sample concentration or injection volume, replacing the column, checking the column temperature, ensuring the correct mobile phase, and adjusting the instrument parameters.

• Extra Peaks: Reasons include other components in the sample, late-eluting peaks from the previous injection, and ghost peaks. Solutions are to confirm the sample components, increase the run time or solvent strength, and check the purity of the mobile phase and use a ghost peak trap.

• Changing Retention Times: Related to factors such as flow rate, insufficient equilibration, poor temperature control, poor column equilibration, changes in column dimensions, changes in the mobile phase environment, improper mobile phase, instrument leaks, and air bubbles in the pump. It is necessary to check the flow rate, extend the equilibration time, ensure accurate temperature, use the correct column and mobile phase, check for leaks, and remove air bubbles.

• Loss of Resolution: May be caused by changes in peak width, retention time, and deterioration or evaporation of the mobile phase. Solutions are to ensure column performance, replace the column, and prepare fresh mobile phase.

• Changes in Sensitivity: Causes involve the injector, sample, detector, column performance, and instrument leaks. Solutions include checking the injector volume, preparing fresh samples, checking the detector parameters and components, testing the column performance, and checking for leaks.

• Baseline Drift: May be caused by column temperature fluctuations, low-quality mobile phase, contamination or air bubbles in the detectng of the detector when the mobile phase is recycled, and incorrect wavelength setting of the detector (UV). Solutions are to control the column temperature, use high-quality mobile phase, flush the flow cell, replace the cell window, optimize the mixing, extend the column flushing, perform strong flushing, auto-zero the detector, and adjust the wavelength.

• Noisy Baseline: Reasons include air, leaks, incomplete mixing, temperature changes, mobile phase contamination, immiscible solvents, air trapped in the system, weak detector lamp, and column leakage. Solutions are to degas, check for leaks and fittings, premix the mobile phase, control the temperature, check the mobile phase, use miscible solvents, flush the flow path, replace the lamp, and replace the column.

• No Pressure Reading, but Flow is Normal: May be due to sensor malfunction or software incompatibility. Solutions are to repair or replace the sensor and use alternative software.

• Low Pressure: Causes include partial leaks in the system, flow rate problems, method problems, incorrect column, too high column temperature, and sensor malfunction. Solutions are to check the connections, ensure the correct flow rate and method, use the correct column, adjust the column temperature, and repair or replace the sensor.

• Fluctuating Pressure: May be caused by air bubbles, worn pump seals, check valve problems, leaks, insufficient degassing, and gradient elution. Solutions are to purge the solvent lines, replace the seals, handle the check valves, check the connections, degas, and adjust the gradient elution.

• High Pressure: Reasons include too high flow rate setting, blocked column, incompatible mobile phase (precipitated buffer or immiscible), improper column, injector blockage, guard column/cartridge blockage, column in-line filter blockage, too low column temperature, sensor malfunction, and pump in-line filter blockage. Solutions are to reduce the flow rate setting, backflush or replace the column, use the correct mobile phase, select the appropriate column, clear the injector blockage, replace or remove the guard column or filter, adjust the column temperature, repair or replace the sensor, and replace the pump in-line filter.

Preventive Measures and Good Practices

• Regularly replace consumables, such as seals, to keep the system running smoothly.
• Avoid keeping aqueous buffers for too long to prevent microbial growth; ensure that the organic mobile phase is capped to prevent compositional changes.
• Use an appropriate solvent grade, such as HPLC-grade solvents, to avoid a large number of particles in the mobile phase.
• Flush the instrument and column with a non-buffered mobile phase to avoid salt precipitation or column degradation.
• Ensure that the detector is not stored in a harsh mobile phase and turn off the lamp when not in use to extend its lifetime.
• Keep good records, including information on instruments, columns, sample and mobile phase preparation.
• Establish the reproducibility of the problem, visually inspect the instrument, ensure the correct method, conduct a system suitability test, change one variable at a time to troubleshoot the problem, and replace the suspected faulty part if necessary.

Liquid chromatography analysis is crucial in scientific research and practical applications, but malfunctions are inevitable. I hope that through today's sharing, everyone can become more confident in dealing with various issues that may arise in liquid chromatography. If you encounter difficulties during the experiment, do not be discouraged, and actively use the knowledge you have learned to troubleshoot and resolve them. At the same time, everyone is also welcome to share their own experiences and insights in the comments section, so that we can grow and progress together. Let's join hands to explore the world of liquid chromatography, and use accurate and reliable experimental results to promote the development of science! Remember to like and share this article, so that more colleagues can benefit!

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