by Brandon Sharp, Ph.D., Technical Science Writer
Troubleshooting is an integral part of maintaining the performance and reliability of your HPLC system, and even the most experienced HPLC user will encounter challenges. Although major repairs will require a professional technician, in many cases the problem can be resolved by the user themselves.
“Typical issues the user can most commonly encounter are pressure fluctuations, baseline noise, peak shape issues, or retention time drift—these issues can be easily handled by the customer to troubleshoot and often fix the problem,” said Cornelia Vad, Senior Product Manager at Agilent Technologies.
In this guide, we’ll explore these and other common HPLC problems, as well as strategies to resolve them.
System Pressure Fluctuations
Pressure fluctuations are one of the most common errors that HPLC users can handle themselves, and a low or high system pressure can be linked to leaks or blockages, respectively.
“For example, when experiencing pressure fluctuations, start with checking for leaks in the system first, followed by inspection of filters for buildup of particulates,” said Tobias Knust, Product Support Manager, LPSD Support, Liquid Phase Separation Division, Agilent Technologies. “Afterward, check for gas bubbles in the system and perform degassing and purging of the pump.”
Columns can be cleaned by washing with a stronger solvent than the mobile phase you’re currently running. Also, make sure you’re filtering your samples before injecting them into the HPLC, and consider installing a guard column.
Peak Tailing
Peak tailing or broadening can arise from various factors, including incorrect sample preparation, or interaction of analytes with the stationary phase. Verify that samples are prepared in a solvent compatible with the mobile phase and column. If appropriate, adjust the pH to ensure that analytes are fully ionized or neutral, depending on the desired separation. Finally, peak tailing may also be caused by column degradation, especially in older columns, so ensure that the column packing is intact by performing a test with a standard compound.
Peak Broadening
Peak broadening can be caused by high flowrates or high column temperatures. If the temperature of the column and mobile phase is different, i.e., a thermal mismatch exists, peak broadening may occur. In this case, a temperature-controlled column oven can be installed to stabilize the temperature.
Baseline Noise
A noisy baseline is commonly caused by contaminated solvents, detector instability, or temperature fluctuations. Start by using freshly prepared, high-purity HPLC-grade solvents and ensuring that all mobile phase components are properly degassed. Verify the detector settings, such as wavelength accuracy for UV detectors. To minimize temperature fluctuations, ensure that the laboratory temperature is stable, or install a column oven.
Retention Time Shifts
Retention time shifts may indicate issues with the mobile phase composition, column degradation, or flow rate consistency, commonly due to pump malfunctions. Check the HPLC pump system for leaks or irregular flow rates. Confirm that the mobile phase preparation is consistent between runs, and ensure the column is properly equilibrated before starting analyses.
Low Signal Intensity
If your peaks are vanishingly small, or not present at all, this is often a result of improper sample preparation, such as extracting too little analyte or not purifying it properly. This is especially problematic when working with samples with complex analytes, complex samples with a low target analyte concentration may require a lengthier extraction step in order to obtain a sufficient amount of analyte. However, be careful – if the extraction step is too aggressive, you may have also extracted impurities that may overpower the signal of the analyte of interest.
Troubleshooting Approach
Identify the problem
Start by looking at your chromatogram for symptoms such as irregular baselines, excessive noise, or peak anomalies (tailing, broadening, etc.).
“The first thing to do is to identify where the problem is stemming from. This can be done by removing one component at a time from the flow path and repeating the test until the issue is resolved,” said Nivesh Mittal, the HPLC Product Manager at Shimadzu Scientific Instruments.
You should also review system logs and method parameters to narrow down potential causes, taking note of the types of samples and solvents that were run through the instrument.
Isolate the cause
Test each of the system’s components one at a time, but keep in mind that some components are more likely to cause issues than others. “Degassers, system controllers, [and] column ovens are much simpler engineering and seldom have issues,” according to Nivesh. Some of the more common culprits include:
- Pumps: Check for leaks, irregular flow, or worn seals. Run the pump with pure solvent to detect pressure irregularities.
- Injectors: Ensure the sample loop is clean and free from blockages. Inspect for proper sealing in the injector valve.
- Columns: Flush the column with strong solvents to remove contaminants. Test with a standard sample to confirm performance.
- Detectors: Evaluate lamp intensity and cleanliness of the flow cell. Replace the lamp if the intensity is insufficient.
Implement solutions
Once the root cause is identified, take corrective measures such as:
- Replacing degraded or contaminated mobile phases.
- Properly filtering your samples.
- Replacing damaged or old columns.
- Tightening loose fittings to eliminate leaks.
- Adjusting parameters such as the flow rate, temperature, or gradient profiles to stabilize retention times.
Preventative Measures
Using in-line filters, a guard column, and filtering your samples and buffered mobile phases can help prevent some issues altogether, particularly pressure fluctuations due to blockages.
Other routine maintenance includes changing:
- inlet solvent filters
- in line solvent filters
- pump seals and plungers
- the lens and lamps on the detectors
- the needle seal in the injection port
- check valves
- valve rotors
“The most frequent and overlooked good chromatography practice is to change the solvents (especially aqueous solvents),” said Mittal. “It is very easy to sideline issues from the mobile phase and troubleshoot everything else. But if users are not changing solvents and buffers as a routine, they are bound to face chromatography issues quickly.”
Performing routine maintenance on a daily or weekly basis can minimize downtime and the need for future troubleshooting.
“Classic examples for daily tasks are replacing solvents and solvent bottles for mobile phases based on water/buffer,” said Knust.
When to Schedule a Service Appointment
Despite your best efforts, troubleshooting sometimes isn’t enough and it’s necessary to consult the equipment manufacturer or an expert technician. If you’re unsure of the best course of action, it’s best to consult a professional to avoid damaging your HPLC.
“If there are any grey areas, and the user is unable to easily access instructions for an activity in a manual, then they don’t want to be working on that themselves,” Nivesh says.
Service appointments may be necessary, with experts from both Shimadzu and Agilent citing pumps as one of the top HPLC components requiring a service appointment.
The tips in this troubleshooting guide are meant to provide guidance for HPLC users to attempt to identify and correct the underlying causes of issues with their HPLC instruments. Regular maintenance like cleaning columns, degassing solvents, and calibrating the system can help minimize downtime and extend the lifespan of your equipment and ensure that you obtain consistent, high-quality data.
About the author
Brandon Sharp, Ph.D., is a freelance technical content writer with experience designing photoresists and other organic materials for advanced lithography applications.
References
https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/analytical-chemistry/small-molecule-hplc/hplc-troubleshooting-guide
https://www.thermofisher.com/tw/zt/home/industrial/chromatography/chromatography-learning-center/high-performance-liquid-chromatography-hplc-support/hplc-troubleshooting.html
https://www.shimadzu.com/an/service-support/technical-support/analysis-basics/tips/abnormalpeakshapes.html
https://www.agilent.com/cs/library/posters/public/Final%20TIPS%20and%20Tricks%20HPLC%20Troubleshooting%20(2).pdf