The 10 Most Common HPLC Problems and Solutions!

Figure 1: Overview of HPLC.Image from shimadzu

01 Causes of Retention Time Drift or Rapid Changes

About Drift:

• Poor Temperature Control: Solution: Use a thermostat to maintain a constant column temperature.

• Changes in the Mobile Phase: Solution: Prevent evaporation, reaction, etc., of the mobile phase.

• Column Not Properly Equilibrated: Solution: Allow longer equilibration time for the column.

About Rapid Changes:

• Flow Rate Variations: Solution: Re-set and stabilize the flow rate.

• Air Bubbles in the Pump: Solution: Eliminate bubbles by degassing.

• Inappropriate Mobile Phase: Solution: Change the mobile phase or mix it properly in a controlled environment.

02 Causes of Tailing or Double Peaks

• Blocked Frit or Column Failure: Solution: Reverse flush the column, replace the frit, or change the column.

• Interfering Peaks: Solution: Use a longer column; change the mobile phase or select a more suitable column.

• Column Overload: Solution: Reduce the injection volume.

03 Main Causes and Solutions for Insufficient HPLC Sensitivity

• Insufficient Sample Amount: Solution: Increase the sample amount.

• Sample Not Eluting from the Column: Solution: Change the mobile phase or the column based on the sample's chemical properties.

• Mismatch Between Sample and Detector: Solution: Adjust the wavelength or change the detector based on the sample's properties.

• Excessive Detector Attenuation: Solution: Adjust the attenuation.

• High Detector Time Constant: Solution: Reduce the time constant.

• Contaminated Detector Cell Window: Solution: Clean the cell window.

• Air Bubbles in the Detector Cell: Solution: Eliminate air bubbles.

• Inappropriate Recorder Voltage Range: Solution: Adjust the voltage range.

• Inappropriate Flow Rate of Mobile Phase: Solution: Adjust the flow rate.

• Detector and Recorder Beyond Calibration Curve: Solution: Check and re-calibrate the recorder and detector.

04 Causes and Solutions for Unstable Column Pressure in HPLC Analysis

• Air in the Pump: Solution: Remove the air and degas the solvent.

• Faulty Proportioning Valve: Solution: Replace the proportioning valve.

• Damaged Pump Seal: Solution: Replace the seal.

• Air Bubbles in the Solvent: Solution: Degas the solvent, and if necessary, change the degassing method.

• System Leakage: Solution: Identify and seal the leak.

• Gradient Elution: Pressure fluctuations are normal during gradient elution.

05 Reasons for Non-reproducible Retention Time After Changing ODS Column Brands

This issue arises due to the potential of analytes to form hydrogen bonds. Despite improvements in filler manufacturing technology, the concentration of silanol groups on ODS fillers varies among different manufacturers. These silanol groups may interact with the sample, affecting the relative retention time of components in the analyte on different brands of ODS columns. Adding a small amount of a competing substance, such as triethylamine (TEA), to the mobile phase can saturate the silanol groups' bonding capability, ensuring better reproducibility of relative retention times across different column brands. If the separation is satisfactory and the system is stable, meeting the system suitability requirements, reproducibility of retention time is not necessary.

06 Causes and Solutions for High Column Pressure During HPLC Column Acceptance Testing

High column pressure is a common issue for HPLC users and can be caused by various factors, often unrelated to the column itself. Check the following steps to identify the cause:

• Remove the Guard Column: Check if the pressure drops; if so, the guard column is the issue.

• Disconnect the Column from the Instrument: Check if the pressure drops; if not, the system plumbing is blocked and needs cleaning.

• Reverse the Column Connection and Flush with Mobile Phase: Use 10 times the column volume of mobile phase without connecting the detector to avoid solid particles entering the flow cell. If the pressure remains high, further inspection is needed; applicable only to used columns.

• Replace the Column Inlet Frit: If pressure drops, impurities in the solvent or sample have blocked the frit, causing pressure rise. If pressure remains high, contact the manufacturer. Typically, using an in-line filter between the injector and guard column can prevent high pressure issues. SGE's Rheodyne 7315 filter is an optimal choice for addressing this issue.

07 Will the Mobile Phase Drain from the Chromatography Column?

Many who conduct chromatography separation experiments encounter situations where the mobile phase in the solvent bottle is accidentally drained by the pump, stopping the HPLC system. This situation generally doesn't damage the column. Even if the pump is full of air, it won’t push the air into the column since the pump is designed to move liquid, not air.

Conversely, another scenario is forgetting to seal both ends of the column or leaving the caps loose, causing the column to dry out. Typically, only the ends of the column may dry out due to evaporation, which takes considerable time. If the column truly dries out, it may still be salvageable. Flush the column with a fully degassed, low surface tension solvent (e.g., helium-degassed methanol) to remove the trapped air. The low surface tension aids in wetting the filler surface, and the degassed solvent should dissolve and remove the trapped air. The column may require about an hour or more of flushing (at 1 mL/min flow rate) to restore to normal.

08 Causes and Solutions for Baseline Drift

Causes

• Temperature Fluctuations: Even minor temperature changes can cause baseline fluctuations, typically affecting differential, conductivity, low-sensitivity UV, or other photodetectors.

• Uneven Mobile Phase: Baseline drift caused by changes in mobile phase conditions is greater than that caused by temperature fluctuations.

• Contaminated Flow Cell or Gas in the Cell

• Blocked Detector Outlet: High pressure can crack the flow cell window, causing a noisy baseline.

• Inappropriate Mobile Phase Ratio or Flow Rate Changes

• Slow Column Equilibration, Especially After Mobile Phase Changes

• Contaminated, Deteriorated Mobile Phase or Made with Low-Quality Solvents

• Strongly Retained Substances in the Sample (High K’ Value) Elute as Broad Peaks, Causing a Gradual Baseline Rise.

• Recycled Solvent Usage (Not Recommended). Detector Not Properly Adjusted.

• Detector Not Set to Maximum Absorption Wavelength.

Solutions

• Control the temperature of the column and mobile phase, and use a heat exchanger before the detector.

• Use HPLC-grade solvents, high-purity salts, and additives. Degas the mobile phase before use, and use online degassing or helium degassing during use.

• Flush the flow cell with methanol or another strong polar solvent. If needed, use 1N nitric acid (do not use hydrochloric acid).

• Remove blockages or replace tubing. Refer to the detector manual to replace the flow cell window.

• Adjust the mobile phase ratio or flow rate. To avoid this issue, regularly check the composition and flow rate of the mobile phase.

• Flush with a medium-strength solvent when changing the mobile phase. Before analysis, flush the column with 10-20 times the volume of the new mobile phase. Pay extra attention to equilibrate the column when using ion-pair reagents or buffer salts.

• Check the composition of the mobile phase. Use high-quality chemicals and HPLC-grade solvents.

• Change the analysis conditions. Use a guard column and, if necessary, periodically flush the column with a strong solvent between injections or during the analysis.

• Re-set the baseline and use fresh mobile phase.

• Adjust the wavelength to the maximum absorption wavelength and re-select the detection wavelength.

09 Causes of Regular Baseline Noise

Causes

• Air in the mobile phase, detector, or pump (sharp peaks)

• Leakage

• Incomplete mixing of the mobile phase

• Temperature effects (column temperature too high, detector not heated)

• Other electronic devices on the same line (occasional noise)

• Pump vibration

Solutions

• Degas the mobile phase. Flush the system to remove air from the detector or pump.

• Check if the fittings are loose, if the pump is leaking, if there is salt precipitation, and if there are unusual noises. Replace pump seals if necessary.

• Shake the mobile phase manually to mix it uniformly or use low-viscosity solvents.

• Reduce temperature differences or use a heat exchanger.

• Disconnect the LC, detector, and recorder, and check if the interference comes from an external source and correct it. Use a precision-grade regulated power supply.

• Add a pulse damper to the system.

10 Causes of Irregular Baseline Noise

Causes

• Leakage

• Contaminated or degraded mobile phase or prepared with low-quality solvents

• Incompatibility of solvents in the mobile phase

• Issues with the detector/recorder electronic components

• Air bubbles in the system

• Air bubbles in the detector

• Contaminated flow cell (even minimal contamination can cause noise)

• Insufficient detector lamp energy

• Loss or blockage of column packing

• Uneven mixing of the mobile phase or malfunctioning mixer

Solutions

• Check if the fittings are loose, if the pump is leaking, if there is salt precipitation, and if there are unusual noises. Replace seals if necessary. Check if the flow cell is leaking.

• Check the composition of the mobile phase.

• Select compatible solvents for the mobile phase.

• Disconnect the power to the detector and recorder, check, and correct.

• Clean the system with a strong polar solvent.

• Clean the detector and install a backpressure regulator after the detector.

• Clean the flow cell with 1N nitric acid (do not use phosphoric acid).

• Replace the lamp.

• Replace the column.

• Repair or replace the mixer. It is recommended not to use the pump's mixing device when the mobile phase is not running in gradient mode.