Mobile Phase Contamination in HPLC: Causes of Baseline Drift and Noise

Issue Summary

Unstable baselines in high-performance liquid chromatography—such as drift, excessive noise, spikes, or waviness—are commonly linked to contamination in the mobile phase. Because the mobile phase continuously flows through every component of the LC system, even trace impurities can accumulate and disrupt detector response. Identifying and correcting mobile phase contamination is therefore a critical step in restoring chromatographic stability and data reliability.

Common Baseline Symptoms Linked to Contaminated Mobile Phases

Mobile phase contamination typically manifests as one or more of the following behaviors:

• Slow upward or downward baseline drift during a run

• High-frequency noise or periodic oscillations

• Sudden positive or negative spikes

• Ghost peaks appearing in blank or sample injections

• Gradual increases in system backpressure

• Decreasing column performance over time

When these symptoms persist in blank runs, the mobile phase should be considered a primary suspect.

Primary Causes of Mobile Phase Contamination

Impure Solvents

Solvents that are not specifically intended for chromatographic use may contain UV-absorbing compounds, stabilizers, or trace organic impurities. These contaminants contribute directly to detector noise and baseline instability, particularly in UV detection at low wavelengths.

Buffer-Related Issues

Incorrect buffer preparation can lead to undissolved salts or precipitation, especially when mobile phases contain high organic content or are stored at low temperatures. These solids may intermittently dissolve during operation, producing baseline disturbances and pressure fluctuations.

Microbial Growth

Aqueous mobile phases that are stored for extended periods are susceptible to microbial contamination. Metabolic byproducts and particulates released by microorganisms introduce background absorbance and random signal variation.

Dissolved Gases and Poor Degassing

Inadequately degassed mobile phases can release gas bubbles inside the pump or detector flow cell. These bubbles interrupt the optical path or flow consistency, causing waviness, spikes, and short-term baseline instability.

System-Derived Contaminants

Mobile phase purity can be compromised by the LC system itself. Worn pump seals, contaminated solvent lines, clogged inlet frits, or residual compounds from previous methods can all leach into the mobile phase and appear as baseline artifacts.

Isolation Strategy: Confirming Mobile Phase Contamination

A structured isolation approach helps verify whether the mobile phase is responsible:

1. Run a blank injection using the current mobile phase.

2. Prepare a fresh batch of mobile phase using new solvents, clean glassware, and freshly prepared buffers.

3. Operate the system without a column to determine whether baseline issues originate upstream.

4. Monitor the baseline while changing solvent composition to identify mixing or composition instability.

5. Replace inlet filters and solvent frits to eliminate particulate sources.

If baseline disturbances disappear after these steps, the root cause is confirmed as mobile phase contamination.

Corrective Actions to Restore Baseline Stability

Improve Mobile Phase Preparation

• Use chromatography-grade solvents only

• Accurately weigh buffer components and ensure complete dissolution

• Filter and degas all mobile phases before use

• Avoid reusing or topping off old solvent bottles

Optimize Degassing and Solvent Handling

• Apply vacuum or ultrasonic degassing consistently

• Minimize solvent exposure to air during operation

• Ensure solvent lines are properly seated and leak-free

Clean and Protect the LC System

• Flush the system with high-organic solvent to remove retained contaminants

• Replace worn pump components that may shed debris

• Install a guard column to intercept contaminants before they reach the analytical column

Preventive Practices for Long-Term Performance

• Label mobile phases with preparation dates and discard aged solutions

• Dedicate solvent bottles and tubing to specific methods

• Maintain stable laboratory temperature conditions

• Perform routine system flushing between methods

These preventive measures significantly reduce baseline artifacts and extend both system and column lifetime.

Conclusion

Mobile phase contamination is a leading cause of baseline drift and noise in HPLC systems. By applying disciplined solvent preparation practices, effective degassing, and systematic isolation techniques, chromatographers can quickly resolve baseline instability and prevent recurrence. Clean mobile phases are foundational to reliable chromatography and reproducible analytical results.