Best Practices for Mobile Phases in HPLC: Preparation, Use, and Storage
Introduction
The mobile phase plays a critical role in high-performance liquid chromatography (HPLC) analyses. An ideal HPLC mobile phase solvent should have low viscosity, good chemical compatibility with both the stationary phase and detector, low toxicity, and reasonable cost and availability.
Despite its importance, the mobile phase is often overlooked, and seemingly minor oversights in its preparation, handling, or storage can lead to unexpected deviations in experimental results. Therefore, proper mobile phase management is essential in ensuring method reproducibility and system stability, as well as extending the lifespan of both the column and the instrument.
Selection and Composition of Mobile Phases
Depending on the system and the analysis itself, different mobile phases should be chosen. In reversed-phase HPLC (RP-LC) systems, methanol-water and acetonitrile-water are the most commonly used mobile phases.
- If UV detection is performed at low wavelengths, acetonitrile-water is generally preferred due to its lower UV absorbance.
- If a buffer is required, then the buffer salt concentration should be low to minimize column contamination and instrument wear.
- If C18-bonded silica columns are used, then the organic content in the mobile phase should not fall below 5% to maintain column efficiency and ensure system stability.
In normal-phase systems, a combination of two or more organic solvents (e.g., dichloromethane and hexane) is typically used as mobile phases.
Mobile phases can be delivered in either isocratic or gradient elution modes. When using gradient elution, a gradient program detailing the composition of solvents over time must be clearly defined, usually documented in tabular format.
Preparation of Mobile Phases
1. Selection of Solvents
When choosing mobile phase solvents, analysts should follow these guidelines:
- Choose solvents with moderate boiling points to avoid excessive evaporation and bubble formation.
- Ensure both physical and chemical compatibility with the detector; when using UV detector, solvents with low UV absorbance are ideal.
- The mobile phase must adequately dissolve all sample components to prevent precipitation or irreversible retention on the column.
- Use low-viscosity solvents to improve resolution, reduce system backpressure, and extend pump life.
- The mobile phase must not react with the analyte, nor degrade the stationary phase.
- In preparative separations, solvents should be easy to be removed, not interfering with the recovery of target compound.
2. Protocols in Preparation
- When mixing organic solvents with aqueous buffers, always add the organic solvent to the buffer solution to reduce the risk of salt precipitation.
- For isocratic methods, pre-mix the organic and aqueous phases thoroughly before use.
- Select an appropriate buffer system and adjust pH accurately.
- Degas and filter the mobile phase using suitable methods such as heating, vacuum, ultrasonication, or helium sparging.
Proper Handling of Mobile Phases During Use
- Mobile phases should be prepared fresh when possible, particularly those containing buffer salts prone to microbial growth. Avoid long-term storage.
- Never "top off" existing mobile phase with a freshly prepared batch. Always replace the entire volume to maintain consistency.
- Avoid using lab sealing films to tightly seal bottles. Instead, opt for dedicated one-way vented bottle caps to allow safe gas exchange.
- Always allow refrigerated mobile phases to reach room temperature before use.
- Avoid using surfactant-based detergents when cleaning mobile phase containers whenever possible.
- Set minimum pressure thresholds or minimum solvent volume alerts, refraining the system from running dry.
- When using buffered mobile phases, activate the plunger wash function or flush the pump heads manually after use to prevent salt buildup.
Mobile Phase Storage
- Mobile phases should be stored in glass or PTFE containers; stainless steel containers may also be used. Never store mobile phases in plastic containers due to the risk of plasticizers leaching into organic solvents.
- All containers must be tightly sealed to prevent solvent evaporation, as it may alter composition, and to minimize absorption of oxygen and carbon dioxide affecting pH levels.
- Phosphate and acetate buffer solutions should be prepared fresh as they are prone to microbial growth and degradation. If they are to be stored, they must be refrigerated for no longer than 3 days and re-filtered prior to use.
- Prepared mobile phases should be sealed tightly to avoid volatilization and contamination, and labeled clearly with solvent composition, ratio, preparation date, and expiration date.
- Light-sensitive solvents should be stored in amber bottles to protect them from degradation.
- Halogenated solvents should be used cautiously. These can contain acidic impurities, therefore corrode stainless steel components and decompose upon contact with water. Avoid storing halogenated solvent mixtures for extended periods, or prepare them fresh before use.
Conclusion
Effective management of mobile phases is critical to the success of HPLC analyses. Analysts should always focus on proper solvent selection, preparation, handling, and storage of mobile phases, and avoid common pitfalls listed in this article to achieve accurate, consistent experiment result.