Introduction:
I'm Bhaskar Napte, and in this blog, we're focusing on the crucial precautions needed for handling and using HPLC/UPLC mobile phases. A correctly prepared mobile phase is fundamental to achieving consistent and reliable results in your chromatography analysis.
1. Selecting the Right Solvents and Chemicals:
The foundation of a successful HPLC analysis lies in using suitable grade solvents and chemicals. For gradient elution modes, it's imperative to opt for ultra-pure HPLC-grade solvents. Even when specific grades aren't available, never compromise on quality, as impurities can lead to additional peaks, or noisy baseline.
2. Importance of Proper Mixing:
Mixing different solvents is essential for achieving homogeneous mobile phase composition. This consistency is key to ensuring equal retention times for each injection. Remember, mixing time doesn't necessarily scale linearly with volume – it's more about achieving homogeneity than sticking to a rigid time frame. Optimize mixing time cautiously if your mobile phase contains viscous solvents such as IPA, OPA, etc. The de-mixing can take place if the solvents have a large polarity difference. Add a co-solvent to overcome phase separation if the mobile phase has solvents that have high polarity differences (e.g., methanol can be added as a co-solvent in case the mobile phase has water and ethyl acetate).
3. Concentration of the Buffer:
The concentration of the buffer or the type of salt in the mobile phase can lead to precipitation over extended periods of storage. Initially, during mobile phase preparation, the solution might appear clear, possibly due to a slightly elevated temperature that enhances the solubility of the salt in the solvents. However, as the solution settles to room temperature over time, the solubility of the salt may decrease, leading to precipitation and rendering the mobile phase unsuitable. Additionally, particular attention is needed when using the mobile phase in gradient elution. The salts, being polar, can become less soluble in the presence of less polar organic solvents. This issue is common in gradient elution, where the mobile phase progressively becomes less polar, potentially causing the salts to precipitate within the HPLC tubing. 4. Consistency in Mixing Techniques:
To avoid errors and variability, use the same mixing procedure every time. Whether you're using mechanical stirring or manual shaking, consistency is crucial for reproducible results.
5. Filtration and Degassing:
Filter your mobile phase using 0.45 or 0.22 Micron filters under a vacuum to remove suspended impurities. Additionally, degassing is vital for removing dissolved air, which can cause flow disturbances and detector noise. However, be cautious of over-degassing, especially with volatile organic solvents.
6. Storage and Microbial Growth Prevention:
Store mobile phases in appropriate containers – borosilicate glass for most, but stainless steel for solutions with pH above 8.0 to avoid metal ion leaching. For long-term storage, particularly with buffered phases, consider adding a small percentage of organic solvent to inhibit microbial growth (This needs to be considered during method development).
7. Container Specifications:
Use containers with narrow openings to limit oxygen intake and solvent evaporation. Incorporate online filters to catch any residual particulates that might form over time.
8. Special Considerations for Certain Solvents:
For oxidizable solvents like chloroform or THF, use an inert gas cover to prevent oxidation. Photosensitive solutions should be stored in amber-colored bottles or covered with aluminum foil to protect from light.
9. Post-Analysis Maintenance:
After completing your analyses, flush the system with water to prevent the deposition of crystalline deposits in the column and pump.
Conclusion:
Managing your HPLC mobile phases effectively is a critical aspect of maintaining your analytical system's integrity. With these guidelines, you can ensure the longevity and accuracy of your HPLC system. Thank you for reading, and continue to expand your knowledge in the field of chromatography!
Very good article. Useful for analytical chemist fraternity.