In this article we delve into the critical aspect of selecting the perfect sample solvent for effective chromatography.
The choice of solvent for dissolving your HPLC sample is crucial because it directly affects the chromatography process.
Impact of Sample Solvent on Chromatography
Ideally, we want the sample solvent to have no effect on the chromatography. However, in reality, the solvent can significantly impact the result. For instance, if your sample is dissolved in a very strong solvent, like 100% methanol, and your mobile phase is relatively weak, 80% water and 20% methanol, the strong solvent can disrupt the chromatography. This often results in poor peak shapes, such as split peaks and shoulders on the peaks.
Best Practices for Solvent Selection
The ideal scenario is to dissolve your sample in the same solvent as your mobile phase. For example, if your mobile phase is 80% water and 20% methanol, you should dissolve your sample in the same 80/20 water-methanol mixture. This ensures that the sample solvent blends seamlessly with the mobile phase, minimizing any potential disruption in the chromatography process.
Alternative Choices and Their Advantages
Dissolving your sample in a solvent weaker than your mobile phase is usually acceptable and can sometimes be advantageous. For example, if your sample is dissolved in 100% water and your mobile phase is 50/50 methanol-water, this is generally fine. In fact, this approach can be beneficial in certain scenarios, such as when you want to make a large volume injection for trace analysis.
Managing Injection Volumes
Injection volume plays a critical role in achieving optimal chromatographic outcomes, especially in trace analysis. A typical injection volume might be around 5 µL, which is great for chromatography, but not always ideal for sensitivity. Increasing the injection volume can improve sensitivity but may also cause peak broadening. To mitigate this, you can use a weaker solvent to focus the sample on the column, enhancing sensitivity without compromising peak shape.
Balancing Solvent Strength with Injection Volume
Striking the right balance between solvent strength and injection volume is key to obtaining accurate and reliable results. If you need to inject a sample dissolved in a strong solvent, such as 100% methanol, consider making a very small volume injection, 1-2 µL, to minimize any negative impact on the chromatography. Modern equipment allows for precise, small volume injections, ensuring high reproducibility and minimal disruption.
Conclusion
Selecting the right solvent for dissolving your HPLC samples is a critical step in ensuring effective chromatography. By following best practices and understanding the impact of solvent strength and injection volume, you can achieve optimal chromatographic results.
Sample prep is where most of the errors occur in the entire analytical procedure. You can also hurt your HPLC. Learn more here.
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