Basics of Mobile Phases in Reverse Phase Chromatography
In reverse-phase chromatography, our stationary phase is non-polar, while the mobile phase is polar. This polarity is typically achieved by incorporating an aqueous component, such as water, into the mobile phase. However, it's crucial to understand the limitations in the amount of water that can be added.
Highly polar molecules, like organic acids (e.g., ascorbic acid), might prompt us to use a highly aqueous mobile phase. However, since our stationary phase is non-polar, it does not retain these polar compounds well. Increasing the water content in the mobile phase can seem like a solution, but there are limits to how much water can be added without affecting the chromatographic process.
Challenges with Highly Aqueous Mobile Phases
Using a mobile phase with high water content (like 90% or more) presents certain challenges. These challenges need to be addressed and overcome for successful chromatography. This article will delve into these challenges and offer solutions.
Understanding C8/C18 Stationary Phases
C8 and C18 stationary phases are non-polar, oil-like materials. Their interaction with water is limited, which can lead to issues. If the mobile phase is too aqueous, it can fail to wet the stationary phase, leading to a phenomenon known as 'stationary phase collapse', where the phase loses its ability to retain compounds effectively.
Visualizing the Issue
Imagine two scenarios: one where the mobile phase contains both water and methanol, and another where it contains only water. In the former, the C8 or C18 stationary phase is properly wetted. In the latter, it becomes 'dewetted', losing its efficacy.
Why Avoid Highly Aqueous Mobile Phases?
1. Stationary Phase Wetting Issues:
When a highly aqueous (very polar) mobile phase is used, it does not interact well with the non-polar stationary phase. Water, being polar, does not effectively "wet" the non-polar surface of the C8 or C18 stationary phases. This lack of proper wetting leads to inadequate interaction between the stationary phase and the analytes (the compounds being separated), affecting the separation efficiency.
2. Stationary Phase Collapse:
Continuous use of a highly aqueous mobile phase can lead to what is known as 'stationary phase collapse'. In this state, the stationary phase fails to retain the analytes effectively. This collapse is due to the high surface tension of water, which prevents it from penetrating the pores of the stationary phase adequately. As a result, the analytes cannot interact effectively with the stationary phase, leading to poor separation and retention issues.
3. Difficulty in Reversing Phase Deactivation:
Once the stationary phase is 'dewetted' due to a highly aqueous mobile phase, reversing this condition can be challenging. It often requires passing a non-polar solvent through the column to reestablish proper wetting of the stationary phase, a process that can be time-consuming and may not fully restore the column's original efficiency.
4. Retention Time Variability and Peak Tailing:
Highly aqueous mobile phases can cause inconsistencies in retention times and increased peak tailing. This is because the interaction between the analytes and the stationary phase is not stable, leading to variable retention times and distorted peak shapes.
Overcoming the Challenges
When using highly aqueous mobile phases, consider these strategies:
Strategy 1:
Use non end-capped columns as they have free silanol groups that interact better with water.
Strategy 2:
Opt for short-chain alkyl phases (C3, C4) which have more free volume and less shielding, enhancing water interaction.
Strategy 3:
Use hydrophilic stationary phases, like those containing amine, amide, phenyl, or pentafluorophenyl groups.
Strategy 4:
Try wide pore diameter phases, as larger pores facilitate better water penetration.
CONCLUSION:
In summary, the use of highly aqueous mobile phases in HPLC with C8/C18 columns is generally avoided because it leads to poor interaction between the stationary phase and the analytes, causing issues like stationary phase collapse, inconsistent retention times, peak tailing, and difficulties in restoring column efficiency.