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The Crucial Role of Water in Normal Phase Liquid Chromatography


Today we're going to dive into a fascinating topic in the world of liquid chromatography: the importance of adding water to the mobile phase in normal-phase liquid chromatography (NPLC). While NPLC typically employs nonpolar mobile phases, water, a highly polar solvent, may seem like an unlikely addition. However, there are compelling reasons for introducing water into the equation, and we're about to explore them in detail

Understanding the Need for Water in NPLC:

In NPLC, the mobile phase is predominantly non-aqueous and usually devoid of water. Instead, it often contains less polar solvents like alcohols. The retention mechanism in this non-aqueous mobile phase primarily relies on adsorption, determining which compounds get absorbed onto the stationary phase. This adsorption occurs due to competition between the sample and the mobile phase for polar adsorption sites on the stationary phase.

Let's consider a bare silica column with its polar silanol groups as an example. When a mobile phase containing a polar organic solvent (e.g., methanol) is used, polar moieties in the solvent compete with each other to adsorb onto the silanol groups. This is where the "like attracts like" rule comes into play, as polar solvents like methanol are preferentially adsorbed onto polar stationary phases, such as silica with hydroxyl groups.

However, polar solvents like methanol can also contain trace amounts of water, which can be absorbed onto the highly polar adsorption centers, such as silanol groups. Water, being an extremely polar solvent, forms hydrogen bonds more readily with these centers than with less polar solvents like methanol or ether. This interaction with water can create a thin layer of absorbed water-rich liquid stationary phase on the surface of the stationary phase, effectively blocking access to the silanol groups.

As the content of absorbed water increases, it progressively deactivates the adsorbent and significantly reduces sample retention time, ultimately affecting the reproducibility of NPLC.

The Reproducibility Challenge:

In NPLC with water-miscible organic mobile phases, achieving consistent retention times poses a significant challenge. This is because the amount of water present in the mobile phase can vary from day to day, from analyst to analyst, and from lab to lab. Consequently, this variability results in inconsistent retention times, making method reproducibility a formidable hurdle.

Two Strategies for Improvement:

1. Removing Water from the Mobile Phase:

One approach to enhance reproducibility is to eliminate water entirely from the non-aqueous mobile phase by using solvents that are completely dehydrated. This ensures that there is no interaction between water and the silica adsorbent sites, making the silanol groups available for interaction with analytes and, in turn, leading to consistent retention times.

2. Adding a Fixed Amount of Water:

Alternatively, deliberately adding a small, fixed amount of water (e.g., 1% or 2%) to the mobile phase can enhance reproducibility. In this scenario, the added water occupies a predictable portion of the silanol groups on the stationary phase. As a result, secondary interactions between analytes and the stationary phase are minimized or even completely suppressed. This leads to improved peak symmetry and increased column efficiency.


In conclusion, the seemingly counterintuitive addition of water to the mobile phase in normal-phase liquid chromatography plays a crucial role in achieving consistent and reproducible results. Whether you opt to eliminate water from the mobile phase or deliberately introduce a fixed amount, the goal is to minimize secondary interactions between analytes and the stationary phase. This not only improves reproducibility but also enhances peak symmetry and column efficiency. So, the next time you embark on an NPLC journey, remember the pivotal role that water can play in ensuring the reliability of your results. Thank you for joining us in unraveling the mysteries of water in NPLC.

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