Mobile Phase in HILIC Mode: Which are Strong and Which are Weak

Imagine a new analyst preparing a mobile phase for a HILIC (hydrophilic interaction liquid chromatography) method. Having learned reversed-phase HPLC, they assume that water is a weak eluent and that increasing its proportion should gradually increase the retention of polar compounds. Therefore, they prepare a mobile phase with more water. However, the peaks begin to elute earlier and crams together, and some components even show no retention at all.

The problem is not from the column or the sample, but from the elution strength of the mobile phase. The relationship within mobile phase composition and elution strength in HILIC mode is completely opposite to that in RP-HPLC: organic solvents such as acetonitrile are relatively weak in HILIC mode, while water is the strong eluent. In other words, the relative elution strength in HILIC follows this order: Acetone < Acetonitrile < Isopropanol < Ethanol < Methanol < Water. 

Note: what is discussed in this article is valid for most, but not all, analytes and scenarios. Counterexamples, which are not discussed due to article length, may exist for the arguments in the article.

The reason is closely related to the HILIC retention mechanism. A water-rich layer forms on the surface of the polar stationary phase. Polar analytes preferentially partition into this layer, resulting in retention. Increasing the water content of the mobile phase strengthens its elution capability, allowing analytes to remain in the mobile phase rather than partition into the stationary-phase water layer. As a consequence, increasing water decreases retention and accelerates elution in HILIC, and excessive water can cause insufficient retention, early elution, or even loss of meaningful separation.

In 2015, Megumi Iwasaki et. al. analyzed kynurenic acid (KYNA) and 7-chlorokynurenic acid (Cl-KYNA) under isocratic HILIC conditions. They found out that the capacity factors of KYNA and Cl-KYNA changes in correspondence to both the concentration of acetonitrile and the pH in the mobile phase. Furthermore, the elution order reversed between acetonitrile-rich and water-rich mobile phases.

In HILIC, Acetonitrile is almost always the main component in the mobile phase for four reasons:

• Aprotic nature: Acetonitrile is a polar aprotic solvent. Unlike alcohols, it does not readily donate hydrogen bonds, helping preserve the water-rich layer on the stationary phase and the associated analyte partitioning mechanism.
• Low viscosity: Acetonitrile's low viscosity helps reduce system backpressure and facilitates efficient chromatographic operation.
• Low UV cutoff: With a UV cutoff of approximately 190 nm, acetonitrile is suitable for UV detection at relatively short wavelengths, making it compatible with a broad range of UV-active analytes.
• Favorable desolvation: Acetonitrile is relatively volatile and has favorable evaporation characteristics, facilitating solvent removal during electrospray ionization mass spectrometry (ESI-MS).

What about polar alcohols (e.g. methanol, ethanol, isopropanol)? While they are also used as mobile phases in HILIC mode, they cannot replace acetonitrile because they can disrupt hydrogen bonding and alter the water-rich layer, and their elution strengths are stronger than acetonitrile. A practical way is to retain acetonitrile as the primary organic phase and replace a portion of water with a polar alcohol to adjust selectivity.

Acetone, in rare cases, may replace acetonitrile. However, its UV cutoff is approximately 330 nm, which voids its capability in most UV methods. Its volatility and desolvation behavior also differ from those of acetonitrile. Therefore, acetone is only reserved for specialized applications.

The relationship between pH and retention in HILIC can also differ substantially from that in reversed-phase HPLC. In reversed-phase, basic compounds are often protonated at low pH, increasing retention; in HILIC they are ionized instead to increase retention (low to medium pH for basic compounds and high pH for acidic compounds).

The reason for this behavior is that, at high pH, silanol groups from the stationary phase is deprotonated and negatively charged, attracting positively charged basic compounds and repelling negatively charged acidic compounds; at low pH it is the reverse. Such behavior cannot reliably be predicted from analyte pKa, and experiment is essential.

Furthermore, since a mobile phase in HILIC mode often contain a high proportion of acetonitrile, the actual pH of the mobile phase shifts toward neutral for around 1 to 1.5 units compared with the original aqueous buffer. For example, an aqueous buffer adjusted to pH 9 may correspond to an actual mobile phase pH of approximately 7.5–8. Meanwhile, the pKa of the target analyte also shifts in the acetonitrile-rich environment. Consequently, its actual ionization state may differ considerably from what would be predicted using its pKa measured in water.

HILIC is frequently coupled with ESI-MS, whole response heavily relies on ionization state. However, a high proportion of acetonitrile inherently promotes droplet formation and desolvation. Ionizing the target analytes further enhances the response.

In fact, a majority of analytes show higher response under high pH conditions than under low pH conditions in HILIC-MS. Only a few basic compounds (morphine, diphenhydramine, etc.) show higher response under low pH conditions. This is opposite to RP-HPLC, where acidic conditions are more commonly used.

In HILIC mode, buffers do more than adjusting pH: they determine peak shape and retention. Without sufficient buffering, charged analytes may undergo unwanted secondary interactions with residual silanol groups or charged functional groups on the stationary phase. These interactions can cause peak tailing, broadening, or loss of retention.

Ammonium formate and ammonium acetate are generally the first choices for HILIC-MS, since they are soluble in high-concentration organic solvent, volatile, and compatible with ESI-MS at suitable concentrations (10 mmol/L for instance).

It should be noted, that when using gradient method in HILIC, both phases should contain buffer. If only the aqueous phase is buffered, the pH and ionization strength would drift during gradient.

Buffer concentration affects acidic and basic compounds in different ways. For basic compounds, increasing buffer concentration facilitates neutral ion pairs, which is more soluble in the hydrophobic mobile phase, resulting in shorter retention and improved peak shape. For acidic compounds, buffer ions can screen electrostatic repulsion from negatively charged silanol group, allowing the analytes to interact more strongly with the stationary phase and extending retention.

An example is Eva Cífková et. al. (2016), where researchers investigated the separation of several acidic lipid classes using HILIC-MS. In the thesis, researchers concluded that a relatively high concentration of ammonium formate (40 mmol/L in the study) is important in obtaining good peak shapes of these acidic lipid classes.

The behaviors of mobile phase in HILIC are opposite to those in RP-HPLC. The following table lists the core properties of the mobile phase in HILIC mode that are different from RP-HPLC.