Quantitative Insights into Nucleoside Retention Mechanisms on Silica via HILIC

Researchers at Fairleigh Dickinson University (Florham Park, New Jersey) published a study in Analytica that investigated the retention mechanisms of 16 native and modified nucleosides on a bare silica column in hydrophilic interaction liquid chromatography (HILIC) (1). While nucleosides are widely studied in HILIC, the retention mechanisms investigated by this team—partitioning, adsorption, and electrostatic interactions—have not been fully elucidated.

The researchers applied a quantitative assessment methodology to determine the contributions of each mechanism and experimentally measured partitioning coefficients (K). Overall, the study offers quantitative insights into nucleoside retention in HILIC and could potentially provide practical guidance for chromatographic method development. The partitioning coefficients measured using the quantitative assessment methodology could serve as a reliable alternative for assessing polarity of polar compounds.

LCGC International spoke to Yong Guo, Professor of Pharmaceutical Science and Chair of the Department of Pharmaceutical Sciences at the Fairleigh Dickinson’s College of Pharmacy and Health Sciences, and corresponding author of the paper, about the group’s findings.

Can you explain the main retention mechanisms in HILIC and how they might simultaneously influence the separation of polar compounds like nucleosides?

The retention mechanisms in HILIC are generally considered to involve hydrophilic partitioning, surface adsorption through polar interactions (such as hydrogen bonding) and electrostatic interactions if both analytes and stationary phases are charged. However, the main retention mechanism may be different for different compounds in a specific system (such as stationary phases and mobile phase). The challenge is to identify what the main retention mechanism is. Most nucleosides investigated in our study are neutral; therefore, both partitioning and adsorption may govern the retention in HILIC. Our study results indicate that the main retention mechanism may be adsorption or partitioning depending on the ammonium acetate concentration in the mobile phase (1).

What factors influence the phase ratio (Φ) in HILIC, and why is it an important parameter when evaluating retention mechanisms?

The adsorbed water layer is the de facto stationary phase as suggested by Pavel Jandera since partitioning occurs between the bulk mobile phase and the adsorbed water layer (2). The phase ratio (Φ = V_S/V_M) represents the volume of the adsorbed water layer in in HILIC. Many factors influence the phase ratio, such as the type of stationary phase (monomeric vs. polymeric phase), bonded phase chemistry, silica support (fully porous vs. superficially porous), acetonitrile content in the mobile phase, salt concentration (ammonium acetate), the third solvent (methanol in the mobile phase), and column temperature. The phase ratio is directly linked to the retention factor (k_par) in the partitioning process by partitioning coefficients (k_par = KΦ). In reversed-phase liquid chromatography (RPLC), the phase ratio is fixed for a specific column. In HILIC, the volume of the adsorbed water layer is influenced by the salt concentration (ammonium acetate) in the mobile phase. Therefore, the phase ratio can be manipulated by salt concentration. This provides a means to vary the phase ratio in a HILIC column by changing the mobile phase.

How does the quantitative assessment methodology (based on k_obs = k_ads + KΦ) help in separating the contributions of different retention mechanisms?

The quantitative assessment methodology starts with the basic thermodynamic principle that the retention factor in a partitioning-driving process is the product of the partitioning coefficient and phase ratio (k_par = KΦ). We hypothesize that the observed retention factor (k_obs) is proportional to the phase ratio for non-ionized compounds without electrostatic interactions assuming that the retention due to surface adsorption (k_ads)is independent of the phase ratio (k_obs = k_ads + KΦ). This hypothesis has been tested and shown to be valid (3). In practice, the observed retention factors (k_obs) are plotted against the phase ratio and linear regression is performed to obtain the slope and intercept. The slope of the k_obs vs. Φ line provides the partitioning coefficient (K) and the intercept the retention contributed by surface adsorption (k_ads). The retention contributed by partitioning is calculated by k_par = KΦ.

In the context of ionized analytes, how is the electrostatic interaction quantified in HILIC? What practical implications does this have for method development?

In addition to partitioning and adsorption, electrostatic interactions are a significant retention mechanism for ionized compounds on charged stationary phases. Like ion chromatography, the electrostatic effect is attenuated by salt concentrations in the mobile phase. It is observed that the electrostatic effectbecomes practically negligible at high salt concentrations in HILIC. If the salt concentration is further increased, the retention of the ionized analytes is controlled only by partitioning and adsorption in the same manner as the non-ionized analytes. Therefore, the equation k_obs = k_ads + KΦ can be applied to the ionized analytes at high salt concentrations if sufficient data points can be obtained within the solubility limit (4). Linear regression of the linear segment of the k_obs vs. Φ plots provides the partitioning coefficient (slope) and the retention contributed by adsorption (intercept), which are assumed to be independent of the phase ratio (the salt concentration). The electrostatic effect (k_elec), either attractive or repulsive at lower salt concentrations is calculated using the equation k_elec = k_obs – (k_ads + KΦ).

Quantitative understanding of the electrostatic effect may potentially assist method development for ionized compounds in HILIC. However, we are still at the beginning of understanding the electrostatic effect and more data is needed. The limited data indicates that electrostatic attraction is strong at low salt concentrations and may be adjusted to achieve desired retention and selectivity by simply changing the salt concentration. If electrostatic repulsion is present, it may be advisable to increase the salt concentration to a moderate level to minimize the repulsive effect and increase retention.

Why might methylation of a nucleoside decrease its polarity, and under what condition could methylation instead increase polarity?

Addition of a non-polar methyl group to native nucleosides generally make the molecule more hydrophobic, decreasing the polarity. Smaller partitioning coefficients of methylated nucleosides obtained in our study are consistent with this trend. In addition, methylation can block the site for hydrogen bonding and reduce overall dipole moment. If methylation results in charges on the nucleosides (for example, 3-methylcytidine and 7-methylguanosine), the polarity of methylated nucleosides increases due to the presence of positive charges.

Why was a bare silica stationary phase used in the study instead of other HILIC phases like amide or zwitterionic phases?

A bare silica stationary phase was selected for this study for three reasons. First, the bare silica phase has been successfully used for nucleoside separation in HILIC. Second, we have used the quantitative assessment methodology to evaluate the retention mechanisms on other HILIC phases such as amide and zwitterionic phases, but not the bare silica phase. Third, we suspect that there would be significant surface adsorption for nucleosides on the bare silica phase due to the presence of surface silanol groups.

How does the quantitative assessment methodology overcome limitations associated with traditional log P values for polar compounds?

The traditional shake flask method may have relatively large errors in the log P values of polar compounds due to very low solubility of polar compounds in the octanol phase and formation of a hydration shell, which hinders the transfer of polar compounds to the octal phase. The HPLC method based on reversed-phase separation may not be suitable for very polar compounds due to insufficient retention. In addition, the conventional log P values are not applicable to ionized compounds and the log D values for ionized compounds are calculated, not directly measured. Quantitative assessment methodology enables direct measurement of partitioning coefficient of polar compounds (both ionized and non-ionized) in HILIC without the limitation of solubility and retention. We are hoping that a correlation between the partitioning coefficient (K) measured using the quantitative assessment methodology and traditional log P values can be established when sufficient compounds are tested. This would allow converting the partitioning coefficients in HILIC to log P values that are more commonly used.

How does temperature influence retention in HILIC, and how would you design an experiment to investigate this effect on nucleosides?

Column temperature influences the retention of polar compounds in HILIC in a variety of ways. First, the partitioning coefficient is temperature dependent, thus column temperature can change the retention due to partitioning. Second, the column temperature can change the volume of the adsorbed water layer, thus the phase ratio. Third, the column temperature may affect direct interactions between analytes and polar stationary phases, possibly leading to an effect on adsorption. To investigate the temperature effect on nucleosides, the retention contributions need to be evaluated in a temperature range, for example, 20 – 70^oC. For this purpose, nucleosides samples need to be run at different salt concentrations at each temperature. Essentially, all the experiments in the published article will be repeated at multiple temperatures. The retention data at each temperature point will be used to generate Van’t Hoff plots. The quantitative assessment methodology provides clear benefits to conventional thermodynamic studies. First, the phase ratio is measured and can be included in the van’t Hoff equation. Second, a clear understanding of the retention mechanisms at each temperature can help with the interpretation of enthalpy and entropy data.

Can you walk me through how you would optimize a HILIC method for separating a complex mixture of modified nucleosides?

Method optimization generally depends on the intended purpose of the method, and different approaches may be taken for different purposes (for example, run time optimization, resolution of critical pair, or isolation of matrix effects). Our study results indicate that most native and modified nucleosides have significantly different partitioning coefficients (K). This implies that there is adequate selectivity (α = K₂/K₁) for most nucleosides. Therefore, it may not be necessary to screen a significant number of columns for selectivity. Retention may be optimized to achieve sufficient resolution by either changing the column or simply changing the salt concentration in the mobile phase. The salt concentration is not a typical chromatographic parameter that is optimized in RPLC; however, it may significantly influence the separation in HILIC. For the nucleosides with similar partitioning coefficients (adenosine and 5-methyluridine), separation may be improved by their difference in adsorption through changing the stationary phase or acetonitrile content in the mobile phase.

References

1. Kleiner, D,; Muscatiello, D.; Gutierrez, Z et al. Quantitative Assessment of Retention Mechanisms of Nucleosides on a Bare Silica Stationary Phase in Hydrophilic Interaction Liquid Chromatography (HILIC). Analytica 2025, 6 (4), 39. DOI: 10.3390/analytica6040039
2. Jandera, P.; Hajek, T. A New Definition of the Stationary Phase Volume in Mixed-Mode Chromatographic Columns in Hydrophilic Liquid Chromatography. Molecules 2021, 26 (16), 4819. DOI: 10.3390/molecules26164819
3. Guo, Y.; Fattal, B. Relative Significance of Hydrophilic Partitioning and Surface Adsorption to the Retention of Polar Compounds in Hydrophilic Interaction Chromatography, Anal. Chim. Acta 2021, 1184, 339025. DOI: 10.1016/j.aca.2021.339025
4. Guo, Y.; Baran, D., Ryan, L. Quantitative Assessment of Retention Mechanisms of Ionized Compounds in Hydrophilic Interaction Chromatography. Anal. Chem. 2025, 97 (7), 4057 – 4065. DOI: 10.1021/acs.analchem.4c05880