Retention and Selectivity of Polar Neutral Molecules in Hydrophilic Interaction Liquid Chromatography (HILIC)

By: David S. Bell, Hugh Cramer, Reporter US Volume 31.2


Analogous to reversed-phase chromatography, polar stationary phases used in hydrophilic interaction chromatography (HILIC) provide different interactions that can be exploited by the chromatographer to retain and separate various components of a mixture. Mechanisms of interaction in HILIC include partitioning, polar interactions and ionic interactions1. Partitioning involves the phase transfer of polar analytes from an organic rich mobile phase into an adsorbed layer of water on the stationary phase. Polar interactions may then occur between the active surface or ligands of the stationary phase, and ionic interactions may occur between charged analytes and oppositely charged moieties on the phase. Stationary phase chemistries can be designed to heighten or attenuate the different mechanisms and thus impart alternative retention and selectivity. For example, bare silica has been shown to adsorb water in the presence of an organic rich mobile phase and thus provides the opportunity for an analyte to partition. Bare silica, under certain pH conditions, also exhibits negatively charged silanol functionalities that may interact strongly with positively charged analytes (ion-exchange). A pentafluorophenyl phase has been shown to exhibit very little partition yet provides a high degree of ion-exchange potential2. On the other end of the spectrum, a pentahydroxy stationary phase, due to the high degree of water retained by the surface, predominantly retains analytes through partition mechanisms and shows relatively little ion-exchange capacity. Through an understanding of the basic interactions stationary phase chemistries provide, one can choose the right blend of interactions that best complements a given separation challenge.

In this study, a set of nucleosides is used to demonstrate the utility of this approach. Nucleosides in general are polar molecules and are weakly basic, thus fall under the category of ‘polar neutrals’. Cytidine, for example, exhibits a basic pKa value of 4.3; whereas, its most acidic pKa is 13.5. Below a pH of 4.3 the compound would predominantly carry a positive charge; however, under most HILIC conditions, the effective pH is such that the compound will be neutral. Figure 1 and Table 1 present the structures of the study analytes and pertinent physicochemical data, respectively. The set of compounds were screened using the stationary phases previously discussed under several HILIC conditions. As one would predict, only those phases exhibiting partition mechanisms proved useful.

Nucleoside Structures

Figure 1. Nucleoside Structures


Table 1. Solubility and Ionization Constants for Nucleoside Probes*

Name pKa
(Most Acidic)
(Most Basic)
LogD at pH
1.7 4.6 6.5 7.4 8
Cytidine 13.5 4.3 -4.02 -2.28 -2.18 -2.18 -2.18
Uridine 9.4 n/a -1.91 -1.91 -1.91 -1.92 -1.93
Inosine - T1** 0 8.7 -4.16 -4.16 -4.16 -4.18 -4.22
Inosine - T2 13.2 3.3 -3.6 -1.98 -1.95 -1.95 -1.95
Inosine - T3 8.9 1.6 -2.37 -2.19 -2.19 -2.21 -2.25
Guanosine - T1** 9.6 2.4 -2.62 -1.88 -1.88 -1.88 -1.89
Guanosine - T2 13.2 3.1 -3.13 -1.66 -1.64 -1.64 -1.64
5-Methyluridine 9.6 n/a -1.49 -1.49 -1.49 -1.49 -1.49
5-Methylcytidine 13.5 4.6 -3.69 -1.97 -1.78 -1.78 -1.78
7-Methylguanosine 6.8 -4 -6.12 -6.11 -5.79 -5.58 -5.54
Pseudouridine 8.5 -4.6 -1.2 -1.2 -1.21 -1.29 -1.48
3-Methylcytidine 13.3 8.9 -3.84 -3.84 -3.72 -3.29 -2.83
2-Thiocytidine 13 2.8 -2.48 -1.41 -1.4 -1.4 -1.4
1-Methyladenosine 13.2 6.1 -4.78 -3.52 -2.19 -2.05 -2.03
2'-O-methylcytidine 13.4 4.3 -3.25 -1.51 -1.38 -1.38 -1.38
*Calculated values from ACD/Percepta, v. 14.0.0
**Inosine and guanosine exist as tautomers


Retention and selectivity data were obtained for a set of twelve nucleosides run using Ascentis® Express OH5 (pentahydroxy), Ascentis Express F5 (pentafluorophenyl), and Ascentis Express HILIC (bare silica) with a variety of mobile phase modifiers and pH values. Note that the pH values listed are measured in aqueous solvent prior to addition of organic. Gradient elution from 95% acetonitrile to 80% acetonitrile was utilized with each modifier condition. Modifiers included 5 mM ammonium acetate at adjusted to pH values of 3, 4, 5 and 6.9 with formic acid and 0.1% formic acid alone. Chromatographic data was obtained at a flow rate of 0.6 mL/min, a temperature of 35 °C and UV detection at a wavelength of 250 nm. Sample mixtures (0.5 µL injections) ranging in individual concentrations from 10–100 µg/mL in water were used.

Results and Discussion

The majority of the chosen nucleosides are neutral within the useful chromatographic pH window and thus cannot interact via ion-exchange. In order to provide retention for the polar neutral molecules, the stationary phase must provide a partitioning mechanism. Both the Ascentis Express OH5 and HILIC phases are polar enough to adsorb water onto their surfaces, thus enabling the potential for partitioning to take place. Figures 2 and 3 show the screening results for the OH5 and HILIC columns, respectively. Both phases provide good retention and selectivity for all of the probes. It is interesting to note that the last three eluting compounds show increased relative retention on the HILIC phase as compared to the OH5 phase. The retention of these late eluters also vary with pH on the HILIC, but are relatively stable using the OH5. Both observations indicate some ion-exchange may be taking place and demonstrates the limited ion-exchange exhibited by the OH5 phase as compared to HILIC. The late eluting compounds were later identified as 1-methyladenosine (basic pKa 6.1), 3- methycytidine (basic pKa 8.9) and the permanently charged 7-methylguanosine.

Screening Results for Nucleosides Using Ascentis Express OH5

Figure 2. Screening Results for Nucleosides Using Ascentis Express OH5


Screening Results for Nucleosides Using Ascentis Express HILIC

Figure 3. Screening Results for Nucleosides Using Ascentis Express HILIC


Figure 4 shows the same experiments run on the Ascentis Express F5. As expected, little or no retention is observed for the polar neutral molecules due to the lack of partitioning provided by this particular phase chemistry. It again appears that a few of the nucleosides are charged enough to retain on the F5 via ion-exchange mechanisms. The screening data indicated that the OH5 column using a pH of 5 provided the most selective and efficient starting point for further method development. These conditions were slightly refined to provide the useful separation shown in Figure 5.

Screening Results for Nucleosides Using Ascentis Express F5

Figure 4. Screening Results for Nucleosides Using Ascentis Express F5



Figure 5. Optimized Separation of Nucleosides Using Ascentis Express OH5
CONDITIONS; column: Ascentis Express OH5, 10 cm x 2.1 mm, 2.7 µm (Product No. 53757-U) mobile phase: (A) 5 mM ammonium acetate, pH 5.0 with acetic acid in 95:5, acetonitrile:water; (B) 5 mM ammonium acetate, pH 5.0 with acetic acid in 80:20, acetonitrile:water; gradient: 0% B held for 1 min; to 100% B in 10 min; held at 100% B for 1 min; flow rate: 0.3 mL/min; column temp.: 25 °C; detector: UV at 250 nm; injection: 2 µL; sample: 10 – 100 µg/mL in 95:5, acetonitrile:water; other information: pH of buffer stock (in water) was adjusted before further dilution with water and/or acetonitrile


HILIC chromatography is a complex system involving partition, polar and ion-exchange interactions. Method development can be greatly facilitated by understanding the interactions that the different stationary phases provide, and applying that knowledge to the separation task at hand. In this study, the Ascentis Express OH5, HILIC and F5 stationary phases are contrasted. The OH5 phase provides primarily partitioning mechanisms, the F5 phase provides primarily ion-exchange and the HILIC phase provides a blend of the two mechanisms. For a neutral set of molecules such as the nucleosides, only partition can be expected to provide interactions that result in retention and selectivity. Indeed, the OH5 and the HILIC phase were shown to be useful and worth the time to investigate.


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  1. W. Naidong, Journal of Chromatography B, 796 (2003) 209-224.
  2. D.S. Bell, Jones, A. Daniel, Journal of Chromatography A, 1073 (2005) 99-109.