Introduction

Hydrophilic Interaction Chromatography (HILIC) is a variant of normal-phase chromatography which is performed using polar stationary phases with partially aqueous eluents. Solutes elute in order of increasing hydrophilicity (polarity), the opposite of reversed-phase.

Mode of Operation

Retention in HILIC is proportional to the amount of organic solvent in the eluent. Typical HILIC eluents contain 65-80% acetonitrile, methanol or propanol. The low proportion of water in the eluent generates a stagnant aqueous layer on the surface of the stationary phase. This enables solutes to partition between the eluent and aqueous layers (Figure 1). In addition, weak electrostatic interactions between solute and stationary phase contribute to overall selectivity

Gradient elution may be performed either with a decreasing organic or increasing salt gradient. Salt is not required for uncharged solutes such as carbohydrates, but 10mM salt is necessary with charged solutes such as peptides.

Ammonium formate and acetate are suitable volatile buffers for LC-MS. Other salts with good solubility in HILIC eluents include potassium methylphosphonate, triethylamine phosphate and sodium perchlorate.

Applications

HILIC phases are particularly useful for compounds that are weakly retained or eluted in the void volume of reversed-phase columns. Typical application areas include carbohydrates, oligonucleotides, peptides and proteins, amino acids, natural products and phosphorylated compounds. HILIC phases do not react with reduceable sugars, making them a good substitute for amino columns in analyzing carbohydrates. Figure 2 illustrates the orthogonal separation selectivity of HILIC compared to reversed-phase.

HILIC Phases

BioBasic AX Cosmosil HILIC Epic HILIC-HC
HALO HILIC Inertsil HILIC
Obelisc N PolyGLYCOPLEX PolyHYDROXYETHYL A PolySULFOETHYL A TSKgel Amide-80 ZIC-HILIC
ZIC-pHILIC
Manufacturer
Thermo Scientific Nacalai Tesque ES Industries
Advanced Materials Technology GL Sciences SIELC
PolyLC
Tosoh Bioscience
SeQuant
Functional Group
Polyethyleneimine Triazole Polyhydroxylated polymer
-Propyl alcohol
Proprietary -Hydroxyethylaspartamide Sulfoethylaspartamide
Carbamoyl Sulfobetaine Sulfobetaine
Particle Size (µm)
5 5 3, 5, 10
2.7
5 5, 10 5, 12
3, 5, 12
3, 5, 12 5, 10 3 3.5, 5, 10 5
Pore Size (Å)
300 120 120 90Å phase on solid core 100 100 -60, 100, 200, 300, 500, 1000, 1500 300, 1000 80 100 100, 200 200