Hydrophilic interaction chromatography (HILIC) is a liquid chromatography mode designed for highly polar analytes. Where reversed-phase HPLC retains peptides by hydrophobicity and can let very polar peptides pass through unretained, HILIC uses a polar stationary phase with a high-organic mobile phase so polar peptides partition into a water-rich layer and are retained.
Principles
Retention in HILIC increases with analyte polarity and decreases as the aqueous fraction of the mobile phase rises. Small differences in polar residues, terminal charges, and modifications shift retention measurably, giving HILIC selectivity that is complementary — often near-orthogonal — to reversed-phase separations.
Laboratory Applications
- Analyzing short, highly polar peptides poorly retained by RP-HPLC
- Separating closely related polar impurities and degradation products
- Coupling to mass spectrometry, since HILIC mobile phases are MS-friendly
- Providing an orthogonal purity method in research peptide quality control
Advantages and Limitations
HILIC offers strong retention for polar species and good MS sensitivity. Limitations include longer equilibration times, sensitivity to sample solvent composition, and method-development complexity — samples must be dissolved in high-organic solvent to avoid peak distortion.
References
Alpert, original HILIC description (Journal of Chromatography, 1990); analytical reviews on HILIC of peptides; USP chromatography general chapters.
For laboratory research education only. Not for human consumption / research use only. No medical, therapeutic, or dosing information is provided or implied.