An improved approach to hydrophilic interaction chromatography of peptides: Salt gradients in the presence of high isocratic acetonitrile concentrations

Abstract

Hydrophilic interaction chromatography (HILIC) for separations of peptides has been employed infrequently, particularly considering that this technique was introduced over 20 years ago. The present manuscript describes a radical departure from the traditional HILIC elution approach, where separations are achieved via increasing salt (sodium perchlorate) gradients in the presence of high isocratic concentrations (>80%) of acetonitrile, denoted HILIC/SALT. This initial study compared to reversed-phase chromatography (RPC), HILIC and HILIC/SALT for the separation of mixtures of synthetic peptide standards varying in structure (amphipathic α-helix, random coil), length (10–26 residues), number of positively charged residues (+1 to +11) and hydrophilicity/hydrophobicity. Results showed a marked superiority of the HILIC/SALT approach compared to traditional HILIC and excellent complementarity to RPC for peptide separations. We believe these initial results offer a new dimension to HILIC, enabling it to transform from an occasional HPLC approach for peptide separations to a more generally applicable method.

Applications of hydrophilic interaction chromatography to amino acids, peptides, and proteins

“…Similar to that observed in HILIC/CEX, the HILIC/SALT approach can also provide good separations of closely related peptides, as well as amphipathic, α‐helical peptides, with substitution in the hydrophilic part . Last but not least, peak shapes were generally improved in HILIC/CEX and HILIC/SALT compared to HILIC . However, these techniques are obviously less MS friendly than pure HILIC due to the presence of nonvolatile additives such as NaClO 4 .…”

“…A linear increasing salt gradient is applied to elute peptides by an increase in the mobile phase polarity via the presence of salt . Similar to that observed in HILIC/CEX, the HILIC/SALT approach can also provide good separations of closely related peptides, as well as amphipathic, α‐helical peptides, with substitution in the hydrophilic part . Last but not least, peak shapes were generally improved in HILIC/CEX and HILIC/SALT compared to HILIC .…”

“…A variant of HILIC/CEX is HILIC/SALT that consists of using a regular HILIC stationary phase instead of CEX column. A linear increasing salt gradient is applied to elute peptides by an increase in the mobile phase polarity via the presence of salt . Similar to that observed in HILIC/CEX, the HILIC/SALT approach can also provide good separations of closely related peptides, as well as amphipathic, α‐helical peptides, with substitution in the hydrophilic part .…”

Multiplexed assay for protein quantitation in the invertebrate Gammarus fossarum by liquid chromatography coupled to tandem mass spectrometry

“…Contaminated waters may cause delays in the molting cycles with adverse outcome on reproduction of these organisms. These results show that the multiplexed SRM protein quantification could be a pertinent method to complement the data obtained from the physiological measures used in the analysis (molt stage, oocyte size) [26]. …”

A novel surface-confined glucaminium-based ionic liquid stationary phase for hydrophilic interaction/anion-exchange mixed-mode chromatography

“…Moreover, charged groups in bonded functionalities could provide the possibility of ion-exchange with oppositely charged solutes while improving hydrophilicity, and thus a HILIC/ion-change mixedmode chromatographic system is obtained. It was reported that HILIC/ion-exchange showed many advantages (such as tunable selectivity, more efficient retention and better separation) for the application of peptides [7], small molecular drugs [8], and proteins [9]. Recently some new HILIC stationary phases were developed and exhibited HILIC/ion-change mixed-mode retention behavior.…”