Separation efficiencies in hydrophilic interaction chromatography

doi:10.1016/j.chroma.2008.01.075

Journal of Chromatography A

Volume 1184, Issues 1–2, 14 March 2008, Pages 474-503

https://doi.org/10.1016/j.chroma.2008.01.075 Get rights and content

Abstract

Hydrophilic interaction chromatography (HILIC) is important for the separation of highly polar substances including biologically active compounds, such as pharmaceutical drugs, neurotransmitters, nucleosides, nucleotides, amino acids, peptides, proteins, oligosaccharides, carbohydrates, etc. In the HILIC mode separation, aqueous organic solvents are used as mobile phases on more polar stationary phases that consist of bare silica, and silica phases modified with amino, amide, zwitterionic functional group, polyols including saccharides and other polar groups. This review discusses the column efficiency of HILIC materials in relation to solute and stationary phase structures, as well as comparisons between particle-packed and monolithic columns. In addition, a literature review consisting of 2006–2007 data is included, as a follow up to the excellent review by Hemström and Irgum.

Introduction

Recently, literature and research on hydrophilic interaction chromatography (HILIC) have been increasing drastically, along with various stationary phases developed for HILIC. HILIC is a kind of normal-phase liquid chromatography (NPLC), and has attracted the attention of researchers that study the separation of polar compounds in a wide variety of scientific fields. In HILIC mode, a mixture of water and organic modifiers in most cases acetonitrile (MeCN) is employed with a polar stationary phase. Structural variations in HILIC type stationary phases are wider than those found in reversed-phase applications. In addition to classical bare silica and aminopropyl-bonded silica, silica gels modified with many polar functionalities such as amide, diol, cyano, derivatives of poly(succinimide), sulfoalkylbetaine, cyclodextrin are applicable to HILIC mode separation. Polymer-based stationary phases such as sulfonated polymers, and diol-containing polymers can also be used. HILIC can be an alternative to reversed-phase chromatographic separation for polar compounds using isocratic and gradient elutions. Both particle-packed columns and monolithic columns have been used in HILIC. HILIC is suitable for electrospray ionization (ESI)-mass spectrometry (MS), because of the compatibility of the aqueous organic mobile phase to ESI-MS, which is a very powerful tool to detect and identify a wide range of polar compounds.

There are many examples of HILIC applications for the analysis of small polar molecules including biomarkers, nucleosides, nucleotides, carbohydrates, amino acids, peptides and proteins, that contribute to the pharmaceutical chemistry, agricultural and food chemistry, medicinal chemistry, proteomics, metabolomics, and glycomics. However, it seems that only limited attention has been paid to the separation efficiencies in HILIC. This review provides a perspective of HILIC type stationary phases, and discusses their separation efficiencies and retention tendencies.

First, various stationary phases for HILIC are introduced, and separation efficiencies of columns packed with particles, and suitable analytes for each stationary phase are discussed. Then, the same discussion is applied to monolithic columns to reveal the differences in these types of support for stationary phases.

Section snippets

Types of stationary phases for hydrophilic interaction chromatography

Although the acronym HILIC was already first suggested by Alpert in 1990 [1], the number of publications regarding HILIC has increased substantially since 2003, as outlined in the well-constructed review by Hemström and Irgum [2]. NPLC has been used widely to separate various compounds from non-polar compounds to highly polar compounds after chromatography was first introduced as a method of separation science [3], [4]. NPLC consists of polar stationary phases, mainly bare silica or alumina,

Separation efficiencies of particle-packed hydrophilic interaction chromatography mode columns

Stationary phases for reversed-phase separation are modified with silanes that possess alkyl chains, between C4 and C30, and mainly C18. Not only “pure” alkyl groups, but also partially polar alkyl groups are employed to increase the retention of polar compounds. This approach does not seem to be so effective for the separation of highly polar compounds. Compared to stationary phases for RPLC, columns for HILIC separation have a wider variety of functional groups and are described as follows.

Separation efficiencies of monolithic hydrophilic interaction chromatography mode columns

Monolithic materials, one-piece structure consisting of skeletons and through-pores have recently attracted the attentions of chromatographers recently. First, organic polymer monolithic columns [159], [160], then silica monolithic columns [161], [162] were reported. Monolithic materials possess several features as supports for chromatographic stationary phases such as higher mechanical stabilities in comparison to particle-packed columns, large through-pores which results in higher

Conclusion

The characteristics of stationary phases for HILIC in terms of the separation efficiencies are summarized in Table 1. Due to the lack of separation examples in the isocratic mode in many cases, the table is not fully completed. HILIC separation targets were roughly separated into eight groups, such as (1) acids, (2) bases, (3) polar small compounds like amides or ureas, (4) Polyols (5) amino acids, (6) peptides, (7) nucleic bases and nucleosides, and (8) oligosaccharides and carbohydrates.

Acknowledgements

We acknowledge the financial support from a Grant-in-Aid for Scientific Research funded by the Ministry of Education, Sports, Culture, Science and Technology, Nos. 17350036 and 19550088.

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¹: Present address: Eisai Co. Ltd., Japan.

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ReviewSeparation efficiencies in hydrophilic interaction chromatography

Abstract

Introduction

Section snippets

Types of stationary phases for hydrophilic interaction chromatography

Separation efficiencies of particle-packed hydrophilic interaction chromatography mode columns

Separation efficiencies of monolithic hydrophilic interaction chromatography mode columns

Conclusion

Acknowledgements

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Review
Separation efficiencies in hydrophilic interaction chromatography