Hydrophilic-interaction chromatography for the separation of peptides, nucleic acids and other polar compounds

Separation of polar compounds on polar stationary phases with partly aqueous eluents is by no means a new separation mode in LC. The first HPLC applications were published more than 30 years ago, and were for a long time mostly confined to carbohydrate analysis. In the early 1990s new phases started to emerge, and the practice was given a name, hydrophilic interaction chromatography (HILIC). Although the use of this separation mode has been relatively limited, we have seen a sudden increase in popularity over the last few years, promoted by the need to analyze polar compounds in increasingly complex mixtures. Another reason for the increase in popularity is the widespread use of MS coupled to LC. The partly aqueous eluents high in ACN with a limited need of adding salt is almost ideal for ESI. The applications now encompass most categories of polar compounds, charged as well as uncharged, although HILIC is particularly well suited for solutes lacking charge where coulombic interactions cannot be used to mediate retention. The review attempts to summarize the ongoing discussion on the separation mechanism and gives an overview of the stationary phases used and the applications addressed with this separation mode in LC.

Hydrophilic interaction chromatography.

Publisher Summary This chapter discusses the composition of reducing sugars in solution. Reducing sugars differ from most other organic compounds in one characteristic property. When a pure organic compound that is not a reducing sugar is dissolved in a solvent, it is certain that the solution will usually contain only one compound; but when a reducing sugar, above an aldotetrose or a 2-pentulose, is dissolved in water, a solution is obtained that always contains at least six compounds: two pyranoses, two furanoses, and an acyclic (open-chain) carbonyl form and its hydrate. There are also minute proportions of septanoses and dimers. Very few of these many compounds that are present in the equilibrium solutions of sugars have ever been isolated. The only method for separating them from the equilibrium mixture is by crystallization and that depends on the fortuitous presence of seed crystals. The chapter details about the pyranose form, the furanose form, and the septanose form. It also discusses the composition of aldoses, ketoses, and substituted and derived sugars in aqueous solution.

The composition of reducing sugars in solution

The recent discovery of two defects (ribose-5-phosphate isomerase deficiency and transaldolase deficiency) in the reversible part of the pentose phosphate pathway (PPP) has stimulated interest in this pathway. In this review we describe the functions of the PPP, its relation to other pathways of carbohydrate metabolism and an overview of the metabolic defects in the reversible part of the PPP.

The biochemistry, metabolism and inherited defects of the pentose phosphate pathway: a review

The retention mechanism and chromatographic behavior for different polar analytes under hydrophilic interaction chromatography (HILIC) conditions have been studied by application of different mobile phases and stationary phases to various analytes at different temperatures. In addition to the commonly accepted mechanism of analyte liquid-liquid partitioning between mobile phase and water-enriched solvent layer which is partially immobilized onto the surface of the stationary phase, hydrogen-bonding, hydrophobic interaction, and ion-exchange interactions may also be involved. The predominant retention mechanism in HILIC separation is not always easily predictable. It can depend not only on the characteristics of the analytes but also on the selection of mobile and stationary phase compositions. The objective of this review is to evaluate the potential application of column temperature and mobile phase composition toward improving HILIC selectivity. The functional groups from analyte structures, stationary phase materials and organic mobile phase solvents will be highlighted.

L.A.Th. Verhaar

Papers

Liquid chromatography of sugars on silica-based stationary phases.

Contribution to the elucidation of the mechanism of sugar retention on amine-modified silica in liquid chromatography

Improved column efficiency in chromatographic analysis of sugars on cation-exchange resins by use of water-triethylamine eluents

The gas chromatographic determination of polyhydroxy monocarbonic acids obtained by oxygenation of-hexoses in aqueous alkaline solutions

Retention behaviour of carbohydrate oligomers in reversed-phase chromatography