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.

Hydrophilic interaction liquid chromatography (HILIC) provides an alternative approach to effectively separate small polar compounds on polar stationary phases. The purpose of this work was to review the options for the characterization of HILIC stationary phases and their applications for separations of polar compounds in complex matrices. The characteristics of the hydrophilic stationary phase may affect and in some cases limit the choices of mobile phase composition, ion strength or buffer pH value available, since mechanisms other than hydrophilic partitioning could potentially occur. Enhancing our understanding of retention behavior in HILIC increases the scope of possible applications of liquid chromatography. One interesting option may also be to use HILIC in orthogonal and/or two-dimensional separations. Bioapplications of HILIC systems are also presented.

/pdf/hydrophilic-interaction-liquid-chromatography-hilic-a-6908wnnc4y.pdf

Hydrophilic interaction liquid chromatography (HILIC)—a powerful separation technique

http://www.protocol-online.org/forums/uploads/monthly_07_2011/msg-6644-082599400%201311927155.ipb

Stationary and mobile phases in hydrophilic interaction chromatography: a review

If an ion-exchange column is eluted with a predominantly organic mobile phase, then solutes can be retained through hydrophilic interaction even if they have the same charge as the stationary phase This combination is termed electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) With mixtures of solutes that differ greatly in charge, repulsion effects can be exploited to selectively antagonize the retention of the solutes that normally would be the best retained This permits the isocratic resolution of mixtures that normally require gradients, including peptides, amino acids, and nucleotides ERLIC affords convenient separations of highly charged peptides that cannot readily be resolved by other means In addition, phosphopeptides can be isolated selectively from a tryptic digest

http://pubs.acs.org/doi/pdf/10.1021/ac070997p

Electrostatic repulsion hydrophilic interaction chromatography for isocratic separation of charged solutes and selective isolation of phosphopeptides.

Liquid chromatography of saccharides

Two-dimensional paper chromatography interspersed with reaction on the paper

Electron microscopic examination of cotton (Gossypium hirsutum L.) and sugarbeet (Beta vulgaris L.) radicles subjected to ethylene at 1, 10, 100 microliters per liter and a saturated ethylene atmosphere showed distinct ultrastructural differences compared to aerobic, control tissue and to anaerobic, nitrogen-treated radicles. Short term ethylene treatments of 1 hour induced the formation of cisternal stacks of rough endoplasmic reticulum in undifferentiated radicle cells. Similar profiles of rough endoplasmic reticulum were not observed in control radicles, but were found in nitrogen-treated tissues. Additionally, ribosomes in all ethylene-treated radicles were larger than ribosomes in control and nitrogen-treated cells. By ribosome density determinations it was found that the ethylene-treated ribosomes were swollen and less dense. The cell walls of the ethylene-treated tissue were also expanded and less dense than those of control tissue.

Ethylene-induced Fine Structure Alterations in Cotton and Sugarbeet Radicle Cells

Glucose yields in the enzymatic hydrolysis of cellulose to glucose are significantly enhanced by treating cellulosic material with ethylene either prior to and/or during the hydrolytic reaction.

Enzymatic method of producing glucose from ethylene treated cellulose

The distribution of 14 C in the various glucose residues of maltotriose was studied as a function of time of photosynthesis of isolated chloroplasts of spinach ( Spinacia oleracea L.) using 14 CO 2 . The distribution of label showed that the reducing-end glucose residue was labeled first and the label subsequently distributed to the second and third glucose residues at approximately equal rates. A mechanism for the distribution of label and the synthesis of maltotriose from the reducing end is presented. The mechanism has postulated to be the same as that for the maltose synthase recently described by Schilling. Maltose biosynthesis from α-d-glucose-1-phosphate was characterized as involving two glucosyl intermediates by a double displacement mechanism with inversion of configuration. The mode of enzymic action by which maltosyl intermediates were transferred to glucosyl intermediates was consistent with the fractional distribution of radioactivity found in each glucose unit of maltotriose during short term photosynthesis experiments.

/pdf/de-novo-maltotriose-biosynthesis-from-the-reducing-end-by-11bd5tsksn.pdf

James C. Linden

Papers

Liquid chromatography of saccharides

Two-dimensional paper chromatography interspersed with reaction on the paper

Ethylene-induced Fine Structure Alterations in Cotton and Sugarbeet Radicle Cells

Enzymatic method of producing glucose from ethylene treated cellulose

De Novo Maltotriose Biosynthesis from the Reducing End by Spinacia oleracea L. Chloroplasts