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

https://www.princeton.edu/~wlu/sunil-2006.pdf

Separation and quantitation of water soluble cellular metabolites by hydrophilic interaction chromatography-tandem mass spectrometry.

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.

Retention behavior of small polar compounds on polar stationary phases in hydrophilic interaction chromatography.

Small organic acids have shown significant retention on various stationary phases, such as amide, amino, aspartamide, silica and sulfobetaine phase commonly used in hydrophilic interaction chromatography (HILIC). This study investigated the effect of chromatographic conditions on the retention behavior of organic acids in HILIC using the tool of design of experiment (DOE). The results of the DOE study indicated that both the content of organic solvent (i.e., acetonitrile) and salt concentration in the mobile phase had significant effects on the retention of organic acids. Higher content of organic solvent in the mobile phase led to a significant increase in retention on all types of stationary phases. Increasing salt concentration also resulted in a moderate increase in retention; however, the effect of salt concentration varied with the type of stationary phase. The study also revealed that column temperature had less impact on retention than organic solvent content and salt concentration in HILIC.

Investigating the Effect of Chromatographic Conditions on Retention of Organic Acids in Hydrophilic Interaction Chromatography Using a Design of Experiment

Peak capacity is the commonly used measure of separation efficiency in gradient elution. This study focuses on the effect of column characteristics (particle size and column length) and operating parameters (gradient time and flow rate) on the peak capacity for small molecule compounds in gradient elution. The goal of this study is to develop a practical strategy to maximize the separation efficiency (i.e., peak capacity) under different constraints (analysis time or pressure limit). Using both experimental data and theoretical modeling, the current study reveals that the peak capacity increases with both gradient time and column length in a non-linear fashion. Marginal peak capacity is proposed to characterize the non-linear increase of peak capacity over the gradient time and column length. This study also attempts to understand the maximum peak capacity achievable under certain pressure limits using Neue’s peak capacity model. The results of this study provide a better understanding of the UPLC technology, and can also help to develop practical strategies to maximize the separation efficiency in gradient elution to meet the separation needs.

Evaluation of The Peak Capacity of Various RP-Columns for Small Molecule Compounds in Gradient Elution

The primary goal of early phase development is to gain a fundamental knowledge of the chemistry of drug substances and drug products to facilitate optimization of synthetic schemes and drug product formulations. At the same time, methods are required for release and stability studies to support clinical trials, and must assure that products are safe in vivo. Ultimately, the knowledge gained during early development translates into designing control methods for commercial supplies that assure patient safety and efficacy. This chapter describes an iterative and systematic approach to HPLC method development, designed to meet the specified goals.

12 HPLC method development in early phase pharmaceutical development

The conventional approach to the measurement of peak capacity of reserved-phase columns under gradient elution assumes that all peaks have constant peak width, but this assumption can lead to inaccurate measurement of peak capacity. An integration approach employing a series of alkylphenones as model compounds was employed to more accurately measure the peak capacity for small molecule compounds under gradient elution. The base peak width of alkylphenones was plotted against retention time and a peak width function over retention time was established by polynomial regression. The peak capacity was then calculated by integrating the inverse of the peak width function over a gradient window. Compared to the conventional method, the integration method is not based on the assumption of equal peak width, thus providing a more accurate measurement of the peak capacity of reserved-phase columns, especially shorter ones packed with sub-2 μm particles under gradient elution.

Sheetal Gaiki

Papers

Retention behavior of small polar compounds on polar stationary phases in hydrophilic interaction chromatography.

Investigating the Effect of Chromatographic Conditions on Retention of Organic Acids in Hydrophilic Interaction Chromatography Using a Design of Experiment

Evaluation of The Peak Capacity of Various RP-Columns for Small Molecule Compounds in Gradient Elution

12 HPLC method development in early phase pharmaceutical development

Measuring Peak Capacity of Reversed-Phase Columns for Small Molecule Compounds Under Gradient Elution