HILIC: An Alternative Approach for Separating Highly Polar Analytes


E-Separation Solutions

E-Separation SolutionsE-Separation Solutions-11-16-2012
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Hydrophilic interaction chromatography (HILIC) is an alternative separation technique for highly polar analytes that are weakly retained in reversed-phase high performance liquid chromatography (HPLC). Participants in this HILIC Technology Forum are William Long of Agilent Technologies, Phil Koerner and Carl Sanchez of Phenomenex, and Nicole M. Cotta of Waters Corp.

Hydrophilic interaction chromatography (HILIC) is an alternative separation technique for highly polar analytes that are weakly retained in reversed-phase high performance liquid chromatography (HPLC). Participants in this HILIC Technology Forum are William Long of Agilent Technologies, Phil Koerner and Carl Sanchez of Phenomenex, and Nicole M. Cotta of Waters Corp.

What are the advantages and limitations of using gradient mobile phase conditions in HILIC mode separations? Do the needs for stringent control of ionic strength and extensive equilibration time make gradient operation secondary to isocratic operation?

Long: With HILIC or reversed phase, gradient conditions can be a useful tool for method scouting or for separation of a wider range of analytes than may be practical when using isocratic analysis. Control of ionic strength as well as pH, temperature, organic type, and concentration, are key to HILIC operation in isocratic or gradient mode; however, HILIC gradients typically require 20 column volumes for reequilibration as opposed to four to five column volumes with gradient reversed-phase HPLC.

Koerner and Sanchez: The advantages of gradient elution in HILIC mode are similar to those for reversed phase, namely the ability to effectively resolve mixtures containing components with large differences in retention behavior in a reasonable amount of time while maintaining good peak shape and detectability for all components. The use of gradient elution in HILIC is generally more costly in terms of time than reversed phase since reequilibration in HILIC typically requires at least 10 column volumes to maintain reasonable reproducibility and retention in subsequent analyses. In reversed phase often five column volumes are sufficient. With today’s automated HPLC systems, full reequilibration is not completely necessary since the time between injections is very reproducible. Control of ionic strength is important in both HILIC and reversed-phase separations, and this is easily accomplished with today’s chromatographic systems. For maximum reproducibility and ruggedness it is recommended to maintain a minimum effective buffer concentration of at least 5 mM (5 mM in the mixed mobile phase).

Cotta: HILIC is a complementary chromatography technique that offers polar analyte retention where reversed phase offers little to no retention of these analytes. It differs from normal-phase chromatography in that it employs an organic–water mobile phase system, thereby overcoming some of the limitations of normal-phase chromatography (dedicated LC system use, poor sample solubility, restricted compatibility with electrospray ionization mass spectrometry [MS], and poor reproducibility). While isocratic operation simplifies all chromatographic methods regardless of mode, HILIC can be very reproducible when used in a gradient manner. For optimal gradient performance it’s recommended that the additive or buffer be added to both the organic and aqueous mobile phase lines so that a constant ionic strength is achieved. For best peak shape and retention, maintain 10 mM buffer or 0.2% additive in the gradient at all times. Adequate initial column equilibration (50 column volumes) and reequilibration between injections (8–10 column volumes) are important to avoid retention time drift.

What are the limits of sample solvents for injection of an analyte onto a HILIC column, in terms of maintaining good peak shape and efficiency?

Long: Solvents that are typically considered “strong for reversed phase” may be ideal injection solvents for HILIC. These typically include acetonitrile or dimethyl sulfoxide (DMSO). The addition of water to an injection solvent can typically cause substantial peak broadening and so should be avoided except when using very small injection volumes. As with reversed phase, best results are achieved when samples are made up in mobile phase or a close approximation.

Koerner and Sanchez: Once again the considerations are similar to those for reversed phase but HILIC is generally more sensitive to sample solvent strength–mobile phase mismatch than reversed phase. As in reversed phase, the effect of this mismatch becomes more problematic as the sample volume increases; that is, a larger mismatch can be tolerated with smaller sample volumes. Ideally the sample would be dissolved in the starting mobile phase, which typically contains 80–95% (v/v) acetonitrile. Sample solubility in high concentrations of acetonitrile can be problematic and some have tried replacing a fraction of the acetonitrile with acetone to improve solubility without sacrificing chromatographic performance. For systems that utilize a needle wash, use of acetonitrile or 80–90% (v/v) acetonitrile in water as the wash solvent is recommended, since some fraction of this solvent typically is injected along with the sample.

Cotta: As with other modes of chromatography, the best chromatographic performance in HILIC is achieved when the sample is dissolved in the initial mobile-phase composition. Peak distortion may occur when the sample is dissolved in a solvent of different polarity and viscosity to that of the mobile phase. A good starting place for a sample diluent is a mixture of 75% acetonitrile and 25% methanol for the best combination of peak shape and sample solubility. 0.2% formic acid or ammonium hydroxide can be added for better solubility.

How can HILIC be effectively combined with other modes to create practical 2-D HPLC formats?

Long: HILIC has been combined with reversed-phase HPLC using on- and off-line modes. In these cases the off-line mode system takes advantage of the peak order reversal and further optimizes the separation using pH and buffer strength. A wide variety of phases allow further varied separations.

Koerner and Sanchez: Several approaches to utilizing HILIC in 2D separations have been described in the literature such as reversed phase–HILIC and ion exchange–HILIC. Most examples involve off-line 2D where fractions are collected from one dimension and reinjected on the other. The advantage of the off-line approach is that the elution strength of the collected fractions can be adjusted before injection on the second dimension. The compatibility of eluate from the first dimension with the mobile phase and column in the second dimension can be overcome with the use of appropriately sized first and second column dimensions as well as appropriate plumbing arrangements. An on-line 2D ion exchange–HILIC separation has been shown in the literature demonstrating that the limits for using HILIC in 2D separations are in the amount of effort and expense required.

Cotta: Since HILIC is an orthogonal technique to reversed phase, the two techniques can be combined in an automated and routine fashion that is capable of processing a large number of complex samples. For example, this practice is currently being employed in the area of lipid profiling. Reversed-phase chromatography separates lipids based on their hydrophobicity, but does not show class differences. HILIC chromatography, on the other hand, provides separation based upon the lipid head group. By exploiting the advantages of both modes of chromatography in a 2D LC–MS method, chromatographers can achieve enhanced resolution, peak shape, and specificity, all of which is possible on a commercially available system.

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